Targeted delivery of TGF-β mRNA to murine lung parenchyma using one-component ionizable amphiphilic Janus Dendrimers

Meshanni, Jaclynn A.; Stevenson, Emily R.; Zhang, Dapeng; Sun, Rachel; Ona, Nathan A.; Reagan, Erin K.; Abramova, Elena; Guo, Chang-Jiang; Wilkinson, Melissa; Baboo, Ishana; Yang, Yuzi; Pan, Liuyan; Maurya, Devendra S.; Percec, Virgil; Li, Yongsheng; Gow, Andrew; Weissman, Drew; Atochina-Vasserman, Elena N.

doi:10.1038/s41467-025-56448-y

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Published: 21 February 2025

Targeted delivery of TGF-β mRNA to murine lung parenchyma using one-component ionizable amphiphilic Janus Dendrimers

Nature Communications volume 16, Article number: 1806 (2025) Cite this article

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Abstract

Current clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways, such as treatment with nebulized instillation and inhalation. However, targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here, we show the development of an mRNA therapeutic for the lower lung in mice using one-component Ionizable Amphiphilic Janus Dendrimers as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA, transforming growth factor-beta, to produce transient protein expression in the lower regions of the lung. This study highlights a method for precise, effective, and safe delivery of TGF-β mRNA to the lung in mice. This delivery system offers a promising approach for targeting therapeutics to the specific tissues, a strategy necessary to fill the current clinical gap in treating parenchymal lung injury and disease.

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Introduction

Acute lung injury (ALI) is a prevalent condition in the United States, with 200,000 new diagnoses each year, resulting in high morbidity and mortality^1,2,3,4. Injury progression is a result of epithelial barrier dysfunction, alveolar damage, pulmonary edema, and surfactant dysfunction brought on by changes in innate immune, epithelial, and endothelial cell dysfunction^1,5,6,7,8. There are 23 generations of division in the lung, where Generations 0–16 are the conductive airways and 16–23 are the respiratory airways. Within the pulmonary circuit, blood flow is matched to gas exchange in the respiratory portion of the lung⁹. Many of the effects of ALI, a result of the liquid leak, are observed in the lung parenchyma beyond the 16^th generation. However, drug delivery to this area is challenging often leading to poor specificity and an uneven distribution^{10,11,12,13,14}. Therefore, there is a therapeutic gap in the treatment of ALI requiring the development of methods to deliver pharmacological agents to the lower lung. This is a particular challenge when administering a complex biologic, such as anti-inflammatory cytokine mRNA, to the site of injury.

Since the beginning of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) pandemic in 2019, there have been a series of major breakthroughs in the development of nucleoside-modified mRNA vaccines by both major research institutions and the pharmaceutical industry^15,16,17,18. Currently, the leading delivery system for mRNA vaccines are four-component lipid nanoparticle (LNP) synthetic delivery systems, which consist of ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-conjugated lipids, which can be assembled by microfluidic or T-tube technology^19,20. Despite their wide adoption of COVID-19 vaccinations, the targeted delivery of mRNA to the lung with four-component LNPs has not proven successful^{21,22,23,24,25,26,27}.

Recently, we reported a novel self-assembling one-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) synthetic delivery system for mRNA, consisting of functional hydrophilic dendrons conjugated to hydrophobic dendrons^28,29. IAJDs and mRNA can be co-assembled into unilamellar and multilamellar onion-like dendrimersome nanoparticles (DNPs) by simple mixing in acetate buffer rather than microfluidic and T-tube devices. Previously we have shown that certain of these IAJDs display organ specificity²⁹. In addition to eliminating structural tissues that can arise from mixtures of four-component LNPs, this technology may allow for programmed delivery to specific organs, such as the lung.

In this study, we explore the potential of one-component IAJDs to deliver therapeutic mRNA encoding the anti-inflammatory cytokine, transforming growth factor-beta (TGF-β) to the lung parenchyma. TGF-β is essential for the resolution of inflammatory events through the regulation of immune cell migration, division, and pro-resolution signal transduction^30,31,32,33. If introduced early in the inflammatory response, TGF-β has the capacity to accelerate resolution, ultimately reducing the incidence of complex lung injury and disease. In addition, we test the nontoxic and transient nature of its delivery, as persistent TGF-β delivery may result in excessive pro-resolution signaling, leading to the development of lung fibrosis over time^32,34,35,36. Here, we confirm that IAJDs can be utilized for the successful and targeted delivery of cytokine mRNA diffusely throughout the lung and that the effects of this delivery are transient, reducing the risk of fibrotic development. Furthermore, we evaluate the potential for IAJD targeted delivery of TGF-β mRNA to limit ALI using an intratracheal bleomycin (ITB) model^3,36,37. These studies contribute to the progress of genetic nanomedicine and raise the possibility of lung-based nanotherapeutics.

Results and discussion

Characterization of lung-specific IAJD34 for targeted mRNA delivery to the lung

Many IAJDs identified are capable of targeting the lung³⁸. With a relatively large chemical structure and a pK_a of 6.74 lending to a strongly lung-specific targeting, IAJD34 was selected for further characterization (Fig. 1A). To evaluate lung-specific delivery, the Luc mRNA-IAJD34 formulation was injected into mice at an initial dose of 10 µg per mouse. Four hours post-injection, whole-body, and organ luminescence were quantified via IVIS. Luciferin intensity in the lungs was strong and several magnitudes higher compared to other organs (Fig. 1B). To evaluate the kinetics of luciferin protein production over time, mice were injected with the Luc mRNA-IAJD34 formulation at a dose of 10 µg per mouse. Live mice were imaged at various time points (4, 24, 48, and 72 h) post-injection, and the luciferase intensity was quantified as a whole-body flux (p/s) (Fig. 1C). Luciferin intensity peaked at 4 h post-injection (3.12 × 10⁷ ± 9.15 × 10⁶), by 24 h, the flux intensity decreased but remained elevated by 37%. At 48 and 72-h post-injection, there was a more significant drop to 3% and 1.9%, respectively (Fig. 1D and Supplementary Table 2).

**Fig. 1: Specificity of IAJD34 for targeted mRNA delivery to the lung.**

The stability of the Luc mRNA-IAJD34 formulation was studied over time at +4 °C. The formulation was stored at +4 °C for 5 days and then injected into mice. Whole-body luciferase intensity did not change significantly compared to the freshly prepared formulation (Supplementary Fig. 2A). Thus, the one-component IAJD34 formulated with Luc mRNA maintains stability and activity at 4 °C and can be stored for at least 5 days prior to use. The stability of particle size and PDI of IAJD34 formulated with TGF-β mRNA was also evaluated and was found to be stable for at least 5 weeks at +4 °C (Supplementary Fig. 2B).

To evaluate the impact of dose on luciferase expression, Luc mRNA-IAJD34 was injected at 10, 20, and 30 µg doses per mouse and imaged at 24 h post-injection. There was a dose-dependent increase in lung-specific luciferase expression, reaching 9.3 × 10⁸ at 30 µg per mouse (Fig. 1E, Supplementary Table 3 and Supplementary Fig. 3). Immunohistochemical staining of lung tissue with the 30 µg dose revealed that expression of luciferase protein was well distributed throughout the alveolar epithelium, with minimal staining in the upper airway compared to the control (Fig. 1F). The mRNA delivery to the alveolar level (i.e., below the 16th generation of the airway tree) had previously been unsuccessful, the expression of luciferase in these lower respiratory zones suggests our approach may serve as a promising therapeutic.

Notably, our evaluation of IAJD34 toxicity and efficacy was limited using this formulation because luciferase is encoded by a large mRNA and has no biological function within the cell. Thus, delivery of clinically relevant mRNA was essential to fully characterize the therapeutic nature of IAJD34.

Evaluation the pulmonary delivery of therapeutic mRNA

Following confirmation of IAJD34 lung specificity, we focused on designing a therapeutic mRNA for lung-targeted delivery. TGF-β, a prominent anti-inflammatory cytokine associated with the resolution of ALI, was selected as our mRNA of interest^31,32. The production and quality of TGF-β mRNA were validated (Supplementary Fig. 4A). Protein expression of TGF-β was confirmed in Human epithelial kidney (HEK293) cells following transfection with TGF-β mRNA (Supplementary Fig. 4B).

To evaluate inflammatory effects in the lung, IAJD34 formulated with TGF-β mRNA (TGF-β mRNA-IAJD34) was injected at 10, 20, and 30 µg per mouse and bronchoalveolar lavage fluid (BAL) markers were measured 24 h post-injection. Increasing doses (10 µg and 20 µg) had no effect on BAL phospholipids or total cell count in the BAL fluid (Fig. 2A). However, a significant increase in BAL fluid protein and neutrophils was observed at the 30 µg dose, indicating epithelial barrier dysfunction (Fig. 2A, B). Although BAL fluid protein was increased, there was no significant increase in BAL IL-6 levels (Fig. 2D), suggesting this may have been a downstream effect of abundant TGF-β delivery rather than a side effect of IAJD34 formulation.

**Fig. 2: Evaluation of dose-dependent toxicity of TGFβ mRNA delivery with IAJD34.**

Histological sections of organs were stained with H&E and evaluated by a board-certified pathologist. No pathological alterations were observed in the liver, spleen, or kidney at any of the tested doses (Supplementary Fig. 5). In the lung histology, a dose-dependent increase in fibrin deposition and lymphocyte infiltration was observed, as was expected at higher doses of TGF-β (Fig. 2C). Liver function was also evaluated by measuring serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), known markers of liver function³⁹. Increased ALT was observed at the 30 µg dose, with no significant change in AST (Fig. 2E). Thus, systemic pathological and toxicological markers are limited in the 10 µg and 20 µg doses of TGF-β mRNA-IAJD34. Even at the highest dose, 30 µg, increases in systemic toxicity markers were minimal. Established four-component LNPs are typically dosed between 1 and 10 μg for vaccinations for SARS-COV2 in mice, indicating that 10 µg would likely be sufficient⁴⁰. In addition, most studies using IAJDs report that 10 µg or less of mRNA to be effective for organ-specific detection³⁸.

To determine alveolar macrophage (AM) activation, BAL cells were evaluated by flow cytometry as previously described³. AMs were defined as viable CD45⁺F4/80⁺SiglecF⁺ cells (Supplementary Fig. 6A). Inflammatory activation was characterized by the expression of Ly6c and CD11c. No dose-dependent difference was observed in the AM phenotype, with a majority of cells being resident AMs (Ly6c⁻/CD11c⁺) under all conditions. This was consistent with the lack of observed acute inflammatory activation (Fig. 3A). There were also no differences in Ly6C/CD206 AM populations at any dose of TGF-β mRNA-IAJD34 compared to the control (Supplemental Table 4). Interstitial macrophages (IMs) were identified in CD45⁺ lung digest cells. IMs were defined as viable being CD45⁺, CD11b⁺, and SiglecF^- cells as previously described (Supplementary Fig. 6B)³. Ly6c⁺ IMs were increased in all doses of TGF-β mRNA-IAJD34 with no increases in either CD11c⁺ or CD206⁺ expression at any dose (Fig. 3B and Supplementary Fig. 7). Ultimately, no acute inflammatory activation was observed in IMs as a result of TGF-β mRNA-IAJD34 treatment when compared to control. It is unclear why treatment with TGF-β mRNA-IAJD34 increased Ly6c in IMs, but a previous study evaluating renal interstitial macrophages in Tgfbr2^fl/fl mice showed a decrease in Ly6c⁺ macrophages indicating that there may be a relationship between Ly6c and TGF-β signaling⁴¹. Further analysis will be necessary to fully understand TGF-β mRNA-IAJD34’s effects on Ly6c⁺ IMs.

**Fig. 3: Absence of inflammatory activation in response to increasing doses of TGF-β mRNA-IAJD34 in alveolar macrophages and interstitial macrophages.**

TGF-β protein exists in an unprocessed form with a latency-associated peptide that is cleaved to form an active protein, which can then initiate signal transduction^42,43. To confirm that TGF-β mRNA-IAJD34 was reaching the lung and being processed, TGF-β protein expression was confirmed in lung tissue digest using western blotting. Cleaved TGF-β was significantly increased at all doses compared to the control, whereas unprocessed and total TGF-β were only significantly increased in the 20 µg and 30 µg groups (Fig. 4).

**Fig. 4: Dose-dependent expression of TGF-β mRNA-IAJD34 in lung tissue.**

We next evaluated the presence of other cytokines and chemokines in the BAL fluid that can be upregulated or downregulated by TGF-β signaling (Supplementary Table 5). Granulocyte-colony stimulating factor (G-CSF), identified as an anti-inflammatory pro-neutrophilic growth factor, was elevated at the 30 µg dose of TGF-β mRNA-IAJD34 compared to the control (Fig. 5A)⁴⁴. By contrast, pro-inflammatory cytokines interleukin-1 alpha (IL-1a), interleukin-9 (IL-9), and tumor necrosis factor (TNFα) were decreased in a dose-dependent manner (Fig. 5B–D)^45,46. Thus, at 24 h post-treatment, TGF-β protein expression alters the downstream signaling of pro- and anti-inflammatory cytokines. These observations are likely directly associated with increased TGF-β signaling and not an off-target effect of IAJD34.

**Fig. 5: TGF-β mRNA-IAJD34-induced alterations in BAL cytokine production and cellular metabolic function.**

Finally, we studied metabolic upregulation, which is predicted to occur with increased protein synthesis and cell signaling stimulated by TGF-β mRNA. CD45⁺ lung-digest cells were evaluated by employing the glycolysis stress test and the mito stress test. For the glycolysis stress test, the extracellular acidification rate (ECAR) was recorded. Glycolysis was determined by the ECAR measurement prior to glucose injection subtracted from the maximum ECAR before oligomycin injection. Injection with TGF-β mRNA-IAJD34 caused a dose-dependent increase in glycolysis, with a significant increase observed in the 20 µg and 30 µg groups compared to the control (Fig. 5E). This increase was associated with increased cytokine production, likely a direct impact of TGF-β signaling cascades^47,48. For the mito stress test, oxygen consumption rate (OCR) was used to determine oxidative phosphorylation. OCR data were first normalized to the post rotenone/antimycin (R/A) injections to control for non-mitochondrial oxygen consumption. Maximal respiration was determined from OCR data as the maximum OCR following the injection of FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), a potent mitochondrial uncoupler. A TGF-β mRNA-IAJD34 induced dose-dependent increase was observed in maximal respiration, though this was not significant (Fig. 5F). Although increased metabolic demand is likely a part of increased translational processes, it has also been hypothesized to be a direct effect of increased TGF-β expression and macrophage activation⁴⁸. Thus, treatment with TGF-β mRNA-IAJD34 successfully alters macrophage cell phenotype and cytokine expression as expected with the production of TGF-β protein.

Evaluating pulmonary delivery of TGF-β mRNA-IAJD34 over time

Due to the lack of toxicity, limited alterations in cell phenotype, and efficient expression of TGF-β protein and downstream signals, the 10 µg dose of TGF-β mRNA-IAJD34 was selected for further development.

The 10 µg dose of empty-IAJD34 or TGF-β mRNA-IAJD34 was further evaluated at 4, 24, and 48 h post-injection. Treatment with TGF-β mRNA-IAJD34 did not increase BAL protein or phospholipids at any time point (Supplementary Fig. 8).

Immunohistochemistry (IHC) staining of lung tissue confirmed that TGF-β protein expression was transiently expressed in lung tissue, peaking at 4 h post-injection, and persisting up to 48 h (Fig. 6). Unprocessed and cleaved TGF-β were also measured in whole lung tissue, showing significant increases in all forms of TGF-β at 4 h post-injection, an increase in cleaved TGF-β at 24 h, and no significant increases in any TGF-β isoforms at 48 h post-injection compared to the control (Fig. 7). Consistent with TGF-β dose-response findings, there were no changes in AM or IM inflammatory activation at any time point compared to control, though slight increases in Ly6c⁺ IMs were observed (Supplementary Fig. 9 and Supplementary Table 6). Thus, a 10 µg dose of TGF-β mRNA-IAJD34 can deliver robust and transient TGF-β to the lung without significant signs of inflammation or toxicity. Transient expression is therapeutically beneficial as prolonged TGF-β expression can lead to fibrin deposition and fibrosis³⁴. However, in the future, it may be necessary to deliver repeat doses of TGF-β mRNA-IAJD34 until resolution is achieved.

**Fig. 6: Cell specific expression of TGF-β mRNA-IAJD34 in the lung over time.**

**Fig. 7: TGF-β protein expression is transiently induced after mRNA delivery to lung via IAJD34.**

TGF-β mRNA-IAJD34 can limit pulmonary injury following exposure to bleomycin

The ultimate goal of lung-targeted delivery of therapeutic mRNA is to generate treatment strategies for pulmonary injury and disease. Intratracheal bleomycin (ITB) is a laboratory model of ALI, characterized by acute pulmonary inflammation occurring over the first 7 days, transitioning to fibrotic development around 14 days, and resolving at 21–28 days post initial exposure^3,4,49. It should be noted that not all models of ITB result in the same level of fibrosis^50,51,52. To have interventive therapeutic potential, treatment with TGF-β is predicted to be most effective during the development of pro-inflammatory cellular activation and pathology.

For initial studies, animals were treated with a 10 µg dose of TGF-β mRNA-IAJD34 or empty-IAJD34 concurrently to instillation of bleomycin or PBS control. To align with initial pulmonary inflammation development and the expression of TGF-β mRNA-IAJD34, animals were euthanized 3 days post ITB or PBS exposure. ITB causes weight loss in mice over the course of the first 7 days⁵¹. Substantial weight loss was observed in groups exposed to ITB, where treatment with TGF-β mRNA-IAJD34 had no impact on ITB-induced decreases in percent body weight (Supplementary Fig. 10).

ITB is also associated with increases in alveolar thickness, immune cell infiltration, and tissue consolidation that variably develop over the first 7 days post-exposure^3,4,49,53. An increase in alveolar epithelial thickness was observed in ITB + empty-IAJD34 animals when compared to PBS control, with no change in percent white space and nuclei count (Fig. 8). Treatment with TGF-β mRNA-IAJD34 alone did not significantly increase epithelial thickness compared to PBS control. However, in the presence of TGF-β mRNA-IAJD34, there was no significant increase in epithelial thickness in response to ITB (Fig. 8). The limited ITB-induced histological alterations are likely a consequence of evaluating injury at such an early time point and a limitation of using BALB/c mice as a model. Notably, BALB/c mice are slightly more resistant to ITB exposure as compared to C57BL6/J mice^50,51,52,54. BALB/c mice were chosen for this study as IAJD targeting and development has primarily been characterized in this strain of mice^29,38,55. The 3-day timepoint was chosen as the peak of inflammatory signaling post ITB injury. Successful delivery of TGF-β is predicted to have its greatest effect on inflammatory cellular phenotype at this time point.

**Fig. 8: Effects of TGF-β mRNA-IAJD34 on bleomycin-induced histological alterations.**

Beyond histopathological alterations, ITB-induced ALI is also associated with increases in pulmonary epithelial injury, marked by increased BAL fluid protein and disrupted epithelial lipid barriers⁵⁶. Exposure to ITB caused a significant increase in BAL fluid protein content as compared to control. TGF-β mRNA-IAJD34 treatment reduced the protein level within the BAL but not significantly (Fig. 9A). Neither ITB nor TGF-β mRNA-IAJD34 had an effect on BAL fluid phospholipid levels, hypothesized to be a limitation of the 3-day time-point (Fig. 9B). As shown previously, ITB induces a loss of BAL cells, a consequence of early pulmonary inflammation and cell death. However, in the presence of TGF-β mRNA-IAJD34, this ITB-induced loss was not observed (Fig. 9C)³.

**Fig. 9: Effects of TGF-β mRNA-IAJD34 bleomycin-induced injury.**

To characterize the loss of BAL cells, AMs (viable CD45⁺Siglec F⁺F4/80⁺ BAL cells) were characterized further as being resident (CD11c⁺CD11b⁻), recruited (CD11c⁻CD11b⁺), or migratory macrophages (CD11c⁺CD11b⁺). In line with previous 7-day studies, exposure to bleomycin reduced resident AMs and increased recruited AMs compared to PBS control (Fig. 9D, E)^3,4. Although treatment with TGF-β mRNA-IAJD34 did not prevent ITB-induced decreases in resident AMs, treatment did mitigate ITB-induced increases in recruited AMs (Fig. 9D, E). In contrast to TGF-β mRNA-IAJD34 dose-response studies, Ly6c⁺ IMs (viable CD45⁺/CD11b⁺SiglecF⁻ lung-digest cells) were decreased in ITB exposed animals irrespective of TGF-β mRNA-IAJD34 treatment (Fig. 9F). ITB-induced changes in AM and IM populations were variable between animals due to the early inflammatory time point and the less sensitive BALB/c model. Treatment with TGF-β mRNA-IAJD34 helped mitigate some AM alterations but had no effect on IMs at this time point. Innate TGF-β expression is expressed more highly in human AMs when compared to other lung cell types³³. There is some evidence that TGF-β plays a less significant role in IM cell maturation and activity, but this remains largely speculative³³. Further model development will be necessary to understand the role that TGF-β plays in modulating ITB-induced AM and IM cell characteristics.

We next evaluated the downstream effects of TGF-β mRNA-IAJD34 on cytokine signaling following exposure to ITB beyond pathological and inflammatory indicators (Fig. 10 and Supplementary Table 7). As anticipated, exposure to ITB elevated levels of G-CSF, IL-6, and CXCL-10 compared to control. These neutrophilic or pro-inflammatory cytokines are elevated following ITB exposure (Fig. 10A–C)^57,58,59,60. Treatment with TGF-β mRNA-IAJD34 prevented ITB-induced increases in all three of these pro-inflammatory cytokines, consistent with the anti-inflammatory signaling of TGF-β (Fig. 10A–C). Exposure to ITB caused a reduction in IL-1α and IL-2 compared to PBS control, irrespective of treatment with TGF-β mRNA-IAJD34 (Fig. 10D, E). Both IL-1α and IL-2 are also pro-inflammatory cytokines, and it is unclear why ITB exposure decreased these cytokines, though it may be a result of AM resident cell loss. Thus, treatment with TGF-β mRNA-IAJD34 can prevent components of ITB-induced pulmonary inflammatory signaling.

**Fig. 10: Effects of TGF-β mRNA-IAJD34 bleomycin-induced cytokine production.**

IAJD34 is a single-component lipid particle designed for the targeted delivery of mRNA to the lung³⁸. The current studies have shown that IAJD34 can successfully deliver luciferase mRNA to the lung, specifically targeting lung parenchyma. This is of particular therapeutic advantage as targeting the smaller airways, especially the respiratory regions beyond the 16^th generation of the respiratory tree, is necessary to treat most lung injury and consequent disease^1,5,7. While necessary, targeting these areas of the lung is challenging and has proven difficult in clinical settings¹.

It is not clear why IAJD34 particles localize to the lung. However, we hypothesize that IAJD34 is able to target these areas due to its lipophilic and charged nature. When injected intravenously, we propose that these characteristics lead to pulmonary accumulation as it is the first pass organ for i.v. administration. TGF-β mRNA-IAJD34 was able to deliver dose-dependent levels of TGF-β mRNA to the lung with limited inflammation and toxicity observed. Once delivered, TGF-β protein was produced, processed, and mediated downstream cytokine signaling. Delivery of TGF-β was transiently expressed over the course of 48 h, which is important as long-term delivery of TGF-β can lead to significant fibrosis.

TGF-β mRNA-IAJD34 was used to treat ITB effects on the lung 3 days post-injury showed only mild improvements in lung histology and barrier function. Although the use of the day 3 timepoint in BALB/c mice meant that these factors were only mildly affected by ITB, our intention was to demonstrate that delivery of TGF-β mRNA-IAJD34 to the lung would reduce inflammatory signaling. Indeed, there was a significant TGF-β mRNA-IAJD34 effect that correlated with TGF-β expression. These results indicate that we could successfully deliver TGF-β to the lower lung and observe a significant signaling effect. Furthermore, this effect appears to be transitory in nature as would be required in a therapeutic setting.

To establish TGF-β mRNA-IAJD34 as a therapeutic in this ALI model, we will need to characterize the effect of TGF-β at 7 days or longer post-injury, which may require multiple dosing regimens. This study establishes the potential use of IAJD34 to treat ALI and other pulmonary ailments that currently require targeted clinical interventions and demonstrates the potential of mRNA delivery for therapeutic use in the lung.

Methods

mRNA production and characterization

Codon-optimized sequences of firefly luciferase (Luc-mRNA, GenBank: MK484108.1) or tumor growth factor-β (TGFβ-mRNA, GenBank: NM_011577.2) were synthesized and cloned into an mRNA production plasmid^25,61. Briefly, nucleoside-modified mRNAs were transcribed to contain 101 nucleotide-long poly(A) tails. mRNAs were modified with m1Ψ-5′-triphosphate (TriLink, # N-1081) instead of UTP and capped cotranscriptionally using the trinucleotide cap1 analog, CleanCap (TriLink, # N-7413). mRNA was purified by cellulose (Sigma-Aldrich, # 11363-250 G) purification²⁵. All mRNA were analyzed for quality control through agarose gel electrophoresis, dsRNA, endotoxin, and IFN-α assays and in vitro transfection. mRNA was stored at −20 °C until ready for use.

TGF-β mRNA transfection in human embryonic kidney 293 cell line

Human Embryonic Kidney (HEK) 293 cells (ATCC CRL-1573) were seeded into a 24-well cell culture plate at a density of 150,000 per well in Dulbecco’s Modified Enriched Medium (DMEM) containing fetal bovine serum (10%), L-glutamine, and penicillin-streptomycin. Cells were allowed to grow for 24 h, and then TGF-β mRNA (500 n per/well) was transfected into cells in triplicate using lipofectamine MessengerMax (Life Technologies, Carlsbad, CA) and OptiMEM serum-free medium (ThermoFisher Scientific, # 31985-062, Rockford, IL) according to manufacturer guidelines. Cells were allowed to grow for an additional 24 h, and then samples collected in RIPA buffer (Sigma Aldrich, # R0278-500mL) containing cOmpleteMini protease cocktail inhibitors (Roche Diagnostics, # 11836153001). Samples were centrifuged (300 × g, 5 min, 4 °C) to remove cellular debris, and supernatant was then collected. Protein concentrations were determined using a Pierce™ BCA Protein Assay (Peirce, # 23227).

Immunoblots

Whole cell lysates samples (10 µg per well) were analyzed for TGF- β by denaturing SDS-PAGE 4–12% Bis-Tris followed by western blot analysis. Samples were transferred to PVDF membrane using an iBlot 2 dry apparatus (ThermoFisher Scientific, Rockford, IL), The membranes were blocked in non-fat dried milk (10% with 5% TTBS), and TGF- β was detected using primary anti TGF-β antibody (1:5000, Abcam ab215715; Waltham, MA) overnight at 4 °C, followed by a secondary goat anti-rabbit IgG HRP at 1:10,000 for 1 h at room temperature. Membranes were stripped and re-probed with anti-GAPDH (Cell Signaling Technology, # 2118S, Danvers, MA) for 1 h at room temperature at a 1:1000 dilution, and a secondary goat anti-rabbit IgG HRP (Bio-Rad, # 170-6515 Bio-Rad, Hercules, CA) at a 1:10,000 for 1 h at room temperature. Membranes were washed in ECL Prime Western Blotting Detection Reagent (Amersham Biosciences, Amersham, UK, # RPN2232) prior to visualization using an Amersham Cytiva imager (Amersham Biosciences, Amersham, UK).

To analyze TGF-β protein expression in lung tissue, immunoblots were performed on collected lung tissue. Tissue from the accessory lobe was mechanically homogenized on dry ice and digested in lysis buffer with protease inhibitors (100 µL/0.33 g tissue). The tissues were sonicated, centrifuged (2 min, 1000 × g), and supernatants were assessed for protein concentration using a Pierce™ BCA Protein Assay. Equal amounts of protein from lung tissue samples (45 µg per well) were analyzed for TGF-β protein expression as described above. Antibodies for TGF-β were used at 1:5000, and goat anti-rabbit HRP at 1:5000. Membranes were washed in ECL Prime Western Blotting Detection Reagent prior to visualization on x-ray film. Uncropped gel images are available in the Source Data file.

Co-assembly of IAJD34 and mRNA

IAJD34 was synthesized as previously described²⁹, and the purity and structural identity of final products and intermediates were determined using various techniques, including thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC), ¹H and ¹³C NMR, and Electrospray Ionization Time-of-Flight (ESI-TOF) mass spectrometry²⁹. Co-assembly of IAJD34 and mRNA was performed^29,55. Nucleoside-modified mRNA encoding Luc mRNA or TGF-β mRNA was dissolved at a concentration of 4 mg/mL in UltraPure DNase/RNase-free PCR-certified water (Teknova, # W3440). IAJD34 was dissolved in ethanol at a concentration of 80 mg/mL. For the co-assembly of IAJD34 with mRNA, the mRNA was mixed with 10 mM acetate buffer (pH 4.0), and this solution was rapidly mixed with IAJD34 in ethanol at an IAJD34/mRNA weight-to-weight ratio of 40 and vortexed for 5 s. The prepared formulation was analyzed for size (nm), polydispersity index (PDI), and zeta potential using dynamic light scattering (DLS) prior to injection of empty-IAJD34, Luc mRNA-IAJD34, and TGF-β mRNA-IAJD34. Prepared formulations were also evaluated for mRNA encapsulation efficiency, defined as the amount of mRNA encapsulated versus free, was determined using a Quant-iT™ RiboGreen™ RNA Assay Kit (Invitrogen) according to manufacturer instructions. Briefly, to measure non-encapsulated mRNA (free mRNA), mRNA-IAJD formulations were diluted with Tris-EDTA buffer (TE) and treated with the RiboGreen™ reagent. To measure total mRNA, mRNA-IAJD formulations were disrupted by treatment with 2% Triton X-100 in TE buffer and treated with RiboGreen™ reagent. Both conditions were performed in triplicate to ensure proper release of encapsulated mRNA. Fluorescence changes were measured using a Qubit 4 Fluorometer (Invitrogen) at 480 nm excitation and 520 nm emission. mRNA encapsulation efficiency was calculated as: (total mRNA – free mRNA)/total mRNA × 100% (EE, %). (Supplementary Fig. 1A).

Cryogenic Transmission Electron Microscopy (Cryo-TEM) imaging was done to characterize the morphologies of mRNA-IAJD34 nanoparticles (Supplementary Fig. 1B). Samples were prepared by deposition of 5 μL sample solution onto a 300-mesh copper TEM grid, and samples were immediately flash-frozen in liquid ethane using a Vitrobot Mark IV system (Thermo Fisher) and maintained at liquid nitrogen temperature. Images were recorded at − 170 °C with an acceleration voltage of 200 kV using a Talos F200C G2 microscope.

Purity of IAJD34 was also confirmed using carbon 13 nuclear magnetic imaging (¹³C NMR), hydrogen nuclear magnetic imaging (¹H NMR), High-performance liquid chromatography (HPLC), and Electrospray Ionization Time-of-Flight (ESI-TOF) (Supplementary Fig. 1C–F). ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance Neo (400 MHz) spectrometer at 298 K. Deuterated chloroform (CDCl₃) was used as the solvent. NMR chemical shifts were calculated with the residual protic solvent of CDCl₃ (1H, δ 7.26 ppm; 13 C, δ 77.16 ppm) as an internal reference. The purity of the products was determined by HPLC. HPLC was performed using a Thermo Fisher Vanquish Core at 298 K on a Chiralpak AD column. Methanol was used as an elute at a flow rate of 1.0 mL/min; λ = 210 nm; tR = 3.122 min. ESI-TOF mass spectrometry was performed on a Thermo Fisher Scientific Exactive^TM. IAJD 34 was dissolved in dichloromethane (3 mg/mL). The report contains the ionization method (ESI in positive mode), chemical formula, [ion]charge, mass to charge ratio (m/z): calculated value, found value.

Animal studies

Male and female wild-type BALB/c (8–10 weeks) mice obtained from Charles River (Luc mRNA study) or Jackson Laboratories (TGF-β studies) were used for all experiments. Studies used male and/or female mice and are indicated in the source data file. Studies were not powered to compare sex. Mice were housed under standard conditions (12 hr light/12 hr dark, 65–75 °F, 40–60% humidity) with food (Lab diet 5053, Irradiated Rodent Diet) and water provided ad libitum. All experiments were conducted in accordance with the University of Pennsylvania and Rutgers University IACUC-approved protocols adhering to the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were housed and cared for in the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facilities. These studies abide by the ARRIVE guidelines. During experimentation, animal welfare was monitored daily, and animals that displayed extensive pain or discomfort beyond the allowance of the study were humanely euthanized via CO₂ in accordance with the IACUC protocols.

Bioluminescence characterization for Luc mRNA delivery to mouse lungs

Female or male 6-8-week-old BALB/c mice were administered 10 μg of Luc mRNA-IAJD34 in a100 μL volume via retro-orbital intravenous injection. Four hours post-injection, mice were injected intraperitoneally (i.p.) with D-luciferin (Regis Technologies) at a dose of 150 mg/kg of body weight. The mice were anesthetized in a ventilated anesthesia chamber with 3% isoflurane (Piramal Healthcare Limited) in oxygen and imaged 10 min post D-luciferin injection using an in vivo imaging system (IVIS, PerkinElmer, Waltham, MA). For organs imaging, mice were sacrificed, organs were collected immediately, and bioluminescence imaging was performed. Bioluminescence was quantified as proton flux (photons/seconds) in each region of interest using Living Image software v3.2 (PerkinElmer)⁵⁵.

TGF-β mRNA-IAJD34 formulation toxicity studies

TGF-β mRNA was co-assembled with IAJD34 as described above. The formulation was then concentrated using a Vivaspin ultrafiltration spin column MWCO 10,000 (Cytiva # 28-9322-47) according to manufacture guidelines. Mice were randomly assigned to control or treated. Mice were anesthetized with isoflurane and received a single retro-orbital intravenous injection of 10 µg, 20 µg, or 30 µg of TGF-β mRNA co-assembled with IAJD34 or empty IAJD34. The following criteria to assess the toxicity of formulated TGF-β mRNA-IAJDs was utilized: mice behavior, serum and BAL fluid cytokine analysis, liver enzyme activities, and main organs’ histological score. Mice were sacrificed 24 h post-injections, and their serum samples and organs were collected. Organs were fixed in 4% paraformaldehyde and further stained with H&E as described below. The liver, spleen, kidney, and lungs were inspected for signs of inflammation and deviations from normal histology.

Enzyme-linked immunosorbent assay (ELISA)

ELISAs for aspartate aminotransferase (AST; Abcam ab263882) and alanine aminotransferase (ALT; Abcam ab282882) were performed in serum in a 1:80 dilution according to manufacture guidelines. Interleukin-6 (IL-6; Abcam ab222503) was used to measure serum and first wash BAL fluid levels in accordance with manufacturer guidelines.

Multiplex cytokine analysis

Cytokine in the first 1 mL of BAL fluid was performed by the University of Pennsylvania Human Immunology Core (RRID: SCR_022380) using a Milliplex Max Mouse Cytokine/Chemokine Magnetic Bead Panel - Premixed 32 Plex - Immunology Multiplex Assay (MCYTMAG-70K-PX32; MilliporeSigma, Burlington, MA). Samples were run in duplicate, and cytokine and chemokine concentrations were determined from a matched standard curve.

Bleomycin mRNA-IAJD34 studies

Mice were randomly assigned to treatment groups. Mice were anesthetized with isoflurane and received a single retro-orbital intravenous injection of 10 µg of TGF-β mRNA formulated with IAJD34 or empty IAJD34. while still under anesthesia, mice immediately received a single intratracheal instillation of either 50 µL PBS or 50 µL of bleomycin (3U/kg of body weight) (Santa Cruz Biotechnology, Inc., Dallas, TX; # sc-200134B) diluted in PBS³. Following injections and treatment, mice were observed to ensure full recovery from anesthesia, and that dose was successfully administered. Animals were weighed daily and provided supportive care when necessary. Mice were sacrificed 3 days post intratracheal administration of bleomycin via a single intraperitoneal injection of ketamine (135 mg/kg of body weight) and xylazine (30 mg/kg of body weight) (Fort Dodge Animal Health, Fort Dodge, IA).

Bronchoalveolar lavage

Lungs were lavaged with 1 mL of ice-cold PBS, followed by five 1 mL washes of ice-cold PBS through a 20-gauge canula inserted into the trachea. Collected BAL fluid was centrifuged at 300 × g for 8 mins. The cell-free supernatant from the first wash was collected for protein and cytokine analysis, and the cell-free supernatant from the five subsequent washes (5 mL) was collected for phospholipid analysis. Cell pellets from both washes were combined and resuspended in 1 mL of staining buffer (5% FBS in PBS, 0.2% sodium azide) and assessed for viability using Trypan Blue Solution (0.4%, ThermoFisher Scientific, Rockford, IL). The total cell count was determined using a Z1 Counter particle counter (Beckman Colter). Approximately 10,000 cells were centrifuged on a Thermo Shandon Cytospin-3 at 750 rpm for 3 min onto a microscope slide, followed by Giemsa staining using a Hema 3™ Stat Pack (Fisherbrand, ThermoFisher Scientific, Rockford, IL). Total and differential cell counts were obtained. Cells were identified as macrophages, eosinophils, neutrophils, and lymphocytes by standard morphology.

Phospholipids

Collected cell-free BAL fluid (5 mL) was centrifuged (20,000 × g, 4 °C, 1 hour) and separated into large and small aggregate surfactant fractions^3,62. Phospholipids were extracted from the large aggregate fraction and resuspended in 30 µL 0.9% sodium chloride. Total phospholipids were measured by light absorbance at 830 nm using a standard curve ranging from 0 to 3.1 µg phosphate^3,63,64.

Lung tissue digest

Lung tissue from the right lobes was incubated at 37 °C for 30 min with intermittent shaking in 5 mL of collagenase buffer (2 mg/mL collagenase type IV (Sigma Aldrich, St. Louis, MO) in RPMI 1640 (ThermoFisher Scientific, Rockford, IL) with 5% FBS (ThermoFisher Scientific, Rockford, IL)). The digested tissue was filtered through a 70 µm strainer, washed with RPMI with 5% FBS, and centrifuged (6 min, 400 × g). The cell pellet was lysed with Red Blood Cell Lysis Buffer (Sigma Aldrich, St. Louis, MO) for 5 min. The purified cell pellet was resuspended at a concentration of 1 × 10⁸ cells/mL PBS with 2% FBS and 1 mM EDTA. CD45 + leukocytes were isolated using the EasySep™ Mouse CD45 Positive Selection Kit (Stemcell Technologies, Cambridge, MA) and prepared for flow cytometry.

Flow cytometry

Cells from the BAL fluid or lung tissue digest were resuspended in 100 µL of staining buffer (PBS with 5% FBS, with 0.2% w/v sodium azide). Cells were incubated with TruStain FcX anti-mouse CD16/32 (Fc Block, 1:100) for 10 min at 4 °C to prevent non-specific antibody binding. Cells were then incubated with antibodies against CD11b, CD206, CD11c, CD45, F4/80, Ly6c, MHC II, and Siglec F at a 1:100 dilution in staining buffer (Supplementary Table 1). After centrifugation and washing, cells were stained with eFluor 780-conjugated fixable viability dye for 30 min at 4 °C, washed with staining buffer, and fixed in paraformaldehyde (3%). Fluorescence was analyzed using a Gallios 10-color flow cytometer (Beckman Colter, Brea, CA). Cells were analyzed after sorting based upon forward and side scatter, doublet discrimination, and viability using Kaluza software v 2.2.1 (Beckman Colter, Brea, CA). Discrete alveolar and interstitial macrophage phenotypes were determined³.

Seahorse real-time cellular metabolic analysis

Positively selected CD45 lung digest cells were plated at 200,000 cells per well in a poly-D-lysine coated Seahorse XF96 Cell Culture Microplate (Agilent, Santa Clara, CA) and incubated at 37̊ °C, 5% CO₂ for 1 h to allow for cell adhesion. The cells were fed with ECAR medium (DMEM media, pH 7.4 (Agilent # 103575-100) with 2 mM L-glutamine) or OCR medium (DMEM media, pH 7.4 with 25 mM glucose, 1 mM pyruvate, and 2 mM L-glutamine). The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using a Seahorse XF96 Analyzer (Agilent Technologies, Santa Clara, CA). For ECAR analysis, CD45 + cells were sequentially treated with 25 mM glucose, 4 μM oligomycin, and 50 mM 2-deoxy-d-glucose (2-DG). For OCR measurement, the cells were sequentially treated with 4 μM oligomycin, 1 μM carbonyl cyanide ptrifluoromethoxyphenylhydrazone (FCCP), 0.5 μM rotenone, and 0.5 μM antimycin A. Data were normalized to µg of protein per well and analyzed using Agilent Wave software.

Histology and immunohistochemistry (IHC)

After BAL fluid collection, the left lung lobe was inflation fixed in 3% paraformaldehyde and embedded in paraffin. Liver, spleen, and kidney tissue were also fixed in 3% paraformaldehyde and embedded in paraffin. Four-micrometer sections were cut, slide-mounted, and left unstained for IHC or stained with hematoxylin and eosin (H&E) to observe histological changes. For the TGF-β dose-response toxicity study, tissues were blindly scored by a board-certified pathologist to determine overt toxicological pathology. For bleomycin studies, scans were blindly scored and quantified via ImageJ (NIH). For ImageJ quantification, samples were analyzed as previously described^3,53. Briefly, randomly selected histological areas (n = 10, 400X) from each sample were captured and used to determine tissue consolidation (% white space), alveolar wall thickness, and cell infiltration (number of nuclei).

Mounted unstained tissue sections were deparaffinized in xylenes followed by decreasing concentrations of ethanol (100–50%) and water. Antigen retrieval was performed in heated citrate buffer (10 mM sodium citrate, pH 6.0) for 30 min followed by quenching of endogenous peroxidase (3% H₂O₂ in methanol). Tissue sections were incubated in blocking buffer (10% normal goat serum in PBS) for 1 h at ambient temperature to prevent non-specific binding. Tissues were incubated at 4 °C for 18 h with anti-firefly luciferase (Abcam ab238448; Waltham, MA 1:100) or TGF-β (Abcam ab215715; Waltham, MA 1:100) antibody in a blocking buffer along with IgG controls (Pro-Sci 3703; Fort Collins, CO, matched concentrations). Sections were washed in decreasing concentrations of Tween-PBS (1%-0.5%) and incubated with biotin-conjugated secondary antibody (Vector Laboratories Vectastain Rabbit Kit; Newark, CA) for 1 h at ambient temperature. Antibody binding was visualized beneath a microscope using a DAB Peroxidase Substrate Kit (Vector Laboratories, Newark, CA). Slides were scanned at 40X magnification using a VS120 Virtual Slide Microscope (Olympus, Waltham, MA) and viewed with OlyVIA software (Olympus, Waltham, MA) at 400X magnification.

Statistical analysis

Statistical analyses were completed using GraphPad Prism version 9 or 10. Results are reported as means ± SE unless otherwise indicated. Data were tested for normal distribution using a Shapiro-Wilks test. If normally distributed, statistical significance for multiple group comparisons was determined using a one-way ANOVA with Tukey’s post-hoc test or Šídák’s multiple comparison test. If not normally distributed, statistical significance for multiple comparisons was determined using a Kruskal-Walli’s test with a Dunn’s multiple comparison test if needed. For parametric single comparisons, statistical significance was determined using an unpaired t-test with Welch’s correction compared to control groups as indicated in figure legends. If single comparisons were nonparametric, statistical significance was determined using a Mann-Whitney U test. All P-values < 0.05 were considered statistically significant. For data presented as Median ± SE, statistical comparisons were made using a Wilcoxon ranked sum test. Statistical tests were conducted using a 5% significance level. n = 3–18 animals/group and is further indicated in figure legends. Studies are represented as at least 2 independent studies.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data that support the findings of this study are included in the manuscript. Source data generated in this study are provided in the Supplementary and Source Data files. Additional data is deposited on figshare [https://doi.org/10.6084/m9.figshare.26128828]. Codon-optimized sequences are available under the following ascension codes for firefly luciferase MK484108.1 [https://www.ncbi.nlm.nih.gov/nuccore/MK484108.1] (Luc) or tumor growth factor-β NM_011577.2 [https://www.ncbi.nlm.nih.gov/nuccore/930697458] (TGFβ), All materials used in this manuscript are available from the corresponding author upon reasonable request. Source data are provided in this paper.

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Acknowledgements

The authors thank Drs. Yangzhu Du, Honghong Sun, and Eline Luning Prak of the Human Immunology Core (supported in part by NIH P30 AI045008 and P30 CA016520. HIC RRID: SCR_022380) at the Perelman School of Medicine at the University of Pennsylvania for their assistance with Luminex assays. They also would also like to thank Drs. Michael Goedken and Marianne Polunas of the Rutgers University Histology Core for their assistance with histological preparation and pathology services; Jessica Cervelli and Rita Hahn of the Rutgers Flow Cytometry Core for assistance with flow cytometry. In addition, they thank Sungjae Lee, Tony Hu, and Houping Ni for their technical assistance. They also express gratitude to Dr. Yongfeng Zhou at Shanghai Jiao Tong University for his assistance with cryo-TEM imaging of formulated IAJD34. Special thanks to Dr. Wook-Jin Park and Jessica A. Vasserman for their help with the characterization of formulated particles (size, PDI, Zeta potential, and encapsulation efficiency). Financial support was provided by the National Institutes of Health National Institute of Environmental Health Sciences [Grant T32-ES01984] and [Grant P30-ES005022] for A.J.G., E.R.S, R.S., M.W., E.A., and C.J.G. NSF-DMR-1807127, 1720530, and 2104554 financially supported V.P. This work was partially supported by the Wellcome Leap R3 program.

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Authors and Affiliations

Penn Institute for RNA Innovation, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
Jaclynn A. Meshanni, Nathan A. Ona, Erin K. Reagan, Ishana Baboo, Drew Weissman & Elena N. Atochina-Vasserman
Infectious Disease Division, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
Jaclynn A. Meshanni, Drew Weissman & Elena N. Atochina-Vasserman
Environmental and Occupational Health Sciences Institute, Rutgers University, Piscataway, NJ, USA
Emily R. Stevenson, Rachel Sun, Elena Abramova, Chang-Jiang Guo, Melissa Wilkinson & Andrew Gow
School of Materials Science and Engineering, East China University of Science and Technology, Shanghai, China
Dapeng Zhang, Yuzi Yang, Liuyan Pan & Yongsheng Li
Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA
Devendra S. Maurya & Virgil Percec

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Jaclynn A. Meshanni
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A.J.G., D.W., and E.N.A.-V. conceived the concept. V.P., D.S.M. conceived and designed the IAJD used in this study. D.Z., Y.Y., L.P., and Y.L. synthesized the IAJD used in this study. J.A.M., E.R.S., D.Z., R.S., N.A.O., E.K.R., E.A., C-J.G., M.W., I.B., and E.N.A-V. performed the experiments. J.A.M., E.R.S., D.Z., R.S., N.A.O., E.K.R., E.A., C-J.G., M.W., I.B., A.J.G., and E.N.A.-V. performed the data analysis. J.A.M., E.R.S., A.J.G., and E.N.A.-V. wrote the manuscript. All authors reviewed and approved the manuscript.

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Correspondence to Elena N. Atochina-Vasserman.

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In accordance with the University of Pennsylvania policies and procedures and our ethical obligations as researchers, we report that D.W., V.P., D.Z., E.N.A.-V, and A.J.G. are named on patents and patent applications relevant to the work done in this publication. We have disclosed those interests fully to the University of Pennsylvania, and we have in place an approved plan for managing any potential conflicts arising from the licensing of our patents. All other authors declare no competing interests.

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Meshanni, J.A., Stevenson, E.R., Zhang, D. et al. Targeted delivery of TGF-β mRNA to murine lung parenchyma using one-component ionizable amphiphilic Janus Dendrimers. Nat Commun 16, 1806 (2025). https://doi.org/10.1038/s41467-025-56448-y

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Received: 28 June 2024
Accepted: 17 January 2025
Published: 21 February 2025
DOI: https://doi.org/10.1038/s41467-025-56448-y

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