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

Targeted delivery of TGF-β mRNA to lung parenchyma using one-component ionizable amphiphilic Janus Dendrimers. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeted delivery of TGF-β mRNA to lung parenchyma using one-component ionizable amphiphilic Janus Dendrimers. Elena Atochina-Vasserman, Jaclynn Meshanni, Emily Stevenson, Dapeng Zhang, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4656663/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Feb, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Current clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways. However, targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here, we have developed an mRNA therapeutic for the lower lung using one-component Ionizable Amphiphilic Janus Dendrimers (IAJDs) as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA, transforming growth factor-beta (TGF-β), to produce transient protein expression in the lower regions of the lung. This study highlights IAJD’s potential for precise, effective, and safe delivery of TGF-β mRNA to the lung. 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. Biological sciences/Immunology/Cytokines/Transforming growth factor beta Biological sciences/Immunology/Immunotherapy Biological sciences/Immunology/Immunogenetics Biological sciences/Drug discovery Biological sciences/Biotechnology/Applied immunology one-component ionizable amphiphilic Janus Dendrimers mRNA targeted delivery anti-inflammatory cytokine TGF-β bleomycin injury respiratory disease lung parenchyma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 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 – 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 – 8 ). Many of the effects of ALI are observed in the lung parenchyma beyond the 16th generation. However, drug delivery to this area is challenging often leading to poor specificity and an uneven distribution ( 9 – 13 ). 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 ( 14 – 17 ). 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 ( 18 , 19 ). Despite their wide adoption for Covid-19 vaccinations, the targeted delivery of mRNA to the lung with four-component LNPs has not proven successful ( 20 – 26 ). 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 ( 27 , 28 ). 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 specificity ( 28 ). In addition to eliminating structural issues 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. Additionally, 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 ( 29 – 32 ). 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 , 32 , 33 ). These studies contribute to the progress of genetic nanomedicine and raise the possibility of lung-based nanotherapeutics. 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 as previously described ( 24 , 34 ). 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, # 11363-250G) purification ( 24 ). 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 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 hours 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 hours 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 (1:5,000, Abcam ab215715; Waltham, MA) overnight at 4°C, followed by a secondary goat anti-rabbit IgG HRP at 1:10,000 for 1 hour at room temperature. Membranes were stripped and re-probed with anti-GAPDH (Cell Signaling Technology, # 2118S, Danvers, MA) for 1 hour at room temperature at a 1:1,000 dilution, and a secondary goat anti-rabbit IgG HRP (Bio-Rad, # 170–6515 Bio-Rad, Hercules, CA) at a 1:10,000 for 1 hour 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, CD45 separated lung digest cells, or the large aggregate fraction from the bronchoalveolar lavage (BAL) fluid. Tissue from the accessory lobe was mechanically homogenized on dry ice and digested in lysis buffer with protease inhibitors (100 µL/0.33g tissue). The tissues were sonicated, centrifuged (2 min, 1,000 × 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 1:5,000 and goat anti-rabbit HRP at 1:5,000. Membranes were washed in ECL Prime Western Blotting Detection Reagent prior to visualization on x-ray film. Co-assembly of IAJD34 and mRNA IAJD34 was synthetized as previously described ( 28 ), 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), 1 H and 13 C NMR, and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry ( 28 ). Co-assembly of IAJD34 and mRNA was performed as previously described ( 28 , 35 ). Briefly, 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) and polydispersity index (PDI) using dynamic light scattering (DLS) prior injection. Animal studies Male and female wild-type BALB/c mice obtained from Charles River (Luc mRNA study) or Jackson Laboratories (TGF-β study) were used for all experiments. Mice were housed under standard conditions with food 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. 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 (PerkinElmer) as previously described ( 35 ). TGF-β mRNA-IAJD34 formulatio n 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 hours 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. 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 serum and 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 PBS as previously described ( 3 ). 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 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). Total cell count was determined using a Z1 Counter particle counter (Beckman Coulter). 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, 1hour) and separated into large and small aggregate surfactant fractions as previously described ( 3 , 36 ). 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 as previously described ( 3 , 37 ) adapted from a previously published method ( 38 ). 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 × 10 8 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 Fat a 1:100 dilution in staining buffer (supplemental 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 Coulter, Brea, CA). Cells were analyzed after sorting based upon forward and side scatter, doublet discrimination, and viability using Kaluza software (Beckman Coulter, Brea, CA). Discrete alveolar and interstitial macrophage phenotypes were determined as previously described ( 3 ). 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% CO 2 for 1 hour 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 2mM 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 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 the Agilent Wave Software v. 2.6.1. Histology and immunohistochemistry (IHC) After BAL fluid collection, the left lung lobe was inflation fixed in 3% of paraformaldehyde and embedded in paraffin. Liver, spleen, and kidney tissue were also fixed in 3% of 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 , 39 ). 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 (numbers 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% H 2 O 2 in methanol). Tissue sections were incubated in blocking buffer (10% normal goat serum in PBS) for 1 hour at ambient temperature to prevent non-specific binding. Tissues were incubated at 4°C for 18 hours with anti-firefly luciferase (Abcam ab238448; Waltham, MA 1:100) or TGF-β (Abcam ab215715; Waltham, MA 1:100) antibody in blocking buffer along with IgG controls (Pro-Sci 3703; Fort Collins, CO, matched concentrations). Sections were washed in decreasing concentrations of Tween-TBS (1%-0.5%) and incubated with biotin-conjugated secondary antibody (Vector Laboratories Vectastain Rabbit Kit; Newark, CA) for 1 hour 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 ± SEM 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. 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 with Data are presented as Median ± SE, statistical comparisons were made using a Wilcoxon ranked sum test. Statistical tests were conducted using a 5% significance level. n = 3–10 animals/group and is further indicated in figure legends. RESULTS AND DISCUSSION Characterization of lung-specific IAJD34 for targeted mRNA delivery to the lung Many IAJDs identified are capable of targeting the lung ( 40 ). 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. 1 A). 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. 1 B). 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 hours) post-injection, and the luciferase intensity was quantified as a whole-body flux (p/s) (Fig. 1 C). Luciferin intensity peaked at 4 hours post-injection (3.12 × 10 7 ± 9.15 × 10 6 ), by 24 hours, the flux intensity decreased but remained elevated by 37%. At 48 and 72-hours post-injection, there was a more significant drop to 3% and 1.9%, respectively (Fig. 1 D, Supplemental Table 2). The stability of the Luc mRNA-IAJD97 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 (Supplemental Fig. 1). 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. 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 hours post-injection. There was a dose-dependent increase in lung-specific luciferase expression, reaching 9.3 × 10 8 at 30 µg per mouse (Fig. 1 E, Supplemental Table 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. 1 F). 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 ( 30 , 41 ). The production and quality of TGF-β mRNA were validated (Supplemental Fig. 2A). Protein expression of TGF-β was confirmed in HEK293 cells following transfection with TGF-β mRNA (Supplemental Fig. 2B). 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 BAL markers were measured 24 hours post-injection. Increasing doses (10 µg and 20 µg) had no effect on BAL phospholipids or total cell count in the BAL fluid (Table 1 ). However, a significant increase in BAL fluid protein and neutrophils was observed at the 30-µg dose, indicating epithelial barrier dysfunction (Table 1 , Fig. 2 A). Although BAL fluid protein was increased, there was no significant increase in BAL IL-6 levels (Fig. 2 C), suggesting this may have been a downstream effect of abundant TGF-β delivery rather than a side effect of IAJD34 formulation. 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 (Supplemental Fig. 3). 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. 2 B). Liver function was also evaluated by measuring serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), known markers of liver function ( 42 ). Increased ALT was observed at the 30 µg dose, with no significant change in AST (Fig. 2 D). 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 sufficient ( 43 ). Additionally, most studies using IAJDs report that 10 µg or less of mRNA to be effective for organ-specific detection ( 40 ). To determine alveolar macrophage (AM) activation, BAL cells were evaluated by flow cytometry as previously described ( 3 ). AMs were defined as viable CD45 + F4/80 + SiglecF + cells (Supplemental Fig. 4). Inflammatory activation was characterized by expression of Ly6c and CD11c. No dose-dependent difference was observed in 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. 3 A). There were also no differences in Ly6C/CD206 AM populations at any dose of TGF-β mRNA-IAJD34 compared to control (Supplemental Table 4). Interstitial macrophages (IMs) were identified in CD45 + lung digest cells. IMs were defined as viable being CD45 + , CD11b + , SiglecF − cells as previously described (Supplemental Fig. 4) ( 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. 3 B, Supplemental Fig. 5). 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 signaling ( 44 ). Further analysis will be necessary to fully understand TGF-β mRNA-IAJD34’s effects on Ly6c + IMs. 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 ( 45 , 46 ). 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 ). We next evaluated the presence of other cytokines and chemokines in the BAL fluid that can be upregulated or downregulated by TGF-β signaling (Supplemental 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. 5 A) ( 47 ). 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. 5 B-D) ( 48 , 49 ). Thus, at 24 hours 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. 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 glycolytic rate assay and the mito stress. For the glycolytic rate assay, extracellular acidification rate (ECAR) was recorded. Glycolysis was determined by the ECAR measurement prior to glucose injection subtracted from the maximum ECAR before oligomycin injections. 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. 5 E). This increase was associated with increased cytokine production, likely a direct impact of TGF-β signaling cascades ( 50 , 51 ). 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. An TGF-β mRNA-IAJD34 induced dose-dependent increase was observed in maximal respiration, though this was not significant (Fig. 5 F). 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 activation ( 51 ). 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 hours post-injection. Treatment with TGF-β mRNA-IAJD34 did not increase BAL protein or phospholipids at any time point (Supplemental Fig. 6). IHC staining of lung tissue confirmed that TGF-β protein expression was transiently expressed in lung tissue, peaking at 4 hours post-injection, and persisting up to 48 hours (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 hours post-injection, an increase in cleaved TGF-β at 24 hours, and no significant increases in any TGF-β isoforms at 48 hours 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 (Supplemental Fig. 7 and Supplemental 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 fibrosis ( 29 ). Additionally, controlled repeat doses of TGF-β mRNA-IAJD34 can be delivered until resolution is obtained with a lower risk of over expressing TGF-β. 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 , 52 ). It should be noted that not all models of ITB result in the same level of fibrosis ( 53 – 55 ). 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 days ( 54 ). 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 (Supplemental Fig. 8). 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 , 39 , 52 ). An increase in alveolar epithelial thickness was observed in ITB + empty-IAJD34 animals, with limited changes in percent white space and nuclei count compared to control (Fig. 8 ). Treatment with TGF-β mRNA-IAJD34 did not significantly increase epithelial thickness compared to the matched control, indicating TGF-β mRNA-IAJD34 can prevent some ITB-induced increases in lung pathology (Fig. 8 ). The limited ITB-induced histological alterations are likely a consequence of evaluating injury at such an early timepoint 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, though the exact reasons for this are not well understood ( 53 – 56 ). BALB/c mice were chosen for this study as IAJD targeting and development has primarily been characterized in this strain of mice ( 28 , 35 , 40 ). 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 barriers ( 57 ). Exposure to ITB caused a significant increase in BAL fluid protein content as compared to control, with TGF-β mRNA-IAJD34 treatment having no effect (Fig. 9 A). 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. 9 B). However, treatment with TGF-β mRNA-IAJD34 prevented ITB-induced loss of cells lining the airway, a consequence of early pulmonary inflammation and cell death (Fig. 9 C) ( 3 ). 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. 9 D-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. 9 D-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. 9 F). 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 types ( 58 ). There is some evidence that TGF-β plays less significant role in IM cell maturation and activity, but this remains largely speculative ( 58 ). 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 , Supplemental 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. 10 A-C) ( 59 – 62 ). Treatment with TGF-β mRNA-IAJD34 prevented ITB-induced increases in all three of these proinflammatory cytokines, consistent with the anti-inflammatory signaling of TGF-β (Fig. 10 A-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. 10 D-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. Conclusion IAJD34 is a single-component lipid particle designed for the targeted delivery of mRNA to the lung ( 40 ). 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 settings ( 1 ). We hypothesize that IAJD34 is able to target these areas due to the large and charged altering ester and amide groups in the head group. When injected intravenously, the large charge density traps the formulated IAJD34 in narrow vascular regions of the lung, ultimately delivering the mRNA of interest to the small pulmonary arteries. 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 hours, which is important as long-term delivery of TGF-b 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-b expression. These results indicate that we could successfully deliver TGF-b 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-b 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 demonstrate the potential of mRNA delivery for therapeutic use in the lung. Declarations 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 assistance with Luminex assays. They also would also like to thank Dr. Michael Goedken and Dr. Marianne Polunas of the Rutgers University Histology Core for their assistance with histological preparation and pathology services. They would also like to thank Jessica Cervelli and Rita Hahn of the Rutgers flow cytometry core for assistance with flow cytometry. Lastly, the authors would like to thank Sungjae Lee, Tony Hu, and Houping Ni for their technical assistance. Author Contributions: A.J.G., D.W., and E.N.A-V. conceived the concept. V.P. 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. Funding: D. Z., and Y.L. were financially supported by the National Key Research and Development Program of China [Grant 2022YFC2403203], National Natural Science Foundation of China [Grant 22305081], and Shanghai Sailing Program [Grant 23YF1408600]. A.J.G., E.R.S, R.S., M.W., E.A., and C.J.G. were financially supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grant T32-ES01984] and [Grant P30-ES005022]. 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Takamizawa et al. , Bleomycin stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activity. The Journal of Immunology 162 , 6200-6208 (1999). Table Table 1 . Inflammatory response to TGF-β mRNA-IAJD34 pulmonary delivery. IAJD34 loaded with TGF-β mRNA at doses of 10, 20, and 30 μg per mouse were delivered to BALB/c mice and compared to naïve animals. At 24 hours post-injection, total BAL protein, differential BAL cell count, and BAL phospholipid levels were quantified as markers of lung injury: MP, Macrophage, NP, neutrophil, LP, Lymphocyte. Data are presented as mean ± SD, n=3-10 per group. Samples were compared using Shapiro-Wilk test for normality followed by an ordinary one-way ANOVA with Tukey’s Multiple comparison where * indicates a significant difference from the control, p<0.05. TGF-β mRNA-IAJD34 per mouse ( μ g) BAL Phospholipids (µg) BAL Protein (mg/mL) BAL Cell Count (x10 4 ) Total MP NP LP Naïve 40.2 ± 17.5 0.23 ± 0.02 26.4 ± 8.3 25.5 ± 7.8 0.2 ± 0.2 0.7 ± 0.4 10 53.1 ± 12.4 0.40 ± 0.27 17.6 ± 11.0 20.2 ± 6.9 0.1 ± 0.2 0.8 ± 0.7 20 42.4 ± 19.2 0.36 ± 0.18 29.9 ± 12.6 28.3 ± 11.8 0.9 ± 1.3 0.8 ± 0.5 30 60.2 ± 15.4 1.25 ± 0.76* 39.8 ± 2.47 35.5 ± 23.6 25.0 ± 25.2* 1.8 ± 1.6 Additional Declarations Yes there is potential Competing Interest. In accordance with the University of Pennsylvania policies and procedures and our ethical obligations as researchers, we report that V.P., D.W., D.Z., E.N.A-V, 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. 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Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Devendra","middleName":"","lastName":"Maurya","suffix":""},{"id":325623062,"identity":"c71855a6-9172-4b7c-aa8e-dbe023f6482a","order_by":14,"name":"Virgil Percec","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Virgil","middleName":"","lastName":"Percec","suffix":""},{"id":325623063,"identity":"88f546f4-bc02-44bb-9ce8-043e1a97dcea","order_by":15,"name":"Yongsheng Li","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yongsheng","middleName":"","lastName":"Li","suffix":""},{"id":325623064,"identity":"db21ee4e-0ad7-4e52-a695-4f84e52dfea6","order_by":16,"name":"Andrew Gow","email":"","orcid":"","institution":"Rutgers University","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Gow","suffix":""},{"id":325623065,"identity":"d00e6014-1df5-4b2c-895f-bcf52f8f0cec","order_by":17,"name":"Drew Weissman","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Drew","middleName":"","lastName":"Weissman","suffix":""}],"badges":[],"createdAt":"2024-06-28 20:30:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4656663/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4656663/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-56448-y","type":"published","date":"2025-02-21T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60124809,"identity":"c5c77b9d-9204-4ce2-9b21-c5cfc20eecb2","added_by":"auto","created_at":"2024-07-12 05:38:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":629579,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecificity of IAJD34 for targeted mRNA delivery to the lung.\u003c/strong\u003e IAJD34 formulated with luciferase mRNA (Luc mRNA-IAJD34) was i.v. injected into BALB/c mice and analyzed for efficient pulmonary mRNA using IVIS. \u003cstrong\u003e(A)\u003c/strong\u003e The chemical structure of one-component IAJD34 (40); \u003cstrong\u003e(B)\u003c/strong\u003e Specificity of Luc mRNA-IAJD34 delivery to the lung. Representative IVIS images of whole-body and organs of mice at 4 hours post-injection of 10 μg Luc mRNA-IAJD34 show luminescent biodistribution of luciferase expression predominantly in the lungs. IVIS images were analyzed, and the photon radiance (Lq) (s\u003csup\u003e−1\u003c/sup\u003esr\u003csup\u003e−1\u003c/sup\u003em\u003csup\u003e−2\u003c/sup\u003e) of each image was quantified. The quantitative bioluminescence imaging (BLI) data are shown as total flux (p/s); \u003cstrong\u003e(C) \u003c/strong\u003eTime course luciferase expression \u003cem\u003ein vivo\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003eRepresentative IVIS images of whole-body mice following 10 μg Luc mRNA-IAJD34, imaged at 4, 24, 48, or 72 hours post-injection; \u003cstrong\u003e(D) \u003c/strong\u003eWhole-body radiant flux (p/s) at various time points following the injection of 10 μg Luc mRNA-IAJD34. Data presented as mean+SEM, with n=12 for 4 hours, n=15 for 24 hours, n=18 for 48 hours, and n=6 for 72 hours.\u003cstrong\u003e\u0026nbsp; (E)\u003c/strong\u003e Dose-dependent pulmonary delivery of Luc mRNA-IAJD34.\u0026nbsp; Representative IVIS images of whole-body and organs of mice injected with Luc mRNA-IAJD34 at doses of 10, 20, or 30 μg, imaged at 24 hrs post-injection. Particle size (nm) and polydispersity index (PDI) for Luc mRNA-IAJD34 at each dose were measured prior injection and are shown above the corresponding mouse IVIS image; \u003cstrong\u003e(F) \u003c/strong\u003eDelivery of Luc mRNA-IAJD34 to lung parenchyma.\u003cstrong\u003e \u003c/strong\u003eAt 24 hours post-injection with 30 μg Luc mRNA-IAJD34, lung tissue was harvested and IHC for luciferase or IgG was performed and compared to naïve animals.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/4087a410ce466564de59261a.png"},{"id":60125407,"identity":"be348c2f-57a2-4975-9800-76533ea968db","added_by":"auto","created_at":"2024-07-12 05:46:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":307093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of dose-dependent toxicity of TGFβ mRNA delivery with IAJD34.\u003c/strong\u003e Various doses of TGFβ mRNA-IAJD34 (10, 20, and 30 μg per mouse) were delivered to BALB/c mice and compared to naïve animals. At 24 hours post-injection, BAL fluid and cells, whole lung tissue, and serum were collected. \u003cstrong\u003e(A)\u003c/strong\u003e BAL cells were fixed and stained using a Hema 3 Stat kit. Representative H\u0026amp;E images are shown; \u003cstrong\u003e(B)\u003c/strong\u003e Formaldehyde-fixed lung tissue was processed, stained with H\u0026amp;E and scored by a board-certified pathologist. \u0026nbsp;Representative H\u0026amp;E images are shown; \u003cstrong\u003e(C)\u003c/strong\u003e IL-6 was measured in BAL fluid via ELISA according to manufacturer’s guidelines; \u003cstrong\u003e(D)\u003c/strong\u003e ALT and AST were measured in serum via ELSIA according to manufacturer’s guidelines. Data are presented as mean ± SE, n=3-10/group from 2 independent experiments. For C and D, samples were compared using Shapiro-Wilk test for normality followed by an ordinary one-way ANOVA with Tukey’s Multiple comparison where *indicates a significant difference from the naïve control, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/030cd6c30953ba65c2f70dbe.png"},{"id":60125409,"identity":"54dc3495-1792-4156-8952-4eece67bbd61","added_by":"auto","created_at":"2024-07-12 05:46:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbsence of inflammatory activation in response to increasing doses of TGF-β mRNA-IAJD34 in alveolar macrophages and interstitial macrophages. \u003c/strong\u003eVarious doses of TGFβ mRNA-IAJD34 (10, 20, and 30 μg per mouse) were delivered to BALB/c mice and compared to naïve animals at 24 hours post-injection. \u003cstrong\u003e(A)\u003c/strong\u003eCells from the BAL were isolated, immunostained, and analyzed by flowcytometry. Cells that were positively stained for both Siglec F and F4/80 were determined to be alveolar macrophages (AMs) and were evaluated for Ly6c and CD11c expression; \u003cstrong\u003e(B)\u003c/strong\u003e Cells from digested lung tissue were immunomagnetically separated based upon CD45 expression. CD45+ cells were isolated, immunostained, and analyzed. Cells that expressed F4/80 and CD11b in the absence of Siglec F were categorized as interstitial macrophages (IMs) and were analyzed for Ly6c and CD206 expression. Data are presented as mean ± SE for n=4-10 per group. Samples were compared using Shapiro-Wilk test for normality followed by an ordinary one-way ANOVA with Šídák's multiple comparisons test where * indicates a significant difference from the naïve control, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/c91bd77864c2b28cd004415e.png"},{"id":60124819,"identity":"352afd10-1de9-4fc6-97d6-0b6590681e3e","added_by":"auto","created_at":"2024-07-12 05:38:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDose-dependent expression of TGF-β mRNA-IAJD34 in lung tissue.\u003c/strong\u003e Various doses of TGF-β mRNA-IAJD34 (10, 20, and 30 μg per mouse) were delivered to BALB/c mice and lung tissue was collected 24 hours post-injection and analyzed for TGF-β protein expression. \u003cstrong\u003e(A)\u003c/strong\u003e Western blots for TGF-β were performed on homogenized whole lung tissue at indicated time points. Representative images are shown; \u003cstrong\u003e(B)\u003c/strong\u003e Western blots results were quantified using densitometry. TGF-β protein expression was normalized to β-actin for each band, and the density was expressed relative to the naïve control. Data are presented as mean ± SE for n=4-9 per group. Statistical comparisons were made using a Mann-Whitney U test where * indicates a significant difference from control, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/636dd0abb9ffdbf882cd61e2.png"},{"id":60125410,"identity":"572c4383-abac-4057-b91a-56373d1773a5","added_by":"auto","created_at":"2024-07-12 05:46:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGF-β mRNA-IAJD34-induced alterations in BAL cytokine production and cellular metabolic function.\u003c/strong\u003e Various doses of TGF-β mRNA-IAJD34 (10, 20, and 30 μg per mouse) were delivered to BALB/c mice and BAL fluid was collected 24 hrs post-injection. \u003cstrong\u003e(A-D)\u003c/strong\u003e Cell- free BAL was evaluated for cytokines using a Milliplex Max Mouse Cytokine/Chemokine Magnetic Bead Panel - Premixed 32 Plex - Immunology Multiplex Assay. The concentration of \u003cstrong\u003e(A)\u003c/strong\u003e G-SCF, \u003cstrong\u003e(B)\u003c/strong\u003e IL-1α, \u003cstrong\u003e(C)\u003c/strong\u003e IL-9, and \u003cstrong\u003e(D)\u003c/strong\u003e TNFα are presented as pg/mL; \u003cstrong\u003e(E-F)\u003c/strong\u003e Cells from digested lung tissue were immunomagnetically separated based upon CD45 expression. CD45+ cells were isolated and analyzed metabolically using an Agilent Seahorse according to manufacturer’s guidelines; \u003cstrong\u003e(E)\u003c/strong\u003e ECAR was measured following injections of glucose, oligomycin (Oligo), and 2-DG, and glycolysis was determined following injection of glucose; \u003cstrong\u003e(F)\u003c/strong\u003e OCR was measured with injections of oligo, FCCP, and rotenone/antimycin (R/A) and normalized to non-mitochondrial energy production. Maximal respiration was determined following FCCP injection. Data are shown as mean ± SE, n = 4-10 per group. Data were evaluated for normality using a Shapiro- Wilk’s test and compared using a one-way ANOVA followed by Tukey's multiple comparisons test. * indicates a significant difference from the control, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/ba302ead86dc53d043ac93f6.png"},{"id":60124817,"identity":"9dbf09df-aac0-4106-9b89-17393eed018c","added_by":"auto","created_at":"2024-07-12 05:38:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":400165,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell specific expression of TGF-β mRNA-IAJD34 in the lung over time.\u003c/strong\u003eEmpty IAJD34 or 10 μg TGF-β mRNA-IAJD34 were delivered to BALB/c mice, and lung tissues were evaluated for TGF-β protein expression at 4, 24, and 48 hrs post-injection. Lung tissues was stained for TGF-β or IgG control via immunohistochemistry. Representative IHC images are shown. The slides are at 100x magnification, and the scale bars presented 100 µm. Increases in TGF-β protein expression were observed in the TGF-β mRNA-IAJD34 group through immunohistochemical staining from 4 to 48 hours.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/5adf0a14c44bf318ed6cd1ec.png"},{"id":60124810,"identity":"a1e8c5fd-a144-46f8-8964-15a0da37ba3b","added_by":"auto","created_at":"2024-07-12 05:38:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":91926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGF-β protein expression is transiently induced after mRNA delivery to lung via IAJD34.\u003c/strong\u003e Empty IAJD34 or 10 ug TGF-β mRNA-IAJD34 were delivered to BALB/c mice and lung tissue homogenates were evaluated for TGF-β protein expression at 4, 24, and 48 hr post-injection. \u003cstrong\u003e(A)\u003c/strong\u003eWestern blots for TGF-β were performed on homogenized whole lung tissue. Representative images are shown. \u0026nbsp;\u003cstrong\u003e(B)\u003c/strong\u003eWestern blots bands were quantified by densitometry and TGF-β protein expression was normalized to β-actin for each band, and the density was expressed relative to the naïve control. Data are presented as mean ± SE, n = 3-5 per group. Statistical comparisons were made using a Mann-Whitney U test, where * indicates a significant difference from the matched empty control, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/2a3660bf03d56851ad4e15c5.png"},{"id":60125889,"identity":"5bc8f4c1-eda3-45b2-9bdc-9b453bc915bf","added_by":"auto","created_at":"2024-07-12 05:54:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":544859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of TGF-β mRNA-IAJD34 on bleomycin-induced histological alterations.\u003c/strong\u003e BALB/c mice were exposed to PBS or bleomycin (ITB) and subsequently received either an empty IAJD34 or 10 µg TGF-β mRNA-IAJD34. Lung tissue was collected 3 days post exposure and injection. Fixed lung tissue was processed and stained with H\u0026amp;E. Representative H\u0026amp;E images are shown. The slides are shown at 400x magnification, with scale bars indicating 50 µm. An inset image shows the data of evaluating alveolar thickness, where 10 fields per slide were randomly selected, and analyzed using ImageJ. Data are presented as Median ± SE, n = 6-10 per group. Statistical comparisons were made using a Wilcoxon ranked sum test, where * indicates a significant difference from the PBS/empty-IAJD34 group, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/3dafc3a47016a42ebdd7d17f.png"},{"id":60124814,"identity":"3f4cc28b-4f24-4595-8b04-08e2c09d8fd5","added_by":"auto","created_at":"2024-07-12 05:38:37","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":54832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of TGF-β mRNA-IAJD34 bleomycin-induced injury.\u003c/strong\u003e BALB/c mice were exposed to PBS or bleomycin (ITB) and subsequently received either an empty IAJD34 or 10 µg TGF-β mRNA-IAJD34. Cell-free BAL fluid, BAL cells, and large aggregate surfactant fractions were collected 3 days post exposure and injection. \u003cstrong\u003e(A)\u003c/strong\u003e Cell-free BAL fluid was evaluated for total protein content using a BCA assay. Data were evaluated for normality using a Shaprio-Wilks normality test. PBS and Bleo were compared using a 2-way ANOVA; \u003cstrong\u003e(B):\u003c/strong\u003e Total phospholipids were determined from the large aggregate surfactant fraction. Data were compared using a 2-way ANOVA; \u003cstrong\u003e(C):\u003c/strong\u003e Total BAL cells were counted using a coulter counter. Data were compared using a 2-way ANOVA with Šídák's multiple comparisons test; \u003cstrong\u003e(D-E):\u003c/strong\u003e BAL cells were immunostained for flow cytometric analysis. Cells that were positively stained for both Siglec F and F4/80 were determined to be alveolar macrophages (AMs). Resident macrophages (CD11c+/CD11b-), can be differentiated from recruited (CD11c-/CD11b+) or migratory macrophages (CD11c+/CD11b+). Resident AMs exposed to PBS and ITB were compared using a 2-way ANOVA. Recruited AMs were compared using a Wilcoxon Signed Rank test. \u003cstrong\u003e(F)\u003c/strong\u003e Cells from digested lung tissue were immunomagnetically separated based upon CD45 expression. CD45+ cells were isolated, immunostained, and analyzed. Cells that expressed F4/80 and CD11b in the absence of Siglec F were categorized as interstitial macrophages (IMs) and were analyzed for Ly6c expression. Ly6c+ IMs exposed to PBS and ITB were compared using a 2-way ANOVA. All data were evaluated for normality using a Shaprio-Wilks normality test and are presented as Mean ± SE, n = 3-10/group. * indicates a significant difference from the control, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/0adb30f09bdb0b2ed725549c.png"},{"id":60125411,"identity":"28283867-49cc-48b8-b4bf-8176665c55f5","added_by":"auto","created_at":"2024-07-12 05:46:37","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":61831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of TGF-β mRNA-IAJD34 bleomycin-induced cytokine production.\u003c/strong\u003e BALB/c mice were exposed to PBS or bleomycin (ITB) and subsequently received either an empty IAJD34 or 10 µg TGF-β mRNA-IAJD34. Cell-free BAL fluid was collected 3 days post exposure and injection. \u003cstrong\u003e(A-E)\u003c/strong\u003e Cell-free BAL fluid was evaluated for cytokines using a Milliplex Max Mouse Cytokine/Chemokine Magnetic Bead Panel - Premixed 32 Plex - Immunology Multiplex Assay. The concentration of \u003cstrong\u003e(A)\u003c/strong\u003eG-SCF, \u003cstrong\u003e(B)\u003c/strong\u003e IL-6, \u003cstrong\u003e(C)\u003c/strong\u003e CXCL10, \u003cstrong\u003e(D)\u003c/strong\u003e IL-1α, and (E) IL-2 are presented as pg/mL. Date shown a mean ± SE, n = 6-10 per group. Data were evaluated for normality using a Shapiro Wilk’s test. If data were normally distributed, they were compared using a 2-way ANOVA followed by Šídák's multiple comparisons test. If data were not normally distributed, they were evaluated using a Kruskal Wallis test. * indicates a significant difference, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/00372f4fa0fe03e6b5d8ed78.png"},{"id":76912829,"identity":"cde912ce-bb24-4a21-850a-80ce69368a60","added_by":"auto","created_at":"2025-02-22 08:06:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3975578,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/32d72371-2a0d-4ee0-8679-e33d0169a239.pdf"},{"id":60124815,"identity":"6672199d-bd54-4557-98a9-bdf2c99c55a6","added_by":"auto","created_at":"2024-07-12 05:38:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4231464,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementalInformation62824.docx","url":"https://assets-eu.researchsquare.com/files/rs-4656663/v1/2faf16bb703b0e93aa5c585d.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nIn accordance with the University of Pennsylvania policies and procedures and our ethical obligations as researchers, we report that V.P., D.W., D.Z., E.N.A-V, 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.","formattedTitle":"Targeted delivery of TGF-β mRNA to lung parenchyma using one-component ionizable amphiphilic Janus Dendrimers.","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAcute lung injury (ALI) is a prevalent condition in the United States, with 200,000 new diagnoses each year, resulting in high morbidity and mortality (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). 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 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Many of the effects of ALI are observed in the lung parenchyma beyond the 16th generation. However, drug delivery to this area is challenging often leading to poor specificity and an uneven distribution (\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eSince 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 (\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). 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 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Despite their wide adoption for Covid-19 vaccinations, the targeted delivery of mRNA to the lung with four-component LNPs has not proven successful (\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecently, 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 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). 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 specificity (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In addition to eliminating structural issues that can arise from mixtures of four-component LNPs, this technology may allow for programmed delivery to specific organs, such as the lung.\u003c/p\u003e \u003cp\u003eIn 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. Additionally, 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 (\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). These studies contribute to the progress of genetic nanomedicine and raise the possibility of lung-based nanotherapeutics.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003emRNA production and characterization\u003c/h2\u003e \u003cp\u003eCodon-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 as previously described (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Briefly, nucleoside-modified mRNAs were transcribed to contain 101 nucleotide-long poly(A) tails. mRNAs were modified with m1Ψ-5\u0026prime;-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, # 11363-250G) purification (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). All mRNA were analyzed for quality control through agarose gel electrophoresis, dsRNA, endotoxin and IFN-α assays and \u003cem\u003ein vitro\u003c/em\u003e transfection. mRNA was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until ready for use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eTGF-β mRNA transfection in human embryonic kidney 293 cell line\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eHuman Embryonic Kidney (HEK) 293 cells were seeded into a 24-well cell culture plate at a density of 150,000 per well in Dulbecco\u0026rsquo;s Modified Enriched Medium (DMEM) containing fetal bovine serum (10%), L-glutamine, and penicillin-streptomycin. Cells were allowed to grow for 24 hours 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 hours and then samples collected in RIPA buffer (Sigma Aldrich, # R0278-500mL) containing cOmpleteMini protease cocktail inhibitors (Roche Diagnostics, # 11836153001). Samples were centrifuged (300 \u003cem\u003eg\u003c/em\u003e, 5 min, 4\u0026deg;C) to remove cellular debris and supernatant was then collected. Protein concentrations were determined using a Pierce\u0026trade; BCA Protein Assay (Peirce, # 23227).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eImmunoblots\u003c/h2\u003e \u003cp\u003eWhole cell lysates samples (10 \u0026micro;g per well) were analyzed for TGF- β by denaturing SDS-PAGE 4\u0026ndash;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 (1:5,000, Abcam ab215715; Waltham, MA) overnight at 4\u0026deg;C, followed by a secondary goat anti-rabbit IgG HRP at 1:10,000 for 1 hour at room temperature. Membranes were stripped and re-probed with anti-GAPDH (Cell Signaling Technology, # 2118S, Danvers, MA) for 1 hour at room temperature at a 1:1,000 dilution, and a secondary goat anti-rabbit IgG HRP (Bio-Rad, # 170\u0026ndash;6515 Bio-Rad, Hercules, CA) at a 1:10,000 for 1 hour 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).\u003c/p\u003e \u003cp\u003eTo analyze TGF-β protein expression in lung tissue, immunoblots were performed on collected lung tissue, CD45 separated lung digest cells, or the large aggregate fraction from the bronchoalveolar lavage (BAL) fluid. Tissue from the accessory lobe was mechanically homogenized on dry ice and digested in lysis buffer with protease inhibitors (100 \u0026micro;L/0.33g tissue). The tissues were sonicated, centrifuged (2 min, 1,000 \u0026times; \u003cem\u003eg\u003c/em\u003e), and supernatants were assessed for protein concentration using a Pierce\u0026trade; BCA Protein Assay. Equal amounts of protein from lung tissue samples (45 \u0026micro;g per well) were analyzed for TGF-β protein expression as described above. Antibodies for TGF-β were used at 1:5,000 and goat anti-rabbit HRP at 1:5,000. Membranes were washed in ECL Prime Western Blotting Detection Reagent prior to visualization on x-ray film.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eCo-assembly of IAJD34 and mRNA\u003c/h2\u003e \u003cp\u003eIAJD34 was synthetized as previously described (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), 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), \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR, and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Co-assembly of IAJD34 and mRNA was performed as previously described (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Briefly, 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) and polydispersity index (PDI) using dynamic light scattering (DLS) prior injection.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnimal studies\u003c/h2\u003e \u003cp\u003eMale and female wild-type BALB/c mice obtained from Charles River (Luc mRNA study) or Jackson Laboratories (TGF-β study) were used for all experiments. Mice were housed under standard conditions with food 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBioluminescence characterization for Luc mRNA delivery to mouse lungs\u003c/h2\u003e \u003cp\u003eFemale or male 6-8-week-old BALB/c mice were administered 10 \u0026micro;g of Luc-mRNA-IAJD34 in a100-\u0026micro;L 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 \u003cem\u003ein vivo\u003c/em\u003e 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 (PerkinElmer) as previously described (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTGF-β mRNA-IAJD34 formulatio\u003c/b\u003en \u003cb\u003etoxicity studies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTGF-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 \u0026micro;g, 20 \u0026micro;g, or 30 \u0026micro;g 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\u0026rsquo; histological score. Mice were sacrificed 24 hours post-injections, and their serum samples and organs were collected. Organs were fixed in 4% paraformaldehyde, and further stained with H\u0026amp;E as described below. Liver, spleen, kidney, and lungs were inspected for signs of inflammation and deviations from normal histology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eELISAs 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMultiplex cytokine analysis\u003c/h2\u003e \u003cp\u003eCytokine in the serum and 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBleomycin mRNA-IAJD34 studies\u003c/h2\u003e \u003cp\u003eMice were randomly assigned to treatment groups. Mice were anesthetized with isoflurane and received a single retro orbital intravenous injection of 10 \u0026micro;g of TGF-β mRNA formulated with IAJD34 or empty IAJD34. while still under anesthesia, mice immediately received a single intratracheal instillation of either 50 \u0026micro;L PBS or 50 \u0026micro;L of bleomycin (3U/kg of body weight) (Santa Cruz Biotechnology, Inc., Dallas, TX; # sc-200134B) diluted in PBS as previously described (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). 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 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBronchoalveolar lavage\u003c/h2\u003e \u003cp\u003e 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 \u0026times; \u003cem\u003eg\u003c/em\u003e 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). Total cell count was determined using a Z1 Counter particle counter (Beckman Coulter). 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\u0026trade; 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePhospholipids\u003c/h2\u003e \u003cp\u003eCollected cell-free BAL fluid (5 mL) was centrifuged (20,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 4\u0026deg; C, 1hour) and separated into large and small aggregate surfactant fractions as previously described (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Phospholipids were extracted from the large aggregate fraction and resuspended in 30 \u0026micro;L 0.9% sodium chloride. Total phospholipids were measured by light absorbance at 830 nm using a standard curve ranging from 0 to 3.1 \u0026micro;g phosphate as previously described (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) adapted from a previously published method (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLung tissue digest\u003c/h2\u003e \u003cp\u003eLung tissue from the right lobes was incubated at 37\u0026deg; 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 \u0026micro;m strainer, washed with RPMI with 5% FBS, and centrifuged (6 min, 400 \u0026times; \u003cem\u003eg\u003c/em\u003e). 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 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells/mL PBS with 2% FBS and 1 mM EDTA. CD45\u0026thinsp;+\u0026thinsp;leukocytes were isolated using the EasySep\u0026trade; Mouse CD45 Positive Selection Kit (Stemcell Technologies, Cambridge, MA) and prepared for flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometry\u003c/h2\u003e \u003cp\u003eCells from the BAL fluid or lung tissue digest were resuspended in 100 \u0026micro;L 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\u0026deg;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 Fat a 1:100 dilution in staining buffer (supplemental Table\u0026nbsp;1). After centrifugation and washing, cells were stained with eFluor 780-conjugated fixable viability dye for 30 min at 4\u0026deg;C, washed with staining buffer, and fixed in paraformaldehyde (3%). Fluorescence was analyzed using a Gallios 10-color flow cytometer (Beckman Coulter, Brea, CA). Cells were analyzed after sorting based upon forward and side scatter, doublet discrimination, and viability using Kaluza software (Beckman Coulter, Brea, CA). Discrete alveolar and interstitial macrophage phenotypes were determined as previously described (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSeahorse real-time cellular metabolic analysis\u003c/h2\u003e \u003cp\u003ePositively 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\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e for 1 hour 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 2mM 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\u0026thinsp;+\u0026thinsp;cells were sequentially treated 25 mM glucose, 4 \u0026micro;M oligomycin, and 50 mM 2-deoxy-d-glucose (2-DG). For OCR measurement, the cells were sequentially treated with 4 \u0026micro;M oligomycin, 1 \u0026micro;M carbonyl cyanide ptrifluoromethoxyphenylhydrazone (FCCP), 0.5 \u0026micro;M rotenone, and 0.5 \u0026micro;M antimycin A. Data were normalized to \u0026micro;g of protein per well and analyzed using the Agilent Wave Software v. 2.6.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHistology and immunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eAfter BAL fluid collection, the left lung lobe was inflation fixed in 3% of paraformaldehyde and embedded in paraffin. Liver, spleen, and kidney tissue were also fixed in 3% of paraformaldehyde and embedded in paraffin. Four-micrometer sections were cut, slide-mounted, and left unstained for IHC or stained with hematoxylin and eosin (H\u0026amp;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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Briefly, randomly selected histological areas (n\u0026thinsp;=\u0026thinsp;10, 400X) from each sample were captured and used to determine tissue consolidation (% white space), alveolar wall thickness, and cell infiltration (numbers of nuclei).\u003c/p\u003e \u003cp\u003eMounted unstained tissue sections were deparaffinized in xylenes followed by decreasing concentrations of ethanol (100\u0026thinsp;\u0026minus;\u0026thinsp;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% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol). Tissue sections were incubated in blocking buffer (10% normal goat serum in PBS) for 1 hour at ambient temperature to prevent non-specific binding. Tissues were incubated at 4\u0026deg;C for 18 hours with anti-firefly luciferase (Abcam ab238448; Waltham, MA 1:100) or TGF-β (Abcam ab215715; Waltham, MA 1:100) antibody in blocking buffer along with IgG controls (Pro-Sci 3703; Fort Collins, CO, matched concentrations). Sections were washed in decreasing concentrations of Tween-TBS (1%-0.5%) and incubated with biotin-conjugated secondary antibody (Vector Laboratories Vectastain Rabbit Kit; Newark, CA) for 1 hour 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were completed using GraphPad Prism version 9 or 10. Results are reported as Means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM 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 Š\u0026iacute;d\u0026aacute;k's multiple comparison test. If not normally distributed, statistical significance for multiple comparisons was determined using a Kruskal-Walli\u0026rsquo;s test. 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\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. For data presented with Data are presented as Median\u0026thinsp;\u0026plusmn;\u0026thinsp;SE, statistical comparisons were made using a Wilcoxon ranked sum test. Statistical tests were conducted using a 5% significance level. n\u0026thinsp;=\u0026thinsp;3\u0026ndash;10 animals/group and is further indicated in figure legends.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of lung-specific IAJD34 for targeted mRNA delivery to the lung\u003c/h2\u003e \u003cp\u003eMany IAJDs identified are capable of targeting the lung (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). With a relatively large chemical structure and a pK\u003csub\u003ea\u003c/sub\u003e of 6.74 lending to a strongly lung specific targeting, IAJD34 was selected for further characterization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To evaluate lung-specific delivery, the Luc mRNA-IAJD34 formulation was injected into mice at an initial dose of 10 \u0026micro;g 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To evaluate the kinetics of luciferin protein production over time, mice were injected with the Luc mRNA-IAJD34 formulation at a dose of 10 \u0026micro;g per mouse. Live mice were imaged at various time points (4, 24, 48, and 72 hours) post-injection, and the luciferase intensity was quantified as a whole-body flux (p/s) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Luciferin intensity peaked at 4 hours post-injection (3.12 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 9.15 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e), by 24 hours, the flux intensity decreased but remained elevated by 37%. At 48 and 72-hours post-injection, there was a more significant drop to 3% and 1.9%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Supplemental Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eThe stability of the Luc mRNA-IAJD97 formulation was studied over time at +\u0026thinsp;4\u0026deg;C. The formulation was stored at +\u0026thinsp;4\u0026deg;C for 5 days and then injected into mice. Whole-body luciferase intensity did not change significantly compared to the freshly prepared formulation (Supplemental Fig.\u0026nbsp;1). Thus, the one-component IAJD34 formulated with Luc mRNA maintains stability and activity at 4\u0026deg;C and can be stored for at least 5 days prior to use.\u003c/p\u003e \u003cp\u003eTo evaluate the impact of dose on luciferase expression, Luc mRNA-IAJD34 was injected at 10, 20, and 30 \u0026micro;g doses per mouse and imaged at 24 hours post-injection. There was a dose-dependent increase in lung-specific luciferase expression, reaching 9.3 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e at 30 \u0026micro;g per mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, Supplemental Table\u0026nbsp;3). Immunohistochemical staining of lung tissue with the 30 \u0026micro;g 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). 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.\u003c/p\u003e \u003cp\u003eNotably, 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation the pulmonary delivery of therapeutic mRNA\u003c/h2\u003e \u003cp\u003eFollowing 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 (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The production and quality of TGF-β mRNA were validated (Supplemental Fig.\u0026nbsp;2A). Protein expression of TGF-β was confirmed in HEK293 cells following transfection with TGF-β mRNA (Supplemental Fig.\u0026nbsp;2B).\u003c/p\u003e \u003cp\u003eTo evaluate inflammatory effects in the lung, IAJD34 formulated with TGF-β mRNA (TGF-β mRNA-IAJD34) was injected at 10, 20, and 30 \u0026micro;g per mouse and BAL markers were measured 24 hours post-injection. Increasing doses (10 \u0026micro;g and 20 \u0026micro;g) had no effect on BAL phospholipids or total cell count in the BAL fluid (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, a significant increase in BAL fluid protein and neutrophils was observed at the 30-\u0026micro;g dose, indicating epithelial barrier dysfunction (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Although BAL fluid protein was increased, there was no significant increase in BAL IL-6 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggesting this may have been a downstream effect of abundant TGF-β delivery rather than a side effect of IAJD34 formulation.\u003c/p\u003e \u003cp\u003eHistological sections of organs were stained with H\u0026amp;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 (Supplemental Fig.\u0026nbsp;3). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Liver function was also evaluated by measuring serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), known markers of liver function (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Increased ALT was observed at the 30 \u0026micro;g dose, with no significant change in AST (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Thus, systemic pathological and toxicological markers are limited in the 10 \u0026micro;g and 20 \u0026micro;g doses of TGF-β mRNA-IAJD34. Even at the highest dose, 30 \u0026micro;g, increases in systemic toxicity markers were minimal. Established four-component LNPs are typically dosed between 1 and 10 \u0026micro;g for vaccinations for SARS-COV2 in mice, indicating that 10 \u0026micro;g would likely be sufficient (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Additionally, most studies using IAJDs report that 10 \u0026micro;g or less of mRNA to be effective for organ-specific detection (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo determine alveolar macrophage (AM) activation, BAL cells were evaluated by flow cytometry as previously described (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). AMs were defined as viable CD45\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eSiglecF\u003csup\u003e+\u003c/sup\u003e cells (Supplemental Fig.\u0026nbsp;4). Inflammatory activation was characterized by expression of Ly6c and CD11c. No dose-dependent difference was observed in 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). There were also no differences in Ly6C/CD206 AM populations at any dose of TGF-β mRNA-IAJD34 compared to control (Supplemental Table\u0026nbsp;4). Interstitial macrophages (IMs) were identified in CD45\u0026thinsp;+\u0026thinsp;lung digest cells. IMs were defined as viable being CD45\u003csup\u003e+\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003e, SiglecF\u003csup\u003e\u0026minus;\u003c/sup\u003e cells as previously described (Supplemental Fig.\u0026nbsp;4) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Ly6c\u003csup\u003e+\u003c/sup\u003e IMs were increased in all doses of TGF-β mRNA-IAJD34 with no increases in either CD11c\u003csup\u003e+\u003c/sup\u003e or CD206\u003csup\u003e+\u003c/sup\u003e expression at any dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplemental Fig.\u0026nbsp;5). 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\u003csup\u003efl/fl\u003c/sup\u003e mice showed a decrease in Ly6c\u003csup\u003e+\u003c/sup\u003e macrophages indicating that there may be a relationship between Ly6c and TGF-β signaling (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Further analysis will be necessary to fully understand TGF-β mRNA-IAJD34\u0026rsquo;s effects on Ly6c\u003csup\u003e+\u003c/sup\u003e IMs.\u003c/p\u003e \u003cp\u003eTGF-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 (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). 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 \u0026micro;g and 30 \u0026micro;g groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe next evaluated the presence of other cytokines and chemokines in the BAL fluid that can be upregulated or downregulated by TGF-β signaling (Supplemental Table\u0026nbsp;5). Granulocyte-colony stimulating factor (G-CSF), identified as an anti-inflammatory pro-neutrophilic growth factor, was elevated at the 30 \u0026micro;g dose of TGF-β mRNA-IAJD34 compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D) (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Thus, at 24 hours 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.\u003c/p\u003e \u003cp\u003eFinally, we studied metabolic upregulation, which is predicted to occur with increased protein synthesis and cell signaling stimulated by TGF-β mRNA. CD45\u003csup\u003e+\u003c/sup\u003e lung-digest cells were evaluated by employing the glycolytic rate assay and the mito stress. For the glycolytic rate assay, extracellular acidification rate (ECAR) was recorded. Glycolysis was determined by the ECAR measurement prior to glucose injection subtracted from the maximum ECAR before oligomycin injections. Injection with TGF-β mRNA-IAJD34 caused a dose-dependent increase in glycolysis, with a significant increase observed in the 20 \u0026micro;g and 30 \u0026micro;g groups compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). This increase was associated with increased cytokine production, likely a direct impact of TGF-β signaling cascades (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). 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. An TGF-β mRNA-IAJD34 induced dose-dependent increase was observed in maximal respiration, though this was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). 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 activation (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Thus, treatment with TGF-β mRNA-IAJD34 successfully alters macrophage cell phenotype and cytokine expression as expected with the production of TGF-β protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEvaluating pulmonary delivery of TGF-β mRNA-IAJD34 over time\u003c/h2\u003e \u003cp\u003eDue to the lack of toxicity, limited alterations in cell phenotype, and efficient expression of TGF-β protein and downstream signals, the 10 \u0026micro;g dose of TGF-β mRNA-IAJD34 was selected for further development.\u003c/p\u003e \u003cp\u003eThe 10 \u0026micro;g dose of empty-IAJD34 or TGF-β mRNA-IAJD34 was further evaluated at 4, 24, and 48 hours post-injection. Treatment with TGF-β mRNA-IAJD34 did not increase BAL protein or phospholipids at any time point (Supplemental Fig.\u0026nbsp;6).\u003c/p\u003e \u003cp\u003eIHC staining of lung tissue confirmed that TGF-β protein expression was transiently expressed in lung tissue, peaking at 4 hours post-injection, and persisting up to 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Unprocessed and cleaved TGF-β were also measured in whole lung tissue, showing significant increases in all forms of TGF-β at 4 hours post-injection, an increase in cleaved TGF-β at 24 hours, and no significant increases in any TGF-β isoforms at 48 hours post-injection compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). 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\u0026thinsp;+\u0026thinsp;IMs were observed (Supplemental Fig.\u0026nbsp;7 and Supplemental Table\u0026nbsp;6). Thus, a 10-\u0026micro;g 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 fibrosis (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Additionally, controlled repeat doses of TGF-β mRNA-IAJD34 can be delivered until resolution is obtained with a lower risk of over expressing TGF-β.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eTGF-β mRNA-IAJD34 can limit pulmonary injury following exposure to bleomycin\u003c/h2\u003e \u003cp\u003eThe 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\u0026ndash;28 days post initial exposure (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). It should be noted that not all models of ITB result in the same level of fibrosis (\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eFor initial studies, animals were treated with a 10-\u0026micro;g 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 days (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). 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 (Supplemental Fig.\u0026nbsp;8).\u003c/p\u003e \u003cp\u003eITB is also associated with increases in alveolar thickness, immune cell infiltration, and tissue consolidation that variably develop over the first 7 days post-exposure (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). An increase in alveolar epithelial thickness was observed in ITB\u0026thinsp;+\u0026thinsp;empty-IAJD34 animals, with limited changes in percent white space and nuclei count compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Treatment with TGF-β mRNA-IAJD34 did not significantly increase epithelial thickness compared to the matched control, indicating TGF-β mRNA-IAJD34 can prevent some ITB-induced increases in lung pathology (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The limited ITB-induced histological alterations are likely a consequence of evaluating injury at such an early timepoint 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, though the exact reasons for this are not well understood (\u003cspan additionalcitationids=\"CR54 CR55\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). BALB/c mice were chosen for this study as IAJD targeting and development has primarily been characterized in this strain of mice (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeyond histopathological alterations, ITB-induced ALI is also associated with increases in pulmonary epithelial injury, marked by increased BAL fluid protein and disrupted epithelial lipid barriers (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Exposure to ITB caused a significant increase in BAL fluid protein content as compared to control, with TGF-β mRNA-IAJD34 treatment having no effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). 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.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). However, treatment with TGF-β mRNA-IAJD34 prevented ITB-induced loss of cells lining the airway, a consequence of early pulmonary inflammation and cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo characterize the loss of BAL cells, AMs (viable CD45\u003csup\u003e+/\u003c/sup\u003eSiglec F\u003csup\u003e+/\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e BAL cells) were characterized further as being resident (CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e\u0026minus;\u003c/sup\u003e), recruited (CD11c\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e), or migratory macrophages (CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e). In line with previous 7-day studies, exposure to bleomycin reduced resident AMs and increased recruited AMs compared to PBS control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD-E) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD-E). In contrast to TGF-β mRNA-IAJD34 dose response studies, Ly6c\u003csup\u003e+\u003c/sup\u003e IMs (viable CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eSiglecF\u003csup\u003e\u0026minus;\u003c/sup\u003e lung-digest cells) were decreased in ITB exposed animals irrespective of TGF-β mRNA-IAJD34 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF). 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 types (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). There is some evidence that TGF-β plays less significant role in IM cell maturation and activity, but this remains largely speculative (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Further model development will be necessary to understand the role that TGF-β plays in modulating ITB-induced AM and IM cell characteristics.\u003c/p\u003e \u003cp\u003eWe next evaluated the downstream effects of TGF-β mRNA-IAJD34 on cytokine signaling following exposure to ITB beyond pathological and inflammatory indicators (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, Supplemental Table\u0026nbsp;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.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA-C) (\u003cspan additionalcitationids=\"CR60 CR61\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Treatment with TGF-β mRNA-IAJD34 prevented ITB-induced increases in all three of these proinflammatory cytokines, consistent with the anti-inflammatory signaling of TGF-β (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA-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.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD-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.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIAJD34 is a single-component lipid particle designed for the targeted delivery of mRNA to the lung (\u003cem\u003e40\u003c/em\u003e). 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\u003csup\u003eth\u003c/sup\u003e generation of the respiratory tree, is necessary to treat most lung injury and consequent disease (\u003cem\u003e1, 5, 7\u003c/em\u003e). While necessary, targeting these areas of the lung is challenging and has proven difficult in clinical settings (\u003cem\u003e1\u003c/em\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe hypothesize that IAJD34 is able to target these areas due to the large and charged altering ester and amide groups in the head group. When injected intravenously, the large charge density traps the formulated IAJD34 in narrow vascular regions of the lung, ultimately delivering the mRNA of interest to the small pulmonary arteries. 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 hours, which is important as long-term delivery of TGF-b\u0026nbsp;can lead to significant fibrosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTGF-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. \u0026nbsp;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. \u0026nbsp;Indeed, there was a significant TGF-β mRNA-IAJD34 effect that correlated with TGF-b\u0026nbsp;expression. These results indicate that we could successfully deliver TGF-b\u0026nbsp;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo establish TGF-β mRNA-IAJD34 as a therapeutic in this ALI model, we will need to characterize the effect of TGF-b\u0026nbsp;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 demonstrate the potential of mRNA delivery for therapeutic use in the lung.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 assistance with Luminex assays. \u0026nbsp;They also would also like to thank Dr. Michael Goedken and Dr. Marianne Polunas of the Rutgers University Histology Core for their assistance with histological preparation and pathology services. They would also like to thank Jessica Cervelli and Rita Hahn of the Rutgers flow cytometry core for assistance with flow cytometry. \u0026nbsp;Lastly, the authors would like to thank Sungjae Lee, Tony Hu, and Houping Ni for their technical assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.J.G., D.W., and E.N.A-V. conceived the concept.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eV.P. conceived and designed the IAJD used in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD.Z., Y.Y., L.P., and Y.L. synthesized the IAJD used in this study.\u003c/p\u003e\n\u003cp\u003eJ.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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJ.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.\u003c/p\u003e\n\u003cp\u003eJ.A.M., E.R.S., A.J.G. and E.N.A-V. wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD. Z., and Y.L. were financially supported by the National Key Research and Development Program of China [Grant 2022YFC2403203], National Natural Science Foundation of China [Grant 22305081], and Shanghai Sailing Program [Grant 23YF1408600].\u003c/p\u003e\n\u003cp\u003eA.J.G., E.R.S, R.S., M.W., E.A., and C.J.G. were financially supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grant T32-ES01984] and [Grant P30-ES005022].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn accordance with the University of Pennsylvania policies and procedures and our ethical obligations as researchers, we report that V.P., D.W., D.Z., E.N.A-V, 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.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eE. R. Johnson, M. A. Matthay, Acute lung injury: epidemiology, pathogenesis, and treatment. \u003cem\u003eJournal of aerosol medicine and pulmonary drug delivery\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 243-252 (2010).\u003c/li\u003e\n \u003cli\u003eA. Dushianthan, M. Grocott, A. Postle, R. Cusack, Acute respiratory distress syndrome and acute lung injury. \u003cem\u003ePostgraduate medical journal\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 612-622 (2011).\u003c/li\u003e\n \u003cli\u003eE. R. Stevenson, M. L. Wilkinson, E. Abramova, C. Guo, A. J. 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Saito\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, Role of interleukin-6 in bleomycin-induced lung inflammatory changes in mice. \u003cem\u003eAmerican journal of respiratory cell and molecular biology\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 566-571 (2008).\u003c/li\u003e\n \u003cli\u003eA. Takamizawa\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, Bleomycin stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activity. \u003cem\u003eThe Journal of Immunology\u003c/em\u003e \u003cstrong\u003e162\u003c/strong\u003e, 6200-6208 (1999).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e.\u0026nbsp;\u003cstrong\u003eInflammatory response to TGF-\u0026beta; mRNA-IAJD34 pulmonary delivery.\u003c/strong\u003e IAJD34 loaded with TGF-\u0026beta; mRNA at doses of 10, 20, and 30 \u0026mu;g per mouse were delivered to BALB/c mice and compared to na\u0026iuml;ve animals. At 24 hours post-injection, total BAL protein, differential BAL cell count, and BAL phospholipid levels were quantified as markers of lung injury: \u0026nbsp; MP, Macrophage, NP, neutrophil, LP, Lymphocyte. Data are presented as mean \u0026plusmn; SD, n=3-10 per group. Samples were compared using Shapiro-Wilk test for normality followed by an ordinary one-way ANOVA with Tukey\u0026rsquo;s Multiple comparison where * indicates a significant difference from the control, p\u0026lt;0.05.\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"767\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.013054830287206%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTGF-\u0026beta; mRNA-IAJD34 per mouse (\u003c/strong\u003e\u003cstrong\u003e\u0026mu;\u003c/strong\u003e\u003cstrong\u003eg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.840731070496084%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAL Phospholipids (\u0026micro;g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.621409921671018%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAL\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eProtein (mg/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.52480417754569%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAL Cell Count (x10\u003csup\u003e4\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.317073170731707%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.365853658536587%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.829268292682926%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.48780487804878%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.013054830287206%\"\u003e\n \u003cp\u003eNa\u0026iuml;ve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.840731070496084%\"\u003e\n \u003cp\u003e40.2 \u0026plusmn; 17.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.621409921671018%\"\u003e\n \u003cp\u003e0.23 \u0026plusmn; 0.02\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.621409921671018%\"\u003e\n \u003cp\u003e26.4\u0026nbsp;\u0026plusmn; 8.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.577023498694517%\"\u003e\n \u003cp\u003e25.5\u0026nbsp;\u0026plusmn; 7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.360313315926893%\"\u003e\n \u003cp\u003e\u0026nbsp;0.2\u0026nbsp;\u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.966057441253264%\"\u003e\n \u003cp\u003e0.7 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.013054830287206%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.840731070496084%\"\u003e\n \u003cp\u003e53.1 \u0026plusmn; 12.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.621409921671018%\"\u003e\n \u003cp\u003e0.40 \u0026plusmn; 0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.621409921671018%\"\u003e\n \u003cp\u003e17.6\u0026nbsp;\u0026plusmn; 11.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.577023498694517%\"\u003e\n \u003cp\u003e20.2\u0026nbsp;\u0026plusmn; 6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.360313315926893%\"\u003e\n \u003cp\u003e0.1\u0026nbsp;\u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.966057441253264%\"\u003e\n \u003cp\u003e0.8\u0026nbsp;\u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.013054830287206%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.840731070496084%\"\u003e\n \u003cp\u003e42.4 \u0026plusmn; 19.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.621409921671018%\"\u003e\n \u003cp\u003e0.36 \u0026plusmn; 0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.621409921671018%\"\u003e\n \u003cp\u003e\u0026nbsp;29.9\u0026nbsp;\u0026plusmn; 12.6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.577023498694517%\"\u003e\n \u003cp\u003e28.3\u0026nbsp;\u0026plusmn; 11.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.360313315926893%\"\u003e\n \u003cp\u003e0.9\u0026nbsp;\u0026plusmn; 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.966057441253264%\"\u003e\n \u003cp\u003e0.8\u0026nbsp;\u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.013054830287206%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.840731070496084%\"\u003e\n \u003cp\u003e\u0026nbsp;60.2 \u0026plusmn; 15.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.621409921671018%\"\u003e\n \u003cp\u003e1.25 \u0026plusmn; 0.76*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.621409921671018%\"\u003e\n \u003cp\u003e39.8\u0026nbsp;\u0026plusmn; 2.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.577023498694517%\"\u003e\n \u003cp\u003e35.5\u0026nbsp;\u0026plusmn; 23.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.360313315926893%\"\u003e\n \u003cp\u003e\u0026nbsp;25.0\u0026nbsp;\u0026plusmn; 25.2*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.966057441253264%\"\u003e\n \u003cp\u003e1.8\u0026nbsp;\u0026plusmn; 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"one-component ionizable amphiphilic Janus Dendrimers, mRNA targeted delivery, anti-inflammatory cytokine, TGF-β, bleomycin injury, respiratory disease, lung parenchyma","lastPublishedDoi":"10.21203/rs.3.rs-4656663/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4656663/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrent clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways. However, targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here, we have developed an mRNA therapeutic for the lower lung using one-component Ionizable Amphiphilic Janus Dendrimers (IAJDs) as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA, transforming growth factor-beta (TGF-β), to produce transient protein expression in the lower regions of the lung. This study highlights IAJD\u0026rsquo;s potential for precise, effective, and safe delivery of TGF-β mRNA to the lung. 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.\u003c/p\u003e","manuscriptTitle":"Targeted delivery of TGF-β mRNA to lung parenchyma using one-component ionizable amphiphilic Janus Dendrimers.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-12 05:38:32","doi":"10.21203/rs.3.rs-4656663/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9731bb1e-83ee-4c2b-be42-c8a86cba097c","owner":[],"postedDate":"July 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34448217,"name":"Biological sciences/Immunology/Cytokines/Transforming growth factor beta"},{"id":34448218,"name":"Biological sciences/Immunology/Immunotherapy"},{"id":34448219,"name":"Biological sciences/Immunology/Immunogenetics"},{"id":34448220,"name":"Biological sciences/Drug discovery"},{"id":34448221,"name":"Biological sciences/Biotechnology/Applied immunology"}],"tags":[],"updatedAt":"2025-02-22T08:06:15+00:00","versionOfRecord":{"articleIdentity":"rs-4656663","link":"https://doi.org/10.1038/s41467-025-56448-y","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-02-21 05:00:00","publishedOnDateReadable":"February 21st, 2025"},"versionCreatedAt":"2024-07-12 05:38:32","video":"","vorDoi":"10.1038/s41467-025-56448-y","vorDoiUrl":"https://doi.org/10.1038/s41467-025-56448-y","workflowStages":[]},"version":"v1","identity":"rs-4656663","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4656663","identity":"rs-4656663","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}