Inhaled PAMAM-based nano-formulation prolonged lung retention for alleviating pulmonary inflammation of COPD

Abstract

Chronic obstructive pulmonary disease (COPD) stands as a predominant respiratory disorder intricately linked with respiratory tract microorganisms and their metabolites. Indole acetic acid (IAA), a derivative of tryptophan produced by Lactobacillus salivarius, possesses notable anti-inflammatory properties. However, the short retention time of the drug in lung still remains a vital obstacle leading to a poor bioavailability. In this study, we innovatively engineer a nano-composite by coupling IAA with generation 4 polyamidoamine (G4 PAMAM) dendrimer to form G4-IAA nano-complex through host-guest interaction. G4-IAA shows significantly improved solubility of IAA and thus enhances its bioavailability. This G4-IAA complex facilitates direct aerosol-based pulmonary administration by inhaled strategy, exhibiting enhanced absorption by respiratory epithelial cells and prolonged lung retention. Our experimental findings reveal that inhalation therapy employing the G4-IAA complex mitigates inflammatory stress and augments pulmonary function in COPD murine models. Single-cell sequencing reveals macrophages may contribute to the functional shifts by G4-IAA, promoting an anti-inflammatory phenotype characteristic of M2 polarization. This research introduces a promising therapeutic strategy, offering improved symptomatic relief and reduced risk of acute exacerbations for individuals afflicted with COPD.

Graphical abstract

1. Introduction

Chronic obstructive pulmonary disease (COPD) is one of the most prevalent respiratory disorders globally, characterized by a multifaceted pathogenesis involving airway and alveolar abnormalities [1,2]. This condition significantly impacts patients' quality of life and imposes a substantial economic burden on healthcare systems. Given the high morbidity and mortality associated with COPD worldwide, there is an urgent need to develop innovative therapeutic strategies to mitigate disease progression.

In COPD patients, the respiratory microbial community undergoes notable changes, with the dynamics and abundance of these microorganisms strongly correlated with COPD severity and pathology [3,4]. For example, an increased prevalence of Prevotella and Streptococcus in airway has been associated with more advanced stages of the disease [4,5]. Our team's research has identified reduced levels of the tryptophan metabolite indole-3-acetic acid (IAA) produced by airway Lactobacillus in COPD patients, suggesting potential therapeutic benefits of IAA [6]. IAA elevation in bronchoalveolar lavage fluid may suppress the secretion of TNF-α, IL-12, IL-13, and CXCL1 by smokers' alveolar macrophages [7]. IAA triggered the aryl hydrocarbon receptor (AhR), inducing interleukin-22 and strengthening intestinal tight junction [[8], [9], [10]], as well as reduced oxidative and inflammatory stress in non-alcoholic fatty liver disease models [11,12]. These findings highlight the therapeutic potential of IAA in host–microbe interactions and its promise for airway-targeted drug development. When administered intraperitoneally, IAA has been shown to reduce airway inflammation, decrease epithelial cell apoptosis, therefore improve lung function in COPD mice [10,12]. However, its efficacy may be limited by issues related to its water solubility and method of administration.

Current clinical therapies for COPD, despite their variety, often provide only short-term relief and require frequent administration to maintain their effects. To address these limitations, nanotechnology has emerged as a promising field in drug research and delivery [13]. Nanomaterials, such as G4 polyamidoamine (PAMAM), show significant potential due to their unique structures and multifunctional properties, offering advances in treating various diseases [[13], [14], [15]]. As a highly branched nano-carrier with diverse biomedical applications, G4 PAMAM is ideal for drug delivery, gene therapy, bioimaging, and tissue engineering due to its physicochemical properties. Previous studies have shown that genenration 2 PAMAM dendrimers can traverse lung epithelial cells via paracellular and transcellular pathways, greatly improving the absorption of peptide and protein drugs like insulin while maintaining low cytotoxicity to the pulmonary membrane [5,16]. In ocular treatments, PAMAM-OH dendrimers have been explored for corneal inflammation by forming complexes with dexamethasone (PAMAM-Dex), functioning as a sustained-release system that maintains effective drug levels and enables controlled release in response to environmental changes [17,18]. G4 PAMAM enhances the intestinal absorption of 5-aminosalicylic acid and mitigates cytokine-induced immune response in the intestinal mucosa [19,20], as well as promotes tissue repair and regeneration by enhancing cell proliferation [21]. These characteristics of PAMAM make it a promising candidate for developing novel COPD therapies that require efficient drug delivery with minimal cytotoxicity and potential for sustained therapeutic effects.

In this study, we constructed a dendrimer-based nano-formulation by encapsulating IAA into G4 PAMAM dendrimers. The formed nano-formulation of G4-IAA was used for pulmonary administration via inhalation for alleviating pulmonary inflammation in COPD. We systematically investigated how G4 PAMAM dendrimers enhance the efficacy of airway microbial metabolite IAA, enabling aerosol delivery to the lungs and potentially slowing the development of COPD while minimizing systemic side effects.

2. Materials and methods

2.1. Synthesis and characterization of G4-IAA

2 mg/mL IAA (HY-18569; MCE, USA) was dissolved in deionized water and then Amino-terminated G4 PAMAM (412449; Sigma-Aldrich, USA) were added to 2 mg/mL. The mixture was sonicated for 2 h to form G4-IAA nanocomplexes until no visible precipitate remained, and then filtered through a 0.22 μm membrane filter (GSWP04700, Sigma, USA). The photos of G4-IAA solution were recorded, and UV–visible (UV–Vis) spectroscopy were used to characterize G4-IAA nano-complex. A dialysis tube with a molecular weight cut-off (MWCO) of 5 kDa was employed to investigate the in vitro release profile of IAA from the G4-IAA complex. At predetermined time intervals, aliquots of the external solution were collected and analyzed by measuring absorbance at 280 nm using a UV–Vis spectrophotometer to quantify the amount of IAA released.

2.2. Cytotoxicity assessment

A549 (CCL-185; ATCC, USA) and BEAS-2B (CRL-3588; ATCC, USA) cells were seeded in 96-well plates, incubated at 37 °C until 30 % confluence. Cytotoxicity was assessed with varying G4 PAMAM concentrations (0–4 mg/mL) using CCK-8 reagent, measuring absorbance at 450 nm. Proliferation was evaluated with 2 mg/mL G4 PAMAM over 1, 3 and 5 days, followed by CCK-8 assay.

2.3. Construction of fluorescent nanocarriers

G4 PAMAM was dissolved in deionized water (100 mg/mL) and mixed with ICG (1:3 M ratio). After 24 h of stirring, the mixture was dialyzed and freeze-dried to obtain G4 PAMAM-ICG. G4-IAA-ICG was then prepared by sonicating 2 mg IAA with 2 mg/mL G4 PAMAM-ICG for 2 hours.

2.4. Cellular uptake assessment

Raw264.7 cells (CRL-3588; ATCC, USA) were cultured in DMEM until 50% confluence, then treated with G4-IAA-ICG for 1, 2, 4 and 8 h. Lysosomal uptake was tracked with Lyso-Tracker Red, and nuclei were stained with DAPI before termination. Cells were washed and imaged using confocal microscopy.

2.5. Animal model establishment and treatment grouping

The experimental protocol of animals was approved by the Health Research Ethic Authority of Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine. C57BL/6 mice (Vital River, China), aged five weeks, were housed in an SPF environment with a 12-h light/dark cycle, 20–25 °C, and 50–70 % humidity. The study comprised four groups: the control group, receiving 40 μL PBS intranasally weekly for 4 weeks; the disease group, induced with 40 μL of 50 μg/kg lipopolysaccharides (L5293; Sigma, USA) and 60 U/kg pancreatin (HY-B2118; MCE, USA) intranasally weekly for 4 weeks; the nebulized inhalation G4-IAA group, treated with nebulized G4-IAA (IAA at 50 mg/kg, G4 PAMAM at 2 mg/mL) every two days for 14 days after lipopolysaccharides and pancreatin treatment; and the intraperitoneal injection IAA group, receiving lipopolysaccharides and pancreatin followed by intraperitoneal IAA injections (50 mg/kg) every two days for 14 days.

2.6. Histopathological assessment

Mice were treated with nebulized G4 PAMAM-ICG loaded with IAA or IAA via intraperitoneal injection, then imaged at 12, 24, and 48 hours using an IVIS 200 system. Lung tissues were fixed, embedded, sectioned, and stained with H&E. Mean linear intercept (MLI) in lung tissue as a measure of the enlargement of the alveolar space or emphysema was determined for 2 regions per mouse studied on a grid consisting of two crossed lines. The number of alveolar intervals (Ns) were counted through the intersection lines and the total length of the lines was measured (Length, L). MLI was calculated: MLI = L/Ns. Alveolar inflammation was scored by the infiltration of inflammatory cells in alveoli and interstitium. No infiltration was scored as 0, mild infiltration was scored as 1, moderate infiltration was scored as 2, and severe infiltration was scored as 3. Alveolar inflammation and emphysema were scored by two independent reviewers.

2.7. Cellular model building and grouping

A549, BEAS-2B and RAW264.7 cells were cultured in DMEM with 10 % fetal bovine serum and penicillin-streptomycin at 37 °C, 5 % CO₂. CSE was prepared from cigarette (Double Happiness, China) smoke and diluted to 10 % CSE, then adjusted to pH 7.4 and filtered. Cells were treated with 10 % CSE with or without LPS, IAA, G4 PAMAM, or G4-IAA for 48 h.

2.8. qPCR analysis of gene expression

Total RNA was extracted from lung tissues, A549, and BEAS-2B cells using the Trizol kit (R411-01; Vazyme, China). cDNA was synthesized with the HiScript III RT SuperMix (R323-01; Vazyme, China). Real-time quantitative PCR was performed using the HiScript III U + One Step qRT-PCR Probe Master Mix with TaqMan technology. Primers were synthesized by Biotnt (Shanghai, China) and used to amplify target genes, with the sequences presented sequences in Table S1 (Supporting information).

2.9. Enzyme-linked immunosorbent assay (ELISA)

Mouse lung tissue homogenates were processed and assayed using ELISA (Dakewe, China). Standards were prepared to generate a standard curve. ELISA plates were coated with specific antibodies, blocked, and then incubated with samples and standards. Detection involved adding a secondary antibody, TMB substrate, and stopping with sulfuric acid. Optical density was measured to quantify cytokine levels against the standard curve.

2.10. Immunofluorescence staining of macrophage polarization

RAW264.7 cells were seeded in confocal dishes and allowed to adhere overnight. Cells were fixed with 4% paraformaldehyde, washed, and incubated with CD206 primary antibody (AG2664; Beyotime, China) overnight at 4°C. After washing, cells were treated with Alexa Fluor 488 secondary antibody (1:250) and Actin-Tracker Red-555 (1:100). Nuclear staining was performed with DAPI. Fluorescence was observed using a confocal laser scanning microscope. The procedure was repeated with CD86 (AG1453; Beyotime, China) antibody in place of CD206.

2.11. Single-cell sequencing of mouse lung tissue

Lung tissues were processed into single-cell suspensions using the Transcriptome Kit (SeekOneDD; Seekgene, China). RNAs were individually labeled with Barcoded Beads, and cells were clustered and identified. Transcriptome libraries were constructed and sequenced on the Illumina platform (Illumina, USA).

2.12. Bacteriostasis experiment

Pseudomonas aeruginosa was recovered on tryptic soy agar (CM0131B; ThermoFisher, USA) plates and cultured in tryptic soy broth (CM0129B; ThermoFisher, USA). After two successive subcultures to ensure activation, the bacterial suspension was diluted 1:100 in fresh TSB. A volume of 200 μL of the diluted suspension was added to each well of a 96-well plate, followed by treatment with various concentrations (0–300 μg/mL) of IAA or G4-IAA. Bacterial growth was monitored by measuring optical density at 600 nm (OD600) using a UV–visible spectrophotometer. For biofilm formation assays, P. aeruginosa was cultured in 96-well plates for 48 h. The biofilms were then fixed with 4 % paraformaldehyde (P0099, Beyotime, China), stained with crystal violet (C0121, Beyotime, China), and subsequently solubilized with 75 % ethanol for quantification.

Statistical analyses: Data were analyzed using GraphPad Prism v7.0. A two-sample t-test compared means between two groups, and one-way ANOVA assessed differences among multiple groups. Results are presented as mean ± standard error of the mean (SEM), with p < 0.05 considered statistically significant.

3. Results and discussion

3.1. Fabrication and characterization of G4-IAA nano-complexes

Previously studies has demonstrated the drug of IAA exhibits poor solubility in water, which makes it challenging to administer effectively to the lungs. This limitation poses a significant obstacle for pulmonary delivery, as achieving the required therapeutic concentration is difficult. Therefore, we propose a facile strategy to enhance the aqueous solubility of IAA (Fig. 1a). PAMAM dendrimers have a large number of hydrophilic surface groups showing excellent aqueous solubility, and possess hydrophobic interior cavity that can easily encapsulate hydrophobic compounds. The hydrophobic IAA drug can be loaded into the hydrophobic interior cavity of G4 PAMAM dendrimer yielding G4-IAA complexes via host-guest interaction to improve the solubility of IAA in aqueous solution. After mixing the G4 PAMAM and IAA by sonication, the G4-IAA mixture presents clearer aqueous solution compared to IAA aqueous solution, indicating that loading IAA into PAMAM dendrimers significantly improved its solubility in water (Fig. 1b). Transmission and scanning electron microscopy (TEM and SEM) both confirmed the spherical morphology of G4-IAA, with approximately 50 nm in diameter per monomer and 2 μm for the polymers, which favored deposition in alveoli and bronchioles and cellular uptake at the same time (Fig. 1c and d) [22,23]. The number-average molecular weight (Mn) and weight-average molecular weight (Mw) were determined by GPC to be 8276 g/mol and 16,809 g/mol respectively, with a polydispersity index (PDI) of 2.03 (Supplementary Fig. 1). UV–visible spectroscopy confirmed the successful encapsulation of IAA into G4 PAMAM, as evidenced by a red shift in IAA absorption peak from 224 nm to 229 nm (Fig. 1e). Further spectroscopic analysis showed that the solubility of IAA nearly reached saturation at a G4-IAA concentration of 2 mg/ml, with no significant difference observed at 4 mg/ml (Fig. 1f and g). Therefore, 2 mg/ml was chosen as the optimal concentration, effectively enhancing the solubility of IAA while maintaining the stability and homogeneity of G4-IAA.

Fig. 1.

Fabrication and Characterization of G4-IAA. (a) Synthesis and application of G4-IAA complex. (b) Photograph of IAA and G4-IAA complex in aqueous solution. (c) TEM image of G4-IAA. (d) SEM image of G4-IAA. (e) UV spectrum of IAA and G4-IAA. (f) UV spectrum of G4-IAA under 185–400 nm. (g) UV spectrum of G4-IAA complex of different concentrations under 229 nm. (h) Cumulative release of IAA from G4-IAA. g–h: Data are presented as means ± SD, with n = 3 experimental replicates per group.

The dendritic architecture of G4 PAMAM offers a distinct advantage for loading small-molecule compounds [11], allowing the otherwise water-insoluble IAA to be effectively incorporated and remain stable for up to 48 hours. Selecting 2 mg/ml as the final concentration maximizes IAA's solubility and minimizes potential adverse effects associated with higher concentrations of G4 PAMAM. IAA was released from G4-IAA in a controlled manner, with complete release occurring after 24 hours (Fig. 1h).

3.2. In vitro anti-inflammatory activity of G4-IAA

In COPD management, current treatments focus on symptom relief and quality of life, with no cure available. Existing therapies, including bronchodilators and anti-inflammatory drugs, offer temporary relief but do not halt lung tissue deterioration. Given the need for new therapeutic strategies to improve long-term outcomes in COPD, we investigated the potential effects of G4-IAA in vitro using lung epithelial cell lines.

We exposed A549 and BEAS-2B to cigarette smoke extract (CSE) and lipopolysaccharide (LPS) to create cellular models commonly used in COPD research for evaluating the efficacy of G4-IAA. Our in-vitro experiments demonstrated that treatment with the G4-IAA significantly reduced the expression of the pro-inflammatory cytokine IL-8 compared to treatments with IAA or G4 PAMAM alone (Fig. 2a–d). Specifically, in the A549 cell line, G4-IAA markedly inhibited the production of IL-6 and IL-8. In the BEAS-2B cell line, G4-IAA was highly effective in lowering IL-1β and IL-8 to the lowest levels among the three treatment groups.

Fig. 2.

The anti-inflammatory effect of IAA, G4 PAMAM and G4-IAA in vitro. (a–b) mRNA level of IL-6 (a) and IL-8 (b) in A549 by RT-qPCR. (c–d) mRNA level of IL-1β (c) and IL-8 (d) in BASE-2B by RT-qPCR. Data are presented as means ± SD, with n = 3 experimental replicates per group. One-way ANOVA with Tukey's multiple comparisons test was performed. Groups were compared to each other. ns: p > 0.05; ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗∗: p < 0.0001. IL-1β: interleukin 1β; IL-6: interleukin 6; IL-8: interleukin 8; TNFα: tumor necrosis factor α; RT-qPCR: real-time polymerase chain reaction; mRNA: messenger ribonucleic acid.

These findings suggest that G4-IAA may exert its anti-inflammatory effects by modulating the expression of inflammatory cytokines. This has important implications for COPD treatment, as inflammation is a pivotal factor in the pathogenesis of the disease. By reducing inflammatory mediators, G4-IAA could potentially alleviate the inflammatory burden and improve clinical symptoms and quality of life for COPD patients.

This also indicated that the anti-inflammatory effects of G4-IAA stemmed from the synergistic interaction between IAA and G4 PAMAM. The dendritic structure of G4 PAMAM interacts with the lipid bilayer of lung epithelial cells through electrostatic attraction, leading to a reduction in lipids within the bilayer and the formation of dendrimer/lipid aggregates in solution [24]. This interaction increases cell membrane permeability temporarily. Moreover, increasing concentrations of G4-IAA did not significantly enhance the anti-inflammatory response within the first 12 hours, which may suggest a sustained and controlled release of IAA from the G4-IAA complex (Supplementary Fig. 2). The cationic G4 PAMAM dendrimer exhibits favorable pulmonary biocompatibility and a slower absorption rate, which helps prolong the retention of IAA in the lungs, enabling controlled release [25]. This established a drug delivery system that was particularly well-suited for COPD treatment, as it ensuresd that IAA delivered its therapeutic effects directly and locally within the lungs, maximizing efficacy at the site of disease.

3.3. Macrophage uptake and polarization dynamics

PAMAM's positive charge may target areas of inflammation, where changes in the lung microenvironment increase its affinity for inflamed tissues [26,27]. This targeting likely enhances epithelial and macrophage membrane permeability, amplifying IAA therapeutic impact. To better understand cellular changes and treatment effects, we performed single-cell RNA sequencing on lung tissue from mice. Intraperitoneal administration of IAA decreased neutrophil counts and increased macrophage populations of mice lungs, suggesting suppression of inflammation and macrophage polarization through AhR activation [28]. The analysis revealed that G4-IAA aerosol-treated mice had significantly fewer macrophages in their lungs compared to intraperitoneal-treated mice, while treatment with IAA alone led to an increase in macrophage numbers (Fig. 3a). We also assessed macrophage clustering and polarization in the three treatment groups. Our findings indicated a reduction in M1 macrophage markers (Fig. 3b), while M2 macrophage polarization was notably influenced (Fig. 3c). CD163 levels, another M2 macrophage marker, declined following IAA injection but recovered with G4-IAA inhalation, whereas CD86 showed an inverse trend (Fig. 3c).

Fig. 3.

Polarization of RAW264.7 induced by G4-IAA. (a) Cell-type composition based on single-cell RNA sequencing analysis. (b) Expression of M1 macrophage-associated genes. (c) Expression of genes associated with M2 macrophage polarization. (d, e) DAPI (blue), CD206 (purple) (d), CD86(purple) (e), G4 PAMAM (green) staining sections of RAW264.7 cells at 1, 2, 4 and 8 hours of co-cultivation of macrophages and IAA-PAMAM complex. (f, g) Relative fluorescence intensity of CD206 (f) and CD86 (g) from quantitative analysis at different time points. Data are presented as means ± SD, with n = 3 experimental replicates per group. One-way ANOVA with Tukey's multiple comparisons test was performed. Adjacent groups were compared. ns: p > 0.05; ∗: p < 0.05; ∗∗: p < 0.01. CD: cluster of differentiation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

We utilized RAW264.7 cell line to verify how the uptake of G4-IAA affects cellular kinetics and induces changes in macrophage polarization. Immunofluorescence imaging with confocal microscopy demonstrated that G4-IAA gradually accumulated within the cells over time. After 4 hours of G4-IAA treatment, there was a significant increase in CD206 expression and a decrease in CD86 expression (Fig. 3d–g). These results suggest that G4-IAA enhances its anti-inflammatory effects by promoting the conversion of macrophages from M1 to M2 polarization.

Moreover, G4-IAA downregulated Plet1 gene expression and modulated macrophage activity by reducing the expression of Ltc4s, suggesting the attenuation of Leukotriene C4 production, critical for anti-inflammatory responses (Supplementary Fig. 3d–e) [29,30]. Concurrently, G4-IAA upregulated genes such as Plac8 and Alas2, which promote tissue repair and angiogenesis (Supplementary Fig. 3e) [31,32]. These findings reveal a complex interaction between IAA and PAMAM in modulating immune responses and macrophage polarization, ultimately mitigating inflammation in COPD.

3.4. Biocompatibility and distribution of G4-IAA

To investigate the distribution and retention of G4-IAA in vivo, we labeled G4 PAMAM with the fluorescent dye ICG. Fluorescence imaging revealed that, following aerosol inhalation, G4-IAA was predominantly localized in the lungs, whereas intraperitoneal injection led to its accumulation in the liver and kidneys (Fig. 4a–d). Notably, the fluorescence intensity in the lungs of the aerosolized group remained significantly higher than in the intraperitoneal group after 48 hours. This suggests that aerosolized inhalation could enhance clinical medication adherence and reduce the need for frequent drug administration. By increasing IAA's solubility, this system may allow IAA to reach the bronchioles and even the alveoli in the form of fine particles, thereby improving its distribution and therapeutic efficacy in the distal pulmonary regions [33].

Fig. 4.

Biocompatibility and distribution of G4-IAA. (a–b) Distribution of G4-IAA in mice (a) or separated organs (b) 12, 24, 48 hours after inhalation (50 mg/kg IAA+2 mg/ml G4 PAMAM) or injection (50 mg/kg IAA+2 mg/ml G4 PAMAM) treatments by fluorescence imaging in vivo. (c–d) Relative intensity of G4-IAA in separated organs 12, 24, 48 hours after inhalation treatment (c) or injection treatment (d) from quantitative analysis of fluorescent images. (e) Representative images of RAW264.7 stained by nucleus (blue), lysosome (red) and G4 PAMAM (green) 2, 4 hours after incubation with G4 PAMAM. Data are presented as means ± SD, with n = 3 experimental (e) or biological (a–d) replicates per group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Immunofluorescence studies on macrophages showed that the green fluorescence of G4-IAA overlapped with the red fluorescence of lysosomes (Fig. 4e), indicating co-localization within macrophage lysosomes. This observation suggests that G4-IAA is internalized by macrophages through phagocytosis and subsequently processed intracellularly, suggesting a mechanism for its intracellular processing and subsequent elimination from the body.

We assessed the biocompatibility of G4 PAMAM at the cellular and murine levels, using two human lung epithelial cell lines, A549 and BEAS-2B, as well as COPD mice. Cell viability assays indicated that G4 PAMAM, at various concentrations, did not significantly affect cell growth over 48 hours. Specifically, at a concentration of 2 mg/mL, cell growth was comparable between groups after 5 days of co-culture with A549 and BEAS-2B cells, demonstrating that both G4 PAMAM and G4-IAA complexes exhibit good biocompatibility and low cytotoxicity (Fig. 5a–d).

Fig. 5.

In vivo and in vitro toxicity of G4-IAA. (a, c) Cell viability of A549 (a) and BEAS-2B (c) incubated with different concentrations of G4 PAMAM for 48 hours using CCK8 assay. (b, d) Cell proliferation of A549 (b) and BEAS-2B (d) incubated with G4 PAMAM (2 mg/ml) or G4-IAA (2 mg/ml). (e–j) The serum level of hepatic (e–g) and renal (h–j) biomarkers in mice 14 days after different treatments (once every 2 days, 50 mg/kg IAA and 2 mg/ml PAMAM G4 per time), including total bilirubin (e), γ-glutamyl transferase (f), alanine aminotransferase (g), uric acid (h), creatinine (i) and urea (j). NC: negative control; TBIL: total bilirubin; γ-GT: γ-glutamyl transferase; ALT: alanine aminotransferase; UA: uric acid; CREA: creatinine; Data are presented as means ± SD, with n = 3 biological replicates per group. One-way ANOVA with Tukey's multiple comparisons test was performed. Treated groups were compared with each other and to the untreated group (labeled as NC in the figure) for statistical significance tests. ns: p > 0.05; ∗: p < 0.05.

Previous studies on G4 PAMAM have reported that it primarily accumulated in the kidney and was excreted renally [34], with additional distribution observed in liver and lung [35]. Our research in vivo toxicity evaluation of G4-IAA further confirmed its safety profile. No significant changes in liver or kidney biomarkers were observed with either aerosol or intraperitoneal administration, indicating a favorable biosafety profile (Fig. 5e–j). Specifically, levels of total bilirubin, γ-glutamyl transferase, alanine aminotransferase, uric acid, creatinine and urea did not differ significantly between treatment groups. Moreover, urea and creatinine levels were lower in the aerosol inhalation group compared to the intraperitoneal group, suggesting reduced renal impact with aerosol delivery (Fig. 5h and i). These results support the potential of G4-IAA as a safe and effective drug delivery system.

3.5. Anti-inflammatory potency of G4-IAA in vivo

In COPD management, current treatments focus on symptom relief and improving quality of life, with no cure available. Existing therapies, including bronchodilators and anti-inflammatory drugs, offer temporary relief but do not halt lung tissue deterioration. Given the need for new therapeutic strategies to improve long-term outcomes in COPD, we investigated the potential effects of G4-IAA using a well-established mouse model of the disease to further validate the anti-inflammatory effects observed in our in-vitro experiments.

Two weeks into the COPD mouse modeling process, continuous aerosol administration of G4-IAA resulted in noticeable improvement in the overall condition of the mice [36]. Histopathological analyses revealed a significant reduction in lung inflammation following G4-IAA treatment, with decreased inflammatory cell infiltration and reduced alveolar destruction (Fig. 6a–c). Functional assessments, like spirometry, demonstrated partial restoration of ventilatory function, with the aerosol delivery modality showing particular efficacy (Fig. 6d). Analysis of inflammatory cytokines indicated significant reductions in key pro-inflammatory mediators, such as TNFα, IL-6, CXCL15, and IL-1β, with aerosol administration exhibiting enhanced efficacy (Fig. 6e–k). Notably, stand-alone G4 PAMAM also displayed inherent anti-inflammatory properties, though to a lesser extent (Fig. 6e–k).

Fig. 6.

The therapeutic function of IAA, G4 PAMAM and G4-IAA in vivo. (a) H&E staining sections of lungs of mice in control, untreated COPD, COPD treatments with G4-IAA (inhalation), IAA (injection) or PAMAM (inhalation) groups. (b) Pulmonary mean linear intercept calculated according to (a). (c) Lung injury score calculated according to (a). (d) Lung function represented by FEV0.1/FVC of mice in control, untreated COPD, COPD treatment with IAA-PAMAM (inhalation) or IAA (injection) groups. (e–g) Concentration of inflammatory cytokines in mice lung tissue by ELISA, including IL-1β (e), IL-6 (f) and CXCL15 (g). (h–k) mRNA levels of inflammatory cytokines in mice lung tissue by RT-qPCR, including IL-1β (h), IL-6 (i) CXCL15 (j) and TNFα (k). Data are presented as means ± SD, with n = 6 biological replicates per group. One-way ANOVA with Sidak's multiple comparisons test was performed. Groups were compared to each other. ns: p > 0.05, not significant; ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001; ∗∗∗∗: p < 0.0001. COPD treatments: 50 mg/kg IAA+2 mg/ml G4 PAMAM (inhalation), 50 mg/kg IAA (intraperitoneal injection), or 2 mg/ml G4 PAMAM (inhalation) per time, once/2 days for 14 days continuously; FEV0.1: forced expiratory volume at 0.1 s; FVC: forced vital capacity.

To further validate these findings, we conducted additional pathological and inflammatory analyses using a cigarette smoke-induced COPD mouse model. Results confirmed that G4-IAA effectively suppressed levels of IL-1β, IL-6, and CXCL15 (Supplementary Fig. 4a). H&E staining of lung tissue also showed reduced inflammation and alveolar destruction, consistent with observations from the initial model (Supplementary Fig. 4b–d).

We further evaluated the antibacterial activity of G4-IAA against Pseudomonas aeruginosa, a common pathogen in COPD-related pneumonia [37,38]. G4-IAA exhibited superior and more sustained inhibition of P. aeruginosa growth compared to free IAA at equivalent concentrations, particularly after 6 hours of incubation (Supplementary Fig. 5a–b). Notably, the inhibitory effect remained significant even after 14 hours (Supplementary Fig. 5c), and it also inhibited the formation of biofilm concentration-dependently at 48 hours (Supplementary Fig. 5d). Given that IAA has been reported to suppress the type III secretion system [39], the enhanced antibacterial effect of G4-IAA may be attributed to the synergistic actions of PAMAM G4, including biofilm disruption and electrostatic interaction with bacterial membranes via its positively charged surface [40].

These results underscore the potential of G4-IAA as a novel therapeutic agent for COPD. G4-IAA not only demonstrates unique advantages over existing treatments but also represents a promising direction for drug development in COPD. The combination strategy of G4-IAA appears to offer a more effective treatment option compared to either G4 PAMAM or IAA alone.

4. Conclusions

In our study, PAMAM dendrimers were employed as a carrier to enhance IAA's anti-inflammatory effects by delivering it directly to the lungs via aerosol. Encapsulated by the dendrimer's branched structure, IAA more effectively reached and persisted in the deep lung tissue, targeting areas of inflammation and enhancing cellular absorption, which improved its anti-inflammatory effects and promoted lung tissue repair with minimal side effects. Both cellular and animal models demonstrated that G4-IAA promoted the transformation of macrophages into an M2 anti-inflammatory phenotype, leading to reduced inflammation and enhanced tissue repair. Our findings highlight the pivotal role of macrophage polarization induced by this nanocomposite in the respiratory system, offering a promising direction for future COPD treatments that harness microbial metabolites and targeted drug delivery.

CRediT authorship contribution statement

Chen Wang: Writing – original draft, Visualization, Investigation, Formal analysis, Data curation, Conceptualization. Xiao-yan Hu: Visualization, Formal analysis, Data curation. Ri Ji: Data curation, Conceptualization. Yi-fan Lu: Visualization, Validation. Xiang Shen: Visualization, Software, Methodology. Zhang Wang: Methodology, Investigation, Conceptualization. Fei Wang: Methodology, Data curation, Conceptualization. Guo-chao Shi: Supervision, Data curation, Conceptualization. Yun Feng: Writing – review & editing, Supervision, Funding acquisition, Data curation, Conceptualization.

Funding

This work was supported by grants from the National Key Research and Development Project of China (2022YFA1304300 (F. Y.)), National Natural Science Foundation of China (No. 82170086) and Shanghai Key Laboratory of Emergency Prevention, Diagnosis and Treatment of Respiratory Infectious Diseases (20dz2261100).

Declaration of competing interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.