Fig. 9: HA-si-TDRD9 NPs reduce HLO susceptibility to PA infection.

A A spherical 3D human lung organoid (HLO) structure was successfully established in Matrigel, and its growth was observed under an optical microscope for 7 days. Representative bright-field images of HLOs cultured for 7 days. Scale bars: 200 μm (top), 100 μm (bottom). n= 3 independent experiments. B Hematoxylin and eosin (HE) staining comparing histological features of native human lung tissue and HLOs. Scale bar: 60 μm. n = 3 independent experiments. C Immunohistochemistry analysis of Ki67, NK2 homeobox 1 (NKX2.1), and SRY-box transcription factor 9 (SOX9) expression in native lung tissue and HLOs. Scale bar: 60 μm. n = 3 independent experiments. D HLOs were incubated with 0.1 μM hyaluronic acid (HA)-coated peptide nanoparticle (NP) system for targeted small interfering RNA (siRNA) delivery against Tudor domain-containing protein 9 (TDRD9) (HA-si-TDRD9 NPs) or HA-si-NC NPs, followed by infection with Pseudomonas aeruginosa (PA) strain PAO1. HE staining showing HA-si-TDRD9-mediated preservation of HLO morphology following PA challenge. n= 3 independent experiments. E Bacterial growth curves in HLO cultures. n = 3. F Quantitative real-time PCR analysis of TDRD9 mRNA levels in HLOs. n = 3 independent experiments. G Western blot analysis of TDRD9, programmed death ligand-1(PD-L1), cluster of differentiation 80 (CD80), and mitogen-activated protein kinase (MAPK) expression. n = 3 independent experiments. H TUNEL assay quantifying apoptotic cells in HLOs. Scale bar: 25 μm. n = 3 independent experiments. I Western blot of cleaved caspase-3, -8, and -9 levels. n = 3 independent experiments. J Enzyme-linked immunosorbent assay (ELISA) quantification of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6 levels in HLO supernatants. n = 3 independent experiments. Data represent median ± interquartile range (IQR), and analyzed by two-sided two-way ANOVA with Tukey’s multiple comparisons test (E), or two-sided one-way ANOVA with Tukey’s multiple comparisons test (F–J). Source data are provided as a Source Data file.