Collagenase 1

Dynabeads conjugated with the anti-CD31 antibody (RRID: AB_2722705) were prepared using the Dynabeads Antibody Coupling Kit (Life Technologies, 14311D) following the manufacturer’s instructions. Mouse lung tissues were obtained immediately after sacrifice, and were washed multiple times with 1X PBS buffer (approximately 3 to 6 times). The lung tissues were then minced into 1–2 mm pieces using a scalpel in the presence of 0.25% collagenase I (Yeasen, Shanghai, China) solution (in PBS + 20% FBS) and were digested in the same solution for 45 min in a 37 °C shaking incubator. After digestion, the cells were filtered through a sterile 70 μm nylon mesh and were washed with cold 1X PBS buffer. The cells were then incubated with the conjugated dynabeads for 30 min at 4 °C to allow binding. The bead-bound cells were washed four times with 1X PBS buffer and collected using a magnet. Finally, all the bead-bound cells were resuspended in DMEM with 20% FBS before being plated.

Fluorescently labeled siIL11@PPGC NPs used in this study were prepared by encapsulating siIL11 into Cy5.5-labeled PPGC NPs as described above. Twenty-four hours after inhalation of the Cy5.5-labeled siIL11@PPGC NPs, bleomycin-induced fibrosis mice were sacrificed and organs were harvested and imaged on an IVIS system (PerkinElmer, USA). To determine the majority cell subtype colocalized with the siIL11@Cy5.5-PPGC NPs, lungs were collected, minced, and digested in PBS containing collagenase I (201.3 U/ml; Yeason), 0.92 M Hepes (Beyotime), and DNase I (50.3 U/ml; Sigma-Aldrich) at 37°C for 1 hour. The obtained digestion was filtered using a 70-μm cell strainer and processed using red blood lysis buffer for 5 min. The samples were then centrifuged at 400g, resuspended in PBS containing 0.5% BSA, and filtered through a 40-μm cell strainer. The samples were subsequently incubated with antibodies against epithelial (EpCAM–Alexa Fluor 647), immune (CD45-phycoerythrin), and endothelial (CD31–Brilliant Violet 421) cell markers at 4°C for 30 min. Flow cytometry (BD Biosciences, USA) was used to determine the internalization of NPs in various cell subtypes.

Primary adipose stem cells (ADSCs) were isolated from GDM mice and normal gestational mice, respectively. Under aseptic conditions, the abdominal cavity of the mice was opened, and AT from the abdominal and inguinal regions was obtained. The AT was then rinsed, minced, and collected in a pre-cooled Hank’s Balanced Salt Solution (Sangon BIOTECH, Shanghai, China). Next, 2 mg/ml collagenase I (Yeason, Shanghai, China) and 3 mM CaCl₂ were added in double volume to the tissue, which was then digested at 37 ℃ for 4 h. Digestion was stopped by adding an equal volume of DMEM/F12 medium (Gibco, GIBCO, NY, USA) containing 10% FBS (Merck KGaA, Darmstadt, Germany), followed by centrifugation at 1200 g for 10 min. The cell precipitates were resuspended, washed with PBS, and cultured in DMEM/F12 medium containing 10% FBS. Mouse normal liver cells, AML12, were purchased from Shanghai Fuheng Biotechnology and cultured in DMEM/F12 medium containing 10% FBS, 1% ITS media supplement (R&D Systems, MN, USA), and 40 ng/ml dexamethasone (Merck KGaA).