Dapi blue

Cy5-labelled hsa-mir-148a-3p mimic and mimic control were purchased from RiboBio (Guangzhou, China). Briefly, Exosomes extracted by the ExoQuick-TC were resuspended in 300μL PBS and transfected with miRNA mimics using Exo-Fect siRNA/miRNA Transfection Kit (System Biosciences, Palo Alto, CA) according to the manufacturer's protocol.
For exosome-uptaking assay, transfected exosomes were labelled with PKH67 (Sigma-Aldric, St. Louis, MO, USA) away from light at room temperature for 4min. After incubation with pre-treated exosomes for 24h, tumor cells were stained with phalloidin-rhodamine (Red, Sigma-Aldric) and DAPI (Blue, Beyotime). Images of tumor cells were captured using a laser confocal microscope (Leica, Germany) and qRT-PCR was performed to validate the miRNA expression level.

HCCLM3 and SMMC-7721 cells were placed in a glass-bottom cell culture dish and treated with 0.1% DMSO or ML-323 (80 μmol/L) for 24 h. The cells were fixed with anhydrous methanol at −20 °C for 25–30 min, blocked with 5% bovine serum albumin, and then with LC3B primary antibody (1:300, overnight at 4 °C; Cell Signaling Technology) and AlexaFluor488® goat anti-rabbit IgG (H+L) secondary antibody (green) (1:500, 2 h at 25 °C in the dark; Beyotime) were incubated separately. The nuclei were stained with DAPI blue (5 μg/ml, 20 min at 25 °C, in the dark; Beyotime). A fluorescence microscope (magnification:200×; OLYMPUS, OLYMPUS Corporation, Japan) was used.

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% triton X-100 (Beyotime, Shanghai, China). After blocking with FBS, cells were incubated with primary antibodies CD31 (1:100, ABclonal; A0378), vWF (1:100, ABclonal; A1335) and DAPI (blue) (Beyotime) and Alexa Fluor594 (red) (1:200, Beyotime) conjugated second antibodies. Images were acquired using fluorescence microscopy (Nikon, Tokyo, Japan).

The lipopolysaccharide (LPS) was isolated from the outer membrane of Gram-negative bacteria to induce acute lung injury models (Wang et al., 2014 (link), 2020 (link)). LPS (5 mg/kg) solution was dropped into mice via intratracheal instillation. After 5 h, the mice were randomly divided into 3 groups and treated with free DiR and different DiR-labeled formulations via tail vein injection. At 24 h, the main organs were isolated for imaging using an IVIS in vivo system (IVIS^® Spectrum, PerkinElmer, USA) at 750/800 nm. Living Image^® software (Caliper, Alameda, CA) was used to quantify the fluorescence signals.
The specific targeting of MM-NLCs to lung inflammation sites was evaluated under CLSM. DiI-labeled NLCs and MM-NLCs were injected into mice via the tail vein. At 12 h, lung tissues from each group were removed, frozen in OCT, and sectioned at 10 μm (RM2016, Leica, Germany). Meanwhile, samples were stained with DAPI (blue, Beyotime) for visualization. The red signals of nanotherapeutics and blue signals of nuclei were recorded to assess the targeting capacity of MM-NLCs to lung inflammation sites in the acute lung injury mouse model.