H 600

The CS/HA nanoparticles were prepared at CS:HA weight ratios of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, and 7:1, as described in the “Preparation of CS/HA/miR-21 nanoparticles” section. The Z-average hydrodynamic diameter and surface charge of the CS/HA nanoparticles were determined by dynamic light scattering using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Malvern, UK) at 25°C. The samples were dried at room temperature after being stained on copper grids and then photographed using a transmission electron microscope (TEM, Hitachi H-600, Hitachi, Tokyo, Japan) to observe the particle morphology. The CS/HA/miR-21 nanoparticles were evaluated by agarose gel electrophoresis. The naked miR-21 and CS/HA/miR-21 nanoparticles with various N/P ratios (1:1, 5:1, 10:1, 15:1, and 20:1) were loaded onto a 2% agarose gel containing ethidium bromide in Tris-borate ethylenediaminetetraacetic acid buffer at pH 8.0. The samples were run on the gel at 120 V for 20 minutes. The gel was then photographed using a GDS-8000 image-acquisition system (UVP, Upland, CA, USA).

Fragments of the right middle lung tissues were cut into 1-mm slices and then fixed in 2.5% glutaraldehyde solution for 2 h at 0–4°C. Afterward, the specimens were rinsed with PBS three times, fixed with 1% osmic acid at room temperature for 1 h, rinsed with PBS three times and stained with 2% uranium acetate solution at room temperature for 30 min, and then dehydrated with dimethyl ketone. The specimens were embedded in Epon-812 at 60°C for 2 days, cut into ultrathin sections (60 nm), and stained at room temperature with 1% uranyl acetate for 30 min and lead citrate for 15 min. The sections were observed under a transmission electron microscope (magnification, ×10,000; Hitachi H-600; Hitachi, Ltd.).

Transplanted liver tissues were obtained from the recipient freshly, cut into 2 × 3 mm samples, and fixed in 2.5% glutaraldehyde solution. Embedded sections were observed under a transmission electron microscopy (Hitachi H-600; Hitachi, Ltd., Tokyo, Japan) for the ultrastructures of the transplanted liver tissue.

Tca-8113 cells were seeded on coverslips and treated with PTS for 1 h. The medium was replaced by fresh medium for a further 24-h incubation. At the end of the incubation period, cells were fixed in 3% glutaraldehyde in PBS for 2 h, washed, and fixed again in 1% osmium tetroxide. Samples were dehydrated in graded ethanol and embedded in epon 812. Serial ultrathin 60-nm sections were stained with uranyl acetate and lead citrate and then observed using a transmission electron microscope (Hitachi H-600; Hitachi, Tokyo, Japan).

Kunming mice were divided into three groups: the negative control group was injected with pure agarose gel, while the others were injected with Fe₃O₄ NPs and HLC Fe₃O₄ NPs. The injected tissue was removed after 7 days postsurgery. Tissue blocks of 1 mm³ were fixed immediately with 2.5% phosphate-buffered glutaraldehyde for 2 hours, rinsed with 0.1 M phosphate-buffered saline for 30 minutes, postfixed in 1% osmium tetroxide for 2 hours, and finally rinsed for 10 minutes using a 0.1 M phosphate buffer. The samples were then dehydrated using an ethanol series. Finally, the samples were embedded in Epon 812. Semithin sections (1–2 mm) were cut and stained with methylthioninium chloride to select appropriate areas for observation. Ultrathin sections (50–70 nm) were stained with 4% uranyl acetate and 0.5% lead citrate and observed under a TEM (Hitachi H-600; Hitachi Ltd., Tokyo, Japan).