Nanoparticles internalization assay

To observe the intracellular localization of HA and HA-Au in hMSCs, the hMSCs were cultured in six-well plate at 37°C with 5% CO₂ for 24 hrs. After refreshing the culture medium containing 100 mg/mL of HA or HA-Au nanoparticles and incubating for 3 days, hMSCs were obtained and fixed using 2.5% glutaraldehyde overnight at 4°C. Then, the cells were post-fixed using 1% osmium tetraoxide for 1 hr at 4°C and then dehydrated stepwise using a series of ethanol concentrations (30%, 50%, 70%, 80%, 90%, 100%). Eventually, the cells were embedded in epoxy resin and sectioned with an ultramicrotome. Ultrathin sections were collected on copper grids to observe the uptake and intracellular distribution of nanoparticles using TEM (Tecnai G20 electron microscope, FEI, USA).