A1r si confocal

NP cells cultured in six‐well plates were fixed with 4% paraformaldehyde for 30 min, and then permeabilized with Triton X‐100 (0.2%, 30 min). The samples were washed in PBS twice, and blocked with 2% goat serum for 1 h. Then the samples were incubated with primary antibodies overnight, and subsequently incubated with FITC or Cy3‐conjugated secondary antibody. After the nuclei were stained for DAPI (0.1 g/ml, 5 min), the fluorescent images were captured using a fluorescence microscope (Olympus, BX53) or a confocal microscope (Nikon A1R SI Confocal, Japan).

NP cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 30 min. The cells in the slides were washed in PBS twice, and blocked with 2% goat serum for 1 h, and then incubated with primary antibodies. Nuclei were stained for 5 min with DAPI (Beyotime). Immunofluorescent images were captured using a fluorescence microscope (Olympus, BX53, United States) or a confocal microscope (Nikon A1R SI Confocal, Japan).

Purified EVs were labeled with 5 μM PKH26 (Sigma-Aldrich, USA) according to the manufacturer's instructions. In order to remove unincorporated dyes, the mixture was washed in PBS and centrifuged at 110,000 × g for 70 min. For internalization assay, EVs (50 μg/ml) were suspended in medium and incubated with NP cells at 37 °C in the EVs group. In the EVs@FEC group, the hydrogel mixed with labelled EVs was placed in a coculture transwell to realize the sustained release. For immunofluorescence analysis, the NP cells were fixed and stained with phalloidin (Beyotime, China) for 1 h and DAPI for 5 min at specific time points. Then, the samples were placed under a fluorescence microscope (Olympus, USA) for image capture. Uptake of EVs was assessed by mean fluorescence intensity (MFI) of red fluorescent signal. For labelled EVs in hydrogels, the hydrogels were transferred to a confocal dish and images were captured via a confocal microscope (Nikon A1R SI Confocal, Japan). For internalization assay through flow cytometry, the treated cells were collected and then applied to FACSCalibur flow cytometer (BD Biosciences, USA). The positive rate of labelled cells was analyzed by FlowJo X software (Tree Star, USA).