Fig. 5: Activation of the transcytosis and transportation of RLS/HS@Au.

a The intercellular transport of Cy5-labeled DNA and transfection of pEGFP by RLS/HS@Au via endocytosis-and-exocytosis. The procedure of the two experiments is similar: (I) Cells (the first batch) were incubated with various lipoplexes for 6 h and observed by CLSM. The culture medium was refreshed for another 12 h culture and harvested as condition medium; (II) Cells (the first batch) were imaged; (III) The condition medium was used to culture new cells (the second batch) for 12 h and observed by CLSM. (Red: Cy5-labeled DNA, Green: EGFP). n = 3 independent experimental cell lines with similar results. b Evaluation of exocytosis and endocytosis through the ratio of Cy5 MFI between the 2nd-batch and 1st-batch of cells (n = 5 fields from 3 independent experimental cell lines), and the EGFP MFI in the 2nd-batch of cells was calculated with ImageJ software (n = 3 fields from 3 independent experimental cell lines). The data are mean ± SD. c The penetration of hybrid lipolexes (containing Cy5-DNA) in HepG2 multicellular tumor spheroid. The multicellular tumor spheroid was cultured with lipoplexes for 6 h and visualized using CLSM in Z-stacks with 20 μm intervals. n = 3 independent experiments with similar results. The Scale bar is 100 μm. d Fluorescence intensity of various lipoplexes contained Cy5-DNA versus depth of tumor spheroid along the yellow arrows in Fig. 4c. e In vivo real-time observation of the lipoplexes contained Cy5-DNA penetration from the blood vessels of tumors through a dorsal window chamber under stereomicroscopy. Representative distribution images of hybrid lipoplexes contained Cy5-DNA in tumors after i.v. injection, observed by a CLSM at different times (20 μg Cy5-DNA per mouse). (Red: Cy5-labeled DNA). n = 3 mice. f Fluorescence intensity changes from the tumor vessel to an in-depth region (along the yellow arrows in Fig. 4e) in the selected area at 2 h post-intravenous injection. Two-sided unpaired Student’s t test was used for the comparisons in (b). p values < 0.05 were considered statistically significant. Source data are provided as a Source Data file.