Fig. 1. Synthesis and characterization of Dox-CBD-SA.
(A) Schematic of CBD-SA mediated drug delivery. (B) Synthesis scheme of Dox-CBD-SA. (C) Affinities [dissociation constant (Kd) values are shown] of CBD-SA and SA against collagen type I and collagen type III were measured by enzyme-linked immunosorbent assay (ELISA). N.D., not determined due to low signal. Graphs with [concentrations] versus [signals] are shown in fig. S2. Two experimental replicates. (D) Dox conjugation ratio per protein is presented. Values were calculated on the basis of the results of bicinchoninic acid assay protein quantification assay (proteins) and absorbance at 495 nm (Dox) (mean ± SD of three experimental replicates). (E) Dox release kinetics from Dox-CBD-SA under three different pH conditions was evaluated by fluorescence (excitation at 495 nm, emission at 590 nm) (n = 3, mean ± SD; two experimental replicates). (F) MMTV-PyMT cells were seeded and incubated overnight. Dox, Dox-SA, or Dox-CBD-SA was added (red). Cells were also stained with LysoTracker (green). Scale bars, 20 μm. Representative pictures are presented. Two experimental replicates. (G and H) Cytotoxicity of Dox variants against MMTV-PyMT cells or MC38 cells in vitro (n = 6, mean ± SEM). Two experimental replicates. IC50, half maximal inhibitory concentration.