Figure 3.

Releasing ligand with NIR light makes mAb-IR700 conjugates hydrophobic causing aggregation and loss of fluorescence. (a) Hypothesized change of chemical structure of mAb-IR700 with NIR light irradiation. NIR light irradiation causes mAb-IR700 to release the ligand (C₁₄H₃₄NO₁₀S₃Si), which makes mAb-IR700 hydrophobic and causes aggregation. Along with the change from hydrophilic to hydrophobic, the blue color of mAb-IR700 conjugate decreases; aggregation appears, and IR700-fluorescence is lost. (b) 3 μM cet-IR700 in PBS was irradiated with NIR light, and imaged in white light and 700 nm fluorescence. Both the blue color in the tube and the 700 nm fluorescence decreased in a light-dose-dependent manner. (c) Mean 700 nm fluorescence intensity of cet-IR700 in PBS was decreased in a dose-dependent manner (n = 3). (d) Absorbance profile of cet-IR700 in PBS with NIR light irradiation measured showed decrease of the absorbance at the Q-band at 690 nm of the SiPcs. (e) Analysis of aggregated particles with an aggregate-sizer was examined at increasing NIR light irradiation 4, 16, and 64 J cm^–2. (f) SDS-PAGE of NIR-light-irradiated cet-IR700 revealed that the protein band of cetuximab disappeared in a dose-dependent manner, and some bands over cetuximab increased with smear. The IR700-fluorecence in the SDS-PAGE decreased in a light-dose-dependent manner. (g) The complex of cet-IR700 and recombinant EGFR protein was irradiated with NIR light and electrophoresed by SDS-PAGE. The protein bands of not only cet-IR700 but also EGFR become thin with a smear over its protein band along with loss of IR700-fluorescence. (h) IT-TOF-MS detected release of the ligand (C₁₄H₃₄NO₁₀S₃Si) from cet-IR700 after NIR light irradiation (16 J cm^–2). With analysis of the isotope peak and product ion scan, the peak was confirmed as the ligand (C₁₄H₃₄NO₁₀S₃Si) (Figure S3).