We tested the cellular uptake of AuNSs and subsequent release of the drugs using confocal laser scanning microscopy (CLSM, Leica TSC SPII/DMi 8). We used a previously published method to confirm drugs release [16]. Since 5Fu and CPT have fluorescence properties (emission spectrum: 405 nm and 490 nm, respectively), we were able track their release and localization within cancer cells. Moreover, no nuclear fluorochrome was applied to tissue sections treated with 5Fu-AuNSs, CPT-AuNSs, free 5Fu and CPT to avoid misinterpretation with the blue fluorescent for of CPT drug. But since EGFR1i does not possess fluorescence emission signal, we stained the targeted receptor using anti-FGFR1 antibody (Abcam, ab10646, USA) at a concentration of 1:100, together with its compatible 2ry antibody Alexa Fluor 488 (Abcam, ab150077, USA). Hoechst 33342 (Sigma Aldrich, 23,491–52-3, Germany) was used as a counterstain for DNA staining. After scarification procedures, we dissected the tumors biopsies and divided each one into 2 equal specimens. We selected the central part of tumoral tissue for all histologic evaluation. We examined 5 different histologic sections by 2 different pathologists for each slide on a magnification power × 63. For autofluorescent cytotoxic drugs, we quantified the differences in the nuclear signals between free and conjugated counterparts using ImageJ software (version 1.52p).

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