Doxorubicin-Incorporated Nanotherapeutic Delivery System Based on Gelatin-Coated Gold Nanoparticles: Formulation, Drug Release, and Multimodal Imaging of Cellular Internalization

摘要

In this work, we developed a new pH- and temperature-responsive nanochemotherapeutic system based on Doxorubicin (DOX) noncovalently bound to biosynthe-sized gelatin-coated gold nanoparticles (DOX-AuNPs@gelatin). The real-time release profile of DOX was evaluated at different pH values (7.4, 5.3, and 4.6) and temperatures (22-45 degrees C) in aqueous solutions, and its therapeutic performance was examined in vitro against MCF-7 breast cancer cells. TEM, dark-field scattering, and wide-field fluorescence microscopy indicated the effective uptake of nanochemotherapeutics with the subsequent release and progressive accumulation of DOX in cell nuclei. MTT assays clearly showed the effectiveness of the treatment by inhibiting the growth of MCF-7 breast cancer cells for a loaded drug concentration of 5 mu g/mL. The most informative data about the dynamic release and localization were provided by scanning confocal microscopy using time-resolved fluorescence and surface-enhanced Raman scattering (SERS) techniques. In particular, fluorescence-lifetime imaging (FLIM) recorded under 485 nm pulsed diode laser excitation revealed the bimodal distribution of DOX in cells. The shorter fluorescence lifetime of DOX localized in nuclei (1.52 ns) than in the cytoplasm (2.4 ns) is consistent with the cytotoxic mechanism induced by DOX DNA intercalation. Remarkably, the few DOX molecules captured between nanoparticles ("electromagnetic hotspbts") after most drug is released act as SERS reporters for the localization of plasmonic nanocarriers in MCF-7 cells. The high drug loading capacity and effective drug release under pH control combined with the advantage of multimodal visualization inside cells clearly indicate the high potential of out DOX-AuNPs@gelatin delivery system for implementation in nanomedicine.