Green-light photodynamic activation of hypericin-synthesized metal nanoparticles for the treatment of 3D cancer models

Photodynamic therapy (PDT) is a minimally invasive and selective cancer treatment based on light-activated photosensitizers generating reactive oxygen species (ROS) to induce tumor cell death. In this work, we report for the first time the synthesis of gold and silver nanoparticles using hypericin (Hyp), a natural photosensitizer. The nanoparticles were obtained through a simple and eco-friendly process, in which Hyp acts simultaneously as reducing and stabilizing agent. The resulting nanoparticles were characterized and assessed for ROS generation under light irradiation using two distinct probes: 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) for singlet oxygen (1O2) detection, and 1,3-diphenylisobenzofuran (DPBF) for total ROS quantification. In both assays, Ag@Hyp exhibited enhanced ROS generation compared to Hyp alone with 99.3% ABDA and 94.1% DPBF disappearance, compared with 90.9% and 88.1% respectively for free Hyp at plateau, whereas Au@Hyp showed a decreased probe degradation (70.3% and 69.4%, respectively). The effect on viability is different depending on the cell lines with a decrease for Ag@Hyp higher respect to Hyp after 7days (increased toxicity respect to Hyp of 13.1%, 21.4% and 7.1% respectively for HT-29, MIA-PaCA-2 and GL-261). The photodynamic effects were further evaluated in two-dimensional (2D) monolayers and three-dimensional (3D) tumor spheroids of three cancer cell lines, human colorectal adenocarcinoma (HT-29), human pancreatic adenocarcinoma (MIA PaCa-2), and murine glioblastoma (GL-261). Only Ag@Hyp induced significant cytotoxicity upon light activation, highlighting the synergistic potential between Hyp's photosensitizing activity and the plasmonic enhancement of silver nanostructures. Overall, this Hyp-mediated synthesis offers a sustainable strategy for developing multifunctional nanotherapeutics for cancer treatment.