From 2D to 3D In Vitro World: Sonodynamically-Induced Prooxidant Proapoptotic Effects of C60-Berberine Nanocomplex on Cancer Cells

Simple Summary: Recently, sonodynamic therapy (SDT) has emerged as a promising non-invasive approach for treating cancer by activating sensitizers with ultrasound (US). In this context, we investigated C-60 fullerene (C-60) as a nanocarrier for the promising drug Berberine (Ber)-both potential aromatic sonosensitizers. The preferential mitochondrial accumulation of C-60 and the proapoptotic effects of Ber also make the C-60-Berberine nanocomplex (C-60-Ber) a good candidate for direct induction of the intrinsic apoptotic cell death under US action. The in vitro research on C-60-Ber can provide insights into novel, non-invasive cancer treatments. These findings lead to the development of targeted therapies with reduced side effects, inspire interdisciplinary collaboration, and open new avenues for drug delivery and cancer therapy research. Objectives: The primary objective of this research targeted the biochemical effects of SDT on human cervix carcinoma (HeLa) and mouse Lewis lung carcinoma (LLC) cells grown in 2D monolayer and 3D spheroid cell culture. Methods: HeLa and LLC monolayers and spheroids were treated with a 20 mu M C-60-Ber for 24 h, followed by irradiation with 1 MHz, 1 W/cm(2) US. To evaluate the efficacy of the proposed treatment on cancer cells, assessments of cell viability, caspase 3/7 activity, ATP levels, and ROS levels were conducted. Results: Our results revealed that US irradiation alone had negligible effects on LLC and HeLa cancer cells. However, both monolayers and spheroids irradiated with US in the presence of the C-60-Ber exhibited a significant decrease in viability (32% and 37%) and ATP levels (42% and 64%), along with a notable increase in ROS levels (398% and 396%) and caspase 3/7 activity (437% and 246%), for HeLa monolayers and spheroids, respectively. Similar tendencies were observed with LLC cells. In addition, the anticancer effects of C-60-Ber surpassed those of C-60, Ber, or their mixture (C-60 + Ber) in both cell lines. Conclusions: The detected intensified ROS generation and ATP level drop point to mitochondria dysfunction, while increased caspase 3/7 activity points on the apoptotic pathway induction. The combination of 1 W/cm(2) US with C-60-Ber showcased a promising platform for synergistic sonodynamic chemotherapy for cancer treatment.