Chitosan-Functionalized Fluorescent Calcium Carbonate Nanoparticle Loaded with Methotrexate: Future Theranostics for Triple Negative Breast Cancer

Herein, fluorescent calcium carbonate nanoclusters encapsulated with methotrexate (Mtx) and surface functionalized with chitosan (25 nm) (@Calmat) have been developed for the imaging and treatment of triple-negative breast cancer (TNBC). These biocompatible, pH-sensitive nanoparticles demonstrate significant potential for targeted therapy and diagnostic applications. The efficacy of nanoparticles (NPs) was evaluated in MDA-MB-231 TNBC cell lines. The enhanced permeability and retention effect facilitated the accumulation of NPs, in tumor-bearing rats, as confirmed by in vivo fluorescence imaging. Treatment with @Calmat resulted in a marked reduction in pro-inflammatory cytokines, with levels of IL-6 (1225 ± 67 pg/mL), IL-1β (379 ± 69 pg/mL), and TNF-α (14.1 ± 2 pg/mL), in contrast to the diseased control group (IL-6: 2223 ± 99; IL-1β: 1632 ± 90; TNF-α: 40 ± 3 pg/mL). A similar trend was observed for liver and kidney function biomarkers. Mechanistic studies revealed that @Calmat treatment activates the Bax/Bcl-2 signaling pathway, leading to cell cycle arrest in the G1 phase and subsequent late-phase apoptosis. As a result, the tumor inhibition rate reached 88%, with 80% of treated rats surviving beyond 100 days. These findings highlight the strong potential of @Calmat as a dual-function theranostic agent for the management of TNBC.