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February 26, 2026Open Access

Chitosan nanoparticles co-delivering curcumin and methotrexate synergistically induce p53/caspase-9-mediated apoptosis in metastatic breast cancer and effectively inhibit tumor growth in vitro and in vivo

HDHamed DadashiTabriz University of Medical Sciences MMMilad MashinchianTabriz University of Medical Sciences AKAlireza Karimian-ShaddelTabriz University of Medical Sciences

Key Points

The aim is to investigate the efficacy of chitosan nanoparticles co-delivering curcumin and methotrexate in inducing apoptosis in metastatic breast cancer.
Developed pH-sensitive chitosan nanoparticles using ionic gelation
Characterized nanoparticles for size, zeta potential, and encapsulation efficiency
Conducted in vitro assays including MTT, flow cytometry, and angiogenesis tests
Examined apoptotic mechanism through western blotting for p53 and caspase-9
Conducted in vivo tests on BALB/c mice bearing 4T1 tumors
Cur-/MTX-Cs nanoparticles induced 62.92% apoptosis in 4T1 breast cancer cells
Achieved 91.12% tumor growth inhibition in vivo, outperforming free curcumin and methotrexate
Reduced cell migration by 56.7% and angiogenesis by 68.9% in vitro
Confirmed minimal systemic toxicity in animal models

Abstract

• pH-sensitive chitosan NPs co-deliver curcumin/MTX with >80% encapsulation efficiency. • Cur-/MTX-Cs NPs induce 62.92% apoptosis via P53/caspase-9 in 4T1 breast cancer. • NPs reduce 4T1 cell migration by 56.7% and angiogenesis by 68.9% in vitro. • Cur-/MTX-Cs NPs achieve 91.12% tumor growth inhibition in vivo, outperforming free drugs. • Biocompatible NPs show minimal systemic toxicity in BALB/c mice. Nanotechnology offers innovative solutions to enhance cancer treatment efficacy through passively- or actively-targeted drug delivery systems. This study developed curcumin (Cur)- and methotrexate (MTX)-loaded chitosan (Cs) nanoparticles (NPs) using ionic gelation to treat 4T1 breast cancer cells, a highly metastatic model. The NPs, characterized by sizes 80%, were biocompatible and significantly enhanced the cytotoxicity of Cur and MTX. Synthesized via a scalable, cost-effective ionic gelation method, these NPs leverage chitosan’s pH-sensitive properties to achieve synergistic drug release in acidic tumor microenvironments, offering a versatile platform for combination therapies. In vitro assays, including MTT, flow cytometry, scratch, mammosphere, and chorioallantoic membrane (CAM) tests, demonstrated that Cur-/MTX-Cs NPs induced 62.92% apoptosis (predominantly late-stage), reduced cell migration by 56.70% at 72 h, disrupted mammospheres by 20.36%, and inhibited angiogenesis by 68.9%. Western blotting revealed upregulated p53 expression and a 72.17% increase in caspase-9 activity, underscoring the apoptotic mechanism. In vivo, Cur-/MTX-Cs NPs achieved 91.12% tumor growth inhibition (TGI) in BALB/c mice bearing 4T1 tumors, significantly (p = 0.001) outperforming free Cur and MTX (38.07% TGI), with minimal systemic toxicity as confirmed by hematological, biochemical, and histopathological analyses. These findings highlight Cur-/MTX-Cs NPs as a safe and effective drug delivery system for breast cancer therapy, with potential for clinical translation to improve patient outcomes across diverse cancer types.

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