Exploring the Therapeutic Potential of Green-fabricated CRL-AgNPs from Catharanthus roseus

• 2 4 full factorial design ensured reproducible and stable nanoparticle formation. • Autoclave-assisted eco-friendly synthesis promoted uniform nucleation and complete reduction. • CRL-AgNPs ( ∼55 nm ) exhibited strong SPR at 436 nm and high colloidal stability ( ζ = –54.5 mV ). • Potent cytotoxicity against MCF-7 cells ( IC₅₀ = 8.03 µg mL -1 ) and cell proliferation activity in an in vivo model. • Apoptosis mechanism validated by p53/BAX upregulation and Bcl2/NFκβ downregulation. Despite the promise of green nanotechnology, the synthesis reproducibility with consistent pharmacological activity remains the key challenge. Therefore, the current project aimed to biosynthesize AgNPs using aqueous leaf extract of Catharanthus roseus as a reducing and capping agent to discover novel therapeutic leads. To attain this target, synthesis of C. roseus leaf-mediated AgNPs (CRL-AgNPs) was optimized using a 2 4 full factorial design, which yielded a highly significant and predictive statistical model. CRL-AgNPs synthesis was confirmed through chromism and UV-Vis spectroscopy. Further characterization by DLS, XRD, FTIR, FE-SEM, and EDX revealed an average particle size of 55.78±13.2 nm with high colloidal stability, scoring a zeta-potential of -54.5±0.57 mV. EDX study validated the elemental composition of synthesized CRL-AgNPs, demonstrating that a strong silver signal accounted for 84.09% of the weight. Cytotoxicity assessments using the MTT assay revealed a dose-dependent inhibition of MCF-7 breast cancer cell proliferation, with an IC 50 value of 8.033μg/mL, indicating their potent anticancer activity. Furthermore, their anti-proliferative efficacy was validated in vivo on EAC cells bearing a mouse model, demonstrating that intra-peritoneal administration of CRL-AgNPs significantly reduced cancer cell growth by 63.20%. Apoptosis was ascertained by nuclear staining and gene expression, with upregulation of p53 and BAX and downregulation of Bcl2 and NF-κβ. Histopathology confirmed selective tissue protection for vital organs in treated mice. Therefore, these findings establish CRL-AgNPs as a sustainable, reproducible, and mechanistically validated nanoparticle that integrates green chemistry with functional biomaterials design, advancing the translational nanotherapeutic development.