Nano drug delivery of a new scfv-based immunotoxin by chitosan nanoparticles upgrades its efficiency against breast cancer.

This study aimed to develop a chitosan‑based nano‑delivery system encapsulating the PE38KDEL‑1F12 immunotoxin (NP‑IMX) and to evaluate its biological performance in breast cancer cells. NP‑IMX nanoparticles were synthesized and characterized, followed by in vitro evaluation in EPHA2‑positive MDA‑MB‑231 breast cancer cells and EPHA2‑negative MCF‑10 A normal breast epithelial cells. Cytotoxicity, cellular uptake, apoptosis, cell‑cycle arrest, migration, drug loading and release, and gene expression (VEGF, p21, Bax, and p53) were assessed. In addition, short‑term in vivo toxicity was evaluated in healthy BALB/c mice. NP‑IMX exhibited very high encapsulation efficiency (~ 99.9%) and pH‑responsive drug release, with approximately 80-85% cumulative release at pH 5.3 compared with 20-25% at pH 7.4. In vitro studies demonstrated enhanced cellular uptake and significantly increased apoptosis (> 80%), G2/M cell‑cycle arrest (~ 59%), and migration inhibition in MDA‑MB‑231 cells compared with the free immunotoxin. NP‑IMX showed higher cytotoxicity toward cancer cells while maintaining minimal toxicity in MCF‑10 A cells. Gene expression analysis revealed upregulation of p53, p21, and Bax and downregulation of VEGF in cancer cells. In vivo toxicity studies indicated reduced systemic toxicity of NP‑IMX compared with free immunotoxin, based on biochemical and histopathological analyses. This study demonstrates that chitosan‑based nano‑encapsulation of the PE38KDEL‑1F12 immunotoxin enhances cancer‑selective in vitro antitumor activity while reducing systemic toxicity in healthy animals. Although these findings support the potential of NP‑IMX as a targeted nano‑immunotoxin platform, further in vivo efficacy studies using tumor‑bearing models are required to confirm its therapeutic relevance.