A pH-Responsive CS-HA-DAP Nanodelivery System Induces Mitotic Catastrophe and Mixed-Type Cell Death via Dual-Targeting of TOP2A/CHK1 for Triple Negative Breast Cancer Therapy
Triple-negative breast cancer (TNBC) is characterized by a highly aggressive nature and poor clinical prognosis, largely due to the absence of effective therapeutic targets. Although natural bioactive peptides hold tremendous promise for anti-tumor therapy, their clinical application is restricted by short in vivo half-lives and insufficient targeting specificity. Herein, we aimed to develop a smart nanodelivery system to deliver deer antler polypeptide (DAP) for the precise treatment of TNBC.We constructed a pH-responsive nanoparticle (CS-HA-DAP) comprising chitosan (CS)-coated hyaluronic acid (HA)-DAP conjugates. Tumor accumulation of the drug was enhanced via HA-mediated active targeting of CD44 receptors and the enhanced permeability and retention (EPR) effect of the nanocarrier. Through RNA sequencing (RNA-seq), molecular docking, and multi-dimensional molecular biology experiments, we systematically elucidated the anti-tumor targets and lethal mechanism of DAP. Furthermore, its biodistribution, therapeutic efficacy, and safety were evaluated in a 4T1 tumor-bearing mouse model. CS-HA-DAP exhibited excellent physicochemical stability and tumor-targeting capability. Mechanistic studies revealed, for the first time, that DAP functions as a topoisomerase II alpha (TOP2A) inhibitor to induce extensive DNA double-strand breaks; concurrently, it inhibits checkpoint kinase 1 (CHK1), leading to the functional loss of the S/G2 cell cycle checkpoint. This synergistic effect of DNA damage and repair blockade forces cancer cells harboring severe genomic injury to undergo mitotic slippage, ultimately driving them into irreversible mitotic catastrophe. Subsequent signaling cascades triggered a mixed-type cell death program. In vivo experiments confirmed that CS-HA-DAP significantly suppressed tumor growth without inducing notable systemic toxicity. In summary, we successfully constructed the CS-HA-DAP nanosystem and validated a novel anti-tumor mechanism driven by TOP2A/CHK1 dual-targeting-induced mitotic catastrophe and mixed-type death. This strategy not only overcomes the barriers to peptide druggability but also provides a promising, innovative solution for the treatment of refractory TNBC. Overall design: 4T1 cells were collected from C_HA_DAP group and the control group. Total RNA pooled was collected from 4T1 cells of three individuals to construct a cDNA library. 4T1 cells transcriptomics were analyzed by Illumina NovaSeq 6000 for high-throughput deep sequencing.