Cell-penetrating peptides (CPPs) are considered ideal carriers for the delivery of drugs through the cell membrane barrier, with enhanced permeation and retention effects. These peptides can increase the bioavailability of drugs and reduce their side effects. In this study, we developed a polypeptide called "cell-penetrating peptide (KRP)", linked it with doxorubicin (DOX), and created a pH-responsive triggered drug delivery system called "KRP-Hyd-DOX".We generated KRP through the application of standard Fmoc solid-phase peptide synthesis (SPPS). We attached DOX through a stable amide bond, using 3-maleimidopropionic acid as a linker, yielding KRP-DOX. With 6-maleimidopropionic acid as a linker, we connected DOX via a pH-sensitive hydrazone bond, resulting in KRP-Hyd-DOX. The pH-responsive drug-release performance of KRP-Hyd-DOX was evaluated through the calculation of the cumulative release of DOX at various time intervals and under different pH conditions via the DOX standard curve in the corresponding buffer. The cell penetration and cytotoxicity of KRP-Hyd-DOX, KRP-DOX, and DOX-hydrochloride (HCL) were compared.The efficiency of KRP-DOX and KRP-Hyd-DOX in releasing free DOX was approximately 30% after 24 hours under pH 7.4. Under pH 5.0, the efficiency of KRP-DOX in releasing free DOX did not change significantly as compared to that at pH 7.4, while the efficiency of KRP-Hyd-DOX in releasing free DOX increased to 65% after 24 hours. After 24-hour exposure to KRP-Hyd-DOX, KRP-DOX, and DOX-HCL across different concentrations, MG63 cell viability exhibited a dose-dependent decline. The half-maximal inhibitory concentration (IC50) of KRP-Hyd-DOX (MG63 cells/5.22 µM) was less than that of KRP-DOX (MG63 cells/7.41 µM).The system we created (KRP-Hyd-DOX) leverages the synergistic effect of KRP and DOX, enhancing efficacy while minimizing side effects.Copyright © 2025 AME Publishing Company. All rights reserved.