Background and Aims Sonodynamic therapy (SDT) is an emerging tumor treatment method that primarily utilizes low-intensity ultrasound (US) to activate sonosensitizers, leading to the production of cytotoxic reactive oxygen species (ROS) that kill cells. This study was conducted with an attempt to develop a novel nanoparticle (NPs) formulation containing a sonosensitizer and the small interfering RNA (siRRS1) of tumor-related gene RRS1 to observe its anticancer effects on colon cancer, to provide new strategies for colon cancer treatment.Methods RRS1 RNA-Seq data were downloaded from the TCGA and GEO databases, and clinical pathological data were collected from 80 patients with colon cancer at Hunan Provincial People's Hospital. The expression of RRS1 in colon cancer and its relationship with prognosis were analyzed. The proliferation and apoptosis of colon cancer cells transfected with siRRS1 were observed. The sonosensitizer tetra (4-carboxyphenyl) porphyrin (TCPP), gene carrier DSPE-PEI, and siRRS1 were assembled into TCPP@DSPE-PEI-siRRS1 NPs (T@D-siRRS1 NPs). After structural characterization and examination of cellular uptake and ROS generation capabilities in vitro, the anticancer effects of T@D-siRRS1 NPs on colon cancer were assessed in cell experiments and tumor-bearing mouse models.Results Both database analysis and clinical specimen testing indicated that RRS1 expression in colon cancer tissues was higher than that in adjacent normal tissues, and high expression was associated with poor prognosis (HR=1.88, 95% CI=1.27-2.79, P=0.002). Univariate and multivariate analyses showed that RRS1 mRNA expression was an independent risk factor for overall survival in colon cancer patients (HR=1.354, 95% CI=1.447-2.215, P=0.031). Knockdown of RRS1 significantly reduced the proliferation capacity of colon cancer cells and increased apoptosis (both P<0.05). Characterization results showed that the particles were uniform and stable; T@D-siRRS1 NPs entered cells through endocytosis, where TCPP could generate singlet oxygen (1O?) under ultrasound irradiation. Subsequent in vitro and in vivo experimental results demonstrated that transfection with NPs carrying siRRS1 or TCPP (T@D-control siRNA, T@D-siRRS1) combined with US resulted in varying degrees of growth inhibition in colon cancer cells, with the T@D-siRRS1 NPs+US treatment showing the strongest effect (all P<0.05). Moreover, no significant damage was observed in the vital organs of mice treated with T@D-siRRS1 NPs+US.Conclusion T@D-siRRS1 NPs plus US integrate SDT and gene therapy, producing an effective synergistic cytotoxic effect on colon cancer cells. Additionally, T@D-siRRS1 NPs demonstrate good safety and biocompatibility, suggesting potential for clinical application.