Cuproptosis, a recently identified form of regulated cell death triggered by excess intracellular copper, has emerged as a promising cytotoxic strategy for cancer therapy. However, the therapeutic efficacy of copper ionophores such as elesclomol (ES) is often hindered by cellular copper homeostasis mechanisms that limit copper influx and cuproptosis induction. To address this challenge, we developed a nanoagent utilizing outer membrane vesicle (OMV) derived from Akkermansia muciniphila (Akk) for co-delivery of antioxidant 1 copper chaperone (Atox1)-targeting siRNA and ES (siAtox1/ES@OMV) to tumors. In vitro, we demonstrated that Atox1 knockdown via siRNA significantly disrupted copper export mechanisms, resulting in elevated intracellular copper levels. Simultaneously, ES facilitated efficient copper influx and mitochondrial transport, leading to Fe–S cluster depletion, increased proteotoxic stress, and robust cuproptosis. In vivo, siAtox1/ES@OMV achieved targeted tumor delivery and induced pronounced cuproptosis. Furthermore, leveraging the immunomodulatory properties of OMVs, siAtox1/ES@OMV promoted T-cell infiltration and the activation of tumor-reactive cytotoxic T cells, enhancing tumor immune responses. The combination of siAtox1/ES-induced cuproptosis and immunogenic cell death synergistically suppressed tumor growth in both subcutaneous breast cancer and orthotopic rectal cancer mouse models. This study highlights the potential of integrating copper homeostasis disruption with a copper ionophore using an immunomodulatory OMV-based vector, offering a promising combinatorial strategy for cancer therapy.

Background: As a novel drug delivery vehicle, transfersomes (TFSs) have great deformability, good biocompatibility, and biodegradability, which can significantly improve drug delivery efficiency. However, their application is limited by their poor stability and vulnerability to leaks. Methods: This paper prepared and assessed the stability of transfersomes co-loaded with Pueraria total flavonoids (PTFs) and tanshinone (TAN), known as PTF/TAN-TFSs, in order to overcome this constraint. Results: The findings showed that the co-loaded system (PTF/TAN-TFSs) had better structural stability than the single-loaded systems (PTF-TFSs and TAN-TFSs), with drug leakage rates decreased by roughly 5% and 7%, respectively, and a more uniform size distribution with a particle size of 44.52 ± 0.47 nm and a PDI of 0.22, while the single-loaded systems had PDI values larger than 0.3. The co-loaded system showed a better sustained-release profile than the suspension with a 15.28% decrease in the maximum cumulative release of the poorly soluble component Tan IIA. Additionally, the co-loaded system demonstrated enhanced solubilization capability, making the poorly soluble component TAN more soluble by 89.52 times compared to the free medication and by 1.97 times compared to the single-loaded system. Furthermore, the co-loaded system significantly improved the intestinal absorption of Tan IIA, increasing the Papp and Ka values in the ileum segment by 2.02-fold and 1.80-fold, respectively. Conclusions: The stability of the system was successfully improved by co-loading many components with varying hydrophobicities. This method provides a fresh way to enhance transferosome stability, broadening their application in the field of drug delivery.