Bufalin (BF), a pivotal active constituent of the traditional Chinese medicine (TCM) cinobufacini, has exhibited potent antitumor effects across various malignancies, including gastric cancer, hepatocellular carcinoma, and osteosarcoma (OS) [[1], [2], [3]]. Nevertheless, the therapeutic potential and clinical application of BF remain hindered by its poor bioavailability and systemic toxicity, particularly the irreversible cardiotoxicity at effective dosages [4]. Recently, nano-drug delivery systems (NDDS) have emerged as promising strategies to optimize pharmacokinetic profiles and tumor-specific accumulation of free drugs [5,6]. Despite several advancements in NDDS for BF delivery, including albumin submicrosphere, metal-organic framework, and liposome [[7], [8], [9]], few platforms cooperatively achieve high drug loading efficiency (LE %), long-term stability, extended circulatory longevity and marked tumor tropism. This scenario encourages us to develop advanced nanostrategies with further improved performances to invert the challenges of BF.

Recently, dimeric prodrug nanoassemblies (DPNAs) have garnered significant interest as promising NDDS for cancer therapy [10]. Compared with monomeric nanosystems, DPNAs markedly improve LE % due to their self-assembly properties. The subsequently introduced homodimeric prodrug nanoassemblies (HPNAs) further elevate this parameter by linking two identical monomers [11]. Moreover, stimuli-responsive linkers, such as glutathione (GSH)-sensitive disulfide bond (SS) and reactive oxygen species (ROS)-responsive thioketal (TK), can endow HPNAs with ingenious drug release capacities [12,13]. Hitherto HPNAs have been successfully investigated to enhance antitumor efficacy and mitigate systemic toxicity in various monomeric components derived from TCM, such as paclitaxel, camptothecin and curcumin [[14], [15], [16]]. However, HPNAs still grapple with challenges in self-assembly ability and stability due to increased steric hindrances and intermolecular interactions [17]. Accordingly, a standalone nanostrategy based on BF HPNAs may not suffice to achieve efficient BF delivery while minimizing its toxicity. This realization hints us to explore preferable approaches to refine the performance of BF HPNAs. Metal-phenolic networks (MPNs) have been increasingly applied in nanomedicine owing to their unique features: extraordinary stability, prolonged circulation, exceptional biocompatibility, and sensitivity to the acidic tumor microenvironment [18,19]. Furthermore, MPNs exhibit high self-assembly capacity via the coordination between polyphenols and metal ions, thereby enabling the co-delivery of loaded drugs and metal ions [20,21]. The potential of Fe-doped MPNs in promoting ferroptosis also has been documented in previous studies [22,23]. Therefore, MPNs may endow HPNAs with their outstanding properties and enhance their anti-tumor efficacies. To our knowledge, no prior study has explored the cooperative co-assembly of these two materials. We reason that incorporating MPNs into BF HPNAs might impart the outstanding properties of MPNs to the latter, thereby creating an ideal NDDS to overcome BF's limitations and unlock its full therapeutic potential.

In this work, we developed a synergistic nano-coassembly strategy integrating MPNs with BF HPNAs to construct advanced BF nanotherapeutics with optimized overall performances. The GSH-responsive BF homodimeric prodrug (SBF) and PEG-functionalized polyphenols were firstly synthesized. Following this, Fe3+ and PEG-polyphenol were introduced to drive the self-assembly of SBF, yielding pH/GSH dual stimuli-activatable nano-coassemblies termed MSBNAs (Fig. 1). The stability perturbation experiment revealed that hydrophobic interactions, electrostatic adsorption, and coordination bonds collectively governed this co-assembly process, endowing MSBNAs with superior self-assembly efficiency and colloidal stability. Pharmacokinetic studies demonstrated the prolonged circulation half-life and excellent tumor-selective accumulation of MSBNAs, which minimized cardiac exposure and alleviated BF-associated cardiotoxicity. In both cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mice models, MSBNAs significantly enhanced the anti-proliferative and anti-metastatic efficacy of BF, achieving 83.7 % tumor growth inhibition and complete suppression of lung metastasis. Mechanistic investigations further revealed that BF could trigger lipid peroxidation and ferroptosis by disrupting ROS, GSH, and iron homeostasis. Critically, excessive cellular irons from MSBNAs inevitably amplified BF-induced ferroptosis, thereby bolstering the antitumor potency of BF. This dual pathway ferroptosis-enhanced nanostrategy positions MSBNAs as an advanced candidate to benefit BF-based cancer theraputics.