Copper-doxorubicin nanodot-based dual catalytic metal prodrug liposome for enhanced chemotherapy and Chemodynamic Combination Therapychemodynamic combination therapy

Owing to their unique properties, metallic therapeutic agents for cancer have over the years garnered considerable scientific interest among the multiple options available. Compared to their organic counterparts, these metallic drugs have demonstrated a greater diversity in their functions and mechanisms of action. For instance, they were reported to disrupt the homeostasis of metal ions in tumor cells and modulate the tumor immune microenvironment [1], [2], [3], [4]. Owing to the aforementioned attributes, multiple clinically approved metallodrugs, including cisplatin, carboplatin, and oxaliplatin have been employed in treating various cancers [5], [6]. However, these agents have been associated with deleterious side effects, including nephrotoxicity, neurotoxicity, ototoxicity, and myelosuppression, hindering their broader application [7], [8]. Notably, developing complexes comprising physiologic metals (such as iron, copper, and calcium) or metal prodrugs could help address these challenges [9], [10].

In recent decades, significant advancements have been realized in the field of antitumor nanomedicine [11], [12], [13]. Nanotechnology offers a promising avenue for advancing metallodrugs, as metal ions can be readily coordinated with polyphenolic compounds, yielding nanodots. These nanodots could facilitate the aggregation of metal ions at the tumor site-a crucial therapeutic step [14], [15], [16]. For instance, to enhance the efficacy of chemodynamic treatment, Duan et al. loaded gallic acid-iron nanodots (GA-Fe) into hollow manganese dioxide [17]. Besides Fe²⁺, Cu²⁺ could also be used to produce nanodots via coordination bonding. In a previous study, copper peroxide nanodots were synthesized by coordinating hydrogen peroxide (H₂O₂) with Cu²⁺ at alkaline pH values, with the resultant demonstrating a pH-dependent generation of hydroxyl radicals (•OH) [18]. Notably, the conditions required for the Fenton reaction of iron-based nanomaterials are more stringent (pH 2–4) [19], [20]. Conversely, copper-based nanomaterials can initiate a Fenton-like reaction with greater efficiency under relatively mild pH conditions, with a catalytic efficiency that is 160 times higher than that of conventional Fe²⁺ [21], [22], [23], [24]. Therefore, Cu-based nanomaterials could be more beneficial in improving the efficacy of Chemodynamic Therapy (CDT).

Doxorubicin (DOX) contains phenolic hydroxyl groups, which could facilitate the formation of metal-DOX complexes via coordination with metal ions [25], [26], [27], [28]. Compared to normal tissues, tumor sites exhibit a lower pH and higher glutathione (GSH, 1–10 mM) levels [29], [30], [31], creating an environment that might render the DOX/Cu²⁺ complex susceptible to dissociation, thus facilitating the liberation of DOX molecules and Cu²⁺ within cancer cells [32]. Furthermore, DOX could activate the production of H₂O₂ by NADPH oxidase 4 (NOX4) in tumor cells, thus enhancing the Fenton-like effect of Cu²⁺ [33]. Moreover, Cu²⁺ could react with intracellular GSH, leading to GSH depletion and intracellular ROS amplification [34], [35]. It is also noteworthy that the synthesis of metal prodrug nanodots by DOX and Cu²⁺ via ligand bonding could mitigate the toxicity of DOX.

Herein, we present a facile strategy for synthesizing Cu-DOX nanodots (CD). To the best of our knowledge, this is the first study to report such a strategy. To facilitate a chemo-chemodynamic synergistic therapeutic effect, CD nanodots were co-loaded with Dihydroartemisinin (DHA) in a liposome nano-delivery system. First, DOX was deprotonated and ligated with Cu²⁺ in an alkaline environment before employing Polyvinylpyrrolidone (PVP) as a stabilizing agent to facilitate the formation of CD nanodots (Scheme 1a). Subsequently, the composite liposome nano-delivery system (CDAL) was prepared with CD nanodots and DHA, which were loaded in the hydrophilic and hydrophobic regions of the liposome, respectively (Scheme 1b). Mechanistically, CD nanodots released DOX and Cu²⁺ in the tumor acidic or high GSH-expressing environment, with the released Cu²⁺ inducing intracellular GSH depletion in tumor cells (Redox reaction I) (Scheme 1c). Notably, the released DOX enhanced the generation of H₂O₂ by upregulation of NOX4, thus facilitating the Cu²⁺-mediated Fenton-like reaction (catalytic reaction II). It could also catalyze the generation of carbon-centered radicals (•R) from DHA, further promoting the generation of Reactive Oxygen Species (ROS; catalytic reaction III) and enhancing the efficacy of tumor therapy. Overall, CDAL with multi-augmented ROS generation via dual-radical production and GSH depletion could effectively inhibit tumor growth, improving the efficacy of chemo-CDT.