Chemotherapy remains the primary strategy for clinical cancer therapy [1], [2]. Doxorubicin (DOX), a classic anthracycline chemotherapy drug, is widely utilized in the treatment of various malignant tumors, including breast cancer, ovarian cancer, leukemia, and lung cancer [3], [4]. DOX exerts its therapeutic effects through intercalation into DNA and inhibition of topoisomerase II, resulting in cell cycle arrest and tumor cell apoptosis [5], [6], [7]. Nevertheless, its clinical application is restricted by nonspecific distribution and severe systemic toxicity, including cardiotoxicity and myelosuppression[8], [9].
Nanodrug delivery systems (NDDS) have shown great potential in prolonging circulation time and enhancing tumor accumulation [10], [11], [12]. For instance, Doxil, first long-circulating liposomal approved by the U.S. Food and Drug Administration (FDA), extended the circulation time of DOX and reduced cardiotoxicity [13], [14], [15]. However, Doxil still faces side effects such as hand-foot syndrome due to the overlong circulation time, which severely restricts the dose elevation of Doxil and its antitumor efficacy [16], [17]. Besides, the high immunogenicity and low biocompatibility of the nanocarrier materials still pose potential safety risks [18], [19]. Human serum albumin (HSA) stands out as an ideal drug carrier due to its non-immunogenicity, excellent biocompatibility and inherent tumor targeting ability [20], [21], [22]. However, due to the low affinity between DOX and HSA, traditional preparation technology did not yield uniformly sized nanoparticles, resulting in large size (∼ 500 nm) and wide size distribution (PDI > 0.6) [23], [24], [25].
Rational design of prodrug to enhance the binding affinity of DOX with HSA hold great potential. The prodrug strategy could improve the undesirable characteristics of chemotherapeutic drugs through suitable structural modifications [26], [27], [28]. Considering the characteristics of HSA as a natural fatty acid carrier, the conjugation of drug molecules with fatty acid could improve the affinity with HSA [29], [30]. Three factors should be considered when designing the structure of prodrugs. Firstly, the chain length should impact the bonding affinity of prodrug to HSA. Secondly, the intelligent release of the active drug from the prodrug is essential to achieve the ideal therapeutic effect [31], [32]. The reduction-sensitive prodrug strategy leverages the pronounced redox difference between the tumor cells and normal cells, particularly the elevated intracellular glutathione (GSH) concentrations in tumor cells [33]. Intelligent intracellular drug release should be achieved by inserting disulfide bond as response modules into the prodrug structure, which has high responsivity with GSH [34], [35], [36]. Finally, the linkage bond between the drug and the response modules also acts as the critical barrier to drug release, and its stability predominantly governs the drug release rate [37].
In this studies, four DOX-fatty alcohol prodrugs were synthesized with different carbon chain lengths (C4, C8, C12, C20) to systematically evaluate the effect of chain length on the binding affinity between DOX and HSA. Disulfide bond was used as response module and conjugated with DOX through a stable carboxamide bond. Notably, when the carbon chain length reached eight, the DOX prodrug (DOX-CO-C8) exhibited significantly enhanced binding affinity to HSA, facilitating the formation of albumin-based nanoparticles (ANPs). However, the cytotoxicity of all prodrugs was extremely weak, suggesting the DOX could not be released on demand due to the stability of carboxamide bond. Subsequently, we further replaced the stable carboxamide bond with relative active carbamate bond (DOX-OCO-C8). Compared to DOX-CO-C8, DOX-OCO-C8 exhibited stronger binding affinity to HSA, enabling the formation of more stable ANPs, which significantly enhanced DOX circulation time and reduced its in vivo clearance rate. Moreover, under the reduction conditions, DOX-OCO-C8 ANPs triggered effective DOX release, leading to superior antitumor efficacy compared to DOX. Additionally, DOX-OCO-C8 ANPs significantly improved DOX safety, with no significant toxicity observed in normal tissues, even at high doses (20 mg/kg equivalent to DOX). Therefore, this albumin-based nano-system enhanced antitumor efficacy and effectively reduced systemic toxicity of DOX, demonstrating great potential as a candidate for clinical application (Fig. 1).