Albumin stands as a crucial and abundantly present plasma protein, pivotal in various physiological functions [1], [2]. It emerges as an ideal candidate for drug transport due to its non-toxic, minimally immunogenic, biocompatible nature, along with an extended circulation period [3]. In the context of tumor proliferation, these neoplasms actively exploit serum proteins, particularly albumin, which preferentially accumulates at the tumor site, serving as a primary source of energy and nitrogen through endocytosis and lysosomal degradation to sustain malignant cell growth [4]. The advent of albumin-based prodrugs marks a significant stride in cancer therapy. Kratz and coworkers pioneered a concept for an acid-sensitive albumin-binding prodrug that, upon intravenous administration, selectively and covalently bind to cysteine34 (Cys34) site of serum albumin [5]. Another two acid-sensitive auristatin E-based albumin-binding prodrugs have been developed, demonstrating convincing antitumor efficacy. However, these two prodrugs exhibited instability in plasma [6]. Notably, the most successful case among acid-sensitive albumin-binding prodrug is the development of INNO-206 (acid-cleavage doxorubicin-based albumin-binding prodrug), currently undergoing phase III studies for sarcoma and gastric cancer treatment [7]. Applying for the high expression of specific enzymes in tumor tissues, Kratz’s group designed and synthesized a large number of enzyme-responsive albumin binding prodrug. These prodrugs were composed of antineoplastic drugs, enzymatically cleaved linkers and maleimide groups that covalently bind to albumin [8], [9], [10], [11], [12], [13], [14]. Further advancements emerged as Papot and coworkers designed and studied β-glucuronidase-sensitive albumin-binding prodrugs for delivering potent compounds like doxorubicin [15] or MMAE [16] to tumor site. These prodrugs undergo cleavage by β-glucuronidase, leading to the release of the cytotoxic agents. Recent reports indicate the development of β-glucuronidase-sensitive double-loaded [17] and triple-loaded [18] albumin-binding prodrugs showing remarkable anti-tumor efficacy and exceptional tolerance in vivo. An albumin-binding prodrug with stimulus-responsive function heralds a burgeoning concept of albumin-binding drug delivery systems, offering promising avenues for tailored and effective cancer treatments.

Metastatic breast cancer poses a formidable clinical challenge, characterized by its near-incorrigible nature, affording patients a mere median survival of 38 months [19]. Notably, the lungs serve as the primary site of metastatic in breast cancer [20]. Intriguingly, individuals with diminished levels of albumin exhibit a significantly heightened propensity for lymph node metastases when compared to their counterparts with elevated albumin content, suggesting that metastatic breast cancer may a higher serum albumin supply to meet its demands for malignant proliferation [21]. SPARC (Secreted protein, acidic and rich in cysteine), a protein renowned for its affinity to albumin [22], [23]. Elevated SPARC expression is a key regulator in the proliferation of the extracellular matrix and cell migration, processes intrinsically linked to tumor invasion, metastasis, and poor prognosis in various malignancies [24], [25]. SPARC protein is positively expressed in 76.1 % the breast cancer cases, predominantly localized within the cytoplasm and interstitium of breast cancer tissues [26]. This conspicuous upregulation of SPARC in breast cancer further underscores the potential utility of an endogenous albumin strategy for the treatment of metastatic breast cancer [27].

Camptothecin (CPT), an inhibitor of topoisomerase I (TOP I), exerts its anticancer activity in tumor cells by capturing TOP I-DNA complexes, disrupting them and inducing apoptosis in single-stranded DNA [28], [29]. Camptothecin derivatives for breast cancer treatment has been investigated in trials. Irinotecan, a derivative of CPT, has demonstrated significant anti-tumor efficacy in clinical trials [30]. The active component of irinotecan is SN38. However, SN38 faces challenges due to its poor physicochemical properties, pharmacokinetic properties and limited tumor targeting capability [31]. Etirinotecan pegol (NKTR-102), a prolonged-action TOP I inhibitor serves as a kind of SN38-releated drug delivery system with a linker susceptible to esterase cleavage [32], [33]. Despite its promising design, the phase III randomized clinical trial revealed no significant disparity in overall survival between Etirinotecan pegol treatment and conventional chemotherapy for patients with metastatic breast cancer.

In our prior study, we developed a β-glucuronidase cleavable albumin-binding SN38 prodrug, which involved linking SN38 to β-glucuronic acid with a hydroxybenzyl self-cleavage group [34]. This prodrug demonstrated selective delivery of SN38 to tumor sites though extracellular β-glucuronidase hydrolysis, exhibited slower plasma clearance, and significantly delayed tumor growth. The design of prodrugs that are cleaved by cathepsin B also deserves to be focused. Cathepsin B is a lysosomal cysteine protease that plays an important role in tumor growth, invasion, metastasis, angiogenesis, overexpressed in various malignant tumors [35]. Cathepsin B-induced mechanism is designed to exploit the releasing of the drug at specific tumor sites which have been overexpressed cathepsin B [36]. Numerous studies have explored prodrugs activated by cathepsin B. Kratz and colleagues utilized cathepsin B as a trigger in their albumin-binding prodrugs of methotrexate and doxorubicin [13], [37]. Similarly, Ghosh et al. conjugated monomethyl auristatin E to recombinant serum albumin using a cathepsin B-sensitive valine-citrulline dipeptide linker [38]. Additionally, Kratz’s team developed an albumin-binding prodrug of CPT, incorporating a cathepsin B-sensitive peptide via an ester bond [8]. These studies underscore the significance of designing cathepsin B as a trigger and connecting it to cytotoxic agents through various ligation sites (e.g., N, O) in albumin-binding prodrugs. The methylene alkoxy carbamate (MAC) self-immolative unit represents a versatile strategy for payload conjugation with different ligation sites [39]. Its initial applications included a 5-fluorouracil release from a glucuronide prodrug using an MAC adapter [40]. Furthermore, Santi et al. utilized an MAC adapter for the β-elimination release of SN38 [41]. In our current work, we have designed a methylene alkoxy carbamate (MAC) self-immolative unit to adapt the valine-alanine (VA) cathepsin B trigger and SN38, which contains a phenolic hydroxyl group, termed Mal-va-mac-SN38 (Fig. 1). The rationale behind the design of the prodrug utilizes leverages the heightened demand for albumin, high expression of SPARC protein, and high expression of cathepsin B of metastatic breast cancer. Through covalent binding with endogenous albumin, Mal-va-mac-SN38 demonstrated remarkable stability during extended circulation. Upon internalization by metastatic breast cancer cells, this prodrug undergoes enzymatic cleavage by cathepsin B, liberating the potent effector molecule SN38, which exerts inhibitory effects on breast cancer lung metastasis (Fig. 1). Our investigation encompasses a comprehensive evaluation of the anti-tumor and anti-metastasis effect of Mal-va-mac-SN38. Additionally, we delve into the elucidation of the cellular uptake mechanism and the mechanisms underlying its anti-metastatic properties.