The reprogrammed energy metabolism pathway of tumor cells, also known as the Warburg effect, has become one of the most used targets for selective cancer treatment [1], [2], [3], [4]. Specifically, the tumor cells consume glucose to meet their intense energy needs through glycolysis, concomitantly producing large amounts of lactate. To adapt such an altered metabolism and avoid self-acidosis, many tumor cells correspondingly tend to upregulate the monocarboxylic acid transporter (MCT) on their cell membrane for controlling the concentrations of intracellular lactate [5], [6], [7], [8], [9], [10]. It has been revealed that the elevated MCT4 expression is mainly responsible for lactate efflux to maintain a stable intracellular pH. Additionally, the flow out of lactate could induce a weak acidic extracellular milieu, which is closely related to the tumor immunosuppression micro-environment [11], [12], [13], [14], [15]. Therefore, the MCT4 has been regarded as an emerged target for cancer therapy. On the other hand, the conversion of lactate to pyruvate and further being decomposed through the tricarboxylic acid (TCA) cycle on mitochondrion could also reduce the intracellular lactate content [16,17], which would attenuate the MCT4 inhibition induced acidosis effect at some extent. Hence, simultaneously inhibiting MCT4 and TCA cycle holds great potential for drastically disrupting the intracellular pH homeostasis of tumor cells. However, the strategy of using single drug to sequentially attack the dual targets of MCT4 and TCA cycle for acidosis anti-tumor therapy has never been reported.

In recent years, supramolecular nanomedicine formed in situ self-assembly has received extensive attention, and has shown a broad clinic application prospect in tumor selective theranostic [18], [19], [20], [21]. Be attributed to the unique self-assembly process, the in situ generated nanomedicine delicately integrates the advantages of small molecular drugs and conventional nanomedicine, including controllable quality, deep penetration, long-term retention and reduced toxicity [22]. The over-expressed enzymes, glutathione and low pH all provide feasible conditions for in situ self-assembly at tumor site [23], [24], [25], [26]. Especially, enzyme-instructed self-assembly (EISA) has attracted popular research interest in controlling tumor cell fate [27,28]. For example, the extracellular alkaline phosphatase (ALP) triggered dephosphorylation and self-assembly on the cell membrane has been applied to selectively degrade membrane proteins of programmed cell death-ligand 1 (PD-L1) in 4T1 cells [29]. Besides, the extracellular ALP combined the intracellular stimulates could also trigger tandem self-assembly to produce more sophisticated nanomaterials for cancer theranostics [30], [31], [32]. Moreover, it was also revealed that EISA as a multi-step process holds great promise for simultaneously interacting with multiple targets of cancer when combined with current anticancer drug [33,34]. These advances have implied that the in situ EISA system holds great promise for precisely controlling the spatiotemporal distribution of one therapeutic agent with multiple targets to produce sequential anticancer effect.

Lonidamine (LND), a small molecular chemotherapeutic drug, has been confirmed possessing multi-targets in tumor cells, including the MCT4 and the mitochondria [35,36]. Although LND has shown quite good anti-cancer efficacy in some preclinical studies, its application still severely hindered by the poor water solubility, low bioavailability and strong hepatotoxicity [37]. To this end, numbers of delivery systems have been fabricated to enhance its therapeutic activity, mainly focused on improving its impaired effect against mitochondria [38], [39], [40], [41], [42], [43]. However, the sequential-dual inhibition effect of LND against different targets has never been explored at present. Inspired by the above background, an enzyme-instructed in situ self-assembly system was constructed to induce acidosis apoptosis for selective cancer therapy. LND, used as a capping group, was conjugated on a D-conformational phosphorylated peptide. Under the catalysis of the high expressed extracellular ALP, the resultant hydrophilic molecule of LND-GDFDFDpY could in situ transformed into hydrophobic molecule of LND-GDFDFDY, and further self-assembled into supramolecular nanomedicine on the cell membrane surface (Scheme 1). With the help of the in situ assembly induced retention effect, the target inhibition of LND against MCT4 could be hopefully realized to block the lactate efflux. After be internalized by the cell, the nanomedicine would further destroy the mitochondrion and TCA cycle, therefore abolish the lactate consumption via disrupting the conversion balance of lactate and pyruvate. As a consequence, through the sequential-dual effect mediated by enzyme instructed in situ self-assembly, lactate would be greatly accumulated in the cell and induce tumor acidosis. Meanwhile, the dysfunctional mitochondria would reduce the oxygen consumption rate (OCR), therefore achieving the purpose of alleviating hypoxia and radiosensitization, which together with acidosis induced apoptosis resulted an effective combined chemo-radiotherapy. This study expanded the application of EISA in inducing tumor acidosis apoptosis and combination chemo-radiotherapy, which could offer an enlightening paradigm for drug delivery and cancer selective therapy.