Nowadays, cancer incidence and associated mortality are still major concerns [1]. However, current treatments are limited to chemotherapy, radiation therapy, and surgical resection [2]. Although a conventional chemotherapy is the most commonly used anti-cancer treatment as it is convenient, inexpensive, and highly available, most chemo-drugs lack tumor-targeting efficacy. In addition, drug resistance could be subsequently developed in advanced tumors during clinical processes [3], [4], [5]. Surgical resection has a high possibility of tumor recurrence. Radiation therapy could cause secondary damage to surrounding normal tissues and induce radiation resistance of tumor cells [6,7]. Especially, single utilization of one of these conventional anticancer methods does not exhibit sufficient levels of therapeutic efficacy for solid tumors. Accordingly, various phototherapeutic approaches with the aid of photo-responsive materials such as photothermal therapy (PTT) and photodynamic therapy (PDT) have been proposed due to their high effectiveness in spatially localized treatment with minimal invasiveness [8], [9], [10]. Moreover, several combinative treatments have recently been adopted to overcome clinical limitations of single anticancer therapy. For example, combination of PTT and chemotherapy can kill drug-resistant cancers [11] and combined use of external stimuli-responsive drugs can reduce side effects such as phototoxicity [12].

Among a series of inorganic photo-responsive reagents, liquid metals (LMs) exhibit unique properties such as metal-like conductivity and shape deformability [13]. They are beneficially applied in soft electronics [14,15], sensors [16,17], and soft robotics [18], [19], [20]. Moreover, gallium (Ga)-based LM has recently been utilized as a new class of metal-based biomaterials due to its biocompatibility, avoidable toxicity [21], [22], [23], [24], [25], [26], and capacity to generate localized heat and stimulate hyperthermic physiological responses [27], [28], [29], [30], [31]. Therefore, Ga-based LMs could be efficient candidate photothermal agents such as gold nanorods, graphene nanomaterials, and molybdenum disulfide nanosheet for implantable antitumor applications to treat solid tumors [32], [33], [34]. 6-mercaptopurine (MP) is a well-known anticancer drug that impedes cell proliferation by interfering with DNA synthesis and transcription processes. MP exerts its influence on cells by inducing misbinding of 6-thioguanine triphosphate into RNA and deoxy-6-thioguanine triphosphate into DNA, in addition to other effects on nucleoside pools and cell signaling [35], [36], [37], [38]. Consequently, MP has been utilized in previous studies as a therapeutic agent to inhibit the growth and metastasis of highly aggressive breast cancer [39,40]. However, the short half-life of MP (less than 2 h) and off-target side effects such as hepatotoxicity necessitate the development of drug carriers for protection and localized MP delivery.

For effective localized administration of multiple reagents at a specific tumor site, injectable hydrogel formulation has been used as an implantable reservoir [41]. The choice of crosslinking chemistry plays a crucial role in determining the properties and performance of these hydrogels. Physical crosslinking relies on non-covalent interactions, while chemical crosslinking involves covalent bonding between polymer chains [42]. In this study, we utilized a chemical crosslinking approach involving free radical polymerization of poly(ethylene glycol) diacrylate (PEGDA) initiated by a redox initiator pair, ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED), and the hydrogel made by this method was used as a reservoir for photothermal agent and chemical drug [43], [44], [45]. 3D hydrogel composites containing photothermal agent and additional chemodrug exhibit outstanding cargo storage, localized delivery of chemo-therapeutic agents, and sufficient heat generation to eliminate fibrous solid tumors [46,47]. Direct localization of dual photothermal- and chemo-reagents into solid tumors could avoid the side effects of systemic administration such as low delivery efficiency, non-specific targeting, and off-site accumulation into other organs, along with elevated antitumor efficacy via combinatory effects. Moreover, it is possible to precisely control intrinsic physico-chemical properties such as injectability and tissue retention by modulating polymeric composition of hydrogels [44]. Particularly, the form of an injectable hydrogel enables easy implantation into the body with localized tumor suppression.

Therefore, in this study, we developed a composite hydrogel-based localized tumor suppression technique via a step-by-step combinatory anticancer therapy (i.e., dual PTT and chemotherapy). Our injectable hybrid formulation was composed of eutectic gallium–indium (EGaIn) particles as an effective PTT inducer and MP as a chemical drug embedded in a crosslinkable interpenetrating network (IPN) hydrogel with thiolated gelatin and PEGDA. Therapeutic modality and associated mechanisms of our hydrogel-mediated combination treatment included the following: (1) body-temperature induced gelation of IPN composite hydrogel at a specific tumor region upon injection, (2) localized preservation of photothermal EGaIn LM and chemodrug MP in tumor-surrounding area, (3) near-infrared (NIR)-responsive localized hypothermia by EGaIn to kill adjacent cancer cells, (4) subsequent release of glutathione from disrupted cancer cell bodies, (5) reductive cleavage of disulfide bonds in conjugated MP with polymeric backbones in the hydrogel, and (6) chemodrug-induced additional cancer cell death (Scheme 1). Our results demonstrated superior antitumor efficacy of such LM-based hybrid composite formulation through a sequential tumor killing process in triple negative breast cancer (TNBC) mouse model via synergistic inorganic EGaIn LM-mediated hypothermia and MP-mediated cancer cell death, specifically in a localized solid tumor region.