Despite exhaustive efforts to defeat cancer throughout multiple disciplines, tumor heterogeneity is one of the major hurdles hindering the development of optimal therapeutic interventions to eradicate cancer [[1], [2], [3]]. The model of clonal evolution, suggested in 1976 by Peter Nowell, infers cancer as a Darwinian system driven by stepwise somatic mutation and clonal selection [3,4]. It is revealed that cancer goes through self-renewal process that creates a complex tumor microenvironment. Furthermore, mutational responses to chemotherapy would lead to drug resistance with poor prognosis and recurrence of cancer via emergence of tumor subclones containing driver mutations leading to, for example, upregulated expression of drug efflux pumps like ATP-binding cassette proteins and ablation of TP53 tumor suppressor gene [5,6]. In fact, immunotherapy gains recent attention as a game changer, introducing immune checkpoint inhibitors and chimeric antigen receptor (CAR) T-cell therapeutic products to the market. Despite the novel concept of eliciting anti-cancer immune responses, antigen escape of the antigen-negative subclones developed as a result of adaptive resistance process of cancer would be a crucial problem that limits the therapeutic efficacy [[7], [8], [9]]. In this respect, combinatorial therapeutics, involving both target-selective chemotherapy with diverse chemical drugs and non-selective physicotherapy employing diverse physical means to disrupt cellular integrity, deserves a special attention to overcome the tumor heterogeneity and drug resistance [10].

Since chemical drugs cause severe side effects and multi-drug resistance to the patients, targeted and simultaneous delivery of multiple drugs to the cancer site has been attempted to enhance their therapeutic efficacy [5,6,11,12]. Combination of drugs is expected to exert additive or synergistic anti-cancer effects through disparate mechanisms of action and reduce the possibility of drug resistance compared to monotherapy that might induce alternative salvage pathways for the cells to survive [13]. For example, combination of paclitaxel (PTX) and doxorubicin (DOX) has been actively studied as first-line chemotherapy for several types of cancer, especially metastatic breast cancer [[14], [15], [16], [17], [18]]. PTX, also known as Taxol, is a taxane drug that interferes microtubule function and restrains cell division, while DOX is an anthracycline medication that intercalates between DNA/RNA base pairs and inhibits topoisomerase II [11]. In clinical trials, the combined administration significantly improved the treatment response rate compared to single-agent therapy, but the outcome on survival or quality of life was minimal along with dose-limiting toxicities [16]. The pronounced adverse effects of the combination therapy might be due to changes in DOX metabolism caused by coexisting solubilizers used to mix poorly water-soluble PTX as observed with the distinct tissue distribution of DOX in animal studies [19,20]. Hence, creative delivery systems that could encompass chemically diverse drugs while minimizing side effects are still needed to enhance therapeutic effectiveness. In addition, physical therapeutics are advantageous for causing indiscriminate cell death in the complex microenvironment of solid tumors comprising not only malignant cells but also stromal cells capable of promoting cancer stemness if the omnipotential physical agents are targeted to the cancer site [21,22]. It is therefore necessary to develop drug delivery system adequate for accommodating both chemotherapeutics and physicotherapeutics in order to annihilate the heterogeneous tumors at once.

In the pursuit of ‘all-at-once’ strategy, the gold nanoparticle (AuNP) microcapsules previously fabricated with a self-assembly protein of α-synuclein (αS) [23] have been employed to encapsulate lipid-based inverted micelles (IMs) which allow co-delivery of multiple drugs with distinctive chemical properties of hydrophilic and hydrophobic substances trapped inside and outside of IMs within the capsules, respectively. The resulting multifactorial microcapsules would therefore exert physicotherapeutic effects by IMs for membrane destabilization and AuNPs causing photothermal effect in addition to the combined chemotherapeutic effects by diverse chemical drugs. For the optimal entrapment of IMs, however, porous poly(lactic-co-glycolic acid) (PLGA) network is required to provide inner skeleton for not only compartmentalizing the internal space of the microcapsules but also preventing the coalescence of IMs which would cause eventual collapse of the microcapsules due to uncontrolled enlargement of IMs. In addition, PLGA is known to exhibit outstanding biocompatibility and biodegradability, making it one of the most widely employed polymers in biomedical applications such as diagnostics [24,25], tissue engineering [26,27], and drug delivery system [28,29].

To selectively discharge all the therapeutic components into the pathological lesion of cancer, proteolytic disintegration of the microcapsules is exploited since the outer shell of the AuNP microcapsules has been constructed via stable αS-αS molecular engagement induced during Pickering emulsion formation of the αS-AuNP conjugates with chloroform [23]. As the αS-mediated outer shell and the internal porous PLGA matrix are disintegrated, therefore, IMs are released near the protease-abundant cancerous environment. It is these IMs that play crucial roles in delivering chemical and physical therapeutics by exhibiting not only the ‘quantal’ release of hydrophilic drugs along with hydrophobic drugs bound to the surface of IMs after their dispersion in chloroform, but also the membrane-disruptive activity toward the heterogeneous cancer cells. In addition, the remnants of αS-AuNP microcapsules, another proteolytic product, could participate in non-selective cell death via photothermal effect once localized either inside or outside the cancer cells in the close vicinity. Taken together, the multifactorial microcapsules fabricated in this study have the following attributes adequate for cancer therapy: (1) tumor-specific release of multiple therapeutic agents by proteases abundant in heterogeneous tumor sites, (2) combinatorial chemotherapy with hydrophilic and hydrophobic drugs, (3) membrane-disruptive activity by IMs as a potent physicotherapeutic agent, and (4) photothermal activity of AuNPs clustered in the fragments of microcapsules.