**1. Introduction**

The ideal nanoparticle-based drug delivery system assures the safe delivery and selective action of a drug to a target site. Indeed, nanomaterials can add further functionality to the conjugated/loaded drug and, taking advantage of their unique size, are able to play a crucial therapeutic role. This has triggered an increased interest in nanopharmaceuticals [1,2] and the development of a wide range of nanoparticle systems, such as liposomes, nanoparticles, micelles, dendrimers, and nanotubes [3–5]. However, only a few nanoparticle-based systems have been FDA-approved for cancer therapy to date [6,7]. Although nanoscience in drug development is in its early stages, the fusion of engineered nanomaterials and nanopharmaceutical research is paving the way for the development of stimuli-responsive drug delivery systems, especially in cancer treatment. Interestingly, several chemically modified polymers [8,9] and cross-linked cyclodextrin-based polymers have been proposed to obtain compounds responsive to the external environment [10]. In this regard, cyclodextrin-based nanosponges are of particular interest [11,12].

Nanosponges are hyper-cross-linked cyclodextrin polymers generally obtained from α, β, and γ cyclodextrins, containing suitable amounts of linear dextrin cross-linked with a proper cross-linking agent. A cage-like structure is obtained via the cross-linking of cyclodextrins, thus creating nanochannels in the polymer matrix that can be modulated employing different types of cross-linking agents and/or varying the amount used [13,14]. It is worth noting that active carbonyl compounds, like carbonyl diimidazole, diphenyl carbonate, and organic dianhydrides such as pyromellitic dianhydride, can be used as cross-linker in the preparation of nanosponges [15,16].

Nanosponges offer several features, such as sustained and controlled release, improvement of aqueous solubility, bioavailability, and stability of the hosted molecules, which could be advantageously exploited for drug delivery [15,17,18]. Indeed, previous research highlighted the capability of nanosponges to encapsulate different active molecules and magnify their activity in either in vitro or in vivo studies [15,19,20]. In particular, several anticancer drugs such as doxorubicin, paclitaxel, and camptothecin have been efficiently incorporated in cyclodextrin-based nanosponges, showing an improved antitumor effect [21–24].

Interestingly, nanosponge-based drug delivery systems can be tuned to form 'stimuli-responsive' nanocarriers that modify their structure in response to external changes, such as pH or redox potential [25–27]. Therefore, glutathione (GSH)-responsive nanocarriers have been developed for targeted intracellular anticancer drug release [28], as the GSH tripeptide has a higher intracellular than extracellular concentration [29]. Several intracellular compartments, such as cytosol, mitochondria, and the cell nucleus, contain higher GSH concentrations than do extracellular fluids and circulation. Moreover, GSH intracellular concentration is further increased in cancer cells and, above all, in chemoresistant cells [30]. Oxidative stress has long been implicated in cancer development and progression [31]. An increase in reactive oxygen species (ROS) usually induces a cell adaptive response and the compensatory up-regulation of antioxidant systems to restore redox homeostasis; GSH/GSH disulfide is the major redox combination in mammalian cells [32]. Moreover, many primary tumors have high levels of overexpression of antioxidant enzymes [33,34].

Trotta et al. have developed a next generation of nanosponges that are bioresponsive to GSH external concentration [35]. This behavior may be an ideal trigger for rapid nanocarrier destabilization inside cells, leading to efficient intracellular drug release through disulfide-bond cleavage [35]. Indeed, the disulfide bridge remains stable in extracellular fluids for long periods before being reduced upon internalization in the cytosol, having a higher GSH concentration, thus improving drug bioavailability [36]. As depleting endogenous antioxidants, like GSH, make cancer cells more chemosensitive, this reduction-sensitive nanosystem is further suited to anticancer therapy owing to its ability to enhance the anticancer activity of such drugs. Previously, the encapsulation of doxorubicin and strigolactone analogues in GSH-responsive nanosponges was in vitro and in vivo evaluated. In vitro release kinetics studies from GSH-NS revealed a GSH concentration-based drug release profile over time. Moreover, the GSH-NSs were able to release the payload as a function of in-cell GSH concentration [37,38]. This behavior might favor the selectively controlled release in target cancer cells and enhance the cytotoxic effect. Indeed, both of the compounds loaded in GSH-NS were more effective in inhibiting the cell viability than the corresponding free drugs, particularly in cancer cells presenting a higher GSH content. In addition, a greater reduction of prostate cancer growth was observed for doxorubicin incorporated in GSH-NS compared with the free drug in xenograft mice models [37].

The fact that GSH-NS may well represent an efficient stimuli-responsive drug delivery system for anticancer drugs prompted in-depth study into their biological effect per se on cell growth reported herein. As preliminary cellular evaluations of nanocarriers are usually carried out on 2D cell cultures, previously, the effects of cyclodextrin-based nanosponges have been widely tested in 2D cell monolayer cultures. However, 3D cell cultures, such as multicellular spheroids (MCS), have various in vivo tissue characteristics including the production of an extracellular matrix [39,40]. This study reports a series of experiments carried out to evaluate the bioeffects of GSH-NS containing two different amounts of disulfide bridges either on 2D cell cultures or on 3D cell cultures of human cancer cells, differing in cancer type and intracellular GSH level, namely, human colorectal, HCT116 and HT-29, and human prostatic, DU145 and PC-3, cancer cell lines.
