Host-guest chemistry describes supramolecular systems composed of two or more molecules that act as molecular building blocks, where intermolecular forces hold their components together [2,3]. These systems typically consist of a macrocyclic molecule, referred to as the host, which is capable of encapsulating a smaller molecule, known as the guest, within its cavity [4]. Host and guest molecules are stabilized by various non-covalent interactions such as hydrogen bonds [5], π-π stacking interactions [6,7], metal-ligand coordination [8,9], van der Waals forces [10,11], and charge transfer interactions [[12], [13], [14]]. One of the most important properties of this system is its dynamic nature, which imparts the resulting architectures with intriguing stimulus-responsive behaviors [2].

Over the years, various macrocycles host, capable of encapsulating smaller molecules within their cavities have been synthesized, resulting in diverse host-guest systems. Examples include crown ethers [15,16], cyclodextrins [17,18], calixarenes [19,20], cucurbiturils [21,22], cyclophanes, [23], and pillararenes [24,25], among others. These systems have been tailored for numerous applications across diverse research areas, such as molecular switches [[26], [27], [28]], kinetic effects, [[29], [30], [31]], and biomedical properties, [[32], [33], [34], [35], [36]], among others.

In particular, within the development of biomedical properties, host-guest macromolecular systems have been designed in which macrocycle hosts are capable of encapsulating small drug molecules. Many of these macromolecular systems serve as passive carriers, with the incorporation of host molecules in pharmaceutical formulations primarily aimed at acting as excipients or enhancing the solubility of drugs [[37], [38], [39]]. In recent years, supramolecular systems have been designed to include drugs, not only improving their pharmacokinetic properties and reducing side effects but also focusing on systems capable of controlled drug delivery to specific target sites, mediated by external stimuli [1,28,[40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50]].

The semi-rigid tetracationic cyclophane, ExBox4+, composed of a 1,4-phenylene-bridged (“extended”) bipyridinium unit, has been synthesized [51]. This cyclophane adopts a box-shaped geometry with dimensions of 3.5 Å (width) × 11.2 Å (length), based on van der Waals radii. The macrocycle is electron-deficient in π-electrons, which enables it to encapsulate hydrophobic, π-electron-rich guest molecules within its cavity, thereby increasing their solubility in water. Strong non-covalent donor-acceptor interactions drive the formation of such a host-guest system [52]. ExBox4+ is stable under physiological conditions, non-cytotoxic, and capable of internalization within lysosomes of living cells, making it an ideal candidate as a cellular delivery vehicle [53].

Due to its characteristics, cyclophane, ExBox4+, can encapsulate the porphyrin 5,15-diphenylporphyrin (DPP) within its cavity. Porphyrins are widely used as photosensitizers in photodynamic cancer therapy (PDT), due to their ability to absorb light energy. Upon light activation, porphyrins generate reactive oxygen species (ROS), which are highly reactive molecules capable of attacking cell membranes and organelles, ultimately inducing cell death in cancer cells through necrosis or apoptosis [54,55].

Specifically, DPP is an aromatic π-electron-rich molecule with a planar conformation [56,57], which facilitates its inclusion in the ExBox4+ cavity. This interaction forms a water-soluble 1:1 host-guest complex, DPP@ExBox4+, with an association constant of Ka = (1.26 ± 0.03) x104 M-1. The fast intermolecular electron transfer within the system enables ExBox4+ to modulate the phototoxicity of DPP, providing photoprotection by inhibiting the generation of ROS. By sequestering DPP, ROS formation is suppressed, which diminishes off-target adverse effects [1].

The cellular environment of a cancer cell is acidic, [58] demonstrating that DPP@ ExBox4+ has the potential as a pH-responsive drug delivery system. The DPP@ExBox4+ complex is sensitive to acidic conditions, as DPP becomes protonated (DPPH22+) at low pH, specifically at its imino nitrogens. This protonation disrupts its planar conformation, which is believed to trigger the release of the porphyrin at the target site. Once released, the porphyrin regains its photosensitizing capabilities, allowing it to generate ROS and act as an effective agent for PDT [1].

In this report, the nature of the interactions between ExBox4+ and DPP will be studied, focusing on their role in enabling the formation of the supramolecular system and contributing to the photoprotection of the porphyrin. Furthermore, the release of DPP from the macrocycle will be analyzed by studying the interactions between ExBox4+ and DPPH2+, as well as their dynamic behaviors. This analysis of the nature of the interactions is relevant for the efficient design of supramolecular prodrugs with potential pharmaceutical applications.

The nature of the host-guest interactions was elucidated through a multi-faceted computational approach. An Energy Decomposition Analysis (EDA) based on the Ziegler-Rauk scheme was employed to quantify the individual energy contributions to the binding energy. To ensure an accurate description of the crucial dispersion forces, Grimme's D3 dispersion correction with Becke–Johnson damping (DFT-D3(BJ)) was applied. Furthermore, charge transfer between the host and guest was assessed via natural population analysis (NPA). Finally, the independent gradient model (IGM) will be used to identify the nature of the non-covalent interactions in both the formation of the host-guest system and the release of DPPH2+.