We further investigated the supramolecular modulation of photodynamic therapy (PDT) through an ex-situ cell-based assay as illustrated in Figure (top schematic). ( ) We first incubated a solution of SIE5 (80 μM in pH 6.5 MES buffer) for 8 h at 37 °C in a vial, which resulted in the release of 7.75 μM MB as quantified by UV-vis measurements (Figure ). An aliquot of this solution was diluted 5-fold with MES buffer, then added to 96-well plate containing HeLa cells (100 μL per well), achieving a final MB concentration of 1.57 μM in the cell culture medium. Subsequently, cells were incubated in this solution at 37 °C for 1 h and then subjected to 10 min photo-irradiation. An MTT colorimetric assay showed <10% cell viability following this PDT treatment (Figure , solid bars). ( ) In contrast to the free SIE5 group, PDT treatment using the 10×CB/SIE5 complex under otherwise identical conditions exhibited minimal photo-induced cytotoxicity (Figure , striped bars), as the concentration of released MB in the CB-inhibited group is below the therapeutic dose (see SI for details). This result underscores the "gating" effect of the supramolecular CB protecting group on the therapeutic effect of the MB-prodrug SIE5. (3) Further, we studied the adamantane-triggered reactivation of the therapeutic PDT effect: 20×adamantane (1.6 mM) was added into a 10×CB/SIE5 solution in a vial to liberate SIE5 through the competitive binding between CB and adamantane, initiating the self-immolation and MB release (9.90 μM MB released for 8 h at 37 °C). This solution was subjected to the same incubation conditions as described above, added to cell media, and the co-incubated cells were subjected to identical photo-irradiation conditions with the cell viability evaluated by MTT. Indeed, this 10×CB/SIE5/20×Ad group exhibited a recovered PDT cell-killing effect, as indicated by < 20% cell viability (Figure , crosshatched bars). Control experiments where cells received identical treatment but without photo-irradiation showed > 60% cell viability (Figure , bars with white base color), confirming that the therapeutic cytotoxic effects observed in the treated cancer cells arise from PDT. The observed viability of 60-80% across the dark-control groups suggests moderately good biocompatibility of SIE5 and its degradation products, while treatments involving only the supramolecular entities without SIE5 demonstrated no cytotoxic effects in HeLa cells (Figure ). Future efforts will aim to mitigate the background toxicity associated with SIE5 and its products. Finally, we demonstrated the supramolecular regulation of PDT through a series of in-situ cellular experiments under optimized conditions (see SI for details). SIE5, 10×CB/SIE5, or 10×CB/SIE5/20×Ad were introduced into the HeLa cell culture media to achieve a final SIE5 concentration of 16 M. After 4 h of co-cultivation, cells were exposed to 10 min photo-irradiation, followed by viability assessment using the MTT assay. Consistent with the ex-situ findings, (1) the SIE5 group exhibited significant PDT cytotoxicity (Figure , solid), (2) the PDT effect was essentially eliminated in the CB-gated 10×CB/SIE5 group (Figure , striped), and (3) the PDT cell-killing effect was restored in the 10×CB/SIE5/20×Ad group (Figure , crosshatched), which included the adamantane as a supramolecular trigger.