Breaking photoswitch activation depth limit using ionizing radiation stimuli adapted to clinical application

Stimulus-triggered therapeutic actions offer precise control and real-time modulation. While endogenous stimuli (pH, redox potential, etc.) are limited by local environment, external stimuli enable high spatiotemporal control. Light-responsive systems, while promising, are severely limited by the shallow penetration depth of the activating photons (UV-near-infrared), restricting their clinical applications to superficial tissues. Photodynamic therapy and photoimmunotherapy, though existing clinical applications, are constrained by this depth limitation. This paper introduces a novel approach to address this limitation by employing high-energy ionizing radiation (IR), widely used in radiotherapy, to activate photosensitive systems deep within tissues. The IR's energy is locally converted into low-energy particles to trigger molecular activation. This concept is applied to azobenzene, a well-established photoswitch, forming a 'radioswitch' capable of activating cytotoxic effects on cancer cells after gamma-ray irradiation. This technique offers a potential breakthrough for expanding the clinical use of photosensitive systems.

Extensive research on photosensitive systems has yielded various applications, from bond cleavage to molecular motors. Initial systems relied on UV photons, but their limited tissue penetration spurred the development of visible and near-infrared light-activated systems, enabling in vivo studies in small animals. Clinical translation, however, is hampered by the limited penetration depth of light in tissue, restricting treatments to superficial cancers or those accessible endoscopically. Several methods have been explored to improve radiotherapy outcomes, such as using iodine-based agents to enhance radiation dose and employing nanoparticles to increase radiation effects through radiosensitization or radioenhancement, sometimes linked to immune system activation. Systems using IR to release cytotoxic agents or break specific bonds have also been developed. These mainly rely on either down-conversion of high-energy photons to UV-vis light or oxidation by reactive oxygen species (ROS). This work explores a novel approach, distinct from the typical applications of IR in radiotherapy, to use IR for specific molecular rearrangements similar to photoactivation.

A radioswitch was designed by combining a high-atomic-number (Z) metal, gadolinium (Gd)-chelate, with an azobenzene photoswitch. Gd efficiently interacts with high-energy photons, releasing lower-energy secondary photons and electrons. These secondary particles interact with the azobenzene moiety, causing isomerization from the *cis* to *trans* form. The Gd-chelate enhances IR interaction, and the azobenzene isomerization enables monitoring via spectrophotometry and HPLC. The radioswitch's activation was evaluated using gamma-ray (GR), X-ray (XR), and electron-beam (E) irradiation, all commonly used in radiotherapy. The irradiation doses were within the clinical range (2-20 Gy). Spectrophotometry and HPLC determined the *cis* to *trans* isomerization efficiency. G-values (number of molecules affected per 100 eV of absorbed energy) were calculated to quantify activation efficacy. The mechanism of activation was investigated using scavengers to identify the involved radicals. The role of hydroxyl radicals (HO•) was assessed using different scavengers, gas-saturated solutions (N2, N2O), and Fenton chemistry. Monte Carlo simulations were performed to analyze the energy distribution of secondary electrons released during IR-Gd interactions. The effect of Gd concentration on HO• production was investigated. The interaction of the *trans*-GdAzo with cell membranes was studied using model phospholipid membranes (DPPC) via small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC). Cell permeabilization and cytotoxicity were evaluated using confocal microscopy and flow cytometry with PANC-1 (cancer cells) and CCRF-CEM ARAC 8 C (gemcitabine-resistant cells). Electron energy-loss spectroscopy coupled with transmission electron microscopy (EELS-TEM) was used to confirm Gd presence in cells. The cytotoxic effects were evaluated in the presence and absence of gemcitabine.

The radioswitch, GdAzo, demonstrated efficient *cis*-*trans* isomerization upon GR irradiation in a dose-dependent manner (33-69% activation at 2-20 Gy). Similar activation was observed with XR and E irradiation, indicating that the activation is driven by low-energy secondary particles and species rather than direct interaction with the primary high-energy particles. Monte Carlo simulations confirmed this hypothesis. Experiments with various scavengers revealed that HO• radicals are crucial for GdAzo activation. Fenton chemistry, mimicking HO• generation, also triggered GdAzo isomerization. SAXS studies showed *trans*-GdAzo insertion into DPPC membranes, increasing membrane spacing. Confocal microscopy and flow cytometry demonstrated that *trans*-GdAzo, but not *cis*-GdAzo, induced significant cell permeabilization and cytotoxicity. EELS-TEM confirmed Gd accumulation in permeabilized cells. The radioswitch showed cytotoxic effects on gemcitabine-resistant cells, enhancing cell death and potentially facilitating drug penetration. No cytotoxicity was observed with a control compound lacking the azobenzene moiety, highlighting the crucial role of the azobenzene radioswitch.

The results demonstrate a successful strategy for deep-tissue photoswitch activation using readily available ionizing radiation. The use of a Gd-chelate to facilitate energy conversion from high-energy photons to the low-energy species required for azobenzene isomerization proved crucial. The findings address the long-standing limitation of photoswitch depth penetration. The HO• radical-mediated mechanism offers insights into the activation process. The ability of this system to permeabilize and induce cytotoxicity in both normal and gemcitabine-resistant cancer cells suggests potential for novel cancer therapies. The potential for theranostics (therapy and diagnostics) exists, as Gd allows for MRI detection.

This research successfully demonstrated deep-tissue activation of a photoswitch using clinically relevant ionizing radiation doses. The radioswitch, GdAzo, efficiently converts IR energy into a cytotoxic effect against cancer cells. This approach overcomes the depth limitations of conventional photoswitch systems, opening new possibilities for clinical translation and therapeutic applications. Future research could focus on optimizing the system's efficacy and exploring its potential in various cancer types and therapeutic strategies.

The study primarily focused on in vitro experiments. Further in vivo studies are needed to confirm the efficacy and safety of the radioswitch. The cytotoxicity observed at high concentrations of GdAzo warrants further investigation to optimize the dosage and minimize potential side effects. The precise mechanism of cell membrane disruption by trans-GdAzo requires further exploration. While the study indicates potential for drug delivery enhancement, dedicated experiments with other drugs are needed to confirm its general applicability.