Photodegradation-Targeting Chimeras and Rhenium Complexes for Precision Cancer Therapy and Immune Activation

Abstract

Effective cancer therapy is often hindered by limited specificity and poor immune activation. Conventional treatments such as chemotherapy and radiotherapy can cause severe side effects and fail to selectively target tumor cells. Photodynamic therapy (PDT), which uses photosensitizers and light to generate reactive oxygen species (ROS) for tumor ablation, offers spatiotemporal control but primarily induces apoptosis – a cell death mode with low immunogenicity that does not elicit strong antitumor immune responses. Consequently, recent strategies focus on inducing immunogenic cell death (ICD) via alternative pathways like pyroptosis (an inflammatory form of programmed cell death) and ferroptosis (iron-dependent, lipid peroxidation-driven cell death) to convert “cold” tumors into immunologically “hot” ones. Moreover, emerging approaches such as proteolysis-targeting chimeras (PROTACs) enable selective protein degradation but require improved tumor targeting and control. This thesis aims to develop novel multi-functional therapeutic strategies that integrate PDT with the induction of pyroptosis and ferroptosis in a tumor-specific manner, as well as a light-triggered targeted protein degradation approach, to enhance treatment specificity and antitumor immunity. A series of zinc(II) phthalocyanine (ZnPc)-based conjugates and hybrid molecules were designed, synthesized, and comprehensively evaluated for their photophysical properties, mechanisms of action, and therapeutic efficacy in vitro and in vivo.

Chapter 2 investigates the anticancer effects of a rhenium(I) isonitrile complex, which is known to induce endoplasmic reticulum stress-mediated apoptosis. This study revealed that TRIP also activates a non-canonical pyroptosis pathway via caspase-3-mediated cleavage of Gasdermin E (GSDME), thereby switching apoptotic cell death into a proinflammatory form. In cancer cell models (e.g., A549 lung carcinoma), TRIP treatment led to GSDME-dependent pyroptosis alongside apoptosis, evidenced by cell swelling and membrane bubbling, as well as the release of immunogenic damage-associated molecular patterns such as extracellular ATP, high-mobility group box 1 (HMGB1), and exposure of calreticulin on the cell surface. These ICD hallmarks indicate that TRIP provokes a strongly immunostimulatory form of tumor cell death. In a murine CT26 colon carcinoma model, prophylactic vaccination with TRIP-treated tumor cells significantly delayed tumor growth and improved tumor-free survival compared to controls, demonstrating a vaccine-like antitumor immune effect in vivo. Thus, TRIP is highlighted as a unique agent capable of concurrently promoting apoptosis and pyroptosis to enhance immunogenic cell death, pointing to a novel strategy for cancer therapy.

Building on these findings, Chapter 3 introduces a glutathione(GSH)-responsive conjugate, Pc-ss-TRIP, which confers tumor-selective induction of pyroptosis and enhanced ICD by combining a photosensitizer with a pyroptotic stimulus. In this design, a Zn(II) phthalocyanine (ZnPc) photosensitizer (Pc-NH₂) is linked to the pyroptosis-inducing moiety TRIP-NH₂ via a cleavable disulfide bond. Under the high-glutathione conditions of the tumor microenvironment, the disulfide linker is cleaved, releasing TRIP-NH₂ inside cancer cells. The freed TRIP triggers endoplasmic reticulum stress and caspase-3/GSDME-mediated pyroptosis, while the ZnPc, upon red-light irradiation, generates ROS that amplify oxidative stress and cell death. In vitro studies demonstrated that Pc-ss-TRIP selectively induces pyroptosis and ICD markers (e.g., calreticulin exposure, HMGB1 release, ATP and interleukin-1β secretion) in cancer cells (A549 and 4T1) while sparing normal cells (HEK293T) due to their lower GSH levels. Notably, light activation further enhanced these effects, confirming a synergistic combination of PDT and pyroptosis induction. Furthermore, in vivo evaluations in a murine 4T1 breast cancer model revealed that Pc-ss-TRIP combined with light irradiation significantly inhibited tumor growth, promoted dendritic cell maturation, and facilitated the profound infiltration of CD8+ T lymphocytes, thereby eliciting a robust systemic antitumor immune response. Collectively, Pc-ss-TRIP exploits the tumor reductive environment to achieve high selectivity in triggering immunogenic cell death, underscoring a promising approach to potentiate anti-tumor immune responses.

Chapter 4 presents an engineered dual-function photosensitizer, Pc-SSZ, which combines a ZnPc core with the anti-inflammatory drug sulfasalazine (SSZ) to concurrently generate ROS and inhibit tumor survival pathways, introducing a new paradigm for ferroptosis-augmented PDT. Two triethylene glycol chains were installed to improve Pc-SSZ’s water dispersibility and cellular uptake. Mechanistically, under red-light irradiation, Pc-SSZ produces ROS as efficiently as free ZnPc; however, unlike conventional ZnPc, it prevents ROS-induced activation of the nuclear factor-κB (NF-κB) pathway by blocking IκB kinase, thereby suppressing downstream pro-survival and antioxidant targets including cyclooxygenase-2, c-Myc, and the Nrf2–GPX4 (NF-E2-related factor 2–glutathione peroxidase 4) axis. The failure to upregulate these defenses leads to unchecked lipid peroxide accumulation and triggers robust ferroptotic cell death. In A549 cells, Pc-SSZ showed potent phototoxicity (IC₅₀ ≈ 0.94 μM) that was significantly attenuated (IC₅₀: 3.92 μM) by the ferroptosis inhibitor ferrostatin-1, confirming ferroptosis as the predominant mode of cell death. By delivering a cytotoxic ROS burst while simultaneously disabling cellular antioxidant defenses, Pc-SSZ circumvents adaptive resistance mechanisms and achieves synergistic cytotoxicity, offering a promising strategy to overcome PDT resistance and enhance anticancer efficacy.

Finally, Chapter 5 explores a novel tumor-targeting photodegradation-targeting chimera (tt-PDTAC) strategy through the design of Pc-ML162-cEBP, which integrates the specificity of targeted protein degradation with the spatiotemporal control of phototherapy. Pc-ML162-cEBP was constructed by conjugating a ZnPc photosensitizer with an alkyne-derivatized GPX4-binding ligand (ML162-yne) and a cyclic epidermal growth factor receptor (EGFR)-targeting peptide. This multifunctional molecule selectively accumulates in EGFR-overexpressing cancer cells and, upon light activation, generates ROS that degrade glutathione peroxidase 4 (GPX4) – a key enzyme suppressing lipid peroxidation – while concurrently depleting intracellular glutathione. The loss of GPX4 disables the cell’s defense against lipid peroxides, thereby inducing ferroptosis, and the ROS-induced damage also activates apoptotic pathways, together yielding a potent dual cell-death effect. Comprehensive in vitro experiments showed that Pc-ML162-cEBP exhibits efficient cellular uptake and, under photoactivation, causes significant GPX4 degradation and cancer cell death with minimal dark toxicity, demonstrating excellent selectivity for light-triggered action. In vivo studies further demonstrated that Pc-ML162-cEBP selectively accumulated in tumors and, upon light irradiation, profoundly halted tumor growth in a 4T1 murine model with minimal systemic toxicity. By harnessing targeted protein degradation alongside photodynamic ROS cytotoxicity, the tt-PDTAC approach greatly enhances the specificity and potency of PDT, highlighting a promising new avenue for precise and effective cancer therapy.

Date of Award	30 Apr 2026
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Pui Chi LO (Supervisor) & Justin J. WILSON (External Co-Supervisor)