PROteolysis-TArgeting Chimeras (PROTACs) are heterobifunctional molecules that induce the degradation of target proteins. They consist of a ligand for the protein of interest (POI), a ligand for an E3 ubiquitin ligase, and a linker connecting them. PROTACs form a ternary complex, resulting in ubiquitination and proteasomal degradation of the POI. This catalytic mechanism allows for efficient targeting at minimal doses and potential circumvention of drug resistance. However, limitations such as inefficient tumor accumulation due to poor pharmacokinetics and systemic side effects due to off-tumor degradation hinder clinical translation. Strategies to improve tumor specificity include ligand modifications (antibody-PROTAC, folate-PROTAC, aptamer-PROTAC), stimuli-responsive methods (photoliable, radiation-triggered, hypoxia-responsive, enzyme-activatable PROTACs), and nanosystem-based delivery. Tumor heterogeneity, particularly the presence of cancer stem-like cells (CSCs) resistant to conventional therapies, poses a further challenge. CSCs reside in hypoxic regions with low ROS levels, contributing to their self-renewal and resistance. Hypoxia-activatable therapeutics target CSCs, but solid tumors also possess normoxic zones. This research aims to address these challenges by developing a region-confined PROTAC nanoplatform that targets both normoxic and hypoxic tumor regions, ensuring holistic tumor cell elimination.

The literature review extensively covers the challenges associated with PROTAC delivery and efficacy, highlighting the limitations of existing strategies to overcome poor pharmacokinetics, off-target effects, and the inherent heterogeneity of solid tumors. Studies on ligand modification strategies for targeted delivery, stimuli-responsive PROTACs for conditional activation, and nanosystem-based delivery approaches for improved tumor accumulation and controlled release are reviewed. The role of cancer stem-like cells (CSCs) in tumorigenesis, their resistance to conventional therapies, and their enrichment in hypoxic regions is discussed, highlighting the need for strategies targeting both normoxic and hypoxic tumor cell populations. The existing literature on hypoxia-activatable therapeutics and their limitations in addressing the overall tumor heterogeneity is critically analyzed. This forms the basis for the rationale behind the design of the novel region-confined PROTAC nanoplatform.

The study involved the synthesis of ROS-responsive and hypoxia-responsive PROTAC prodrugs. The ROS-responsive prodrug, ARV771-TK, was created by incorporating a thioketal (TK) group into the BRD4 PROTAC (ARV771). ARV771-TK was copolymerized with an acid-responsive monomer (DPA) via RAFT polymerization. A separate polymer containing a photosensitizer (PPa) and DPA monomers was also synthesized. A hypoxia-responsive prodrug, ARV771-Nb, was created by modifying ARV771 with a nitrobenzyl group. Both prodrugs were incorporated into self-assembling PROTAC nanoparticles (PGDAT and PGDAT@N). The nanoparticles' characteristics were analyzed using DLS, TEM, HPLC, and other techniques. In vitro studies assessed BRD4 degradation, ROS generation, cellular uptake, and cytotoxicity. MMP-2-mediated PEG shedding was evaluated to enhance tumor targeting. In vivo studies using MDA-MB-231 breast cancer and HN30 head and neck squamous cell carcinoma xenograft models evaluated tumor accumulation, antitumor efficacy, and the effect on CSCs. Techniques employed included photoacoustic imaging, fluorescence imaging, immunohistochemistry, western blotting, flow cytometry, RNA sequencing, and qPCR. Statistical analyses were performed to compare treatment groups.

The synthesized ROS-activatable PROTAC prodrug ARV771-TK showed limited POI degradation capacity alone but was effectively released upon ROS generation via PDT. The acid-activatable and ROS-responsive PROTAC nanoparticles (PGDAT) were successfully synthesized and characterized, demonstrating acid-induced disaggregation and ROS generation upon laser irradiation. MMP-2-mediated PEG unshielding significantly enhanced cellular uptake. In vitro studies showed that PGDAT nanoparticles, upon laser irradiation, efficiently degraded BRD4 via the ubiquitin-proteasome pathway, demonstrating synergistic cytotoxicity of BRD4 degradation and PDT. In vivo, MMP-2-sensitive PGDAT nanoparticles showed enhanced tumor accumulation and penetration compared to MMP-2-insensitive controls. Laser irradiation triggered ROS generation at the tumor site, leading to increased ARV771 release and BRD4 degradation. The PGDAT + laser group exhibited significant tumor growth inhibition and prolonged survival in MDA-MB-231 tumor-bearing mice. However, tumor relapse occurred, attributed to the persistence of CSCs. The hypoxia-responsive PROTAC prodrug ARV771-Nb was effectively activated in hypoxic conditions and degraded BRD4 in CSCs in vitro, inhibiting their self-renewal capacity. The region-confined PROTAC nanoparticles (PGDAT@N) combining both ROS- and hypoxia-responsive prodrugs resulted in superior tumor growth inhibition and prevention of recurrence in both MDA-MB-231 and HN30 tumor models. This was achieved through simultaneous degradation of BRD4 in both normoxic and hypoxic regions, eliminating both non-CSCs and CSCs.

The study successfully demonstrated the concept of a region-confined PROTAC nanoplatform for spatiotemporally tunable protein degradation. The enhanced tumor accumulation and penetration achieved through MMP-2-mediated PEG shedding address a major limitation of PROTAC delivery. The stimuli-responsive design allows for precise control of PROTAC release, minimizing off-target effects. The inclusion of both ROS- and hypoxia-responsive components effectively targets both normoxic and hypoxic tumor regions, mitigating the issue of tumor heterogeneity and CSC resistance. The superior antitumor efficacy and prevention of recurrence observed in the PGDAT@N + laser group highlights the potential of this approach for enhanced cancer therapy. The findings support the development of more sophisticated PROTAC delivery systems for improved cancer treatment.

This study presents a novel region-confined PROTAC nanoplatform that effectively targets and degrades BRD4 in both normoxic and hypoxic tumor regions, leading to significant tumor regression and prevention of recurrence. The platform's unique design addresses key limitations of PROTAC therapy, including poor tumor specificity, off-target effects, and the challenge of tumor heterogeneity. The results suggest that this approach holds significant promise for improving cancer treatment outcomes. Future research could explore additional endogenous stimuli for controlled release, optimize nanoparticle design for improved biodistribution and penetration, and evaluate the platform's efficacy in various cancer types and preclinical models.

While the study demonstrated significant antitumor efficacy, limitations exist. The study primarily focused on two specific cancer models. Further research is needed to determine the generalizability of these findings across a broader range of cancer types. The long-term effects and potential toxicity of the nanoplatform require further investigation. The exact mechanisms underlying the synergistic effects between BRD4 degradation and PDT warrant additional study. Finally, clinical translation requires additional preclinical work and thorough safety assessment.