Cancer is a terrifying public health threat with low survival rates and a global prevalence. Therefore, exploring effective diagnostic and therapeutic methods is crucial. Traditional cancer treatments, including surgery, radiotherapy, and chemotherapy, often prove to be limited in efficacy and significant in side effects. Fortunately, fluorescence imaging-guided photodynamic therapy (FLI-guided PDT) offers a non-invasive strategy that minimizes resistance and reduces side effects. Notably, FLI technology has high sensitivity, real-time monitoring, and in situ response capabilities, effectively providing doctors with an additional pair of “eyes” for cancer treatment. On the other hand, photosensitizers (PSs) can convert tissue oxygen into cytotoxic reactive oxygen species (ROS) under light irradiation, thereby inducing cancer cell death. Thus, integrating FLI and PDT into a single system represents a powerful weapon against cancer.
Based on this, The Hong Kong University of Science and TechnologyAcademician Tang Benzhen,Professor Jacky W. Y. Lam, and Sichuan UniversityProfessor Liu Xiaoheng proposed an effective molecular design paradigm that combines anion-π+ interactions with inherent crowded conformations to enhance fluorescence efficiency and ROS generation. Mechanistically, the Hippo signaling pathway is involved in the death of melanoma cells after sensitization, promoting the nuclear localization of its downstream factor Yes-associated protein, thereby regulating the transcription and expression of apoptosis-related genes. The findings of this study will trigger the development of high-performance, multifunctional AIE PSs based on clearly regulated mechanisms for precise cancer treatment. The related work was published in ACS NANO under the title “Integrating Anion−π+ Interaction and Crowded Conformation to Develop Multifunctional NIR AIEgen for Effective Tumor Theranostics via Hippo–YAP Pathway”.
Figure 1. Schematic diagram of the designed AIE PSs, nanofabrication, phototherapy applications, and related molecular biological mechanisms after PDT treatment.
Design, Synthesis, and Characterization
As a proof of concept, DPA or TPA was used as the electron donor, and positively charged heteroaromatic rings were used as the acceptor to construct a charged D−a system (Figure 1a). A one-pot multi-component reaction was used to efficiently synthesize four cationic oxygens. The results show that the combination of anion-π+ interactions and crowded conformations enables PSs to have tighter molecular stacking and stable triplet exciton aggregation, thereby enhancing fluorescence and ROS generation capabilities. By controlling the position and number of DPA units, the optimal AIEgen DBQ-2DPA-4TPA was obtained. Its NPs exhibited the highest near-infrared emission, excellent ROS generation capacity, selective mitochondrial targeting, and prolonged tumor retention time, ultimately promoting the expected anti-tumor effect (Figure 1). At the same time, the potential mechanisms of cell death after PDT treatment were systematically studied. Global gene expression profiling showed that apoptosis is the main type of cell death in melanoma cells, regulated by the Hippo signaling pathway (Figure 1b).
Figure 2. Optical properties of DBQ-2DPA, DBQ-2TPA, DBQ-4TPA, and DBQ-2DPA-4TPA.
Subsequently, their photophysical properties were studied. At the molecular level, DBQ-2DPA, DBQ-2TPA, DBQ-4TPA, and DBQ-2DPA-4TPA exhibited typical charge transfer absorption bands at around 560 nm in tetrahydrofuran (THF) solution, with molar absorptivities (ε) of 2.74 × 104, 0.34 × 104, 0.80 × 104, and 2.51 × 104 M−1 cm−1 (Figure 2a). Then, their photoluminescence (PL) spectra were measured in the solid state (Figure 2b).
Figure 3. Crystal characterization of DBQ-2DPA.
As shown in Figure 3a, there is an anion-π+ interaction with a distance of 2.901 Å between the fluorine atoms (−F) of hexafluorophosphate anion (PF6−) and the positively charged dibenzophenanthridine (DBQ) core of DBQ-2DPA, which can avoid emission quenching caused by π−π stacking in the solid state. More F··π+ interactions between PF6− and the DBQ unit can be determined within a range of 2.963 to 3.367 Å (Figure 3b). In addition, abundant intramolecular and intermolecular hydrogen bonds with F···H interactions at distances of 2.518 ~ 2.865 Å, and C – H··π interactions at distances of 3.222 and 3.530 Å were also found in the lattice of DBQ-2DPA (Figure 3b−d). There is a large dihedral angle of 48.00°~ 71.13° between the phenyl ring or DPA group and the DBQ core, proving the existence of a twisted structure (Figure 3e).
Figure 4. ROS generation of DBQ-2DPA, DBQ-2TPA, DBQ-4TPA, and DBQ-2DPA-4TPA under white light irradiation at different times (12.0 mW cm−2).
Reactive Oxygen Species Production, Assessment, and Theoretical Explanation
As shown in Figure 4a, the change in fluorescence signals of the DCFH group alone was negligible after white light irradiation, while the fluorescence was significantly enhanced in the presence of PSs. Under the same conditions, the total ROS production capacity compared to commercially available Ce6 and RB PSs was Ce6 ≈ DBQ-2TPA < DBQ-4TPA < RB < DBQ-2DPA < DBQ-2DPA-4TPA. Clearly, DBQ-2DPA-4TPA has the highest ROS generation capacity compared to other PSs, with its PL intensity increasing nearly 62 times after 50 seconds of light exposure (Figure 4a).
Figure 5. Theoretical explanation of reactive oxygen species production.
To determine the high ROS generation capacity of AIE PSs, theoretical calculations were performed based on time-dependent density functional theory methods. First, the highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) distributions of these AIE PSs in the first excited state (S1) were optimized (Figure 5a). The results show that the HOMO is almost concentrated in the electron-rich DPA or TPA part, while the LUMO is almost distributed on the DBQ core. These results indicate a significant impact of information and communication technology. Considering the sufficient separation of HOMO and LUMO, we determined the energy band gaps (ΔES1‐T1) between S1 and T1 (first excited state) for DBQ-2DPA, DBQ-2TPA, DBQ-4TPA, and DBQ-2DPA-4TPA to be 0.3689, 0.0509, 0.0501, and 0.2045 eV, respectively (Figure 5b).
Figure 6. Cellular imaging of DBQ-2DPA-4TPA NPs.
Cell Imaging and Phototherapeutic Performance of DBQ-2DPA-4TPA NPs
As shown in Figure 6a, DBQ-2DPA-4TPA NPs can selectively accumulate in cancer cells (A375, HeLa, U87, and HepG2 cells) rather than in normal cells (ECs and SMCs). To further confirm its selectivity, flow cytometry was performed. As expected, the cellular uptake of DBQ-2DPA-4TPA NPs in cancer cells was stronger, with higher fluorescence intensity compared to normal cells. Subsequently, commercially available probes for tracking mitochondria, endoplasmic reticulum, and lysosomes were used to explore the subcellular localization of DBQ-2DPA-4TPA NPs in A375 (melanoma) cells.
Figure 7. Phototherapeutic performance of DBQ-2DPA-4TPA NPs.
Next, based on a melanoma mouse model, the in vivo phototherapeutic performance of DBQ-2DPA-4TPA NPs was evaluated. First, the FLI capability of DBQ-2DPA-4TPA NPs was studied. After tumor injection, the fluorescence signal gradually increased over time and could still be detected after 12 days (Figure 7a). Moreover, except for tumor tissue, no organ distribution of DBQ-2DPA-4TPA NPs was observed (Figure 7b).
[Conclusion]
In this study, the researchers proposed a credible molecular design concept that combines anion-π+ interactions and inherent crowded conformations to suppress non-radiative transitions and demonstrated the enhancement of fluorescence efficiency and ROS generation capacity. By introducing multiple donors through a concise method, it possesses compact molecular packing, dense energy levels, more possible ISC channels, and relatively stable triplet excitons, laying a solid foundation for FLI-guided PDT. Impressively, the optimal AIE PS DBQ-2DPA-4TPA and its NPs exhibit the ability to selectively distinguish cancer cells from normal cells, generate efficient ROS, target mitochondria specifically, and prolong tumor retention time, thereby promoting the expected anti-tumor effect. Systematic mechanistic studies found that the Hippo signaling pathway was shut down after PDT, and the apoptosis pathway was activated, which was related to the elevated expression and nuclear accumulation of YAP. Therefore, nuclear YAP initiated the transcription and high expression of apoptosis-related genes. Thus, constructing reliable and efficient strategies for multifunctional NIR AIE PSs with clearly regulated mechanisms is expected to inspire the development of ideal PSs for precise cancer treatment.
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Original link:
https://pubs.acs.org/doi/10.1021/acsnano.3c05080
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