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DOI: 10.1039/D5CC03925H (Communication) Chem. Commun., 2025, Advance Article

Molecular design of NIR type I phenothiazine-based photosensitizers with aggregation-enhanced ROS generation for effective photodynamic therapy applications†

Ziqi Zoua, Dong Liub, Jiaxing Wana, Kun Zhangac, Meiying Liub, Shaorong Huang*c, Jianwen Tian*a, Xiaoyong Zhang*a and Yen Weid
aDepartment of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China. E-mail: tjwen820@126.com; zhangxiaoyong@ncu.edu.cn
bKey Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
cInstitute of Geriatrics, Jiangxi Provincial People's Hospital &, the First Affiliated Hospital of Nanchang Medical College, Nanchang 330006, Jiangxi, People's Republic of China. E-mail: huangshaorong@ncmc.edu.cn
dDepartment of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing, 100084, P. R. China

Received 14th July 2025 , Accepted 23rd July 2025

First published on 5th August 2025

Abstract

In this work, four phenothiazine (PHE)-based PSs (termed PPI1, PPI2, TPI1, and TPI2) were designed and synthesized by integrating various electron donor groups (pyrene, phenanthrene, terphenyl, and tetraphenylene) with PHE to regulate their donor structure. These PSs exhibit near-infrared fluorescence and aggregation-enhanced reactive oxygen species generation ability through a type I pathway, offering a potential strategy to enhance the therapeutic efficacy of PDT in cancer treatment.

Photodynamic therapy (PDT), as a clinically approved alternative antitumor modality, has demonstrated distinct advantages over conventional cancer treatments including chemotherapy, radiotherapy, and surgical intervention.1 Its superior spatiotemporal precision, minimal invasiveness, and reduced drug resistance profiles make it particularly valuable in oncological applications.2 The therapeutic mechanism of PDT involves the generation of reactive oxygen species (ROS) by photosensitizers (PSs) under light irradiation, thereby inducing oxidative damage to cellular components such as proteins, nucleic acids, and organelles, ultimately leading to tumor cell apoptosis. Based on the photochemical pathways, they are generally divided into type I and type II PSs, which can generate different types of ROS. Compared with type II PSs, type I PSs exhibit superior tolerance to hypoxia through two synergistic mechanisms: (1) reduced oxygen demand in electron-transfer-dominated processes enables ROS generation at pO2 levels ≤2 mmHg;3 (2) metabolic recycling of oxygen via enzymatic cascades occurs where O2˙− participates in the superoxide dismutase (SOD)-catalyzed disproportionation reaction.4 Meanwhile, H2O2 can further generate highly toxic ˙OH via Fenton chemistry,5 thereby establishing a self-sustaining oxygen cycle. However, to the best of our knowledge, current PSs predominantly generate singlet oxygen (1O2) through a type II photochemical pathway. The PDT efficacy of these type II PSs is often restricted by some critical factors, such as strong dependence on oxygen concentrations and the excitation wavelength in the visible region (≤650 nm). Therefore, the development of near-infrared (NIR) light-activated type I PSs is critical for highly efficient PDT.6

The structural design of PSs plays a pivotal role in determining their photophysical properties and efficiency in PDT. Various organic PSs such as porphyrins, phthalocyanines, chlorin e6, phenothiazine (PHE) derivatives, and aggregation-induced emission (AIE) PSs have been reported thus far.7 Among them, PHE derivatives have indeed shown promise as efficient PSs, possibly ascribed to the nitrogen (N) atom in their molecular structure with lone-pair electrons. These lone pairs play a crucial role in the photophysical processes involved in PDT. Therefore, incorporating PHE as a molecular motif holds significant potential advantages for the precise design of high-performance type I PSs for PDT. Our recent report demonstrated that the ROS generation capability of PHE-based PSs can be adjusted by changing their acceptor moieties, while their PDT efficacy is not associated with their ROS generation capabilities.8 The acceptor with four cyano groups exhibited the best PDT efficacy, possibly attributed to the reaction between the thiol groups of GSH and the cyano groups of PSs, thereby disrupting the cellular redox homeostasis. However, the effects of donor structures on the ROS generation capability and PDT efficiencies of PHE-based PSs have not been explored thus far.

In this contribution, four PHE-based donor molecules were meticulously designed and synthesized. These donors were created by conjugating four distinct polycyclic units—pyrene, phenanthrene, terphenyl, and tetraphenylene—which exhibit varying degrees of planarity, with PHE in order to fine-tune the structural characteristics of the donor moieties. Subsequently, the four designed donors were conjugated with the acceptor 1,3-bis(dicyanomethylene)indane, resulting in the formation of four PHE-based photosensitizers (PSs) with D–D–A structures, which are designated as PPI1, PPI2, TPI1, and TPI2. Notably, these PSs exhibit NIR fluorescence and aggregation-enhanced ROS generation capability, highlighting their potential for applications in PDT. To enhance their suitability for biomedical applications, two of these PSs, which demonstrated superior ROS generation capabilities, were encapsulated using the non-ionic, commercially available polymer F127. This encapsulation facilitated the self-assembly of PPI2 nanoparticles (NPs) and TPI1 NPs for intracellular imaging and PDT (Scheme S1, ESI†).

The synthetic routes and characterization results of the chemical structures of PPI1, PPI2, TPI1, and TPI2 and their intermediates are shown in the ESI† (Scheme S2 and Fig. S1–S12). The optical properties of these PSs were investigated using UV-vis absorption and fluorescence spectroscopy. Two regions of absorption peaks were found in the UV-vis spectrum of PPI2, in which the short absorption peak at 360 nm is assigned to the π → π* transition of aromatic structures, while the longer absorption peak at 631 nm originates from the intramolecular charge transfer (ICT) absorption (Fig. 1A). Compared with PPI2, PPI1 and TPI1, TPI2 exhibits NIR absorption in both DMSO and H2O (Fig. S13–S15, ESI†). The fluorescence spectra show that there are two emission peaks in the range of 650–850 nm for the four PSs (Fig. 1B and Fig. S16–S19, ESI†). The strong absorption and fluorescence in the NIR region indicate the successful synthesis of PSs with a D–D–A structure, which is crucial for PDT applications. Density-functional theory and time-dependent density-functional theory calculations were performed to gain insights into the physical and electronic properties of PSs in the ground and excited states. The optimized ground state molecular geometries calculated using Gaussian 16W9 and visualized using GaussView 6.0 are shown in Fig. 1C.


image file: d5cc03925h-f1.tif
Fig. 1 (A) UV-vis spectra of PPI2 in distilled water and DMSO solvent; (B) fluorescence intensity of PPI2 in DMF, EA and THF. (C) Configurations of PPI1, PPI2, TPI1 and TPI2; (D) their electronic distribution in the frontier orbitals, calculated using density-functional theory at the B3LYP level, with the 6-31G(d,p) basis set, as implemented in the Gaussian 16W package.

The frontier molecular orbitals reveal the electronic distribution of the PSs. As shown in Fig. 1D, the highest occupied molecular orbital (HOMO) is mainly located in the PHE and thickened ring primitive part, while the lowest occupied molecular orbital (LUMO) is mainly located in the 1,3-bis(dicyanomethylene)indene and π-bridge part. The energy of the HOMO gradually increases as the electron-donating capacity of the donor increases. The energy gaps between the HOMO and LUMO states (ΔE) are calculated to be 2.17 eV, 2.15 eV, 2.18 eV, and 2.07 eV for PPI1, PPI2, TPI1, and TPI2, respectively, while those between the S1 and T2 states (ΔEST) are 0.15 eV, 0.15 eV, 0.16 eV, and 0.12 eV for PPI1, PPI2, TPI1, and TPI2, respectively (Fig. S20, ESI†). The calculation results suggest that the four PSs exhibit a significant ICT effect, which helps reduce the energy splitting between the singlet and triplet states, thereby promoting ISC and enhancing the triplet state population.

To test the calculation results, dichlorofluorescein (DCFH) was employed to measure the total ROS levels generated by the four PSs. As shown in Fig. 2A and Fig. S21 (ESI†), the magnitude of the change in fluorescence intensity after the addition of PSs is very small, indicating that these four PSs hardly produce ROS in the pure organic solvent DMSO (in the single-molecule state). However, in water, all PSs generated ROS in the aggregated state (Fig. 2B and Fig. S22, S23, ESI†). Compared with the control commercially available PS Ce6, the fluorescence intensity of PPI2, TPI1, and TPI2 increased more significantly, indicating that all of them were capable of generating ROS. Therefore, we conclude that the four PSs have an aggregation-enhanced ROS effect. Interestingly, when the ABDA probe was used to monitor the production of 1O2, there was almost no decrease in the absorption intensity after the addition of PSs and white light irradiation (Fig. 2C and Fig. S24, ESI†). However, a notable increase in the fluorescence intensity of DHR123 upon irradiation of these PSs indicates their efficient O2˙− production (Fig. 2D and Fig. S25, ESI†). The above results suggest that all PSs can effectively generate type I ROS in the aggregate state. This characteristic is critical for achieving optimal therapeutic outcomes in PDT.


image file: d5cc03925h-f2.tif
Fig. 2 ROS produced by the four PSs (500 μg mL−1) under white light irradiation at 541 nm and 525 nm versus irradiation time using H2DCF-DA (1 μM) as an indicator: (A) in DMSO and (B) in H2O; (C) absorption intensity of ABDA (4 mM) solution containing PSs (1 mg ml−1) at 401 nm vs. irradiation time; (D) fluorescence intensity of DHR123 (1 μM) solution containing the four PSs (200 μg mL−1) at 526 nm versus irradiation time; white light irradiance: 50 mW cm−2.

F127, a commercially available non-ionic polymeric surfactant, was used to prepare PS NPs. When coated with surfactants, PPI2 and TPI1 will self-assemble into NPs in aqueous solutions and exhibit excellent water solubility. Therefore, the average particle size and physical morphology of PPI2 and TPI1 NPs in aqueous dispersions were evaluated using dynamic light scattering (DLS) and scanning electron microscopy (SEM). The DLS results showed that the average particle size of PPI2 NPs and TPI1 NPs was 204 nm and 336 nm with PDIs of 0.385 and 0.497, respectively (Fig. 3A and B). In agreement with the DLS results, SEM images show that the NPs were all spherical in shape and the particle size was also around 200 nm (Fig. S26, ESI†). In addition, surface potential measurements showed that the zeta potential of PPI2 NPs was −29.43 ± 0.43 mV, while that of TPI1 NPs was −12.2 ± 0.29 mV (Fig. S27, ESI†). Fig. 3C and D show that PPI2 NPs and TPI1 NPs exhibit obvious NIR fluorescence and UV absorption in aqueous solution. All the above results indicate that PPI2 NPs and TPI1 NPs have a small particle size, good aqueous dispersion, and NIR absorbance, which are favourable for cellular uptake into the cells for NIR light irradiation PDT. The ROS-producing ability of these two NPs was further evaluated and the results are displayed in Fig. 3E and Fig. S29 (ESI†). All these four NPs still have the ability to produce ROS with high efficiency, following the order: PPI2 NPs > TPI1 NPs > PPI1 NPs > TPI2 NPs. The O2˙− generation by these four NPs was detected using DHR123 and all four NPs were able to generate O2˙− efficiently (Fig. 3F and Fig. S30, ESI†). In order to visualise ROS generation at the cellular level, we used DCFH and dihydroethidium (DHE) to detect the total ROS and O2˙−, respectively. As evidenced by inverted fluorescence microscopy, bright green and red fluorescence signals were observed after MDA-MB-231 cells were co-incubated with PPI2 NPs and TPI1 NPs with light irradiation of these PSs, demonstrating that the two PSs can significantly elevate intracellular ROS levels and are promising for PDT applications through a type I pathway (Fig. S31 and S32, ESI†).


image file: d5cc03925h-f3.tif
Fig. 3 Hydrodynamic size distribution of (A) PPI2 NPs, (B) TPI1 NPs in THF/H2O (v/v = 1/9); (C) excitation and emission spectra of PPI2 NPs in distilled water; (D) UV-vis spectra of PPI2 and TPI1 NPs in distilled water; (E) ROS generation by PS NPs (100 μg mL−1) versus the irradiation time upon white light irradiation using H2DCF-DA (1 μM) as an indicator; (F) fluorescence intensity of DHR123 (1 μM) solution containing PS NPs (100 μg mL−1) versus the irradiation time. White light irradiance: 50 mW cm−2.

Based on the good ROS-generating ability of PPI2 NPs and TPI1 NPs, the intracellular dark toxicity and phototoxicity of PPI2 NPs and TPI1 NPs were evaluated using MDA-MB-231 cancer cells. As shown in Fig. 4A and Fig. S33 (ESI†), the cell viability values are still more than 90% under dark conditions, reflecting negligible dark toxicity. The cell viability gradually decreased with the increase of PS concentration and light density. Under optimal conditions (80 μg mL−1 and 5 mW cm−2), almost all the cells were killed. In vitro studies revealed that PPI2 NPs and TPI1 NPs emitted distinct red fluorescence in solution. Their smaller particle sizes enabled cellular uptake, making them suitable for cellular imaging. To assess their imaging potential, MDA-MB-231 cancer cells were incubated with 30 μg mL−1 PPI2 NPs and TPI1 NPs for 4 h, then analysed via CLSM. As shown in Fig. 4B and Fig. S34 (ESI†), the cells displayed bright red fluorescence (cytoplasm) and blue fluorescence (nucleus), confirming effective cellular uptake and imaging capability. To further confirm the PDT efficiency, cells were stained with Calcein-AM and propidium iodide (PI) to indicate live and dead cells, respectively. As shown in Fig. 4C and Fig. S35 (ESI†), only green fluorescence signals were detected after the cells were incubated with PBS or PSs under dark conditions, further verifying the good biocompatibility of the PSs. However, no green fluorescence signals were observed, while the corresponding red fluorescence signals emerged when the cells were exposed to PSs after 3 min of light exposure. The above results further demonstrated the desirable PDT efficacy of PPI2 NPs and TPI1 NPs.


image file: d5cc03925h-f4.tif
Fig. 4 (A) Light/dark cell survival at different concentrations of PPI2 NPs; (B) CLSM observations of MDA-MB-231 cancer cells incubated with 80 μg mL−1 PPI2 NPs: (a) bright field, (b) DAPI staining of nuclei, (c) fluorescence field, and (d) superimposed image (scale bar = 50 μm); (C) after co-incubation of PPI2 NPs and Calcein-AM/PI in MDA-MB-231 cancer cells, co-staining inverted fluorescence imaging with different amounts of light (left: PBS; middle: unilluminated; right: light exposure for 3 min). 5 mW cm−2, energy dose: 0.9 J cm−2: measured concentration: 80 μg mL−1; scale bar = 100 μm.

In this study, we have successfully developed a series of novel NIR type I PSs using a rational D–D–A molecular design strategy. These PSs represent a significant advancement in the field of PDT, as they exhibit several desirable properties that address key challenges in current PDT applications. All synthesized PSs demonstrated strong NIR fluorescence and exceptional ROS generation efficiency in aqueous solutions. These characteristics are primarily attributed to their robust ICT and efficient ISC effects. The ICT process facilitates the transfer of an electron within the molecule, enhancing the formation of the triplet state, which is crucial for ROS generation. The efficient ISC ensures that a significant portion of the S1 population is converted to T1, thereby maximizing ROS production. To further explore the potential of these PSs for PDT, we encapsulated PPI2 and TPI1 with amphiphilic polymers, formulating them into water-dispersible NPs. This approach not only improved the solubility and stability of the PSs in aqueous environments but also facilitated their cellular uptake and distribution. Cell viability assays and live/dead cell staining experiments demonstrated that PPI2 and TPI1 NPs were highly effective for PDT under light irradiation. This study provides valuable insights into the design of NIR type I PSs with anoxia-resistant properties, which could help overcome the limitations of conventional hypoxic PDT. Future work will focus on further optimizing the molecular structures of these PSs to enhance their photophysical properties and ROS generation efficiency. Additionally, in vivo studies will be conducted to evaluate the therapeutic efficacy and biocompatibility of these PSs in animal models. The development of targeted delivery systems and the exploration of combination therapies with other cancer treatments are also planned to maximize the therapeutic potential of these PSs. In summary, the present study not only introduces a new class of NIR type I PSs with promising properties for PDT but also lays the foundation for the development of next-generation PDT agents with improved performance and broader clinical applicability.

This research was supported by the National Natural Science Foundation of China (No. 21967016, 22261035, and 22265019).

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the ESI.†

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: The 1H NMR, 13C NMR, and ROS generation results of samples. See DOI: https://doi.org/10.1039/d5cc03925h
This journal is © The Royal Society of Chemistry 2025
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