Phthalocyanine Aggregates as “Semiconductor-like” Photocatalysts for Hypoxic-Tumor Photodynamic Immunotherapy

Phthalocyanine Aggregates as “Semiconductor-like” Photocatalysts for Hypoxic-Tumor Photodynamic Immunotherapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Phthalocyanine Aggregates as “Semiconductor-like” Photocatalysts for Hypoxic-Tumor Photodynamic Immunotherapy Xingshu Li, Hao Liu, Ziqing Li, Xiaojun Zhang, Yihui Xu, Guoyan Tang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3933352/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Photodynamic immunotherapy (PIT) has emerged as a promising approach for efficient eradication of primary tumors and inhibition of tumor metastasis. However, most of photosensitizers (PSs) for PIT exhibit notable oxygen dependence. Herein, a concept emphasizing on transition from molecular PSs into “semiconductor-like” photocatalysts is proposed, which converts the PSs from type II photoreaction to efficient type I photoreaction. Detailed mechanism studies reveal that the nanostructured phthalocyanine aggregate (NanoNMe) generates radical ion pairs through a photoinduced symmetry breaking charge separation process, achieving charge separation through a “self-substrate” approach and leading to exceptional photocatalytic charge transfer activity. Additionally, a reformed phthalocyanine aggregate (NanoNMO) is fabricated to improve the stability in physiological environments. NanoNMO showcases outstanding photocytotoxicities under both normoxic and hypoxic conditions and exhibits remarkable tumor targeting ability. Notably, the photodynamic effect mediated by NanoNMO not only triggers the systemic anti-tumor immune response but also synergizes with PD-1 antibodies to enhance the infiltration of cytotoxic T lymphocytes into tumor sites, leading to the effective inhibition of tumor growth. Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Physical sciences/Chemistry/Photochemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cancer is a devastating disease, with one of the most fatal aspects lying in the metastasis of cancer cells 1–5 . In recent years, photodynamic immunotherapy (PIT), which combines photodynamic therapy (PDT) with tumor immunotherapy, especially immune checkpoint blocking therapy, has emerged as a prominent hotspot in the field of tumor therapy 6–10 . However, the current research is still in its early stages. Most of photosensitizers (PSs) used for PIT mainly inhibit tumor through the oxygen-dependent type II photoreaction 3, 11–13 . The hypoxic tumor microenvironment (TME) limits the therapeutic effect of this type of PDT, which limits the synergistic effect of PIT accordingly 6, 14 . Moreover, the hypoxic TME promotes cancer cell metastasis and tumor immunosuppression 15, 16 , while the consumption of oxygen during type II PDT further exacerbates tumor hypoxia, thereby impeding the effectiveness of PIT against tumor metastasis 17, 18 . Consequently, a crucial aspect for advancing the clinical application of PIT lies in the development of PSs that exhibit less oxygen dependency. Type I PSs have been proved to possess the advantage of less oxygen dependency 19–21 . However, most of the existing type I PSs primarily rely on various substrates as electron transfer agents to enhance electron transfer efficiency, which impose stringent requirements on the type and concentration of substrates, as well as their redox potential and binding affinity with PSs 22–25 . Therefore, the research on type I PSs remains relatively limited. Our prior research has discovered that a phenoxy-linked polyamine mono-substituted zinc (II) phthalocyanine (Pc) can self-assemble into nanodots in water and generate reactive oxygen species (ROS) through type I photoreaction 26 . Nevertheless, it is urgent to delve deeply into the quantitative structure activity relationship (QSAR) between the structural characteristics of zinc (II) Pc and its type I photoreaction. Additionally, the internal mechanisms underlying the efficient type I photoreaction of the nanodots need to be elucidated as well. To tackle these challenges, a series of tetrasubstituted zinc (II) Pcs incorporating various electron donor/acceptor groups were designed and synthesized herein 27, 28 . Furthermore, the Pc aggregates and the Pc monomers were fabricated in aqueous solution 29, 30 to investigate the QSAR between the electronic characteristics of the substituents and the ROS generations of the Pc aggregates and Pc monomers. The obtained results revealed that the Pc aggregates substituted with electron donor group (such as NanoNMe) exhibited a significant type I photoactivity. The superoxide anion (O 2 •− ) production of NanoNMe was found to be 15-fold higher than that of methylene blue (MB). Detailed mechanism (Scheme 1a ) studies revealed that NanoNMe underwent a photoinduced symmetry breaking charge separation (SBCS) process, achieving charge separation and formating Pc •+ and Pc •− ion pairs through a “self-substrate” approach. The products of SBCS exhibited similarities to electron-hole pairs 31, 32 , enabling to consider NanoNMe as a semiconductor to analyze its charge transfer properties. Band analysis revealed that NanoNMe possessed the capability to reduce O 2 to O 2 •− and exhibited remarkable photocatalytic charge transfer activity, making NanoNMe a “semiconductor-like” photocatalyst to facilitate efficient charge transfer. Additionally, a reformed Pc aggregate (NanoNMO) was fabricated to improve the stability and the type I photoactivity in physiological environments by incorporating octaethylene glycol and electron donor amino groups into the Pc structure to improve its amphipathy. The results of in vitro experiments demonstrated an outstanding photocytotoxicity of NanoNMO under both normoxic and hypoxic conditions. NanoNMO also exhibited excellent tumor targeting ability and significant phototherapeutic efficacy in 4T1 tumor-bearing mice. Notably, the PDT effect mediated by NanoNMO not only triggered the systemic immune response but also synergized with PD-1 antibody to inhibit the growth of primary and distant tumor (Scheme 1b ). This work introduces a groundbreaking concept transforming molecular PSs into “semiconductor-like” photocatalysts, providing a novel perspective to design less oxygen dependent PSs and presenting a practical example converting molecular PSs to “semiconductor-like” photocatalysts. Additionally, this work pioneers the investigation of combining high-efficiency PSs with immune checkpoint inhibitors (ICIs) under hypoxic conditions, which serves as a reference for the development of PSs combining immunotherapy to suppress the growth and metastasis of hypoxic tumors. Results Preparation of Pc aggregates and Pc monomers and their ROS generations Zinc (II) phthalocyanine is a functional dye with notable advantages, including strong absorption within the phototherapeutic window (650–850 nm), high ROS yield, and tunable photochemical properties 33, 34 . These attributes render it a promising PS for PDT. However, most of the reported zinc (II) Pcs are oxygen dependent type II PSs 35–37 . Besides, the hydrophobic plane makes Pcs prone to aggregate, leading to the quenching of photoactivity 38–40 . Our prior work has demonstrated a zinc (II) Pc nanodot capable of generating ROS through type I mechanism 26 . To investigate the QSAR between the structural characteristics of zinc (II) Pc and its type I photoreaction, ten tetrasubstituted zinc (II) Pcs (Fig. 1 a) were meticulously designed and synthesized herein. The structural modification involved replacing the para position of the phenoxy group connected to the Pc with various electron donor/acceptor substituents (Fig. 1 b). Specifically, the Pcs were named by the abbreviation of their substituents successively as NMe, NH, OMe, Bu, H, Cl, OCF, COP, CN and NO. The detailed synthesis and characterization of these Pcs are provided in the supporting information (Fig. S1 -S20). Pc aggregates (NanoPcs) with a size range of 200 ± 50 nm and Pc monomers (MonoPcs) were fabricated in H 2 O through different methods (Fig. 1 c). Specifically, NanoPcs were obtained by diluting a tetrahydrofuran (THF) solution of the Pc into H 2 O, with or without the application of ultrasound. For NMe, NH, and OMe, NanoPcs were prepared by diluting the THF solution of the Pc into H 2 O under ultrasound conditions. For other structures, NanoPcs were prepared by diluting the THF solution of the Pc into H 2 O directly. The detailed preparation steps are described in the Methods section. The dynamic light scattering (DLS) results (Fig. S21) revealed that all of NanoPcs exhibited uniform dispersion in H 2 O, with particle sizes ranging of 200 ± 50 nm. Transmission electron microscope (TEM) images of NanoPcs (Fig. S22) demonstrated that their morphology resembled of regular spherical shapes, with a diameter of approximate 200 nm, which was consistent with the DLS results. The steady-state absorption spectra (Fig. S23) revealed that the absorption spectra of NanoPcs exhibited broadening and decreased intensity compared to the absorption features of the Pc monomers in N, N-dimethylformamide (DMF). Additionally, the steady-state fluorescence emission of NanoPcs (Fig. S24) attenuated significantly compared to that of Pc monomers in DMF. These findings indicated the significantly changed light absorption and fluorescence emission properties of NanoPcs compared to Pc monomers. Besides, MonoPcs were obtained by mixing a THF solution of Pc with surfactant Cremophor EL (CEL) in advance, followed by H 2 O dilution. DLS results (Fig. S25a) and TEM images (Fig. S25b) demonstrated that all of MonoPcs possessed particle sizes near 10 nm, with no observable nanostructure, indicating the uniform disperation of MonoPcs in H 2 O. Moreover, the steady-state absorption and fluorescence emission revealed that the absorption and fluorescence emission of MonoPcs exhibited similar features with those of Pc monomers in DMF, suggesting that the CEL-regulated MonoPcs could exist as monomers in H 2 O. The ROS efficiencies for both NanoPcs and MonoPcs were initially assessed to conduct a QSAR analysis. The ROS generations were evaluated using the dihydrodichlorofluorescein (DCFH) probe (Fig. S26, S27), whose fluorescence at 525 nm can be produced by various ROS including O 2 •− , hydroxyl radical ( • OH) and singlet oxygen ( 1 O 2 ). To enable a quantative comparison, the relative ROS generations of NanoPcs and MonoPcs were determined comparing them with the reference samples (Fig. 1 d). For NanoPcs, an equal concentration of MB was used as the reference sample, while for MonoPcs, a mixture of CEL and MB was used as the reference sample. The observations indicate that as the value of σρ decreases, both NanoPcs and MonoPcs exhibit higher ROS generation. Moreover, when the σρ value is more than 0, NanoPcs demonstrate lower ROS generation than MonoPcs, and when the σρ value is less than 0, NanoPcs demonstrate higher ROS generation than MonoPcs, surpassing that of MB significantly. To assess the specific contributions, the O 2 •− generation was evaluated using dihydrohexidine (DHE) probe (Fig. S28, S29), the • OH generation was evaluated using aminophenyl fluorescein (APF) probe (Fig. S30, S31), and the 1 O 2 generation was evaluated using singlet oxygen sensing green (SOSG) probe (Fig. S32, S33). The O 2 •− generation results were presented in Fig. 1 e. The observations indicate that as the value of σρ decreases, both NanoPcs and MonoPcs display higher O 2 •− generation. Moreover, when the σρ value is more than 0, NanoPcs demonstrate lower O 2 •− generation than MonoPcs, and when the σρ value is less than 0, NanoPcs demonstrate significantly higher O 2 •− generation than MonoPcs. The O 2 •− generation of NanoPcs can reach up to 15-fold higher than that of MB at most. The • OH generation results were depicted in Fig. 1 f. The observations indicate that as the value of σρ increases, both NanoPcs and MonoPcs demonstrate decreased • OH generations. Notably, NanoPcs demonstrate lower • OH generation compared to MonoPcs. Moreover, the • OH generations of both NanoPcs and MonoPcs are relatively inefficient, which are either comparable or lower than that of MB. The 1 O 2 generation results were depicted in Fig. 1 g. The observations indicate that as the value of σρ increases, MonoPcs demonstrate a mild decrease in 1 O 2 generation. Notably, NanoPcs demonstrate lower 1 O 2 generation than MonoPcs. Moreover, both NanoPcs and MonoPcs display low level of 1 O 2 generations, which are consistently lower than that of MB. To sum up, all the QSAR results demonstrate that both NanoPcs and MonoPcs exhibit higher ROS generation when the σρ value is less than 0. Notably, the generations of both ROS and O 2 •− by NanoPcs (σρ < 0) demonstrate remarkably high level, making them excellent ROS and O 2 •− generators. Investigation on intersystem crossing process of MonoPcs Generally, the generation of ROS is closely related to the intersystem crossing (ISC) process of PSs 21, 41 . In order to gain insights into the ROS generation mechanism of MonoPcs, the ISC process was analyzed. Initially, time-dependent density functional theory (TD-DFT) calculations were conducted to analyze the energy levels of excited state involved in the ISC process. The results revealed that as the σρ value increased, the energy gap (∆E ST ) between singlet state (S1) and triplet state (T1) of Pcs gradually widened (Fig. 2 a), indicating an increased energy interval of the ISC process. The increased ∆E ST are unfavorable for the ROS generations, which explain the observed trend where, as the σρ values increased, the ROS generations of MonoPcs decreased. Furthermore, the excited state dynamics of MonoPcs were investigated using femtosecond transient absorption (fs-TA) spectroscopy 41 . To conduct specific analysis, NMe and CN were chosen as representative examples. As depicted in Fig. 2 b and 2 c, the contour maps illustrated two-dimensional color-coded fs-TA spectra of NMe and CN upon excitation at 694 and 685 nm, respectively, providing insights into the evolution of excited states. The negative absorption was attributed to the ground state bleaching (outlined with white dotted lines). The positive absorption was attributed to the excited state absorption (ESA, outlined with blue and red dotted lines), where the blue dotted lines indicated the ESA of singlet state, and the red dotted line indicated the ESA of triplet state. To distinguish singlet ESA from triplet ESA, fs-TA plots at different delay times were extracted from the contour map and presented at the bottom of the contour map. For NMe, after a 90 fs timeframe following excitation, a series of ESA grew in intensity with prolonged delay time within the 500–750 nm range. Among these absorptions, the dynamic decay trace at 510 nm exhibited a decay lifetime of 3.18 ns, which was consistent with the fluorescence lifetime (3.98 ns) determined by time correlated single photon counting (Fig. S34a). Based on these evidences, the ESA around 510 nm was assigned to singlet ESA. Over time, the ESA near 560 nm exhibited a continuously growing peak after 500 ps with no attenuation trend observed within the maximum range of 3.08 ns. Furthermore, the fs-TA plots revealed the occurrence of an obvious isosbestic point between the ESA near 560 nm and the ESA near 510 nm, indicating the occurrence of a singlet-to-triplet ISC process. Eventually, the hysteresis and prolonged lifetime of the ESA near 560 nm was attributed to triplet ESA. Likewise, for CN, a series of ESA grew in intensity within the 500–750 nm range as well. The dynamic decay trace at 510 nm aligned with the fluorescence lifetime (Fig. S34b). Moreover, an isosbestic point was observed between the ESA near 560 nm and the ESA near 510 nm, indicating the occurrence of ISC process and triplet state. Afterwards, the parameters related to ISC were calculated based on the singlet and triplet ESA (Fig. 2 d). The occurrence times of ISC (t ISC ) were estimated by determining the populated time of triplet state, which were found to be approximately 500 ps and 566 ps for NMe and CN, respectively. The rates of ISC (k ISC ) were calculated as 0.20 and 0.16 (×10 10 s − 1 , 1/t ISC ) for NMe and CN, respectively. Subsequently, the efficiencies of ISC (Φ ISC ) were estimated according to the previous studies 41, 42 (Φ ISC = [1/t (T1, rise) ]/[1/t (S1, decay) ]) as 16.4% and 8.6% for NMe and CN, respectively. The excited state dynamics results were consistent with the theoretical calculations, indicating that as the σρ value decreased, the Φ ISC of Pcs increased, result in a higher generation of ROS. “Self-substrate” photoinduced electron transfer Unlike MonoPcs, NanoPcs generate ROS through type I photoreaction involved electron transfer. The crucial step in this mechanism is the intermolecular photoinduced electron transfer (PET) of NanoPcs 45, 46 . Accordingly, the mechanism of the ROS generation by NanoPcs is proposed. As shown in Fig. 3 a, upon photon excitation, partial molecules of NanoPcs are excited, resulting in the formation of an energy asymmetric local excited state (Pc* + Pc). The local excited state is unstable and promptly undergoes symmetry breaking charge separation (SBCS) to reduce its asymmetry. As a result, a pair of free radical ions is formed (Pc •+ + Pc •− ). To analyze the intermolecular SBCS, the excited state photophysical processes of NMe and NanoNMe were analyzed using fs-TA. The results revealed a significant difference in the photophysical process between NMe and NanoNMe. As shown in Fig. 3 b, the ESA of NMe in the 450–600 nm range exhibited uniformly decreased vibration peaks within 500 ps, indicating the absence of intermediate species during excited state decay process of NMe. For NanoNMe (Fig. 3 c), the ESA transformed into mixed species features in the 450–600 nm range within 1 ps, suggesting the generation of intermediate species. Specifically, the ESA at 450–550 nm showed uniformly decreased vibration peaks, with an absorption range consistent with the ESA of NMe, indicating the formation of excited states. Furthermore, after approximately 0.58 ps, different species features appeared near 600 nm, indicating the generation of other species. The SBCS has been extensively reported in various organic molecular aggregates, including perylenediimide (PDI) and its derivatives 47, 48 . The occurrences of SBCS (τ SBCS ) for PDI derivatives ranges from sub-ps to hundreds-ps and are closely related to molecular structure, distance, and solvent polarity 49 . Based on the mechanism and the reported results, the ESA near 600 nm was attribute to the absorption signal of the Pc •− species resulting from SBCS. The dynamic decay trace (Fig. 3 d) indicated that Pc •− gradually formed after the decay of the excited state, yet it exhibited a longer lifetime compared to the excited state, demonstrating a distinct kinetic property. Conversely, no observation of any species was detected in the fs-TA analysis of NanoCN (Fig. 3 e). To investigate the feasibility of a photoinduced SBCS, the Gibbs free energy (∆G) was calculated. Typically, the condition for SBCS process to be possible is that ∆G is negative 50 . Therefore, the cyclic voltammetry was employed to determine the redox potentials of all the Pcs (Fig. S35, Table S2). The ∆G of Pcs was calculated based on the Rehm-Weller Eq. 5 1 (Eq. 1 ) (Fig. 3 f). \(\varDelta G=e\left({E}_{ox}-{E}_{red}\right)-{E}^{*}-\frac{{e}^{2}}{2\pi {\epsilon }_{0}{\epsilon }_{s}d}\approx e\left({E}_{ox}-{E}_{red}\right)-{E}^{*}\) (Eq. 1 ) Where e represents the elementary charge; E ox is the potential for one-electron oxidation of Pcs, and E red is the potential for one-electron reduction of Pcs; E* is the excitation energy obtained from TD-DFT. The last term accounts for the coulombic interactions between two ions produced at a distance d and screened by the solvent with a static dielectric constant ε s . The results indicated that for substituents with σρ < 0, the ∆G of Pcs was negative, suggesting that Pcs with σp < 0 were capable of undergoing photoinduced SBCS. However, as the σp value increased, the ∆G of Pcs increased, indicating a gradual decrease in charge separation ability of Pcs. Consequently, the electron transfer mechanism of NanoPcs can be described as follows: the local excited state of NanoPcs undergoes a SBCS process, resulting in the generation of Pc •+ and Pc •− free radical species, thereby achieving charge separation through a way of “self-substrate”. “Semiconductor-like” photocatalysis for O 2 •− generation The free radical ion pairs of SBCS (Pc •+ and Pc •− ) in NanoPcs can exhibit spatial overlap and extend from localized orbitals to delocalized orbitals 31, 32 , which can be described as free electron-hole pairs within a semiconductor (band theory) framework (Fig. 4 a). Therefore, the redox properties of NanoPcs were investigated through band theory 52 to analyze the generations of O 2 •− and • OH. The reduction of O 2 depends on the conduction band (CB) potential of NanoNMe. Consequently, the flat band potential of NanoNMe was determined using the Mott-Schottky plots (Fig. 4 b). The results revealed that the flat band potential of NanoNMe was − 0.47 V, which was approximately equal to the CB potential and less negative than the reduction potential of O 2 (-0.33 V), indicating that NanoNMe could photoreduce O 2 to generate O 2 •− . Additionally, the oxidation of H 2 O depends on the valence band (VB) potential of NanoNMe. The VB potential of NanoNMe was determined by ultraviolet photoelectron spectroscopy (UPS) valence band spectra (Fig. 4 c). The edge of the valence band maximum energy (E f ) for NanoNMe was found to be 0.18 eV, and the work function (Φ) was calculated to be 5.47 eV. The VB potential of NanoNMe (vs NHE) was concluded to be 1.21 V, which is less than the oxidation potential of H 2 O (1.99 V), indicating that NanoNMe cannot photooxidize H 2 O to generate • OH. Additionally, the optical bandgap energy determined from absorption spectra (Eg = 1.69 eV, Fig. S36) matched closely with the calculated bandgap (1.68 eV, Fig. 4 d), providing convincing evidence of the redox capability of NanoNMe. Furthermore, the charge transfer abilities of NanoNMe were further investigated through transient photocurrent response and charge transfer resistance, with NanoCN used as a comparison. Interestingly, NanoNMe exhibited a significantly enhanced photocurrent under light illumination (Fig. 4 e), while the photocurrent of NanoCN was negligible. Electrochemical impedance spectroscopy (Fig. 4 f) revealed that the charge transfer resistance of NanoNMe was significantly smaller than that of NanoCN. Additionally, the charge flow abilities in the aqueous solution were studied by analyzing the conductivity changes (Fig. 4 g). Over the course of illumination, the conductivity of NanoNMe showed a significant enhancement compared to NMe or NanoCN. These results indicate that NanoNMe exhibits a “semiconductor-like” photocatalytic activity, leading to a significant enhancement of charge transfer under illumination. Based on these findings, the present study proposes a novel design strategy for Type I PSs by converting molecular PSs to “semiconductor-like” photocatalysts, offering new insights for addressing PDT in hypoxic tumors. Fabrication of physiologically stable NanoPcs and their in vitro phototherapeutic efficacy Motivated by the efficient photocatalytic generation of O 2 •− by NanoNMe, we further investigated the potential of NanoNMe for biomedical applications. The stability is a crucial requirement for biomedical application of NanoPcs. Therefore, we initially examined the stability of NanoNMe in physiological conditions. In a phosphate-buffered saline (PBS) solution containing fetal bovine serum (FBS), the particle size of NanoNMe gradually increased over time (Fig. S37a), suggesting an unstable nanostructure of NanoNMe. Additionally, the absorption spectra of NanoNMe (Fig. S37b, S37c) demonstrated a gradual decreased intensity in PBS containing FBS over time, indicating the decreased light-harvesting properties. Conversely, an increment in fluorescence intensity was observed for NanoNMe as the concentration of FBS increased (Fig. S37d, S37e), indicating the gradually restored fluorescence emission of NanoNMe under the influence of FBS. The significant changes in nanostructure and optical activity collectively demonstrated the poor stability of NanoNMe in a physiological environment. To facilitate the stable dispersion of NanoPc in physiological environments, a novel amphiphilic Pc (NMO) was designed by replacing one substituent of NMe with octaethylene glycol. The detailed synthesis and characterization of NMO are presented in the Supporting Information (Fig. S38, S39). The nanostructured aggregate of NMO (NanoNMO) was prepared through a nano precipitation method (Fig. 5 a). The detailed fabrication steps of NanoNMO are provided in the Methods section. DLS and TEM results exhibited that the average size of NanoNMO was approximately 110 nm (Fig. 5 b), exhibiting a morphology resembled of regular spherical shapes. As expected, NanoNMO exhibited improved stability in physiological environments compared to NanoNMe. During the initial 12 h period upon the addition of FBS, the size distribution of NanoNMO remained relatively unchanged, along with consistent absorption and fluorescence emission (Fig. S37f-S37j). Afterwards, the size of NanoNMO started to increase, accompanied by an intensified fluorescence emission, indicating the instability of NanoNMO after 24 h. Additionally, the ROS generations of NanoNMO were further evaluated with NMO, Chlorin e6 (Ce6), and MB serving as control (Fig. S40). The results demonstrated that NanoNMO exhibited robust ROS generations, higher than that of NMO and Ce6, and approximately 1.8-fold higher than that of MB (Fig. 5 c). Specifically, the O 2 •− generation of NanoNMO was approximately 7.9-fold higher than that of MB (Fig. 5 d), while no significant generation of • OH and 1 O 2 was observed (Fig. S41). These results indicate that NanoNMO is an efficient O 2 •− generator. Further investigation was conducted to assess the in vitro PDT activity of NanoNMO. Initially, the intracellular ROS generations in mouse breast cancer cells (4T1) were evaluated using confocal laser scanning microscopy (CLSM) under both normoxic and hypoxic conditions. As shown in Fig. S42, the fluorescence intensities of the oxygen stress indicator (ROS-ID) under both normoxic and hypoxic conditions were significantly higher than the negative control and comparable to the positive control, indicating the successful establishment of normoxic and hypoxic conditions. Subsequently, the intracellular ROS generation ability was assessed using DCFH as a fluorescent indicator. As depicted in Fig. 5 e, negligible fluorescence was observed in cells without laser irradiation. However, after 685 nm laser irradiation, significant fluorescence was observed in the Ce6, NMO, and NanoNMO treated cells under normoxic conditions. Notably, NanoNMO group exhibited the highest fluorescence emission (Fig. 5 f), indicating its superior ROS production ability. However, under hypoxic conditions, only NanoNMO group displayed bright fluorescence signals comparable to those observed under normoxic conditions, suggesting that NanoNMO could effectively generate ROS in hypoxic conditions as well. Additionally, the intracellular O 2 •− generation capacity was assessed using DHE as a fluorescent indicator. As shown in Fig. 5 g, minimal fluorescence was observed in cells without laser irradiation. After 685 nm laser irradiation, neither Ce6 nor NMO displayed significant fluorescence signals under normoxic or hypoxic conditions, suggesting limited intracellular O 2 •− generation. In contrast, NanoNMO displayed robust fluorescence signals under both normoxic or hypoxic conditions (Fig. 5 h), indicating efficient O 2 •− generation of NanoNMO under both normoxic or hypoxic conditions. Next, the in vitro phototherapeutic effects of Ce6, NMO, and NanoNMO were evaluated through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Fig. 5 i and 5 j, no obvious toxicity was observed for Ce6, NMO, and NanoNMO under dark conditions. Under normoxic conditions, Ce6, NMO, and NanoNMO exhibited significant concentration-dependent cytotoxicities following 685 nm laser irradiation (30 min, 15 mW·cm -2 ), with 50% inhibitory concentrations (IC 50 ) of 2.35, 0.40, and 0.31 µM, respectively. However, under hypoxic conditions, the photocytotoxicity of Ce6 and NMO decreased significantly, leading to an increase in IC 50 to 5.74 and 2.60 µM, respectively. In contrast, NanoNMO maintained a potent photocytotoxicity, with an IC 50 of 0.41 µM. The live/dead cell staining assay (Fig. 5 k, S43) also demonstrated the effective cell damage induced by NanoNMO under both normoxic and hypoxic conditions. These findings suggest that O 2 concentration barely affect the photocytotoxicity of NanoNMO. In vivo biodistribution and phototherapeutic efficacy of NanoNMO To explore the biodistribution, Balb/c mice bearing a 4T1 tumor were administered with an intravenous injection of NanoNMO (200 µM, 100 µL), employing NMO (200 µM, 100 µL) as a control. Fluorescence and photoacoustic dual-modal imaging was used to evaluate the in vivo biodistribution. As shown in Fig. 6 a, after intravenous injection, a significent fluorescence signal of NMO rapidly disseminated throughout the body and diminished gradually over time, with no significant tumor accumulation observed. Conversely, faint fluorescence signal was only observed at the tumor site within 24 h after injection of NanoNMO. Moreover, photoacoustic imaging (Fig. 6 b) revealed that NanoNMO predominantly exhibited photoacoustic signals at the tumor site after intravenous injection and reached a maximum at 8 h (Fig. 6 c), indicating the superior tumor targeting ability of NanoNMO. Notably, at 24 h post intravenous injection, the fluorescence signal of NanoNMO at the tumor site significantly increased, whereas the photoacoustic signal noticeably diminished. Since the photoacoustic signal of NanoNMO in H 2 O containing 10% FBS decreased significantly compared to that in pure water (Fig. S44), we speculated that the tumor accumulated NanoNMO may gradually disassemble, leading to enhanced fluorescence and reduced photoacoustic signals after 24 h. As a control, mice injected with NMO exhibited low systemic photoacoustic signals, indicating negligible tumor accumulation. To evaluate the distribution of NMO and NanoNMO in different organs, mice were sacrificed 36 h postinjection and the fluorescence intensities of the tumor and major organs including heart, liver, spleen, lung, kidney and skin were assessed. As shown in Fig. 6 d and 6 e, the fluorescence signal of NanoNMO at the tumor site was significantly higher than that of other organs, registering 5.2-fold higher signal than that in the liver. In contrast, the fluorescence signal of NMO at the tumor site was only 0.8-fold than that of the liver, revealing an outstanding tumor targeting capability of NanoNMO. Consequently, NanoNMO can remain stable in vivo for 12 h, thereby facilitating the effective implementation of PDT. Subsequently, the in vivo phototherapeutic efficacy of NanoNMO was further conducted on 4T1 tumor-bearing mice. Once the volume of tumor reached 100 mm 3 , the mice were divided into four groups (five mice per group): ( 1 ) treated with saline (control); ( 2 ) treated with saline followed by laser irradiation (685 nm, 100 mW·cm -2 ) 8 h after injection; ( 3 ) treated with NanoNMO (200 µM, 100 µL); ( 4 ) treated with NanoNMO (200 µM, 100 µL) followed by laser irradiation (685 nm, 100 mW·cm -2 ) 8 h after injection. As depicted in Fig. 6 f, all the control groups (including Saline, Saline + Laser, and NanoNMO only) exhibited significant level of tumor growth. In contrast, the tumor growth in NanoNMO + laser group was inhibited obviously. After 14 days of treatment, the tumor inhibition rate reached 96 ± 4% compared to the control group. Additionally, the body weight of mice in different groups displayed no significant differences and maintained a natural growth during the course of treatments (Fig. 6 g). These results suggest that NanoNMO possesses exceptional in vivo phototherapeutic efficacy and excellent biocompatibility. In vivo PIT efficiency of NanoNMO in combination with PD-1 antibody To investigate the potential of type I PSs mediated PDT to induce an anti-tumor immune response, the level of immune cell cytokines in the plasma was studied on NanoNMO injected 4T1 tumor-bearing mice. The mice were divided into four groups (five mice per group): ( 1 ) treated with saline (control); ( 2 ) treated with saline followed by laser irradiation (685 nm, 100 mW·cm − 2 ) 8 h after injection; ( 3 ) treated with NanoNMO (200 µM, 100 µL); ( 4 ) treated with NanoNMO (200 µM, 100 µL) followed by laser irradiation (685 nm, 100 mW·cm − 2 ) 8 h after injection. Compared to the control group (including Saline, Saline + Laser, and NanoNMO only), the level of TNF-α (Fig. 7 a) and IFN-γ (Fig. 7 b) cytokines of NanoNMO + laser group showed a significant enhancement, suggesting the occurrence of acute inflammation, which was a very crucial mechanism in inducing tumor-specific immunity through PDT. These results suggest that NanoNMO mediated PDT can induce the acute inflammation in the host, leading to a systemic immune response. Further investigation was conducted to explore the synergistic anti-tumor potential of NanoNMO mediated PDT in combination with immune checkpoint inhibitors (ICIs). A 4T1 bilateral tumor model of Balb/c mouse was established by injecting 4T1 cells into the right flank of the mice (primary tumor), followed by injecting 4T1 cells into the left flank of mice (distant tumor) after 6 d (Fig. 7 c). Once the volume of the distant tumor reached 100 mm 3 , the mice were divided into six groups (five mice per group) to monitor tumor growth following different treatments. Group 1: treated with saline (control); Group 2: treated with saline followed by laser irradiation (685 nm, 100 mW·cm − 2 ) 8 h after injection; Group 3: treated with NanoNMO (200 µM, 100 µL); Group 4: treated with NanoNMO (200 µM, 100 µL) followed by laser irradiation (685 nm, 100 mW·cm − 2 ) on the primary tumor 8 h after injection; Group 5: treated with αPD-1 twice (2.5 mg·kg − 1 ) on day 1 and day 3, respectively; Group 6: treated with NanoNMO (200 µM, 100 µL) followed by laser irradiation (685 nm, 100 mW·cm − 2 ) on the primary tumor 8 h after injection, and then treated with αPD-1 twice (2.5 mg·kg − 1 ) on day 1 and day 3, respectively. The distant tumor received no laser irradiation. The tumor volume of both tumors and the weight of mice were monitored throughout the process. As shown in Fig. 7 d, the control groups (including Saline and Saline + Laser) exhibited rapid growth of the primary tumor. In addition, neither NanoNMO group nor αPD-1 group exhibited significant tumor inhibition on the primary tumor. In contrast, both the NanoNMO + Laser group and the NanoNMO + Laser + αPD-1 group demonstrated a significant suppression of primary tumor growth. Notably, the NanoNMO + Laser + αPD-1 group demonstrated the highest treatment efficacy, with a tumor inhibition rate of 82 ± 3% compared to Group 1 after 14 days of treatment, surpassing that of the NanoNMO + Laser group (73 ± 2%). These results indicate an enhanced inhibitory effect on the primary tumor through the synergistic therapy. Additionally, as shown in Fig. 7 e, the control groups of the distant tumor (Saline, Saline + Laser, and NanoNMO) exhibited a rapid increase. Moreover, the NanoNMO + Laser group showed minimal distant tumor inhibition as well, suggesting that the immune response triggered by PDT was inferior and unable to suppress the growth of the distant tumor. The αPD-1 group showed a slight distant tumor inhibitory effect, which was attributed to the mild anti-tumor immune response induced by αPD-1. In comparison, the NanoNMO + Laser + αPD-1 group demonstrated a significant distant tumor inhibitory effect, achieving a tumor inhibition rate of 71 ± 2% after 14 days of treatment. These results indicate that the combination of NanoNMO and αPD-1 could significantly enhance the systemic anti-tumor immune response, thereby suppressing the growth of the distant tumor effectively. After 14 days of treatment, the average weight of the primary tumors in the NanoNMO + Laser + αPD-1 group was 0.15 g (Fig. S45a), which was 4.5-fold lower than that of Saline group (0.68 g), 4.3-fold lower than that of Saline + Laser group (0.64 g), 5.0-fold lower than that of NanoNMO group (0.75 g), 3.4-fold lower than that of αPD-1 group (0.51 g) and 2.2-fold lower than that of NanoNMO + Laser group (0.33 g). Furthermore, the average weight of the distant tumors in the NanoNMO + Laser + αPD-1 group (0.01 g) was almost diminished (Fig. S45b), which was 27-fold lower than that of Saline group (0.27 g), 21-fold lower than that of Saline + Laser group (0.21 g), 21-fold lower than that of NanoNMO group (0.21 g), 15-fold lower than that of NanoNMO + Laser group (0.15 g), and 5-fold lower than that of αPD-1 group (0.05 g). The total results demonstrate that the synergistic treatment combining NanoNMO mediated PDT and αPD-1 not only improves the inhibition effect on primary tumors but also triggers a significient systemic anti-tumor immune response to suppress distant tumors. The excellent synergistic anti-tumor efficacy of the NanoNMO + Laser + αPD-1 group was further confirmed by ex vivo photos of the tumor tissues (Fig. 7 f). Notably, the body weight of mice displayed a natural growth during the treatment period (Fig. S45c), indicating the excellent biocompatibility of all treatments. Inspired by the remarkable synergistic anti-tumor effect of NanoNMO and PD-1 antibodies, we proceeded to investigate the antitumor immunity evoked by NanoNMO mediated PDT combining PD-1 antibody in 4T1 mouse model. The infiltration of cytotoxic T lymphocytes (CTLs) at tumor sites can evoke an anti-tumor immune response, leading to the elimination of tumor cells. Therefore, we collected lymphocytes from the primary tumors, distant tumors, and spleens of 4T1 mice and determined the frequency of infiltrated CTLs by flow cytometry after staining the sample with anti-CD3, anti-CD4, and anti-CD8 antibodies. A 4T1 bilateral tumor model of Balb/c mouse was established as outlined in Fig. 7 c and the mice were divided into six groups as mentioned earlier. After 12 h, the frequency of CTL cells in primary tumors, distant tumors, and spleens of mice with different treatments were evaluated. As shown in Fig. 7 g- 7 i and Fig. S46, the frequency of CD4 + T cells of the NanoNMO + Laser + αPD-1 group (primary tumor: 8.2%, distant tumor: 7.9%, spleen: 12.8%) increased significantly compared to Saline group (primary tumor: 1.9%, distant tumor: 3.7%, spleen: 7.8%), which was higher than the NanoNMO + Laser group (primary tumor: 3.9%, distant tumor: 3.8%, spleen: 8.8%) and the αPD-1 group (primary tumor: 4.2%, distant tumor: 4.9%, spleen: 10.0%). These findings indicate that the synergistic treatment of NanoNMO and αPD-1 promotes the infiltration of CD4 + T cells in tumor sites significantly, resulting in an effective anti-tumor immune response. Moreover, the frequency of CD8 + T cells (Fig. 7 j- 7 l and Fig. S47) of the NanoNMO + Laser + αPD-1 group (primary tumor: 14.5%, distant tumor: 8.9%, spleen: 5.8%) exhibited a significant increase compared to Saline group (primary tumor: 1.9%, distant tumor: 3.6%, spleen: 2.1%). Notably, these values surpassed those observed in the NanoNMO + Laser group (primary tumor: 6.7%, distant tumor: 4.3%, spleen: 4.1%) and the αPD-1 group (primary tumor: 10.1%, distant tumor: 6.8%, spleen: 4.2%), demonstrating significant improvement in immune response. These results are consistent with the anti-tumor activity studies, indicating the effective infiltration of CTL cells in the NanoNMO + Laser + αPD-1 group. As a result, a remarkable anti-tumor immune response was observed, thereby inhibiting the growth and metastasis of systemic tumors. In summary, the combination therapy of NanoNMO mediated type I PDT with αPD-1 synergistically improves PDT efficiency and triggers the body’s anti-tumor immune response (Fig. 7 m), thereby promoting the infiltration of CTL cells at tumor sites to achieve a synergistic treatment for systemic tumors. Discussion This work presents a novel concept that converts molecular PSs into “semiconductor-like” photocatalysts, facilitating the shift of PSs from the traditional type II photoreaction to highly efficient type I photoreaction. We offer an explanation of why the self-assembled PSs can significantly stimulate electron transfer activity from two perspectives. On the one hand, the understanding of electron excitation mechanism of PS aggregates undergoes a transformation from a molecular PS model to a semiconductor photocatalyst model. On this basis, the electron transfer and redox capabilities of PS aggregates are successfully analyzed through band theory. On the other hand, the efficient “self-substrate” SBCS mechanism has been employed to elucidate the ROS generations of PS aggregates. This “self-substrate” photoinduced charge separation approach not only minimizes photon energy loss 32, 48 but also eliminates the dependence on external substrates. Compared with previous works 22–25, 52 , the “self-substrate” design for Type I PS does not require consideration on the type and concentration of substrates, as well as their redox potential and binding affinity with PSs. This design strategy offers a more convenient and efficient pathway for constructing Type I PSs. To facilitate biomedical applications, an amphiphilic Pc molecular structure has been designed to prepare a relatively stable Pc aggregate (NanoNMO) under physiological conditions. The results of in vitro studies have demonstrated the excellent photocytotoxicity of NanoNMO under both normoxic and hypoxic conditions. The results of in vivo experiments have revealed the remarkable tumor targeting ability and phototherapeutic efficacy of NanoNMO. Notably, NanoNMO mediated PDT not only triggers the anti-tumor immune response but also synergizes with PD-1 antibody to inhibit systemic tumor growth through enhancing the infiltration of CTL cells in tumor sites. This contribution pioneers the investigation of combining highly efficient PSs with ICIs under hypoxic conditions, providing a reference for the design of PSs combined with immunotherapy to effectively inhibit the growth and metastasis of hypoxic tumors, garnering significant implications. Methods Materials Reactions were conducted under a nitrogen atmosphere to minimize side reaction. Methylene blue (MB) was brought from TCI, Shanghai, China. 4-trifluoromethoxyphenol, Cremophor EL (CEL), p-hydroxybenzophenone, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and chlorin e6 (Ce6) were brought from Alfa, Shanghai, China. Dihydroethidium (DHE), ctDNA, aminophenyl fluorescein (APF) were purchased from Sigma-Aldrich China. Singlet oxygen sensor green (SOSG) and ROS-ID™ hypoxia/oxidative stress detection kit were purchased from Enzo Life Sciences Inc. Fetal bovine serum (FBS) was bought from HyClone, Shanghai, China. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was bought from Sigma, Shanghai, China. Calcein-AM/PI cell flow cytometry kit and Annexia V-FITC/PI cell flow cytometry kit were obtained from Solarbio, Beijing, China. The TNF-α and IFN-γ ELISA kits were obtained from Shanghai Enzyme Biotechnology Co., Ltd. αPD-1 antibodies were purchased from BioX cell Co., Ltd. USA. The anti-CD45-FITC, anti-CD8-PerCP-Cy5.5, anti-CD4-APC and anti-CD3-PE were purchased from BD Biosciences, USA. Fabrication of phthalocyanine aggregates (NanoPcs) and phthalocyanine monomers (MonoPcs) Fabrication of NanoPcs. NanoPcs were prepared using two different methods. Method 1, a 2 mM solution of Pc in THF was directly added to 1995 µL of water. The mixture underwent 6 pumping cycles at a constant speed using a 1000 µL pipette gun. Method 2, the THF solution of Pc (2 mM) was added to 1995 µL of water under ultrasonic conditions and kept under ultrasonic action for 10 s without any other actions. The final concentration of NanoPcs was 5 µM. Specifically, NanoNMe, NanoNH, NanoOMe and NanoNMO were fabricated using Method 2, while the other NanoPcs were fabricated using Method 1. Fabrication of MonoPcs. MonoPcs were prepared using the following method: A 2 mM solution of Pc in THF was initially mixed with 20 µL of CEL. The resulting mixture was further diluted with 1975 µL of water and underwent 6 pumping cycles at a constant speed using a 1000 µL pipette gun. The final concentration of MonoPcs was 5 µM, with the final proportion of CEL being 1% (v:v). Measurement of ROS properties ROS detection. The ROS generation efficiencies of photosensitizers were evaluated by the specific capture agent DCFH. Photosensitizers (5 µM) were dissolved with DCFH (10 µM) in a water solution. The mixture was than irradiated with > 610 nm light (5 mW·cm − 2 ). After various irradiation times, fluorescence intensity (excited at 488 nm) of the mixture was recorded. The fluorescence intensities at 525 nm of different irradiation time were recorded to obtain the slope of fluorescence emission. The relative ROS generations of NanoPcs or MonoPcs were determined comparing them with the reference samples (MB). The probe slope of MB was considered as 1, and the slope ratios were served as the ordinate. O 2 •− detection. The O 2 •− generation efficiencies of photosensitizers were evaluated by the specific capture agent DHE and ctDNA. Photosensitizers (5 µM) were dissolved with DHE (50 µM) and ctDNA (250 µg·mL − 1 ) in a water solution. The mixture was than irradiated with > 610 nm light (5 mW·cm − 2 ). After different irradiation times, fluorescence intensity (excited at 510 nm) of the mixture was recorded. The fluorescence intensities at 599 nm of different irradiation time were recorded to obtain the slope of fluorescence emission. The relative O 2 •− generations of NanoPcs or MonoPcs were determined comparing them with the reference samples (MB). The probe slope of MB was considered as 1, and the slope ratios were served as the ordinate. • OH detection. The • OH generation efficiencies of photosensitizers were evaluated by the specific capture agent APF. Photosensitizers (5 µM) were dissolved with APF (10 µM) in a water solution. The mixture was than irradiated with > 610 nm light (5 mW·cm − 2 ). After different irradiation times, fluorescence intensity (excited at 490 nm) of the mixture was recorded. The fluorescence intensities at 515 nm of different irradiation time were recorded to obtain the slope of fluorescence emission. The relative • OH generations of NanoPcs or MonoPcs were determined comparing them with the reference samples (MB). The probe slope of MB was considered as 1, and the slope ratios were served as the ordinate. 1 O 2 detection. The 1 O 2 generation efficiencies of photosensitizers were evaluated by the specific capture agent SOSG. Photosensitizers (5 µM) were dissolved with SOSG (5 µM) in a water solution. The mixture was than irradiated with > 610 nm light (5 mW·cm − 2 ). After different irradiation times, fluorescence intensity (excited at 488 nm) of the mixture was recorded. The fluorescence intensities at 520 nm of different irradiation time were recorded to obtain the slope of fluorescence emission. The relative 1 O 2 generations of NanoPcs or MonoPcs were determined comparing them with the reference samples (MB). The probe slope of MB was considered as 1, and the slope ratios were served as the ordinate. Measurement of fs-TA The ultrafast fs-TA measurements were conducted under ambient conditions using a fs pump-probe system in conjunction with an amplified laser setup 53 . The setup included a mode-locked Ti: sapphire seed laser (Spectra Physics, Maitai) coupled with a regenerative amplifier (Spitfire Pro, Spectra Physics) and a high-powered laser (3 mJ, ~ 35 fs, Empower, Spectra Physics) for pumping and amplification. The amplified 800 nm output was divided into two parts, with the majority of the beam (~ 85%) passing through an optical parametric amplifier (TOPAS prime, Spectra Physics) to generate pump pulses with tunable wavelengths. The remaining portion of the beam was used to generate the white light continuum probe and reference pulses (400–600 nm) after traversing an optical delay line and passing through photosensitizer solutions. In this setup, the probe beam passed through the sample, while the reference beam went straight to the reference spectrometer. To obtain fs-TA spectra with and without the pump pulses, a chopper modulating the pump pulse was used. An optical fiber, connected to a multichannel spectrometer equipped with a CMOS sensor, captured the changes in intensity of the probe/reference beam caused by the pump pulses. The optical delay line, with a maximum delay of approximately 3 ns, was adjusted during the measurements. The acquired spectral profiles were subsequently processed using Surface Xplorer software. Measurement of electrochemical properties Cyclic voltammetry measurement. Cyclic voltammetry experiment was performed using a three-electrode system, where the working electrode was a glassy carbon electrode, the counter electrode was a Pt wire electrode, and the reference electrode was an Ag/AgCl electrode. Measurements were carried out in DMF containing 0.1 M (n-Bu) 4 N + PF 6– . The scan rate used was 100 mV·s − 1 . Ferrocene was employed as an external reference. The feasibility of photoinduced symmetry breaking charge separation (SBCS) can be estimated by evaluating the Gibbs-free energy (∆G). The calculation of ∆G can be accomplished using the Rehm-Weller equation (Eq. 1 ). Mott − Schottky (MS) analysis. Mott-Schottky analysis was performed using a three-electrode system, where the working electrode consisted of an indium tin oxide (ITO) glass electrode adhered with lyophilized NanoNMO or NanoCN, the counter electrode was a Pt wire electrode, and the reference electrode was an Ag/AgCl electrode. The measurements were carried out in an aqueous solution of 0.2 M Na 2 SO 4 . Transient photocurrent and electrochemical impedance spectroscopy measurement. The transient photocurrent and electrochemical impedance spectroscopy measurement were performed using a three-electrode system, where the working electrode consisted of an ITO glass electrode adhered with lyophilized NanoNMO or NanoCN, the counter electrode was a Pt wire electrode, and the reference electrode was an Ag/AgCl electrode. The measurements were carried out in an aqueous solution of 0.2 M Na 2 SO 4 . For the transient photocurrent measurements, the working electrode was irradiated with a white light (180 mW·cm − 2 ), with an interval of 20 s between each irradiation. For the electrochemical impedance spectroscopy measurements, the working electrode was consistently irradiated with a white light (180 mW·cm − 2 ). Conductivity measurement. The conductivity measurement was performed in aqueous solutions containing NMe, NanoNMe, and NanoCN at a concentration of 1 mg·mL − 1 , respectively. As a control, water was used as the reference solution. The solution was irradiated with a white light (180 mW·cm − 2 ), and the conductivity values were recorded with an interval of 30 s. Measurement of redox properties Valence band (VB) measurement. The UPS measurement was performed using the He excitation energy (hν = 21.22 eV). The value of VB was determined by fitting a straight line into the leading edge of the spectra. The VB (vs NHE) of NanoNMe can be calculated according to the equation S1. $${E}_{NHE}={E}_{f}+{\Phi }-4.44$$ S1 Where E NHE is the VB potential of NanoNMe; E f is the binding energy of Fermi edge (E f ); Φ is the work function. Φ can be calculated according to Φ = hν - (cutoff - E f ), where the value of cutoff was obtained as 15.93 eV. Φ was calculated as 5.47 eV. The E NHE of NanoNMe was calculated as 1.21 eV (vs NHE). Computational Details Ground-state geometries optimizations and time-dependent density functional theory (TD-DFT) calculations were carried out at B3LYP 43 /6-31G* method. All calculations were carried out using the Gaussian 16 package. The polarizable continuum model 44 with default parameters was used to implicitly consider solvation effects of water solution. In vitro study Cell Culture. The 4T1 cells were grown in DMEM at 37 ℃ in a humidified 5% CO 2 atmosphere, with the medium supplemented with 10% FBS, Bispecific Antibody (0.02 units·mL − 1 ), and Amphotericin (0.25 µg·mL − 1 ). Measurement of the intracellular ROS production 54 . Approximately 5 × 10 4 4T1 cells suspension in DMEM cell culture medium (0.4 mL) were inoculated on confocal dishes. To construct the in vitro normoxic and hypoxic conditions, a part of 4T1 cells was cultured in an incubator chamber at 37 ℃ with a humidified, 21% O 2 , 5% CO 2 , and 74% N 2 atmosphere for 12 h. Another 4T1 cells were cultured in an incubator chamber at 37 ℃ with a humidified, 2% O 2 , 5% CO 2 , and 93% N 2 atmosphere for 12 h. After the removal of the medium, the cells were incubated in fresh medium containing Ce6 or NMO or NanoNMO (4 µM) in darkness for 1.5 h. Subsequently, DCFH-DA was added at a final concentration of 10 µM and incubated for 30 min. The cells were then washed three times with PBS, replenished with 500 µL of culture medium, and utilized a 685 nm laser (15 mW·cm − 2 ) for 5 min. Upon removing the culture medium, the cells underwent a PBS rinse before imaging via a LEICA TCS SPE confocal microscope. The probe was excited at 488 nm and monitored at 500–600 nm. The images were then digitized and analyzed using SPE ROI Fluorescence Statistics software. The average intracellular fluorescence intensities were recorded for a total of 50 cells in each sample. Measurement of the intracellular O 2 •− production 54 . Approximately 5 × 10 4 4T1 cells suspension in DMEM cell culture medium (0.4 mL) were inoculated on confocal dishes. 4T1 cells were devided two parts to culture under both normoxic and hypoxic conditions for 12 h. After the removal of the medium, the cells were incubated in fresh medium containing Ce6 or NMO or NanoNMO (4 µM) in darkness for 1.5 h. DHE was added at a final concentration of 10 µM and incubated for 30 min. The cells were then washed three times with PBS, replenished with 500 µL of culture medium, and utilized a 685 nm laser (15 mW·cm − 2 ) for 5 min. Upon removing the culture medium, the cells underwent a PBS rinse before imaging via a LEICA TCS SPE confocal microscope. The probe was excited at 532 nm and monitored at 540–650 nm. The images were then digitized and analyzed by SPE ROI Fluorescence Statistics software. The average intracellular fluorescence intensities were recorded for a total of 50 cells in each sample. In vitro photodynamic activity assay. 4T1 cells (about 1 × 10 4 cells per well) were cultured in 96-wellplates and devided two parts to culture under both normoxic and hypoxic conditions for 12 h at 37 ℃ in a humidified 5% CO 2 atmosphere. The previous medium was aspirated and discarded, and 100 µL per well of Ce6 or NMO or NanoNMO with the specified concentration gradient was subsequently added. After 2 h of cultivation and removal of the culture medium, the cells were washed with PBS and replenished with 100 µL of culture medium. Subsequently, the cells were exposed to ambient temperature illumination for 0.5 h. The light source utilized a 685 nm laser (30 min, 15 mW·cm − 2 ), the total fluence was 27 J·cm − 2 . Cell viability was determined by the MTT assay. Following illumination, the cells were incubated overnight at 37 ℃ with 5% CO 2 . A 40 µL MTT solution in PBS (2.5 mg mL − 1 ) was added to each well, and the cells were subsequently incubated for 4 h under the same conditions. After removing the medium, 150 µL of DMSO was added to each well. The 96-well plate was gently agitated on a Tecan M200Pro microplate reader at room temperature for 20 s prior to measuring the absorbance at 490 nm. The cell viability was then detected by the equation S2: Where Ai is the absorbance of the i th data ( i = 1, 2, …, n), Ā control is the average absorbance of the control wells, in which the Pc is absent, and n (≥3) is the number of the data points. Live/Dead cell co-staining. 4T1 cells (about 1 × 10 4 cells per well) were cultured in 96-wellplates and devided two parts to culture under both normoxic and hypoxic conditions for 12 h at 37 ℃ in a humidified 5% CO 2 atmosphere. The previous medium was aspirated and discarded, and 100 µL per well of NMO or NanoNMO were added at concentrations of 0.4 or 0.8 µM, respectively. After 2 h of cultivation and removal of the culture medium, the cells were washed with PBS and replenished with 100 µL of culture medium. Subsequently, the cells were exposed to ambient temperature illumination for 0.5 h. The light source utilized a 685 nm laser (30 min, 15 mW·cm − 2 ), the total fluence was 27 J·cm − 2 . After the aforementioned treatments, 4T1 cells were additionally stained using the Calcein-AM/PI Double Stain Kit 54 following the instructions provided in the manual. The excitation wavelength was 488 nm, and the emission wavelength was set to 505–545 nm for the green channel and 600–700 nm for the red channel. In vivo study Mouse models. Mouse breast cancer (4T1) cells were acquired from the China Center for Type Culture Collection (CCTCC, Shanghai, China), and the mice were procured from Wushi Animal Co. Ltd. (Fu Zhou, China). All animal studies were carried out in compliance with guidelines of the Animal Ethics Committee of Fuzhou University (2023-SG-001), and also approved by the committee. To initiate a subcutaneous tumor model, approximately 5 × 10 6 4T1 cells in 200 µL were subcutaneously inoculated on the flank of mice weighing 18–20 g. Fluorescence imaging. The mice received an intravenous injection of either NMO or NanoNMO (200 µM, 100 µL) in H 2 O. In vivo fluorescence images of the mice were performed on a IVIS Lumina III imaging system. Both NMO and NanoNMO were excited at 640 nm and monitored at 660–750 nm. After the in vivo imaging studies, the mice were euthanized 36 h after the injection. The heart, liver, spleen, lung, kidney, tumor and skin were extracted and imaged. Data was processed through the IVIS system. Three mice were utilized for each experimental group. Photoacoustic imaging. The mice received an intravenous injection of either NMO or NanoNMO (200 µM, 100 µL) in H 2 O. In vivo photoacoustic images of the mice were performed on a photoacoustic computerized tomography scanner (MSOT 256-TF, Germany). Data was processed through the iThera Medical system. Three mice were utilized for each experimental group. In vivo PDT effect assay. To evaluate the in vivo PDT effects of NanoNMO, the mice received an intravenous injection of NanoNMO (200 µM, 100 µL) in H 2 O. 8 h post-injection, the mice underwent irradiation at the tumor site using a 685 nm laser (100 mW·cm − 2 ) for a duration of 5 min. As a control, blank mice were treated with saline only (100 µL). To evaluate the therapeutic efficacy, the tumor size was measured using a digital caliper every alternate day. Daily photographs were taken for a duration of 14 d. Tumor volumes were calculated as 0.5 × length × width × width. In vivo cytokine release. The tumor-bearing mice received an intravenous injection of NanoNMO (200 µM, 100 µL) in H 2 O. 8 h post-injection, the mice underwent irradiation at the tumor site using a 685 nm laser (100 mW·cm − 2 ) for a duration of 5 min. The blood was collected 12 h after PDT and the levels of serum TNF-α and IFN-γ were determined by enzyme-linked immunosorbent assay (ELISA) to evaluate the acute inflammation induced by the treatment 8, 55 . In vivo photodynamic immunotherapy (PIT) effect assay. To evaluate the in vivo PIT effects of NanoNMO, the mice received an intravenous injection of NanoNMO (200 µM, 100 µL) in H 2 O firstly. 8 h post-injection, the mice underwent irradiation at the tumor site using a 685 nm laser (100 mW·cm − 2 ) for a duration of 5 min. Then αPD-1 was intravenously injected (2.5 mg·kg − 1 ) at 24 h and 72 h after injection, respectively. To evaluate the therapeutic efficacy, the tumor size was measured using a digital caliper every alternate day. Daily photographs were taken for a duration of 14 d. Tumor volumes were calculated using the formula 0.5 × length × width × width. Flow cytometry assay for immune response. All mice were euthanized on the 7th day post-treatment, and the bilateral tumors and spleens were excised. The tumors and spleens were subsequently digested using a DMEM mixture that included 0.2% collagenase IV, 0.1% hyaluronidase, and 0.002% DNase I at 37°C for 1 h, followed by mechanical grinding using the rubber end of a syringe. The resulting cell suspension was sieved through nylon mesh filters and rinsed with PBS. The single-cell suspension was then incubated with anti-CD45-FITC, anti-CD8-APC, anti-CD4-PerCP-Cy5.5, and anti-CD3-PE, followed by shaking for 30 min at 4°C 8 . After centrifugation at 1000 rpm for 5 min, the supernatant was discarded, and the cell suspension was diluted with sterilized PBS. Cell acquisition was performed using a NovoCyte Flow Cytometer (ACEA Biosciences, USA), and data analysis was conducted using the FlowJo software. Statistical information Statistical comparisons were made using two-tailed Student’s t test (between two groups). All statistical analysis was performed using SPSS. p < 0.05 was considered to be statistically difference; p < 0.01 was considered to be significant statistically difference; p < 0.001 was considered to be extremely significant statistically difference. Quantitative data were presented as mean ± standard deviation (SD). Declarations Data Availability All data generated in this study are available within the Supplementary Information. Acknowledgements The authors thank the National Natural Science Foundation of China (Grant Nos. 22078066, T2322004 and 22178065). Competing interests The authors declare no competing financial interest. Contributions H. L.: Performed the synthesis and the in vivo test, analyzed the data, and prepared the manuscript draft; Z. L.: Performed the in vitro test and analyzed the data; X. Z.: Performed the in vivo test and analyzed the data; Y. X.: Assisted the in vivo test; G. T.: Assisted the in vitro test; Z. W.: Performed the computational calculation; Y.-Y. 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Ed. 59 , 11591-11599 (2020). Markovic, V., Villamaina, D., Barabanov, I., Lawson Daku, L.M. & Vauthey, E. Photoinduced symmetry‐breaking charge separation: The direction of the charge transfer. Angew. Chem. Int. Ed. 50 , 7596-7598 (2011). Lin, C., Kim, T., Schultz, J.D., Young, R.M. & Wasielewski, M.R. Accelerating symmetry-breaking charge separation in a perylenediimide trimer through a vibronically coherent dimer intermediate. Nat. Chem. 14 , 786-793 (2022). Zhao, X., et al. Near-infrared self-assembled hydroxyl radical generator based on photoinduced cascade electron transfer for hypoxic tumor phototherapy. Adv. Mater. 35 , 2305163 (2023). Vauthey, E. Photoinduced symmetry-breaking charge separation. ChemPhysChem 13 , 2001-2011 (2012). Weller, A. Photoinduced electron transfer in solution: Exciplex and radical ion pair formation free enthalpies and their solvent dependence. Phys. Chem. 133 , 93-98 (1982). Teng, K.-X., Niu, L.-Y. & Yang, Q.-Z. Supramolecular photosensitizer enables oxygen-independent generation of hydroxyl radicals for photodynamic therapy. J. Am. Chem. Soc. 145 , 4081-4087 (2023). Zou, S., et al. From nonluminescent to blue-emitting Cs4PbBr6 nanocrystals: Tailoring the insulator bandgap of 0d perovskite through Sn cation doping. Adv. Mater. 31 , 1900606 (2019). Zhao, Y.Y., et al. Nanostructured phthalocyanine assemblies with efficient synergistic effect of type I photoreaction and photothermal action to overcome tumor hypoxia in photodynamic therapy. J. Am. Chem. Soc. 143 , 13980-13989 (2021). Zheng, B.-Y., et al. Phthalocyanine-based photosensitizers combined with anti-PD-L1 for highly efficient photodynamic immunotherapy. Dyes Pigm. 185 , 108907-108917 (2021). Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation.docx Supplementary Information Scheme1.png Scheme 1. (a) Schematic illustration of the mechanism of molecular PSs and the charge separation and photocatalytic mechanism of “semiconductor-like”photocatalysts. (b) Schematic illustration of the PIT synergistic process of NanoNMO and PD-1 antibody. Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3933352","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":275648833,"identity":"a00a0775-9ced-4d39-9f13-f4b403c84d2d","order_by":0,"name":"Xingshu Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDACZiBmbGCQYWNvfMAAYROphYeN57ABkVqgyngYJJKJ1GJwnPfwy587DvPwST5m/szDYCO74QDzswf4tEg286VZ8545zMMmncxgzMOQZrzhAJu5AT4t/Mw8ZsaMbSAt+QeSeRgOJ244wMMmgU8LG1CL4U+QFsnDDId5GP4T1gK0xfgBL0iLBDNjMw/DAcJaJJt5zJh529KBgZzMzDjHINl45mE2M7xaDM6fMf74s81aTr79MPOHNxV2sn3Hm5/h1QLyDpICUFAxE1APUvKBsJpRMApGwSgY0QAAWQU9lYih2kQAAAAASUVORK5CYII=","orcid":"","institution":"Fuzhou University","correspondingAuthor":true,"prefix":"","firstName":"Xingshu","middleName":"","lastName":"Li","suffix":""},{"id":275648834,"identity":"444ee2cd-69d0-4aab-aa84-44764828a481","order_by":1,"name":"Hao Liu","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Liu","suffix":""},{"id":275648835,"identity":"cce6f69f-21dd-4fcf-a2a3-7b55c862f1d6","order_by":2,"name":"Ziqing Li","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Ziqing","middleName":"","lastName":"Li","suffix":""},{"id":275648836,"identity":"69fe0235-426f-4c55-bfaa-32eaeb02fcd8","order_by":3,"name":"Xiaojun Zhang","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojun","middleName":"","lastName":"Zhang","suffix":""},{"id":275648837,"identity":"2d394f1c-5b55-4bbf-b47d-8bd75856d7c8","order_by":4,"name":"Yihui Xu","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yihui","middleName":"","lastName":"Xu","suffix":""},{"id":275648838,"identity":"7c52d8c8-1733-44bb-9f93-9bf5e46c46ac","order_by":5,"name":"Guoyan Tang","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Guoyan","middleName":"","lastName":"Tang","suffix":""},{"id":275648839,"identity":"6f1dbdd3-97bc-49f6-81dc-dd857eee2a24","order_by":6,"name":"Zhaoxin Wang","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhaoxin","middleName":"","lastName":"Wang","suffix":""},{"id":275648840,"identity":"bed92597-bb0a-4014-8064-bb7682950447","order_by":7,"name":"Yuan-Yuan Zhao","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yuan-Yuan","middleName":"","lastName":"Zhao","suffix":""},{"id":275648841,"identity":"b50a5716-dcab-4c6f-bdba-c2027e6f783e","order_by":8,"name":"Mei-Rong Ke","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Mei-Rong","middleName":"","lastName":"Ke","suffix":""},{"id":275648842,"identity":"01f8712c-11d7-490e-8ceb-81f54e18c9db","order_by":9,"name":"Bi-Yuan Zheng","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Bi-Yuan","middleName":"","lastName":"Zheng","suffix":""},{"id":275648843,"identity":"155bbf04-7b43-4635-abca-21b75adf2c85","order_by":10,"name":"Shuping Huang","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Shuping","middleName":"","lastName":"Huang","suffix":""},{"id":275648844,"identity":"e17d836c-c0d7-4e0f-b5d2-00c5c633586d","order_by":11,"name":"Jian-Dong Huang","email":"","orcid":"","institution":"Fuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jian-Dong","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-02-06 09:07:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3933352/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3933352/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-55575-2","type":"published","date":"2025-01-02T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52418757,"identity":"b5b29610-4155-4c86-9215-2922f812f555","added_by":"auto","created_at":"2024-03-11 12:47:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":259701,"visible":true,"origin":"","legend":"\u003cp\u003eFabrication of NanoPcs and MonoPcs and their ROS generations. (a) The structure of tetrasubstituted zinc (II) Pcs and (b) quantitative electron properties of the substituent group (σρ) on zinc (II) Pcs. The σρ value is a substituent constant derived from the Hammett equation\u003csup\u003e27\u003c/sup\u003e. The σρ value of electron donor substituents are negative, while the σρ value of electron accept substituents are positive. The absolute value of the σρ indicates the electron donating or accepting ability of substituent. (c) Schematic illustration of the fabrication of NanoPcs and MonoPcs. (d) Relative ROS generations of NanoPcs and MonoPcs. (e) Relative O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e generations of NanoPcs and MonoPcs. (f) Relative \u003csup\u003e·\u003c/sup\u003eOH generations of NanoPcs and MonoPcs. (g) Relative \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generations of NanoPcs and MonoPcs. The relative ROS, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e, \u003csup\u003e·\u003c/sup\u003eOH, and \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generations of NanoPcs or MonoPcs were determined comparing them with the reference samples (MB). The probe slope of MB was considered as 1, and the slope ratios were served as the ordinate.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3933352/v1/d322a1249110077638d162c7.png"},{"id":52418762,"identity":"8dbabe92-c566-4067-95b2-55fd1cc4bd46","added_by":"auto","created_at":"2024-03-11 12:47:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":265038,"visible":true,"origin":"","legend":"\u003cp\u003eInvestigation on ISC process of MonoPcs. (a) The calculated S1 and T1 energy levels of Pcs. Ground-state geometries optimizations and TD-DFT calculations were carried out at B3LYP\u003csup\u003e43\u003c/sup\u003e/6-31G* method. All calculations were carried out using the Gaussian 16 package. The polarizable continuum model\u003csup\u003e44\u003c/sup\u003e with default parameters was used to implicitly consider solvation effects of H\u003csub\u003e2\u003c/sub\u003eO. 2D pseudo-color fs-TA spectra of (b) NMe and (c) CN obtained with time delays from 0 to 3080 ps (NMe: 694 nm, 47 μJ; CN：685 nm, 77 μJ) and plots at different pump-probe delay times. The upward arrow symbolizes the population of triplet states while the downward arrow represents the depopulation of singlet states. The presence of an isosbestic point (marked with a black solid lines) indicates the appearance of both singlet and triplet species, which represents the occurrence of ISC. (d) The excitation energy data calculated by B3LYP/6-31G* and the ISC parameters obtained from the fs-TA results of NMe and CN.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3933352/v1/14012108b377ca4f8e57d797.png"},{"id":52418760,"identity":"702787f6-9681-49c2-8df4-dbb57fd24640","added_by":"auto","created_at":"2024-03-11 12:47:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":312557,"visible":true,"origin":"","legend":"\u003cp\u003e“Self-substrate” PET mechanism of NanoPcs. (a) Schematic illustration of the electron transfer mechanism of NanoPcs. fs-TA spectra of (b) NMe in THF and (c) NanoNMe in H\u003csub\u003e2\u003c/sub\u003eO upon excitation by 694 nm pulses. * represents excited state species, ▲ represents Pc\u003csup\u003e·-\u003c/sup\u003e. (d) The dynamic decay trace and lifetimes of excited states and Pc\u003csup\u003e·-\u003c/sup\u003e species of NanoNMe within a 3 ns range. (e) fs-TA spectra of NanoCN in H\u003csub\u003e2\u003c/sub\u003eO at different time delays upon excitation by 685 nm pulses. (f) Difference between the oxidation and reduction potentials (shown as e(E\u003csub\u003eox\u003c/sub\u003e-E\u003csub\u003ered\u003c/sub\u003e)) and the calculated excited energy (E*) of Pcs. The redox potentials of Pcs were measured through cyclic voltammetry in DMF containing 0.1 M (n-Bu)\u003csub\u003e4\u003c/sub\u003eN\u003csup\u003e+\u003c/sup\u003ePF\u003csup\u003e6–\u003c/sup\u003e, using glassy carbon as the working electrode, Ag/AgCl as the reference electrode, Pt wire as the counter electrode, with a scan rate of 100 mV·s\u003csup\u003e-1\u003c/sup\u003e. Ferrocene was used as an external reference. The excited energy of Pcs was calculated by B3LYP/6-31G* method.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3933352/v1/e1e593d542258f09112cd5ef.png"},{"id":52421889,"identity":"c76b7904-b0d6-43f9-af6c-372e02fde124","added_by":"auto","created_at":"2024-03-11 12:55:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":241587,"visible":true,"origin":"","legend":"\u003cp\u003e“Semiconductor-like” photocatalysis for O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e generation. (a) Schematic illustration of the molecular orbital diagram of Pcs and the band diagram of NanoPcs. The excited Pcs transfer energy to O\u003csub\u003e2\u003c/sub\u003e, generating \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. The excited NanoPcs transfer electron to O\u003csub\u003e2\u003c/sub\u003e, generating O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e. (b) Mott-Schottky plots of NanoNMe under dark conditions (100 Hz). The lyophilized NanoNMe adhered ITO glass was served as the working electrode, Ag/AgCl as the reference electrode, and platinum wire as the counter electrode, with 0.2 M Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e as the supporting electrolyte. (c) UPS valence band spectrum of NanoNMe. The work function (Φ) was calculated through the formula Φ = hν - (cutoff - E\u003csub\u003ef\u003c/sub\u003e), where the excitation energy of the He light source was 21.22 eV. The VB potential was calculated through the formula E\u003csub\u003eVB \u003c/sub\u003e(NHE) = E\u003csub\u003ef\u003c/sub\u003e + Φ - 4.44. (d) Band diagram of NanoNMe, including CB, VB potentials and the band gap. (e) Representative photocurrent response of NanoNMe and NanoCN on an ITO glass electrode with the interval of 20 s. (f) Electrochemical impedance spectroscopy of NanoNMe and NanoCN. Ag/AgCl served as the reference electrode, platinum wire as the counter electrode, and 0.2 M Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e as the supporting electrolyte with a bias of -0.2 V, white light, 180 mW·cm\u003csup\u003e-2\u003c/sup\u003e. (g) Changes in conductivity of NanoNMe, NanoCN, and NMe (all at 1 mg·mL\u003csup\u003e-1\u003c/sup\u003e) over time with the interval of 30 s, white light, 180 mW·cm\u003csup\u003e-2\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3933352/v1/fcc152dff73359c8571b6100.png"},{"id":52418763,"identity":"7c6f7e57-0459-4f7f-880b-57eedcd8c959","added_by":"auto","created_at":"2024-03-11 12:47:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":592754,"visible":true,"origin":"","legend":"\u003cp\u003eROS generations and \u003cem\u003ein vitro\u003c/em\u003e phototherapeutic efficacy of NanoNMO. (a) Structure of NMO and schematic illustration of the fabrication of NanoNMO. (b) Size distribution and morphology of NanoNMO (5 μM) measured by DLS and TEM. (c) Relative ROS generations and (d) relative O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e generation of NanoNMO. The relative ROS and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e generations of NanoNMO, NMO and Ce6 were determined comparing them with the reference samples (MB). The probe slope of MB was considered as 1, and the slope ratios were served as the ordinate. (e) CLSM images of intracellular DCFH fluorescence and (f) quantitative fluorescence emission of 4T1 cells incubated with Ce6, NMO, and NanoNMO under both normoxic and hypoxic conditions. (g) CLSM images of intracellular DHE fluorescence and (h) quantitative fluorescence emission of 4T1 cells incubated with Ce6, NMO, and NanoNMO under both normoxic and hypoxic conditions. Cells were irradiated with 685 nm laser for 5 min (15 mW·cm\u003csup\u003e-2\u003c/sup\u003e). F.I., Fluorescence intensity. Cytotoxic effects of Ce6, NMO, and NanoNMO on 4T1 cells under (i) normoxic and (j) hypoxic conditions in dark or with 685 nm laser irradiation (30 min, 15 mW·cm\u003csup\u003e-2\u003c/sup\u003e). (k) Calcein-AM/PI costaining images detected by CLSM, with Calcein-AM staining displayed in the green channel and PI staining in the red channel (scale bar = 100 μm). Data were expressed as mean ± SD *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. NS, not significant, determined by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3933352/v1/870409c1baa32def90ec8e90.png"},{"id":52418764,"identity":"82eb4b6a-3e42-46dc-bba3-38f9ba584350","added_by":"auto","created_at":"2024-03-11 12:47:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":594444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e biodistribution and phototherapeutic efficacy of NanoNMO. (a) \u003cem\u003eIn vivo\u003c/em\u003e fluorescence imaging (excited at 640 nm, ×10\u003csup\u003e7\u003c/sup\u003e ps\u003csup\u003e−1\u003c/sup\u003e·cm\u003csup\u003e−1\u003c/sup\u003e·sr\u003csup\u003e−1\u003c/sup\u003e·μW\u003csup\u003e−1\u003c/sup\u003e·cm\u003csup\u003e2\u003c/sup\u003e, white ellipses representing the tumor area) and (b) \u003cem\u003ein vivo\u003c/em\u003e photoacoustic imaging (excited at 690 and 700 nm, yellow ellipses representing the tumor area) of 4T1 tumor bearing mice before and after intravenous injection of NMO or NanoNMO. (c) Quantitative fluorescence and photoacoustic intensities of the tumor sites after intravenous injection of NanoNMO (n = 3). F.I., fluorescence intensity; PA.I., photoacoustic intensity. (d) \u003cem\u003eEx vivo\u003c/em\u003e fluorescence imaging of 4T1 tumor bearing mice injected with NMO and NanoNMO (×10\u003csup\u003e7\u003c/sup\u003e ps\u003csup\u003e−1\u003c/sup\u003e·cm\u003csup\u003e−1\u003c/sup\u003e·sr\u003csup\u003e−1\u003c/sup\u003e·μW\u003csup\u003e−1\u003c/sup\u003e·cm\u003csup\u003e2\u003c/sup\u003e) and (e) quantitative fluorescence intensities. H., heart; Li., liver; Sp., spleen; Lu., lung; K., kidney; T., tumor; Sk., skin. (f) Tumor growth plots of 4T1 tumor bearing mice following various treatments (n = 5). (g) Average body weight changes of mice after indicated treatments. Data were expressed as mean ± SD. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, determined by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3933352/v1/5508bd8ba30c0731d84a177f.png"},{"id":52418765,"identity":"0b8936ea-a9fe-48d1-838f-b736a4d1cf3b","added_by":"auto","created_at":"2024-03-11 12:47:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":515703,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo \u003c/em\u003ePIT efficiency of NanoNMO in combination with PD-1 antibody. Biochemical analysis of (a) TNF-α and (b) IFN-γ in the serum of mice after 24 h of NanoNMO treatment. (c) Schematic outline of the establishment of a 4T1 bilateral tumor model and the the treatment steps and procedures for different treatments. PDT treatment was performed 8 h after intravenous injection of NanoNMO with a 685 nm laser (100 mW·cm\u003csup\u003e−2\u003c/sup\u003e, 5 min), followed by intravenous injection of αPD-1 (2.5 mg·kg\u003csup\u003e-1\u003c/sup\u003e) on the first and third days, respectively. Tumor volume was continuously monitored until day 14, after which mice were sacrificed for further biochemical studies. Tumor growth plots of (d) primary tumors and (e) distant tumors in 4T1 tumor-bearing mice following the indicated treatments (n = 5). (f) Representative tumor images of primary tumors and distant tumors of mice after 14 d of indicated treatments. The quantitative data of CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes in (g) primary tumors, (h) distant tumors and (i) spleen of mice after the indicated treatments. The quantitative data of CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes in (j) primary tumors, (k) distant tumors and (l) spleen of mice after the indicated treatments. (m) Schematic illustration of the PIT synergistic process of NanoNMO and αPD-1. The illustration was created with \u003cu\u003eBioRender.com\u003c/u\u003e. Data were expressed as mean ± SD *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. NS, not significant, determined by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-3933352/v1/98be1d9f3e58de1beced0fc8.png"},{"id":72874819,"identity":"f7661951-3ad6-4cbb-a60f-b35f0585aa6f","added_by":"auto","created_at":"2025-01-03 08:07:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3584365,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3933352/v1/a608b9ab-8fd0-4f88-99b2-a027c3dcb489.pdf"},{"id":52418759,"identity":"05a1209f-fa9f-4c7e-a01e-c1116016df03","added_by":"auto","created_at":"2024-03-11 12:47:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4880201,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Information\u003c/p\u003e","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3933352/v1/99c50dd4844218d110f15f87.docx"},{"id":52418758,"identity":"36e8d25d-9c47-4612-a663-de3571a3f607","added_by":"auto","created_at":"2024-03-11 12:47:44","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":315751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. (a) Schematic illustration of the mechanism of molecular PSs and the charge separation and photocatalytic mechanism of “semiconductor-like”photocatalysts. (b) Schematic illustration of the PIT synergistic process of NanoNMO and PD-1 antibody.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-3933352/v1/35b139b414147c7e65666874.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Phthalocyanine Aggregates as “Semiconductor-like” Photocatalysts for Hypoxic-Tumor Photodynamic Immunotherapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer is a devastating disease, with one of the most fatal aspects lying in the metastasis of cancer cells\u003csup\u003e1\u0026ndash;5\u003c/sup\u003e. In recent years, photodynamic immunotherapy (PIT), which combines photodynamic therapy (PDT) with tumor immunotherapy, especially immune checkpoint blocking therapy, has emerged as a prominent hotspot in the field of tumor therapy\u003csup\u003e6\u0026ndash;10\u003c/sup\u003e. However, the current research is still in its early stages. Most of photosensitizers (PSs) used for PIT mainly inhibit tumor through the oxygen-dependent type II photoreaction\u003csup\u003e3, 11\u0026ndash;13\u003c/sup\u003e. The hypoxic tumor microenvironment (TME) limits the therapeutic effect of this type of PDT, which limits the synergistic effect of PIT accordingly\u003csup\u003e6, 14\u003c/sup\u003e. Moreover, the hypoxic TME promotes cancer cell metastasis and tumor immunosuppression\u003csup\u003e15, 16\u003c/sup\u003e, while the consumption of oxygen during type II PDT further exacerbates tumor hypoxia, thereby impeding the effectiveness of PIT against tumor metastasis\u003csup\u003e17, 18\u003c/sup\u003e. Consequently, a crucial aspect for advancing the clinical application of PIT lies in the development of PSs that exhibit less oxygen dependency.\u003c/p\u003e \u003cp\u003eType I PSs have been proved to possess the advantage of less oxygen dependency\u003csup\u003e19\u0026ndash;21\u003c/sup\u003e. However, most of the existing type I PSs primarily rely on various substrates as electron transfer agents to enhance electron transfer efficiency, which impose stringent requirements on the type and concentration of substrates, as well as their redox potential and binding affinity with PSs\u003csup\u003e22\u0026ndash;25\u003c/sup\u003e. Therefore, the research on type I PSs remains relatively limited. Our prior research has discovered that a phenoxy-linked polyamine mono-substituted zinc (II) phthalocyanine (Pc) can self-assemble into nanodots in water and generate reactive oxygen species (ROS) through type I photoreaction\u003csup\u003e26\u003c/sup\u003e. Nevertheless, it is urgent to delve deeply into the quantitative structure activity relationship (QSAR) between the structural characteristics of zinc (II) Pc and its type I photoreaction. Additionally, the internal mechanisms underlying the efficient type I photoreaction of the nanodots need to be elucidated as well.\u003c/p\u003e \u003cp\u003eTo tackle these challenges, a series of tetrasubstituted zinc (II) Pcs incorporating various electron donor/acceptor groups were designed and synthesized herein\u003csup\u003e27, 28\u003c/sup\u003e. Furthermore, the Pc aggregates and the Pc monomers were fabricated in aqueous solution\u003csup\u003e29, 30\u003c/sup\u003e to investigate the QSAR between the electronic characteristics of the substituents and the ROS generations of the Pc aggregates and Pc monomers. The obtained results revealed that the Pc aggregates substituted with electron donor group (such as NanoNMe) exhibited a significant type I photoactivity. The superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) production of NanoNMe was found to be 15-fold higher than that of methylene blue (MB). Detailed mechanism (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1a\u003c/span\u003e) studies revealed that NanoNMe underwent a photoinduced symmetry breaking charge separation (SBCS) process, achieving charge separation and formating Pc\u003csup\u003e\u0026bull;+\u003c/sup\u003e and Pc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e ion pairs through a \u0026ldquo;self-substrate\u0026rdquo; approach. The products of SBCS exhibited similarities to electron-hole pairs\u003csup\u003e31, 32\u003c/sup\u003e, enabling to consider NanoNMe as a semiconductor to analyze its charge transfer properties. Band analysis revealed that NanoNMe possessed the capability to reduce O\u003csub\u003e2\u003c/sub\u003e to O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and exhibited remarkable photocatalytic charge transfer activity, making NanoNMe a \u0026ldquo;semiconductor-like\u0026rdquo; photocatalyst to facilitate efficient charge transfer. Additionally, a reformed Pc aggregate (NanoNMO) was fabricated to improve the stability and the type I photoactivity in physiological environments by incorporating octaethylene glycol and electron donor amino groups into the Pc structure to improve its amphipathy. The results of \u003cem\u003ein vitro\u003c/em\u003e experiments demonstrated an outstanding photocytotoxicity of NanoNMO under both normoxic and hypoxic conditions. NanoNMO also exhibited excellent tumor targeting ability and significant phototherapeutic efficacy in 4T1 tumor-bearing mice. Notably, the PDT effect mediated by NanoNMO not only triggered the systemic immune response but also synergized with PD-1 antibody to inhibit the growth of primary and distant tumor (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1b\u003c/span\u003e). This work introduces a groundbreaking concept transforming molecular PSs into \u0026ldquo;semiconductor-like\u0026rdquo; photocatalysts, providing a novel perspective to design less oxygen dependent PSs and presenting a practical example converting molecular PSs to \u0026ldquo;semiconductor-like\u0026rdquo; photocatalysts. Additionally, this work pioneers the investigation of combining high-efficiency PSs with immune checkpoint inhibitors (ICIs) under hypoxic conditions, which serves as a reference for the development of PSs combining immunotherapy to suppress the growth and metastasis of hypoxic tumors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003ePreparation of Pc aggregates and Pc monomers and their ROS generations\u003c/p\u003e\n\u003cp\u003eZinc (II) phthalocyanine is a functional dye with notable advantages, including strong absorption within the phototherapeutic window (650\u0026ndash;850 nm), high ROS yield, and tunable photochemical properties\u003csup\u003e33, 34\u003c/sup\u003e. These attributes render it a promising PS for PDT. However, most of the reported zinc (II) Pcs are oxygen dependent type II PSs\u003csup\u003e35\u0026ndash;37\u003c/sup\u003e. Besides, the hydrophobic plane makes Pcs prone to aggregate, leading to the quenching of photoactivity\u003csup\u003e38\u0026ndash;40\u003c/sup\u003e. Our prior work has demonstrated a zinc (II) Pc nanodot capable of generating ROS through type I mechanism\u003csup\u003e26\u003c/sup\u003e. To investigate the QSAR between the structural characteristics of zinc (II) Pc and its type I photoreaction, ten tetrasubstituted zinc (II) Pcs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea) were meticulously designed and synthesized herein. The structural modification involved replacing the para position of the phenoxy group connected to the Pc with various electron donor/acceptor substituents (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). Specifically, the Pcs were named by the abbreviation of their substituents successively as NMe, NH, OMe, Bu, H, Cl, OCF, COP, CN and NO. The detailed synthesis and characterization of these Pcs are provided in the supporting information (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e-S20).\u003c/p\u003e\n\u003cp\u003ePc aggregates (NanoPcs) with a size range of 200\u0026thinsp;\u0026plusmn;\u0026thinsp;50 nm and Pc monomers (MonoPcs) were fabricated in H\u003csub\u003e2\u003c/sub\u003eO through different methods (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). Specifically, NanoPcs were obtained by diluting a tetrahydrofuran (THF) solution of the Pc into H\u003csub\u003e2\u003c/sub\u003eO, with or without the application of ultrasound. For NMe, NH, and OMe, NanoPcs were prepared by diluting the THF solution of the Pc into H\u003csub\u003e2\u003c/sub\u003eO under ultrasound conditions. For other structures, NanoPcs were prepared by diluting the THF solution of the Pc into H\u003csub\u003e2\u003c/sub\u003eO directly. The detailed preparation steps are described in the \u003cspan class=\"InternalRef\"\u003eMethods\u003c/span\u003e section. The dynamic light scattering (DLS) results (Fig. S21) revealed that all of NanoPcs exhibited uniform dispersion in H\u003csub\u003e2\u003c/sub\u003eO, with particle sizes ranging of 200\u0026thinsp;\u0026plusmn;\u0026thinsp;50 nm. Transmission electron microscope (TEM) images of NanoPcs (Fig. S22) demonstrated that their morphology resembled of regular spherical shapes, with a diameter of approximate 200 nm, which was consistent with the DLS results. The steady-state absorption spectra (Fig. S23) revealed that the absorption spectra of NanoPcs exhibited broadening and decreased intensity compared to the absorption features of the Pc monomers in N, N-dimethylformamide (DMF). Additionally, the steady-state fluorescence emission of NanoPcs (Fig. S24) attenuated significantly compared to that of Pc monomers in DMF. These findings indicated the significantly changed light absorption and fluorescence emission properties of NanoPcs compared to Pc monomers. Besides, MonoPcs were obtained by mixing a THF solution of Pc with surfactant Cremophor EL (CEL) in advance, followed by H\u003csub\u003e2\u003c/sub\u003eO dilution. DLS results (Fig. S25a) and TEM images (Fig. S25b) demonstrated that all of MonoPcs possessed particle sizes near 10 nm, with no observable nanostructure, indicating the uniform disperation of MonoPcs in H\u003csub\u003e2\u003c/sub\u003eO. Moreover, the steady-state absorption and fluorescence emission revealed that the absorption and fluorescence emission of MonoPcs exhibited similar features with those of Pc monomers in DMF, suggesting that the CEL-regulated MonoPcs could exist as monomers in H\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e\n\u003cp\u003eThe ROS efficiencies for both NanoPcs and MonoPcs were initially assessed to conduct a QSAR analysis. The ROS generations were evaluated using the dihydrodichlorofluorescein (DCFH) probe (Fig. S26, S27), whose fluorescence at 525 nm can be produced by various ROS including O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, hydroxyl radical (\u003csup\u003e\u0026bull;\u003c/sup\u003eOH) and singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e). To enable a quantative comparison, the relative ROS generations of NanoPcs and MonoPcs were determined comparing them with the reference samples (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). For NanoPcs, an equal concentration of MB was used as the reference sample, while for MonoPcs, a mixture of CEL and MB was used as the reference sample. The observations indicate that as the value of \u0026sigma;\u0026rho; decreases, both NanoPcs and MonoPcs exhibit higher ROS generation. Moreover, when the \u0026sigma;\u0026rho; value is more than 0, NanoPcs demonstrate lower ROS generation than MonoPcs, and when the \u0026sigma;\u0026rho; value is less than 0, NanoPcs demonstrate higher ROS generation than MonoPcs, surpassing that of MB significantly.\u003c/p\u003e\n\u003cp\u003eTo assess the specific contributions, the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation was evaluated using dihydrohexidine (DHE) probe (Fig. S28, S29), the \u003csup\u003e\u0026bull;\u003c/sup\u003eOH generation was evaluated using aminophenyl fluorescein (APF) probe (Fig. S30, S31), and the \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation was evaluated using singlet oxygen sensing green (SOSG) probe (Fig. S32, S33). The O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation results were presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee. The observations indicate that as the value of \u0026sigma;\u0026rho; decreases, both NanoPcs and MonoPcs display higher O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation. Moreover, when the \u0026sigma;\u0026rho; value is more than 0, NanoPcs demonstrate lower O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation than MonoPcs, and when the \u0026sigma;\u0026rho; value is less than 0, NanoPcs demonstrate significantly higher O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation than MonoPcs. The O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation of NanoPcs can reach up to 15-fold higher than that of MB at most.\u003c/p\u003e\n\u003cp\u003eThe \u003csup\u003e\u0026bull;\u003c/sup\u003eOH generation results were depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef. The observations indicate that as the value of \u0026sigma;\u0026rho; increases, both NanoPcs and MonoPcs demonstrate decreased \u003csup\u003e\u0026bull;\u003c/sup\u003eOH generations. Notably, NanoPcs demonstrate lower \u003csup\u003e\u0026bull;\u003c/sup\u003eOH generation compared to MonoPcs. Moreover, the \u003csup\u003e\u0026bull;\u003c/sup\u003eOH generations of both NanoPcs and MonoPcs are relatively inefficient, which are either comparable or lower than that of MB. The \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation results were depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg. The observations indicate that as the value of \u0026sigma;\u0026rho; increases, MonoPcs demonstrate a mild decrease in \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation. Notably, NanoPcs demonstrate lower \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation than MonoPcs. Moreover, both NanoPcs and MonoPcs display low level of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generations, which are consistently lower than that of MB. To sum up, all the QSAR results demonstrate that both NanoPcs and MonoPcs exhibit higher ROS generation when the \u0026sigma;\u0026rho; value is less than 0. Notably, the generations of both ROS and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e by NanoPcs (\u0026sigma;\u0026rho;\u0026thinsp;\u0026lt;\u0026thinsp;0) demonstrate remarkably high level, making them excellent ROS and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generators.\u003c/p\u003e\n\u003cp\u003eInvestigation on intersystem crossing process of MonoPcs\u003c/p\u003e\n\u003cp\u003eGenerally, the generation of ROS is closely related to the intersystem crossing (ISC) process of PSs\u003csup\u003e21, 41\u003c/sup\u003e. In order to gain insights into the ROS generation mechanism of MonoPcs, the ISC process was analyzed. Initially, time-dependent density functional theory (TD-DFT) calculations were conducted to analyze the energy levels of excited state involved in the ISC process. The results revealed that as the \u0026sigma;\u0026rho; value increased, the energy gap (∆E\u003csub\u003eST\u003c/sub\u003e) between singlet state (S1) and triplet state (T1) of Pcs gradually widened (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea), indicating an increased energy interval of the ISC process. The increased ∆E\u003csub\u003eST\u003c/sub\u003e are unfavorable for the ROS generations, which explain the observed trend where, as the \u0026sigma;\u0026rho; values increased, the ROS generations of MonoPcs decreased.\u003c/p\u003e\n\u003cp\u003eFurthermore, the excited state dynamics of MonoPcs were investigated using femtosecond transient absorption (fs-TA) spectroscopy\u003csup\u003e41\u003c/sup\u003e. To conduct specific analysis, NMe and CN were chosen as representative examples. As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec, the contour maps illustrated two-dimensional color-coded fs-TA spectra of NMe and CN upon excitation at 694 and 685 nm, respectively, providing insights into the evolution of excited states. The negative absorption was attributed to the ground state bleaching (outlined with white dotted lines). The positive absorption was attributed to the excited state absorption (ESA, outlined with blue and red dotted lines), where the blue dotted lines indicated the ESA of singlet state, and the red dotted line indicated the ESA of triplet state. To distinguish singlet ESA from triplet ESA, fs-TA plots at different delay times were extracted from the contour map and presented at the bottom of the contour map.\u003c/p\u003e\n\u003cp\u003eFor NMe, after a 90 fs timeframe following excitation, a series of ESA grew in intensity with prolonged delay time within the 500\u0026ndash;750 nm range. Among these absorptions, the dynamic decay trace at 510 nm exhibited a decay lifetime of 3.18 ns, which was consistent with the fluorescence lifetime (3.98 ns) determined by time correlated single photon counting (Fig. S34a). Based on these evidences, the ESA around 510 nm was assigned to singlet ESA. Over time, the ESA near 560 nm exhibited a continuously growing peak after 500 ps with no attenuation trend observed within the maximum range of 3.08 ns. Furthermore, the fs-TA plots revealed the occurrence of an obvious isosbestic point between the ESA near 560 nm and the ESA near 510 nm, indicating the occurrence of a singlet-to-triplet ISC process. Eventually, the hysteresis and prolonged lifetime of the ESA near 560 nm was attributed to triplet ESA. Likewise, for CN, a series of ESA grew in intensity within the 500\u0026ndash;750 nm range as well. The dynamic decay trace at 510 nm aligned with the fluorescence lifetime (Fig. S34b). Moreover, an isosbestic point was observed between the ESA near 560 nm and the ESA near 510 nm, indicating the occurrence of ISC process and triplet state.\u003c/p\u003e\n\u003cp\u003eAfterwards, the parameters related to ISC were calculated based on the singlet and triplet ESA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). The occurrence times of ISC (t\u003csub\u003eISC\u003c/sub\u003e) were estimated by determining the populated time of triplet state, which were found to be approximately 500 ps and 566 ps for NMe and CN, respectively. The rates of ISC (k\u003csub\u003eISC\u003c/sub\u003e) were calculated as 0.20 and 0.16 (\u0026times;10\u003csup\u003e10\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1/t\u003csub\u003eISC\u003c/sub\u003e) for NMe and CN, respectively. Subsequently, the efficiencies of ISC (\u0026Phi;\u003csub\u003eISC\u003c/sub\u003e) were estimated according to the previous studies\u003csup\u003e41, 42\u003c/sup\u003e (\u0026Phi;\u003csub\u003eISC\u003c/sub\u003e = [1/t\u003csub\u003e(T1, rise)\u003c/sub\u003e]/[1/t\u003csub\u003e(S1, decay)\u003c/sub\u003e]) as 16.4% and 8.6% for NMe and CN, respectively. The excited state dynamics results were consistent with the theoretical calculations, indicating that as the \u0026sigma;\u0026rho; value decreased, the \u0026Phi;\u003csub\u003eISC\u003c/sub\u003e of Pcs increased, result in a higher generation of ROS.\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;Self-substrate\u0026rdquo; photoinduced electron transfer\u003c/p\u003e\n\u003cp\u003eUnlike MonoPcs, NanoPcs generate ROS through type I photoreaction involved electron transfer. The crucial step in this mechanism is the intermolecular photoinduced electron transfer (PET) of NanoPcs\u003csup\u003e45, 46\u003c/sup\u003e. Accordingly, the mechanism of the ROS generation by NanoPcs is proposed. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, upon photon excitation, partial molecules of NanoPcs are excited, resulting in the formation of an energy asymmetric local excited state (Pc* + Pc). The local excited state is unstable and promptly undergoes symmetry breaking charge separation (SBCS) to reduce its asymmetry. As a result, a pair of free radical ions is formed (Pc\u003csup\u003e\u0026bull;+\u003c/sup\u003e + Pc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003eTo analyze the intermolecular SBCS, the excited state photophysical processes of NMe and NanoNMe were analyzed using fs-TA. The results revealed a significant difference in the photophysical process between NMe and NanoNMe. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb, the ESA of NMe in the 450\u0026ndash;600 nm range exhibited uniformly decreased vibration peaks within 500 ps, indicating the absence of intermediate species during excited state decay process of NMe. For NanoNMe (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec), the ESA transformed into mixed species features in the 450\u0026ndash;600 nm range within 1 ps, suggesting the generation of intermediate species. Specifically, the ESA at 450\u0026ndash;550 nm showed uniformly decreased vibration peaks, with an absorption range consistent with the ESA of NMe, indicating the formation of excited states. Furthermore, after approximately 0.58 ps, different species features appeared near 600 nm, indicating the generation of other species. The SBCS has been extensively reported in various organic molecular aggregates, including perylenediimide (PDI) and its derivatives\u003csup\u003e47, 48\u003c/sup\u003e. The occurrences of SBCS (\u0026tau;\u003csub\u003eSBCS\u003c/sub\u003e) for PDI derivatives ranges from sub-ps to hundreds-ps and are closely related to molecular structure, distance, and solvent polarity\u003csup\u003e49\u003c/sup\u003e. Based on the mechanism and the reported results, the ESA near 600 nm was attribute to the absorption signal of the Pc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e species resulting from SBCS. The dynamic decay trace (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed) indicated that Pc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e gradually formed after the decay of the excited state, yet it exhibited a longer lifetime compared to the excited state, demonstrating a distinct kinetic property. Conversely, no observation of any species was detected in the fs-TA analysis of NanoCN (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e\n\u003cp\u003eTo investigate the feasibility of a photoinduced SBCS, the Gibbs free energy (∆G) was calculated. Typically, the condition for SBCS process to be possible is that ∆G is negative\u003csup\u003e50\u003c/sup\u003e. Therefore, the cyclic voltammetry was employed to determine the redox potentials of all the Pcs (Fig. S35, Table S2). The ∆G of Pcs was calculated based on the Rehm-Weller Eq.\u0026nbsp;5\u003csup\u003e1\u003c/sup\u003e (Eq.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\varDelta G=e\\left({E}_{ox}-{E}_{red}\\right)-{E}^{*}-\\frac{{e}^{2}}{2\\pi {\\epsilon }_{0}{\\epsilon }_{s}d}\\approx e\\left({E}_{ox}-{E}_{red}\\right)-{E}^{*}\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eWhere \u003cem\u003ee\u003c/em\u003e represents the elementary charge; \u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003eox\u003c/em\u003e\u003c/sub\u003e is the potential for one-electron oxidation of Pcs, and \u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003ered\u003c/em\u003e\u003c/sub\u003e is the potential for one-electron reduction of Pcs; \u003cem\u003eE*\u003c/em\u003e is the excitation energy obtained from TD-DFT. The last term accounts for the coulombic interactions between two ions produced at a distance \u003cem\u003ed\u003c/em\u003e and screened by the solvent with a static dielectric constant \u003cem\u003e\u0026epsilon;\u003c/em\u003e\u003csub\u003e\u003cem\u003es\u003c/em\u003e\u003c/sub\u003e. The results indicated that for substituents with \u0026sigma;\u0026rho;\u0026thinsp;\u0026lt;\u0026thinsp;0, the ∆G of Pcs was negative, suggesting that Pcs with \u0026sigma;p\u0026thinsp;\u0026lt;\u0026thinsp;0 were capable of undergoing photoinduced SBCS. However, as the \u0026sigma;p value increased, the ∆G of Pcs increased, indicating a gradual decrease in charge separation ability of Pcs. Consequently, the electron transfer mechanism of NanoPcs can be described as follows: the local excited state of NanoPcs undergoes a SBCS process, resulting in the generation of Pc\u003csup\u003e\u0026bull;+\u003c/sup\u003e and Pc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e free radical species, thereby achieving charge separation through a way of \u0026ldquo;self-substrate\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;Semiconductor-like\u0026rdquo; photocatalysis for O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation\u003c/p\u003e\n\u003cp\u003eThe free radical ion pairs of SBCS (Pc\u003csup\u003e\u0026bull;+\u003c/sup\u003e and Pc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) in NanoPcs can exhibit spatial overlap and extend from localized orbitals to delocalized orbitals\u003csup\u003e31, 32\u003c/sup\u003e, which can be described as free electron-hole pairs within a semiconductor (band theory) framework (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). Therefore, the redox properties of NanoPcs were investigated through band theory\u003csup\u003e52\u003c/sup\u003e to analyze the generations of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and \u003csup\u003e\u0026bull;\u003c/sup\u003eOH. The reduction of O\u003csub\u003e2\u003c/sub\u003e depends on the conduction band (CB) potential of NanoNMe. Consequently, the flat band potential of NanoNMe was determined using the Mott-Schottky plots (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). The results revealed that the flat band potential of NanoNMe was \u0026minus;\u0026thinsp;0.47 V, which was approximately equal to the CB potential and less negative than the reduction potential of O\u003csub\u003e2\u003c/sub\u003e (-0.33 V), indicating that NanoNMe could photoreduce O\u003csub\u003e2\u003c/sub\u003e to generate O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e. Additionally, the oxidation of H\u003csub\u003e2\u003c/sub\u003eO depends on the valence band (VB) potential of NanoNMe. The VB potential of NanoNMe was determined by ultraviolet photoelectron spectroscopy (UPS) valence band spectra (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). The edge of the valence band maximum energy (E\u003csub\u003ef\u003c/sub\u003e) for NanoNMe was found to be 0.18 eV, and the work function (\u0026Phi;) was calculated to be 5.47 eV. The VB potential of NanoNMe (vs NHE) was concluded to be 1.21 V, which is less than the oxidation potential of H\u003csub\u003e2\u003c/sub\u003eO (1.99 V), indicating that NanoNMe cannot photooxidize H\u003csub\u003e2\u003c/sub\u003eO to generate \u003csup\u003e\u0026bull;\u003c/sup\u003eOH. Additionally, the optical bandgap energy determined from absorption spectra (Eg\u0026thinsp;=\u0026thinsp;1.69 eV, Fig. S36) matched closely with the calculated bandgap (1.68 eV, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed), providing convincing evidence of the redox capability of NanoNMe.\u003c/p\u003e\n\u003cp\u003eFurthermore, the charge transfer abilities of NanoNMe were further investigated through transient photocurrent response and charge transfer resistance, with NanoCN used as a comparison. Interestingly, NanoNMe exhibited a significantly enhanced photocurrent under light illumination (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee), while the photocurrent of NanoCN was negligible. Electrochemical impedance spectroscopy (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef) revealed that the charge transfer resistance of NanoNMe was significantly smaller than that of NanoCN. Additionally, the charge flow abilities in the aqueous solution were studied by analyzing the conductivity changes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg). Over the course of illumination, the conductivity of NanoNMe showed a significant enhancement compared to NMe or NanoCN. These results indicate that NanoNMe exhibits a \u0026ldquo;semiconductor-like\u0026rdquo; photocatalytic activity, leading to a significant enhancement of charge transfer under illumination. Based on these findings, the present study proposes a novel design strategy for Type I PSs by converting molecular PSs to \u0026ldquo;semiconductor-like\u0026rdquo; photocatalysts, offering new insights for addressing PDT in hypoxic tumors.\u003c/p\u003e\n\u003cp\u003eFabrication of physiologically stable NanoPcs and their \u003cem\u003ein vitro\u003c/em\u003e phototherapeutic efficacy\u003c/p\u003e\n\u003cp\u003eMotivated by the efficient photocatalytic generation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e by NanoNMe, we further investigated the potential of NanoNMe for biomedical applications. The stability is a crucial requirement for biomedical application of NanoPcs. Therefore, we initially examined the stability of NanoNMe in physiological conditions. In a phosphate-buffered saline (PBS) solution containing fetal bovine serum (FBS), the particle size of NanoNMe gradually increased over time (Fig. S37a), suggesting an unstable nanostructure of NanoNMe. Additionally, the absorption spectra of NanoNMe (Fig. S37b, S37c) demonstrated a gradual decreased intensity in PBS containing FBS over time, indicating the decreased light-harvesting properties. Conversely, an increment in fluorescence intensity was observed for NanoNMe as the concentration of FBS increased (Fig. S37d, S37e), indicating the gradually restored fluorescence emission of NanoNMe under the influence of FBS. The significant changes in nanostructure and optical activity collectively demonstrated the poor stability of NanoNMe in a physiological environment. To facilitate the stable dispersion of NanoPc in physiological environments, a novel amphiphilic Pc (NMO) was designed by replacing one substituent of NMe with octaethylene glycol. The detailed synthesis and characterization of NMO are presented in the Supporting Information (Fig. S38, S39).\u003c/p\u003e\n\u003cp\u003eThe nanostructured aggregate of NMO (NanoNMO) was prepared through a nano precipitation method (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). The detailed fabrication steps of NanoNMO are provided in the \u003cspan class=\"InternalRef\"\u003eMethods\u003c/span\u003e section. DLS and TEM results exhibited that the average size of NanoNMO was approximately 110 nm (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb), exhibiting a morphology resembled of regular spherical shapes. As expected, NanoNMO exhibited improved stability in physiological environments compared to NanoNMe. During the initial 12 h period upon the addition of FBS, the size distribution of NanoNMO remained relatively unchanged, along with consistent absorption and fluorescence emission (Fig. S37f-S37j). Afterwards, the size of NanoNMO started to increase, accompanied by an intensified fluorescence emission, indicating the instability of NanoNMO after 24 h. Additionally, the ROS generations of NanoNMO were further evaluated with NMO, Chlorin e6 (Ce6), and MB serving as control (Fig. S40). The results demonstrated that NanoNMO exhibited robust ROS generations, higher than that of NMO and Ce6, and approximately 1.8-fold higher than that of MB (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). Specifically, the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation of NanoNMO was approximately 7.9-fold higher than that of MB (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed), while no significant generation of \u003csup\u003e\u0026bull;\u003c/sup\u003eOH and \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e was observed (Fig. S41). These results indicate that NanoNMO is an efficient O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generator.\u003c/p\u003e\n\u003cp\u003eFurther investigation was conducted to assess the \u003cem\u003ein vitro\u003c/em\u003e PDT activity of NanoNMO. Initially, the intracellular ROS generations in mouse breast cancer cells (4T1) were evaluated using confocal laser scanning microscopy (CLSM) under both normoxic and hypoxic conditions. As shown in Fig. S42, the fluorescence intensities of the oxygen stress indicator (ROS-ID) under both normoxic and hypoxic conditions were significantly higher than the negative control and comparable to the positive control, indicating the successful establishment of normoxic and hypoxic conditions. Subsequently, the intracellular ROS generation ability was assessed using DCFH as a fluorescent indicator. As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee, negligible fluorescence was observed in cells without laser irradiation. However, after 685 nm laser irradiation, significant fluorescence was observed in the Ce6, NMO, and NanoNMO treated cells under normoxic conditions. Notably, NanoNMO group exhibited the highest fluorescence emission (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef), indicating its superior ROS production ability. However, under hypoxic conditions, only NanoNMO group displayed bright fluorescence signals comparable to those observed under normoxic conditions, suggesting that NanoNMO could effectively generate ROS in hypoxic conditions as well. Additionally, the intracellular O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation capacity was assessed using DHE as a fluorescent indicator. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eg, minimal fluorescence was observed in cells without laser irradiation. After 685 nm laser irradiation, neither Ce6 nor NMO displayed significant fluorescence signals under normoxic or hypoxic conditions, suggesting limited intracellular O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation. In contrast, NanoNMO displayed robust fluorescence signals under both normoxic or hypoxic conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eh), indicating efficient O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation of NanoNMO under both normoxic or hypoxic conditions.\u003c/p\u003e\n\u003cp\u003eNext, the \u003cem\u003ein vitro\u003c/em\u003e phototherapeutic effects of Ce6, NMO, and NanoNMO were evaluated through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ei and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ej, no obvious toxicity was observed for Ce6, NMO, and NanoNMO under dark conditions. Under normoxic conditions, Ce6, NMO, and NanoNMO exhibited significant concentration-dependent cytotoxicities following 685 nm laser irradiation (30 min, 15 mW\u0026middot;cm\u003csup\u003e-2\u003c/sup\u003e), with 50% inhibitory concentrations (IC\u003csub\u003e50\u003c/sub\u003e) of 2.35, 0.40, and 0.31 \u0026micro;M, respectively. However, under hypoxic conditions, the photocytotoxicity of Ce6 and NMO decreased significantly, leading to an increase in IC\u003csub\u003e50\u003c/sub\u003e to 5.74 and 2.60 \u0026micro;M, respectively. In contrast, NanoNMO maintained a potent photocytotoxicity, with an IC\u003csub\u003e50\u003c/sub\u003e of 0.41 \u0026micro;M. The live/dead cell staining assay (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ek, S43) also demonstrated the effective cell damage induced by NanoNMO under both normoxic and hypoxic conditions. These findings suggest that O\u003csub\u003e2\u003c/sub\u003e concentration barely affect the photocytotoxicity of NanoNMO.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e biodistribution and phototherapeutic efficacy of NanoNMO\u003c/p\u003e\n\u003cp\u003eTo explore the biodistribution, Balb/c mice bearing a 4T1 tumor were administered with an intravenous injection of NanoNMO (200 \u0026micro;M, 100 \u0026micro;L), employing NMO (200 \u0026micro;M, 100 \u0026micro;L) as a control. Fluorescence and photoacoustic dual-modal imaging was used to evaluate the \u003cem\u003ein vivo\u003c/em\u003e biodistribution. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, after intravenous injection, a significent fluorescence signal of NMO rapidly disseminated throughout the body and diminished gradually over time, with no significant tumor accumulation observed. Conversely, faint fluorescence signal was only observed at the tumor site within 24 h after injection of NanoNMO. Moreover, photoacoustic imaging (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb) revealed that NanoNMO predominantly exhibited photoacoustic signals at the tumor site after intravenous injection and reached a maximum at 8 h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec), indicating the superior tumor targeting ability of NanoNMO. Notably, at 24 h post intravenous injection, the fluorescence signal of NanoNMO at the tumor site significantly increased, whereas the photoacoustic signal noticeably diminished. Since the photoacoustic signal of NanoNMO in H\u003csub\u003e2\u003c/sub\u003eO containing 10% FBS decreased significantly compared to that in pure water (Fig. S44), we speculated that the tumor accumulated NanoNMO may gradually disassemble, leading to enhanced fluorescence and reduced photoacoustic signals after 24 h. As a control, mice injected with NMO exhibited low systemic photoacoustic signals, indicating negligible tumor accumulation. To evaluate the distribution of NMO and NanoNMO in different organs, mice were sacrificed 36 h postinjection and the fluorescence intensities of the tumor and major organs including heart, liver, spleen, lung, kidney and skin were assessed. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ee, the fluorescence signal of NanoNMO at the tumor site was significantly higher than that of other organs, registering 5.2-fold higher signal than that in the liver. In contrast, the fluorescence signal of NMO at the tumor site was only 0.8-fold than that of the liver, revealing an outstanding tumor targeting capability of NanoNMO. Consequently, NanoNMO can remain stable \u003cem\u003ein vivo\u003c/em\u003e for 12 h, thereby facilitating the effective implementation of PDT.\u003c/p\u003e\n\u003cp\u003eSubsequently, the \u003cem\u003ein vivo\u003c/em\u003e phototherapeutic efficacy of NanoNMO was further conducted on 4T1 tumor-bearing mice. Once the volume of tumor reached 100 mm\u003csup\u003e3\u003c/sup\u003e, the mice were divided into four groups (five mice per group): (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) treated with saline (control); (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) treated with saline followed by laser irradiation (685 nm, 100 mW\u0026middot;cm\u003csup\u003e-2\u003c/sup\u003e) 8 h after injection; (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e) treated with NanoNMO (200 \u0026micro;M, 100 \u0026micro;L); (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e) treated with NanoNMO (200 \u0026micro;M, 100 \u0026micro;L) followed by laser irradiation (685 nm, 100 mW\u0026middot;cm\u003csup\u003e-2\u003c/sup\u003e) 8 h after injection. As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef, all the control groups (including Saline, Saline\u0026thinsp;+\u0026thinsp;Laser, and NanoNMO only) exhibited significant level of tumor growth. In contrast, the tumor growth in NanoNMO\u0026thinsp;+\u0026thinsp;laser group was inhibited obviously. After 14 days of treatment, the tumor inhibition rate reached 96\u0026thinsp;\u0026plusmn;\u0026thinsp;4% compared to the control group. Additionally, the body weight of mice in different groups displayed no significant differences and maintained a natural growth during the course of treatments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg). These results suggest that NanoNMO possesses exceptional \u003cem\u003ein vivo\u003c/em\u003e phototherapeutic efficacy and excellent biocompatibility.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e PIT efficiency of NanoNMO in combination with PD-1 antibody\u003c/p\u003e\n\u003cp\u003eTo investigate the potential of type I PSs mediated PDT to induce an anti-tumor immune response, the level of immune cell cytokines in the plasma was studied on NanoNMO injected 4T1 tumor-bearing mice. The mice were divided into four groups (five mice per group): (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) treated with saline (control); (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) treated with saline followed by laser irradiation (685 nm, 100 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) 8 h after injection; (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e) treated with NanoNMO (200 \u0026micro;M, 100 \u0026micro;L); (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e) treated with NanoNMO (200 \u0026micro;M, 100 \u0026micro;L) followed by laser irradiation (685 nm, 100 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) 8 h after injection. Compared to the control group (including Saline, Saline\u0026thinsp;+\u0026thinsp;Laser, and NanoNMO only), the level of TNF-\u0026alpha; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea) and IFN-\u0026gamma; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb) cytokines of NanoNMO\u0026thinsp;+\u0026thinsp;laser group showed a significant enhancement, suggesting the occurrence of acute inflammation, which was a very crucial mechanism in inducing tumor-specific immunity through PDT. These results suggest that NanoNMO mediated PDT can induce the acute inflammation in the host, leading to a systemic immune response.\u003c/p\u003e\n\u003cp\u003eFurther investigation was conducted to explore the synergistic anti-tumor potential of NanoNMO mediated PDT in combination with immune checkpoint inhibitors (ICIs). A 4T1 bilateral tumor model of Balb/c mouse was established by injecting 4T1 cells into the right flank of the mice (primary tumor), followed by injecting 4T1 cells into the left flank of mice (distant tumor) after 6 d (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec). Once the volume of the distant tumor reached 100 mm\u003csup\u003e3\u003c/sup\u003e, the mice were divided into six groups (five mice per group) to monitor tumor growth following different treatments. Group 1: treated with saline (control); Group 2: treated with saline followed by laser irradiation (685 nm, 100 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) 8 h after injection; Group 3: treated with NanoNMO (200 \u0026micro;M, 100 \u0026micro;L); Group 4: treated with NanoNMO (200 \u0026micro;M, 100 \u0026micro;L) followed by laser irradiation (685 nm, 100 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) on the primary tumor 8 h after injection; Group 5: treated with \u0026alpha;PD-1 twice (2.5 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on day 1 and day 3, respectively; Group 6: treated with NanoNMO (200 \u0026micro;M, 100 \u0026micro;L) followed by laser irradiation (685 nm, 100 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) on the primary tumor 8 h after injection, and then treated with \u0026alpha;PD-1 twice (2.5 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on day 1 and day 3, respectively. The distant tumor received no laser irradiation. The tumor volume of both tumors and the weight of mice were monitored throughout the process.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed, the control groups (including Saline and Saline\u0026thinsp;+\u0026thinsp;Laser) exhibited rapid growth of the primary tumor. In addition, neither NanoNMO group nor \u0026alpha;PD-1 group exhibited significant tumor inhibition on the primary tumor. In contrast, both the NanoNMO\u0026thinsp;+\u0026thinsp;Laser group and the NanoNMO\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;\u0026alpha;PD-1 group demonstrated a significant suppression of primary tumor growth. Notably, the NanoNMO\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;\u0026alpha;PD-1 group demonstrated the highest treatment efficacy, with a tumor inhibition rate of 82\u0026thinsp;\u0026plusmn;\u0026thinsp;3% compared to Group 1 after 14 days of treatment, surpassing that of the NanoNMO\u0026thinsp;+\u0026thinsp;Laser group (73\u0026thinsp;\u0026plusmn;\u0026thinsp;2%). These results indicate an enhanced inhibitory effect on the primary tumor through the synergistic therapy. Additionally, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee, the control groups of the distant tumor (Saline, Saline\u0026thinsp;+\u0026thinsp;Laser, and NanoNMO) exhibited a rapid increase. Moreover, the NanoNMO\u0026thinsp;+\u0026thinsp;Laser group showed minimal distant tumor inhibition as well, suggesting that the immune response triggered by PDT was inferior and unable to suppress the growth of the distant tumor. The \u0026alpha;PD-1 group showed a slight distant tumor inhibitory effect, which was attributed to the mild anti-tumor immune response induced by \u0026alpha;PD-1. In comparison, the NanoNMO\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;\u0026alpha;PD-1 group demonstrated a significant distant tumor inhibitory effect, achieving a tumor inhibition rate of 71\u0026thinsp;\u0026plusmn;\u0026thinsp;2% after 14 days of treatment. These results indicate that the combination of NanoNMO and \u0026alpha;PD-1 could significantly enhance the systemic anti-tumor immune response, thereby suppressing the growth of the distant tumor effectively.\u003c/p\u003e\n\u003cp\u003eAfter 14 days of treatment, the average weight of the primary tumors in the NanoNMO\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;\u0026alpha;PD-1 group was 0.15 g (Fig. S45a), which was 4.5-fold lower than that of Saline group (0.68 g), 4.3-fold lower than that of Saline\u0026thinsp;+\u0026thinsp;Laser group (0.64 g), 5.0-fold lower than that of NanoNMO group (0.75 g), 3.4-fold lower than that of \u0026alpha;PD-1 group (0.51 g) and 2.2-fold lower than that of NanoNMO\u0026thinsp;+\u0026thinsp;Laser group (0.33 g). Furthermore, the average weight of the distant tumors in the NanoNMO\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;\u0026alpha;PD-1 group (0.01 g) was almost diminished (Fig. S45b), which was 27-fold lower than that of Saline group (0.27 g), 21-fold lower than that of Saline\u0026thinsp;+\u0026thinsp;Laser group (0.21 g), 21-fold lower than that of NanoNMO group (0.21 g), 15-fold lower than that of NanoNMO\u0026thinsp;+\u0026thinsp;Laser group (0.15 g), and 5-fold lower than that of \u0026alpha;PD-1 group (0.05 g). The total results demonstrate that the synergistic treatment combining NanoNMO mediated PDT and \u0026alpha;PD-1 not only improves the inhibition effect on primary tumors but also triggers a significient systemic anti-tumor immune response to suppress distant tumors. The excellent synergistic anti-tumor efficacy of the NanoNMO\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;\u0026alpha;PD-1 group was further confirmed by \u003cem\u003eex vivo\u003c/em\u003e photos of the tumor tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ef). Notably, the body weight of mice displayed a natural growth during the treatment period (Fig. S45c), indicating the excellent biocompatibility of all treatments.\u003c/p\u003e\n\u003cp\u003eInspired by the remarkable synergistic anti-tumor effect of NanoNMO and PD-1 antibodies, we proceeded to investigate the antitumor immunity evoked by NanoNMO mediated PDT combining PD-1 antibody in 4T1 mouse model. The infiltration of cytotoxic T lymphocytes (CTLs) at tumor sites can evoke an anti-tumor immune response, leading to the elimination of tumor cells. Therefore, we collected lymphocytes from the primary tumors, distant tumors, and spleens of 4T1 mice and determined the frequency of infiltrated CTLs by flow cytometry after staining the sample with anti-CD3, anti-CD4, and anti-CD8 antibodies. A 4T1 bilateral tumor model of Balb/c mouse was established as outlined in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec and the mice were divided into six groups as mentioned earlier. After 12 h, the frequency of CTL cells in primary tumors, distant tumors, and spleens of mice with different treatments were evaluated. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eg-\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ei and Fig. S46, the frequency of CD4\u003csup\u003e+\u003c/sup\u003e T cells of the NanoNMO\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;\u0026alpha;PD-1 group (primary tumor: 8.2%, distant tumor: 7.9%, spleen: 12.8%) increased significantly compared to Saline group (primary tumor: 1.9%, distant tumor: 3.7%, spleen: 7.8%), which was higher than the NanoNMO\u0026thinsp;+\u0026thinsp;Laser group (primary tumor: 3.9%, distant tumor: 3.8%, spleen: 8.8%) and the \u0026alpha;PD-1 group (primary tumor: 4.2%, distant tumor: 4.9%, spleen: 10.0%). These findings indicate that the synergistic treatment of NanoNMO and \u0026alpha;PD-1 promotes the infiltration of CD4\u003csup\u003e+\u003c/sup\u003e T cells in tumor sites significantly, resulting in an effective anti-tumor immune response. Moreover, the frequency of CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ej-\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003el and Fig. S47) of the NanoNMO\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;\u0026alpha;PD-1 group (primary tumor: 14.5%, distant tumor: 8.9%, spleen: 5.8%) exhibited a significant increase compared to Saline group (primary tumor: 1.9%, distant tumor: 3.6%, spleen: 2.1%). Notably, these values surpassed those observed in the NanoNMO\u0026thinsp;+\u0026thinsp;Laser group (primary tumor: 6.7%, distant tumor: 4.3%, spleen: 4.1%) and the \u0026alpha;PD-1 group (primary tumor: 10.1%, distant tumor: 6.8%, spleen: 4.2%), demonstrating significant improvement in immune response. These results are consistent with the anti-tumor activity studies, indicating the effective infiltration of CTL cells in the NanoNMO\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;\u0026alpha;PD-1 group. As a result, a remarkable anti-tumor immune response was observed, thereby inhibiting the growth and metastasis of systemic tumors. In summary, the combination therapy of NanoNMO mediated type I PDT with \u0026alpha;PD-1 synergistically improves PDT efficiency and triggers the body\u0026rsquo;s anti-tumor immune response (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003em), thereby promoting the infiltration of CTL cells at tumor sites to achieve a synergistic treatment for systemic tumors.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis work presents a novel concept that converts molecular PSs into \u0026ldquo;semiconductor-like\u0026rdquo; photocatalysts, facilitating the shift of PSs from the traditional type II photoreaction to highly efficient type I photoreaction. We offer an explanation of why the self-assembled PSs can significantly stimulate electron transfer activity from two perspectives. On the one hand, the understanding of electron excitation mechanism of PS aggregates undergoes a transformation from a molecular PS model to a semiconductor photocatalyst model. On this basis, the electron transfer and redox capabilities of PS aggregates are successfully analyzed through band theory. On the other hand, the efficient \u0026ldquo;self-substrate\u0026rdquo; SBCS mechanism has been employed to elucidate the ROS generations of PS aggregates. This \u0026ldquo;self-substrate\u0026rdquo; photoinduced charge separation approach not only minimizes photon energy loss\u003csup\u003e32, 48\u003c/sup\u003e but also eliminates the dependence on external substrates. Compared with previous works\u003csup\u003e22\u0026ndash;25, 52\u003c/sup\u003e, the \u0026ldquo;self-substrate\u0026rdquo; design for Type I PS does not require consideration on the type and concentration of substrates, as well as their redox potential and binding affinity with PSs. This design strategy offers a more convenient and efficient pathway for constructing Type I PSs.\u003c/p\u003e \u003cp\u003eTo facilitate biomedical applications, an amphiphilic Pc molecular structure has been designed to prepare a relatively stable Pc aggregate (NanoNMO) under physiological conditions. The results of \u003cem\u003ein vitro\u003c/em\u003e studies have demonstrated the excellent photocytotoxicity of NanoNMO under both normoxic and hypoxic conditions. The results of \u003cem\u003ein vivo\u003c/em\u003e experiments have revealed the remarkable tumor targeting ability and phototherapeutic efficacy of NanoNMO. Notably, NanoNMO mediated PDT not only triggers the anti-tumor immune response but also synergizes with PD-1 antibody to inhibit systemic tumor growth through enhancing the infiltration of CTL cells in tumor sites. This contribution pioneers the investigation of combining highly efficient PSs with ICIs under hypoxic conditions, providing a reference for the design of PSs combined with immunotherapy to effectively inhibit the growth and metastasis of hypoxic tumors, garnering significant implications.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eMaterials\u003c/p\u003e\n\u003cp\u003eReactions were conducted under a nitrogen atmosphere to minimize side reaction. Methylene blue (MB) was brought from TCI, Shanghai, China. 4-trifluoromethoxyphenol, Cremophor EL (CEL), p-hydroxybenzophenone, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and chlorin e6 (Ce6) were brought from Alfa, Shanghai, China. Dihydroethidium (DHE), ctDNA, aminophenyl fluorescein (APF) were purchased from Sigma-Aldrich China. Singlet oxygen sensor green (SOSG) and ROS-ID\u0026trade; hypoxia/oxidative stress detection kit were purchased from Enzo Life Sciences Inc. Fetal bovine serum (FBS) was bought from HyClone, Shanghai, China. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was bought from Sigma, Shanghai, China. Calcein-AM/PI cell flow cytometry kit and Annexia V-FITC/PI cell flow cytometry kit were obtained from Solarbio, Beijing, China. The TNF-\u0026alpha; and IFN-\u0026gamma; ELISA kits were obtained from Shanghai Enzyme Biotechnology Co., Ltd. \u0026alpha;PD-1 antibodies were purchased from BioX cell Co., Ltd. USA. The anti-CD45-FITC, anti-CD8-PerCP-Cy5.5, anti-CD4-APC and anti-CD3-PE were purchased from BD Biosciences, USA.\u003c/p\u003e\n\u003cp\u003eFabrication of phthalocyanine aggregates (NanoPcs) and phthalocyanine monomers (MonoPcs)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFabrication of NanoPcs.\u003c/strong\u003e NanoPcs were prepared using two different methods. Method 1, a 2 mM solution of Pc in THF was directly added to 1995 \u0026micro;L of water. The mixture underwent 6 pumping cycles at a constant speed using a 1000 \u0026micro;L pipette gun. Method 2, the THF solution of Pc (2 mM) was added to 1995 \u0026micro;L of water under ultrasonic conditions and kept under ultrasonic action for 10 s without any other actions. The final concentration of NanoPcs was 5 \u0026micro;M. Specifically, NanoNMe, NanoNH, NanoOMe and NanoNMO were fabricated using Method 2, while the other NanoPcs were fabricated using Method 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFabrication of MonoPcs.\u003c/strong\u003e MonoPcs were prepared using the following method: A 2 mM solution of Pc in THF was initially mixed with 20 \u0026micro;L of CEL. The resulting mixture was further diluted with 1975 \u0026micro;L of water and underwent 6 pumping cycles at a constant speed using a 1000 \u0026micro;L pipette gun. The final concentration of MonoPcs was 5 \u0026micro;M, with the final proportion of CEL being 1% (v:v).\u003c/p\u003e\n\u003cp\u003eMeasurement of ROS properties\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS detection.\u003c/strong\u003e The ROS generation efficiencies of photosensitizers were evaluated by the specific capture agent DCFH. Photosensitizers (5 \u0026micro;M) were dissolved with DCFH (10 \u0026micro;M) in a water solution. The mixture was than irradiated with \u0026gt;\u0026thinsp;610 nm light (5 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). After various irradiation times, fluorescence intensity (excited at 488 nm) of the mixture was recorded. The fluorescence intensities at 525 nm of different irradiation time were recorded to obtain the slope of fluorescence emission. The relative ROS generations of NanoPcs or MonoPcs were determined comparing them with the reference samples (MB). The probe slope of MB was considered as 1, and the slope ratios were served as the ordinate.\u003c/p\u003e\n\u003cp\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e \u003cstrong\u003edetection.\u003c/strong\u003e The O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation efficiencies of photosensitizers were evaluated by the specific capture agent DHE and ctDNA. Photosensitizers (5 \u0026micro;M) were dissolved with DHE (50 \u0026micro;M) and ctDNA (250 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in a water solution. The mixture was than irradiated with \u0026gt;\u0026thinsp;610 nm light (5 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). After different irradiation times, fluorescence intensity (excited at 510 nm) of the mixture was recorded. The fluorescence intensities at 599 nm of different irradiation time were recorded to obtain the slope of fluorescence emission. The relative O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generations of NanoPcs or MonoPcs were determined comparing them with the reference samples (MB). The probe slope of MB was considered as 1, and the slope ratios were served as the ordinate.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u0026nbsp;\u003cstrong\u003e\u0026bull;\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eOH detection.\u003c/strong\u003e The \u003csup\u003e\u0026bull;\u003c/sup\u003eOH generation efficiencies of photosensitizers were evaluated by the specific capture agent APF. Photosensitizers (5 \u0026micro;M) were dissolved with APF (10 \u0026micro;M) in a water solution. The mixture was than irradiated with \u0026gt;\u0026thinsp;610 nm light (5 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). After different irradiation times, fluorescence intensity (excited at 490 nm) of the mixture was recorded. The fluorescence intensities at 515 nm of different irradiation time were recorded to obtain the slope of fluorescence emission. The relative \u003csup\u003e\u0026bull;\u003c/sup\u003eOH generations of NanoPcs or MonoPcs were determined comparing them with the reference samples (MB). The probe slope of MB was considered as 1, and the slope ratios were served as the ordinate.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u0026nbsp;\u003cstrong\u003e1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u0026nbsp;\u003c/sub\u003e \u003cstrong\u003edetection.\u003c/strong\u003e The \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation efficiencies of photosensitizers were evaluated by the specific capture agent SOSG. Photosensitizers (5 \u0026micro;M) were dissolved with SOSG (5 \u0026micro;M) in a water solution. The mixture was than irradiated with \u0026gt;\u0026thinsp;610 nm light (5 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). After different irradiation times, fluorescence intensity (excited at 488 nm) of the mixture was recorded. The fluorescence intensities at 520 nm of different irradiation time were recorded to obtain the slope of fluorescence emission. The relative \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generations of NanoPcs or MonoPcs were determined comparing them with the reference samples (MB). The probe slope of MB was considered as 1, and the slope ratios were served as the ordinate.\u003c/p\u003e\n\u003cp\u003eMeasurement of fs-TA\u003c/p\u003e\n\u003cp\u003eThe ultrafast fs-TA measurements were conducted under ambient conditions using a fs pump-probe system in conjunction with an amplified laser setup\u003csup\u003e53\u003c/sup\u003e. The setup included a mode-locked Ti: sapphire seed laser (Spectra Physics, Maitai) coupled with a regenerative amplifier (Spitfire Pro, Spectra Physics) and a high-powered laser (3 mJ, ~\u0026thinsp;35 fs, Empower, Spectra Physics) for pumping and amplification. The amplified 800 nm output was divided into two parts, with the majority of the beam (~\u0026thinsp;85%) passing through an optical parametric amplifier (TOPAS prime, Spectra Physics) to generate pump pulses with tunable wavelengths. The remaining portion of the beam was used to generate the white light continuum probe and reference pulses (400\u0026ndash;600 nm) after traversing an optical delay line and passing through photosensitizer solutions. In this setup, the probe beam passed through the sample, while the reference beam went straight to the reference spectrometer. To obtain fs-TA spectra with and without the pump pulses, a chopper modulating the pump pulse was used. An optical fiber, connected to a multichannel spectrometer equipped with a CMOS sensor, captured the changes in intensity of the probe/reference beam caused by the pump pulses. The optical delay line, with a maximum delay of approximately 3 ns, was adjusted during the measurements. The acquired spectral profiles were subsequently processed using Surface Xplorer software.\u003c/p\u003e\n\u003cp\u003eMeasurement of electrochemical properties\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCyclic voltammetry measurement.\u003c/strong\u003e Cyclic voltammetry experiment was performed using a three-electrode system, where the working electrode was a glassy carbon electrode, the counter electrode was a Pt wire electrode, and the reference electrode was an Ag/AgCl electrode. Measurements were carried out in DMF containing 0.1 M (n-Bu)\u003csub\u003e4\u003c/sub\u003eN\u003csup\u003e+\u003c/sup\u003ePF\u003csup\u003e6\u0026ndash;\u003c/sup\u003e. The scan rate used was 100 mV\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Ferrocene was employed as an external reference. The feasibility of photoinduced symmetry breaking charge separation (SBCS) can be estimated by evaluating the Gibbs-free energy (∆G). The calculation of ∆G can be accomplished using the Rehm-Weller equation (Eq. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMott\u0026thinsp;\u0026minus;\u0026thinsp;Schottky (MS) analysis.\u003c/strong\u003e Mott-Schottky analysis was performed using a three-electrode system, where the working electrode consisted of an indium tin oxide (ITO) glass electrode adhered with lyophilized NanoNMO or NanoCN, the counter electrode was a Pt wire electrode, and the reference electrode was an Ag/AgCl electrode. The measurements were carried out in an aqueous solution of 0.2 M Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransient photocurrent and electrochemical impedance spectroscopy measurement.\u003c/strong\u003e The transient photocurrent and electrochemical impedance spectroscopy measurement were performed using a three-electrode system, where the working electrode consisted of an ITO glass electrode adhered with lyophilized NanoNMO or NanoCN, the counter electrode was a Pt wire electrode, and the reference electrode was an Ag/AgCl electrode. The measurements were carried out in an aqueous solution of 0.2 M Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. For the transient photocurrent measurements, the working electrode was irradiated with a white light (180 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), with an interval of 20 s between each irradiation. For the electrochemical impedance spectroscopy measurements, the working electrode was consistently irradiated with a white light (180 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConductivity measurement.\u003c/strong\u003e The conductivity measurement was performed in aqueous solutions containing NMe, NanoNMe, and NanoCN at a concentration of 1 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. As a control, water was used as the reference solution. The solution was irradiated with a white light (180 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), and the conductivity values were recorded with an interval of 30 s.\u003c/p\u003e\n\u003cp\u003eMeasurement of redox properties\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValence band (VB) measurement.\u003c/strong\u003e The UPS measurement was performed using the He excitation energy (h\u0026nu;\u0026thinsp;=\u0026thinsp;21.22 eV). The value of VB was determined by fitting a straight line into the leading edge of the spectra. The VB (vs NHE) of NanoNMe can be calculated according to the equation S1.\u003c/p\u003e\n\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv id=\"FileID_Equ1\" class=\"mathdisplay\"\u003e$${E}_{NHE}={E}_{f}+{\\Phi }-4.44$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003eS1\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eWhere \u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003eNHE\u003c/em\u003e\u003c/sub\u003e is the VB potential of NanoNMe; \u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e is the binding energy of Fermi edge (E\u003csub\u003ef\u003c/sub\u003e); \u003cem\u003e\u0026Phi;\u003c/em\u003e is the work function. \u003cem\u003e\u0026Phi;\u003c/em\u003e can be calculated according to \u0026Phi;\u0026thinsp;=\u0026thinsp;h\u0026nu; - (cutoff - E\u003csub\u003ef\u003c/sub\u003e), where the value of \u003cem\u003ecutoff\u003c/em\u003e was obtained as 15.93 eV. \u003cem\u003e\u0026Phi;\u003c/em\u003e was calculated as 5.47 eV. The \u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003eNHE\u003c/em\u003e\u003c/sub\u003e of NanoNMe was calculated as 1.21 eV (vs NHE).\u003c/p\u003e\n\u003cp\u003eComputational Details\u003c/p\u003e\n\u003cp\u003eGround-state geometries optimizations and time-dependent density functional theory (TD-DFT) calculations were carried out at B3LYP\u003csup\u003e43\u003c/sup\u003e/6-31G* method. All calculations were carried out using the Gaussian 16 package. The polarizable continuum model\u003csup\u003e44\u003c/sup\u003e with default parameters was used to implicitly consider solvation effects of water solution.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture.\u003c/strong\u003e The 4T1 cells were grown in DMEM at 37 ℃ in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere, with the medium supplemented with 10% FBS, Bispecific Antibody (0.02 units\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and Amphotericin (0.25 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of the intracellular ROS production\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e54\u003c/strong\u003e\u0026nbsp;\u003c/sup\u003e. Approximately 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e 4T1 cells suspension in DMEM cell culture medium (0.4 mL) were inoculated on confocal dishes. To construct the \u003cem\u003ein vitro\u003c/em\u003e normoxic and hypoxic conditions, a part of 4T1 cells was cultured in an incubator chamber at 37 ℃ with a humidified, 21% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 74% N\u003csub\u003e2\u003c/sub\u003e atmosphere for 12 h. Another 4T1 cells were cultured in an incubator chamber at 37 ℃ with a humidified, 2% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 93% N\u003csub\u003e2\u003c/sub\u003e atmosphere for 12 h. After the removal of the medium, the cells were incubated in fresh medium containing Ce6 or NMO or NanoNMO (4 \u0026micro;M) in darkness for 1.5 h. Subsequently, DCFH-DA was added at a final concentration of 10 \u0026micro;M and incubated for 30 min. The cells were then washed three times with PBS, replenished with 500 \u0026micro;L of culture medium, and utilized a 685 nm laser (15 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for 5 min. Upon removing the culture medium, the cells underwent a PBS rinse before imaging via a LEICA TCS SPE confocal microscope. The probe was excited at 488 nm and monitored at 500\u0026ndash;600 nm. The images were then digitized and analyzed using SPE ROI Fluorescence Statistics software. The average intracellular fluorescence intensities were recorded for a total of 50 cells in each sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of the intracellular O\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003csup\u003e\u003cstrong\u003e\u0026bull;\u0026minus;\u003c/strong\u003e\u0026nbsp;\u003c/sup\u003e \u003cstrong\u003eproduction\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e54\u003c/strong\u003e\u003c/sup\u003e. Approximately 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e 4T1 cells suspension in DMEM cell culture medium (0.4 mL) were inoculated on confocal dishes. 4T1 cells were devided two parts to culture under both normoxic and hypoxic conditions for 12 h. After the removal of the medium, the cells were incubated in fresh medium containing Ce6 or NMO or NanoNMO (4 \u0026micro;M) in darkness for 1.5 h. DHE was added at a final concentration of 10 \u0026micro;M and incubated for 30 min. The cells were then washed three times with PBS, replenished with 500 \u0026micro;L of culture medium, and utilized a 685 nm laser (15 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for 5 min. Upon removing the culture medium, the cells underwent a PBS rinse before imaging via a LEICA TCS SPE confocal microscope. The probe was excited at 532 nm and monitored at 540\u0026ndash;650 nm. The images were then digitized and analyzed by SPE ROI Fluorescence Statistics software. The average intracellular fluorescence intensities were recorded for a total of 50 cells in each sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e \u003cstrong\u003ephotodynamic activity assay.\u003c/strong\u003e 4T1 cells (about 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well) were cultured in 96-wellplates and devided two parts to culture under both normoxic and hypoxic conditions for 12 h at 37 ℃ in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The previous medium was aspirated and discarded, and 100 \u0026micro;L per well of Ce6 or NMO or NanoNMO with the specified concentration gradient was subsequently added. After 2 h of cultivation and removal of the culture medium, the cells were washed with PBS and replenished with 100 \u0026micro;L of culture medium. Subsequently, the cells were exposed to ambient temperature illumination for 0.5 h. The light source utilized a 685 nm laser (30 min, 15 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), the total fluence was 27 J\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. Cell viability was determined by the MTT assay. Following illumination, the cells were incubated overnight at 37 ℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. A 40 \u0026micro;L MTT solution in PBS (2.5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was added to each well, and the cells were subsequently incubated for 4 h under the same conditions. After removing the medium, 150 \u0026micro;L of DMSO was added to each well. The 96-well plate was gently agitated on a Tecan M200Pro microplate reader at room temperature for 20 s prior to measuring the absorbance at 490 nm. The cell viability was then detected by the equation S2:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"550\" height=\"48\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere \u003cem\u003eAi\u003c/em\u003e is the absorbance of the \u003cem\u003ei\u003c/em\u003eth data (\u003cem\u003ei\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1, 2, \u0026hellip;, n), \u003cem\u003eĀ\u003c/em\u003e\u003csub\u003e\u003cem\u003econtrol\u003c/em\u003e\u003c/sub\u003e is the average absorbance of the control wells, in which the Pc is absent, and \u003cem\u003en\u003c/em\u003e (\u0026ge;3) is the number of the data points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLive/Dead cell co-staining.\u003c/strong\u003e 4T1 cells (about 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well) were cultured in 96-wellplates and devided two parts to culture under both normoxic and hypoxic conditions for 12 h at 37 ℃ in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The previous medium was aspirated and discarded, and 100 \u0026micro;L per well of NMO or NanoNMO were added at concentrations of 0.4 or 0.8 \u0026micro;M, respectively. After 2 h of cultivation and removal of the culture medium, the cells were washed with PBS and replenished with 100 \u0026micro;L of culture medium. Subsequently, the cells were exposed to ambient temperature illumination for 0.5 h. The light source utilized a 685 nm laser (30 min, 15 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), the total fluence was 27 J\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. After the aforementioned treatments, 4T1 cells were additionally stained using the Calcein-AM/PI Double Stain Kit\u003csup\u003e54\u003c/sup\u003e following the instructions provided in the manual. The excitation wavelength was 488 nm, and the emission wavelength was set to 505\u0026ndash;545 nm for the green channel and 600\u0026ndash;700 nm for the red channel.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse models.\u003c/strong\u003e Mouse breast cancer (4T1) cells were acquired from the China Center for Type Culture Collection (CCTCC, Shanghai, China), and the mice were procured from Wushi Animal Co. Ltd. (Fu Zhou, China). All animal studies were carried out in compliance with guidelines of the Animal Ethics Committee of Fuzhou University (2023-SG-001), and also approved by the committee. To initiate a subcutaneous tumor model, approximately 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e 4T1 cells in 200 \u0026micro;L were subcutaneously inoculated on the flank of mice weighing 18\u0026ndash;20 g.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence imaging.\u003c/strong\u003e The mice received an intravenous injection of either NMO or NanoNMO (200 \u0026micro;M, 100 \u0026micro;L) in H\u003csub\u003e2\u003c/sub\u003eO. \u003cem\u003eIn vivo\u003c/em\u003e fluorescence images of the mice were performed on a IVIS Lumina III imaging system. Both NMO and NanoNMO were excited at 640 nm and monitored at 660\u0026ndash;750 nm. After the \u003cem\u003ein vivo\u003c/em\u003e imaging studies, the mice were euthanized 36 h after the injection. The heart, liver, spleen, lung, kidney, tumor and skin were extracted and imaged. Data was processed through the IVIS system. Three mice were utilized for each experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhotoacoustic imaging.\u003c/strong\u003e The mice received an intravenous injection of either NMO or NanoNMO (200 \u0026micro;M, 100 \u0026micro;L) in H\u003csub\u003e2\u003c/sub\u003eO. \u003cem\u003eIn vivo\u003c/em\u003e photoacoustic images of the mice were performed on a photoacoustic computerized tomography scanner (MSOT 256-TF, Germany). Data was processed through the iThera Medical system. Three mice were utilized for each experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003ePDT effect assay.\u003c/strong\u003e To evaluate the \u003cem\u003ein vivo\u003c/em\u003e PDT effects of NanoNMO, the mice received an intravenous injection of NanoNMO (200 \u0026micro;M, 100 \u0026micro;L) in H\u003csub\u003e2\u003c/sub\u003eO. 8 h post-injection, the mice underwent irradiation at the tumor site using a 685 nm laser (100 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for a duration of 5 min. As a control, blank mice were treated with saline only (100 \u0026micro;L). To evaluate the therapeutic efficacy, the tumor size was measured using a digital caliper every alternate day. Daily photographs were taken for a duration of 14 d. Tumor volumes were calculated as 0.5 \u0026times; length \u0026times; width \u0026times; width.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003ecytokine release.\u003c/strong\u003e The tumor-bearing mice received an intravenous injection of NanoNMO (200 \u0026micro;M, 100 \u0026micro;L) in H\u003csub\u003e2\u003c/sub\u003eO. 8 h post-injection, the mice underwent irradiation at the tumor site using a 685 nm laser (100 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for a duration of 5 min. The blood was collected 12 h after PDT and the levels of serum TNF-\u0026alpha; and IFN-\u0026gamma; were determined by enzyme-linked immunosorbent assay (ELISA) to evaluate the acute inflammation induced by the treatment\u003csup\u003e8, 55\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003ephotodynamic immunotherapy (PIT) effect assay.\u003c/strong\u003e To evaluate the \u003cem\u003ein vivo\u003c/em\u003e PIT effects of NanoNMO, the mice received an intravenous injection of NanoNMO (200 \u0026micro;M, 100 \u0026micro;L) in H\u003csub\u003e2\u003c/sub\u003eO firstly. 8 h post-injection, the mice underwent irradiation at the tumor site using a 685 nm laser (100 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for a duration of 5 min. Then \u0026alpha;PD-1 was intravenously injected (2.5 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 24 h and 72 h after injection, respectively. To evaluate the therapeutic efficacy, the tumor size was measured using a digital caliper every alternate day. Daily photographs were taken for a duration of 14 d. Tumor volumes were calculated using the formula 0.5 \u0026times; length \u0026times; width \u0026times; width.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry assay for immune response.\u003c/strong\u003e All mice were euthanized on the 7th day post-treatment, and the bilateral tumors and spleens were excised. The tumors and spleens were subsequently digested using a DMEM mixture that included 0.2% collagenase IV, 0.1% hyaluronidase, and 0.002% DNase I at 37\u0026deg;C for 1 h, followed by mechanical grinding using the rubber end of a syringe. The resulting cell suspension was sieved through nylon mesh filters and rinsed with PBS. The single-cell suspension was then incubated with anti-CD45-FITC, anti-CD8-APC, anti-CD4-PerCP-Cy5.5, and anti-CD3-PE, followed by shaking for 30 min at 4\u0026deg;C\u003csup\u003e8\u003c/sup\u003e. After centrifugation at 1000 rpm for 5 min, the supernatant was discarded, and the cell suspension was diluted with sterilized PBS. Cell acquisition was performed using a NovoCyte Flow Cytometer (ACEA Biosciences, USA), and data analysis was conducted using the FlowJo software.\u003c/p\u003e\n\u003cp\u003eStatistical information\u003c/p\u003e\n\u003cp\u003eStatistical comparisons were made using two-tailed Student\u0026rsquo;s t test (between two groups). All statistical analysis was performed using SPSS. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically difference; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 was considered to be significant statistically difference; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 was considered to be extremely significant statistically difference. Quantitative data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated in this study are available within the Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the National Natural Science Foundation of China (Grant Nos. 22078066, T2322004 and 22178065).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH. L.: Performed the synthesis and the \u003cem\u003ein vivo\u003c/em\u003e test, analyzed the data, and prepared the manuscript draft; Z. L.: Performed the \u003cem\u003ein vitro\u003c/em\u003e test and analyzed the data; X. Z.: Performed the \u003cem\u003ein vivo\u003c/em\u003e test and analyzed the data; Y. X.: Assisted the \u003cem\u003ein vivo\u003c/em\u003e test; G. T.: Assisted the \u003cem\u003ein vitro\u003c/em\u003e test; Z. W.: Performed the computational calculation; Y.-Y. Z.: Assisted the \u003cem\u003ein vivo\u003c/em\u003e test; M.-R. K.: Assisted the synthesis; B.-Y. Z.: Assisted the biological test; S. H., Assisted the computational calculation; J.-D. H.: Conceptualization of the \u0026ldquo;Semiconductor-like\u0026rdquo; photocatalysts, design of the project, supervision, review \u0026amp; editing, funding acquisition; X. L.: Conceptualization, design of the project and experiments, analysis of data, writing, funding acquisition.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFelsher, D.W. Cancer revoked: Oncogenes as therapeutic targets. \u003cem\u003eNat. Rev. 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Soc.\u003c/em\u003e \u003cstrong\u003e143\u003c/strong\u003e, 13980-13989 (2021).\u003c/li\u003e\n\u003cli\u003eZheng, B.-Y., et al. Phthalocyanine-based photosensitizers combined with anti-PD-L1 for highly efficient photodynamic immunotherapy. \u003cem\u003eDyes Pigm.\u003c/em\u003e \u003cstrong\u003e185\u003c/strong\u003e, 108907-108917 (2021).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3933352/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3933352/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhotodynamic immunotherapy (PIT) has emerged as a promising approach for efficient eradication of primary tumors and inhibition of tumor metastasis. However, most of photosensitizers (PSs) for PIT exhibit notable oxygen dependence. Herein, a concept emphasizing on transition from molecular PSs into \u0026ldquo;semiconductor-like\u0026rdquo; photocatalysts is proposed, which converts the PSs from type II photoreaction to efficient type I photoreaction. Detailed mechanism studies reveal that the nanostructured phthalocyanine aggregate (NanoNMe) generates radical ion pairs through a photoinduced symmetry breaking charge separation process, achieving charge separation through a \u0026ldquo;self-substrate\u0026rdquo; approach and leading to exceptional photocatalytic charge transfer activity. Additionally, a reformed phthalocyanine aggregate (NanoNMO) is fabricated to improve the stability in physiological environments. NanoNMO showcases outstanding photocytotoxicities under both normoxic and hypoxic conditions and exhibits remarkable tumor targeting ability. Notably, the photodynamic effect mediated by NanoNMO not only triggers the systemic anti-tumor immune response but also synergizes with PD-1 antibodies to enhance the infiltration of cytotoxic T lymphocytes into tumor sites, leading to the effective inhibition of tumor growth.\u003c/p\u003e","manuscriptTitle":"Phthalocyanine Aggregates as “Semiconductor-like” Photocatalysts for Hypoxic-Tumor Photodynamic Immunotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-11 12:47:39","doi":"10.21203/rs.3.rs-3933352/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"740c10e1-057f-4ca1-80f6-726846bd6f88","owner":[],"postedDate":"March 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29060862,"name":"Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":29060863,"name":"Physical sciences/Chemistry/Photochemistry"}],"tags":[],"updatedAt":"2025-01-03T08:07:06+00:00","versionOfRecord":{"articleIdentity":"rs-3933352","link":"https://doi.org/10.1038/s41467-024-55575-2","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-01-02 05:00:00","publishedOnDateReadable":"January 2nd, 2025"},"versionCreatedAt":"2024-03-11 12:47:39","video":"","vorDoi":"10.1038/s41467-024-55575-2","vorDoiUrl":"https://doi.org/10.1038/s41467-024-55575-2","workflowStages":[]},"version":"v1","identity":"rs-3933352","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3933352","identity":"rs-3933352","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}