Advanced searchCitation search
Skip main navigation
Open AccessCCS ChemistryRESEARCH ARTICLES21 Feb 2025

Transferrin-Mediated Nanophotosensitizer to Enhance Phototherapy with Photoacoustic Imaging Under Hypoxia

    https://doi.org/10.31635/ccschem.025.202505468

    Various obstacles [poor tissue penetration, hypoxic tumor microenvironment, and reactive oxygen species (ROS) production quenching due to aggregation] obstruct imaging-guided high-efficiency phototherapy targeting tumors. Herein, nanophthalocyanine (ZnPcN4-TF) is elaborately designed based on a synthetic phthalocyanine derivative (ZnPcN4) and nonimmunogenic transferrin (TF) through multiple noncovalent interactions for photoacoustic (PA) imaging-guided phototherapy. By conjugating electron-rich amino groups effectively suppressed fluorescence and ROS generation due to the strong photoinduced electron transfer effect, ZnPcN4 became an ideal photothermal and PA imaging agent. ZnPcN4-TF not only effectively targeted tumor sites and accumulated there, but also, surprisingly, facilitated the enhancement of ROS production via a type I process in an aggregate compared to ZnPcN4 itself, potentially due to accelerated electron transfer. Simultaneously, ZnPcN4-TF had a substantially more powerful photothermal effect than ZnPcN4 itself. Thus, this design effectively overcomes the obstacles to photodynamic therapy (aggregation-related ROS generation quenching in a hypoxic environment). Furthermore, PA imaging solves the tissue penetration challenge in optical imaging. This study provides a broader base for designing novel photosensitizers to improve phototherapy under hypoxia.

    Introduction

    Cancer remains one of the most common concerns threatening the health and life of humans worldwide. Imaging-guided therapy has attracted tremendous attention in biomedicine and precision therapy because it can precisely target lesions, reducing damage to normal tissues.1,2 In particular, there have been great advances in optical imaging-guided therapy.3,4 However, tissues heavily scatter photons, attenuating the optical signal proportionally to tissue depth, generally limiting its penetration to less than 1 mm.5 This limit severely hinders further clinical applications. Benefiting from an acoustic property that provides a deeper tissue penetration,6,7 photoacoustic (PA) imaging allows deeper imaging (above the optical diffusion limit) by resolving acoustic signal generated by a high-absorbing chromophore emitted by its thermal expansion after a short-pulse laser excitation.8–10 The sudden local increase in temperature generates an acoustic pressure wave (called the PA wave), which is detected by an external ultrasonic transducer and then resolved into an image.11 PA imaging enables high-resolution imaging over tissue that is as yet unreachable by other optical imaging modalities in vivo,12–14 since the scattering of phonons in biological tissue is weaker than that of photons.15 Meanwhile, various intrinsic chromophores,16,17 mostly hemoglobin,18 are targetable imaging objects, the administration of extrinsic PA contrast agents helps extending the imaging depth by increasing imaging contrast.19 However, many reported PA reagents are plagued by low biocompatibility, poor photostability, or insufficient extinction coefficient and photothermal conversion efficiency under the near-infrared (NIR) window illumination.

    Phototherapy refers to photodynamic therapy (PDT), photothermal therapy (PTT), or the combination of both.20 The energy of the excited photosensitizers (PSs) decays through fluorescence, reactive oxygen species (ROS) production, or thermal energy, depending on the inherent structure of each PS. ROS includes cytotoxic type I oxygen radicals [such as hydroxyl radicals and superoxide ions (O2•−)], produced by electron/hydrogen transfer and type II singlet oxygen (1O2), produced by energy transfer.21,22 However, most of the conventional PSs generate ROS through the type II mechanism, which heavily depends on oxygen concentration.23 Thus, the hypoxic character of the tumor microenvironment severely limits the therapeutic effectiveness of PDT.24–26 Although some studies have used carriers coloading oxygen or hydrogen peroxide, the preparation process is complicated and imposes a burden on delivery. Relatively speaking, type I PSs hold greater potential than type II PSs for tumor treatment since they depend less on oxygen concentration.27–29 Unfortunately, although there are some studies on type I ROS, the aggregation of PSs often restrains ROS production.30,31 Meanwhile, PTT can also overcome the oxygen dependence.32–34 However, the photothermal conversion efficiency of PSs is usually weakened by fluorescence and intersystem crossing and it is rare for PSs to have both excellent type I PDT and PTT.35 Therefore, designing a PS integrating efficient photothermal performance and enhanced type I ROS generation in aggregation with NIR absorption is warranted.

    Phthalocyanine has good tissue penetration due to its highly conjugated structure through π–π* transition.36,37 Further, phthalocyanine has various substitution sites allowing the facile modification of its optical properties.29,38 However, the strong hydrophobicity of most phthalocyanine molecules that absorb NIR light seriously impedes their use in biological applications.39 With the recent development of nanotechnology, nanoplatforms can serve as an effective solution. Self-assembly, as a “bottom-up” nanofabrication method, can solve the problem of delivery and targeting.40,41 In addition, the dense electronic structure of energy levels, resulting from the overlapping of the intermolecular orbitals of the building blocks, often alters the energy decay pathway in unexpected ways.42

    In this study, we successfully constructed a nanophthalocyanine (ZnPcN4-TF) based on the self-assembly of a zinc(II) phthalocyanine (ZnPc) derivative (ZnPcN4) and transferrin (TF) (Scheme 1a). ZnPcN4 was engineered by conjugating ZnPc with an electron-rich amino group, endowing it with a strong absorbance in the NIR tissue transparency window, together with a low fluorescence quantum yield due to photoinduced electron transfer (PET),43 allowing the absorbed light energy to be converted into heat instead of fluorescence emission. Thus, ZnPcN4 is an ideal photothermal agent for PTT and PA imaging. Inspired by natural chromoproteins, We used TF to assemble with ZnPcN4 through noncovalent interactions (hydrogen bonding, π–π stacking, and hydrophobic interaction), providing the advantages of nontoxicity, nonimmunogenicity and biodegradability. TF not only solves the dilemma of the aggregation of ZnPcN4 molecules in the physiological environment caused by strong hydrophobicity, but it also prevents ROS generation quenching in the aggregated state. In addition, ZnPcN4-TF displayed good tumor accumulation and biocompatibility. Surprisingly, ZnPcN4-TF significanatly enhanced the production of type I ROS and exhibited an excellent PTT effect, thus effectively avoiding the “Archilles’s heel” of traditional PDT. Overall, the in vitro and in vivo results demonstrated that ZnPcN4-TF had an excellent PA imaging-guided phototherapy effect under hypoxia (Scheme 1b).

    Scheme 1

    Scheme 1 | Schematic illustration of nanophthalocyanine (ZnPcN4-TF) use for antitumor phototherapy. (a) Construction of ZnPcN4-TF from ZnPcN4 and TF. (b) Activation of ZnPcN4-TF to generate type I ROS production and heat for phototherapy with PA imaging under hypoxia.

    Experimental Methods

    Materials and instruments

    apo-Transferrin human (TF), 2,7-dichlorofluorescin diacetate (DCFHDA), 9,10-anthracenedipropanoic acid (ABDA), hydroethidine (DHE), methylene blue (MB), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and dimethylsulfoxide (DMSO) were provided by Sigma-Aldrich Korea (Seoul, South Korea). calf thymus DNA (ctDNA) was obtained from Invitrogen (Carlsbad, California, United States). All reagents were used without further purification. ZnPcN4 was obtained via previously described procedures.44

    All absorption spectra were measured with a Thermo Scientific Evolution 201 UV–Visible spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, United States), and fluorescence measurements were recorded using an FS-2 fluorescence spectrometer (Scinco, Seoul, South Korea). The dynamic light scattering (DLS) measurement was performed using a Nano-ZS instrument (Malvern, Worcestershire, United Kingdom). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images were captured using a JMS-6700F (JEOL) and an electron microscope (JEOL-2100F) (JEOL Ltd., Tokyo, Japan) operating at 200 kV, respectively. All confocal laser scanning microscopy images were obtained using an Olympus Fluoview FV1200 confocal laser scanning microscope (Olympus, Tokyo, Japan).

    Preparation of ZnPcN4-TF

    ZnPcN4 (1 mM in DMSO) and TF [0.2 mM in phosphate buffer solution (PBS)] were first prepared as stock solutions. 330 μL of TF stock solution was diluted in 470 μL PBS, and then 200 μL of the ZnPcN4 stock solution was added under ultrasonic stirring, yielding uniformly dispersed ZnPcN4-TF.

    Photothermal properties

    A ZnPcN4-TF aqueous solution (1 mL, 10 μM) was placed in a quartz cuvette and then irradiated for 10 min with a 655 nm laser (1.5 W/cm2). Alternatively, aqueous solutions containing different concentrations were irradiated with a 655 nm laser at various power values. The temperature was tested with a thermocouple probe combined with a digital thermometer or thermal imager. Pure water was used as a control. The photothermal conversion efficiency was calculated as our previously described procedure.45

    Analyses of ROS generation

    DCFHDA was used as the fluorescence probe for ROS generation in water. Samples (ZnPcN4-TF, ZnPcN4, MB, and TF, 10 μM) were mixed separately with 20 μM DCFHDA. Then, the mixed solutions were irradiated with red light for various lengths of time and the fluorescence intensity was recorded.

    Analyses of singlet oxygen generation

    ABDA was chosen as a probe for the detection of 1O2 generation. Samples (ZnPcN4-TF, ZnPcN4, MB and TF, 10 μM) were mixed separately with 10 μM ABDA. Then, the mixed solutions were irradiated with red light for various times, and the absorbance was recorded.

    Analyses of superoxide anion generation

    DHE was used as a probe for superoxide anion generation. Since DHE can be oxidized by superoxide to form ethidium, which can emit red fluorescence at approximately 570 nm upon DNA intercalation. Samples (ZnPcN4-TF, ZnPcN4, MB, and TF, 10 μM) and DHE (50 μM) were dissolved in an aqueous DNA solution (250 μg/mL). The fluorescence intensity was record after various irradiation.

    Cell culture and live/dead cell staining

    MCF-7 cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in EMEM (Eagle’s Minimum Essential Medium containing non-essential amino acid) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.

    Live/dead cell staining. MCF-7 cells were preincubated with ZnPcN4-TF for 24 h and then subjected to light irradiation. After that, the cells were added with calcein AM (1 μM) and propidium iodide (PI) (5 μM) further incubated for 0.5 h. Then the cells were observed under a confocal microscope.

    PA imaging equipment

    A multispectral acoustic-resolution PA imaging system was used for both in vitro and in vivo PA imaging.46 A wavelength-tunable, 10-Hz Q-switched pulsed laser system (Surelite OPO Plus; Continuum, Milpitas, California, United States) excited the target and a single-element 5-MHz ultrasound transducer (V308; Olympus NDT Inc., Waltham, Massachusetts, Unites States) received a time-resolved PA signal. Objects were scanned in the X and Y directions with a 0.1 mm step.

    PA in vitro experiments

    PA spectra were obtained for ZnPcN4-TF, ZnPcN4, and MB as controls. Samples were prepared in 10% DMSO, sonicated for 10 min, and injected into 0.3-mm silicone microtubings (Silastic™; Dow Chemical Company, Wilmington, Delaware, United States) fixed in a holder. The holder was submerged in water, and cross-sectional PA images were acquired at wavelengths from 680 to 800 nm, in 10 nm increments. From the maximum amplitude projection (MAP) images, tubing regions were selected, and the mean and standard deviation of the top 10% PA amplitudes were calculated to generate the PA spectrum.

    To generate concentration-PA yield curves, the procedure was repeated at 710 nm for various concentrations of ZnPcN4-TF or ZnPcN4. To assess dye solubility effects on PA imaging, 200 μM solutions of ZnPcN4 or ZnPcN4-TF were imaged in a 96-microwell plate at 710 nm. Pixel amplitudes from individual wells were extracted and presented as PA amplitude histograms.

    72-hour longitudinal in vivo PA imaging

    All animal treatments and experimental procedures complied with the Institutional Animal Care and Use Committee (IACUC) protocols of Pohang University of Science and Technology (POSTECH) (Approval No.: POSTECH-2022-0052-C3-R1). Five-week-old nude Balb-c mice were subcutaneously inoculated with 1 × 107 MCF-7 human breast cancer cells in their right dorsal flank. The tumors were cultured for three weeks at minimum, and the xenograft models with tumor volumes exceeding 200 mm3 were selected for the in vivo PA experiment. For PA imaging, mice were anesthetized with 5% isoflurane initially, then maintained at 1% isoflurane with a 0.4 L/min flow rate throughout the imaging procedure. Each mouse was placed on a heating pad in right lateral recumbent position, exposing its right flank for imaging. The scanning field-of-view was set to cover the whole range of the body, typically 40 × 60 mm. The whole-body PA images were acquired under two wavelengths: 710 nm as peak absorption (PA710nm) and 800 nm as background signal-equivalent wavelength (PA800nm). After preinjection imaging, 100 μL of 200 μM ZnPcN4-TF or ZnPcN4 was intravenously injected via tail vein injection, and whole-body imaging was repeated at each imaging timepoint (1, 4, 8, 24, 48, and 72 h postinjection). We used PA ratiometry (PA710nm/PA800nm) to effectively demonstrate the whole-body dye accumulation. From the MAP images, liver and tumor sites were segmented, and the mean and standard error of the upper 10% pixel amplitudes were longitudinally monitored over time.

    In vivo photothermal efficacy experiments

    All the animal experiments complied with the POSTECH IACUC protocols. The 1 × 107 MCF-7 cells suspended in 100 μL of cell culture media were inoculated subcutaneously at the right flank of Balb/c nu/nu (five-week-old, female). Mice were randomly divided into five groups (n = 4 for each group). When the average tumor volume reached about 200 mm3, 200 μL of samples (PBS, 200 μM ZnPcN4, 200 μM ZnPcN4-TF) were intravenously injected. The tumor sites were irradiated with a 660 nm laser (SDL-660-LM-1000T: Shanghai Dream Lasers Technology, Shanghai, China) for 10 min after 8 h sample injection (low power: 0.2 W/cm2, high power: 2.0 W/cm2). During the laser irradiation, the temperature of the mice was monitored by a thermal imaging camera (forward-looking infrared (FLIR), FLIR-E6390, Wilsonville, Oregon, United States). The data were presented as the mean ± SD, and one-way analysis of variance (ANOVA) was used for statistical analyses by GraphPad Prism 7 (San Diego, California, United States).

    In vivo phototherapeutic experiments

    The MCF-7 cells (1 × 107) were inoculated subcutaneously into the right flank of five -week-old Balb/c nu/nu female mice. Mice were randomly divided into eight groups (five mice per group) when the average tumor volume reached about 200 mm3. Then each sample (PBS, 200 μM ZnPcN4 (suspended in 10% DMSO and 90% PBS), 200 μM ZnPcN4-TF (suspended in PBS), all at 200 μL) was intravenously injected. For the laser irradiation groups, 660 nm laser was irradiated on the tumor site for 10 min after 8 h sample injection. At this point, the laser power was controlled for the expected therapeutic effect, and low power (0.2 W/cm2) was used to yield only the PDT effect, while high power (2.0 W/cm2) was used to provide the combined PDT and PTT effect. The tumor volume and body weight were monitored for three weeks. The tumor volume was calculated by the formula ab2/2, where a is the longest dimension and b is the shortest dimension. Three weeks later, the tumor-mice were sacrificed and the tumors were dissected and fixed in 10% neutral buffered formalin. The data were presented as the mean ± SD of independent experiments. All results were analyzed by performing one-way or two-way ANOVA (*P > 0.05, **P > 0.01, ***P > 0.001, and ****P > 0.0001) in GraphPad Prism 7.

    Biosafety assessment of ZnPcN4-TF

    Each sample (PBS, ZnPcN4, and ZnPcN4-TF) was intravenously injected into Balb/c nu/nu mice (5–6-week-old, female). After 21 days, mice were sacrificed and major organs (heart, lung, liver, kidney, spleen) were collected. The organs were fixed in 10% neutral buffered formalin and stained with hematoxylin and eosin.

    Statistical analysis

    All statistical analyses were performed using Excel, and all results were analyzed by performing one-way or two-way ANOVA (P > 0.05) in GraphPad Prism 7.

    Data were presented as the means ± SD of independent experiments. Statistical significance was assessed using an independent-samples t-test. A P-value >0.05 was considered statistically significant.

    Results and Discussion

    Photophysical properties of phthalocyanine derivatives

    Phthalocyanine is a good PS candidate for phototherapy due to its tunable structure, long absorbance wavelength, and high extinction coefficient. The structure-inherent strategy can regulate the pathway of photochemical properties of PSs, which can be utilized for phototherapy.47 The phthalocyanine derivative (ZnPcN4) ( Supporting Information Figure S1a) was synthesized by conjugating ZnPc with an electron-rich amino group based on the structure-inherent regulation of the energy pathway strategy.48 We previously reported the synthesis scheme and characterizations of this compound.44,49

    We first investigated the photophysical properties of ZnPcN4. ZnPcN4 has a maximum absorption peak around 700 nm ( Supporting Information Figure S1b) with a molar extinction coefficient of 2.65 × 105 cm−1 L mol−1, confirming that ZnPcN4 has strong absorbance in the NIR tissue transparency window. Meanwhile, the fluorescence of ZnPcN4 is very low ( Supporting Information Figure S1c), which can be attributed to the PET effect. Since the pathway of fluorescence competes with heat generation, this compound can convert the absorbed light energy into heat instead of fluorescence emission. Thus, ZnPcN4 is an ideal photothermal agent for PTT and PA imaging.

    Preparation and characterization of ZnPcN4-TF

    Due to its strong hydrophobicity, ZnPcN4 easily aggregates and precipitates in PBS ( Supporting Information Figure S2a), which severely limits its application in the physiological environment. The development of nanoscience in phototherapy has been a major stride forward in the resolution of some of the challenges associated with classic PSs, like severe aggregation. Since TF provides multiple noncovalent bond interaction sites, allowing the assembly with hydrophobic PSs, it was used to assemble with ZnPcN4. The optical imaging demonstrated that TF prevented the aggregation and precipitation of ZnPcN4 ( Supporting Information Figure S2a). To confirm the formation of well-dispersed nanoparticles, electronic absorbance and DLS assays were performed ( Supporting Information Figure S2b–d). After adjusting the feed ratio of ZnPcN4 to TF, the absorption peak of ZnPcN4 remained unaffected. However, when the feed ratio of ZnPcN4 to TF was increased to 1:1 ( Supporting Information Figure S2b), the absorption peak at approximately 280 nm intensified. Furthermore, when the ZnPcN4:TF ratio reached 1:3, an obvious size distribution of TF was observed ( Supporting Information Figure S2c,d), which could be attributed to the presence of free TF. To determine the optimal ratio of ZnPcN4 to TF, we measured the fluorescence of TF at different ZnPcN4 concentrations ( Supporting Information Figure S3a). The fluorescence intensity decreased linearly as the ZnPcN4:TF ratio increased, until the ratio reached 3, indicating the presence of free TF before the ratio was reached ( Supporting Information Figure S3b). Beyond a ratio of 3, the fluorescence intensity was affected by the weak interactions between ZnPcN4 and TF molecules. Therefore, we supposed that the optimal ZnPcN4:TF ratio would be 3:1, and adopted it as ZnPcN4-TF in this study. The binding driving force can be attributed to the interaction of the native hydrophobic region of TF with the ZnPcN4 hydrophobic backbone and π–π stacking.

    To further understand the assembly of ZnPcN4-TF, the property of ZnPcN4-TF was measured. Compared with the free ZnPcN4, the characteristic absorption peaks of ZnPcN4-TF at around 700 nm were smaller and broader, which is mainly attributed to hydrophobic interactions and π–π stacking (Figure 1a).50,51 The DLS showed that the nanoparticles were about 100 nm, with a narrow size distribution (PdI: 0.159) (Figure 1b). Further, the morphology of ZnPcN4-TF was observed by SEM (Figure 1c) and TEM (Figure 1d). The results were consistent with the DLS results. These size distributions are suitable for an enhanced permeability and retention effect. Since the stability of nanoparticles is an essential requirement for application in a biological environment, we then verified the stability of ZnPcN4-TF. After one day, the nanoparticle size at five times dilution barely changed ( Supporting Information Figure S4a), indicating that ZnPcN4-TF has good resistance to dilution over time. Next, we added 10% fatal bovine serum to simulate the physiological environment ( Supporting Information Figure S4b). There was almost no discernible change, confirming the good stability of ZnPcN4-TF in a biological environment.

    Figure 1

    Figure 1 | Preparation of ZnPcN4-TF. (a) Electronic absorption of ZnPcN4-TF and monomeric ZnPcN4/TF. (b) DLS, (c) SEM images, and (d) TEM images of ZnPcN4-TF. The ratio of ZnPcN4-TF is 3:1.

    Photochemical and photothermal properties of ZnPcN4-TF

    Subsequently, the photochemical and photothermal properties of ZnPcN4-TF were determined to assess the potential for phototherapy. After energy excitation, PSs usually return to the ground state through emitting fluorescence, generating ROS, or producing heat. The weak fluorescence of ZnPcN4 suggests that its energy may decay as heat. To directly evaluate the photothermal properties of ZnPcN4-TF, we evaluated the thermal behaviors of ZnPcN4-TF with different concentrations and under laser irradiation at different intensities. After 5 min of irradiation, the temperature of ZnPcN4-TF increased by about 25 °C, while those of MB and ZnPcN4 increased by only 8 °C and 11 °C, respectively (Figure 2a). This result indicates that ZnPcN4-TF converts light energy into heat effectively and the self-assembly with TF enhanced the heat conversion.52

    Figure 2

    Figure 2 | Photochemical and photothermal properties of ZnPcN4-TF. (a) Temperature elevation of different samples in PBS (10 μM) under laser irradiation at 1.5 W/cm2. (b) Photothermal effect of ZnPcN4-TF at various concentrations under laser irradiation at 1.5 W/cm2. (c) Photothermal effect of 10 μM ZnPcN4-TF at various powers of laser irradiation. (d) Irradiation–cooling cycle profiles of ZnPcN4-TF. The 655 nm laser was turned on for 10 min and off for 10 min. (e) Profile of the cooling time as a function of the negative natural logarithm of the temperature. (f) Photothermal stability of ZnPcN4-TF through three irradiation cycles. (g) Total ROS generation, (h) 1O2 generation, and (i) O2•− generation by ZnPcN4-TF in PBS after irradiation with a white light source. All at 10 μM, DCFH-DA as ROS probe, ABDA as 1O2 probe, and DHE as O2•− probe.

    We then varied the concentrations and the irradiation power (Figure 2b,c). The higher the ZnPcN4-TF concentration and the stronger the irradiation intensity, the higher the temperature it reaches, proving that the photothermal effect of ZnPcN4-TF is dependent on both concentration and light irradiation intensity. To determine the photothermal conversion efficiency of ZnPcN4-TF, a heating and cooling curve was plotted (Figure 2d). The photothermal conversion efficiency was 38.2% as calculated using an existing method, which is higher than many reported compounds (Figure 2e).53 The temperature elevation of ZnPcN4-TF remained stable through three consecutive heating and cooling cycles (Figure 2f), proving that ZnPcN4-TF has excellent photothermal stability. Taken together, these results indicate that ZnPcN4-TF has an excellent potential for PTT and PA imaging.

    Subsequently, ROS generation was assessed using DCFHDA as a fluorescence probe (Figure 2g and Supporting Information Figure S5). ZnPcN4-TF enhanced ROS production more than ZnPcN4 did. To determine the type of ROS produced, ABDA (Figure 2h and Supporting Information Figures S6–S7) and DHE was used as the probe to detect 1O2 and O2•−, respectively. However, the results displayed that there was almost no 1O2 generation for ZnPcN4-TF. Fortunately, ZnPcN4-TF exhibited a good ability to generate O2•− under both normal oxygen and hypoxic conditions (Figure 2i and Supporting Information Figures S8–S9). Compared with ZnPcN4 alone, the presence of TF facilitated the production of O2•−. This is likely due to the fact that the well-defined nanostructure, self-assembled through the electron-rich surface of amine groups and unique properties of TF, accelerated the electron transfer.54–56 To further determine the O2•− generation of ZnPcN4-TF, electron paramagnetic resonance (EPR) spectroscopy was carried out using 5,5-dimethyl-1-pyrroline-N-oxide as the spin-trap agent for O2•−. As shown in Supporting Information Figure S10, neither ZnPcN4-TF in dark nor light irradiation alone had no EPR signals. In contrast, for ZnPcN4-TF under NIR light irradiation, a characteristic paramagnetic adduct was observed, indicating the production of O2•−. This intriguing finding is very helpful for phototherapy under hypoxia since the production of type I ROS is less dependent on oxygen concentration.57

    In vitro phototherapy effect of ZnPcN4-TF in MCF-7 cells

    Encouraged by its good properties in solution, the applicability of ZnPcN4-TF in cells was investigated. The breast cancer cell line, such as MCF-7, expresses significantly higher levels of TF receptors than normal cell lines, so MCF-7 was selected for this study.58 First, we incubated MCF-7 cells with ZnPcN4-TF or ZnPcN4 for 24 h. The bright images revealed numerous aggregates in MCF-7 cells that were incubated solely with ZnPcN4 ( Supporting Information Figure S11). This was due to the strong hydrophobicity of free ZnPcN4, indicating the importance of self-assembly with TF. To visually evaluate intracellular ROS generation in MCF-7 cells, DCFHDA was used as a fluorescence probe (Figure 3a). Unsurprisingly, there was no fluorescence generation in MCF-7 cells treated with PBS in the presence or absence of light irradiation and in cells incubated with ZnPcN4-TF in the absence of light irradiation. However, cells cultured with ZnPcN4-TF displayed the strong fluorescence after longer light irradiation under lower power, indicating that ZnPcN4-TF effectively generated ROS in the cells under light irradiation. In addition, intracellular ROS production also depends on the time and intensity of light irradiation. To confirm the type of intracellular ROS produced, we detected 1O2 and O2•− using the fluorescence probes singlet oxygen-sensor-green (SOSG) and DHE, respectively ( Supporting Information Figure S12). Without light irradiation, there was no fluorescence inside the cells. Only the red fluorescence of DHE appeared after light exposure, indicating that the cells produced O2•−.

    Figure 3

    Figure 3 | Effect of ZnPcN4-TF with irradiation on cell viability. (a) Intracellular ROS generation using DCFHDA as a fluorescence probe. (b) Fluorescence microscope images of MCF-7 cells costained with calcein AM (2 μM, live cell marker), and PI (1 μM, dead cell marker). The control is MCF-7 cells treated with EMEM medium. Scale bars: 50 μm.

    The production of intracellular ROS under hypoxia was further observed by confocal fluorescence imaging. DCFHDA, SOSG, and DHE were also used as the fluorescent probes for total ROS production, 1O2 and O2•−, respectively. We saw that even under hypoxia, a large number of ROS are still produced by the ZnPcN4-TF with light irradiation ( Supporting Information Figure S13). Supporting Information Figure S14 displayed the generation of O2•− but no 1O2 by ZnPcN4-Tf with light irradiation under hypoxia, which is consistent with the result under normoxia, further proving the generation of type I ROS. Furthermore, the live/dead cells staining assay (costaining MCF-7 cells with calcein AM and PI) revealed that, although the cells exhibited partial red fluorescence under low-power (0.5 W/cm2) light irradiation, the cancer cell-killing efficiency was low ( Supporting Information Figure S15). Then the increased light irradiation power (1.5 W/cm2) was used. MCF-7 cells that were not incubated with ZnPcN4 and those incubated with ZnPcN4-TF in the absence of light irradiation displayed green fluorescence, indicating good cell viability, confirming the good biocompatibility of ZnPcN4-TF. However, obvious red fluorescence appeared without no green fluorescence in MCF-7 cells incubated with ZnPcN4-TF and irradiated, indicating effective cell death (Figure 3b). Besides, the colorimetric MTT assay also revealed that ZnPcN4-TF combined with light irradiation decreased cell viability ( Supporting Information Figure S16). This is consistent with the result of the live/dead cell staining assay. All these results demonstrate that ZnPcN4-TF with light irradiation can effectively kill tumor cells.

    In vitro and in vivo PA imaging

    PA imaging allows deep tissue penetration and high resolution in living organisms, which is of great significance to visualize physiological changes at the lesion site.59,60 Hence, the feasibility of using ZnPcN4-TF as a PA contrast agent was assessed by conducting in vitro experiments (Figure 4). First, we recorded the PA spectra of various samples (ZnPcN4-TF, ZnPcN4, MB, and 10% DMSO as control) under various NIR wavelength illumination. Parallel to the PA peak matching the optical absorption peak in general, ZnPcN4-TF showed the highest PA signal at 710 nm excitation, which was ∼2.25-fold greater than that of equimolar ZnPcN4 (Figure 4a). Next, we plotted the PA signal amplitude against concentration for ZnPcN4-TF and ZnPcN4 under 710 nm laser excitation (Figure 4b). The PA signal of ZnPcN4-TF was stronger than that of ZnPcN4 at the same concentration, probably because of the hydrophobic aggregation of ZnPcN4.61 To confirm this, PA images from the cells filled with ZnPcN4 and ZnPcN4-TF in a 96-microwell plate were recorded (Figure 4c). As anticipated, the photograph clearly showed the aggregation of ZnPcN4, while ZnPcN4-TF was stable in solution. Simultaneously, the resultant PA image of ZnPcN4 displayed mottled PA intensity all over the cell surface in a speckle-like pattern, while the cell filled with ZnPcN4-TF glowed uniformly (Figure 4d). Next, the PA pixel intensity was plotted into a PA amplitude histogram (Figure 4e), showing the variance of PA signal distribution. This plot showed that ZnPcN4-TF signals were located at a high amplitude band with a narrow variance, while ZnPcN4 signals were widely dispersed. Indeed, they were spread out from very low intensity to high levels equivalent to those of ZnPcN4-TF, resulting in a lower mean PA signal intensity than that of ZnPcN4-TF. All these results indicated that the lower PA signal of ZnPcN4 was due to the hydrophobic aggregation, while ZnPcN4-TF exhibited outstanding water solubility and yielded a decent PA signal in vitro.

    Figure 4

    Figure 4 | In vitro PA properties of ZnPcN4 and ZnPcN4-TF. (a) Normalized PA spectrum of various samples. The presented values are normalized to the PA amplitude of ZnPcN4-TF at 680 nm. (b) PA signal amplitude against ZnPcN4-TF and ZnPcN4 concentrations at 710 nm laser excitation. (c) In vitro hydrosolubility of ZnPcN4 and ZnPcN4-TF in a microwell plate. (d) PA MAP acquired at 710 nm. (e) Quantified PA amplitude histogram of (d) the upper 500,000 pixels from ZnPcN4 and ZnPcN4-TF.

    Next, the in vivo PA imaging was triplicated in an MCF-7 xenograft tumor model to explore the biodistribution of ZnPcN4-TF. The MCF-7 tumor-bearing mice received ZnPcN4-TF or ZnPcN4 via a tail vein injection and the whole-body PA images were acquired over 72 h (including the preinjection state) (Figure 5). We acquired an image series under two nominal wavelengths, 710 nm for the identical PA signal peak of ZnPcN4-TF and ZnPcN4, and 800 nm as a nominal wavelength to observe background-equivalent absorption. To highlight the agents’ entire body distribution from ambient PA signals generated from intrinsic chromophores such as hemoglobin, the dual-wavelength images were mapped into single composite MAP with two different complementary colormaps (Figure 5a). Meanwhile, there was a very slight change in mouse posture between two acquisition events, the PA MAP at 710 nm highlighted the dye accumulation to the organ, which was unidentifiable in the 800 nm PA MAP. Further, blood vessels appeared in stronger contrast in the 800 nm MAP than in the 710 nm MAP because the abundant oxygenated hemoglobin in the blood has a stronger absorption coefficient at 800 nm than at 710 nm. Next, we divided the pixel values of a pair of PA MAP images by each other, resulting in a 2D PA ratio (PA710nm/PA800nm) image allowing the visualization of the drug accumulation over the body. The quantitative analysis showed the time-dependent accumulation and clearance for both ZnPcN4-TF and ZnPcN4 at the liver and peritumoral sites (Figure 5b,c). Compared with the rapid accumulation of ZnPcN4-TF at the liver, which reached its maximum of 147.4% ± 17.1% signal increase at 1 h after the injection, ZnPcN4 gradually increased to its maximum at 24 h, which was ∼2.53-fold lower than that of ZnPcN4-TF. At all time points, the accumulation of ZnPcN4-TF was higher than that of ZnPcN4, demonstrating the more effective dispersion of ZnPcN4-TF through systemic circulation thanks to enhanced solubility. Further, around the tumor site, at 8 h postinjection, the ZnPcN4-TF PA signal reached a 53.9% ± 17.5% increase, while the ZnPcN4 PA signal was unchanged due to the absence of targeted delivery. This result confirmed that TF conjugation conferred ZnPcN4-TF with a targeting ability toward TF receptor-rich tumor cells. Furthermore, we used the 8 h time point in the later phototherapeutic experiment to target the timing at which most of the nanodrug had accumulated at the tumor site.

    Figure 5

    Figure 5 | In vivo whole-body PA imaging of MCF-7 tumor-bearing mouse monitored for 72 h and PA contrast agent distribution mapping using dual-wavelength ratio quantification. (a) Illustration of composite PA XY-MAP images acquired at 710 nm (green) and 800 nm (purple) excitation and their representative ratio (PA710nm/PA810nm, orange) MAP 8 h after injection. White dashed lines indicate the whole-body skin contour of the mouse; the liver and tumor are marked with the red and yellow dotted lines, respectively. Scale bar: 10 mm. Right column: magnified view of the XZ-MAP dual-wavelength ratio image over the tumor region. The yellow box outlines the PA710nm/PA810nm cross-sectional images of the tumor region. White dashed lines indicate the skin surface, yellow dashed lines indicate subcutaneous tumor mass, and red arrows indicate deep liver locations. Scale bar: 2 mm. Quantified PA ratio variation in the (b) liver region and (c) tumor region before and 72 h after the injection of ZnPcN4-TF (n = 3), and ZnPcN4 (n = 3). Error bar: standard error.

    In vivo phototherapy effect of ZnPcN4-TF

    The biosafety of ZnPcN4-TF was evaluated before conducting the in vivo phototherapeutic experiments. The hemolysis activity of ZnPcN4 and ZnPcN4-TF is 3.75% and 3.92%, respectively. Both of them meet the clinical safety threshold of less than 5%, thus ensuring the biosafety of intravenous administration ( Supporting Information Figure S17).62,63 Furthermore, histologic examination of major organs following ZnPcN4 and ZnPcN4-TF tail-vein injection showed no significant morphological differences compared to the PBS group ( Supporting Information Figure S18). These results indicate that ZnPcN4-TF is suitable for bioapplications.

    To evaluate the in vivo phototherapeutic effect of ZnPcN4-TF on tumors, we formed eight groups of mice bearing MCF-7 tumors: (1) mice treated with PBS only, (2) mice treated with high-power laser irradiation only (2 W/cm2), (3) mice treated with ZnPcN4 only, (4) mice treated with ZnPcN4 and low-power laser irradiation (0.2 W/cm2), (5) mice treated with ZnPcN4 and high-power laser irradiation, (6) mice treated with ZnPcN4-TF only, (7) mice treated with ZnPcN4-TF and low-power laser irradiation, and (8) mice treated with ZnPcN4-TF and high-power laser irradiation. Based on the in vivo PA results, light irradiation was performed 8 h after the injection (Figure 6a). Based on previous experiments, low-power irradiation with PSs can only generate ROS to verify the PDT effect, whereas high-power irradiation with PSs can generate both ROS and heat to destroy the tumor. For mice submitted to light irradiation, thermal images were recorded using an infrared thermal imager (Figure 6b). The mean temperature of mice treated with ZnPcN4-TF and high-power irradiation increased to 49.4 °C, compared to 38.9 and 39.9 °C for mice treated with ZnPcN4 and high-power irradiation or high-power irradiation only, respectively (Figure 6c). This result demonstrats that ZnPcN4-TF exhibits efficient photothermal conversion under high-power light irradiation in vivo and also underscores the significance of TF. In contrast, the average temperature of mice treated with ZnPcN4-TF and low-power irradiation increased only to 31.8 °C, further emphasizing the synergistic effect of PTT and PDT at high-power irradiation, as well as the isolated effect of PDT alone at low-power irradiation. After the treatment, we monitored tumor volume and body weight in all the mice. The different treatments barely affected the average body weight of the mice (Figure 6d), which maintained a steady increase, demonstrating the biocompatibility of ZnPcN4-TF for PDT and PTT. Meanwhile, the treatment regimens differently affected the tumor volumes (Figure 6e). Mice treated with ZnPcN4-TF and at low-power irradiation had significantly smaller tumors than the control groups (ZnPcN4-TF without light irradiation, light irradiation only, or ZnPcN4 and various light irradiation). Since this treatment (ZnPcN4-TF + low-power irradiation) did not significantly increase the temperature, these results revealed that PDT had an antitumor effect. Nevertheless, ZnPcN4-TF had a much greater antitumor effect when coupled with high-power irradiation than with low-power irradiation, demonstrating the excellent synergy of PDT and PTT. Tumor images (Figure 6f) and average tumor weight (Figure 6g) further confirmed that ZnPcN4-TF had significant antitumor efficacy under light irradiation. Overall, these results indicated that ZnPcN4-TF exhibited excellent antitumor capability under hypoxia through the combination of PTT and type I PDT.

    Figure 6

    Figure 6 | In vivo antitumor efficacy of ZnPcN4-TF intravenous injection on tumor-bearing mice. (a) Schematic illustration of the treatment of ZnPcN4-TF on tumor-bearing mice. (b) Thermal infrared imaging of tumor-bearing mice submitted to the indicated drug and irradiation treatments. (c) Temperature curves of the tumor-bearing mice submitted to the indicated treatments. Error bars represent the SD (n = 4, ****P > 0.0001). (d) Changes in the mean body weight of mice submitted to the indicated treatments. (e) Tumor growth curve of tumor-bearing mice after various treatments. (f) Tumor images and (g) average tumor weights after 21 days of the indicated treatments. Data are expressed as mean ± SEM (n = 5, *P > 0.05, **P > 0.01, ***P > 0.001, and ****P > 0.0001 determined by two-way ANOVA).

    Conclusion

    We constructed a nanophthalocyanine (ZnPcN4-TF) based on the self-assembly of TF and ZnPcN4, which was synthesized by conjugating ZnPc with an electron-rich amino group based on the structure-inherent regulation of the energy pathway strategy. Due to the PET effect, ZnPcN4 emits almost no fluorescence, making it an ideal candidate for PTT and PA imaging. ZnPcN4-TF not only solves the low bioavailability issue of ZnPcN4 (due to its strong hydrophobicity), but it also effectively addresses the aggregation-induced quenching of ROS production of traditional PSs. Further, the integration of type I ROS and the photothermal effect successfully overcomes the drawbacks of hypoxia in the tumor environment. Both the in vitro and in vivo results demonstrate that ZnPcN4-TF is an excellent PS for enhancing phototherapy with PA imaging under hypoxia.

    Supporting Information

    Supporting Information is available and includes the supporting figures ( Figures S1–S18).

    Conflict of Interest

    There is no conflict of interest to report.

    Funding Information

    This work was supported by the National Natural Science Foundation of China (grant nos. T2322004 and 22078066), Science Research Project of Hebei Education Department (grant no. QN2025122), the Hebei Natural Science Foundation (grant no. B2024208046), the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT (MSIT)) (grant no. 2022R1A2C3005420), the Basic Science Research Program through the NRF funded by the Ministry of Education (grant no. 2020R1A6A1A03047902), National R&D Program through the NRF funded by the MSIT (grant no. 2023R1A2C3004880), BK21 FOUR projects (POSTECH), and the NRF grant funded by the Korean government (MSIT) (grant no. 2022R1A3B1077354). TEM and NMR measurements were performed using a JEM-2100F electron microscope (JEOL) and an NMR 300 MHz spectrometer at the National Research Facilities and Equipment Center (NanoBioEnergy Materials Center) at Ewha Womans University.

    References

    • 1. Smith B. R.; Gambhir S. S.Nanomaterials for In Vivo Imaging.Chem. Rev.2017, 117, 901–986. Google Scholar
    • 2. Kang X.; Zhang Y.; Song J.; Wang L.; Li W.; Qi J.; Tang B. Z.A Photo-Triggered Self-Accelerated Nanoplatform for Multifunctional Image-Guided Combination Cancer Immunotherapy.Nat. Commun.2023, 14, 5216. Google Scholar
    • 3. Cheng M. H. Y.; Harmatys K. M.; Charron D. M.; Chen J.; Zheng G.Stable J-Aggregation of an aza-BODIPY-Lipid in a Liposome for Optical Cancer Imaging.Angew. Chem. Int. Ed. Engl.2019, 58, 13394–13399. Google Scholar
    • 4. Li C.; Chen G.; Zhang Y.; Wu F.; Wang Q.Advanced Fluorescence Imaging Technology in the Near-Infrared-II Window for Biomedical Applications.J. Am. Chem. Soc.2020, 142, 14789–14804. Google Scholar
    • 5. Ntziachristos V.Going Deeper than Microscopy: The Optical Imaging Frontier in Biology.Nat. Methods2010, 7, 603–614. Google Scholar
    • 6. Oh D.; Lee D.; Heo J.; Kweon J.; Yong U.; Jang J.; Ahn Y. J.; Kim C.Contrast Agent-Free 3D Renal Ultrafast Doppler Imaging Reveals Vascular Dysfunction in Acute and Diabetic Kidney Diseases.Adv. Sci.2023, 10, e2303966. Google Scholar
    • 7. Li X.; Park E. Y.; Kang Y.; Kwon N.; Yang M.; Lee S.; Kim W. J.; Kim C.; Yoon J.Supramolecular Phthalocyanine Assemblies for Improved Photoacoustic Imaging and Photothermal Therapy.Angew. Chem. Int. Ed. Engl.2020, 59, 8630–8634. Google Scholar
    • 8. Hao Y.; Wang J.; Wang J.; Guo Z.; Liu X.; Lv S.; Hu Q.; Huo K.; Yao Q.; Jiang J.; Zeng S.; Kang H.; Peng X.; Yoon J.; Li H.Leveraging an Electron-Acceptor Engineering Strategy to Regulate Excitation Dynamics of Dyes for Devising Ideal Phototherapeutic Agents in Synergistic Photodynamic/Mild-Photothermal Tumor Therapy.CCS Chem.2024, 6, 1–17. Google Scholar
    • 9. Choi W.; Park B.; Choi S.; Oh D.; Kim J.; Kim C.Recent Advances in Contrast-Enhanced Photoacoustic Imaging: Overcoming the Physical and Practical Challenges.Chem. Rev.2023, 123, 7379–7419. Google Scholar
    • 10. Park E.-Y.; Park S.; Lee H.; Kang M.; Kim C.; Kim J.Simultaneous Dual-Modal Multispectral Photoacoustic and Ultrasound Macroscopy for Three-Dimensional Whole-Body Imaging of Small Animals.Photonics2021, 8, 13. Google Scholar
    • 11. Yang J.; Choi S.; Kim C.Practical Review on Photoacoustic Computed Tomography Using Curved Ultrasound Array Transducer.Biomed. Eng. Lett.2022, 12, 19–35. Google Scholar
    • 12. Choi W.; Park E. Y.; Jeon S.; Yang Y.; Park B.; Ahn J.; Cho S.; Lee C.; Seo D. K.; Cho J. H.; Kim C.Three-Dimensional Multistructural Quantitative Photoacoustic and US Imaging of Human Feet in Vivo.Radiology2022, 303, 467–473. Google Scholar
    • 13. Park J.; Park B.; Kim T. Y.; Jung S.; Choi W. J.; Ahn J.; Yoon D. H.; Kim J.; Jeon S.; Lee D.; Yong U.; Jang J.; Kim W. J.; Kim H. K.; Jeong U.; Kim H. H.; Kim C.Quadruple Ultrasound, Photoacoustic, Optical Coherence, and Fluorescence Fusion Imaging with a Transparent Ultrasound Transducer.Proc. Natl. Acad. Sci. U.S.A.2021, 118, e1920879118. Google Scholar
    • 14. Park B.; Oh D.; Kim J.; Kim C.Functional Photoacoustic Imaging: From Nano- and Micro- to Macro-Scale.Nano Converg.2023, 10, 29. Google Scholar
    • 15. Zou Q.; Abbas M.; Zhao L.; Li S.; Shen G.; Yan X.Biological Photothermal Nanodots Based on Self-Assembly of Peptide-Porphyrin Conjugates for Antitumor Therapy.J. Am. Chem. Soc.2017, 139, 1921–1927. Google Scholar
    • 16. Baik J. W.; Kim H.; Son M.; Choi J.; Kim K. G.; Baek J. H.; Park Y. H.; An J.; Choi H. Y.; Ryu S. Y.; Kim J. Y.; Byun K.; Kim C.Intraoperative Label-Free Photoacoustic Histopathology of Clinical Specimens.Laser Photonics Rev.2021, 15, 2100124. Google Scholar
    • 17. Kim D.; Park E.; Park J.; Perleberg B.; Jeon S.; Ahn J.; Ha M.; Kim H. H.; Kim J. Y.; Jung C. K.; Kim C.An Ultraviolet-Transparent Ultrasound Transducer Enables High-Resolution Label-Free Photoacoustic Histopathology.Laser Photonics Rev.2023, 18, 2300652. Google Scholar
    • 18. Kim J.; Kim G.; Li L.; Zhang P.; Kim J. Y.; Kim Y.; Kim H. H.; Wang L. V.; Lee S.; Kim C.Deep Learning Acceleration of Multiscale Superresolution Localization Photoacoustic Imaging.Light-Sci. Appl.2022, 11, 131. Google Scholar
    • 19. Park E. Y.; Oh D.; Park S.; Kim W.; Kim C.New Contrast Agents for Photoacoustic Imaging and Theranostics: Recent 5-Year Overview on Phthalocyanine/Naphthalocyanine-Based Nanoparticles.APL Bioeng.2021, 5, 031510. Google Scholar
    • 20. Xiao P.; Xie W.; Zhang J.; Wu Q.; Shen Z.; Guo C.; Wu Y.; Wang F.; Tang B. Z.; Wang D.De Novo Design of Reversibly pH-Switchable NIR-II Aggregation-Induced Emission Luminogens for Efficient Phototheranostics of Patient-Derived Tumor Xenografts.J. Am. Chem. Soc.2023, 145, 334–344. Google Scholar
    • 21. Yang M.; Kim Y.; Youn S.-Y.; Jeong H.; Shirbhate M. E.; Uhm C.; Kim G.; Nam K. T.; Cha S.-S.; Kim K. M.; Yoon J.Conversion of Albumin into a BODIPY-Like Photosensitizer by a Flick Reaction, Tumor Accumulation and Photodynamic Therapy.Biomaterials2025, 313, 122792. Google Scholar
    • 22. Yang M.; Li X.; Yoon J.Activatable Supramolecular Photosensitizers: Advanced Design Strategies.Mater. Chem. Front.2021, 5, 1683–1693. Google Scholar
    • 23. Liu S.; Wang B.; Yu Y.; Liu Y.; Zhuang Z.; Zhao Z.; Feng G.; Qin A.; Tang B. Z.Cationization-Enhanced Type I and Type II ROS Generation for Photodynamic Treatment of Drug-Resistant Bacteria.ACS Nano2022, 16, 9130–9141. Google Scholar
    • 24. Chen D.; Wang Z.; Dai H.; Lv X.; Ma Q.; Yang D. P.; Shao J.; Xu Z.; Dong X.Boosting O2•− Photogeneration via Promoting Intersystem-Crossing and Electron-Donating Efficiency of Aza-BODIPY-Based Nanoplatforms for Hypoxic-Tumor Photodynamic Therapy.Small Methods2020, 4, 2000013. Google Scholar
    • 25. Li M.; Shao Y.; Kim J. H.; Pu Z.; Zhao X.; Huang H.; Xiong T.; Kang Y.; Li G.; Shao K.; Fan J.; Foley J. W.; Kim J. S.; Peng X.Unimolecular Photodynamic O2-Economizer to Overcome Hypoxia Resistance in Phototherapeutics.J. Am. Chem. Soc.2020, 142, 5380–5388. Google Scholar
    • 26. Li M.; Xia J.; Tian R.; Wang J.; Fan J.; Du J.; Long S.; Song X.; Foley J. W.; Peng X.Near-Infrared Light-Initiated Molecular Superoxide Radical Generator: Rejuvenating Photodynamic Therapy Against Hypoxic Tumors.J. Am. Chem. Soc.2018, 140, 14851–14859. Google Scholar
    • 27. Yao J.; Cheng Y.; Zhou M.; Zhao S.; Lin S.; Wang X.; Wu J.; Li S.; Wei H.ROS Scavenging Mn3O4 Nanozymes for in Vivo Anti-Inflammation.Chem. Sci.2018, 9, 2927–2933. Google Scholar
    • 28. Samuel E. L.; Marcano D. C.; Berka V.; Bitner B. R.; Wu G.; Potter A.; Fabian R. H.; Pautler R. G.; Kent T. A.; Tsai A. L.; Tour J. M.Highly Efficient Conversion of Superoxide to Oxygen Using Hydrophilic Carbon Clusters.Proc. Natl. Acad. Sci. U.S.A.2015, 112, 2343–2348. Google Scholar
    • 29. Li X.; Lee D.; Huang J. D.; Yoon J.Phthalocyanine-Assembled Nanodots as Photosensitizers for Highly Efficient Type I Photoreactions in Photodynamic Therapy.Angew. Chem. Int. Ed. Engl.2018, 57, 9885–9890. Google Scholar
    • 30. Yu Y.; Wu S.; Zhang L.; Xu S.; Dai C.; Gan S.; Xie G.; Feng G.; Tang B. Z.Cationization to Boost Both Type I and Type II ROS Generation for Photodynamic Therapy.Biomaterials2022, 280, 121255. Google Scholar
    • 31. Qu R.; Zhen X.; Jiang X.Emerging Designs of Aggregation-Induced Emission Agents for Enhanced Phototherapy Applications.CCS Chem.2022, 4, 401–419. Link, Google Scholar
    • 32. Yang M.; Li X.; Kim G.; Wang R.; Hong S. J.; Lee C. H.; Yoon J.A J-Aggregated Nanoporphyrin Overcoming Phototoxic Side Effects in Superior Phototherapy with Two-Pronged Effects.Chem. Sci.2022, 13, 12738–12746. Crossref, Google Scholar
    • 33. Li X.; Lovell J. F.; Yoon J.; Chen X.Clinical Development and Potential of Photothermal and Photodynamic Therapies for Cancer.Nat. Rev. Clin. Oncol.2020, 17, 657–674. Google Scholar
    • 34. Hu H.; Zhang Y.-Y.; Ma H.; Yang Y.; Mei S.; Li J.; Xu J.-F.; Zhang X.A Supramolecular Naphthalene Diimide Radical Anion with Efficient NIR-II Photothermal Conversion for E. coli-Responsive Photothermal Therapy.Angew. Chem. Int. Ed.2023, 62, e202308513. Google Scholar
    • 35. Wen K.; Tan H.; Peng Q.; Chen H.; Ma H.; Wang L.; Peng A.; Shi Q.; Cai X.; Huang H.Achieving Efficient NIR-II Type-I Photosensitizers for Photodynamic/Photothermal Therapy Upon Regulating Chalcogen Elements.Adv. Mater.2022, 34, e2108146. Google Scholar
    • 36. Li X.; Zheng B.-D.; Peng X.-H.; Li S.-Z.; Ying J.-W.; Zhao Y.; Huang J.-D.; Yoon J.Phthalocyanines as Medicinal Photosensitizers: Developments in the Last Five Years.Coord. Chem. Rev.2019, 379, 147–160. Google Scholar
    • 37. Zhao L.; Li S.; Liu Y.; Xing R.; Yan X.Kinetically Controlled Self-Assembly of Phthalocyanine-Peptide Conjugate Nanofibrils Enabling Super-Large Redshifted Absorption.CCS Chem.2019, 1, 173–180. Link, Google Scholar
    • 38. Li X.; Peng X. H.; Zheng B. D.; Tang J.; Zhao Y.; Zheng B. Y.; Ke M. R.; Huang J. D.New Application of Phthalocyanine Molecules: From Photodynamic Therapy to Photothermal Therapy by Means of Structural Regulation Rather than Formation of Aggregates.Chem. Sci.2018, 9, 2098–2104. Google Scholar
    • 39. Li X.; Yu S.; Lee Y.; Guo T.; Kwon N.; Lee D.; Yeom S. C.; Cho Y.; Kim G.; Huang J. D.; Choi S.; Nam K. T.; Yoon J.In Vivo Albumin Traps Photosensitizer Monomers from Self-Assembled Phthalocyanine Nanovesicles: A Facile and Switchable Theranostic Approach.J. Am. Chem. Soc.2019, 141, 1366–1372. Google Scholar
    • 40. Yuan C.; Ji W.; Xing R.; Li J.; Gazit E.; Yan X.Hierarchically Oriented Organization in Supramolecular Peptide Crystals.Nat. Rev. Chem.2019, 3, 567–588. Google Scholar
    • 41. Chang R.; Zou Q.; Zhao L.; Liu Y.; Xing R.; Yan X.Amino-Acid-Encoded Supramolecular Photothermal Nanomedicine for Enhanced Cancer Therapy.Adv. Mater.2022, 34, e2200139. Google Scholar
    • 42. Xu G.; Li C.; Chi C.; Wu L.; Sun Y.; Zhao J.; Xia X. H.; Gou S.A Supramolecular Photosensitizer Derived from an Arene-Ru(II) Complex Self-Assembly for NIR Activated Photodynamic and Photothermal Therapy.Nat. Commun.2022, 13, 3064. Google Scholar
    • 43. Zhuang J.; Li N.; Zhang Y.; Li B.; Wen H.; Zhang X.; Zhang T.; Zhao N.; Tang B. Z.Esterase-Activated Theranostic Prodrug for Dual Organelles-Targeted Imaging and Synergetic Chemo-Photodynamic Cancer Therapy.CCS Chem.2022, 4, 1028–1043. Link, Google Scholar
    • 44. Lee E.; Li X.; Oh J.; Kwon N.; Kim G.; Kim D.; Yoon J.A Boronic Acid-Functionalized Phthalocyanine with an Aggregation-Enhanced Photodynamic Effect for Combating Antibiotic-Resistant Bacteria.Chem. Sci.2020, 11, 5735–5739. Google Scholar
    • 45. Yang M.; Özdemir Z.; Kim H.; Nah S.; Andris E.; Li X.; Wimmer Z.; Yoon J.Acid-Responsive Nanoporphyrin Evolution for Near-Infrared Fluorescence-Guided Photo-Ablation of Biofilm.Adv. Healthcare Mater.2022, 11, 2200529. Google Scholar
    • 46. Jeon M.; Kim J.; Kim C.Multiplane Spectroscopic Whole-Body Photoacoustic Imaging of Small Animals in Vivo.Med. Biol. Eng. Comput.2016, 54, 283–294. Google Scholar
    • 47. Tan X.; Luo S.; Long L.; Wang Y.; Wang D.; Fang S.; Ouyang Q.; Su Y.; Cheng T.; Shi C.Structure-Guided Design and Synthesis of a Mitochondria-Targeting Near-Infrared Fluorophore with Multimodal Therapeutic Activities.Adv. Mater.2017, 29, 1704196. Google Scholar
    • 48. Li M.; Long S.; Kang Y.; Guo L.; Wang J.; Fan J.; Du J.; Peng X.De Novo Design of Phototheranostic Sensitizers Based on Structure-Inherent Targeting for Enhanced Cancer Ablation.J. Am. Chem. Soc.2018, 140, 15820–15826. Google Scholar
    • 49. Li X.; Jeong K.; Lee Y.; Guo T.; Lee D.; Park J.; Kwon N.; Na J. H.; Hong S. K.; Cha S. S.; Huang J. D.; Choi S.; Kim S.; Yoon J.Water-Soluble Phthalocyanines Selectively Bind to Albumin Dimers: A Green Approach Toward Enhancing Tumor-Targeted Photodynamic Therapy.Theranostics2019, 9, 6412–6423. Google Scholar
    • 50. Yuan C.; Xing R.; Cui J.; Fan W.; Li J.; Yan X.Multistep Desolvation as a Fundamental Principle Governing Peptide Self-Assembly Through Liquid–Liquid Phase Separation.CCS Chem.2024, 6, 255–265. Link, Google Scholar
    • 51. Wu G.; Li F.; Tang B.; Zhang X.Molecular Engineering of Noncovalent Dimerization.J. Am. Chem. Soc.2022, 144, 14962–14975. Crossref, Google Scholar
    • 52. Li S.; Zhang W.; Xing R.; Yuan C.; Xue H.; Yan X.Supramolecular Nanofibrils Formed by Coassembly of Clinically Approved Drugs for Tumor Photothermal Immunotherapy.Adv. Mater.2021, 33, 2100595. Google Scholar
    • 53. Xing R.; Zou Q.; Yuan C.; Zhao L.; Chang R.; Yan X.Self-Assembling Endogenous Biliverdin as a Versatile Near-Infrared Photothermal Nanoagent for Cancer Theranostics.Adv. Mater.2019, 31, 1900822. Google Scholar
    • 54. Chen W.; Wang Z.; Tian M.; Hong G.; Wu Y.; Sui M.; Chen M.; An J.; Song F.; Peng X.Integration of TADF Photosensitizer as “Electron Pump” and BSA as “Electron Reservoir” for Boosting Type I Photodynamic Therapy.J. Am. Chem. Soc.2023, 145, 8130–8140. Google Scholar
    • 55. Zhou J.; Xu D.; Tian G.; He Q.; Zhang X.; Liao J.; Mei L.; Chen L.; Gao L.; Zhao L.; Yang G.; Yin W.; Nie G.; Zhao Y.Coordination-Driven Self-Assembly Strategy-Activated Cu Single-Atom Nanozymes for Catalytic Tumor-Specific Therapy.J. Am. Chem. Soc.2023, 145, 4279–4293. Google Scholar
    • 56. Zhang N.; Wang L.; Wang H.; Cao R.; Wang J.; Bai F.; Fan H.Self-Assembled One-Dimensional Porphyrin Nanostructures with Enhanced Photocatalytic Hydrogen Generation.Nano Lett.2018, 18, 560–566. Google Scholar
    • 57. Shigemitsu H.; Ohkubo K.; Sato K.; Bunno A.; Mori T.; Osakada Y.; Fujitsuka M.; Kida T.Fluorescein-Based Type I Supramolecular Photosensitizer via Induction of Charge Separation by Self-Assembly.JACS Au2022, 2, 1472–1478. Google Scholar
    • 58. Zhang Q.; Wang N.; Ma M.; Luo Y.; Chen H.Transferrin Receptor-Mediated Sequential Intercellular Nanoparticles Relay for Tumor Deep Penetration and Sonodynamic Therapy.Adv. Ther.2019, 2, 1800152. Google Scholar
    • 59. Choi S.; Yang J.; Lee S. Y.; Kim J.; Lee J.; Kim W. J.; Lee S.; Kim C.Deep Learning Enhances Multiparametric Dynamic Volumetric Photoacoustic Computed Tomography in Vivo (DL-PACT).Adv. Sci.2022, 10, 2202089. Google Scholar
    • 60. Kim J.; Park B.; Ha J.; Steinberg I.; Hooper S. M.; Jeong C.; Park E. Y.; Choi W.; Liang T.; Bae J. S.; Managuli R.; Kim Y.; Gambhir S. S.; Lim D. J.; Kim C.Multiparametric Photoacoustic Analysis of Human Thyroid Cancers In Vivo.Cancer Res.2021, 81, 4849–4860. Google Scholar
    • 61. Wang Y.; Gao Z.; Han Z.; Liu Y.; Yang H.; Akkin T.; Hogan C. J.; Bischof J. C.Aggregation Affects Optical Properties and Photothermal Heating of Gold Nanospheres.Sci. Rep.2021, 11, 898. Google Scholar
    • 62. Tong X.; Shi Z.; Xu L.; Lin J.; Zhang D.; Wang K.; Li Y.; Wen C.Degradation Behavior, Cytotoxicity, Hemolysis, and Antibacterial Properties of Electro-Deposited Zn–Cu Metal Foams as Potential Biodegradable Bone Implants.Acta Biomater.2020, 102, 481–492. Google Scholar
    • 63. Liu J.; Shi L.; Wang Y.; Li M.; Zhou C.; Zhang L.; Yao C.; Yuan Y.; Fu D.; Deng Y.; Liu M.; Wang G.; Wang L.; Wang Z.Ruthenium-Based Metal-Organic Framework with Reactive Oxygen and Nitrogen Species Scavenging Activities for Alleviating Inflammation Diseases.Nano Today2022, 47, 101627. Google Scholar
    Previous article
    Next article