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• Cancer is an important and persistent problem that seriously endangers global public health, necessitating the pursuit of safe and effective treatment options [1]. Conventional clinical treatment methods, such as surgery, radiotherapy, and chemotherapy, are restricted in their wider applications due to the well-known limitations, including insufficient efficacy, unavoidable side effects and high recurrence rates [2–4]. Therefore, many researchers have been committed to developing more effective cancer treatment options [5–7]. Among them, multi-mode nano therapeutic platforms stand out as a potential and promising treatment approach [8–12].

  Phototherapy, due to its non-invasive nature, minimal side effects, and good therapeutic performance, has been extensively utilized for the treatment of tumors [13–16]. However, it has been demonstrated that single phototherapy mode has some obvious defects, leading to unsatisfactory efficacy. For example, the heat of photothermal therapy (PTT) will promote the expression of heat shock proteins such as heat shock protein 90 (HSP90) which considerably reduces the efficacy of PTT. Photodynamic therapy (PDT) is also limited by the hypoxic tumor microenvironment, resulting in decrease in efficiency of reactive oxygen species (ROS) production [17,18]. Furthermore, the damage to normal tissues is in many cases unavoidable, when patients are exposed to natural sunlight or visible light after PDT treatment [19]. More effective photosensitizers/photothermal agents are thus in intense and consistent demand.

  Compared to the always-on photosensitizers, an activatable photosensitizer is an apparently safer and more effective choice, as the background phototoxicity is effectively suppressed. PDT could be activated under specific conditions and at specific area by stimulations such as light, enzyme, and tumor microenvironment (TME) [20–24]. In addition, PDT can inhibit the expression of heat shock proteins and reduce the heat resistance of tumor [25]. Correspondingly, PTT can increase blood flow rate and oxygen supply in blood vessels specifically at the tumor site which enhances the effect of PDT. The design of an intelligent therapeutic agent which can combine photoactivatable PDT and PTT to improve the efficiency of photon utilization and thus the therapeutic performance shows clear advantage [26–28].

  Gas therapy is a therapeutic modality based on the utilization of specialized signaling gas molecules that can synergistically enhance the efficacy of treatment in cancer cells [29,30]. The common signaling molecules employed in gas therapy encompassed hydrogen sulfide (H₂S), nitric oxide (NO), carbon monoxide (CO) and hydrogen (H₂) [31–34]. H₂S, as one of the typical gas signaling molecules, maintains the cellular homeostasis by repairing hyperactive inflammation [35]. However, excess concentrations of exogenous H₂S can interfere with mitochondrial respiration, deactivate energy supply to cancer cells, and thus inhibit tumor growth [36]. In addition, the presence of H₂S can also inhibit the expression of heat shock proteins [37]. Therefore, integrating H₂S gas therapy with phototherapy provides a new approach to improve the effectiveness of treating malignant tumors. One thing needs to be addressed is that due to the drawbacks of traditional gas therapy, such as premature gas release and rapid systemic clearance, rapid and effective release of H₂S in tumor microenvironment is highly anticipated [38].

  As a promising optical diagnostic technology, fluorescence imaging provides several advantages such as non-invasiveness, low cost and excellent spatiotemporal resolution [39]. Compared to the visible (400–700 nm) and near-infrared-Ⅰ (NIR-Ⅰ) (700–900 nm) regions with obvious photon scattering and background interference that can considerably reduce the imaging quality, NIR-Ⅱ fluorescence imaging shows more attraction in tumor imaging and diagnosis [40]. Currently, widely used organic NIR-Ⅱ fluorescence probes mainly include benzobisthiadiazole (BBTD) dyes, cyanine dyes, boron dipyrromethene (BODIPY) derivatives and conjugated polymer dyes, etc. [41–44]. Perylene diimide (PDI) and its derivatives, with excellent photophysical and photochemical properties, have been utilized in tumor therapy and in vivo fluorescence imaging [45–48]. However, PDI-based NIR-Ⅱ fluorescence probes with emission maximum in the NIR-Ⅱ region, have been rarely reported [49], thus developing a PDI-based probe that integrates phototherapy and in vivo NIR-Ⅱ fluorescence imaging is a topic worth exploring.

  Taking all the above considerations into account, we have developed a PDI based NIR-Ⅱ fluorescence imaging guided multi-mode therapeutic platform (Scheme 1). Firstly, we synthesized a novel PDI compound called PDI-DBU by introducing an electron donating group DBU. The absorption of PDI-DBU was in the range of 700–900 nm, and the fluorescence emission was red shifted to the NIR-Ⅱ region. Under 808 nm laser irradiation, PDI-DBU exhibited good photothermal effects. In addition, 808 nm laser irradiation could lead to oxidation of PDI-DBU, resulting in destruction of the donor-acceptor (D-A) structure and a significant blue shift in absorption and turn-on visible region fluorescence emission. The oxidized PDI-DBU was proved to be an effective photosensitizer. After photothermal therapy, utilizing a 530 nm laser could turn on the photodynamic therapy [50,51]. Meanwhile, a polysulfide-based H₂S donor DPS was synthesized, which could be triggered by the abundant glutathione (GSH) in TME, rapidly generating H₂S. The generated H₂S not only achieved gas therapy, but also inhibited the expression of HSP90 and relieve hypoxic environment by disrupting mitochondrial function, thereby promoting the effectiveness of phototherapy. We integrated PDI-DBU and DPS to obtain PDI-DS nanoparticles (NPs). Excellent in vivo NIR-Ⅱ fluorescence imaging capability and in vivo tumor suppression efficiency of the therapeutic platform have been demonstrated.

  Scheme 1

  

  Scheme 1.  Schematic illustration of the construction of PDI-DS NPs as the synergistic theranostic application.

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  The synthetic scheme of PDI-DBU is shown in Fig. S1 (Supporting information). 3, 4, 9, 10-Perylenetetracarboxylic dianhydride and 3, 6, 9-trioxadecane-1-amine were dissolved in imidazole and heated to 160 ℃ under an argon atmosphere to obtain PDI-PEG. Then, PDI-PEG reacted with DBU in anhydrous toluene to obtain PDI-DBU. The obtained PDI-PEG and PDI-DBU were verified by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry analyses (Figs. S2–S6 in Supporting information). The obtained PDI-DBU was encapsulated into NPs using DPS (Figs. S7–S9 in Supporting information) and a co-precipitation method, which were named as PDI-DS NPs. The NPs containing only PDI-DBU (named as PDI-D NPs) were synthesized using the same method as the control. Size and morphology of PDI-DS NPs and PDI-D NPs were analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). As shown in Figs. 1a and b, DLS and TEM images confirmed that the two NPs were both spherical with average sizes of 105.3 and 111.1 nm for PDI-DS NPs and PDI-D NPs, respectively. PDI-DS NPs and PDI-D NPs presented zeta potential values of −13.7 and −15.3 mV, respectively (Fig. 1c). The sufficient surface negative charge ensured good colloid stability of the NPs. The sizes of PDI-DS NPs and PDI-D NPs in phosphate buffer saline (PBS) solution had no obvious fluctuations within 14 days of storage, proving superior colloidal stability of the materials (Fig. S10 in Supporting information). UV-vis-NIR absorption spectra (Fig. 1d) showed that PDI-DBU exhibited significant absorption in the near-infrared region (700–900 nm), and the maximum absorption peak of DPS was located at 280 nm with almost no absorption beyond 400 nm. PDI-D NPs retained the characteristic absorption band of PDI-DBU, and PDI-DS NPs exhibited characteristic absorption bands of DPS and PDI-DBU, confirming successful encapsulation of DPS and PDI-DS NPs. The loading rates of PDI-DBU and DPS in PDI-DS NPs were calculated to be 7.3% and 4.7% by the standard calibration curves, respectively (Fig. S11 in Supporting information). Fluorescence spectrum displayed in Fig. 1e showed that the emission of PDI-DS NPs peaked at 930 nm and extended well into the NIR-Ⅱ region (Fig. S12 in Supporting information). The fluorescence quantum yield of the material in water was calculated to be 0.3%, using indocyanine green (ICG) as reference (13% in DMSO) (Fig. S13 in Supporting information) [52]. After 6 min of irradiation with 808 nm laser, the absorption of PDI-DS NPs decreased in the NIR region, accompanied by an increase in absorption at 530 nm (Fig. 1f), resulting in decrease in NIR-Ⅱ fluorescence and activation of visible region fluorescence (Fig. 1g and Fig. S14 in Supporting information) which could be utilized in subsequent analysis of internalization of the NIR-Ⅱ fluorescence probe into cells by confocal microscopy. The oxidation and loss of electron donating ability of the DBU functional unit was clearly seen when comparing the changes in absorption spectra (Figs. S15 and S16 in Supporting information). The ¹H NMR spectrum and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum confirmed that PDI-DBU was oxidized under 808 nm laser irradiation, which in turn destroyed the D-A structure of PDI-DBU and restored the inherent visible fluorescence of the PDI compound (Figs. S17–S19 in Supporting information).

  Figure 1

  

  Figure 1.  DLS size distribution of (a) PDI-DS NPs and (b) PDI-D NPs in aqueous solution (Inset: TEM images of PDI-DS NPs and PDI-D NPs). Scale bar: 100 µm. (c) Zeta potential of PDI-DS NPs and PDI-D NPs. (d) UV-vis absorption spectra of PDI-DBU (in dichloromethane), DPS (in dichloromethane), PDI-D NPs (in water) and PDI-DS NPs (in water). (e) Fluorescence emission spectrum of PDI-DS NPs in water. (f, g) Changes in absorption (f) and emission spectra (g) of PDI-DS NPs under 808 nm laser irradiation. (h) H₂S release kinetics of PDI-DS NPs incubated with GSH (30 equiv.). (i) H₂S generation efficiency of PDI-DS NPs with GSH (10 min, 0–60 equiv. to DPS). Data are presented as mean ± standard deviation (SD) (n = 3).

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  The generation of H₂S from DPS could be triggered by GSH (Fig. S20 in Supporting information). The efficiency of H₂S generation was estimated using a H₂S colorimetric assay kit, which could quantitatively detect the presence of H₂S (Fig. S21 in Supporting information) [53]. As shown in Fig. 1h, 10 min after the addition of 30 equiv. of GSH (0.9 mmol/L) to PDI-DS NPs, the release of H₂S reached a plateau, verifying rapid response of PDI-DS NPs to GSH. In addition, we also investigated the effect of GSH concentration on the release of H₂S, it was found that the release of H₂S was dependent on GSH concentration and reached maximum value at a GSH concentration of 30 equiv. (0.9 mmol/L) (Fig. 1i). The experiments mentioned above verified the potential of PDI-DS NPs to be effectively stimulated by endogenous millimolar concentrations of GSH in cancer cells to produce H₂S, providing a feasibility for in vivo H₂S gas therapy.

  Due to the existence of broad absorption in NIR region, photothermal properties of PDI-DS NPs were investigated. We first evaluated heat generation behavior of PDI-DS NPs under 808 nm irradiation at 1 W/cm². As depicted in Fig. 2a, temperature of PDI-DS NPs solution increased from 23 ℃ to 45 ℃ at a material concentration of 200 µg/mL after laser irradiation for 300 s, while temperature of the PBS solution only rose by 3 ℃, and temperature changes were positively correlated with material concentration. Then, the influence of laser power density on the photothermal effect was also investigated. Temperature changes of PDI-DS NPs aqueous solution increased with the increase of laser power (Fig. 2b). The temperature showed a slight decrease after 6 irradiation-cooling cycles, but considerably more stable than ICG (Fig. 2c), as a result of the 808 nm laser activated oxidation of PDI. Photothermal conversion efficiency (PCE) of PDI-DS NPs was calculated to be 29.6% based on the heating and cooling profile (Fig. 2d) and the linear curve (Fig. 2e). As shown in Fig. S22 (Supporting information), photothermal images collected by an infrared thermal imager showed rapid heat generation to the plateau within 180 s, also proving satisfactory photothermal effect. Such properties endowed PDI-DS NPs with the ability to provide mild photothermal therapy, which could avoid the excessive thermal damage to healthy tissues [54].

  Figure 2

  

  Figure 2.  (a) Photothermal effect of PDI-DS NPs under 808 nm laser irradiation (1 W/cm², 6 min). Concentrations of PDI-DS NPs: 0–250 µg/mL. (b) Photothermal effect of PDI-DS NPs under 808 nm laser irradiation (200 µg/mL, 6 min). Laser power: 0.4–1.2 W/cm². (c) Temperature curves of five irradiation/cooling cycles of PDI-DS NPs and ICG in water. (d) Temperature curve of PDI-DS NPs (200 µg/mL) under 808 nm laser irradiation (1 W/cm², 6 min) followed by natural cooling and (e) the linear curve of cooling time versus temperature. (f) The absorption responses of ABDA in the presence of PDI-DS NPs under 808 nm laser irradiation (1 W/cm²) in water. (g) The absorption responses of ABDA in the presence of PDI-DS NPs under 530 nm laser irradiation (1 W/cm²) in water. (h) The absorption responses of ABDA in the presence of PDI-DS NPs under 808 nm laser irradiation (1 W/cm², 6 min), followed by 530 nm laser irradiation (1 W/cm²) in water. (i) Electron spin resonance (ESR) measurements of the generation of ¹O₂ using 2,2,6,6-tetramethylpiperidine (TEMP) as the spin-trapping adduct in the dark, under 808 nm laser irradiation (6 min, 1 W/cm²), or under 808 nm laser irradiation (6 min, 1 W/cm²) followed by 530 nm laser irradiation (10 min, 1 W/cm²).

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  We then investigated photodynamic properties of PDI-DS NPs. A singlet oxygen (¹O₂) sensing agent 9, 10-anthracenedipropanoic acid (ABDA) was used to validate the generation of ¹O₂. As displayed in Figs. 2f and g, there was no significant absorption decrease of ABDA (~400 nm), indicating that PDI-DS NPs could not produce ¹O₂ whether irradiated with 808 nm laser or 530 nm laser. Surprisingly, when PDI-DS NPs were first irradiated with 808 nm laser for 6 min, and subsequently exposed to 530 nm laser, a significant decrease in the absorption band of ABDA was observed (Fig. 2h). This clearly indicated the generation of a considerable amount of ¹O₂, as further confirmed by EPR shown in Fig. 2i. Therefore, it could be concluded that 808 nm laser irradiation resulted in oxidation of PDI-DBU and subsequent destruction of the D-A structure, leading to increase in absorption in the visible region and activation of the photodynamic property of the material. Superoxide anion radical (O₂^•−) probe DHR123 and hydroxyl radical (OH^•) indicator terephthalic acid (TA) were also used to test whether PDI-DS NPs could generate type Ⅰ ROS. The changes in fluorescence intensity displayed in Figs. S23 and S24 (Supporting information) demonstrated that PDI-DS NPs could only generate ¹O₂ as the type Ⅱ photosensitizer after being activated via 808 nm laser.

  Benefit from the rapid H₂S release capability triggered by excess GSH in TME, satisfactory photothermal effect, and 808 nm laser-activatable photodynamic property, we explored cytotoxicity and therapeutic effects at the cellular level of PDI-DS NPs. After incubated with PDI-DS NPs for 8 h, bright green fluorescence in cells activated by 808 nm laser indicated excellent cellular internalization efficiency of the material (Fig. S25 in Supporting information). We then investigated toxicity of PDI-DS NPs and PDI-D NPs using L929 cells. As could be seen from Fig. 3a, PDI-DS NPs and PDI-D NPs both exhibited negligible cytotoxicity at the tested concentration range, which in part could be a result of low levels of GSH in L929 cells that could not trigger the release of H₂S. The material thus proved excellent biological safety. Toxicity of PDI-DS NPs and PDI-D NPs on 4T1 cells were studied. Cytotoxicity patterns shown in Fig. 3b indicated higher cell survival rate when incubated with PDI-D NPs, while PDI-DS NPs groups exhibited, to a certain degree, dose-dependent cytotoxicity without laser treatment. However, after applied 808 nm laser at 1 W/cm² for 6 min, the survival rate of 4T1 cells decreased significantly, which verified the killing effect of photothermal and H₂S synergistic therapy. It is not surprising that 4T1 cells incubated with PDI-DS NPs exhibited the lowest survival rate when irradiated with 808 nm laser for 6 min followed by 530 nm laser for 10 min. The calculated combination index (CI) values also proved satisfactory synergistic effects of phototherapy and gas therapy (Fig. S26 in Supporting information) [55]. The above results clearly demonstrate efficient synergistic killing capability of the material towards 4T1 cancer cells.

  Figure 3

  

  Figure 3.  (a) Viability of L929 cells when incubated with PDI-DS NPs and PDI-D NPs of different concentrations. (b) Viability of 4T1 cells under different treatment methods (PDI-D NPs, PDI-DS NPs, PDI-DS NPs + 808 nm laser, PDI-D NPs + 808 nm laser + 530 nm laser and PDI-DS NPs + 808 nm laser + 530 nm laser). (c) Live/dead staining of 4T1 cells under different treatments. Red fluorescence, PI-stained dead cells; green fluorescence, calcein AM-stained living cells. (d) Flow cytometry analysis of apoptosis levels under different treatments. (e) CLSM images of ROS generated in 4T1 cells (DCFH-DA stained) under different treatments. The different groups in the figure were as follows: Ⅰ: PBS, Ⅱ: PBS + 808 nm laser + 530 nm laser, Ⅲ: PDI-DS NPs, Ⅳ: PDI-DS NPs + 808 nm laser, Ⅴ: PDI-D NPs + 808 nm laser + 530 nm laser, Ⅵ: PDI-DS NPs + 808 nm laser + 530 nm laser. Scale bar: 100 µm. Data are presented as mean ± SD (n = 3).

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  Then, live-dead cell co-staining experiments were carried out to more intuitively verify the killing effect of PDI-DS NPs towards 4T1 cells. As indicated in Fig. 3c, cells incubated with PDI-DS NPs without laser irradiation appeared small amount of localized red fluorescence, while the PBS and PDI-D NPs group emitted obvious green fluorescence. Meanwhile, the 4T1 cells treated with the "PDI-DS NPs + 808 nm laser", and the "PDI-D NPs + 808 nm laser + 530 nm laser" groups were further stained with red fluorescence, indicating more cell death. The PDI-DS NPs + 808 nm laser + 530 nm laser group showed the best killing effect towards 4T1 cells. Meanwhile, the flow cytometry experiment has also been applied to quantitatively verify the efficiency of synergistic therapy (Fig. 3d). Then 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was applied to evaluate the generation of intracellular ROS (Fig. 3e). 4T1 cells treated with PDI-D NPs or PDI-DS NPs, could both produce significant ROS when irradiated with 808 nm laser followed by 530 nm laser, while the other 4 groups did not show significant green fluorescence, suggesting that photodynamic properties of the NPs were effectively activated.

  H₂S produced by PDI-DS NPs in 4T1 cells was tracked by Washington State Probe-1 (WSP-1) [56]. The obvious green fluorescence given in Fig. 4a revealed the production of H₂S within the cells, which has also been validated by cell flow cytometry experiments (Fig. S27 in Supporting information). N-Ethylmaleimide (NEM), an effective thiol scavenger, reduced the green fluorescence of WSP-1 significantly by reducing intracellular GSH levels, further confirming accumulation of H₂S. Elevated H₂S could interfere with the respiratory chain of mitochondria, thereby block the nutritional supply of tumors [57]. We subsequently quantified intracellular ATP content to validate such property of PDI-DS NPs. After treatment with PDI-DS NPs or NaHS (external H₂S donor), the production of ATP was significantly restricted, demonstrating the inhibitory effect of H₂S (Fig. 4b). Regarding the downstream events, we explored whether the reduction of energy supply caused by H₂S could suppress the expression of heat shock protein HSP90 [58]. Western blot analysis confirmed the changes in intracellular HSP90 levels under different treatments. As given in Fig. 4c and Fig. S28 (Supporting information), HSP90 levels of 4T1 cells treated with PDI-D NPs + 808 nm laser increased by 22%, compared to the PBS group, while the PDI-DS NPs + 808 nm laser reduced by 45%, proving that PDI-DS NPs could inhibit the expression of HSP90, thereby promoting the photothermal effect. Then, we used JC-1 probe to visualize the disruption of mitochondria membrane potential by H₂S. In cells of normal vitality, JC-1 exists in the form of aggregates and emits red fluorescence. When the mitochondrial membrane potential decreases, JC-1 can emit green fluorescence in the form of monomer molecules. In Fig. 4d, significant increase in green fluorescence indicated that H₂S released by PDI-DS NPs could reduce the mitochondrial membrane potential and destruct their normal functions. Taking all of these into consideration, it became evident that PDI-DS NPs could effectively generate H₂S in 4T1 cells activated by GSH, disrupt the normal function of mitochondria, block the energy supply of tumor, inhibit the overexpression of HSP90, and therefore promote the therapeutic effect of photothermal therapy.

  Figure 4

  

  Figure 4.  (a) Imaging of cellular H₂S generation from PDI-DS NPs with or without NEM. (b) Intracellular ATP levels of 4T1 cells treated with PDI-D NPs, PDI-DS NPs (200 µg/mL), or NaHS (100 µmol/L). (c) HSP90 protein expression in 4T1 cells treated with PBS, PDI-D NPs + 808 nm laser or PDI-DS NPs + 808 nm laser. (d) Changes in mitochondria membrane potential of 4T1 cells visualized using the JC-1 probe (green fluorescence channel for abnormal mitochondria and red fluorescence channel for normal mitochondria). Different treatment groups: Ⅰ: PBS, Ⅱ: PBS + 808 nm laser + 530 nm laser, Ⅲ: PDI-DS NPs, Ⅳ: PDI-DS NPs + 808 nm laser, Ⅴ: PDI-D NPs + 808 nm laser + 530 nm laser, Ⅵ: PDI-DS NPs + 808 nm laser + 530 nm laser. Scale bar: 100 µm. Data are presented as mean ± SD (n = 3). The significance of the difference of more than two groups was determined via ANOVA-LSD post hoc test. ***P < 0.001.

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  Benefit from the satisfactory in vitro NIR-Ⅱ imaging performance shown in Fig. S12, the in vivo NIR-Ⅱ imaging ability of PDI-DS NPs was investigated. All animal experiments were approved by the guidelines of the Committee on Animal Use and Care of Changchun Institute of Applied Chemistry, Chinese Academy of Sciences (IACUC Issue No. CIAC 2024 [0089]). Prior to this, we conducted an in vivo biosafety assessment by measuring the hemolysis rate. The results indicated that hemolysis rates of both PDI-DS NPs and PDI-D NPs remained below 5% even at a high material concentration of 800 µg/mL, demonstrating their superior biocompatibility (Fig. S29 in Supporting information). As depicted in Fig. S30 (Supporting information), blood vessels in the fore and hind limbs of the nude mice could be clearly observed, following injection of PDI-DS NPs. Additionally, we observed that PDI-DS NPs also enabled clear imaging of the spine of the mice. Abdomen imaging revealed more pronounced whole-body vascular structures, even some minute blood vessels could be clearly observed (Fig. 5a and Fig. S31 in Supporting information). Through imaging of the mice using different longpass filters (900, 1000 and 1100 nm) and at multiple orientations, it was discovered that PDI-DS NPs exhibited the most effective imaging performance under a 1100 nm longpass filter, and the imaging quality was much superior to that of ICG (Figs. S32 and S33 in Supporting information). For instance, when comparing images of the blood vessels of lower limbs of the mice, PDI-DS NPs exhibited much superior signal-to-background ratio (SBR) compared to ICG (Fig. 5b and Fig. S34 in Supporting information). Meanwhile, popliteal lymph node (PLN) and sacral lymph node (SLN) could all be clearly imaged after injection of PDI-DS NPs, again showing considerably better imaging performance compared to ICG (Fig. 5c). As a result of the excellent imaging penetration, blood vessels in the head of the mice could also be clearly captured (Figs. 5d and e). Given the superb imaging performance described above, PDI-DS NPs were administered into tumor-bearing mice via the tail vein, fluorescence intensity in the tumor area increased over time and reached maximum enrichment at 16 h (Figs. 5f and g). SBR was calculated to be 3.9 according to the fluorescence intensity on the tangent line depicted in Fig. S35 (Supporting information). Therefore, we set 16 h after injection as the optimal treatment point. Furthermore, we euthanized the tumor-bearing mice 16 h and 24 h post-injection and harvested the primary organs and tumors. As shown in Figs. 5h and i, PDI-DS NPs were predominantly concentrated in the liver, spleen and tumor tissues.

  Figure 5

  

  Figure 5.  (a) NIR-Ⅱ fluorescence imaging of mouse abdomen and enlarged image of hindlimb blood vessels after injection of PDI-DS NPs or ICG via the tail vein. (b) NIR-Ⅱ fluorescence intensity profiles along the cross-sectional lines shown in (a). (c) NIR-Ⅱ fluorescence imaging of the lymph nodes (left: with PDI-DS NPs, right: with ICG). (d) NIR-Ⅱ fluorescence imaging of brain blood vessels. (e) NIR-Ⅱ fluorescence intensity profile along the cross-sectional line shown in (d). (f) NIR-Ⅱ fluorescence imaging of the 4T1 tumor-bearing mice at different time point post-injection of PDI-DS NPs. (g) Fluorescence image signal intensity changes of the tumor area shown in (f). (h) NIR-Ⅱ fluorescence imaging and (i) statistical analysis of fluorescence signal intensity of tumor and primary organs of the 4T1 tumor-bearing mice (collected at 0, 16 and 24 h). Data are presented as mean ± SD (n = 3).

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  Pharmacokinetic parameters are also crucial for proper evaluation of the therapeutic agents. The pharmacokinetics of PDI-DS NPs were studied using healthy mice. Concentration of PDI-DS NPs in the blood decreased significantly over time, with a calculated blood elimination half-life of 2.8 h, which could be attributed to the rapid accumulation of NPs in tumor and high rate of metabolism (Fig. S36 in Supporting information). In addition, the collected feces showed significant NIR-Ⅱ fluorescence, whilst the collected urine contained almost no fluorescence, indicating satisfactory liver metabolic capability of PDI-DS NPs, which was consistent with the tissue distribution images shown in Fig. 5h (Fig. S37 in Supporting information). In light of the aforementioned, PDI-DS NPs could therefore be regarded as an outstanding NIR-Ⅱ fluorescence imaging agent.

  Given the superb turn-on phototoxicity, exceptional NIR-Ⅱ imaging capability and rapid release of H₂S, in vivo therapeutic experiments were conducted. Initially, we utilized an infrared thermal imager to capture the in vivo photothermal images of PDI-DS NPs. As shown in Figs. 6a and b, temperature of the PBS + 808 nm laser group did not show significantly rise, whereas the tumor area treated with PDI-DS NPs + 808 nm laser experienced a rapid increase in temperature at the tumor, reaching 46 ℃ within 6 min. When tumor volume reached 50–100 mm³, 4T1 tumor-bearing mice were randomly divided into 6 groups: Ⅰ, PBS; Ⅱ, PBS + 808 nm laser + 530 nm laser; Ⅲ, PDI-DS NPs; Ⅳ, PDI-DS NPs + 808 nm laser; Ⅴ, PDI-D NPs + 808 nm laser + 530 nm laser; Ⅵ, PDI-DS NPs + 808 nm laser + 530 nm laser. As presented in Fig. 6c, tumor growth in PBS group and PBS + 808 nm laser + 530 nm laser group was not inhibited. The growth of tumor in other groups was inhibited to various degrees. Among them, the PDI-DS NPs group demonstrated the least tumor growth inhibition due to the H₂S gas therapy and lack of phototherapy. Not surprisingly, the PDI-DS NPs + 808 nm laser + 530 nm laser group enhanced tumor ablation most effectively, attributed to the synergistic effects of phototherapy and gas therapy. There were no significant differences in body weight among all 6 groups of tumor-bearing mice, indicating satisfactory biosafety of the materials (Fig. 6d). After 14 days of treatment, all tumor-bearing mice were euthanized. Photograph of tumors and tumor weight changes across all 6 groups presented in Figs. 6e and f further validated superior therapeutic efficacy of the material. Subsequently, we conducted histological examination using the collected tumor tissues (Fig. 6g). The hematoxylin and eosin (H&E) stained tumor tissues indicated the most severe tumor cell damage and most pronounced therapeutic effect of the PDI-DS NPs + 808 nm laser + 530 nm laser group compared to the other control groups. The most significant downregulation of the proliferation marker Ki-67 also indicated significant inhibition of tumor cell proliferation. H&E images given in Fig. S38 (Supporting information) revealed that there was no noticeable abnormality in major organs of the 6 groups. Blood routine test data (Fig. S39 in Supporting information) indicated that all parameters were within normal limits. As a result, PDI-DS NPs can serve as an efficient and safe therapeutic agent for combined phototherapy and gas therapy.

  Figure 6

  

  Figure 6.  (a) Photothermal images and (b) photothermal curves of 4T1 tumor-bearing mice under 808 nm laser irradiation at different time points. (c) Relative tumor volumes of different treatment groups. (d) Average weights of the mice of different treatment groups. (e) Photograph of the dissected 4T1 tumors from each group after the 14-day therapy. (f) Mean body weights of the tumors of different treatment groups. (g) H&E staining and Ki67 staining of the dissected 4T1 tumors after the 14-day treatment. The different groups in the figure are as follows: Ⅰ: PBS; Ⅱ: PBS + 808 nm laser + 530 nm laser; Ⅲ: PDI-DS NPs; Ⅳ: PDI-DS NPs + 808 nm laser; Ⅴ: PDI-D NPs + 808 nm laser + 530 nm laser; Ⅵ: PDI-D NPs + 808 nm laser + 530 nm laser. Scale bar: 100 µm. Data are presented as mean ± SD (n = 5). The significance of the difference of more than two groups was determined via ANOVA-LSD post hoc test. P < 0.05, **P < 0.01, ***P < 0.001.

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  In summary, we have developed a NIR-Ⅱ fluorescence imaging guided multi-mode therapeutic platform (PDI-DS NPs) using an organic phototherapuetic agent PDI-DBU, and a H₂S donor DPS. PDI DBU exhibits satisfactory photothermal effect and NIR-Ⅱ fluorescence emission properties. Furthermore, PDI-DBU can be oxidized under 808 nm laser irradiation which destroys the extended conjugated structure, resulting in activation of the photodynamic therapy. PDI-DBU can thus be converted from a photothermal agent into an effective photosensitizer. Additionally, when triggered by the abundant GSH within TME, PDI-DS NPs rapidly releases H₂S. The generated H₂S not only inhibits tumor growth through gas therapy but also suppresses the expression of HSP90, thereby reversing tumor resistance to heat, achieving a three in one synergistic therapy. The in vivo NIR-Ⅱ fluorescence imaging capabilities of PDI-DS NPs, such as visualizing the tumors, blood vessels, spine and lymph nodes have been successfully demonstrated. We also confirm the outstanding therapeutic efficacy of PDI-DS NPs both in vitro and in vivo. Therefore, this nano-platform, based on a PDI derivative, and the integration NIR-Ⅱ fluorescence imaging with multimodal therapy, demonstrate excellent potential of PDI compounds in synergistic therapy.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Wei Zhou: Writing – original draft, Methodology, Data curation, Conceptualization. Di He: Writing – review & editing, Investigation, Conceptualization. Ning Liu: Writing – review & editing, Methodology, Investigation, Conceptualization. Ying Li: Writing – review & editing, Methodology, Data curation. Wenzhao Han: Writing – review & editing, Data curation, Conceptualization. Weiping Zhou: Methodology, Conceptualization. Siyu Zhang: Methodology, Investigation. Cong Yu: Writing – review & editing, Funding acquisition, Data curation, Conceptualization.

  Acknowledgments

  This work was supported by the National Natural Science Foundation of China (No. 22274148), the Science and Technology Development Foundation of Jilin Province (Nos. 20220204098YY, 20230402045GH, 20230402018GH, YDZJ202201ZYTS359, YDZJ202201ZYTS351, 20240404070ZP, SKL202302030), and the Jilin Province Development and Reform Commission's Innovation Capacity Building Program (No. 2023C041-8). The in vivo NIR-Ⅱ fluorescence imaging experiment in this paper were conducted in the Joint Laboratory of Opto-Functional Theranostics in Medicine and Chemistry, First Hospital of Jilin University.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.110854.

  
  
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  Scheme 1  Schematic illustration of the construction of PDI-DS NPs as the synergistic theranostic application.

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  Figure 1  DLS size distribution of (a) PDI-DS NPs and (b) PDI-D NPs in aqueous solution (Inset: TEM images of PDI-DS NPs and PDI-D NPs). Scale bar: 100 µm. (c) Zeta potential of PDI-DS NPs and PDI-D NPs. (d) UV-vis absorption spectra of PDI-DBU (in dichloromethane), DPS (in dichloromethane), PDI-D NPs (in water) and PDI-DS NPs (in water). (e) Fluorescence emission spectrum of PDI-DS NPs in water. (f, g) Changes in absorption (f) and emission spectra (g) of PDI-DS NPs under 808 nm laser irradiation. (h) H₂S release kinetics of PDI-DS NPs incubated with GSH (30 equiv.). (i) H₂S generation efficiency of PDI-DS NPs with GSH (10 min, 0–60 equiv. to DPS). Data are presented as mean ± standard deviation (SD) (n = 3).

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  Figure 2  (a) Photothermal effect of PDI-DS NPs under 808 nm laser irradiation (1 W/cm², 6 min). Concentrations of PDI-DS NPs: 0–250 µg/mL. (b) Photothermal effect of PDI-DS NPs under 808 nm laser irradiation (200 µg/mL, 6 min). Laser power: 0.4–1.2 W/cm². (c) Temperature curves of five irradiation/cooling cycles of PDI-DS NPs and ICG in water. (d) Temperature curve of PDI-DS NPs (200 µg/mL) under 808 nm laser irradiation (1 W/cm², 6 min) followed by natural cooling and (e) the linear curve of cooling time versus temperature. (f) The absorption responses of ABDA in the presence of PDI-DS NPs under 808 nm laser irradiation (1 W/cm²) in water. (g) The absorption responses of ABDA in the presence of PDI-DS NPs under 530 nm laser irradiation (1 W/cm²) in water. (h) The absorption responses of ABDA in the presence of PDI-DS NPs under 808 nm laser irradiation (1 W/cm², 6 min), followed by 530 nm laser irradiation (1 W/cm²) in water. (i) Electron spin resonance (ESR) measurements of the generation of ¹O₂ using 2,2,6,6-tetramethylpiperidine (TEMP) as the spin-trapping adduct in the dark, under 808 nm laser irradiation (6 min, 1 W/cm²), or under 808 nm laser irradiation (6 min, 1 W/cm²) followed by 530 nm laser irradiation (10 min, 1 W/cm²).

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  Figure 3  (a) Viability of L929 cells when incubated with PDI-DS NPs and PDI-D NPs of different concentrations. (b) Viability of 4T1 cells under different treatment methods (PDI-D NPs, PDI-DS NPs, PDI-DS NPs + 808 nm laser, PDI-D NPs + 808 nm laser + 530 nm laser and PDI-DS NPs + 808 nm laser + 530 nm laser). (c) Live/dead staining of 4T1 cells under different treatments. Red fluorescence, PI-stained dead cells; green fluorescence, calcein AM-stained living cells. (d) Flow cytometry analysis of apoptosis levels under different treatments. (e) CLSM images of ROS generated in 4T1 cells (DCFH-DA stained) under different treatments. The different groups in the figure were as follows: Ⅰ: PBS, Ⅱ: PBS + 808 nm laser + 530 nm laser, Ⅲ: PDI-DS NPs, Ⅳ: PDI-DS NPs + 808 nm laser, Ⅴ: PDI-D NPs + 808 nm laser + 530 nm laser, Ⅵ: PDI-DS NPs + 808 nm laser + 530 nm laser. Scale bar: 100 µm. Data are presented as mean ± SD (n = 3).

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  Figure 4  (a) Imaging of cellular H₂S generation from PDI-DS NPs with or without NEM. (b) Intracellular ATP levels of 4T1 cells treated with PDI-D NPs, PDI-DS NPs (200 µg/mL), or NaHS (100 µmol/L). (c) HSP90 protein expression in 4T1 cells treated with PBS, PDI-D NPs + 808 nm laser or PDI-DS NPs + 808 nm laser. (d) Changes in mitochondria membrane potential of 4T1 cells visualized using the JC-1 probe (green fluorescence channel for abnormal mitochondria and red fluorescence channel for normal mitochondria). Different treatment groups: Ⅰ: PBS, Ⅱ: PBS + 808 nm laser + 530 nm laser, Ⅲ: PDI-DS NPs, Ⅳ: PDI-DS NPs + 808 nm laser, Ⅴ: PDI-D NPs + 808 nm laser + 530 nm laser, Ⅵ: PDI-DS NPs + 808 nm laser + 530 nm laser. Scale bar: 100 µm. Data are presented as mean ± SD (n = 3). The significance of the difference of more than two groups was determined via ANOVA-LSD post hoc test. ***P < 0.001.

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  Figure 5  (a) NIR-Ⅱ fluorescence imaging of mouse abdomen and enlarged image of hindlimb blood vessels after injection of PDI-DS NPs or ICG via the tail vein. (b) NIR-Ⅱ fluorescence intensity profiles along the cross-sectional lines shown in (a). (c) NIR-Ⅱ fluorescence imaging of the lymph nodes (left: with PDI-DS NPs, right: with ICG). (d) NIR-Ⅱ fluorescence imaging of brain blood vessels. (e) NIR-Ⅱ fluorescence intensity profile along the cross-sectional line shown in (d). (f) NIR-Ⅱ fluorescence imaging of the 4T1 tumor-bearing mice at different time point post-injection of PDI-DS NPs. (g) Fluorescence image signal intensity changes of the tumor area shown in (f). (h) NIR-Ⅱ fluorescence imaging and (i) statistical analysis of fluorescence signal intensity of tumor and primary organs of the 4T1 tumor-bearing mice (collected at 0, 16 and 24 h). Data are presented as mean ± SD (n = 3).

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  Figure 6  (a) Photothermal images and (b) photothermal curves of 4T1 tumor-bearing mice under 808 nm laser irradiation at different time points. (c) Relative tumor volumes of different treatment groups. (d) Average weights of the mice of different treatment groups. (e) Photograph of the dissected 4T1 tumors from each group after the 14-day therapy. (f) Mean body weights of the tumors of different treatment groups. (g) H&E staining and Ki67 staining of the dissected 4T1 tumors after the 14-day treatment. The different groups in the figure are as follows: Ⅰ: PBS; Ⅱ: PBS + 808 nm laser + 530 nm laser; Ⅲ: PDI-DS NPs; Ⅳ: PDI-DS NPs + 808 nm laser; Ⅴ: PDI-D NPs + 808 nm laser + 530 nm laser; Ⅵ: PDI-D NPs + 808 nm laser + 530 nm laser. Scale bar: 100 µm. Data are presented as mean ± SD (n = 5). The significance of the difference of more than two groups was determined via ANOVA-LSD post hoc test. P < 0.05, **P < 0.01, ***P < 0.001.

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