Abstract Gas therapy (GT) and/or phototherapy have been recently employed as immunogenic cell death (ICD) agents for activating immunotherapy, whereas the effective activation of sufficient immune responses remains an enormous challenge in such single therapeutic modality. In this study, a near-infrared (NIR)-triggered programmable nanomotor with hydrogen sulfide (H[2]S) and nitric oxide (NO) generation is well designed to achieve oncotherapy by cascading mild photothermal, gas, and reactive oxygen species (ROS)-reinforced immunogenic cell death. In brief, a gas signal molecule donor NOSH with H[2]S and NO capable of on-demand H[2]S and NO release was synthesized and then loaded into hollow mesoporous copper sulfide nanoparticles (termed as HCuSNPs) with an inherent NIR absorption and surface modification activity to obtain the programmable nanomotor (termed as NOSH@PEG-HCuSNPs). In particular, NOSH@PEG-HCuSNPs can effectively achieve the simultaneous spatiotemporal co-delivery of NOSH and HCuSNPs, thereby exerting the synergistic effects of GT and mild photothermal therapy (mPTT). It is worth noting that the anti-tumor response of mPTT is effectively enhanced by GT by disrupting the mitochondrial respiratory chain, inhibiting ATP production, and promoting tumor cell apoptosis. One by one, a large number of peroxynitrite anion (ONOO^−) radicals are generated by the interactions of ROS from mPTT and NO from NOSH. Meanwhile, the unique protective mechanism of H[2]S is utilized to induce tumor thermal ablation by reducing the overexpression of heat shock protein 90 (HSP 90) and minimize the unnecessary damage toward normal tissues. Finally, ICD is markedly augmented by the cascading effects of mPTT, ONOO⁻radicals, and H[2]S. Concurrently, the immunosuppressive tumor microenvironment is reprogrammed, effectively inhibiting distant tumor tissues and preventing metastasis and tumor recurrence. Taken together, this study provides a new perspective for innovation in the field of oncotherapy. Keywords: Gas signal molecule, Nanomotor, Mild photothermal therapy, Immunogenic cell death, Oncortherapy Graphical abstract Image 1 [39]Open in a new tab 1. Introduction Gas signaling molecules are important transmitters in living organisms. Such molecules can precisely bind to the corresponding receptors and trigger specific biological effects to transmit information or induce specific physiological reactions, and thus play an important regulatory role [[40]1,[41]2]. In recent years, gas therapy (GT) based on in situ generation of gaseous signaling molecules such as nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H[2]S), and hydrogen (H[2]) has attracted great interest in oncotherapy due to its excellent therapeutic effect and biosafety [[42]3]. Typically, H[2]S can activate intracellular acidification, induce cell cycle arrest, inhibit promoting cell survival pathways, and ultimately result in tumor apoptosis [[43]4]. In addition, H[2]S can disrupt mitochondrial homeostasis by reducing the activation of cytochrome c oxidase (COX IV), interfere with the respiratory chain, and inhibit cellular energy supply to hinder tumor proliferation [[44]5]. Notably, H[2]S also has a cytoprotective effect, which can be conducive to repairing overactive inflammation, accelerating wound healing, maintaining intracellular redox homeostasis [[45]6]. Therefore, H[2]S as a natural repair agent can reduce the damage of invasive anti-cancer methods. A large number of studies have found that heat shock protein 90 (HSP 90) as a key protein can induce cell heat resistance [[46]7], and its expression is positively correlated with ATP levels [[47]8]. Besides, H[2]S-induced depletion of intracellular ATP pools is considered to block the production of HSP 90, thereby reversing resistance to invasive oncotherapy [[48]9]. It is also found that NO can exert its tumor-killing effect by inducing mitochondrial/DNA damage, blocking DNA synthesis and repair, and inhibiting cell respiration [[49]10]. In addition, NO as a natural regulator can also change the immunosuppressive tumor microenvironment. On the one hand, NO itself can overcome resistance resulted from oncotherapy by activating immune cells, inhibiting immunosuppressive factors, promoting immune cell infiltration, and promoting tumor vascular normalization [[50]11,[51]12]. On the other hand, the latest studies have confirmed that NO can react with reactive oxygen species (ROS) generated during various phototherapy processes to generate peroxynitrite anion (ONOO^−), which is a reactive nitrogen species (RNS) that has a stronger inhibitory effect on tumor growth than traditional •OH and •O[2]^− [[52]13,[53]14]. What's more, the generation of RNS can effectively reverse the immunosuppressive tumor microenvironment by regulating the polarization of macrophages to an anti-tumor state, thereby enhancing the therapeutic efficacy of various tumors [[54]15,[55]16]. Therefore, GT involving multiple gas signaling molecules may become a promising direct therapy and indirect auxiliary anti-tumor methods. Photothermal therapy (PTT) is as an emerging and promising strategy for various diseases can convert light energy into heat energy to directly kill tumor cells by thermal ablation of tumor tissues [[56]17]. In order to achieve the tumor ablation, the pathological site is generally heated to more than 50 °C by the characteristics of photothermal agents. Although the increase in local temperature is effective for tumor elimination, it will inevitably lead to inevitable damage toward adjacent normal tissues through thermal diffusion [[57]18]. In addition, skin thermal swelling induced by photothermal hyperthermia also may induce long-term inflammation and hyperimmunogenicity, thereby inducing serious side effects [[58]19]. In contrast with conventional PTT, mild photothermal therapy (mPTT) (the operating temperature <45 °C) can significantly reduce the damage risk toward normal skin [[59]20]. However, due to the limited radiation efficiency of the therapeutic laser used in mPTT, the blood flow in the therapeutic region rapidly dissipates heat. Hence, mPPT are difficult to kill tumor cells at the tumor edge and around blood vessels beyond the laser penetration limit, which will result in this fact that tumor are not thoroughly cleared and further developed into invasive tumor to continue proliferation and recurrence of tumor [[60]21]. In addition, some authoritative studies have revealed that the immunosuppressive microenvironment of tumor can be beneficial for tumor cells to escape the attack of the immune system and promote the growth, invasion, and metastasis of tumor cells [[61]22]. Although mPTT can reshape the tumor immunosuppressive microenvironment by inducing immunogenic cell death (ICD) during treatment, the interaction between the tumor microenvironment and the immune response after treatment strengthens the tumor's immunosuppressive state. As a result, the tumor areas that have not been completely cleared bypass the host's immune response, thereby increasing the risk of mPTT recurrence [[62]9]. Therefore, how to explore some effective methods to regulate the tumor immunosuppressive microenvironment, break through the tumor immune biological barrier, reduce the risk of metastasis and recurrence, and thus improve the therapeutic effect and maximize the clinical potential of mPTT. For example, how to skillfully combine mPPT with other therapeutic methods (photodynamic therapy (PDT), chemotherapy, chemodynamic therapy (CDT), ferroptosis) is expected to become a promising oncotherapy strategy. In recent years, the combination strategy of GT and mPTT has attracted widespread attention in oncotherapy [[63]23]. Regrettably, existing treatment methods are simply a complex superposition of photothermal therapy and other treatments, rather than a simple treatment strategy that cascades the synergy between photothermal therapy and various treatment methods [[64]24]. Therefore, to further improve the potential application value of mPTT with a simple system as much as possible and achieve ideal therapeutic effects and harmless therapeutic processes, how to explore mPTT-based other new strategies and develop nanoplatforms is urgent It should be pointed out that the long-term exposure to H[2]S or NO does not impair the viability of normal cells, which provides feasibility for the synergistic targeted oncotherapy with H[2]S and NO [[65]25,[66]26]. Although the importance of H[2]S and NO has becoming increasingly apparent in anti-tumor occurrence on account of their unstable chemical properties, it is difficult to effectively deliver therapeutic levels of H[2]S and NO to tumor tissues [[67]6,[68]27]. On the one hand, this delivery difficulty derived from the obvious dose-dependent activity of gas signal molecules. In sharp contrast to high concentrations, low levels of H[2]S and NO stimulate tumor occurrence by accelerating the cell cycle, promoting proliferation, and inhibiting apoptosis [[69]28,[70]29]. On the other hand, it is a great challenge to ensure that the two gas signal molecules are simultaneously targeted and delivered to the tumor site [[71]30]. Therefore, a gas donor that can release H[2]S and NO continuously and simultaneously in sufficient quantity is introduced into the photothermal nanoplatform to reverse the immunosuppressive microenvironment and achieve precise coordination of GT and mPTT. Using a single regulation to achieve efficient anti-cancer effects has very important application prospects. In this paper, we first designed and synthesized a gas signal molecule donor NOSH that releases H[2]S and NO on demand to enhance the near-infrared-induced mPTT effect [[72]31]. Subsequently, large-cavity mesoporous copper sulfide nanoparticles HCuSNPs with fixed near-infrared (NIR) absorption and surface modification activity were constructed, which not only exhibited excellent photothermal conversion efficiency but were also considered to be one of the promising biodegradable and biocompatible photothermal conversion agents [[73]32]. On this basis, we loaded the gas donor NOSH onto the photothermal agent HCuSNPs and modified it with polyethylene glycol (PEG) derivatives that can improve its biocompatibility and tumor targeting [[74]33],constructing a programmable nanomotor NOSH@PEG-HCuSNPs. This nanomotor can not only achieve the synchronous delivery of NOSH and HCuSNPs, but also play an effective synergistic role between GT and mPTT. NOSH@PEG-HCuSNPs can induce additional mitochondrial dysfunction during tumor thermal ablation under near-infrared laser irradiation, thereby enhancing tumor cell apoptosis. In addition, by utilizing the unique protective mechanism of H[2]S, the overexpression of HSP 90 during tumor thermal ablation was reduced, which greatly reduced the unnecessary damage to normal tissues during laser irradiation. A large number of ONOO^− free radicals with strong tumor inhibition were generated by the interaction between ROS in mPTT and NO in NOSH. Therefore, the cascade reaction of mPPT, ONOO^− free radicals and H[2]S enhanced tumor ICD, reversed the tumor immune microenvironment, and effectively prevented tumor recurrence and metastasis. This strategy confirmed the feasibility of non-invasive mPTT to cure tumors and showed great application potential. 2. Materials and methods 2.1. Reagents and instruments Unless otherwise specified, the reagents used in this experiment are analytically pure and do not require further purification. All reagents were purchased from legal commercial channels. Copper (II) chloride dihydrate (CuCl[2]·2H[2]O) and Sodium sulfide nonahydrate (Na[2]S·9H[2]O) were obtained from Chengdu Kelong Chemical Co., Ltd. Poly (vinylpyrrolidone) (PVP K40, Mw = 40,000) was purchased from Sigma-Aldrich. Hydrazine hydrate aqueous solution (N[2]H[4]·H[2]O) was provided by Shanghai Zhongqin Chemical Reagent Co., Ltd. L-Arg, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysulfosuccinimide sodium salt (NHS), Fluorescein isothiocyanate (FITC) and Indocyanine green (ICG) were supplied by Macklin. NH[2]-PEG-NH[2] came from Carbohydrates Technology. Fetal bovine serum (FBS), Trypsin, RPMI-1640 medium, Streptomycin Penicillin, and Cell counting kit-8 (CCK-8) were purchased from Wilber. Lyso-Tracker Red Probe, Apoptosis Kit, NO Detection Kit, and ROS detection kit were provided by Beyotime. Cell Live Dead Staining Kit and ONOO^− Detection Kit were supplied by Bestbio. Hoechst 33342 came from Biosharp. ATP Detection Kit was obtained from Solarbio. 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), anti-CD11c-FITC, anti-CD80-APC, anti-CD86-PE, anti-CD3-FITC, anti-CD4-APC, anti-CD8a-PE, anti-CD86-PE, anti-CD206-PE, anti-F4/80-APC, anti-CD11b-FITC, and anti-CD11c-FITC were obtained from Elabscience. All ELISA kits (IFN-γ, IL-10, IL-12p40, GZMS-B, PGE[2] and TNF-α) were purchased from Boster. 4T1 cells were provided by the cell bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. NMR spectra were measured using a superconducting nuclear magnetic resonance spectrometer AVANCE Ⅲ HD (Switzerland); Fluorescence emission spectra were recorded on a fluorescence spectrometer RF-5301pc (Japan); Absorption spectra were measured on a UV–visible spectrophotometer TU-1810 (China); High-resolution mass spectra were measured using a high-resolution time-of-flight mass spectrometer MicroTof Q II (USA); Fluorescence images of cells were taken using a fluorescence microscope Axio Imager.Z2 (Germany); Cell viability and cytokines were recorded using a microplate reader Flash3001 (USA); The morphology of the samples was analyzed by a scanning electron microscope JSM-5601LV (Japan); The structure of the samples was characterized using an X-ray diffractometer Smartlab-SE (Japan), an X-ray photoelectron spectrometer ESCALAB 250Xi (USA), and a Fourier transform infrared spectrometer VERTEX 70v (Germany); Zeta potential and particle size analyzer 90Plus Pals were used (USA) to characterize samples; Thermogravimetric changes of samples were recorded using a synchronous thermal analyzer STA449F3 (Germany); Temperature changes of samples and living bodies were recorded using an infrared thermal imager Fotric 325L (China); In vivo photoacoustic data were recorded using an ultrasonic imaging instrument Vevo F2 LAZR-X (Japan); In vivo fluorescence imaging data were recorded using an in vivo imager VISQUE Invivo Smart-LF (Singapore); Flow cytometry analysis was performed using a flow cytometer CytoFLEX (USA). 2.2. Synthesis process 2.2.1. Synthesis of hollow copper sulfide nanoparticles (HCuSNPs) CuCl[2]·2H[2]O solution (100 μL) and polyvinyl pyrrolidone (0.24 g) were mixed in deionized water (25 mL) and stirred at room temperature. NaOH (pH 9, 25 mL) solution was added to the stirred mixed solution, and then hydrazine solution (6.4 μL) was added. The color of the solution turned to bright yellow to form a Cu[2]O ball suspension. Subsequently, 200 μL of Na[2]S (320 mg mL^−1) aqueous solution was added to the Cu[2]O suspension, stirred at 60 °C for 2 h to ensure sufficient reaction until the solution turned dark green, cooled to room temperature, centrifuged (11000 rpm, 10 min), washed 3 times with deionized water, and HCuSNPs were obtained by freeze-drying. 2.2.2. Synthesis of NOSH Compound 1: Under nitrogen, DCC (845 mg, 4.09 mmol), DMAP (50 mg, 0.409 mmol) and salicylaldehyde (500 mg, 4.09 mmol) were added to 4-bromobutyric acid (683 mg, 4.09 mmol) in CH[2]Cl[2], and the whole reaction mixture was stirred at room temperature overnight. After the reaction was completed (checked by TLC), the precipitate was filtered off, and the organic phase was extracted into CH[2]Cl[2] (2x15 mL) after adding water. The organic solvent was removed by reduced pressure to obtain the crude product. The product was purified by column chromatography with a yield of 80 %. Compound 2: Compound 1 (500 mg, 1.84 mmol) was mixed with AgNO[3] (780 mg, 4.6 mmol) in CH[3]CN (20 mL), stirred at 70 °C for 7 h, filtered through celite and concentrated under reduced pressure. The organic layer was separated after adding CH[2]Cl[2] (20 mL) and H[2]O (20 mL), and the aqueous layer was extracted 3 times with CH[2]Cl[2] (20 mL). All organic layers were dried and concentrated under reduced pressure. The crude product was purified by flash chromatography. Eluent [90:10 PE/EtOAc (v/v)]. The product was a light yellow oil with a yield of 84 %. Compound 3: KMnO[4] (692 mg, 4.38 mmol) was added to a stirred solution of Compound 2 (2.96 g, 11.74 mmol) in acetone (20 mL) at 0 °C. The reaction mixture was heated to room temperature and stirred for 3 h. After the reaction was completed (checked by TLC), oxalic acid was added and the precipitate was filtered off. The filtrate was diluted with dichloromethane, washed with water, dried, concentrated under reduced pressure, and the crude product was purified by crystallization to obtain the product. White solid, yield 83 %. Compound NOSH: DCC (201.0 mg, 0.97 mmol) and DMAP (10.8 mg, 0.09 mmol) were added to CH[2]Cl[2] of Compound 3 (238.0 mg, 0.88 mmol). Then ADT-OH (200.0 mg, 0.88 mmol) was added and stirred at room temperature overnight. After the reaction was completed by TLC, the precipitate was filtered off, water was added, and then extracted into dichloromethane (2x50 mL). The organic solvent was removed under reduced pressure to obtain a crude product. Further purification by column chromatography gave a pure orange solid with a yield of 78 %. 2.2.3. Synthesis of NOSH@HCuSNPs Take 10 mL of HCuSNPs (1 mg mL^−1) in deionized water and stir with 1 mL of NOSH (10 mg mL^−1) in DMSO at room temperature, and stir overnight. The mixture was centrifuged at 10000 g. The crude product was washed three times with DMSO. Finally, it was dispersed in deionized water for later use. 2.2.4. Synthesis of NH[2]-PEG-FA Folic acid FA (44.1 mg, 0.1 mmol) was dissolved in DMSO (3 mL). Then EDC·HCl (28.8 mg, 0.15 mmol) and hydroxysuccinimide NHS (17.3 mg. 0.15 mmol) were added to the above solution and stirred in the dark for 12 h. Finally, NH[2]-PEG-NH[2] (400 mg) was dissolved in CHCI[3] (1 mL) and DMSO (2 mL), and FA was added to the above reaction solution and stirred for 24 h. The product was precipitated in ether and dried under vacuum. 2.2.5. Synthesis of NOSH@PEG-HCuSNPs NH[2]-PEG-FA (200 mg) and NOSH@HCuSNPs (20 mg) were dissolved in 20 mL of deionized water and stirred at room temperature for 12 h. The material was washed three times with deionized water and freeze-dried. Finally, it was dispersed in deionized water for use. 2.2.6. Synthesis of FITC-NOSH@PEG-HCuSNPs FITC (0.1 mg mL^−1) was mixed with an ethanol solution of NOSH@PEG-HCuSNPs (1 mg mL^−1, 10 mL) and stirred at room temperature for 12 h. Subsequently, it was centrifuged and washed to obtain FITC-labeled NOSH@PEG-HCuSNPs, which were redispersed in deionized water for cell imaging. 2.2.7. Synthesis of ICG-NOSH@PEG-HCuSNPs ICG and NOSH@PEG-HCuSNPs were dissolved in 10 mL of ethanol solution at a ratio of 1:2 and vigorously stirred at room temperature for 12 h, followed by centrifugation. After washing with ethanol, ICG-labeled NOSH@PEG-HCuSNPs were obtained and finally redispersed in deionized water for in vivo imaging. 2.3. In vitro performance characterization 2.3.1. In vitro photothermal performance First, the NOSH@PEG-HCuSNP deionized water solutions with different concentrations (0, 25, 50, 100, 200, 400 and 800 mg L^−1) in the EP tube were irradiated with an 808 nm near-infrared laser with a power density of 1.5 W cm^−2 for 10 min. Secondly, the NOSH@PEG-HCuSNP deionized water solution with a concentration of 200 mg L^−1 was irradiated with a near-infrared laser with several power densities (0, 0.5, 1.0, 1.5 and 2.0 W cm^−2) for 10 min. Finally, in order to investigate the thermal stability of NOSH@PEG-HCuSNP, the solution was irradiated with an 808 nm laser (1.5 W cm^−2) for 10 min, and then cooled naturally for 15 min. The temperature change curves of five consecutive cycles were recorded, and the photothermal conversion efficiency (η) was evaluated according to the previously reported formula. Specifically,The photothermal conversion efficiency was calculated using the following equation: Among them, hAs (Tmax−Ts): Total heat generated from photothermal conversion; Q[dis]: Heat dissipation power from non-photothermal processes, estimated from the control experiments. 2.3.2. In vitro NO release assay We chose the GRIESS reagent method as the method to detect the release of NO in NOSH@PEG-HCuSNPs. The GRIESS reagent was prepared as follows: 6 mL of concentrated phosphoric acid (85 %), 70 mL of deionized water and 1.0 g of anhydrous p-aminobenzenesulfonic acid were fully dissolved and then diluted to 100 mL to prepare reagent A; 0.1 g of N-1-naphthylethylenediamine hydrochloride was dissolved in hydrogen ion water and diluted to 100 mL to prepare reagent B. During the detection process, reagent A and reagent B were mixed in a ratio of 1:1 to prepare the GRIESS reagent for detection. First, the standard curve of GRIESS reagent for detecting NO was drawn by adding different concentrations of NaNO[2] (0, 12.5, 25, 50, 100, 200, 400, 800 μM). Then, NOSH@PEG-HCuSNPs and GRIESS reagent were added to PBS solution at the same time. By adding different concentrations (0, 25, 50, 100, 200, and 400 μg mL^−1) of NOSH@PEG-HCuSNPs and laser irradiation of different powers (0, 0.5, 0.75, 1, 1.25, and 1.5 W cm^−2), the UV absorption at 540 nm was recorded by UV spectrophotometer after a period of time under different pretreatment conditions. Finally, the amount of NO released under different pretreatment conditions was calculated by the standard curve. 2.3.3. In vitro H[2]S release assay We selected probe NP-N[3] as the detection of H[2]S release in NOSH@PEG-HCuSNPs. First, by adding different concentrations (0, 0.25, 0.5, 1, 2, 5, 10, 20, 40 μM) of NaHS, the standard curve of probe NP-N[3] for detecting H[2]S was drawn. Then, NOSH@PEG-HCuSNPs and NP-N[3] were added to DMSO: PBS (v/v = 4:1) solution at the same time. By adding different concentrations (0, 25, 50, 100, 200, and 400 μg mL^−1) of NOSH@PEG-HCuSNPs and laser irradiation of different powers (0, 0.5, 0.75, 1, 1.25, and 1.5 W cm^−2), the fluorescence emission intensity at the emission wavelength of 520 nm under different pretreatment conditions was recorded by fluorescence spectrophotometer. Finally, the amount of H[2]S released under different pretreatment conditions was calculated by the standard curve. 2.3.4. Stability evaluation NOSH@PEG-HCuSNPs were dispersed in Aqueous, PBS, Normal Saline and 10 % FBS 1640 medium to evaluate their biostability under different simulated environments. The experimental solution was kept at 37 °C and stirred at 100 rpm to simulate the in vivo environment. Then, the experimental solution was collected from each sample at a given time, and the biodegradation behavior of NOSH@PEG-HCuSNPs was directly studied by transmission electron microscopy and the particle size change of the nanomaterials was recorded. NOSH@PEG-HCuSNPs were incubated in PBS solution and 10 % FBS 1640 medium under 808 nm laser irradiation, and TEM images after different incubation times were observed by transmission electron microscopy. 2.4. Cell experiment 2.4.1. Cell culture Breast cancer cells 4T1 were used for cell verification and in vivo modeling experiments in this study. 4T1 cells were cultured at 37 °C in a humidified atmosphere containing 5 % carbon dioxide (CO[2]) using RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS) and 1 % streptomycin/penicillin. 2.4.2. Cytotoxicity The cytotoxicity of the synthesized materials was evaluated using the CCK-8 method. First, 4T1 cells were inoculated in a 96-well plate with 5000 cells per well and cultured in RPMI-1640 medium containing 10 % FBS at 37 °C and 5 % CO[2] for 12 h. Then, different concentrations of HCuSNP, NOSH and NOSH@HCuSNPs were added and incubated for 12 h, 24 h and 48 h, respectively. Subsequently, CCK-8 (10 μL) solution was added to each well and further incubated for 2 h. The absorbance value of each well was measured at 450 nm using a microplate reader, and there were 6 parallel groups for each concentration. The cell survival rate was calculated by VR = A/A[0] × 100 %, where A was the absorbance value of the experimental group at different concentrations, and A[0] was the UV absorbance value of the blank control group. 2.4.3. Cell uptake 4T1 cells were seeded in 12-well plates and incubated with RPMI-1640 medium containing 10 % FBS for 24 h under standard conditions (37 °C, 5 % CO[2]). Then, Hoechst 33342 dye for labeling cell nuclei, Lyso-Tracker Red dye for labeling lysosomes, and FITC-NOSH@PEG-HCuSNPs for labeling materials were added to the cells. The cells were incubated for appropriate times (0, 0.5 h, 1 h, 2 h, and 4 h). Fluorescence microscopy and flow cytometry were used to observe the uptake of materials by cells. 2.4.4. Anticancer activity 4T1 cells were seeded in 96-well plates, with 5000 cells per well, and cultured in RPMI-1640 medium containing 10 % FBS at 37 °C and 5 % CO[2] for 12 h. The cells were grouped and pretreated with different treatments: (1) Blank, (2) Laser (1.0 W cm^−2), (3) HCuSNPs, (4) NOSH, (5) NOSH@PEG-HCuSNPs, (6) HCuSNPs + Laser(1.0 W cm^−2), (7) HCuSNPs + Laser(0.5 W cm^−2), (8) NOSH@PEG-HCuSNPs + Laser(1.0 W cm^−2) and (9) NOSH@PEG-HCuSNPs + Laser(0.5 W cm^−2). The experimental group without laser treatment was directly incubated for 12 h after administration, and the group requiring laser treatment was irradiated with 808 nm laser for 10 min and then continued to be incubated for 12 h. All experimental groups had 6 parallel groups, and the anti-tumor activity was evaluated by CCK-8 method. 2.4.5. Live and dead cell staining The Calcein-AM/PI live and dead cell staining kit was used to detect cell apoptosis. 4T1 cells were seeded in 12-well plates and incubated with RPMI-1640 medium containing 10 % FBS for 24 h under standard conditions (37 °C, 5 % CO[2]). The cells were grouped and pretreated differently: (1) Blank, (2) Laser (1.0 W cm^−2), (3) HCuSNPs, (4) NOSH, (5) NOSH@PEG-HCuSNPs, (6) HCuSNPs + Laser(1.0 W cm^−2), (7) HCuSNPs + Laser(0.5 W cm^−2), (8) NOSH@PEG-HCuSNPs + Laser(1.0 W cm^−2) and (9) NOSH@PEG-HCuSNPs + Laser(0.5 W cm^−2). The laser-treated group was irradiated with 808 nm laser for 10 min. Afterwards, the cells were moved to standard conditions (37 °C, 5 % CO[2]) for 1 h, washed twice with pre-cooled PBS, and incubated with Calcein-AM and PI double staining reagent for 15 min. After washing the cells with PBS, the cells were immediately imaged by fluorescence microscopy. 2.4.6. Apoptosis Annexin V-FITC/PI apoptosis detection kit was used to detect cell apoptosis. 4T1 cells were seeded in 12-well plates and incubated with RPMI-1640 medium containing 10 % FBS for 24 h under standard conditions (37 °C, 5 % CO[2]). The cells were grouped and subjected to different pretreatments: (1) Blank, (2) Laser (1.0 W cm^−2), (3) HCuSNPs, (4) NOSH, (5) NOSH@PEG-HCuSNPs, (6) HCuSNPs + Laser(1.0 W cm^−2), (7) HCuSNPs + Laser(0.5 W cm^−2), (8) NOSH@PEG-HCuSNPs + Laser(1.0 W cm^−2) and (9) NOSH@PEG-HCuSNPs + Laser(0.5 W cm^−2). The group requiring laser treatment was irradiated with 808 nm laser for 10 min. Afterwards, the cells were moved to standard conditions (37 °C, 5 % CO[2]) for 1 h, washed twice with pre-cooled PBS, and then resuspended in 100 μL of PBS buffer. The proportion of apoptotic cells was detected by flow cytometry using the method described in the Annexin V-FITC/PI kit. 2.4.7. Detection of mitochondrial membrane potential The JC-1 mitochondrial membrane potential detection kit was used to observe the changes in mitochondrial membrane potential in 4T1 cells after different pretreatments. 4T1 cells were seeded in 12-well plates and incubated with RPMI-1640 medium containing 10 % FBS for 24 h under standard conditions (37 °C, 5 % CO[2]). The cells were grouped and subjected to different pretreatments: (1) Blank, (2) Laser (1.0 W cm^−2), (3) HCuSNPs, (4) NOSH, (5) NOSH@PEG-HCuSNPs, (6) HCuSNPs + Laser(1.0 W cm^−2), (7) HCuSNPs + Laser(0.5 W cm^−2), (8) NOSH@PEG-HCuSNPs + Laser(1.0 W cm^−2) and (9) NOSH@PEG-HCuSNPs + Laser(0.5 W cm^−2). The laser-treated group was irradiated with 808 nm laser for 10 min. Afterwards, the cells were moved to standard conditions (37 °C, 5 % CO[2]) for 1 h and then incubated with JC-1 working solution at 37 °C for 30 min. Subsequently, the cells were washed with PBS, and the changes in mitochondrial membrane potential in the cells were observed by fluorescence microscopy (red fluorescence, ex = 585 nm, em = 590 nm; green fluorescence, ex = 514 nm, em = 529 nm). 2.4.8. Intracellular H[2]S, NO, ROS and ONOO^− detection NP-N[3] probe, DAF-FM DA kit, DCFH-DA probe and ONOO^− detection kit were used to detect changes in intracellular H[2]S, NO, ROS and ONOO^− content. 4T1 cells were seeded in 12-well plates and incubated with RPMI-1640 medium containing 10 % FBS for 24 h under standard conditions (37 °C, 5 % CO[2]). The cells were grouped and pretreated differently: (1) Blank, (2) Laser (1.0 W cm^−2), (3) HCuSNPs, (4) NOSH, (5) NOSH@PEG-HCuSNPs, (6) HCuSNPs + Laser(1.0 W cm^−2), (7) HCuSNPs + Laser(0.5 W cm^−2), (8) NOSH@PEG-HCuSNPs + Laser(1.0 W cm^−2) and (9) NOSH@PEG-HCuSNPs + Laser(0.5 W cm^−2). The laser-treated group was irradiated with 808 nm laser for 10 min. Afterwards, the cells were incubated with different working solutions at 37 °C according to the instructions of different probes and kits. After washing the cells with PBS, the cell images were immediately recorded by fluorescence microscopy. 2.4.9. ATP detection The ATP content in 4T1 tumor cells was determined using an ATP detection kit. 4T1 cells were seeded in 6-well plates and incubated with RPMI-1640 medium containing 10 % FBS for 24 h under standard conditions (37 °C, 5 % CO[2]). The cells were divided into groups and subjected to different pretreatments: (1) Blank, (2) Laser (1.0 W cm^−2), (3) HCuSNPs, (4) NOSH, (5) NOSH@PEG-HCuSNPs, (6) HCuSNPs + Laser(1.0 W cm^−2), (7) HCuSNPs + Laser(0.5 W cm^−2), (8) NOSH@PEG-HCuSNPs + Laser(1.0 W cm^−2) and (9) NOSH@PEG-HCuSNPs + Laser(0.5 W cm^−2). The laser-treated groups were irradiated with 808 nm laser for 10 min. Afterwards, ATP content was determined according to the ATP Assay Kit procedure. Operate on ice, add 500 μL of extract to each well, ultrasonically disrupt, and centrifuge. Take the supernatant and add 500 μL of chloroform to mix, centrifuge, and collect the supernatant. Use an enzyme reader to measure the absorbance value at 340 nm, and calculate the ATP content in each group through the standard curve. 2.4.10. Western blotting 4T1 cells were inoculated in a 6-well plate and incubated with RPMI-1640 medium containing 10 % FBS for 24 h under standard conditions (37 °C, 5 % CO[2]). The group to be laser treated was irradiated with 808 nm laser for 10 min. Wash the cells three times with cold PBS, and lyse the cells after 12 h to determine the total protein using the BCA protein assay kit. The same mass of protein (40 μg) was separated by 12 % or 10 % tris-succinyl gel electrophoresis, and then the separated proteins were transferred to polyvinylidene difluoride membrane (PVDF) and sealed with 5 % skim milk for 1 h to reduce nonspecific binding. Finally, the primary and secondary antibodies were used for detection, and the enhanced chemiluminescence detection kit was used for analysis in a chemiluminescence imaging system. Western blotting was used to detect the expression levels of heat shock protein 90 (Hsp 90) and cytochrome c oxidase subunit IV (COX IV) in cells after different treatments. 2.4.11. mRNAs profiling analysis Total RNA was extracted using TRIzol reagent according to the instructions. RNA purity and quantification were identified using NanoDrop 2000 spectrophotometer, and RNA integrity was assessed using Agilent 2100 Bioanalyzer. Transcriptome libraries were constructed using VAHTS Universal V5 RNA-seq Library Prep Kit according to the instructions. Transcriptome sequencing and analysis were performed by Shanghai Ouyi Biotechnology Co., Ltd. First, the Illumina Novaseq 6000 sequencer was used to obtain raw reads data after image recognition and base recognition. Next, fastp software was used to remove adapters and low-quality reads to obtain high-quality clean reads for subsequent data analysis. We used DESeq2 software to perform differentially expressed gene analysis, where genes that met the thresholds of q value < 0.05 and foldchange >2 or foldchange <0.5 were defined as differentially expressed genes (DEGs). GO and KEGG pathway analysis were performed on host genes of differentially expressed mRNA to show the expression changes of up-regulated or down-regulated genes. The Search Tool for The Retrieval of Interacting Genes/Proteins (STRING) algorithm was used to analyze the functional interaction network of regulatory proteins involved in signaling pathways. 2.5. In vivo experiment 2.5.1. Hemolysis test Blood was collected from mice by orbital venous plexus blood sampling and sodium heparin was added. Red blood cells were separated from the blood samples by centrifugation. 1 mL of separated red blood cells was diluted in 50 mL PBS and treated with the same volume of NOSH@PEG-HCuSNPs at 37 °C for 60 min. In addition, DI water and PBS were added to the same volume of diluted separated red blood cells as positive and negative controls, respectively. After incubation, all samples were centrifuged at 1000 rpm for 3 min, 100 μL of supernatant was transferred to a 96-well plate, and the absorbance at a wavelength of 590 nm was measured using an ELISA reader. 2.5.2. In vivo biosafety evaluation Healthy male Balb/c mice (six weeks old, n = 4) were randomly divided into groups for the experiment. Each mouse was injected with a single dose of PBS, HCuSNPs, and NOSH@PEG-HCuSNPs through the tail vein at a dose of 20 mg kg^−1. Mice were killed on the 14th day after administration. Then, the mice were dissected, and the main tissues and organs (heart, liver, spleen, lung, and kidney) were removed, fixed with 4 % paraformaldehyde, embedded in paraffin, and tissue sections were prepared. These tissue sections will be stained with hematoxylin and eosin (H&E) to observe pathological characteristics, evaluate the possible toxicity of combined treatment on major organs, and capture images. 2.5.3. Establishment of mouse tumor model Male Balb/c mice were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. All experiments were carried out in accordance with the guidelines of the Ethics Committee of Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, and the experiments were approved by the Ethics Committee of Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences. Primary tumor model: 4T1 tumor cells (2 × 10^6 cells) suspended in PBS (100 μL) were subcutaneously injected into the right axilla of mice to establish a tumor model. The tumor was allowed to grow for about 18 days, and the experiment was performed when the tumor volume reached about 100 mm^3. Double tumor model: 4T1 tumor cells (2 × 10^6 cells) suspended in PBS (100 μL) were subcutaneously injected into the right axilla of mice. When the tumor volume reached about 50 mm^3, 4T1 tumor cells (2 × 10^6 cells) suspended in PBS (50 μL) were subcutaneously injected into the left axilla of mice. The experiment was started when the right axillary tumor volume of all mice reached about 100 mm^3. 2.5.4. In vivo imaging Fluorescence imaging: ICG-NOSH@PEG-HCuSNPs (200 μL, 20 mg kg^−1) modified with ICG were injected into tumor-bearing mice (n = 4) through the tail vein, and then the fluorescence images at 0, 1, 3, 6, 12, and 24 h were captured using an in vivo fluorescence imager. Photoacoustic imaging: NOSH@PEG-HCuSNPs (200 μL, 20 mg kg^−1) were injected into tumor-bearing mice (n = 4) through the tail vein, and the photoacoustic signals of the respective tumor areas of the mice administered at different time points (0, 1, 3, 6, 12, 24 h) were recorded. 2.5.5. In vivo photothermal performance PBS (200 μL), HCuSNPs (200 μL, 20 mg kg^−1) and NOSH@PEG-HCuSNPs (200 μL, 20 mg kg^−1) were injected into tumor-bearing mice (n = 4) through the tail vein. 6 h after injection, the mouse tumor was irradiated with 808 nm laser (0.5 W cm^−2/1.0 W cm^−2) for 10 min. The tumor temperature was monitored and the infrared thermal image was recorded using an infrared thermal imager. 2.5.6. In vivo antitumor effect The tumor-bearing mice were randomly divided into 9 groups, namely (1) Blank, (2) Laser (1.0 W cm^−2), (3) HCuSNPs, (4) NOSH, (5) NOSH@PEG-HCuSNPs, (6) HCuSNPs + Laser(1.0 W cm^−2), (7) HCuSNPs + Laser(0.5 W cm^−2), (8) NOSH@PEG-HCuSNPs + Laser(1.0 W cm^−2) and (9) NOSH@PEG-HCuSNPs + Laser(0.5 W cm^−2). Laser (808 nm, 10 min) was applied 6 h after administration. The tumor volume and mouse weight of each group were measured every 3 days to evaluate the treatment results. Tumor volume (V mm^3) = (a × b^2)/2, where a and b are the length and width of the tumor, respectively. 2.5.7. Immune response evaluation The mice in each group were dissected after treatment, and the tumors, spleens, and tumor-draining lymph node tissues of the mice were collected and dissociated into single cell suspensions using tissue lysis buffer. After centrifugation, the cells were resuspended in culture medium and washed with PBS, stained at room temperature for 1 h, and finally the cell content was detected by flow cytometry. In the experiment, different antibodies were used to stain different cell types, among which DC cells were stained with anti-CD11c-FITC, anti-CD80-APC, and anti-CD86-PE antibodies; for T cells, anti-CD3-FITC, anti-CD4APC, and anti-CD8a-PE antibodies were used for staining; M1 macrophages/M2 macrophages were stained with anti-CD86-PE/anti-CD206-PE, anti-F4/80-APC, and anti-CD11bFITC antibodies. In addition, tumor sections of each group were further studied by hematoxylin and eosin (H&E) staining, TUNEL (TdT-mediated dUTP Nick-End Labeling) method and ki67 immunohistochemical staining. Serum samples of mice after various treatments were collected and diluted for analysis. Cytokines INF-γ, TNF-α, GZMS-B, IL-12p40, IL-10, and PGE[2] were analyzed using ELISA kits according to the supplier's protocol. 2.6. Statistical analysis The data obtained in the experiment were verified at least 3 times and are expressed as mean ± standard deviation (SD). One-way ANOVA was performed using SPSS statistical software to determine multiple comparisons between groups. ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05 were considered statistically significant. 3. Results 3.1. Design, synthesis, and characterization of NOSH and NOSH@PEG-HCuSNPs In order to synthetize the gas donor NOSH with NO and H[2]S, as shown in [75]Scheme 1, the nitrate (-ONO[2]) groups as donors were employed to release NO and then attach them to salicylaldehyde by esterification. Afterwards, the H[2]S releasing group 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH) was also directly coupled to the salicylaldehyde skeleton via esterification to obtain NOSH [[76]34]. The structure of NOSH was characterized by ^1H NMR, ^13N NMR, and high-resolution mass spectrometry. The spectra shown in [77]Figure S1 and Figure S2 showed the characteristic peaks of NOSH, and [78]Fig. S3 showed the characteristic peak of M + Na at m/z 499.99, which means that the synthesis was successful. Scheme 1. [79]Scheme 1 [80]Open in a new tab (A) Construction process of NOSH@PEG-HCuSNPs. (B) Schematic diagram of NIR-triggered programmable NOSH@PEG-HCuSNPs for cascading oncotherapy by mPTT/gas/ROS-reinforced ICD. Next, hollow mesoporous copper sulfide nanoparticles (termed as HCuSNPs) were synthesized by a simple two-step synthesis procedure according to previous literature reports [[81]32]. Afterwards, enlightened by the interfacial assembly, the gas donor NOSH was loaded into the hollow nanostructure of HCuSNPs and then modified by a polyethylene glycol derivative containing with folic acid (NH[2]-PEG[2000]-FA) to obtain NIR-triggered programmable targeting nanomotor (termed as NOSH@PEG-HCuSNPs) ([82]Scheme 1). As shown in [83]Fig. 1A and [84]Fig. S4, the field emission transmission electron microscopy (TEM) showed that as-constructed NOSH@PEG-HCuSNPs had an appropriate spherical with hollow mesoporous cavities. In addition, mapping analysis illustrated the uniform distribution of Cu, S, and N elements in the hollow NOSH@PEG-HCuSNPs framework ([85]Fig. 1B–F). As observed from the dynamic light scattering (DLS), the mean diameters of HCuSNPs, NOSH@HCuSNPs, and NOSH@PEG-HCuSNPs were 212.1, 230.2, and 242.0 nm, respectively, which was well consistent with TEM results ([86]Fig. 1G). The ζ potentials of HCuSNPs, NOSH@HCuSNPs, and NOSH@PEG-HCuSNPs were −15.1 ± 2.1, −10.3 ± 2.3, and −3.3 ± 0.6 mV, respectively ([87]Fig. 1H). It should be pointed out that these changes in diameter and ζ potential might be mainly resulted in HCuSNPs loaded and modified via positively charged NOSH and NH[2]-PEG[2000]-FA. Fig. 1. [88]Fig. 1 [89]Open in a new tab Physicochemical characterization of HCuSNPs, NOSH@HCuSNPs, and NOSH@PEG-HCuSNPs. (A) TEM images and (B–F) TEM-mapping images of NOSH@PEG-HCuSNPs. (G–I) Size distribution, ζ potential, and FT-IR spectra of HCuSNPs, NOSH@HCuSNPs, and NOSH@PEG-HCuSNPs. (J-K)XRD spectra and High-resolution XPS spectra of NOSH@PEG-HCuSNPs. (L) TGA curves of HCuSNPs, NOSH@HCuSNPs, and NOSH@PEG-HCuSNPs. Data are expressed as the mean ± SD. In order to further confirm whether gas donor NOSH was successfully loaded into HCuSNPs, Fourier transform infrared (FT-IR) spectroscopy analysis, the powder X-ray diffraction (XRD) pattern, and X-ray photoelectron spectroscopy (XPS) were conducted. As depcited in the FT-IR of [90]Fig. 1I, the constructed NOSH@PEG-HCuSNPs showed unique characteristic peaks of NOSH, such as the characteristic peak of R[2]CH[2] group at around 1700 cm^−1 and the characteristic peak of ester bond at around 2900 cm^−1. As shown in [91]Fig. 1J, the characteristic peaks at 2 θ = 26.76, 29.58, 31.86, 48.07, 52.85, and 59.18 corresponds to the crystal planes (101), (102), (103), (108), (110) and (116) of the hexagonal Cu crystal, respectively. Notably, the XRD spectra of HCuSNPs, NOSH@HCuSNPs, and NOSH@PEG-HCuSNPs possessed the similar Cu crystal structures ([92]Fig. S5) In order to further clarify the formation mechanism of NOSH@PEG-HCuSNPs, the chemical states and binding energies of the associated elements were detected. As shown in [93]Fig. 1K, the chemical states of Cu and S were analyzed by XPS. The peaks at 930.8 and 950.7 eV were attributed to the binding energies of Cu 2p3/2 and Cu 2p1/2, and the peaks observed in the range of 158–168 eV corresponded to the binding energies of S2p3/2 and S2p1/2. These above results were consistent with the XPS data of CuS nanocrystals reported previously [[94]35]. Moreover, the effective payload of NOSH was confirmed by thermogravimetric analysis (TGA) of HCuSNPs and NOSH@HCuSNPs ([95]Fig. 1L). The payload of the gas donor NOSH was determined to be 24.03 %. In a word, the above results are consistent with the successful construction of NOSH@PEG-HCuSNPs. Besides, in order to evaluate the physiological stability of NOSH@PEG-HCuSNPs, they were dispersed in water phosphate-buffered saline (PBS), saline, and 10 % FBS for one week to monitor size and morphology fluctuations. As could be seen in [96]Fig. S6, NOSH@PEG-HCuSNPs showed no obvious size and morphology changes, evidencing that they had a good physiological stability. It was also found that 200 μg mL^−1 of NOSH@PEG-HCuSNPs had a low hemolysis, indicating that they had an good biocompatibility and can be used in further biological applications ([97]Fig. S7). 3.2. The photothermal effect induced by NOSH@PEG-HCuSNPs As shown in [98]Fig. S8, the as-prepared NOSH@PEG-HCuSNPs have strong near-infrared absorption ability, indicating that NOSH@PEG-HCuSNPs have high application potential in NIR laser photothermal conversion. In vitro photothermal performance of NOSH@PEG-HCuSNPs under 808 nm laser irradiation dispersed in water was measured by an infrared thermal imager. The aqueous dispersions of NOSH@PEG-HCuSNPs with different concentrations were exposed to laser (1.5 W cm^−2) for 10 min, and the results showed a concentration-dependent change trend. Unlike the blank group, which was insensitive to the thermal response from 808 laser irradiation, the temperature of NOSH@PEG-HCuSNPs in the aqueous solution at 200 μg mL^−1 increased tp ca. 45 °C within 10 min ([99]Fig. 2A). As depicted in [100]Fig. 2B, a similar temperature increase trend could also be observed in the centrifuge tube through real-time thermal imaging image recording. In addition, the temperature change of NOSH@PEG-HCuSNPs in aqueous solution under different laser intensity was quantified ([101]Fig. 2C). At a specific concentration (200 μg mL^−1), the temperature of NOSH@PEG-HCuSNPs in aqueous solution could be adjusted from 47.3 °C to 92.5 °C when the irradiation power varied from 0.5 W cm^−2 to 2.0 W cm^−2. The change of the thermal imaging image in [102]Fig. 2D also further evidence that the photothermal conversion performance of NOSH@PEG-HCuSNPs was highly dependent on the laser intensity density. In order to explore the photothermal stability of NOSH@PEG-HCuSNPs, the temperature change was monitored during the consecutive cycles of 5 laser on/off. As shown in [103]Fig. 2E, the photothermal performance of NOSH@PEG-HCuSNPs didn't show obvious degradation under the repeated 808 nm laser irradiation, indicating that NOSH@PEG-HCuSNPs had an excellent photothermal stability. According to the calculation formula of the previous report [[104]36], the photothermal conversion efficiency (η) of NOSH@PEG-HCuSNPs nanoparticles was also calculated to be η = 27.8 % based on the measured data in [105]Fig. 2F and G. This value is slightly lower than some nanomaterials with photothermal conversion function in the NIR-I window, but it can meet the conditions for photothermal therapy [[106]37,[107]38]. Therefore, the above results indicated the feasibility of NOSH@PEG-HCuSNPs as an ideal photothermal agent with potential thermal therapy. Fig. 2. [108]Fig. 2 [109]Open in a new tab In vitro property valuation of NOSH@PEG-HCuSNPs. (A) Photothermal effect and (B) Infrared thermal images of NOSH@PEG-HCuSNPs with different concentrations (0–800 μg mL^−1) under 808 nm laser irradiation (1.5 W cm^−2). (C) Photothermal effect and (D) infrared thermal images of NOSH@PEG-HCuSNPs (200 μg mL^−1) under 808 nm laser irradiation with different laser intensities (0–2.0 W cm^−2). (E) Temperature change curve of NOSH@PEG-HCuSNPs (200 μg mL^−1) under repeated 808 nm laser irradiation for 5 cycles. (F) Temperature curve of NOSH@PEG-HCuSNPs (laser turned off after 10 min). (G) Time constant calculated from the cooling period. (H–I) NO and H[2]S release amount of NOSH@PEG-HCuSNPs (100 μg mL^−1) under different 808 nm laser intensities (0–1.5 W cm^−2) for 10 min. All experiments were performed at least three times. Data are expressed the mean ± SD (n = 3). 3.3. In vitro NO and H[2]S release of NOSH@PEG-HCuSNPs According to the design strategy of NOSH@PEG-HCuSNPs, the loaded gas donor can simultaneously release H[2]S and NO. In order to ensure the effect on laser-mediated NO and H[2]S-assisted PTT, the NO and H[2]S release of gas donor NOSH in NOSH@PEG-HCuSNPs should be measured. The morphological changes of NOSH@PEG-HCuSNPs under 808 nm laser irradiation was observed and recorded by TEM. As shown in [110]Fig. S9, after 808 nm laser irradiation of PBS or 10 % FBS culture medium for 12 h, it was found that the framework of NOSH@PEG-HCuSNPs was collapsed and its morphology was almost disappeared, meaning that laser-mediated NO and H[2]S release could be accelerated in NOSH@PEG-HCuSNPs.In order to further verify the NO release of NOSH@PEG-HCuSNPs, the Griess reagent was chosen as the detection method. As shown in [111]Fig. S10, the nitrate (-ONO[2]) group could continuously release NO when dissolved in water. Therefore, in order to quantify the NO release from NOSH@PEG-HCuSNPs, the Griess reagent standard curve was established [[112]31]. As shown in [113]Fig. S11, under 808 nm laser irradiation, the release of NO continued to increase with the increase of laser treatment time, temperature and NOSH@PEG-HCuSNPs concentration. In addition, the NO release amount of NOSH@PEG-HCuSNPs treated with different laser intensities also had an obvious difference ([114]Fig. 2H). Next, the release of the H[2]S in NOSH@PEG-HCuSNPs was further evaluated by the H[2]S-specific fluorescent probe NP-N[3] (this probe was synthesized according to previous work [[115]39], the detection mechanism is shown in [116]Fig. S12). Unlike other H[2]S donors, the exact mechanism of H[2]S release from ADT-OH and its derivatives is still unclear. However, it is hypothesized that cellular enzymes can lead to the H[2]S release from ADT-OH. In addition, previous studies have confirmed that ADT-OH can release H[2]S in rat liver homogenate, rat plasma, and cell lysate, but rarely releases H[2]S in buffer alone [[117]5]. Based on this assumption, TCEP is usually added to promote the release of H[2]S from ADT-OH and its derivatives to achieve in vitro release detection. Based on the standard curve of NP-N[3] for detecting H[2]S ([118]Fig. S13), it can be found in [119]Figs. S14 and 2I that the release amount of H[2]S in NOSH@PEG-HCuSNPs significantly changed and the photothermal effect further enhanced the release of H[2]S under different NOSH@PEG-HCuSNPs concentrations and different laser intensity irradiation. The increase in NOSH@PEG-HCuSNPs concentration and laser intensity led to an increase in the temperature in the aqueous solution, which promoted the hydrolysis process of the donor NOSH, enabling it to release NO and H[2]S faster and more. In addition, the in vitro release experiment conducted above also proves that the donor NOSH can avoid the problem of premature release. The release of NO and H[2]S under non-laser triggered conditions showed that the release rate of NOSH from NOSH@PEG-HCuSNPs was still small without 808 nm laser irradiation, with less than 10 % released within 12 h, indicating strong retention under physiological conditions. This can be attributed to the stabilizing effect of PEG modification, which minimizes premature release during systemic circulation. These findings collectively indicate that NOSH@PEG-HCuSNPs are able to maintain stability and minimize premature release under physiological conditions while ensuring effective release of therapeutic gas molecules at the tumor site after laser activation. This shows the operational feasibility of the proposed GT/PTT combination therapy. 3.4. In vitro anti-tumor and molecular mechanism of NOSH@PEG-HCuSNPs Encouraged by the good physicochemical properties, the anti-tumor effect and the associated molecular mechanism of NOSH@PEG-HCuSNPs were further evaluated and investigated on a cellular level ([120]Fig. 3A). In detail, NOSH@PEG-HCuSNPs were labeled with Fluorescein Isothiocyanate (FITC) probe, and their intracellular uptake efficiency was qualitatively evaluated by confocal laser scanning microscopy (CLSM). As shown in [121]Fig. 3B, the fluorescence intensity of FITC in 4T1 cells obviously increased upon time prolonging from 0 h to 4 h, and reached a peak value at 1 h, indicating that NOSH@PEG-HCuSNPs could be quickly and effectively internalized into 4T1 cells. Notably, the co-localization tendency of lysosomes and NOSH@PEG-HCuSNPs gradually increased. It should be pointed out that the above mentioned qualitative CLSM results were well line with the quantitative flow cytometry ([122]Fig. S15). These phenomena could be explained by the mature theory that folic acid (FA)-modified nanoparticles could be specifically internalized into tumor cells and then delivered into the acidic lysosomes through FA receptor-transduced endocytosis. Fig. 3. [123]Fig. 3 [124]Open in a new tab In vitro cellular experiments of NOSH@PEG-HCuSNPs. (A) Schematic illustration of NOSH@PEG-HCuSNPs for cascading oncotherapy by GT and mPTT. (B) CLSM images of cellular endocytosis of FITC-labeled NOSH@PEG-HCuSNPs in 4T1 cells at different time points (green channel: FITC, red channel: Lyso-Tracker Red, blue channel: Hoechst 33342, scale: 20 μm). (C) cell viability and (D) Calcein-AM/PI staining images (green channel: Calcein AM, red channel: PI, scale: 500 μm) of 4T1 cells after different treatments. (E) The staining images of DAF-FM DA kit, NP-N[3] probe, DCFH-DA probe, and ONOO^− detection kit was employed to evaluate the NO release, H[2]S release, ROS release, and ONOO^− generation after different treatments (scale: 20 μm), respectively. (F) The JC-1 staining images (green channel: JC-1 monomer, red channel: JC-1 aggregate, scale: 20 μm) of 4T1 cells after different treatments. (G) COX IV and HSP 90 expressions and (H) Flow cytometry after Annexin V-FITC/PI staining of 4T1 cells after different treatments. Data are expressed as the mean ± SD (n = 3) and ∗∗∗P < 0.001. (For interpretation of the references to color in this