A pH-responsive protein self-assembly-based targeted nanodrug and a preparation method thereof

Abstract

The application provides a kind of targeted nano-drug based on pH response protein self-assembly and a preparation method thereof, and belongs to the technical field of nanomaterials.The application first activates photosensitizer Ce6 and peptide segment cNGQGEQc respectively, then couples with CAT respectively, to prepare photosensitizer-protein conjugate and photosensitizer-peptide segment conjugate, and then obtains the nano-probe through the self-assembly behavior of photosensitizer-protein conjugate, photosensitizer-peptide segment conjugate, hydrophilic silver sulfide quantum dots and HSP90 inhibitor.The nano-probe not only realizes the drug release and NIR-II imaging guidance of tumor microenvironment response, but also relieves hypoxia and inhibits heat resistance synchronously, truly realizes the synergistic amplification effect of PDT / PTT, and provides a promising strategy for precise and efficient treatment of NSCLC.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, and in particular to a targeted nanomedicine based on pH-responsive protein self-assembly and its preparation method. Background Technology

[0002] Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancer cases. Traditional systemic chemotherapy is often accompanied by severe non-specific toxic side effects, while surgery and radiotherapy are usually only applicable to localized or resectable lesions and have limited effectiveness against tumors that have metastasized extensively. Therefore, developing treatment strategies that combine high precision and higher therapeutic efficiency is of great significance for improving the clinical prognosis of NSCLC.

[0003] Photodynamic therapy (PDT) and photothermal therapy (PTT) are considered highly promising minimally invasive tumor treatments due to their good spatiotemporal controllability and relatively low systemic toxicity. PDT induces tumor cell death by generating reactive oxygen species (ROS) under light irradiation using photosensitizers, while PTT relies on photothermal materials to convert light energy into heat energy for local thermal ablation. However, the efficacy of both treatments is significantly constrained by the tumor microenvironment. On the one hand, the prevalent hypoxia in solid tumors severely limits the continuous generation of ROS, thus weakening the therapeutic effect of PDT; on the other hand, the thermal stress induced by PTT often leads to the high expression of heat shock proteins (HSPs), especially heat shock protein 90 (HSP90), which makes tumor cells resistant to thermal damage, thereby reducing treatment efficiency. Therefore, simultaneously alleviating tumor hypoxia and overcoming HSP-mediated heat resistance are key scientific issues that urgently need to be addressed to achieve highly efficient PDT / PTT synergistic therapy.

[0004] To address the aforementioned issues, researchers have developed various PDT / PTT combined therapy platforms based on nanomaterials. However, most reported nanosystems still have limitations in terms of structural design and functional integration. Many systems rely on simple physical encapsulation or mixing of functional components, making it difficult to precisely control the assembly process, often resulting in insufficient structural stability, significant batch-to-batch variability, and unsatisfactory synergistic therapeutic effects. Furthermore, introducing complex polymeric carriers or inorganic frameworks to achieve multifunctionality often increases the structural complexity and metabolic burden of the system, posing potential biosafety risks and limiting its further translational applications. Although tumor microenvironment-responsive nanotherapeutic systems have been widely proposed, their response behavior is often imprecise, prone to problems such as stimulation threshold mismatch, premature drug leakage, or uncontrollable release kinetics, thus hindering truly on-demand, precise treatment. Summary of the Invention

[0005] In view of this, the present invention aims to provide a targeted nanomedicine based on pH-responsive protein self-assembly and a method for its preparation.

[0006] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is a method for preparing a pH-responsive protein self-assembly-based nanoprobe, comprising the following steps: The photosensitizer Ce6 was activated in an organic solvent in the presence of condensing agent 1 and activator 1, and then mixed with CAT for a coupling reaction 1. Finally, the resulting reaction solution 1 was purified 1 to obtain the photosensitizer-protein conjugate. The peptide cNGQGEQc was activated in an organic solvent in the presence of condensing agent 2 and activator 2, then mixed with CAT for a coupling reaction 2, and finally the resulting reaction solution 2 was purified 2 to obtain the targeted peptide-protein conjugate. Hydrophilic silver sulfide quantum dots, photosensitizer-protein conjugates, photosensitizer-peptide conjugates, and HSP90 inhibitors were mixed evenly in solvent 2, and then self-assembled under light-protected conditions to obtain the nanoprobe.

[0007] The second technical solution of the present invention is a nanoprobe based on pH-responsive protein self-assembly prepared by the above preparation method.

[0008] The third technical solution of the present invention is the application of the above-mentioned pH-responsive protein self-assembly-based nanoprobe in the preparation of a drug for treating non-small cell lung cancer.

[0009] The present invention discloses the following technical effects: This invention proposes a controllable protein self-assembly strategy to construct a multifunctional NIR-II fluorescent nanoplatform, Ag2S@CAT-Ce6-cNGQGEQc@GM (A@CcN@G), for imaging-guided targeted synergistic therapy in non-small cell lung cancer (NSCLC). In this carrier-free supramolecular system, multiple non-covalent interactions ensure its stability under physiological conditions, while the acidic environment of the tumor microenvironment triggers controlled dissociation, enabling precise drug release. Within the system, Ag2S quantum dots provide NIR-II fluorescence imaging and photothermal functions, CAT decomposes endogenous H2O2 to alleviate tumor hypoxia, and Ce6 enhances ROS generation. The NSCLC-targeting peptide cNGQGEQc endows the platform with active targeting capabilities, while the HSP90 inhibitor geldanamycin (GM) effectively inhibits PTT-induced thermostableness and further amplifies the synergistic therapeutic effect of PDT / PTT. This carrier-free supramolecular nanoplatform not only achieves drug release in response to the tumor microenvironment and NIR-II imaging guidance, but also truly realizes the synergistic amplification effect of PDT / PTT by simultaneously alleviating hypoxia and inhibiting thermostability, providing a promising strategy for the precise and efficient treatment of NSCLC. Attached Figure Description

[0010] Figure 1 (a) TEM images (from left to right) of Ag2S QDs, Ag2S–NH2QDs, and A@CcN@G NPs; (ii) inset of (a) shows the lattice fringes of Ag2S QDs; (b) DLS particle size distribution of A@CcN@G NPs assembled at pH 7.4; (c) XRD pattern of Ag2S QDs (monoclinic, JCPDS No. 14-0072); (d) FTIR spectrum of Ag2S–NH2QDs with amino (–NH2) functional groups; (e) ζ-potential values ​​of Ag2S–NH2, CAT, CAT-Ce6, CAT-Ce6-cNGQGEQc, and A@CcN@G NPs; (f) Ce6, GM, and A@CcN@G (g) UV-Vis absorption spectra of NPs; (h) UV-Vis spectra and standard calibration curves of GM solutions at different concentrations (inset); (i) HPLC chromatogram of cNGQGEQc; (j) ESI-MS mass spectrum of cNGQGEQc; (h) fluorescence spectra of assembled Ag2S QDs and A@CcN@G NPs.

[0011] Figure 2(a) shows the isoelectric surface properties of CAT under different pH conditions; (b) shows the molecular docking between geldanamycin (GM) and catalytic enzyme (CAT); (c) shows the TEM image of the nanoparticle assembly process in PBS (pH 7.4), where i–iii correspond to 0, 10, and 30 min, respectively; (d) shows the particle size distribution of A@CcN@G nanoparticles in DMEM, PBS, and 10% FBS; (e) shows the fluorescence intensity changes of Ag2S and A@CcN@G nanoparticles within 72 h and the corresponding ζ-potential changes in (f); (g) shows the TEM image of the nanoparticle dissociation process under PBS (pH 6.5), where i–iii correspond to 0, 1, and 3 h, respectively. (h) shows the change in nanoparticle size after treatment at pH 7.4 and pH 6.5 for 60 min; (i) shows the time-dependent release curves of GM in A@CcN@G nanoparticles under different pH conditions.

[0012] Figure 3 (a) shows the oxygen production capacity of A@CcN@G nanoparticles in solutions with different H2O2 concentrations; (b) shows the catalytic oxygen production performance of A@CcN@G nanoparticles under different pH conditions; (c) shows the change in dissolved oxygen levels before and after near-infrared light irradiation; (d) shows the oxygen production capacity of A@CcN@G or A@BnN@G nanoparticles containing H2O2 under normal or hypoxic conditions. 1 O2 generation was detected using a DPBF probe (650 nm, 300 mW cm⁻¹). -2 (e) shows the photothermal heating curves of different concentrations of A@CcN@G nanoparticles under near-infrared laser irradiation (808 nm, 300 mW cm⁻¹). -2 (f) shows the infrared thermal imaging image obtained within 3 min of illumination; (g) shows the photothermal stability of A@CcN@G nanoparticles under the same conditions for six heating-cooling cycles.

[0013] Figure 4 (a) Cell viability of A549 cells after co-incubation with different concentrations of Ag2S nanoparticles for 12 h under light-protected conditions; (b) Cell viability of A549 cells after incubation with A@CcN@G nanoparticles under light-protected conditions; (c) Cell viability of different treatment groups (G1–G5) after irradiation with a 650 nm laser (300 mW cm⁻¹). -2(d) and (e) are confocal laser scanning microscopy (CLSM) images of A549 cells after incubation with nanoparticles for 0–120 min; cell nuclei are stained with DAPI (blue), and the near-infrared II (NIR-II) fluorescence signal of Ag2S is shown as red; (f) and (g) are CLSM images of A549 cells after incubation with A@CcN@G or A@Cc@G nanoparticles, respectively; (h) and (i) are CLSM images of cellular uptake of A@CcN@G nanoparticles in A549, 4T1 and HepG2 cells.

[0014] Figure 5 (a) shows the effects of irradiation with a 650 nm laser (300 mW cm⁻¹) under normal and hypoxic conditions. -2 (a) Confocal laser scanning microscopy (CLSM) images of reactive oxygen species (ROS) induced in A549 cells by different nanoparticle treatment groups (e.g., A@CcN@G, A@BnN@G, etc.); (b) Quantitative analysis of ROS fluorescence intensity in the corresponding treatment groups; (c) Singlet oxygen (ROS) in different treatment groups (G1–G5). 1 The quantitative results of singlet oxygen (O2) generation were detected using the SOSG probe; (d) shows the fluorescence quantitative results of singlet oxygen (O2) generated by A549 cells under different treatment conditions. 1 CLSM images of O2; (e) images of different laser power densities (100, 300 and 600 mW cm⁻¹). -2 (a) Calcein-AM / PI double staining images of A549 cells treated with PBS, Ag2S, or Ag2S@GM under irradiation; (f) and (g) Western blot analysis results of caspase-9 and survivin in A549 cells under different treatment conditions; (h) Western blot analysis of HSP90 expression level in A549 cells under different treatment conditions; (i) Western blot analysis of HIF-1α expression level in A549 cells after different treatments under normoxic and hypoxic conditions; (j) Schematic diagram of the synergistic therapeutic mechanism of A@CcN@G nanoparticles integrating photodynamic therapy (PDT), mild photothermal therapy (mild PTT), and chemotherapy.

[0015] Figure 6 (a) shows the effect of irradiation with a 650 nm laser (300 mW cm⁻¹). -2Under the conditions of 3 min, Calcein-AM / PI fluorescence staining images of A549 cells after different treatment groups: (G1) PBS, (G2) A@C, (G3) A@Cc, (G4) A@CcN and (G5) A@CcN@G; (b) fluorescence quantitative analysis of the ratio of live cells / dead cells in the corresponding treatment groups; (c) flow cytometry analysis of apoptosis of A549 cells under different treatment conditions; (d) and (e) JC-1 fluorescence images and quantitative analysis of mitochondrial membrane potential (ΔΨm) of A549 cells after laser irradiation.

[0016] Figure 7 (a) shows the treatment regimen: intravenous injection (iv) was administered on days 1, 3, and 8, followed by 650 nm laser irradiation (300 mW·cm²) 2 hours after administration. -2 (a) NIR-II fluorescence imaging images and fluorescence imaging of major organs in A549 tumor-bearing mice at 0–480 min after drug administration; (b) Infrared thermal imaging images of mice in the PBS group and A@CcN@G group under near-infrared light irradiation; (c) In vivo fluorescence imaging results of different treatment groups (G1–G5) 2 h after treatment; (d) Physical photographs of tumors peeled off in different treatment groups (G1–G5) after 14 days of treatment; (e) Serial NIR-II fluorescence imaging results of different treatment groups (G1, G5) on days 1, 8 and 14; (g)–(i) Time changes in tumor volume, body weight and tumor weight in different treatment groups (G1–G5) during the 14-day monitoring period.

[0017] Figure 8 To perform staining analysis on tumor tissue sections from the (G1)Ag2S, (G2)A@C, (G3)A@Cc, (G4)A@CcN, and (G5)A@CcN@G treatment groups 14 days after treatment: the first column is H&E staining; the second column is Ki-67 staining to assess tumor cell proliferation; the third column is TUNEL staining to detect apoptosis; and the fourth and fifth columns are immunofluorescence staining for HSP90 and HIF-1α, respectively.

[0018] Figure 9 Characterization of the components of the A@CcN@G nanoplatform, including: (a): FFT and IFFT analysis of HRTEM images to confirm the crystal structure of Ag2S quantum dots (QDs); (b): UV-Vis absorption spectrum of A@CcN@G nanoparticles; (c): TEM image of catalase (CAT).

[0019] Figure 10The assembly of A@CcN@G nanoparticles was optimized and characterized. (a) Molecular docking analysis of the ligand-protein complex was performed. The calculated binding energy was −6.22 kcal / mol, and the key interaction parameters were as follows: inhibition constant (Ki) = 27.82 μM; van der Waals (VDW) energy = −7.62 kcal / mol; hydrogen bond (HB) energy = 0.24 kcal / mol; electrostatic energy = −0.24 kcal / mol; desolvation energy = −1.64 kcal / mol; torsional energy = 1.65 kcal / mol; (b) UV-Vis absorption spectra of the nanoparticles at different time points during dialysis (0.5, 1, 1.5, 2, 2.5, 3.5, 4 h).

[0020] Figure 11 For the hemolysis experiment and observation of tumor-bearing mice during treatment, (a): hemolysis test of nanoparticle formulation, confirming its excellent blood compatibility; (b): representative photos of A549 tumor-bearing nude mice in different treatment groups on days 1, 4, 10 and 14: (G1) A; (G2) A@C; (G3) A@Cc; (G4) A@CcN; (G5) A@CcN@G.

[0021] Figure 12 Representative H&E staining images of major organs (heart, liver, spleen, lung, and kidney) from different groups (G1–G5) of mice. No obvious histopathological abnormalities were observed in any treatment group, suggesting negligible systemic toxicity. Scale bar = 50 μm. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] In this invention, room temperature is defined as 25±5 °C.

[0024] This invention first activates the photosensitizer Ce6 and the peptide cNGQGEQc, respectively, and then conjugates them with CAT to prepare photosensitizer-protein conjugates and photosensitizer-peptide conjugates. Subsequently, well-dispersed spherical nanoparticles (i.e., pH-responsive protein self-assembly-based nanoprobes) are formed through the self-assembly of the photosensitizer-protein conjugates, photosensitizer-peptide conjugates, hydrophilic silver sulfide quantum dots, and HSP90 inhibitors. These nanoparticles are then used for imaging-guided targeted synergistic therapy of non-small cell lung cancer.

[0025] Specifically, the first aspect of this invention provides a method for preparing a pH-responsive protein self-assembled nanoprobe, comprising the following steps: In the presence of condensing agent 1 and activator 1, photosensitizer Ce6 (photosensitizer chlorin e6) was activated in organic solvent 1, then mixed with CAT (catalase) for coupling reaction 1, and finally the resulting reaction solution 1 was purified 1 to obtain photosensitizer-protein conjugate. The peptide cNGQGEQc was activated in an organic solvent in the presence of condensing agent 2 and activator 2, then mixed with CAT for a coupling reaction 2, and finally the resulting reaction solution 2 was purified 2 to obtain the photosensitizer-peptide conjugate. Hydrophilic silver sulfide quantum dots, photosensitizer-protein conjugates, photosensitizer-peptide conjugates, and HSP90 inhibitors (HSP90 inhibitor geldanamycin, GM) were mixed evenly in solvent 2, and then self-assembled under light-protected conditions to obtain the pH-responsive protein-based self-assembled nanoprobe.

[0026] In a preferred embodiment of the present invention, the condensing agent 1 and condensing agent 2 are the same, both being 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); the activator 1 and activator 2 are the same, both being N-hydroxysuccinimide (NHS); and the organic solvent 1 and organic solvent 2 are the same, both being dimethyl sulfoxide (DMSO).

[0027] In a preferred embodiment of the present invention, the molar ratio of condensing agent 1 to activator 1 is 1:(1.1~1.6), and the molar ratio of condensing agent 1 to photosensitizer Ce6 is 0.5~1.5 mmol:0.049. g The conditions for activation 1 are: room temperature and light protection reaction for 0.5~1h.

[0028] More preferably, the molar ratio of condensing agent 1 to activator 1 is 1:1.4, and the molar ratio of condensing agent 1 to photosensitizer Ce6 is 0.0814 mmol:0.049 mmol. g。

[0029] In a preferred embodiment of the present invention, the mass ratio of the photosensitizer Ce6 to CAT is (0.02~0.06):0.01; the conditions for the coupling reaction 1 are: room temperature and light protection reaction for 12~24 h.

[0030] More preferably, the mass ratio of the photosensitizer Ce6 to CAT is 0.049:0.01.

[0031] In a preferred embodiment of the present invention, purification 1 is performed by centrifuging reaction solution 1, collecting the supernatant, and then dialyzing the supernatant to remove unreacted small molecule reagents; the dialyzing is performed sequentially in 50 mM, 30 mM, and 10 mM PBS at pH=7.4 and in deionized water.

[0032] In a preferred embodiment of the present invention, the molar ratio of condensing agent 2 to activator 2 is 1:(1.1~1.6), and the molar ratio of condensing agent 2 to peptide cNGQGEQc is 0.1~0.5 mmol:0.01. g The activation conditions for Activation 2 are: room temperature and light protection for 0.5-1 h.

[0033] More preferably, the molar ratio of condensing agent 2 to activator 2 is 1:1.4, and the molar ratio of condensing agent 2 to peptide cNGQGEQc is 0.04 mmol:0.01 mmol. g。

[0034] In a preferred embodiment of the present invention, the mass ratio of the peptide cNGQGEQc to CAT is (0.8~1.1):(0.8~1.1); the conditions for the coupling reaction 2 are: room temperature and light protection reaction for 12~24 h.

[0035] More preferably, the mass ratio of the peptide cNGQGEQc to CAT is 1:1.

[0036] In a preferred embodiment of the present invention, purification 2 is performed by centrifuging reaction solution 2, collecting the supernatant, and then dialyzing the supernatant to remove unreacted small molecule reagents; the dialyzing is performed by sequentially dialyzing in 50 mM, 30 mM, and 10 mM PBS buffer and deionized water in a dialysis bag with a molecular weight cutoff of 8 kDa.

[0037] In a preferred embodiment of the present invention, the mass ratio of the hydrophilic silver sulfide quantum dots, the photosensitizer-protein conjugate, the photosensitizer-peptide conjugate, and the HSP90 inhibitor is 1:(20~25):(20~25):(60~70). The solvent 2 is a mixture of ethanol and water.

[0038] More preferably, the mass ratio of the hydrophilic silver sulfide quantum dots, photosensitizer-protein conjugate, photosensitizer-peptide conjugate, and HSP90 inhibitor is 1:20:20:60.

[0039] The method for preparing the hydrophilic silver sulfide quantum dots in this invention includes the following steps: The silver precursor was mixed with an organothiol and heated under an inert atmosphere. After the reaction was completed, anhydrous ethanol was added to the resulting reaction solution to induce precipitation. Then, solid-liquid separation was performed, and the precipitate was collected to obtain hydrophobic silver sulfide quantum dots. The hydrophobic silver sulfide quantum dots were dispersed in anhydrous chloroform, then β-mercaptoethylamine was added, and a ligand exchange reaction was carried out in the dark. After the ligand exchange reaction was completed, hydrophilic silver sulfide quantum dots were obtained. The silver precursor is silver diethyldithiocarbamate (DDTC), the organothiol is dodecanethiol (DT), and the mass-to-volume ratio of the silver precursor to the organothiol is 0.02~0.07 g:22~25 mL (more preferably 0.0512 g:24.4 mL); the heating reaction is carried out at a temperature of 210~230 °C for 1.5~3 h. The mass-to-volume ratio of the silver precursor to anhydrous chloroform is 0.02~0.07 g:15~20 mL (more preferably 0.0512 g:15 mL), and the mass ratio of the silver precursor to β-mercaptoethylamine is 0.02~0.07:0.25 (more preferably 0.0512:0.25). The parameters for the ligand exchange reaction are set as follows: shaking at room temperature for 8~12 h. After the ligand exchange reaction is completed, the chloroform is removed, and the separated Ag2S QDs are dissolved in acetic acid solution and stored in the dark at 4 ℃ for subsequent experiments and characterization.

[0040] In a preferred embodiment of the present invention, the self-assembly reaction is carried out at room temperature for 1.5 to 3 hours.

[0041] In a preferred embodiment of the present invention, after the self-assembly reaction is completed, the process further includes a solid-liquid separation step and a collection of the solid product.

[0042] In this invention, the reactions involved (such as activation reactions, coupling reactions, and self-assembly reactions) also include stirring, sonication, and other operations during the reaction process to promote uniform mixing of materials and accelerate the reaction process.

[0043] A second aspect of the present invention provides a pH-responsive protein self-assembly-based nanoprobe prepared by the above preparation method.

[0044] A third aspect of the present invention provides the application of the above-mentioned pH-responsive protein self-assembly-based nanoprobe in the preparation of a drug for treating non-small cell lung cancer.

[0045] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0046] In this embodiment, all animal experiments strictly followed the "Regulations for the Management of Experimental Animals" issued by Hainan Medical University and were approved by the Animal Ethics Committee of Hainan Medical University. The BALB / c nude mice used in the experiments were male and aged 3–4 weeks.

[0047] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0048] Example 1 (1) Synthesis of NIR-II region Ag2S quantum dots (QDs): 0.0512 g of silver diethyldithiocarbamate (DDTC) and 24.4 mL of dodecyl mercaptan (DT) were added to a three-necked flask equipped with a magnetic stirrer. The system was then subjected to alternating vacuum and argon purging three times (30 s each time) to thoroughly remove oxygen and moisture. Under argon protection, the reaction system was heated to 210 °C and maintained for 1.5 h. During this process, the solution color gradually changed from light to black, indicating the nucleation and growth of Ag₂S nanocrystals. After the reaction, the system was allowed to cool naturally to room temperature, and 50 mL of anhydrous ethanol was added to induce precipitation. The black precipitate was collected by centrifugation at 6000 rpm for 20 min and washed twice with ethanol to remove residual organic impurities. The final product was dispersed in 15 mL of anhydrous chloroform to obtain hydrophobic Ag₂S QDs.

[0049] To achieve aqueous phase transfer of quantum dots, the hydrophobic Ag₂S QDs prepared above and dissolved in chloroform were transferred to a glass vial, and 250 mg of β-mercaptoethylamine was added. The vial was wrapped in aluminum foil to protect it from light, and a magnetic stir bar was added. The two-phase system was shaken overnight to complete the ligand exchange reaction. After the reaction, the hydrophilic Ag₂S QDs were observed to have separated and adhered to the vial. The chloroform was removed, and the separated Ag₂S QDs were dissolved in 3 mL of acetic acid solution at pH = 2.99. The solution was stored in a light-protected state at 4°C for subsequent experiments and characterization.

[0050] (2) Preparation of photosensitizer-protein conjugate (CAT–Ce6): Ce6 (0.049 g), EDC (0.156 g), and NHS (0.129 g) were dissolved in 2 mL of DMSO and activated by sonication for 0.5 h in the dark. 10 mg of CAT was weighed and dissolved in 2 mL of pure water. The activated Ce6–NHS solution was then slowly added dropwise to the CAT solution, and the mixture was stirred for 12 h in the dark. After the reaction, the product was centrifuged at 8000 rpm for 10 min to remove insoluble impurities, and the supernatant was collected. The product was then placed in an 8 kDa dialysis bag and dialyzed sequentially for 6 h each in 50 mM, 30 mM, and 10 mM PBS (pH 7.4) and deionized water (with the solution changed every 2 h) to remove unreacted small molecule reagents. The final product was aliquoted and stored at -20 °C for subsequent experiments.

[0051] (3) Preparation of photosensitizer-peptide conjugate (CAT–cNGQGEQc): 0.01 g of cNGQGEQc peptide, 0.078 g of EDC, and 0.065 g of NHS were dissolved in 2 mL of DMSO and activated by sonication for 0.5 h under light-protected conditions. 10 mg of CAT was weighed and dissolved in 2 mL of pure water. Subsequently, the activated peptide solution was slowly added dropwise to the CAT solution, and the reaction was stirred for 12 h under light-protected conditions. After the reaction, the above products were centrifuged at 8000 rpm for 10 min to remove insoluble impurities, and the supernatant was collected. The resulting solution was then placed in a dialysis bag with a molecular weight cutoff of 8 kDa and dialyzed sequentially in 50 mM, 30 mM, and 10 mM PBS buffer and deionized water for 6 h each, changing the buffer every 2 h to remove unreacted small molecule reagents. The final product was aliquoted and stored at -20℃ for subsequent experiments.

[0052] (4) Preparation of A@CcN@G nanoparticles: First, galdromycin (GM) was dissolved in ethanol to prepare a stock solution with a concentration of 0.2 mg / mL. Then, 2 μL of 1.0 mg / mL Ag₂S QDs solution, 200 μL of 0.2 mg / mL CAT–Ce₆ solution, and 200 μL of 0.2 mg / mL CAT–cNGQGEQc solution were sequentially added to 600 μL of 0.2 mg / mL GM stock solution, and diluted with pure water to a total volume of 3 mL. The mixture was sonicated for 2 h in the dark to promote self-assembly among the components. After the reaction, the mixture was centrifuged at 12,000 rpm for 10 min to remove unbound free CAT–Ce₆, CAT–cNGQGEQc, Ag₂S QDs, and free GM, finally obtaining Ag₂S@CAT–Ce₆–cNGQGEQc@GM nanoparticles, denoted as A@CcN@G NPs.

[0053] As controls, nanoparticles (A@C (Ag2S@Ce6), A@Cc (Ag2S@CAT-Ce6), and A@CcN (Ag2S@CAT-Ce6-cNGQGEQc)) were prepared using the same method. The morphology and assembly state of the obtained nanoparticles were characterized by transmission electron microscopy (TEM), and the zeta potential and hydrated particle size were determined by dynamic light scattering (DLS). The crystal structure of the Ag2S QDs was analyzed by X-ray diffraction (XRD). UV–Vis–NIR absorption spectra were measured using a UN-600 spectrophotometer, and fluorescence spectra were acquired using an FL-QM fluorescence spectrophotometer.

[0054] Dynamic light scattering (DLS) was used to determine the hydrated particle size distribution of nanoparticles in solution. The prepared A@CcN@G nanoparticle solution was adjusted to pH ≈ 6.5, and its particle size distribution changes were monitored by DLS at different time points (0, 10, 20, 30, 40, 50, and 60 min). Furthermore, transmission electron microscopy (TEM) was used to visualize the morphological changes of the nanoparticles during dissociation, obtaining information on their particle size, shape, and aggregation state. Samples were taken at different time points, and the evolution of the nanoparticle morphology was analyzed by TEM.

[0055] To evaluate the production of reactive singlet oxygen by A@CcN@G NPs under normoxic and hypoxic conditions ( 1 The oxygen-generating capacity was tested as follows. Bovine serum albumin (BSA) was used instead of CAT to prepare control nanoparticles without oxygen-generating function. At the same Ce6 concentration (12 μg / mL)... -1 Under the conditions described, A@CcN@G NPs and A@BcN@G NPs were co-incubated with SOSG probes (3 μg, dissolved in methanol) in a buffer solution containing the same concentration of H2O2. The samples were irradiated with a 650 nm laser (300 mW cm⁻¹) under nitrogen purging (N₂ replacement) and non-nitrogen purging conditions, respectively. -2 The H2O2 concentration was set at 100 μM to simulate the actual H2O2 level in the hypoxic tumor microenvironment (TME). The fluorescence intensity change of SOSG at 528 nm under different conditions was quantitatively analyzed by monitoring the fluorescence intensity change of SOSG at an excitation wavelength of 494 nm. 1 O2 generation.

[0056] After culturing A549 cells overnight, the culture medium was replaced with fresh medium, and A@CcN@G nanoparticles (Ce6 concentration of 12 μg / mL) were added. -1 Cells were incubated with sterile PBS for 5, 60, 90, and 120 min. Then, 4′,6-diamidinyl-2-phenylindole (DAPI, 200 μL) was added to each culture dish for nuclear staining, and the cells were incubated in the dark for 10 min, followed by washing with PBS three times. Finally, confocal laser scanning microscopy (CLSM) was used to observe the uptake of nanoparticles within the cells.

[0057] A549 cells were seeded in confocal culture dishes and cultured for 24 h to achieve appropriate cell density. Subsequently, different treatment groups of nanoparticle formulations (PBS, A@c, A@Cc, A@CcN, and A@CcN@G NPs, all with a Ce6 concentration of 12 μg / mL) were added. -1The nanoparticles were then co-incubated with cells for 4 hours to ensure sufficient interaction between the nanoparticles and cells. After incubation, the cells were irradiated with a 650 nm laser (300 mW cm⁻¹). -2 (3 min) to activate the photosensitizer in the nanoparticles and induce 1 O2 generation was then observed. SOSG (10 μM) was subsequently added, and the reaction was allowed to proceed for 10 min. Cells were then washed three times with PBS. Finally, changes in SOSG fluorescence intensity were observed using confocal laser scanning microscopy (CLSM) to characterize the cells. 1 The generation of O2 and its distribution within cells.

[0058] To assess cell viability, A549 cells were treated with PBS, A@c, A@Cc, A@CcN, and A@CcN@G NPs (Ce6 concentration, all 12 μg / mL). -1 The cells were incubated for 12 hours, followed by irradiation with a 650 nm laser (300 mW cm⁻¹). -2 After irradiation, cells were incubated for another 4 h, followed by live / dead staining with Calcein-AM / PI for 30 min. After staining, cells were washed three times with PBS and imaged under CLSM, with excitation wavelengths of 488 nm and 561 nm for Calcein-AM and PI, respectively.

[0059] When the tumor volume in A549 tumor-bearing mice grew to approximately 60 mm 3 Mice were randomly divided into five groups (n=3 per group). Each group received a different treatment via tail vein injection, including a control group (Ag2S: 1 mg / kg). -1 A@C, A@Cc, A@CcN, and A@CcN@G nanoparticles, with the GM dosage of A@CcN@G being 5 mg / kg. -1 The dosage of Ce6 is 1 mg / kg. -1 The total injection volume was 200 μL.

[0060] Twelve hours after injection, mice were anesthetized with sodium pentobarbital (6 mg / mL). -1 10 μL g -1 Subsequently, in vivo fluorescence imaging was performed using the IVIS Lumina Series XR imaging system. During imaging, the excitation wavelength was set to 600 nm, and the emission signal was acquired at 800 nm to evaluate the enrichment behavior of nanoparticles at tumor sites and to further analyze their synergistic therapeutic potential.

[0061] Throughout the treatment process, different formulations, including Ag2S (control group), A@c, A@Cc, A@CcN, and A@CcN@G nanoparticles, were administered via tail vein injection in each experimental group on days 1, 3, and 8. The dosage of Ag2S was 1 mg / kg. -1 GM is 5 mg / kg -1 Ce6 is 1 mg / kg -1 The total injection volume was 200 μL. During treatment, the weight changes and tumor volume of each nude mouse were recorded every 4–5 days. The tumor volume (G5) was calculated using the following formula: V = length × width × height × π / 6.

[0062] To assess apoptosis in A549 cells, flow cytometry analysis was performed according to the kit instructions. After treatment, A549 cells were collected, washed with PBS, and resuspended in binding buffer. Annexin V-FITC was then added, and the cells were incubated in the dark for 15 min; followed by propidium iodide (PI) staining for 5 min as per the instructions. After staining, the data were analyzed using a flow cytometer (CyFlow Cube 6, Sysmex, Japan). The obtained flow cytometry data were processed and analyzed using FlowJo software to quantitatively assess apoptotic events in A549 cells.

[0063] All experiments were performed independently in triplicate. Data analysis and plotting were performed using GraphPad Prism software (version 8.0), Excel 2016 (Microsoft), and Origin 2021, respectively. Experimental results are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using one-way ANOVA; multiple comparisons between groups were performed using two-way ANOVA. The criteria for statistical significance were: p<0.05, p<0.01, p<0.001.

[0064] Characterization and effect verification results: (1) The microstructure and crystal properties of Ag2S were characterized by high-resolution transmission electron microscopy (HR-TEM), and the results showed that the sample was composed of granular nanocrystals. Locally ordered and clear lattice fringes could be observed within the particles. Figure 1 (a) (ii) Illustration). Lattice spacing measurement results ( Figure 9Figure (a) shows that the interplanar spacing is 0.238 nm, corresponding to the (-103) crystal plane of monoclinic Ag2S (JCPDS card: 14-0072), indicating that Ag2S nanocrystals with good crystallinity were successfully prepared.

[0065] The overall particle size of A@CcN@G is approximately 100 nm, consistent with the results of dynamic light scattering (DLS) testing. Figure 1 (b)). Furthermore, X-ray diffraction (XRD) analysis further confirmed the crystal structure of Ag₂S QDs (b). Figure 1 (c) Two distinct absorption peaks were observed in the FTIR spectrum of Ag2S QDs ( Figure 1 (d) also confirms that the –NH2 group was successfully modified on its surface.

[0066] The successful assembly of A@CcN@G NPs primarily relies on electrostatic interactions. We measured the zeta potential of each individual component and before and after assembly, and the results were consistent with expectations: Ag2S-NH2 exhibited a significant positive charge, while the remaining functional components carried a moderate to strong negative charge. This charge difference facilitated the electrostatic self-assembly between the functional modules, resulting in a slightly positively charged surface on the final nanoparticles. Figure 1 (e) thus ensures efficient integration of each component. To further verify whether Ce6 and GM were successfully introduced into the system, we analyzed the UV-vis absorption spectrum of A@CcN@G, where the characteristic absorption peaks clearly corresponded to Ce6 and GM, indicating that both were successfully loaded (e). Figure 1 (f)). Furthermore, by establishing standard curves for UV-vis absorbance of GM at different concentrations (f) Figure 1 In the middle (g), the loading efficiency of GM in nanoparticles was calculated to be 23% (g). Figure 9 (b)

[0067] The introduction of catalase (CAT) into the system aims to improve the therapeutic efficiency of photodynamic therapy (PDT). In the tumor microenvironment, CAT catalyzes the decomposition of H2O2 to generate O2, thereby increasing local oxygen levels and enhancing Ce6-mediated photodynamic effects. Negative staining TEM images show that CAT exhibits an irregular, large-molecule protein structure. Figure 9 (c) Considering the potential non-specific distribution of nanoparticles after entering the bloodstream, we further introduced a small molecule peptide, cNGQGEQc, that can specifically target non-small cell lung cancer. The molecular composition and purity of this targeting peptide were verified by HPLC and mass spectrometry analysis. Figure 1 (h), (i)).

[0068] To confirm that the self-assembly process does not affect the fluorescence performance of the system, we compared the fluorescence spectra of Ag2S QDs before assembly with those of A@CcN@G after complete assembly. Figure 1 (j)). The results showed that the fluorescence intensity of the two was basically consistent, indicating that the optical properties of Ag2S QDs were well preserved after assembly. In summary, the above results jointly demonstrate that A@CcN@G can be successfully constructed under physiological conditions, all functional components are effectively integrated, and it simultaneously possesses ideal optical properties, structural features, and targeting capabilities.

[0069] (2) To further verify the response characteristics of A@CcN@G to the acidic tumor microenvironment, we analyzed multiple representative sets of data obtained from CAT isosurface simulations under different pH conditions. Under simulated physiological conditions (pH = 7.4), the CAT protein surface exhibited a significant negative charge. As the pH decreased to 6.5 and further to 5.5 (close to the acidic range of the tumor microenvironment), the charge on the protein surface gradually weakened and eventually reversed, becoming positively charged. Figure 2 (a)

[0070] During the assembly of nanoparticles, the HSP90 inhibitor geldanamycin (GM) was designed to bind to catalase (CAT) via non-covalent interactions, thereby achieving its stable introduction into the nanosystem. For example... Figure 2 As shown in (b), molecular docking results indicate that GM can be inserted into the hydrophobic pocket of CAT and achieves stable binding through multiple hydrogen bonds with key amino acid residues ARG126 and GLU255, with bond lengths of 2.0 Å, 2.8 Å, and 2.3 Å, respectively. The calculated binding energy is −6.22 kcal mol. -1 The inhibition constant (Ki) was 27.82 µM, indicating a relatively stable and favorable binding relationship between GM and CAT. Further intermolecular interaction energy analysis showed a total interaction energy of −7.86 kcal mol. -1 The main source of this is van der Waals interaction (−7.62 kcal mol). -1 While electrostatic interaction contributes relatively little (−0.24 kcal mol), -1 This indicates that hydrophobic interactions play a dominant role in the stability of the complex. Figure 10 (a)). These results collectively demonstrate that GM can be effectively anchored within the CAT framework during nanoparticle assembly, providing a molecular-level basis for its stable encapsulation and subsequent controllable release.

[0071] Based on the above results, we propose the following mechanism of action: Under physiological pH conditions (7.4), negatively charged protein components can bind with positively charged Ag2S-NH2 via electrostatic interactions to form stable multifunctional A@CcN@G nanoparticles. However, when A@CcN@G reaches the acidic tumor microenvironment, the protein surface charge changes, thereby disrupting the electrostatic interactions that maintain the stability of the nanoparticles. This weakening of interactions leads to the gradual dissociation of the nanoparticles, achieving sustained and controlled drug release. This pH-responsive behavior ensures that the drug is released primarily at the tumor site, effectively avoiding premature leakage that may occur during systemic circulation. Notably, this mechanism directly utilizes the protein's inherent pH-sensitive properties without the need for additional responsive materials, demonstrating the simplicity of the system design and the rationality of its function.

[0072] The assembly process was dynamically observed at different time points under physiological pH conditions (7.4) using TEM. In the initial stage (0 min), mainly dispersed Ag₂S QDs (approximately 7 nm) were observed; at 10 min, partially assembled structures with a particle size of approximately 60 nm were observed; by 30 min, complete A@CcN@G nanoparticles with a particle size of approximately 100 nm had formed. Figure 2 (c)). These results validate the proposed stepwise self-assembly mechanism and demonstrate that the system can exhibit dynamically adjustable assembly behavior depending on environmental conditions.

[0073] Stability is a crucial prerequisite for the in vivo application of nanoparticles. After confirming the successful construction of A@CcN@G NPs, we systematically evaluated their stability under various physiologically relevant conditions. The nanoparticles were dispersed in different buffer systems, including 50 mM PBS, 10% fetal bovine serum (FBS), and DMEM medium, and their particle size changes were monitored over 24 h. Dynamic light scattering (DLS) results showed that A@CcN@G maintained good dispersibility and structural stability in all the above media. Figure 2 (d) Besides colloidal stability, optical stability is also a key factor for NIR-II fluorescence imaging-guided therapy. Therefore, we further investigated the fluorescence stability of A@CcN@G. Compared with free Ag2S-NH2QDs, A@CcN@G maintained a stable fluorescence signal throughout 72 h, demonstrating good optical performance and suitability for in vivo imaging applications. Figure 2 (e)). Meanwhile, within the same timescale, its zeta potential showed almost no significant change, indicating that the surface charge of the nanoparticles and the electrostatic interactions upon which they depend were well maintained. Figure 2 (f)). In summary, these characterization results demonstrate that A@CcN@G can maintain structural and functional stability in complex biological environments, showing good potential for biomedical applications.

[0074] To further evaluate the response behavior of A@CcN@G NPs under acidic conditions, we systematically investigated their dissociation kinetics. After incubation at pH 6.5, transmission electron microscopy (TEM) images were acquired at 0 h, 1 h, and 3 h. The results showed that A@CcN@G gradually transformed from initially well-defined spherical nanoparticles with a particle size of approximately 100 nm into dispersed Ag₂S-NH₂ particles with a particle size of approximately 10 nm. This morphological change visually indicates that A@CcN@G underwent a time-progressive dissociation process in an acidic environment, visually verifying its pH-triggered disassembly and assembly mechanism. Figure 2 (g)

[0075] To further quantify its pH response characteristics, A@CcN@G NPs were incubated for 1 h under simulated physiological conditions (pH 7.4) and acidic conditions (pH 6.5) simulating the tumor microenvironment, respectively. Particle size changes were then monitored by dynamic light scattering (DLS). Under acidic conditions, the average particle size of the nanoparticles significantly decreased from approximately 148 nm to approximately 20 nm within 1 h, indicating a significant dissociation process in the system. Figure 2 (h)). This result is highly consistent with the morphological changes observed by TEM. Furthermore, the drug release efficiency under acidic conditions was quantitatively analyzed using standard calculation methods. The results showed that at pH 6.5, the system achieved almost complete drug release within 3 h (h). Figure 2 Zhong (i) and Figure 10 (b)

[0076] The results above demonstrate that A@CcN@G NPs can selectively respond to the acidic tumor microenvironment and achieve targeted and efficient drug release through controlled dissociation, thus providing strong support for precision treatment at the tumor site.

[0077] (3) We systematically studied the optical and photothermal properties of A@CcN@G NPs in aqueous solution. The prevalent hypoxia in the tumor microenvironment (TME) is one of the important factors restricting the application of photodynamic therapy (PDT) in solid tumors; at the same time, tumor tissue is usually accompanied by high levels of hydrogen peroxide (H2O2). Utilizing catalase (CAT) to efficiently catalyze the decomposition of H2O2 to generate water and oxygen is considered an effective strategy to alleviate tumor hypoxia and thus enhance the efficacy of PDT. Given that CAT has been successfully introduced into the A@CcN@G nanosystem, it is necessary to systematically evaluate the oxygen-generating catalytic ability of this system. To this end, we conducted enzymatic reaction experiments under different H2O2 concentrations and different pH conditions, and quantitatively analyzed the amount of oxygen generated by the nanoparticles. The results showed that even under low H2O2 concentrations, A@CcN@G still exhibited significant catalytic activity and could effectively convert H2O2 into O2. Meanwhile, the amount of oxygen generated was significantly dependent on the H2O2 concentration ( Figure 3 (a) indicates that the abundant H2O2 in the tumor microenvironment can serve as an effective substrate source for reactive oxygen species (ROS) generation during PDT. Furthermore, CAT exhibits the highest catalytic oxygen production efficiency at pH 6.5, suggesting that A@CcN@G performs better under the weakly acidic conditions characteristic of the tumor microenvironment. Figure 3 (b)). Theoretically, an increase in oxygen levels should help enhance the effect of PDT. Further comparison of O2 content in the solution before and after near-infrared (NIR) irradiation revealed a significant decrease in oxygen after irradiation, indicating that the oxygen produced by CAT was effectively consumed during PDT, thus verifying the key auxiliary role of CAT in enhancing photodynamic therapy. Figure 3 (c)

[0078] The therapeutic effect of PDT largely depends on the generation of singlet oxygen by the photosensitizer Ce6 under light conditions. 1 To evaluate the PDT performance of nanoparticles under different oxygen environments, we quantitatively analyzed their ability to react with oxygen (O2). 1 O2 generation capacity. 1,3-Diphenylisobenzofuran (DPBF) was used as a specific probe, and its generation capacity was monitored by UV-vis absorption spectroscopy. To construct a control system without oxygen production capacity, bovine serum albumin (BSA) was used instead of CAT to prepare A@BnN@G NPs. Subsequently, A@CcN@G and A@BnN@G were co-incubated with A549 cells for 12 h under normoxic or hypoxic conditions before laser irradiation. O2 generation capacity was measured under 650 nm laser irradiation (300 mW / cm²). 2 Both types of nanoparticles exhibited significant photo-triggered behavior in a normal oxygen environment (3 min). 1O2 generation capacity; under the same Ce6 concentration, the introduction of CAT did not significantly affect the PDT effect. However, under hypoxic conditions, A@BnN@G's 1 O2 generation capacity decreased significantly, while A@CcN@G still maintained high efficiency. 1 O2 production is achieved through CAT catalysis of endogenous H2O2 to oxygen, thereby compensating for the adverse effects of an oxygen-deficient environment. Figure 3 (d)). The above results fully demonstrate the key role of CAT in enhancing the effect of PDT under hypoxic conditions, and further highlight the advantages of A@CcN@G in overcoming tumor hypoxia limitation.

[0079] The realization of photothermal therapy (PTT) in this system relies on the excellent photothermal conversion capability of the NIR-II Ag2S quantum dots integrated within A@CcN@G. In this part of the experiment, we systematically evaluated the temperature rise performance of A@CcN@G under near-infrared laser irradiation to determine the optimal nanoparticle concentration for achieving mild and effective photothermal therapy. The results show a significant positive correlation between laser power density and system temperature rise. At an 808 nm laser and a power density of 300 mW / cm², the system achieved optimal temperature rise. 2 Under these conditions, A@CcN@G can heat to approximately 43 °C within 3 minutes. This temperature range is considered to effectively kill tumor cells while minimizing damage to surrounding normal tissues. Figure 3 (e)). Based on the above results, subsequent experiments selected a nanoparticle concentration of 100 μg / mL and used 300 mW / cm². 2 A 3-minute laser irradiation was used as the standard treatment. Infrared thermal imaging results further verified its photothermal performance: within 3 minutes of 808 nm laser irradiation, the temperature of A@CcN@G rapidly increased to approximately 44 °C and remained stable during the irradiation process, meeting the expected treatment design requirements. Figure 3 (f)). Furthermore, A@CcN@G underwent six heating-cooling cycles under the same laser conditions, and the results showed no significant attenuation in its photothermal performance, indicating that the system possesses excellent photothermal stability. Figure 3 (g)). In summary, A@CcN@G exhibits stable and efficient photothermal properties, laying the foundation for its use as a multifunctional, mild, and targeted tumor therapy platform.

[0080] (4) Based on the excellent photothermal conversion performance and singlet oxygen (A@CcN@G NPs exhibited in aqueous solution) 1In addition to its O2 generation capacity, we further evaluated its biomedical application potential at in vitro and in vivo levels. First, the cytotoxicity of A@CcN@G was evaluated using the standard CCK-8 assay. Under light-protected conditions, even when A549 cells were in contact with Ag2S (200 μg / mL),... Figure 4 (a) or A@CcN@G (200 μg / mL, Figure 4 In (b) after 12 h of co-incubation, the cells maintained a high survival rate (>60%), indicating that the nanosystem itself has extremely low inherent cytotoxicity and good biocompatibility. However, under 650 nm laser irradiation (power density 300 mW / cm²), the cells showed a lower survival rate. 2 After 3 min, the cell viability of the A@CcN@G treatment group rapidly decreased to 18%, indicating that this system can produce a significant phototoxic effect through the synergistic effect of photothermal and photodynamic under light conditions. Figure 4 (c)

[0081] After confirming the good cellular biocompatibility of the nanoparticles, we further investigated the intracellular uptake behavior of A@CcN@GNPs in vitro. Utilizing the NIR-II fluorescence properties of Ag2S QDs and combining this with DAPI staining of cell nuclei, the intracellular distribution of the nanoparticles was visualized using confocal laser scanning microscopy (CLSM). CLSM images showed that the fluorescence signal in the cytoplasm gradually increased with prolonged incubation time, indicating continuous uptake of the nanoparticles. A significant intracellular fluorescence signal was observed at 60 min, reaching its maximum accumulation level at 90 min; however, after 2 h, most of the nanoparticles had been metabolized and cleared by the cells. Figure 4 (d) and (e)). Based on the above results, 90 min was uniformly selected as the optimal co-incubation time for subsequent experiments.

[0082] Besides the high permeability and retention effect (EPR) of tumor tissue, the efficient uptake of A@CcN@G NPs in A549 cells is mainly attributed to the cNGQGEQc targeting peptide introduced on their surface. This peptide specifically recognizes and binds to relevant receptors on the surface of A549 cells. To verify this targeting effect, A@Cc@G NPs without cNGQGEQc were used as a control. The results showed that the uptake of A@Cc@G in A549 cells was significantly lower than that of A@CcN@G, demonstrating that cNGQGEQc plays a key role in promoting endocytosis. Figure 4(f), (g)). Further, under the same nanoparticle concentration and incubation conditions, A@CcN@G was co-incubated with three tumor cell lines: A549, 4T1, and HepG2. The results showed that the strongest fluorescence signal was detected in A549 cells, indicating the highest uptake efficiency of the nanoparticles; while 4T1 and HepG2 cells showed only weaker fluorescence signals, presumably mainly due to non-specific EPR-mediated uptake. Figure 4 (h), (i)).

[0083] (5) Based on the excellent singlet oxygen exhibited by A@CcN@G NPs in solution ( 1 To further evaluate its O2 generation capacity and photothermal conversion performance, we assessed its potential for in vitro tumor cell killing via photothermal decomposition (PDT). To verify CAT's catalytic oxygen production function, bovine serum albumin (BSA) was used to replace CAT to prepare control nanoparticles A@BnN@G, which lacked oxygen production capacity. Subsequently, A@CcN@G and A@BnN@G were co-incubated with A549 cells under normoxic and hypoxic conditions, respectively, and intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA probe. The results were consistent with those in the solution system: under light conditions, even in a hypoxic environment, A@CcN@G still exhibited significant O2 generation capacity. 1 O2 generation capacity; while A@BnN@G only has comparable PDT effects under normoxic conditions, and its efficacy is significantly reduced under hypoxic conditions. Figure 5 (a) and (b)). These results indicate that CAT in A@CcN@G can effectively catalyze the decomposition of H2O2 to generate O2, thereby alleviating hypoxia and maintaining the continuous PDT.

[0084] The above conclusions were further validated by Western blot analysis of hypoxia-inducible factor-1α (HIF-1α). As a classic biomarker of hypoxic stress, the expression level of HIF-1α in the A@C group was significantly higher than that in the control group, indicating that significant oxygen depletion occurred during PDT. In contrast, the expression of HIF-1α was significantly reduced in all treatment groups containing CAT, indicating that the introduction of CAT (and the synergistic effect of the targeting peptide) can effectively alleviate tumor hypoxia, thereby enhancing the therapeutic effect of PDT (Figure 5(i)).

[0085] After confirming that CAT has a stable oxygen production and hypoxia-alleviating effect, the enhancement mechanism of PDT and the possible regulatory role of GM were further investigated. SOSG probes were used under laser irradiation... 1 The generation of O2 was visualized and analyzed. CLSM images show that under NIR illumination, group G2 (A@C) begins to generate O2 due to the activation of Ce6. 1 O2; while in group G3 (A@Cc)1 The significantly enhanced O2 generation demonstrates the catalytic amplification effect of CAT. 1 The most significant O2 generation was observed in group G5, specifically A@CcN@G NPs. Figure 5 (d), (c)). We hypothesize that this enhancement effect is related to the regulation of survivin by GM. Survivin is an anti-apoptotic client protein of HSP90 and can inhibit caspase-9 activation; therefore, GM indirectly promotes caspase-9 expression and activation by inhibiting survivin. Western blot results confirmed this hypothesis: compared with other treatment groups, caspase-9 expression was significantly upregulated in the A@CcN@G group, indicating that GM promotes PDT-induced apoptosis by regulating survivin. Figure 5 (f) Meanwhile, survivin expression was significantly downregulated in the A@CcN@G group, further demonstrating that GM can inhibit survivin, thereby relieving its inhibitory effect on caspase-9. Figure 5 (g)

[0086] In addition to photothermal therapy (PDT), the performance of A@CcN@G in gentle photothermal therapy (PTT) was systematically evaluated. Traditional PTT often causes non-specific tissue damage due to high temperatures and induces overexpression of heat shock protein 90 (HSP90), leading to heat resistance in tumor cells. To overcome this problem, this study introduced the HSP90 inhibitor GM into a nanoplatform. Specifically, A549 cells were co-incubated with PBS, Ag2S, or Ag2S@GM for 90 min, respectively, and then subjected to different power densities (100, 300, and 600 mW / cm²). -2 Laser irradiation was performed under these conditions. Live / dead cell staining (Calcein-AM / PI) results showed that 100 mW cm⁻¹… -2 It causes almost no cell death, while 600 mW cm -2 Even in the PBS group, it caused extensive cell necrosis, demonstrating poor biosafety. In contrast, at 300 mW cm⁻¹, it caused extensive cell necrosis. -2 Under mild PTT conditions, the PBS group showed almost no significant cytotoxicity, while the Ag2S group exhibited only moderate cytotoxicity, presumably related to heat stress-induced upregulation of HSP90. Notably, under the same conditions, the Ag2S@GM group achieved approximately 60% cell death. Figure 5 (e) indicates that GM can effectively inhibit HSP90-mediated thermostability. Correspondingly, Western blot results also show that, compared with the control group without GM, the expression level of HSP90 in the A@CcN@G group was significantly reduced ( Figure 5 (h)

[0087] Based on the above experimental results, we constructed a schematic diagram to summarize the synergistic therapeutic mechanism of A@CcN@G NPs ( Figure 5 (j) Among them, GM, as an HSP90 inhibitor, not only reduces heat tolerance and enhances PTT efficacy, but also promotes the degradation of the HSP90 client protein survivin, thereby relieving its inhibitory effect on caspase-9 and further amplifying the PDT-induced apoptosis effect. Ultimately, A@CcN@G achieves multiple synergistic therapeutic effects of PDT, mild PTT, and chemotherapy by synergistically regulating the HSP90 / survivin / caspase-9 signaling axis.

[0088] (6) Subsequently, the therapeutic effects of A@CcN@G NPs under in vitro conditions were further evaluated. Five treatment groups were set up in this experiment: (G1) PBS; (G2) Ag2S@Ce6 (denoted as A@c); (G3) Ag2S@CAT-Ce6 (denoted as A@Cc); (G4) Ag2S@CAT-Ce6-cNGQGEQc (denoted as A@CcN); (G5) Ag2S@CAT-Ce6-cNGQGEQc@GM (denoted as A@CcN@G).

[0089] AM / PI staining results showed that almost no red fluorescence was observed in groups (G1) and (G2), indicating that the killing effect on tumor cells was limited by both PTT and PDT treatment alone. In contrast, red fluorescence was significantly enhanced in group (G3), suggesting an increased degree of cell death; red fluorescence was further enhanced in group (G4); and almost no green fluorescence was observed in group (G5), indicating large-scale tumor cell death. To simulate the severe hypoxic state commonly found in the tumor microenvironment, we co-incubated A@CcN@G NPs with A549 cells under hypoxic culture conditions and performed AM / PI staining. The results showed that even under hypoxic conditions, the A@CcN@G treatment group (v+ hypoxia) still maintained significant tumor cell killing ability. Figure 6 (a) Further quantitative fluorescence analysis showed that, under NIR-II imaging guidance, A@CcN@G NPs achieved highly efficient tumor cell clearance through PTT / PDT synergistic therapy, with a killing efficiency of up to approximately 90%. Figure 6 (b)

[0090] Given that PDT primarily induces apoptosis by activating caspase-9, we further validated the therapeutic effect of A@CcN@G NPs using flow cytometry. Following the previously described experimental groupings, significant differences in the apoptosis rate were observed among the groups after laser irradiation, with the overall trend highly consistent with AM / PI staining results. Specifically, the apoptosis rate induced by the A@CcN@G NPs treatment group was approximately 62.6%, indicating that under NIR imaging guidance, A@CcN@G-mediated phototherapy primarily achieves tumor cell killing by inducing apoptosis rather than necrosis. Figure 6 (c)

[0091] Previous studies have shown that the accumulation of excessive reactive oxygen species (ROS) leads to oxidative damage to mitochondrial DNA (mtDNA). Therefore, further assessment of changes in mitochondrial function is needed to indirectly reflect the level of intracellular ROS generation. JC-1 (also known as CBIC2(3)) is a fluorescent probe commonly used to detect mitochondrial membrane potential (ΔΨm). After co-incubation with A549 cells in each treatment group and light treatment, JC-1 staining results showed a mitochondrial damage trend consistent with AM / PI staining and flow cytometry (Figure 6(d), (e)). Among them, the introduction of CAT significantly enhanced ROS generation during PDT through continuous oxygen supply, while cNGQGEQc promoted the targeted cell uptake of nanoparticles, resulting in a significant increase in PDT efficiency in groups (G3) and (G4), accompanied by higher levels of ROS generation and mitochondrial damage. A@CcN@G NPs (G5), which integrates all functional modules, showed the most significant mitochondrial dysfunction and the strongest tumor cell killing effect.

[0092] (7) To evaluate the synergistic therapeutic effect of A@CcN@G in vivo, we constructed a BALB / c mouse A549 tumor model. To fully utilize the imaging capabilities of Ag2S to monitor tumor progression, the tumor volume was approximately 150 mm. 3 Mice were randomly divided into five groups: (G1) Ag2S, (G2) A@c, (G3) A@Cc, (G4) A@CcN, and (G5) A@CcN@G. Before treatment, the biocompatibility of the nanoparticles was assessed using an in vivo hemolysis assay, which showed a hemolysis rate of less than 1%. Figure 11 (a) indicates that administration via tail vein injection has good safety.

[0093] Subsequently, the distribution and metabolic behavior of the nanoparticles in vivo were systematically evaluated. In A549 tumor-bearing mice, after intravenous injection of A@CcN@G via the tail vein, in vivo NIR-II fluorescence imaging was performed at 5 min, 30 min, 1 h, 2 h, 5 h, and 12 h. After imaging, major organs (heart, liver, spleen, lung, and kidney) and tumor tissue were collected for ex vivo fluorescence imaging analysis. The results showed that the fluorescence signal at the tumor site could be detected 5 min after injection and gradually increased over time, reaching a peak at approximately 2 h. Therefore, 2 h after injection was selected as the optimal time window for laser irradiation in subsequent treatment experiments. By 12 h, the in vivo fluorescence signal had essentially disappeared. The ex vivo imaging results further indicated that NIR-II fluorescence was mainly distributed in the liver and tumor tissue, suggesting that the nanoparticles have good tumor accumulation ability and are metabolically cleared through the liver pathway. Figure 7 (b)

[0094] Based on the above results, we developed an in vivo treatment protocol: treatment began approximately 10 days after subcutaneous inoculation of A549 cells into immunodeficient nude mice, which was defined as day 0 of treatment; subsequent administrations were given on days 1, 3, and 8. Each treatment involved an intravenous injection of 100 μL of nanoparticles, followed by 650 nm near-infrared laser irradiation (300 mW / cm²) on the tumor site 2 hours after injection. 2 (3 min) Figure 7 (a)

[0095] Subsequently, the in vivo photothermal therapy (PTT) efficacy of A@CcN@G was validated. A549 tumor-bearing nude mice were divided into a PBS control group and an A@CcN@G treatment group. Two hours after injection, the tumor area was irradiated with a 650 nm laser for 3 minutes, and the temperature change at the tumor site was monitored in real time using infrared thermography. The results showed that the tumor temperature in the A@CcN@G group rapidly increased to approximately 42 °C within 3 minutes and remained stable during irradiation; in contrast, the tumor temperature in the PBS group showed almost no significant change, remaining at a baseline level of approximately 35 °C. These results clearly demonstrate that A@CcN@G nanoparticles can achieve efficient photothermal conversion in vivo and produce a controllable local heating effect under near-infrared irradiation. Figure 7 (c)

[0096] A 14-day systemic treatment experiment was then conducted. To verify its targeting ability, in vivo fluorescence imaging was performed on all A549 tumor-bearing nude mice 2 hours after the first treatment. The results showed that drug distribution could be observed in all groups of mice, with the treatment group containing the cNGQGEQc targeting peptide showing more significant enrichment at the tumor site. Figure 7(d)). Furthermore, in vivo fluorescence imaging was performed on days 1, 8, and 14 throughout the treatment cycle for both groups (G1) and (G5). Results showed that tumor volume significantly increased in the Ag2S group within 14 days, while tumor growth was significantly inhibited in the A@CcN@G group (d). Figure 7 (f)

[0097] During treatment, bright field photographs of mice were taken every 3 days. Figure 11 (b) ), and the tumor volume was measured and recorded using vernier calipers. Figure 7 (g)). Meanwhile, the body weight of mice in each group was monitored, and the results showed that the body weight remained within the normal range, and no obvious systemic toxicity was observed. Figure 7 (h)). Mice were sacrificed at the end of day 14 of treatment, subcutaneous tumors were removed and photographed. The results clearly showed that, compared with the control group, all treatment groups exhibited varying degrees of tumor inhibition, with the A@CcN@G group showing the most significant efficacy, and two mice even showing complete tumor regression. Figure 7 (e)). The above in vivo results fully demonstrate that A@CcN@G-mediated PDT / PTT synergistic therapy under NIR-II imaging guidance has significant therapeutic advantages for A549 tumors.

[0098] To further assess in vivo biocompatibility, histological analysis was performed on the major organs (heart, liver, spleen, lung, and kidney) of mice in each treatment group. H&E staining results showed that all organs maintained normal tissue morphology and structure, and no obvious tissue damage or inflammatory response was observed, indicating that A@CcN@G combined with phototherapy did not cause significant organ toxicity. Figure 12 ).

[0099] (8) The apoptosis of tumor cells was further evaluated by H&E staining and TUNEL analysis of tumor tissue sections. The A@CcN@G treatment group showed obvious nuclear condensation and significant green fluorescence signal, indicating that a large number of cells underwent apoptosis. At the same time, the proliferation activity of tumor cells was detected by Ki-67 antibody staining. The results showed that the A@CcN@G group had the strongest inhibitory effect on tumor cell proliferation, while the inhibitory effect of other treatment groups was relatively weak. Immunofluorescence staining results further showed that the expression levels of HSP90 and HIF-1 in the (G5) group were significantly reduced, indicating that A@CcN@G-mediated PTT / PDT synergistic therapy can effectively inhibit tumor growth at the microscopic level by regulating a variety of tumor-related proteins. Figure 8 In summary, A@CcN@G is a highly efficient nanoplatform for multimodal targeted therapy of A549 tumors.

[0100] In summary, this invention presents the first NIR-II fluorescence imaging-guided phototherapy nanoplatform based on the self-assembly of proteins and fluorescent quantum dots. This platform, through the rational integration of functional modules such as Ag2S, CAT, cNGQGEQc, Ce6, and GM, organically combines imaging guidance, active targeting, and PDT / PTT synergistic therapy, achieving multi-level therapeutic enhancement. In the design process, biosafety was a core consideration, and the HSP90 inhibitor GM was introduced to achieve mild photothermal therapy (mildPTT), thereby simultaneously addressing two key therapeutic bottlenecks: the limitation of PDT efficacy due to tumor hypoxia and the risk of tissue damage caused by the high temperatures generated in traditional PTT. Through this strategy, the platform achieves synergistic amplification across multiple treatment modalities, rather than simple superposition. Furthermore, Ag2S quantum dots endow the system with stable NIR-II fluorescence imaging capabilities, enabling real-time monitoring of the treatment process and providing intuitive evidence for precision treatment.

[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a pH-responsive protein self-assembly-based nanoprobe, characterized in that, Includes the following steps: The photosensitizer Ce6 was activated in an organic solvent in the presence of condensing agent 1 and activator 1, and then mixed with CAT for a coupling reaction 1. Finally, the resulting reaction solution 1 was purified 1 to obtain the photosensitizer-protein conjugate. The peptide cNGQGEQc was activated in an organic solvent in the presence of condensing agent 2 and activator 2, then mixed with CAT for a coupling reaction 2, and finally the resulting reaction solution 2 was purified 2 to obtain the targeted peptide-protein conjugate. Hydrophilic silver sulfide quantum dots, photosensitizer-protein conjugates, photosensitizer-peptide conjugates, and HSP90 inhibitors were mixed evenly in solvent 2, and then self-assembled under light-protected conditions to obtain the nanoprobe.

2. The preparation method according to claim 1, characterized in that, The condensing agent 1 and condensing agent 2 are the same, both being 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the activator 1 and activator 2 are the same, both being N-hydroxysuccinimide; the organic solvent 1 and organic solvent 2 are the same, both being dimethyl sulfoxide.

3. The preparation method according to claim 1, characterized in that, The molar ratio of condensing agent 1 to activator 1 is 1:(1.1~1.6), and the molar ratio of condensing agent 1 to photosensitizer Ce6 is 0.5~1.5 mmol:0.

049. g The conditions for activation 1 are: room temperature and light protection reaction for 0.5~1 h.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the photosensitizer Ce6 to CAT is (0.02~0.06):0.01; the conditions for the coupling reaction 1 are: room temperature and light protection for 12~24 h.

5. The preparation method according to claim 1, characterized in that, The molar ratio of condensing agent 2 to activator 2 is 1:(1.1~1.6), and the molar ratio of condensing agent 2 to peptide cNGQGEQc is 0.1~0.5 mmol:0.

01. g The activation conditions for Activation 2 are: room temperature and light protection for 0.5-1 h.

6. The preparation method according to claim 1, characterized in that, The mass ratio of the peptide cNGQGEQc to CAT is (0.8~1.1):(0.8~1.1); the conditions for the coupling reaction 2 are: room temperature and light protection for 12~24 h.

7. The preparation method according to claim 1, characterized in that, The mass ratio of the hydrophilic silver sulfide quantum dots, photosensitizer-protein conjugate, photosensitizer-peptide conjugate and HSP90 inhibitor is 1:(20~25):(20~25):(60~70).

8. The preparation method according to claim 1, characterized in that, The self-assembly reaction was carried out at room temperature for 1.5 to 3 hours.

9. The pH-responsive protein self-assembly-based nanoprobe prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the pH-responsive protein self-assembly-based nanoprobe as described in claim 9 in the preparation of a drug for treating non-small cell lung cancer.

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