Preparation method and application of 8-hydroxyquinoline functionalized polypeptide molecule and metal complex thereof

Abstract

This invention discloses a method for preparing and applying 8-hydroxyquinoline-functionalized polypeptide molecules and their metal complexes. HQ-FFGGRGD (HQD) is prepared via solid-phase polypeptide synthesis. Using 2-chlorotriphenylmethylchloro resin as a carrier, amino acids and 8-hydroxyquinoline-7-carboxylic acid are sequentially coupled, followed by cleavage and purification to obtain the HQD molecule. This molecule can self-assemble into a stable nanostructure through coordination and multiple non-covalent interactions under the drive of transition metal ions, yielding the HQD metal complex. The HQD metal complex can efficiently load chemotherapeutic drugs to form drug-loaded nanosystems, exhibiting pH-responsive and laser-responsive drug release characteristics, significantly enhancing intracellular drug uptake. This invention integrates chemokinetic therapy, photothermal therapy, DNA damage, and chemotherapy into a single nanoplatform, achieving multimodal synergistic anti-tumor therapy with advantages such as good biocompatibility, strong targeting, simple preparation, and high therapeutic efficiency.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing and applying an 8-hydroxyquinoline-functionalized polypeptide molecule and its metal complex. Background Technology

[0002] 8-Hydroxyquinoline, as a monoproton bidentate metal chelate ligand, and its derivatives and metal complexes have been widely used in functional materials fields such as sensing and catalysis due to their excellent metal coordination ability. Furthermore, its metal complexes can exert potential antitumor activity through DNA binding and oxidative stress induction. However, in current technologies, 8-hydroxyquinoline-related active substances mostly exist in small molecule form or are merely physically loaded or lightly modified onto other carriers as simple functional units. Researchers have not been able to fully utilize its strong metal coordination properties to construct stable supramolecular nanoassemblies, resulting in problems such as single function, lack of targeting, and limited therapeutic efficiency in tumor treatment. This makes it difficult to achieve the synergistic integration of multiple therapeutic mechanisms such as photothermal therapy, chemokinetic therapy, DNA damage induction, and chemotherapy.

[0003] Peptide molecules, especially short peptides containing aromatic amino acids, are widely used to construct nanocarrier structures such as nanofibers and nanospheres due to their excellent biocompatibility, biodegradability, and programmable supramolecular self-assembly properties. In existing technologies, these peptide-based materials are mostly used as bioinert carriers, loading chemotherapeutic drugs or nucleic acids solely through physical embedding, without endowing the carrier with inherent therapeutic functions. Some studies have attempted to introduce amino acids with weak coordinating ability, such as histidine, into the peptide sequence to bind metal ions; however, this design is only used to regulate the macroscopic morphology of the assembly or to construct metal ion sensing probes, failing to deeply integrate metal ions as core components that endow the carrier with powerful and diverse therapeutic functions. There is a lack of effective correlation between the structural construction role of metal ions and their biotherapeutic function, failing to achieve a synergistic unity of structure and function.

[0004] Currently, the construction of existing multifunctional anti-tumor nanoplatforms mostly employs inorganic nanoparticles as photothermal or catalytic cores, followed by multi-step surface chemical modification to couple targeting molecules and physically load chemotherapeutic drugs. These systems have significant drawbacks: First, the synthesis and assembly processes involve multiple components and steps, resulting in complex structures, significant batch-to-batch variability, and difficulty in large-scale preparation, as well as poor functional flexibility and controllability. Second, the long-term biosafety of inorganic nanomaterials is questionable; their in vivo metabolic pathways are not yet clear, and they are prone to accumulation in vivo and causing biotoxicity. Third, the integration between different therapeutic modules is merely a simple physical process, lacking the design of intrinsic synergistic mechanisms at the molecular level, thus limiting the efficiency of synergistic therapy. Fourth, some systems lack effective tumor-targeting structures, easily leading to non-specific drug distribution and severe off-target toxicity, further limiting their clinical translational potential.

[0005] Therefore, there is an urgent need to develop a functional nanomaterial with a reasonable structural design, simple preparation process, and excellent biocompatibility and precise tumor targeting ability, so as to achieve efficient multimodal synergistic treatment of tumors. Summary of the Invention

[0006] The purpose of this invention is to address the problems of existing anti-tumor therapy systems based on metal coordination or nanomaterials, such as limited functionality, difficulty in synergistic treatment modalities, poor biocompatibility, and complex preparation processes. This invention provides a method for preparing and applying 8-hydroxyquinoline-functionalized peptide molecules and their metal complexes. This material is a metal-coordinated self-assembled nanomaterial with programmable structure, simple preparation, excellent biocompatibility, and tumor-targeting capabilities. Through rational molecular design, the metal coordination function of 8-hydroxyquinoline, the supramolecular construction ability of self-assembled peptides, and the targeting function of bioactive sequences are organically combined. This enables multi-modal synergistic tumor therapy within a single nanoplatform, effectively overcoming the problems of low functional integration, limited treatment efficiency, and significant off-target toxicity in existing technologies.

[0007] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0008] The first aspect of this invention provides a method for preparing an 8-hydroxyquinoline-functionalized polypeptide molecule, comprising the following steps:

[0009] S1. Using 2-chlorotriphenylmethyl chloride resin as a solid support, after swelling and washing, it is sequentially coupled with fluorene methoxycarbonyl-aspartic acid-β-tert-butyl ester (Fmoc-L-aspartic acid beta-tert-butyl ester, i.e., Fmoc-Asp(OtBu)-OH), Fmoc-glycine (Fmoc-Gly-OH), Fmoc-Pbf-arginine (Fmoc-Arg(Pbf)-OH), Fmoc-L-phenylalanine (Fmoc-Phe-OH), and Fmoc-L-phenylalanine (Fmoc-Phe-OH). After each coupling reaction, the Fmoc protecting group is removed to obtain polypeptide molecules.

[0010] S2. The polypeptide molecule obtained in S1 is coupled with 8-hydroxyquinoline-7-carboxylic acid. Then, the product is cleaved and dissociated from the 2-chlorotriphenylmethyl chloride resin using a cleavage fluid. After post-treatment, the 8-hydroxyquinoline-functionalized polypeptide molecule is obtained.

[0011] This invention employs a solid-phase peptide synthesis method, using 2-chlorotriphenylmethyl chloride resin as a carrier to synthesize 8-hydroxyquinoline-functionalized peptide molecules (denoted as HQ-FFGGRGD, or HQD for short). This method has the advantages of simple operation, mild reaction conditions, strong controllability of peptide chain elongation, and easy control of product purity. The resulting 8-hydroxyquinoline-functionalized peptide molecules combine the biocompatibility and targeting properties of peptides with the metal coordination ability of 8-hydroxyquinoline, providing a core molecular basis for the subsequent preparation of metal complexes, supramolecular self-assembly, and the construction of multimodal antitumor nanosystems.

[0012] Further, in S1, the swelling is performed using dichloromethane (DCM) for 20-40 min; the washing process is performed using N,N-dimethylformamide (DMF) for 3-5 washes.

[0013] Furthermore, in S1, the specific operation of the coupling reaction with fluorenemethyloxycarbonyl-aspartic acid-β-tert-butyl ester is as follows: N,N-diisopropylethylamine (DIEA) is added to the fluorenemethyloxycarbonyl-aspartic acid-β-tert-butyl ester solution, and the resulting mixed solution is added to the 2-chlorotriphenylmethyl chloride resin after swelling and washing treatment, and the reaction is carried out for 1-2 h.

[0014] Furthermore, the solvent for the fluorenemethyloxycarbonyl-aspartic acid-β-tert-butyl ester solution is DMF.

[0015] Furthermore, in S1, after the coupling reaction with fluorenemethyloxycarbonyl-aspartic acid-β-tert-butyl ester, the resin after the coupling reaction is further treated with a blocking solution; the blocking solution is a mixed solution of dichloromethane, methanol (MeOH) and N,N-diisopropylethylamine, and the volume ratio of dichloromethane, methanol and N,N-diisopropylethylamine is (14-18):(1-5):1.

[0016] Further, in S1, the specific operation of the coupling reaction with Fmoc-glycine is as follows: Fmoc-glycine and benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate (HBTU) are dissolved in N,N-dimethylformamide, and then N,N-diisopropylethylamine is added. The resulting mixed solution is added to 2-chlorotriphenylmethyl chloride resin and reacted for 1-2 h.

[0017] Furthermore, the mass ratio of Fmoc-glycine to benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate is 1:(0.8-1).

[0018] Furthermore, in S1, the specific operation of the coupling reaction with Fmoc-Pbf-arginine is as follows: Fmoc-Pbf-arginine and benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate are dissolved in N,N-dimethylformamide, and then N,N-diisopropylethylamine is added. The resulting mixed solution is added to 2-chlorotriphenylmethyl chloride resin and reacted for 1-2 h.

[0019] Furthermore, the mass ratio of Fmoc-Pbf-arginine to benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate is 1:(0.8-1).

[0020] Furthermore, in S1, the specific operation for the coupling reaction with Fmoc-L-phenylalanine is as follows: Fmoc-L-phenylalanine and benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate are dissolved in N,N-dimethylformamide, and then N,N-diisopropylethylamine is added. The resulting mixed solution is then added to 2-chlorotriphenylmethyl chloride resin and reacted for 1-2 h.

[0021] Furthermore, the mass ratio of Fmoc-L-phenylalanine to benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate is 1:(0.8-1).

[0022] Furthermore, in S1, all coupling reactions are carried out under nitrogen protection.

[0023] Further, in S1, the specific operation for removing the Fmoc protecting group is as follows: the resin after coupling reaction is reacted with the Fmoc-removing protective solution, wherein the Fmoc-removing protective solution is a mixed solution of piperidine and N,N-dimethylformamide, and the volume ratio of piperidine to N,N-dimethylformamide is 1:(3-5).

[0024] Further, in S2, the specific operation of the coupling reaction is as follows: 8-hydroxyquinoline-7-carboxylic acid and benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate are dissolved in N,N-dimethylformamide, and then N,N-diisopropylethylamine is added. The resulting mixed solution is added to 2-chlorotriphenylmethyl chloride resin and reacted for 1.5-2.5 h.

[0025] Further, in S2, the cutting fluid is a mixed solution of trifluoroacetic acid (TFA) and water, and the volume ratio of trifluoroacetic acid to water is (90-98):(2-10).

[0026] Furthermore, in S2, the post-processing includes the steps of precipitation, purification, and freeze-drying.

[0027] Furthermore, the precipitation is carried out using diethyl ether, and the precipitation is allowed to stand at -15°C to -25°C for 8-16 hours; the purification is carried out using high performance liquid chromatography.

[0028] The second aspect of the present invention provides a polypeptide molecule functionalized with 8-hydroxyquinoline prepared by the preparation method described in the first aspect.

[0029] In the 8-hydroxyquinoline-functionalized polypeptide molecule (HQ-FFGGRGD) provided by this invention, HQ is an 8-hydroxyquinoline-7-carboxylic acid group, which serves as a monoproton bidentate ligand to provide a coordination site for metal ions; FF is a phenylalanine-phenylalanine dipeptide fragment used to enhance molecular rigidity and promote supramolecular self-assembly through π-π stacking; GGRGD is a functional peptide segment containing an arginine-glycine-aspartic acid sequence, wherein the RGD sequence serves as a targeting ligand that can specifically recognize and bind to integrin receptors on the surface of tumor cells, thereby promoting cellular uptake.

[0030] A third aspect of this invention provides a method for preparing a metal complex of a polypeptide molecule functionalized with 8-hydroxyquinoline, comprising the following steps:

[0031] The 8-hydroxyquinoline-functionalized polypeptide molecule described in the second aspect is dissolved in a solvent, a transition metal salt is added, the pH of the system is adjusted to neutral, and a coordination reaction is carried out to obtain a metal complex of the 8-hydroxyquinoline-functionalized polypeptide molecule.

[0032] This invention utilizes the coordination between the 8-hydroxyquinoline group and metal ions, combined with non-covalent interactions such as hydrogen bonding, electrostatic interactions, and π-π stacking, to drive the supramolecular self-assembly of HQD molecules, thereby forming structurally stable metal-coordinated hybrid nanoparticles, nanofibers, or nanoaggregates.

[0033] Furthermore, the molar ratio of the 8-hydroxyquinoline-functionalized polypeptide molecule to the metal salt is (2-3):1.

[0034] Furthermore, the transition metal salt includes copper salt, iron salt, or manganese salt, preferably divalent copper salt, trivalent iron salt, or divalent manganese salt.

[0035] Cu 2+ It can effectively promote the generation of reactive oxygen species (ROS), accelerate the consumption of glutathione (GSH), and exacerbate DNA damage; Fe 3+ / Mn 2+ It can provide photothermal conversion or catalytic assistance functions, achieving functional complementarity and synergistic enhancement of multiple metal ions.

[0036] Furthermore, the pH of the system is adjusted using HCl solution or NaOH solution, with a pH adjustment range of 7.0-7.8.

[0037] Furthermore, the coordination reaction is carried out at a temperature of 20-25 °C for 1-3 h.

[0038] The fourth aspect of this invention provides a metal complex of an 8-hydroxyquinoline-functionalized polypeptide molecule prepared by the preparation method described in the third aspect.

[0039] The 8-hydroxyquinoline-functionalized peptide-metal complexes provided by this invention achieve a high degree of coupling between metal coordination function and supramolecular self-assembly capability at the molecular level. The metal ions play multiple key roles in the nanostructure, achieving synergistic unity in structural construction and therapeutic function: acting as a self-assembly driving force and structural regulation center to stabilize the nanostructure; acting as a Fenton-like catalytic center to mediate ROS generation and oxidative stress; and acting as a photothermal absorption and energy conversion center to achieve near-infrared photothermal therapy.

[0040] The fifth aspect of this invention provides the use of the 8-hydroxyquinoline-functionalized polypeptide molecule described in the second aspect or the 8-hydroxyquinoline-functionalized polypeptide molecule metal complex described in the fourth aspect in the preparation of a drug for treating tumors.

[0041] Furthermore, the 8-hydroxyquinoline-functionalized polypeptide molecule metal complex is loaded with doxorubicin (DOX).

[0042] 8-hydroxyquinoline-functionalized peptide molecules and metal complexes are loaded with chemotherapeutic drugs to form drug-loaded nanosystems. These drug-loaded nanosystems achieve efficient encapsulation of chemotherapeutic drugs through supramolecular interactions, thereby significantly improving drug delivery efficiency within cells.

[0043] The HQD molecule and its metal complex provided by this invention can be used for cancer treatment. Its therapeutic mechanisms include, but are not limited to: metal ion-mediated Fenton-like reaction to generate ROS and consume GSH, achieving chemokinetic therapy; efficient photothermal conversion under near-infrared light irradiation to achieve photothermal therapy; HQD metal complexes binding to DNA through both groove binding and intercalation binding modes, inducing DNA breaks and inhibiting tumor cell proliferation; and loading chemotherapeutic drugs to achieve targeted delivery and synergistic chemotherapy.

[0044] The above-described technical solution of the present invention has the following beneficial effects:

[0045] 1. The 8-hydroxyquinoline-functionalized polypeptide molecules prepared in this invention can self-assemble into uniformly sized spherical nanoparticles with an average diameter of approximately 43 nm in aqueous solution under physiological conditions and at a low concentration (0.2 wt%) through multiple non-covalent interactions. Based on the excellent metal coordination ability of 8-hydroxyquinoline, Cu is further introduced... 2+ Fe 3+ Mn2+ Different transition metal ions can self-assemble into larger nanoparticles driven by multiple non-covalent interactions and metal coordination, and the resulting nanoparticles all exhibit good stability and dispersibility. HQD metal complexes show significant and broad near-infrared absorption characteristics, demonstrating excellent photothermal conversion efficiency and photothermal stability, and the photothermal performance is positively correlated with material concentration and laser power.

[0046] 2. The HQD metal complex nanomaterials prepared in this invention, which self-assemble in aqueous solution, exhibit certain peroxidase-like (POD) and glutathione peroxidase-like (GPx) activities under specific conditions. Specifically, the HQD copper complex nanoparticles significantly promote ROS generation and GSH consumption, exacerbating cellular oxidative stress. Spectroscopic methods confirmed that the HQD metal complex, due to its planar structure and charge properties, possesses strong DNA-binding affinity, exhibiting both groove binding and intercalation binding modes. It can also cleave supercoiled DNA via an oxidative pathway, with its cleavage ability consistent with its reactive oxygen species generation capacity. The HQD metal complex nanoparticles demonstrate good cell compatibility and biosafety, exhibiting a concentration-dependent inhibitory effect on 4T1 cell proliferation, with significantly better results than HQD ligands alone.

[0047] 3. This invention utilizes multiple intermolecular physical interactions to efficiently load the chemotherapeutic drug DOX onto HQD metal complex nanoparticles, achieving drug loading and encapsulation efficiencies of 53.02% and 34.64%, respectively. In vitro drug release experiments show that this drug-loaded nanosystem exhibits significant pH-responsive release characteristics. The cumulative drug release under simulated tumor acidic environment (pH=5.0 or pH=6.5) is significantly higher than under normal physiological conditions (pH=7.4). Under exogenous stimulation with 808 nm near-infrared laser, the drug release rate is further significantly enhanced, with a cumulative release rate reaching 47.34% under intermittent laser irradiation. Furthermore, this drug-loaded nanosystem can significantly enhance the uptake and internalization of DOX in 4T1 cells through time-dependent endocytosis.

[0048] 4. This invention is based on the HQD metal complex self-assembled nanosystem, which simultaneously integrates and synergistically exerts multiple anti-tumor therapeutic mechanisms, including metal ion-mediated ROS generation and GSH consumption (chemokinetic therapy), near-infrared photothermal conversion effect (photothermal therapy), HQD metal complex binding and breaking of DNA, and efficient loading and targeted delivery of chemotherapeutic drugs. The above multi-mode synergistic effect significantly improves the anti-tumor therapeutic effect. Attached Figure Description

[0049] Figure 1 This is a synthetic route diagram of the HQD molecule in Example 1.

[0050] Figure 2 The HQD molecules prepared in Example 1 1 H NMR spectrum.

[0051] Figure 3 The image shows the MALDI-TOF MS spectrum of the HQD molecule prepared in Example 1.

[0052] Figure 4 These are solution phase diagrams and TEM images of HQD molecules and their metal complexes prepared in Examples 1-4; the upper image shows the HQD solution and HQD... Cu Solution, HQD Fe Solution, HQD Mn Solution state diagrams (from left to right), the bottom image shows HQD molecules and HQD... Cu HQD Fe HQD Mn TEM images (from left to right).

[0053] Figure 5 The images show the UV-Vis spectra of HQD molecules and their metal complexes prepared in Examples 1-4; where A represents HQD, HQD2, and HQD3. Cu HQD Fe With HQD Mn The UV-Vis spectra are shown in Figure 1. B is the absorbance data of HQD metal complexes obtained with different HQD ligand-metal molar ratios at 420 nm.

[0054] Figure 6 The infrared spectra of HQD molecules and their metal complexes prepared in Examples 1-4 are shown.

[0055] Figure 7 The CD spectra of HQD molecules and their metal complexes prepared in Examples 1-4 are shown.

[0056] Figure 8 The figures show the particle size distribution and Zeta potential diagrams of the HQD molecules and their metal complexes prepared in Examples 1-4; where A is the particle size distribution of the HQD molecules, and B is the particle size distribution of the HQD molecules. Cu Particle size distribution diagram, C represents HQD Fe Particle size distribution diagram, D is HQD Mn Particle size distribution diagram, E represents HQD, HQD Cu HQD Fe With HQD Mn Zeta potential diagram.

[0057] Figure 9 The graphs show the particle size and PDI variations of the HQD molecules and their metal complexes prepared in Examples 1-4; where A represents the HQD molecule, B represents the HQD...Cu C stands for HQD Fe D stands for HQD Mn .

[0058] Figure 10 The absorption spectra of solutions of HQD molecules and their metal complexes prepared in Examples 1-4, and temperature-time curves under different parameters are shown. In this figure, A represents the absorption spectrum in the near-infrared region (650-900 nm), and B represents the absorption spectrum under an 808 nm laser (1.0 W / cm²). 2 Temperature-time curves of HQD molecules and their metal complex solutions (50 μg / mL) under irradiation, where C represents HQD. Fe Solutions (50, 100, and 200 μg / mL) were subjected to an 808 nm laser (1.0 W / cm²). 2 The temperature-time curve of irradiation, where D represents HQD. Fe The solution (50 μg / mL) was subjected to 808 nm lasers (0.5, 1.0, and 1.5 W / cm²). 2 Temperature-time variation curve of irradiation.

[0059] Figure 11 The graph shows the photothermal stability test results of the HQD metal complexes prepared in Examples 2-4; where A represents HQD. Fe The solution under 808 nm laser (1.0 W / cm) 2 The temperature-time curve after four cycles of switching irradiation, where B represents HQD. Fe The heating and cooling curves of the solution under one irradiation cycle, and the linear relationship between cooling time and -lnθ, where C represents HQD. Cu A linear relationship between the cooling time of the solution under one irradiation cycle and -lnθ, where D is the HQD. Mn The linear relationship between the cooling time of the solution under one cycle of irradiation and -lnθ.

[0060] Figure 12 The figures show the peroxidase-like catalytic performance test results of the HQD molecules and their metal complexes prepared in Examples 1-4; where A is a physical image of the color change of the solution when TMB is oxidized to oxTMB, B is the UV absorption curve of TMB solutions containing different components at 650 nm, C is the absorbance change of the product of TMB catalyzed by HQD metal complexes at 650 nm under different pH conditions, and D is the absorbance change of the product of TMB catalyzed by HQD metal complexes at 650 nm at different temperatures.

[0061] Figure 13 HQD in the presence of free radical scavengers CuAbsorbance-time curve at 650 nm during the oxidation of TMB.

[0062] Figure 14 The figures show the glutathione peroxidase catalytic performance test results of the HQD metal complexes prepared in Examples 2-4; where A is the UV-Vis absorption spectrum of the HQD metal complex consuming GSH at the same concentration (0.1 mM), and B is the HQD... Cu Absorbance changes at 412 nm for solution-catalyzed oxidation of GSH at different times (inset showing HQD) Cu Color changes of the solution before and after oxidation of GSH), C represents different concentrations of HQD. Cu Absorbance changes at 412 nm for the catalytic oxidation of GSH by solutions (0.05, 0.1, 0.15, and 0.2 mM), where D represents the HQD at different temperatures (25, 37, and 50 °C). Cu Absorbance change curve at 412 nm for solution-catalyzed oxidation of GSH.

[0063] Figure 15 HQD Cu UV-Vis absorption spectra of the solution (50 μM) and mixed solutions containing different concentrations of DNA (0-100 μM); where A represents DNA and HQD. Cu HQD Cu DNA = 1:0.2 (molar ratio), HQD Cu DNA = 1:0.5, HQD Cu DNA = 1:0.8, HQD Cu DNA = 1:1.0, B is HQD Cu DNA = 1:1.0, HQD Cu DNA = 1:1.2, HQD Cu DNA = 1:1.4, HQD Cu DNA = 1:1.6, HQD Cu DNA = 1:2.0.

[0064] Figure 16 The left image shows the fluorescence spectrum (left panel) and Stern-Volmer curve (right panel) of Hoechst binding to ct-DNA in the presence and absence of HQD metal complexes; where A represents HQD. Cu B is HQD Mn C stands for HQD Fe .

[0065] Figure 17The left image shows the fluorescence spectrum (left panel) and Stern-Volmer curve (right panel) of EB binding to ct-DNA in the presence and absence of HQD metal complexes; where A represents HQD. Cu B is HQD Mn C stands for HQD Fe .

[0066] Figure 18 The image shows the results of the pBR322 DNA cleavage activity assay using HQD metal complexes; where A is an agarose gel electrophoresis image of different components cleaving pBR322 DNA under physiological conditions, lane 1: blank DNA; lane 2: DNA + HQD + H2O2; lane 3: DNA + HQD Cu Lane 4: DNA + HQD Mn + H2O2; Lane 5: DNA + HQD Fe + H2O2; Lane 6: DNA + HQD Cu + H2O2; Lane 7: DNA + HQD Cu + H2O2 + TBA; Lane 8: DNA + HQD Cu + H2O2 + SOD ([H2O2] = 0.1 mM, [TBA] = 4 mM, [SOD] = 2 μM); B represents different concentrations of HQD. Cu Agarose gel electrophoresis image of pBR322 DNA fragments: Lane 1: blank DNA; Lanes 2-8: DNA + H2O2 + HQD Cu (Concentrations were 0.05 mM, 0.08 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, and 0.5 mM, respectively).

[0067] Figure 19 HQD Cu Characterization and in vitro release test results of the drug-loaded nanoparticle system with DOX supported on nanoparticles (HQDCuD); where A represents HQDCuD, DOX, and HQD. Cu B is the UV-Vis absorption spectrum of HQDCuD, C is the TEM image of HQDCuD, D is the Zeta potential diagram of HQDCuD and HQDCuD, E is the standard curve of DOX solution, E is the cumulative release rate-time curve of DOX within 34 h under different pH conditions (5.0, 6.5 and 7.4), and F is the cumulative release rate-time curve of DOX within 34 h with / without laser irradiation.

[0068] Figure 20 HQD Cu HQD FeHQD Mn A graph showing cell viability data after 24 hours of co-incubation with HUVECs.

[0069] Figure 21 The results of the HQDCuD uptake assay for 4T1 cells are shown in Figure A. A is a confocal image of 4T1 cells after co-incubation with free DOX and HQDCuD for 3 h and 6 h. B is a histogram of fluorescence intensity distribution of DOX and HQDCuD groups detected by flow cytometry. C is a comparison of fluorescence intensity of DOX and HQDCuD groups at different incubation times.

[0070] Figure 22 The graph shows the intracellular ROS levels of HQD molecules and their metal complexes, and HQDCuD prepared in Examples 1-4; where A represents 4T1 cells with / without laser irradiation and PBS, HQD, and HQD. Fe HQD Mn HQD Cu Confocal image of ROS fluorescence generated in cells after 6 h of incubation with HQDCuD (scale bar is 100 µm), B is a graph of ROS content in 4T1 cells quantitatively measured by flow cytometry.

[0071] Figure 23 The figures show the intracellular GSH levels of HQD molecules and their metal complexes, and HQDCuD prepared in Examples 1-4; where A represents 4T1 cells with / without laser irradiation and PBS, HQD, and HQD. Fe HQD Mn HQD Cu Confocal image of GSH fluorescence generated in cells after 6 h of incubation with HQDCuD (scale bar is 100 µm). B is a graph of GSH content in 4T1 cells as measured by flow cytometry.

[0072] Figure 24 The graph shows the in vitro antitumor activity test results of HQD molecules and their metal complexes, and HQDCuD prepared in Examples 1-4; where A represents 4T1 cells with HQD and HQDuD. Cu HQD Fe and HQD Mn Cell viability data after 24 h of co-incubation: B shows the cell viability data of 4T1 cells after 24 h of co-incubation with free DOX and HQDCuD; C shows the cell viability data of 4T1 cells with HQD and HQDCuD. Fe HQD Mn HQD Cu Plot of cell viability data for HQDCuD and co-incubated for 24 h under both light and dark conditions.

[0073] Figure 25 4T1 cells and HQD Fe HQD Mn HQD Cu Cell staining after co-incubation with HQDCuD (100 μg / mL) for 24 h (green represents live cells, red represents dead cells), scale bar is 100 µm.

[0074] Figure 26 To investigate the interaction between 4T1 cells and HQD under 808 nm laser irradiation conditions Fe HQD Mn HQD Cu Cell staining after co-incubation with HQDCuD (100 μg / mL) for 24 h (green represents live cells, red represents dead cells), scale bar is 100µm.

[0075] Figure 27 4T1 cells with PBS and HQD Fe HQD Mn HQD Cu Flow cytometry plots of cell apoptosis after co-incubation with and without laser irradiation of HQDCuD for 24 h. Detailed Implementation

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0077] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0078] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0079] In the following test cases, mouse breast cancer (4T1) cells and human umbilical vein endothelial cells (HUVECs) were purchased from Beijing BioBio Biotechnology Co., Ltd. Cell culture was performed using high-glucose DMEM basal medium. 10% heat-inactivated fetal bovine serum and penicillin-streptomycin antibiotic solution were added to the DMEM to prepare a complete medium. Cells were cultured in a 37 ℃, 5% CO2 incubator. Cell resuscitation: The cryovials were removed from liquid nitrogen and immediately placed in a 37 ℃ water bath with rapid shaking until completely thawed (1 min). In a biosafety cabinet, the cell suspension was transferred to a 15 mL centrifuge tube containing 2 mL of complete medium and gently mixed. The cells were centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The cell pellet was gently resuspended in 1 mL of complete medium and transferred to a T25 culture flask containing 9 mL of fresh complete medium, and gently shaken. The medium was replaced the next day to remove residual DMSO. Cell passage: When the cells reach 80%-90% confluence, passage them; discard the old culture medium, gently wash the cells twice with 1× PBS sterile buffer; add 1 mL of trypsin solution and digest in a 37 ℃ incubator for 2 minutes; after confirming under a microscope that most cells have become rounded and detached, immediately add 2 mL of complete culture medium to stop the digestion; gently pipette the bottom of the culture flask, collect the cell suspension into a centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, resuspend the cells in fresh complete culture medium, and passage and plate the cells as needed.

[0080] Example 1

[0081] A method for preparing an 8-hydroxyquinoline-functionalized polypeptide molecule HQ–FFGGRGD includes the following steps:

[0082] (1) Weigh 0.5 g of 2-chlorotriphenylmethyl chloride resin and place it in a solid-phase reactor. Purge with nitrogen and add 10 mL of anhydrous dichloromethane (DCM) to allow the resin to swell fully for 30 min. After the reaction is complete, blow off the residual DCM with nitrogen and then wash the resin four times with anhydrous N,N-dimethylformamide (DMF) to remove residual solvent and impurities.

[0083] (2) Under nitrogen protection, 718.9 mg of Fmoc-Asp(OtBu)-OH was dissolved in 10 mL of anhydrous DMF and sonicated to ensure complete dissolution. After the resin swelled, 775 µL of N,N-diisopropylethylamine (DIEA) was added to the Fmoc-Asp(OtBu)-OH solution, mixed well, and then added to the treated resin. The reaction was allowed to proceed for approximately 1.5 h. After the reaction was complete, the resin was washed four times with anhydrous DMF.

[0084] (3) Prepare a blocking solution by volume ratio of DCM:methanol (MeOH):DIEA = 16:3:1. Add 10 mL of the blocking solution to the solid-phase reactor and treat the resin under a nitrogen atmosphere for 10 min to quench unreacted active sites on the resin. Repeat the treatment twice. Then wash the resin with anhydrous DMF 5 times.

[0085] (4) Mix piperidine and DMF at a volume ratio of 1:4 to prepare a 20% de-Fmoc protection solution. Under nitrogen flow, allow the resin to react with the de-Fmoc protection solution for 30 min. After draining the reaction solution, wash the resin three times with the de-Fmoc protection solution and finally wash it four times with anhydrous DMF.

[0086] (5) Under nitrogen protection, 771.47 mg Fmoc-Gly-OH and 711.08 mg benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate (HBTU) were dissolved in anhydrous DMF and dissolved by sonication. When the reaction was in its last 5 min, 775 µL of DIEA was added and mixed thoroughly. This activated mixture was then added to the resin system and reacted for 1 h. After draining the reaction solution, the resin was washed four times consecutively with anhydrous DMF. Step (4) was then repeated to remove the Fmoc protecting group.

[0087] (6) Under nitrogen protection, 771.47 mg Fmoc-Arg(Pbf)-OH and 711.08 mg HBTU were dissolved in anhydrous DMF and dissolved by sonication. When the reaction was in progress for the last 5 minutes, 775 µL of DIEA was added and mixed thoroughly. This activated mixture was then added to the resin system and reacted for 1 hour. After draining the reaction solution, the resin was washed four times consecutively with anhydrous DMF. Step (4) was then repeated to remove the Fmoc protecting groups.

[0088] (7) Under nitrogen protection, 771.47 mg Fmoc-Phe-OH and 711.08 mg HBTU were dissolved in anhydrous DMF and dissolved by sonication. When the reaction was in its last 5 min, 775 µL of DIEA was added and mixed thoroughly. This activated mixture was then added to the resin system and reacted for 1 h. After draining the reaction solution, the resin was washed four times consecutively with anhydrous DMF. Step (4) was then repeated to remove the Fmoc protecting groups.

[0089] (8) Under nitrogen protection, 771.47 mg Fmoc-Phe-OH and 711.08 mg HBTU were dissolved in anhydrous DMF and dissolved by sonication. When the reaction was in its last 5 min, 775 µL of DIEA was added and mixed thoroughly. This activated mixture was then added to the resin system and reacted for 1 h. After draining the reaction solution, the resin was washed four times consecutively with anhydrous DMF. Step (4) was then repeated to remove the Fmoc protecting groups.

[0090] (9) Under nitrogen protection, 771.47 mg of 8-hydroxyquinoline-7-carboxylic acid and 711.08 mg of HBTU were dissolved in anhydrous DMF, and the dissolution was assisted by sonication. When the reaction was in progress for the last 5 minutes, 775 µL of DIEA was added and mixed well. Then the activated mixture was added to the resin system and reacted for 2 hours. After the reaction solution was drained, the resin was washed 5 times each with anhydrous DCM, anhydrous MeOH and n-hexane, and the resin was dried with nitrogen.

[0091] (10) Trifluoroacetic acid (TFA) and deionized water were mixed at a volume ratio of 95:5 to prepare a cleavage solution, which was then added to the solid-phase reactor along with the resin system. The reaction was carried out under nitrogen protection and at room temperature for 2 h to cleave the peptide from the resin and release it into the solution. After draining the solution containing the product, the cleavage solution was added again and reacted for 10 min. This operation was repeated three times, and all drained solutions containing the product were collected.

[0092] (11) After drying the product solution with an air pump, pre-cooled anhydrous diethyl ether was added, and the solution was placed in a -20 °C refrigerator for precipitation overnight. The crude product was obtained by filtration the next day. After purification by gradient elution (water:acetonitrile from 80:20 to 20:80) by high performance liquid chromatography (HPLC), the yield was 87%. The product was then freeze-dried under vacuum to obtain the 8-hydroxyquinoline-functionalized polypeptide molecule HQ-FFGGRGD, denoted as HQD molecule.

[0093] Example 1: HQD molecules were successfully prepared using 8-hydroxyquinoline-7-carboxylic acid (HQ) as the functionalization unit via solid-phase peptide synthesis. The synthetic route is as follows: Figure 1 As shown.

[0094] Example 2

[0095] A metal complex of an 8-hydroxyquinoline-functionalized polypeptide molecule (HQD) Cu The preparation method of ) includes the following steps:

[0096] Weigh the HQD molecules prepared in Example 1 and dissolve them in ultrapure water to prepare a 5 mM stock solution. Use ultrasound to assist in the complete dissolution. Add CuCl2·2H2O to the stock solution to control the reaction between the HQD ligands and Cu.2+ The molar ratio was 2:1. The pH was adjusted to 7.4 using 1M NaOH solution. After mixing thoroughly, the mixture was allowed to stand at room temperature to allow the coordination reaction to proceed completely, yielding HQD. Cu Solution. Add HQD Cu The solution was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and purified by dialysis in deionized water. Dialysis was performed under light-protected conditions, with the external dialysate replaced every 4 hours for a total of 48 hours to thoroughly remove salt impurities. The purified solution was stored at 4 °C in the dark for later use. The purified solution was then freeze-dried under vacuum to obtain HQD. Cu .

[0097] Example 3

[0098] A metal complex of an 8-hydroxyquinoline-functionalized polypeptide molecule (HQD) Fe The preparation method of ) includes the following steps:

[0099] Weigh the HQD molecules prepared in Example 1 and dissolve them in ultrapure water to prepare a 5 mM stock solution. Use ultrasound to assist in complete dissolution. Add FeCl3·6H2O to the stock solution to control the reaction between the HQD ligands and Fe... 3+ The molar ratio was 3:1. The pH was adjusted to 7.4 using 1M NaOH solution. After thorough mixing, the mixture was allowed to stand at room temperature to allow the coordination reaction to proceed fully, yielding HQD. Fe Solution. Add HQD Fe The solution was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and purified by dialysis in deionized water. Dialysis was performed under light-protected conditions, with the external dialysate replaced every 4 hours for a total of 48 hours to thoroughly remove salt impurities. The purified solution was stored at 4 °C in the dark for later use. The purified solution was then freeze-dried under vacuum to obtain HQD. Fe .

[0100] Example 4

[0101] A metal complex of an 8-hydroxyquinoline-functionalized polypeptide molecule (HQD) Mn The preparation method of ) includes the following steps:

[0102] Weigh the HQD molecules prepared in Example 1 and dissolve them in ultrapure water to prepare a 5 mM stock solution. Use ultrasound to assist in complete dissolution. Add MnCl2 to the stock solution to control the reaction between the HQD ligand and Mn. 2+ The molar ratio was 2:1. The pH was adjusted to 7.4 using 1 M NaOH solution. After mixing thoroughly, the mixture was allowed to stand at room temperature to allow the coordination reaction to proceed completely, yielding HQD. Mn Solution. Add HQD MnThe solution was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and purified by dialysis in deionized water. Dialysis was performed under light-protected conditions, with the external dialysate replaced every 4 hours for a total of 48 hours to thoroughly remove salt impurities. The purified solution was stored at 4 °C in the dark for later use. The purified solution was then freeze-dried under vacuum to obtain HQD. Mn .

[0103] Test Example 1

[0104] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 The chemical structure of the HQD molecule prepared in Example 1 was systematically characterized and confirmed using 1H NMR and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). The testing method was as follows: 5 mg of purified HQD molecule was placed in an NMR tube, and 0.6 mL of deuterated dimethyl sulfoxide (DMSO-d6) was added. The mixture was shaken until completely dissolved, and its proton NMR spectrum was measured using an INOVA 400 MHz NMR spectrometer. Separately, a suitable concentration of peptide sample solution and α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution were prepared. Sample preparation was performed using a layered spotting method: a small amount of matrix solution was first added to the mass spectrometry target plate and allowed to dry naturally at room temperature to form a substrate; then, the sample solution was added to the substrate, and after drying, another layer of matrix solution was applied to the sample layer surface and allowed to dry completely. After sample preparation, the molecular weight and structure of the target molecule were characterized using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0105] The HQD molecules prepared in Example 1 1 H NMR image as follows Figure 2 As shown, the NMR data are: 1 H NMR (400MHz, DMSO- d6) δ 9.30 (s,1H), 8.56 (dd, J = 55.2, 44.9 Hz,3H), 8.19 (dd, J =31.7, 8.1 Hz,2H), 8.12 – 7.88 (m,4H), 7.83 (t, J = 8.3 Hz,1H), 7.45 (dd, J =8.2, 4.3 Hz,1H), 7.37 (dd, J = 7.9, 4.3 Hz,1H), 7.27 (t, J = 7.9 Hz,2H), 7.24– 7.17 (m,3H), 7.11 (dt, J = 18.5, 9.1 Hz,3H), 6.67 (s,1H), 6.57 (d, J = 8.8Hz,1H), 4.67 (s,1H), 4.48 (s,1H), 4.29 (s,1H), 4.14 (s,1H), 3.79 (ddd, J =43.9, 21.8, 8.7 Hz,5H), 3.50 (d, J = 16.5 Hz,1H), 3.20 – 2.97 (m,4H), 2.85(dd, J = 22.6, 9.3 Hz,2H), 2.34 (d, J = 10.0 Hz,1H), 2.06 – 1.95 (m,1H), 1.85(dd, J=12.7,7.0 Hz,1H),1.50 (t,J=47.1 Hz,4H). The MALDI-TOF MS spectrum of the HQD molecule prepared in Example 1 is shown below. Figure 3 As shown, the mass spectrometry data are: MS: calcdM=925.37, obsd(M+H) + =926.75, obsd(M+Na) + =948.72.

[0106] Test Example 2

[0107] Testing HQD molecules and their interaction with transition metal ions (Cu) 2+ Fe 3+ and Mn 2+ The self-assembly ability and microstructure of the coordinated HQD molecules and their metal complexes prepared in Examples 1-4 were dissolved in deionized water at pH 7.4 to prepare a homogeneous sample solution (HQD solution, HQD) with a concentration of 0.2 wt%. Cu Solution, HQD Fe Solution, HQD Mn All sample solutions were clear and transparent, such as... Figure 4 As shown in Figure A.

[0108] The sample preparation and testing process for transmission electron microscopy (TEM) is as follows: 4 μL of HQD solution and HQD... Cu Solution, HQD Fe Solution, HQD Mn The solution was dropped onto the surface of a 200-mesh copper mesh and allowed to stand at room temperature for approximately 30 seconds to promote sample adsorption. Excess liquid was then gently blotted away along the edge of the copper mesh with filter paper, and the mesh was allowed to air dry at room temperature. After drying, 5 μL of 2.0% (w / v) phosphotungstic acid staining solution (pH=7.0) was dropped onto the copper mesh surface under light-protected conditions. After staining for 10 minutes, residual staining solution was again blotted away with filter paper, and the mesh was allowed to dry thoroughly at room temperature. Finally, the microstructure of the sample was observed using a transmission electron microscope (Hitachi HT7700) with an accelerating voltage of 120 kV.

[0109] TEM characterization results as follows Figure 4 As shown in Figure B, individual HQD molecules spontaneously assemble into well-dispersed nanoparticles in aqueous solution, with an average particle size of approximately 39 nm. This is because the π-π stacking and hydrophilic-hydrophobic interactions between the 8-hydroxyquinoline heterocycle and the phenylalanine dipeptide fragment jointly drive the supramolecular assembly process. After the introduction of transition metal ions, 8-hydroxyquinoline acts as a strong chelating bidentate ligand to coordinate with the metal ions, maintaining high system stability while the size of the nanostructures undergoes a significant change: HQD Cu NPs, HQD Fe NPs and HQD Mn The average particle sizes of the NPs increased to 52 nm, 82 nm, and 63 nm, respectively. This phenomenon is mainly due to the coordination between the 8-hydroxyquinoline structural unit and the metal ion. The metal ion, as a coordination crosslinking node, effectively enhances the intermolecular interaction, thereby promoting the formation of a more compact organic-inorganic hybrid nanostructure.

[0110] Test Example 3

[0111] The formation process of HQD metal complexes was analyzed using ultraviolet-visible spectroscopy to determine the stoichiometric ratio of HQD binding to transition metal ions. The testing method was as follows: First, HQD molecules were weighed and dissolved in ultrapure water to prepare a 5 mM stock solution, which was then sonicated to ensure complete dissolution. To determine the stoichiometric ratio of the metal ion to the peptide ligand, the molar ratio method was used for monitoring: Under a fixed metal ion concentration ([CuCl2·2H2O] / [FeCl3·6H2O] / [MnCl2] = 0.5 mM), the molar ratio of the ligand to the metal ion was gradually changed (0:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1). The pH was adjusted to 7.4 using 1 M HCl / NaOH solution. After thorough mixing, the mixture was allowed to stand at room temperature, and the change in absorbance at the characteristic absorption peak (420 nm) was monitored. Subsequently, HQD and its metal complex solutions (50 μM) were prepared according to the determined stoichiometric ratio, placed in quartz cuvettes, and scanned in the range of 200-900 nm using a UV-Vis spectrophotometer (UV-1900i). Before testing, baseline correction was performed using a blank solvent, and then each sample solution was measured sequentially.

[0112] Test results are as follows Figure 5 As shown, from Figure 5 As shown in Figure A, the HQD ligand exhibits absorption peaks at 273 nm and 342 nm, which can be attributed to electronic transitions within the ligand. After coordination with metal ions, HQD… Cu HQD Fe and HQD Mn The low-energy absorption bands red-shifted to 392 nm, 347 nm, and 350 nm, respectively; simultaneously, a new broad absorption band appeared in the 400-500 nm region. This feature is attributed to ligand-metal charge transfer (LMCT) transitions, directly proving that HQD successfully formed coordination bonds with metal ions. Further titration experiments with fixed metal ion concentrations and varying ligand-metal molar ratios were conducted, and the changes in the characteristic absorption peak (420 nm) were monitored using UV-Vis absorption spectroscopy. The results showed that the coordination stoichiometry ratios of HQD with Cu(II), Fe(III), and Mn(II) were 2:1, 3:1, and 2:1, respectively, corresponding to the formation of stable HQD. Cu HQD Fe and HQD Mn ,like Figure 5 As shown in B.

[0113] Test Example 4

[0114] Fourier transform infrared (FT-IR) analysis was performed on the HQD molecules and their metal complexes prepared in Examples 1-4 using a Thermo Fisher Scientific Nicolet 6700 FT-IR spectrometer equipped with an attenuated total reflectance (ATR) accessory to further verify the metal ion (Cu) 2+ Fe 3+ and Mn 2+ The coordination interaction between the sample and HQD molecules was investigated. The test method involved placing the lyophilized solid sample powder directly onto the surface of an ATR crystal, without any additional pretreatment. The spectral scanning range was set to 4000-400 cm⁻¹. -1 The resolution is 4 cm. -1 Each sample was scanned a total of 32 times. Background spectra were acquired and automatically subtracted before testing, and all tests were performed at room temperature.

[0115] Test results are as follows Figure 6 As shown, the HQD molecule is at 3415 cm⁻¹ -1 A distinct and broad absorption peak is observed nearby, which can be attributed to the stretching vibration of the phenolic hydroxyl group (-OH) in the quinoline ring, indicating that the hydroxyl group exists in a non-coordinated, free state. After the introduction of metal ions and the formation of a coordination structure, the characteristic peak of the -OH stretching vibration almost completely disappears, indicating that the hydroxyl group undergoes deprotonation and participates in the metal coordination process, thereby disrupting the original OH bond vibration mode. Furthermore, the quinoline ring skeleton of the HQD molecule shows a peak at 1604 cm⁻¹. -1 1382 cm -1 and 1190 cm -1 The peaks at these locations correspond to the characteristic stretching vibration absorption bands of the C=N, CN, and CO groups, respectively. Upon the introduction of metal ions, these characteristic peaks exhibit varying degrees of redshift towards lower wavenumbers, while their peak intensities significantly decrease. This decrease in wavenumber and change in absorption intensity typically stems from the redistribution of electron clouds caused by coordination interactions, indicating that the N and O atoms in the quinoline ring act as electron donors and effectively interact with the metal ions. These infrared spectral changes confirm that the metal ions successfully constructed a stable metal-ligand coordination structure through chelation with the N and O coordination sites in the hydroxyquinoline group.

[0116] Test Example 5

[0117] The secondary structures of the HQD molecules and their metal complexes prepared in Examples 1-4 were systematically characterized using circular dichroism spectroscopy (CD) to evaluate the effect of metal coordination on polypeptide chain conformation. The testing method was as follows: the HQD molecules and their metal complexes prepared in Examples 1-4 were dissolved in deionized water at pH 7.4 to prepare sample solutions with a concentration of 0.2 wt% (HQD solution, HQD...). Cu Solution, HQDFe Solution, HQD Mn (Solution); The sample solution was placed in a quartz cuvette with an optical path of 1 mm and tested using a Jasco J-815 spectrometer at room temperature. The spectral scanning range was set to 185-300 nm, the scanning rate to 50 nm / min, and the scanning interval to 1 nm. Each sample was scanned at least three times, and the average value was taken to improve the signal-to-noise ratio. Baseline correction was performed using the same solvent as a blank before testing, and the obtained spectral data were smoothed before being used for subsequent analysis.

[0118] Test results are as follows Figure 7 As shown, the HQD molecule exhibits a distinct positive peak at 192 nm and a typical negative Cotton effect at 213 nm. This characteristic spectral pattern is consistent with the β-sheet structure, indicating that the HQD polypeptide chain in solution is predominantly β-sheet oriented. In contrast, the CD spectrum of the HQD metal complex shows significant changes: its positive Cotton peak shifts to shorter wavelengths, located in the 185-189 nm range, while the corresponding negative peak shifts slightly to 210-212 nm, and its intensity is significantly enhanced. This peak shift and signal enhancement typically reflect further ordering of the polypeptide backbone conformation, indicating that the introduction of metal ions restricts the conformational freedom of the polypeptide chain through coordination, thereby promoting the formation of a more compact and stable folded structure.

[0119] Test Example 6

[0120] The particle size distribution and Zeta potential of the HQD molecules and their metal complexes prepared in Examples 1-4 were measured using a Zetasizer Nano-ZS dynamic light scattering spectrometer (DLS, Malvern, UK) to further determine the particle size and surface charge characteristics of the nanoparticles formed by the HQD molecules and their metal complexes. The testing method was as follows: the HQD molecules and their metal complexes prepared in Examples 1-4 were dissolved in deionized water at pH 7.4 to prepare a 0.2 wt% sample solution (HQD solution, HQD...). Cu Solution, HQD Fe Solution, HQD Mn The solution was left to stand at room temperature for 12 h. Through non-covalent interactions between HQD peptide molecules, spontaneous assembly of molecules formed nanoparticles. After assembly, a short-term, mild sonication was performed to ensure uniform particle size, resulting in a dispersion of HQD molecules and their metal complexes as nanoparticles. 200 µL of the nanoparticle dispersion was injected into a double-sided translucent quartz cuvette at room temperature. The hydrated particle size and polydispersity index (PDI) of each sample were measured at least three times, and the average value was taken. The Zeta potential was measured using a Malvern Zeta potential sample cell. All tests were performed under the same conditions.

[0121] Test results are as follows Figure 8 As shown in the figure, the average kinetic diameter of HQD NPs is approximately 43.44 nm, and the PDI is 0.268, indicating that HQD molecules form well-dispersed nanoparticles in solution. The HQD metal complex maintains good dispersibility, and the nanoparticle size has increased. Cu HQD Fe and HQD Mn Nanoparticles with diameters of approximately 53.61 nm, 85.65 nm, and 67.31 nm were formed, respectively. The particle size measured by DLS was slightly larger than that measured by TEM, mainly due to the difference in sample preparation method and testing conditions: DLS measures the kinetic diameter in the hydrated state, while TEM observes the particle core size in the dry state.

[0122] also, Figure 8 Figure E shows the change in Zeta potential of the HQD molecule before and after coordination with metal ions. The Zeta potential of the HQD molecule is -45.63 mV. Cu HQD Fe and HQD Mn The potentials increased to -41.20 mV, -35.66 mV and -38.56 mV, respectively, indicating that it has good biocompatibility with cell membranes.

[0123] Test Example 7

[0124] The stability of the HQD molecules and their metal complexes prepared in Examples 1-4 under physiological conditions was tested. The test method was as follows: the HQD molecules and their metal complexes prepared in Examples 1-4 were dissolved in deionized water at pH 7.4 to prepare a sample solution with a concentration of 0.2 wt% (HQD solution, HQD...). Cu Solution, HQD Fe Solution, HQD Mn (Solution); The sample solution was placed in a 37 ℃ constant temperature incubator. 200 µL of the sample solution was injected into a quartz cuvette every day, and the changes in particle size and PDI were continuously monitored over 7 days using a dynamic light scattering (DLS) instrument.

[0125] Test results are as follows Figure 9 As shown, during the 7-day monitoring period, all nanoparticles (HQD NPs, HQD...) Cu NPs, HQD Fe NPs, HQD MnThe particle size of the NPs did not fluctuate significantly, and the PDI remained consistently within the range of 0.1–0.3. This result indicates that the nanoparticles formed by HQD and its metal complexes exhibit excellent in vitro stability under physiological conditions.

[0126] Test Example 8

[0127] The photothermal properties of the HQD molecules and their metal complexes prepared in Examples 1-4 were tested using the following methods:

[0128] (1) Concentration dependence test of photothermal properties: Solutions of HQD molecules and their metal complexes at different concentrations (50, 100 and 200 µg / mL) were prepared and added to 96-well plates. An 808 nm near-infrared laser (light power density: 1.0 W / cm²) was used for the test. 2 Laser irradiation distance: 15 cm, spot size: 1 cm 2 The sample was continuously irradiated for 8 minutes, and the temperature change was monitored in real time using an infrared thermal imager (Fotric 225s). The temperature was recorded every 30 seconds during the process.

[0129] (2) Dependence of photothermal properties on laser intensity: HQD molecules and their metal complex solutions (50 µg / mL) were added to a 96-well plate, and the laser power of the 808 nm near-infrared laser was set to 0.5, 1.0, and 1.5 W / cm², respectively. 2 The sample was continuously irradiated for 8 minutes, and the temperature change was monitored and recorded in real time using an infrared thermal imager.

[0130] (3) Photothermal stability test: HQD molecules and their metal complex solutions (50 µg / mL) were placed in 96-well plates and subjected to photothermal stability testing at 1.0 W / cm². 2 The sample was irradiated with an 808 nm laser for 8 min at a laser intensity, and then the solution was allowed to cool naturally to room temperature. This heating-cooling cycle was repeated four times, and the temperature change during the process was recorded using an infrared imager.

[0131] The photothermal conversion efficiency (η) is calculated based on the temperature-time change curves of the sample during laser irradiation heating and natural cooling. The calculation formula is as follows: Among them, T max T represents the highest temperature reached by the sample during laser irradiation. surr Q represents the ambient temperature. s The solvent contributes thermally to the system, I is the laser power density, and A is the laser power density. λ is the absorbance of the sample at 808 nm. hS is the heat dissipation coefficient, calculated from the natural cooling curve after laser shutdown, and its calculation formula is: m is the mass of the solution, C H2O The specific heat capacity of water, is the system time constant. The calculation formula is: Where t is the time during the cooling process, θ is the thermal driving constant, and T is the real-time temperature.

[0132] The test results show the dependence of photothermal performance on concentration and laser intensity as follows: Figure 10 As shown, from Figure 10 As can be seen from Figure A, the HQD metal complex exhibits significant and broad near-infrared absorption characteristics in the 650-900 nm range. From... Figure 10 As can be seen from B, all three HQD metal complexes can rapidly convert light energy into heat energy under near-infrared laser irradiation, causing a significant increase in solution temperature. Among them, HQD Fe It exhibits the most outstanding photothermal performance, with its solution temperature rapidly increasing from an initial 28.1 °C to 47.7 °C within 8 minutes, compared to HQD. Mn and HQD Cu Under the same conditions, the temperature increase was slightly lower, with the solution temperature rising to 45.0 ℃ and 43.1 ℃ respectively, but it still exhibited good photothermal conversion behavior. From Figure 10 As can be seen from C, further improving HQD Fe When the concentration was increased to 100 μg / mL or 200 μg / mL, the temperature rise of the system further increased under continuous irradiation with an 808 nm laser, indicating that its photothermal effect has a significant concentration dependence. Figure 10 As can be seen from D, at a lower power density (0.5 W / cm²), 2 HQD Fe The temperature of the solution (50 μg / mL) slowly increased from 28.2 °C to 39.5 °C within 8 min, exhibiting a mild and controllable photothermal response; while when the laser power density was increased to 1.5 W / cm², the temperature remained stable. 2 At the same concentration of HQD Fe The solution rapidly heated to 57.8 °C within the same irradiation time, indicating that the system can flexibly switch from gentle photothermal therapy to efficient thermal ablation by adjusting the laser power.

[0133] The results of the photothermal stability test are as follows Figure 11 As shown, from Figure 11 As can be seen from A, after four consecutive "laser on-off" heating and cooling cycles under 808 nm laser irradiation, the HQD Fe The maximum temperature and heating rate of the solution remained almost unchanged, indicating that the nanosystem possesses excellent structural stability and energy conversion reliability during repeated photothermal conversion. Based on HQD...Fe The temperature rise and fall curves of the solution are used to further calculate its photothermal conversion efficiency (η). Figure 11 As shown in Figure B, the results show HQD. Fe The η value is as high as 37.3%. In comparison, from Figure 11 As can be seen from C and D, HQD Mn Solution and HQD Cu The photothermal conversion efficiencies of the solutions were 32.6% and 30.3%, respectively. The η values ​​of HQD metal complexes were significantly higher than those of many commonly used inorganic photothermal reagents, such as Cu9S5 (25.7%), MoS2 (26.9%), and gold nanorods (21%), which fully highlights the significant advantages and application potential of HQD metal complex nanoparticles in the field of near-infrared photothermal therapy.

[0134] Test Example 9

[0135] The peroxidase-like (POD) catalytic performance of the HQD molecules and their metal complexes prepared in Examples 1-4 was tested using the following methods:

[0136] (1) Assay of basic peroxidase activity

[0137] A reaction system was constructed by adding 10 µL of 3,3',5,5'-tetramethylbenzidine (TMB) solution and 20 µL of H2O2 to 930 µL of 10 mM phosphate-buffered saline (PBS), using TMB as the chromogenic substrate. The solution was briefly vortexed to ensure thorough mixing. Subsequently, 40 µL of HQD molecules or their metal complex solution was added to initiate the catalytic reaction. The resulting mixed solution contained 0.2 mM TMB, 5 mM H2O2, and 0.2 mM HQD molecules or their metal complex. An illumination experimental group was set up using an 808 nm near-infrared laser (power density 1.0 W / cm²). 2 The oxidant was continuously irradiated for 8 minutes, and the characteristic absorption peak of the oxidation product oxTMB was monitored in real time at a wavelength of 650 nm using a UV-Vis spectrophotometer to reflect the peroxidase-like catalytic activity of HQD molecules and their HQD metal complexes.

[0138] (2) Effect of pH on catalytic activity

[0139] 10 µL of TMB solution and 20 µL of H2O2 were added to 930 µL of 10 mM PBS buffer at pH 5.0, 6.5, and 7.4, respectively, and mixed thoroughly. Then, 40 µL of HQD metal complex solution was added to initiate the reaction. The resulting mixed solutions contained 0.2 mM TMB, 5 mM H2O2, and 0.2 mM HQD metal complex. After 2 min, the characteristic absorption peak of oxTMB was measured at 650 nm, and the absorbance value was recorded.

[0140] (3) Effect of temperature on catalytic activity

[0141] 10 µL of TMB solution and 20 µL of H2O2 were added to 10 mM PBS buffer. The reaction system was placed under constant temperature conditions of 25 °C, 37 °C, and 50 °C, respectively. After thorough mixing, 40 µL of HQD metal complex solution was added to initiate the reaction. The resulting mixed solution contained 0.2 mM TMB, 5 mM H2O2, and 0.2 mM HQD metal complex. After 2 min, the characteristic absorption peak of oxTMB was detected at 650 nm and the absorbance was recorded.

[0142] Test results are as follows Figure 12 As shown, from Figure 12 As can be seen in Figure A, the color change of the solution when TMB is oxidized to oxTMB is observed. From... Figure 12 As shown in Figure B, no significant absorption signal was observed at 650 nm in either the system containing only HQD + TMB + H2O2 or the blank control group without the HQD metal complex, and the solution color remained unchanged. This indicates that TMB is difficult to be effectively oxidized in the absence of metal active sites. In contrast, when HQD is introduced into the TMB + H2O2 system... Cu Subsequently, the solution rapidly turned a distinct blue color and exhibited a strong characteristic absorption peak at 650 nm, clearly indicating the formation of oxidized TMB (oxTMB). This result fully confirms that HQD... Cu It can act as a highly efficient peroxidase-like catalyst, significantly promoting the oxidation reaction of TMB. Under the same experimental conditions, HQD... Mn and HQD Fe It can also trigger the conversion of TMB to oxTMB, exhibiting certain peroxidase-like catalytic activity. However, its corresponding absorbance intensity is significantly lower than that of HQD. Cu This indicates that there are significant differences in catalytic efficiency among different metal centers in the HQD coordination environment, with Cu-based complexes exhibiting superior catalytic performance. This difference may be closely related to the redox potential, electron transfer ability, and interaction mechanisms of different metal ions with H₂O₂. Figure 12 As can be seen from C, HQD Cu It exhibits significantly enhanced catalytic activity under weakly acidic conditions (e.g., pH=5.0), while its catalytic efficiency decreases markedly in a near-physiological neutral environment (pH=7.4). This phenomenon is consistent with the characteristic of natural peroxidases exhibiting higher activity in acidic microenvironments, indicating that HQD... Cu It exhibits certain biomimetic catalytic behavior. From... Figure 12 As can be seen from Figure D, compared with room temperature (25 °C), the absorbance of the system at 650 nm significantly increased with increasing reaction temperature to 37 °C and 50 °C, indicating that heating can effectively accelerate the catalytic oxidation rate of TMB. This result suggests that the local temperature increase caused by the photothermal effect may further amplify the POD-like activity of HQD metal complexes, providing a favorable basis for their application in photothermal synergistic catalysis or tumor microenvironment-responsive therapy.

[0143] Test Case 10

[0144] Free radical scavenging experiments were used to study HQD. Cu The types of reactive oxygen species (ROS) generated during the catalytic oxidation of TMB were determined, and superoxide dismutase (SOD) was selected as the superoxide anion free radical (O2·) - tert-butanol (TBA), a specific scavenger of HQD, and an effective scavenger of hydroxyl radicals (·OH), were introduced into the HQD-containing sample in Test Example 9. Cu In the TMB + H2O2 catalytic system, the absorbance of the oxidation product oxTMB as a function of reaction time was monitored in real time at a wavelength of 650 nm using a UV-Vis spectrophotometer. The formation of oxTMB was quantitatively analyzed, and the type of reactive oxygen species in the system was inferred based on the effect of the scavenger on the reaction rate.

[0145] The measurement results are as follows Figure 13 As shown, compared with the blank system without scavengers, the absorbance of the system at 650 nm decreased significantly after the addition of SOD (100 μM) or TBA (150 mM), and the formation rate of oxTMB was significantly slowed down. This result indicates that O2· - Both ·OH and HQD are involved in HQD. Cu The POD-like catalytic reaction mediated by this type of peroxidase is a key active intermediate in the catalytic process of this type of peroxidase.

[0146] Test Example 11

[0147] The glutathione peroxidase (GPx) catalytic performance of the HQD metal complexes prepared in Examples 2-4 was tested. The test method was as follows: HQD metal complex solutions of different concentrations (0, 0.05, 0.10, 0.15 and 0.20 mM) were mixed with 1 mM reduced glutathione (GSH) solution at a volume ratio of 1:1 and incubated together at constant temperatures of 25 ℃, 37 ℃ and 60 ℃, respectively. At the preset time point (8 min), an equal amount of the reaction system sample was taken and reacted with 10 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). DTNB, as a specific chromogenic probe, can be reduced by the thiol group in GSH molecules to generate 2-nitro-5-thiobenzoic acid (TNB). This product has a distinct characteristic absorption peak at 412 nm. Therefore, by measuring the absorbance at 412 nm using a UV-Vis spectrophotometer, the content of residual GSH in the system can be quantitatively analyzed, thereby evaluating the GSH oxidation kinetics mediated by HQD metal complexes.

[0148] Test results are as follows Figure 14 As shown, from Figure 14 As can be seen from A and B, adding HQD to a solution containing DTNB and GSH... Cu Subsequently, as the reaction time increased, the absorbance at 412 nm gradually decreased, and the solution color gradually changed from yellow to light yellow, indicating that HQD... Cu It can effectively catalyze the oxidation of GSH, thereby consuming the GSH in the system. In contrast, HQD... Fe and HQD Mn Under the same conditions, its catalytic oxidation ability for GSH is significantly weaker, showing a much lower level than that for HQD. Cu The GPx-like catalytic activity. Furthermore, HQD... Cu The GPx-like catalytic activity also exhibited a significant concentration- and temperature-dependent characteristic. Figure 14 As can be seen from C and D, with HQD Cu Increasing the concentration from 0.05 mM to 0.2 mM and the reaction temperature from 25 °C to 50 °C significantly accelerated the GSH oxidation rate, further indicating that HQD… Cu It exhibits superior GPx-like catalytic performance under higher catalyst concentrations and heating conditions.

[0149] Test Example 12

[0150] The DNA binding and cleavage activities of the HQD molecules and their metal complexes prepared in Examples 1-4 were tested. First, buffer solutions were prepared: 10 mM Tris-HCl buffer (10 mM, NaCl, pH=7.4) for dissolving calf thymus DNA (ct-DNA): 1 mL of 1 M Tris-HCl stock solution was measured, and 58.40 mg of NaCl was weighed. The solution was dissolved in an appropriate amount of ultrapure water, and the pH was adjusted to 7.4 using a pH meter. The final volume was brought to 100 mL, stirred thoroughly, filtered, sterilized, and stored at 4 °C. Tris-boric acid (TBE) electrophoresis buffer was prepared: 21.5620 g of tris(hydroxymethyl)aminomethane, 11.00574 g of boric acid, and 1.412 g of EDTA were dissolved in ultrapure water, and the pH was adjusted to 8.0 to prepare 200 mL of 10×TBE buffer solution. This solution was then diluted tenfold for use as the electrophoresis buffer. Next, the ct-DNA solution was prepared and its concentration determined: 3 mg of ct-DNA was weighed and dissolved overnight in Tris-HCl buffer at 4 °C to form a homogeneous solution. A small amount of the stock solution was diluted a certain factor, and its absorbance at 260 nm was measured. The absorbance A was also observed. 260 / A 280 The ratio of α to β is checked to ensure the purity meets requirements. The concentration of ct-DNA and the molar absorptivity ε of the double-stranded DNA are calculated using Beer-Lambert's law. 260 = 6600 L·mol -1 ·cm -1 The methods for testing DNA binding and cleavage activity are as follows:

[0151] (1) DNA binding studies based on ultraviolet-visible absorption spectroscopy: HQD Cu HQD Fe and HQD Mn A series of samples with concentration gradients of 10, 25, 40, 50, 60, 70, 80, and 100 μM were prepared by sequentially adding ct-DNA to a fixed solution concentration of 50 μM. All mixed solutions were incubated at room temperature for 40 minutes to reach binding equilibrium before spectral analysis. UV-Vis absorption spectra were performed using the corresponding concentrations of ct-DNA solution as a reference to subtract the background absorption of the DNA itself. The binding characteristics between HQDCu and ct-DNA were investigated by analyzing the spectral changes.

[0152] (2) DNA binding study based on fluorescence spectroscopy: To investigate the ability of HQD metal complexes to competitively displace DNA and bind fluorescent dyes, a competitive fluorescence titration method was used. First, ct-DNA solution (100 μM, dissolved in Tris-HCl buffer, pH=7.4) was incubated with either ethidium bromide (EB) or Hoechst 33342 (Hoechst) (20 μM) to form stable fluorescently labeled DNA complexes. Subsequently, HQD metal complexes (concentration range 0-100 μM) were added to the system in a gradient and incubated at room temperature until binding equilibrium was reached. The binding was then measured at the corresponding excitation wavelengths (EB:λ). ex = 510 nm, λ em =605 nm; Hoechst 33342:λ ex = 350 nm, λ em Fluorescence spectra were recorded at 485 nm and based on the modified Stern-Volmer equation F0 / F=1+K. SV [Q] The fluorescence quenching effect was analyzed. Binding affinity was quantitatively evaluated by fitting the relationship between the fluorescence intensity ratio F0 / F and the complex concentration, where F0 and F represent the fluorescence intensity before and after complex addition, respectively, and K... SV [Q] represents the Stern-Volmer quenching constant of the HQD metal complex for Hoechst / EB in ct-DNA solution, where [Q] is the final concentration of the complex.

[0153] (3) Agarose gel electrophoresis experiment of plasmid pBR322 DNA: The DNA cleavage activity of HQD metal complexes was evaluated using supercoiled pBR322 DNA. In different HQD metal complex solutions (0.5 mM), pBR322 DNA (0.5 g / L, 0.5 μL) was added in the presence of reducing agent H2O2 (0.05 mM) and diluted to 15 μL with Tris-HCl buffer. After mixing thoroughly, the mixture was reacted in a constant temperature water bath at 37 ℃ for 60 min. 10 μL of the reaction solution was added to 1 μL of loading buffer, pipetted thoroughly, and then added to a 0.8% agarose gel containing 0.01% GoldView nucleic acid dye. Electrophoresis analysis was performed in TBE electrophoresis buffer (Sub-Cell® GT, 80 V, 80 min). The gel was then developed and analyzed using UV light.

[0154] HQD Cu The UV-Vis absorption spectra of the solution (50 μM) and its mixtures with different concentrations of DNA (0-100 μM) are shown in the figure. Figure 15 As shown, the comparison between blank ct-DNA and HQD alone...Cu The absorption spectrum of ct-DNA shows a characteristic absorption peak at 260 nm, originating from π-π* base transitions; while HQD... Cu It exhibits a ligand-related absorption band at 272 nm. With the gradual addition of ct-DNA to HQD... Cu In solution, significant spectral changes were observed in the 250-280 nm wavelength range (corresponding to the absorption region of the complex ligands themselves), indicating a significant intermolecular interaction between the two molecules, possibly involving perturbations in electronic structure or changes in packing state. Further analysis of different HQDs... Cu The effect of the HQD / DNA molar ratio on the spectrum was observed to be that when the ratio was in the range of 0–1.0, the absorption intensity at 272 nm gradually increased, accompanied by a blue shift of approximately 2 nm (to 270 nm). This hyperchromatic and blue shift phenomenon was attributed to HQD. Cu HQD binds to DNA phosphate groups via electrostatic interactions and van der Waals forces through groove binding. As DNA concentration continues to increase, HQD... Cu When the HQD / DNA molar ratio exceeds 1:1, the spectral behavior undergoes a significant change: the absorption band intensity at 270 nm gradually decreases and exhibits a red shift, eventually stabilizing at approximately 273 nm. This hypochromic and red-shift effect is a typical characteristic of insertion binding, primarily driven by the π-π stacking interaction between the aromatic chromophore of the complex and the DNA base pairs. In summary, the spectroscopic titration results indicate that HQD... Cu The interaction with DNA exhibits a concentration-dependent bimodal characteristic: groove binding predominates at low DNA concentrations, while intercalation binding switches to intercalation binding at high DNA concentrations. This reveals that the complex can bind to DNA through different non-covalent mechanisms, providing important evidence for subsequent research on its biological activity and mechanism of action.

[0155] The fluorescence spectra of Hoechst / EB binding to ct-DNA in the presence and absence of HQD metal complexes are as follows: Figure 16 and 17 As shown, free Hoechst and EB exhibit weak fluorescence signals, but their fluorescence emission significantly increases after binding to ct-DNA, displaying characteristic peaks at 485 nm and 605 nm, respectively. With the gradual addition of the HQD metal complex to the aforementioned DNA-probe complex system, the fluorescence intensity of both probes decreases in a concentration-dependent manner, i.e., fluorescence quenching occurs. This phenomenon directly indicates that the HQD metal complex can competitively replace Hoechst or EB pre-bound to DNA, thus confirming that the complex interacts with DNA through both groove binding (competing with Hoechst) and intercalation binding (competing with EB).

[0156] Stern-Volmer analysis was performed on the fluorescence titration data, and K was calculated. SV The display shows that HQD Cu Through the trench bonding mode (characterized by Hoechst displacement experiments, K) SV = 1.31 × 10 4 M -1 The affinity of ) was significantly higher than that of its binding via intercalation (characterized by EB substitution experiments, Ksv = 3.43 × 10 3 M -1 This dual-mode combination feature in HQD Fe and HQD Mn This has also been verified in HQD. Fe and HQD Mn Similarly exhibiting groove bonding (K) SV They are 1.08 × 10 4 M -1 and 1.01 × 10 4 M -1 ) and intercalation combination (K SV They are 2.40 × 10 3 M -1 and 2.54 × 10 3 M -1 Both exhibit stronger groove-bonding affinity.

[0157] The results of the pBR322 DNA cleavage activity assay for HQD metal complexes are as follows: Figure 18 As shown in Figure A, under physiological conditions (10 mM Tris-HCl buffer, pH=7.4, 37 ℃), pBR322 in the control group mainly maintained a supercoiled morphology (Form I, lane 1). In the system containing HQD ligand and H2O2 (lane 2) and in the system without H2O2, pBR322 mainly maintained a supercoiled morphology. Cu Similar electrophoretic bands were observed in the system (lane 3), indicating that no significant DNA cleavage was detected under these conditions. In contrast, when H2O2 was added as a reducing agent, 0.5 mM HQD... Cu (Lane 6) can induce almost complete conversion of Form I into nicked circular DNA (Form II) and linear DNA (Form III), exhibiting significant DNA cleavage activity. Furthermore, HQD... Cu The cleavage activity gradually increases with increasing concentration, such as Figure 18 (As shown in B). In contrast, HQD Mn (Lane 4) and HQD Fe The cleavage efficiency in lane 5 was significantly weaker, with only a small amount of DNA being converted to Form II. To further elucidate HQD...Cu The DNA cleavage mechanism, respectively in HQD Cu The H2O2 system incorporates the ·OH scavenger TBA (lane 7) and O2· - Scavenger SOD (lane 8). Both scavengers significantly inhibited DNA cleavage, accompanied by a marked recovery of form I. These results indicate that HQD... Cu DNA cleavage activity occurs via a reactive oxygen species-mediated oxidation pathway, involving ·OH and O2· - The formation of species.

[0158] Test Example 13

[0159] Using HQD Cu Nanoparticles were used as drug carriers to construct a drug-loaded nanosystem (HQDCuD) containing doxorubicin (DOX), and in vitro release tests were conducted. The test method involved preparing a series of DOX solutions at concentrations (5, 12.5, 50, 100, and 200 μg / mL), measuring their characteristic absorbance at 480 nm using a UV-Vis spectrophotometer, and plotting a standard curve for the DOX solutions. The encapsulation efficiency of the unencapsulated DOX dialysate was measured at 480 nm. The encapsulation efficiency and drug loading of DOX were calculated using the following formulas: Encapsulation efficiency (%) = Mass of loaded DOX / (Mass of carrier + Mass of loaded DOX); Drug loading (%) = Mass of loaded DOX / Mass of added DOX. In vitro release assays were performed using dialysis. 1 mL of HQDCuD solution was added to a dialysis bag (molecular weight cutoff 3.5 kDa), sealed, and then the bag was immersed in a centrifuge tube containing 12 mL of PBS solution (pH 7.4, 6.5, and 5.0). The centrifuge tubes were placed in a shaker (37 °C, 100 rpm). At predetermined time points, 1.5 mL of solution was removed and replaced with the same volume of freshly prepared PBS solution. The absorbance of the solution at 480 nm was measured using UV-Vis spectroscopy, and the cumulative release was calculated using the following formula: Where Er is the cumulative release of DOX, and V e C is the volume of the replacement medium. i Let V0 be the concentration of DOX during the i-th sampling, and C be the initial total volume of the release medium. n The concentration of DOX in the sample is n, where n is the number of times the replacement medium is used, and m is the number of times the replacement medium is used. drug The mass of DOX in the drug-loaded nanoparticles used for release.

[0160] Test results are as follows Figure 19 As shown, the HQDCuD solution after dialysis was tested using a UV-Vis spectrophotometer. Figure 19As can be seen from Figure A, the HQDCuD solution exhibits a characteristic absorption peak for DOX at 480 nm, indicating that DOX was successfully loaded. This can be further confirmed by plotting... Figure 19 Quantitative analysis of the standard curve of DOX solution in HQD showed a drug loading of up to 53.02% and an encapsulation efficiency of 34.64%, indicating that HQD... Cu Nanoscale systems can efficiently load DOX molecules. After loading DOX, HQD... Cu Through synergistic self-assembly with DOX, a denser nanoparticle structure is formed, increasing the particle size from 52 nm to 89 nm. Figure 19 As shown in B. Meanwhile, from... Figure 19 As shown in Figure C, the Zeta potential of HQDCuD increased from -41.2 mV to -36.67 mV. This change may be due to the introduction of positively charged amino groups in the DOX molecule, which partially neutralizes the surface charge of the nanoparticles, helping to maintain the colloidal stability of the system and improve its biocompatibility. Further investigation into the in vitro drug release behavior of HQDCuD revealed that the system exhibits a significant pH-responsive release characteristic. Figure 19 As can be seen from the results, under simulated normal physiological conditions (pH=7.4), the release process of DOX is relatively slow, with a cumulative release rate of only 16.56% within 34 hours, which is beneficial for reducing non-specific drug release in normal tissues. In contrast, under weakly acidic conditions (pH=6.5) and stronger acidic conditions (pH=5.0), the cumulative release rate of DOX increased to 24.8% and 44.7%, respectively, indicating that the HQDCuD assembly structure can undergo partial dissociation in the acidic tumor microenvironment, thereby promoting drug release. Furthermore, given HQD... Cu The excellent near-infrared photothermal conversion performance of the nanosystem, when combined with 808 nm near-infrared laser as an external stimulus, significantly improved the release rate of DOX in HQDCuD, achieving a cumulative release rate of 47.34% under intermittent laser irradiation. Figure 19 As shown in Figure F, this enhanced release behavior is mainly attributed to the local temperature increase caused by the photothermal effect, which accelerates the relaxation of the nanostructure and the diffusion process of drug molecules. In summary, the HQDCuD nanosystem can achieve precise and controllable DOX release under the dual stimulation of tumor-associated acidic microenvironment and near-infrared laser irradiation, providing a promising nanodelivery platform for photothermal-chemotherapy synergistic antitumor therapy.

[0161] Test Example 14

[0162] The cytotoxicity of HQD metal complex nanoparticles to human umbilical vein endothelial cells (HUVECs) was detected using the Cell Counting Kit-8 (CCK-8) assay to verify the cell compatibility of the material. The test method was as follows: healthy HUVECs were seeded into 96-well plates at a density of 8 × 10³ cells / well and cultured in an incubator for 12 h. Then, HQD nanoparticles were added to each well. Cu HQD Fe HQD Mn The solution was incubated with cells at different concentrations (0, 10, 25, 50, 75, 100, 150, and 200 μg / mL) for 24 h. Untreated cells served as the control group, and cell-free culture medium served as the blank group. On the second day, the liquid in the well plate was aspirated and replaced with 100 μL of culture medium containing 10% CCK-8. The plates were then incubated in the dark for 30 min, and the absorbance at 450 nm was measured using a Synergy NEO microplate reader.

[0163] Test results are as follows Figure 20 As shown, HUVECs are subjected to HQD Cu HQD Fe HQD Mn After 24 h of treatment with nanoparticles, high cell viability was observed across the concentration range of 0-200 μg / mL. With increasing nanoparticle concentration, cell activity in all three treatment groups showed a slight concentration-dependent decrease; however, at low concentrations (≤ 25 μg / mL), cell viability remained above 90%, showing no significant difference compared to the control group (0 μg / mL). Even at higher concentrations (100-200 μg / mL), HUVECs viability remained within the range of approximately 75-85%, with no significant cytotoxicity observed. These results indicate that HQD metal complex nanoparticles exhibit good cell compatibility with normal HUVECs over a wide concentration range without inducing significant cytotoxic reactions, suggesting a sound safety basis for this system in biomedical applications.

[0164] Test Example 15

[0165] The uptake efficiency and intracellular distribution characteristics of a drug-loaded nanosystem (HQDCuD) containing doxorubicin (DOX) by 4T1 cells were tested to evaluate its endocytic behavior. The test method was as follows: 4T1 cells were loaded with 1.0 × 10⁻⁶ cells of doxorubicin (DOX) at a concentration of 1.0 × 10⁻⁶ cells / cells. 5Cells were seeded per well in confocal culture dishes and incubated at a constant temperature for 12 h. Culture medium containing DOX solution (3.46 μg / mL) and HQDCuD solution (100 μg / mL) were added, and the cells were incubated at 37 ℃ for 3 h and 6 h, respectively. Cells were washed three times with PBS and stained with Hoechst 33342 (10 μg / mL) in the dark for 15 min. After a second PBS wash, cell uptake was observed using a laser scanning confocal microscope (FV 1200). Cells were then prepared into six-well plates using the same method. After incubation, cells were collected, stained, and their uptake efficiency of HQDCuD and DOX was quantitatively analyzed using flow cytometry (FACSCalibur). (DOX: E x = 480 nm, E m = 570 nm; Hoechst33342: E x = 350 nm, E m = 460 nm).

[0166] Test results are as follows Figure 21 As shown, from Figure 21 As shown in Figure A, the Hoechst 33342 channel represents nuclear staining, while the DOX channel indicates DOX uptake. A stronger red fluorescence signal indicates greater DOX uptake by the cell. Compared to DOX alone, 4T1 cells treated with HQDCuD exhibited a more significant red fluorescence signal. DOX showed only weak intracellular fluorescence at both 3 h and 6 h incubation times, indicating limited efficiency in transmembrane entry and intracellular accumulation. In contrast, HQDCuD showed detectable red fluorescence at 3 h incubation, although the intensity was relatively weak, suggesting the beginning of endocytosis. When the incubation time was extended to 6 h, the red fluorescence significantly increased and became widely distributed within the cell, showing a clear time-dependent uptake characteristic. Flow cytometry analysis results are as follows... Figure 21 As shown in Figures B and C, this trend was further quantitatively confirmed: the mean fluorescence intensity of cells in the HQDCuD-treated group was significantly higher than that in the free DOX group at both 3 h and 6 h, and approximately 1.6 times that of free DOX at 6 h, indicating that the nanodelivery system significantly promoted intracellular accumulation of DOX. These results demonstrate that HQDCuD nanoparticles can significantly improve the uptake and internalization efficiency of DOX in 4T1 cells through a time-dependent endocytosis process, effectively improving the intracellular delivery behavior of the drug and providing important cellular evidence for enhancing the efficacy of subsequent antitumor therapy.

[0167] Test Example 16

[0168] Intracellular ROS levels were measured using HQD molecules and their metal complexes, as well as HQDCuD, prepared in Examples 1-4. The potential of HQD molecules, their metal complexes, and HQDCuD solutions to exacerbate intracellular oxidative stress was assessed, and the effect of photothermal stimulation on ROS generation behavior was further investigated. The testing method involved preparing 4T1 cells at 1.2 × 10⁻⁶ ppm. 5 Seeds were inoculated into confocal dishes per well and incubated in an incubator for 12 h. The culture medium was then discarded, and HQD and HQD were added to the dishes respectively. Cu HQD Fe HQD Mn Co-cultured with HQDCuD (100 μg / mL) solution for 6 h. During this period, 1 W / cm² was used. 2 Cells were continuously irradiated with an 808 nm laser for 8 min. After culture, the culture medium was discarded, and cell debris was washed away with PBS. 1 mL of 10 μM 2',7'-dihydrodichlorofluorescein diacetate (DCFH-DA) probe solution was added. DCFH-DA is oxidized by intracellular ROS to generate green fluorescent 2',7'-dichlorofluorescein (DCF). The cells were stained in an incubator in the dark for 30 min. After staining, the cells were washed three times with pre-cooled PBS, and 200 μL of PBS solution was added. The fluorescence signal of intracellular ROS was captured using a confocal microscope. Under the same conditions, flow cytometry was used to quantitatively analyze ROS levels (E). x = 480 nm, E m = 525 nm).

[0169] Test results are as follows Figure 22 As shown, from Figure 22 As shown in Figure A, under conditions without laser irradiation, the blank control group (Control, PBS group) and the free HQD treatment group only exhibited weak green fluorescence signals, indicating low intracellular ROS levels and that the cells were in a normal redox balance state. In contrast, the green fluorescence in cells was significantly enhanced after treatment with HQD metal complexes, indicating that metal coordination significantly improved ROS generation capacity. Specifically, HQD... Cu The fluorescence intensity was most significant in the treatment group, while HQD... Fe With HQD Mn The treatment group showed a moderate enhancement trend, indicating significant differences in the catalytic ROS generation catalysis of different metal centers. Figure 22 As can be seen from B, the quantitative analysis results of flow cytometry are consistent with the results of confocal imaging, further confirming HQD. Cu It exhibits the strongest ability to promote ROS generation. The fluorescence intensity of the HQDCuD-treated group further increased, suggesting that the introduction of DOX can interact with HQD. CuThe catalytic activity synergistically amplifies the level of intracellular oxidative stress. Furthermore, under 808 nm laser irradiation (1 W / cm²), 2 After 8 min, the ROS fluorescence signal in all treatment groups was significantly enhanced, indicating that the photothermal effect can effectively promote the catalytic reaction kinetics, thereby continuously amplifying ROS generation. This multi-synergistic mechanism provides important cellular evidence for the efficient amplification of oxidative stress by HQDCuD in tumor therapy.

[0170] Test Example 17

[0171] Intracellular GSH levels of HQD molecules and their metal complexes, as well as HQDCuD, prepared in Examples 1-4 were measured using a glutathione assay kit (ThiolTrace). TM The Violet assay was used to investigate the ability of different sample solutions to simulate glutathione oxidase activity and consume GSH. The assay method was as follows: prepared 4T1 cells were incubated at 1.2 × 10⁻⁶ cells per cubic meter of water. 5 Seeds were inoculated into confocal dishes per well and incubated in an incubator for 12 h. The culture medium was then discarded, and HQD and HQD were added to the dishes respectively. Cu HQD Fe HQD Mn Co-cultured with HQDCuD (100 μg / mL) solution for 6 h. During this period, 1 W / cm² was used. 2 Cells were continuously irradiated with an 808 nm laser for 8 min. After culture, the culture medium was discarded, and cell debris was washed away with PBS. 1 mL of 20 μM Thiol Trace was added. TM Violet probe solution was used for staining in an incubator in the dark for 30 min. After staining, the cells were washed three times with pre-cooled PBS, and 200 μL of PBS solution was added. The intracellular fluorescence signal (E) was then captured using a confocal microscope. x = 405 nm, E m = 525 nm).

[0172] Test results are as follows Figure 23 As shown, the untreated control cells exhibited strong green fluorescence, reflecting a high basal GSH concentration and undisturbed redox homeostasis. HQD Cu The significantly weakened fluorescence signal in the HQDCuD-treated group indicates that these two nanomaterials can effectively catalyze the consumption of intracellular GSH and disrupt the antioxidant defense system of tumor cells. Irradiation with an 808 nm near-infrared laser (1 W / cm²) further confirmed this effect. 2 After 8 minutes, HQD CuThe fluorescence intensity of the +laser and HQDCuD +laser groups further decreased. This phenomenon is consistent with the mechanism of enhanced catalytic activity of materials under photothermal heating conditions: local temperature rise not only accelerates the electron transfer process but may also promote the redox reaction between Cu active sites and GSH, thereby depleting the GSH library more efficiently and continuously. In contrast, HQD... Fe With HQD Mn Although some degree of fluorescence attenuation was also observed in the treatment group, its signal intensity was significantly higher than that of HQD. Cu The results indicate that the two materials have relatively limited ability to consume GSH, and their catalytic activity and oxidative stress-induced efficiency are lower than those of the copper-based system. These results highlight the limitations of HQD. Cu It exhibits excellent catalytic activity in both ROS generation and GSH consumption. The DOX-loaded HQDCuD combines this advantage with chemotherapy, effectively enhancing the induction efficiency of oxidative stress in tumor cells and achieving synergistic effects between catalytic therapy and chemotherapy.

[0173] Test Example 18

[0174] The HQD molecules and their metal complexes, as well as HQDCuD, prepared in Examples 1-4 were tested for in vitro antitumor activity. Their cytotoxicity against 4T1 cells was evaluated using a CCK-8 assay system. The assay method was as follows: 8 × 10⁶ prepared 4T1 cells were cultured in each well. 3 100 μL of cell suspension was seeded into 96-well plates and cultured overnight. The old culture medium was then discarded, and 100 μL of different concentrations of DOX (0, 0.35, 0.87, 1.73, 2.6, 3.46, 5.19, and 6.92 μg / mL), HQDCuD, HQD, and HQD2 were added. Cu HQD Fe and HQD Mn Cells were co-cultured with culture media solutions of (0, 10, 25, 50, 75, 100, 150, and 200 μg / mL) for 24 h. Untreated cells served as the control group, and cell-free culture media served as the blank group. The concentration gradients and cell compatibility assays were consistent. Cell viability was measured using a CCK-8 assay kit on the second day. Cell viability under laser irradiation was also investigated; for cells placed for 5 h, 1.0 W / cm² laser was used. 2 Cells were irradiated with an 808 nm laser for 7 min, followed by 19 h of culture, during which the concentration of HQD and HQD metal complex solutions was maintained at 100 μg / mL. Cell viability was then measured using the same method.

[0175] Test results are as follows Figure 24 As shown, from Figure 24As shown in Figure A, HQD metal complex nanoparticles exhibited significant anti-proliferative effects against 4T1 cells, showing a clear concentration-dependent inhibitory trend. Compared with HQD ligands alone, all HQD metal complex nanoparticles showed stronger cytotoxicity, indicating that the introduction of metal ions significantly enhanced their antitumor activity. Among them, HQD... Cu Nanoparticles exhibited the most pronounced inhibitory effect: at a concentration of 100 μg / mL, 4T1 cell viability was reduced to 40.53%; when the concentration was further increased to 200 μg / mL, cell viability plummeted to only 7.9%. In contrast, HQD... Fe With HQD Mn The inhibitory effect of nanoparticles on 4T1 cells was relatively mild. This difference may be related to HQD. Cu The stronger reactive oxygen species (ROS) generation capacity and more efficient DNA damage induction capacity of nanoparticles are closely related. From Figure 24 As shown in Figure B, under the same DOX dosage, HQDCuD exhibited stronger anti-proliferative activity against 4T1 cells compared to DOX, indicating that the nanodelivery system can effectively increase intracellular drug accumulation and enhance its therapeutic effect. Figure 24 As can be seen from C, under 808 nm near-infrared laser irradiation conditions, after HQD Cu The viability of 4T1 cells treated with 100 μg / mL and HQDCuD (100 μg / mL) was further reduced to 20.69% and 11.60%, respectively. These results fully demonstrate that photothermal therapy can produce a significant synergistic effect with chemotherapy and enzyme-like catalytic therapy, thereby achieving a multimodal synergistic enhancement of anti-tumor therapeutic effects.

[0176] Test Example 19

[0177] The cytotoxicity of the HQD metal complexes and HQDCuD prepared in Examples 2-4 against tumor cells was tested, and their cytotoxicity in 4T1 cells was evaluated using a live / dead cell double staining method. The test method was as follows: prepared 4T1 cells were inoculated at 1.5 × 10⁻⁶ cells per cell line. 5 Inoculate each well with HQD and seed it into a confocal dish. Incubate for 24 h in an incubator, discard the culture medium, and add HQD to each dish. Cu HQD Fe HQD Mn Co-cultured with HQDCuD (100 μg / mL) solution for 24 h. During this period, 1 W / cm² was used. 2Cells were continuously irradiated with an 808 nm laser for 8 min. After culture, the culture medium was discarded, and cell debris was washed away with PBS. A standardized Calcein-AM / propidium iodide (PI) staining system was used, with 1 µL of Calcein-AM (4 mM) and 3 µL of PI (1.5 mM) double staining solution added, and staining was performed in the dark for 15 min. In viable cells, esterases can convert non-fluorescent Calcein-AM into green fluorescent Calcein, while damaged or dead cells are labeled with PI dye due to increased membrane permeability, showing red fluorescence, thus effectively distinguishing between cell viability and death. After staining, the cells were washed three times with pre-cooled PBS, and 200 μL of PBS solution was added. The intracellular fluorescence signal (Caicein-AM: E) was captured using a confocal microscope. x = 490 nm, E m = 515 nm; PI:E x = 488 nm, E m = 630 nm).

[0178] Test results are as follows Figure 25 As shown, the untreated control cells exhibited uniform green fluorescence with almost no detectable red fluorescence signal, indicating good cell condition and high viability. In contrast, HQD... Fe Only a small amount of scattered red fluorescence was observed in the treatment group, indicating that it induced death in only a small number of cells, and the overall cytotoxicity was weak. HQD Mn The treatment group showed a significant increase in red fluorescence signal and a decrease in green fluorescence, reflecting a moderate killing effect on 4T1 cells. In HQD... Cu In the treatment group, the significantly enhanced red fluorescence and the drastically reduced green fluorescence indicated a large number of cell deaths, and its cytotoxicity was the most prominent among all HQD metal complex nanoparticle treatments. Furthermore, the HQDCuD treatment group exhibited a higher density of red fluorescence signal, indicating that the introduction of DOX further amplified the killing effect on tumor cells, consistent with its synergistic chemotherapy effect.

[0179] HQD after irradiation with 808 nm near-infrared laser Fe + laser, HQD Mn + laser and HQD Cu In the laser-treated group, the red fluorescence signal was further enhanced, indicating that the photothermal effect can promote cell death to some extent. Figure 26As shown in the figure, the HQDCuD + laser treatment group showed almost no green fluorescence, only strong and widespread red fluorescence, indicating that cell viability was reduced to a minimum. In summary, the above results clearly demonstrate that HQD... Cu The synergistic effect of nanoparticles with the chemotherapy drug DOX and photothermal therapy can significantly enhance the killing effect on tumor cells, verifying the significant advantages of this multimodal synergistic treatment strategy in tumor treatment.

[0180] Test Case 20

[0181] The apoptosis level of 4T1 cells after co-incubation with HQD metal complexes prepared in Examples 2-4 and HQDCuD was assessed using Annexin V-FITC / PI double staining flow cytometry. The test method was as follows: the prepared 4T1 cells were incubated at 1.5 × 10⁻⁶ cells per cell line. 5 Inoculate each well of the culture medium into a six-well plate and incubate for 24 hours. Discard the culture medium and add HQD to each well. Fe HQD Mn HQD Cu Co-cultured with HQDCuD (100 μg / mL) solution for 24 h. During this period, 1 W / cm² was used. 2 Cells were continuously irradiated with an 808 nm laser for 8 min. After culture, the culture medium was collected, cell debris was washed away with PBS, and cells were digested with trypsin digestion solution. Cells were collected by centrifugation at 1000 rpm for 5 min, washed with pre-cooled PBS, and resuspended in 200 μL Annexin V-FITC binding buffer. 4 μL Annexin V-FITC and 8 μL PI were added and incubated in the dark for 15 min. The apoptosis induced in each group was quantitatively analyzed by flow cytometry. In this method, Annexin V-FITC specifically recognizes and binds to phosphatidylserine (PS) residues that turn outward during apoptosis, thereby labeling apoptotic cells; propidium iodide (PI) can only enter cells that have lost membrane integrity and is used to indicate necrotic populations. Through two-parameter analysis, the cells were divided into four subpopulations: Annexin V⁻ / PI⁻ (LL, live cells), Annexin V⁺ / PI⁻ (LR, early apoptotic cells), Annexin V⁺ / PI⁺ (UR, late apoptotic / necrotic cells), and Annexin V⁻ / PI⁺ (UL, necrotic cells).

[0182] Test results are as follows Figure 27As shown, the untreated control groups (Control, PBS group, and PBS + laser group) exhibited only extremely low levels of apoptosis (2.25% and 3.14%, respectively), indicating that spontaneous cell death was negligible. In contrast, treatment with HQD metal complex nanoparticles significantly induced apoptosis, and the effect was metal-dependent. Cu The induced apoptosis rate was the highest (40.8%), significantly higher than that of HQD. Fe (29.5%) and HQD Mn (30.3%). After loading with doxorubicin (DOX), the apoptosis rate in the HQDCuD treatment group further increased to 56.8%, confirming the chemosensitizing effect of this nanoplatform. Photothermal activation under 808 nm laser irradiation further amplified the apoptosis effect: HQD Fe +laser group, HQD Mn +laser group and HQD Cu The apoptosis rates in the +laser group increased to 42.5%, 40.4%, and 48.58%, respectively. HQDCuD combined with laser irradiation produced the highest apoptosis rate (62.3%), accompanied by the lowest cell viability. These quantitative apoptosis data are highly consistent with the qualitative live / dead cell staining results, jointly demonstrating that HQDCuD combined with laser irradiation resulted in the highest apoptosis rate (62.3%). Cu The multimodal synergistic strategy of nanoparticles, chemotherapy, and photothermal therapy achieved significant pro-apoptotic and anti-tumor effects through a synergistic mechanism.

[0183] In summary, this invention successfully designed and synthesized the functional molecule HQD, which possesses both biological activity and self-assembly properties, by covalently coupling 8-hydroxyquinoline with the functional peptide sequence FFGGRGD. Leveraging the excellent bidentate coordination ability and inherent biological activity of the 8-hydroxyquinoline unit, HQD can be synthesized in transition metal ions (Fe2+, Fe ... 3+ Cu 2+ Mn 2+ Driven by coordination with HQD, ordered supramolecular self-assembly occurs through multiple non-covalent interactions, forming multifunctional metal-coordination hybrid nanomaterials with excellent biocompatibility with normal cells. A systematic review of the metal ion-dependent functions indicates that HQD… Fe and HQD Mn The nanoparticles exhibited moderate levels of thermal conversion efficiency and enzyme-like catalytic activity. In comparison, HQD... Cu Nanoparticles exhibit superior multifunctional synergistic properties: they can efficiently catalyze ROS generation, consume intracellular GSH, and induce DNA breaks through a dual mechanism of DNA intercalation and groove binding, achieving highly efficient inhibition of tumor cells from both redox homeostasis imbalance and DNA oxidative damage perspectives. Meanwhile, HQD… CuIt also exhibits strong near-infrared absorption and high photothermal conversion efficiency, generating localized high temperatures under near-infrared laser irradiation, further exacerbating cellular oxidative stress and promoting tumor cell apoptosis. Based on HQD... Cu With its excellent drug loading performance, the DOX-loaded drug-carrying nanosystem (HQDCuD) can significantly improve the efficiency of drug uptake and accumulation in cells. Through the synergistic effect of chemotherapy, photothermal therapy and ROS-mediated oxidative damage, it ultimately achieves a potent anti-tumor therapeutic effect.

[0184] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art should understand that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing an 8-hydroxyquinoline-functionalized polypeptide molecule, characterized in that, Includes the following steps: S1. Using 2-chlorotriphenylmethyl chloride resin as a solid support, after swelling and washing, it is sequentially coupled with fluorenemethyloxycarbonyl-aspartic acid-β-tert-butyl ester, Fmoc-glycine, Fmoc-Pbf-arginine, Fmoc-L-phenylalanine, and Fmoc-L-phenylalanine. After each coupling reaction, the Fmoc protecting group is removed to obtain polypeptide molecules. S2. The polypeptide molecule obtained in S1 is coupled with 8-hydroxyquinoline-7-carboxylic acid. Then, the product is cleaved and dissociated from the 2-chlorotriphenylmethyl chloride resin using a cleavage fluid. After post-treatment, the 8-hydroxyquinoline-functionalized polypeptide molecule is obtained.

2. The preparation method according to claim 1, characterized in that, In S1, the specific operation for the coupling reaction with fluorenemethyloxycarbonyl-aspartic acid-β-tert-butyl ester is as follows: N,N-diisopropylethylamine is added to the fluorenemethyloxycarbonyl-aspartic acid-β-tert-butyl ester solution, and the resulting mixed solution is added to the 2-chlorotriphenylmethyl chloride resin after swelling and washing treatment, and the reaction is carried out for 1-2 h. And / or, in S1, the specific procedure for the coupling reaction with Fmoc-glycine is as follows: Fmoc-glycine and benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate are dissolved in N,N-dimethylformamide, then N,N-diisopropylethylamine is added, and the resulting mixed solution is added to 2-chlorotriphenylmethyl chloride resin and reacted for 1-2 h; And / or, in S1, the specific operation of the coupling reaction with Fmoc-Pbf-arginine is as follows: Fmoc-Pbf-arginine and benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate are dissolved in N,N-dimethylformamide, then N,N-diisopropylethylamine is added, and the resulting mixed solution is added to 2-chlorotriphenylmethyl chloride resin and reacted for 1-2 h; And / or, in S1, the specific operation for the coupling reaction with Fmoc-L-phenylalanine is as follows: Fmoc-L-phenylalanine and benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate are dissolved in N,N-dimethylformamide, and then N,N-diisopropylethylamine is added. The resulting mixed solution is added to 2-chlorotriphenylmethyl chloride resin and reacted for 1-2 h.

3. The preparation method according to claim 1, characterized in that, In S1, the specific operation for removing the Fmoc protecting group is as follows: the resin after coupling reaction is reacted with the Fmoc-removing protective solution, which is a mixed solution of piperidine and N,N-dimethylformamide, and the volume ratio of piperidine to N,N-dimethylformamide is 1:(3-5).

4. The preparation method according to claim 1, characterized in that, In S2, the specific operation of the coupling reaction is as follows: 8-hydroxyquinoline-7-carboxylic acid and benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate are dissolved in N,N-dimethylformamide, and then N,N-diisopropylethylamine is added. The resulting mixed solution is added to 2-chlorotriphenylmethyl chloride resin and reacted for 1.5-2.5 h. And / or, in S2, the cutting fluid is a mixed solution of trifluoroacetic acid and water, wherein the volume ratio of trifluoroacetic acid to water is (90-98):(2-10).

5. An 8-hydroxyquinoline-functionalized polypeptide molecule prepared by the preparation method according to any one of claims 1-4.

6. A method for preparing a metal complex of an 8-hydroxyquinoline-functionalized polypeptide molecule, characterized in that, Includes the following steps: The 8-hydroxyquinoline-functionalized polypeptide molecule described in claim 5 is dissolved in a solvent, a transition metal salt is added, the pH of the system is adjusted to neutral, and a coordination reaction is carried out to obtain a metal complex of the 8-hydroxyquinoline-functionalized polypeptide molecule.

7. The preparation method according to claim 6, characterized in that, The molar ratio of the 8-hydroxyquinoline-functionalized polypeptide molecule to the metal salt is (2-3):1; the transition metal salt includes copper salt, iron salt or manganese salt.

8. A metal complex of an 8-hydroxyquinoline-functionalized polypeptide molecule prepared by the preparation method of claim 6 or 7.

9. The use of a polypeptide molecule functionalized with 8-hydroxyquinoline as described in claim 5 or a metal complex of a polypeptide molecule functionalized with 8-hydroxyquinoline as described in claim 8 in the preparation of a drug for treating tumors.

10. The application according to claim 9, characterized in that, The 8-hydroxyquinoline-functionalized polypeptide molecule metal complex is loaded with doxorubicin.

Images