A metal-polypeptide based hydrogel scaffold and its preparation method and application

Abstract

The application provides a metal-polypeptide-based hydrogel support, comprising a hydrogel formed by coordination of polypeptides and metal ions, wherein the polypeptides are Ac-(FX) n -NH2, wherein n is a natural number, Ac represents a carboxyl group, NH2 represents an amino group, (FX) n represents n FX repeating units, wherein F is a phenylalanine residue, and X is selected from a glutamic acid residue or an aspartic acid residue. The hydrogel support is formed based on coordination-mediated supramolecular self-assembly of metals and polypeptides, has stable structure and good mechanical strength, and can realize excellent antibacterial or immunotherapy effect through slow release of metal ions.

Description

Technical Field

[0001] This invention belongs to the field of polymer materials technology, specifically relating to a metal-peptide-based hydrogel scaffold, its preparation method, and its application. Background Technology

[0002] The interaction between peptides / proteins and metal ions is widespread in nature and involves complex mechanisms. Peptides contribute to a complete structure, while metal ions can participate in various physiological and pathological processes, including bone repair, wound healing, antibacterial activity, cell adhesion, and immune regulation. Because peptides provide a wide range of functional groups, they exhibit variable interactions with metal ions. Metal-peptide coordination is considered a promising approach for fabricating organic-inorganic composite biomaterials.

[0003] However, since metal ions are typically introduced via surface adsorption or simple chelation (e.g., chelation with carboxyl or histidine side chains), this approach makes it difficult to control release kinetics and affects long-term performance. Therefore, functionalization strategies are urgently needed to achieve stable loading, controlled release, and tight binding of metal ions to the support structure. Summary of the Invention

[0004] To address the problems of the prior art, this invention provides a metal-peptide-based hydrogel scaffold, its preparation method, and its application. This hydrogel scaffold is formed based on supramolecular self-assembly mediated by the coordination of metal and peptide, exhibiting structural stability and good mechanical strength. It can achieve excellent antibacterial or immunotherapeutic effects through the sustained release of metal ions.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A metal-peptide-based hydrogel scaffold comprises a hydrogel formed by coordination of a peptide with metal ions, wherein the peptide is Ac-(FX). n -NH2, the metal ion is selected from Cu 2+ Mn 2+ Mn 4+ Zn 2+ Ca 2+ 、Sr 2+ or Ce 4+ Where n is a natural number, Ac represents a carboxyl group, NH2 represents an amino group, and (FX) n This represents n repeating units of FX, where F is a phenylalanine residue and X is selected from glutamic acid (E) residues or aspartic acid (D) residues.

[0006] Preferably, n is 3 or 4. Preferably, X is a glutamic acid (E) residue.

[0007] Preferably, the hydrogel scaffold comprises a polymer scaffold, and the hydrogel is attached to the surface of the scaffold.

[0008] Preferably, the polymer scaffold is formed by 3D printing.

[0009] Preferably, the polymer scaffold is PCL.

[0010] A second objective of this invention is to provide a method for preparing the hydrogel scaffold, comprising: The polypeptide and the metal ions are mixed in solution and allowed to stand to self-assemble into the hydrogel.

[0011] Preferably, the pH of the solution is 6.8 to 7.2.

[0012] Preferably, the preparation method further includes: impregnating the hydrogel with the polymer scaffold.

[0013] A third objective of this invention is to provide the application of the hydrogel scaffold in the preparation of antibacterial materials, bone defect repair materials, or antitumor materials.

[0014] The beneficial effects of this invention are as follows: Unlike traditional strategies that involve the physical adsorption or weak chelation of metal ions onto material surfaces, this invention utilizes metal ions (Cu) 2+ Mn 2+ Zn 2+ Ca 2+ 、Sr 2+ Ce 4+ It is directly integrated into Ac-(FX) as a coordination node. n The β-sheet framework of -NH2 peptides (n = 3 or 4) was used to construct a stable three-dimensional nanofiber network in which metal ions were simultaneously involved in structural cross-linking and bioactivity. The resulting hydrogel exhibited pH-responsive sustained release of metal ions, particularly under weakly acidic infection conditions, with a significantly enhanced release effect, thus resolving the usual trade-off between structural integrity and controllable metal ion release. This concept can be extended to other metal-peptide systems for constructing multifunctional biomaterials. Particular attention should be paid to the interaction of Mn and other metal-peptide ions. 2+ When interacting with Ac-(FX)4-NH2, Mn 2+ It will be slowly oxidized to Mn in solution. 4+ This can produce additional biological effects.

[0015] In addition, with Cu 2+ The CPSH-PCL scaffold (CPSH@PCL) for node coordination also exhibited long-term antibacterial and anti-biofilm effects different from burst-release systems. Sustained low-dose Cu 2+Exposure to CPSH's superhydrophilic surface effectively inhibits initial bacterial adhesion and disrupts mature biofilms, enabling the scaffold to maintain an anti-infection balance in vivo. Simultaneously, the scaffold remodels the oxidative microenvironment through a dual antioxidant mechanism: firstly, it decomposes exogenous reactive oxygen species (ROS) like catalase; secondly, it activates the intracellular SIRT1-SOD2-CAT pathway. This synergistic regulation mitigates infection-induced oxidative damage and creates a favorable environment for tissue regeneration.

[0016] Importantly, CPSH@PCL promotes osteogenic growth not solely through a single osteoinductive signal, but through systemic metabolic regulation. Sustained release of Cu... 2+ The HIF-1α signaling pathway was upregulated, and metabolic reprogramming towards glycolysis was inhibited, thereby promoting ATP production and the generation of biosynthetic precursors required for bone matrix synthesis. Therefore, metabolism becomes a central convergence point connecting infection control, oxidative stress relief, and osteogenic differentiation. An infectious cranial defect model confirmed that this multifunctional synergistic effect was preserved in vivo, resulting in significant and high-quality bone regeneration under infectious conditions.

[0017] CPSH@PCL implements Cu 2+ Synchronous release control, sustained antibacterial and antibiofilm activity, regulation of oxidative stress, and promotion of osteogenic differentiation. Mechanistic studies show that CPSH@PCL partially couples infection control with bone regeneration through HIF-1α-mediated metabolic reprogramming (towards glycolysis). This structure-function integrated strategy provides a general blueprint for designing infection-resistant bone repair materials, broadening the application potential of multifunctional biomaterials in regenerative medicine.

[0018] And with Mn 2+ Mn 4+ The manganese-based coordination peptide hydrogel (MnPSH) system can perform tumor immunotherapy through two mechanisms: activating innate immunity and disrupting the tumor microenvironment (TME). This manganese-based hydrogel can be injected in situ into the tumor site. In the weakly acidic environment of the body, the coordination between manganese ions and peptides gradually weakens, and the self-assembled three-dimensional network structure of the hydrogel gradually collapses, thus slowly releasing manganese ions at the tumor site. 4+ It can activate innate immunity through the cGAS-STING pathway and directly consume excess GSH and H2O2 in the TME to produce oxygen and reactive oxygen species (ROS), which can weaken the proliferation and invasion of tumors, regulate the inhibition of the immune environment, and thus enhance the sensitivity to anticancer treatments (such as chemotherapy, radiotherapy and immunotherapy). Attached Figure Description

[0019] Figure 1 It is the Cu obtained in Example 1 (2) 2+The image shows the appearance of the coordination peptide hydrogels at the nodes, where from left to right are CPSH-0.5, CPSH-1, and CPSH-1.5.

[0020] Figure 2 The hydrogel scaffold appearance diagram in Example 1 (2) shows the appearance diagrams of the PCL scaffold, CPSH@PCL, and P@PCL from left to right.

[0021] Figure 3 This is a TEM image of CPSH in Example 1 (3).

[0022] Figure 4 These are SEM images of several samples in Example 1 (3), from left to right: CPSH, PCL, and CPSH@PCL.

[0023] Figure 5 The rheological test results of CPSH in Example 1 (3) are shown. The left figure is the storage modulus and loss modulus diagram, and the right figure is the composite viscosity diagram.

[0024] Figure 6 The infrared spectra of FE3 and CPSH in Example 1 (3) are shown.

[0025] Figure 7 In Example 1 (3), Cu in CPSH@PCL at different pH values 2+ The release curve.

[0026] Figure 8 This is the matrix-assisted laser desorption / ionization time-of-flight mass spectrum of CPSH in Example 1 (3).

[0027] Figure 9 This is a qualitative analysis diagram of the dilution coating plate method in Example 1 (4) of the broad-spectrum antibacterial properties of CPSH@PCL.

[0028] Figure 10 The qualitative analysis of CPSH@PCL in the biofilm formation inhibition experiment in Example 1 (4) is performed by crystal violet staining.

[0029] Figure 11 This is the qualitative analysis of crystal violet staining in the CPSH@PCL mature biofilm clearance experiment in Example 1 (4).

[0030] Figure 12 The results of in vitro H2O2 removal of CPSH@PCL obtained by TMB colorimetric method in Example 1 (5) are as follows.

[0031] Figure 13 The results are quantitative results of the cell streaking experiment in Example 1 (6), where the left figure shows BMSCs and the right figure shows OBs.

[0032] Figure 14 Osteogenesis-related genes in the PCL group, P@PCL group, and CPSH@PCL group of Example 1 (7) after 7 days of culture. ALP, OPN, Runx2, OCN and glycolysis-related genes GLUT1, HK2, PDK1, LDHA, HIF-1α The relative expression results.

[0033] Figure 15 The results are from the qualitative analysis of a smear plate one week after surgery for infectious bone defects in rats in Example 1 (8).

[0034] Figure 16 These are micro-CT images reconstructed from rats 4 weeks after surgery in Example 1 (8).

[0035] Figure 17 Mn is introduced in Example 2 (2) 2+ FE3-Mn on day 1 after ionization 2+ and FE4-Mn on day 10 4+ Macroscopic view of hydrogels.

[0036] Figure 18 It is FE3-Mn in Example 2 (3) 2+ FE4-Mn 4+ Zeta potential analysis results for MnCl2, FE3 and FE4.

[0037] Figure 19 It is FE3-Mn in Example 2 (3) 2+ X-ray photoelectron Mn2p at 1 day (left image) and 10 days (right image) 3 / 2 Multiple split peak fitting spectrum.

[0038] Figure 20 It is FE4-Mn in Example 2 (3) 4+ X-ray photoelectron Mn2p at 1 day (left image) and 10 days (right image) 3 / 2 Multiple split peak fitting spectrum.

[0039] Figure 21 It is FE4-Mn in Example 2 (3) 4+ X-ray photoelectrons of Mn2p oxidation in January (left) and June (right) 3 / 2 Multiple split peak fitting spectrum.

[0040] Figure 22 It is FE3-Mn in Example 2 (3) 2+ (Left image) and FE4-Mn 4+ (Right figure) Matrix-assisted laser desorption / ionization time-of-flight mass spectra.

[0041] Figure 23It is FE3-Mn in Example 2 (3) 2+ (Yellow), FE4-Mn 4+ Infrared spectra of FE3 (blue), FE4 (red), and FE5 (green).

[0042] Figure 24 It is FE3-Mn in Example 2 (3) 2+ With FE4-Mn 4+ SEM and TEM images.

[0043] Figure 25 It is FE3-Mn in Example 2 (3) 2+ With FE4-Mn 4+ The rheological analysis diagrams are shown. The left diagram shows the storage modulus versus loss modulus, and the right diagram shows the composite viscosity.

[0044] Figure 26 It is FE3-Mn in Example 2 (3) 2+ With FE4-Mn 4+ Mn 2+ Release kinetics analysis.

[0045] Figure 27 It is FE3-Mn in Example 2 (3) 2+ With FE4-Mn 4+ Effects on the viability of DC2.4 and 4T1 cells.

[0046] Figure 28 It is (a)FE3, (b)FE4, (c)FE3-Mn in Example 2 (4) 2+ and (d)FE4-Mn 4+ Schematic diagram of the reaction with H2O2; e is the quantitative determination of MnCl2 and FE3-Mn by TMB colorimetric method. 2+ and FE4-Mn 4+ H2O2 consumption capacity diagram.

[0047] Figure 29 It is MnCl2, FE3, FE4, and FE3-Mn in Example 2 (4) 2+ and FE4-Mn 4+ Flow cytometry analysis of activated DC2.4 cells.

[0048] Figure 30 This is a statistical chart of the average fluorescence intensity (MFI) of CD80 and CD86 in Example 2 (4).

[0049] Figure 31 It is MnCl2, FE3, FE4, and FE3-Mn in Example 2 (4) 2+ and FE4-Mn 4+Flow cytometry analysis of macrophages polarized CD163- / CD86+.

[0050] Figure 32 It is MnCl2, FE3, FE4, and FE3-Mn in Example 2 (4) 2+ and FE4-Mn 4+ Flow cytometry analysis of macrophage polarization of CD206- / CD86+.

[0051] Figure 33 This is a statistical chart of the average fluorescence intensity (MFI) of CD206, CD163 and CD86 in Example 2 (4).

[0052] Figure 34 The different treatment methods in Example 2 (5) are Saline, FE3-Mn 2+ and FE4-Mn 4+ A diagram of mouse tumor volume.

[0053] Figure 35 The groups (a) Saline and (b) FE3-Mn in Example 2 (5) are 2+ and (c)FE4-Mn 4+ Gross observation of mice, (d) Saline, FE3-Mn 2+ and FE4-Mn 4+ Gross observation of the tumor, and its relationship with (e)Saline and FE3-Mn 2+ and FE4-Mn 4+ A graph showing the tumor weight in mice.

[0054] Figure 36 It is the F4 / 80 macrophages in the 4T1 tumor tissue of Example 2 (5) + / CD11b + Flow cytometry analysis.

[0055] Figure 37 The groups (a) Saline and (b) FE3-Mn in Example 2 (5) are 2+ and (c)FE4-Mn 4+ H&E staining image of lung tissue (scale bar 500 μm).

[0056] Figure 38 The groups (a) Saline and (b) FE3-Mn in Example 2 (5) are 2+ and (c)FE4-Mn 4+ Immunofluorescence staining image of 4T1 tumor tissue. The scale bar in the image represents 200 μm.

[0057] Figure 39This is a diagram showing the relative expression results of T cell-related genes, oxidation-related genes, and STING pathway-related genes in the 4T1 tumor tissue in Example 2 (5). Detailed Implementation

[0058] Example 1 (1) Obtaining FE3 polypeptide: Ac-(FE)3-NH2 was purchased externally, and its quality was determined by mass spectrometry and high performance liquid chromatography. It is referred to as FE3 below.

[0059] (2) Preparation of CPSH and CPSH@PCL: Weigh out FE3 peptide and dissolve it in sterile water to prepare a 20 mM solution. Adjust the pH to 7.0 ± 0.2 using NaOH. Add 10, 20, and 30 mM copper chloride solutions to this solution respectively, mix well, and let stand at room temperature for 30 minutes to self-assemble into hydrogels, which are labeled as CPSH-0.5, CPSH-1, and CPSH-1.5 respectively. Figure 1 CPSH-1.5 forms a stable, invertible gel, which will be referred to as CPSH in subsequent experiments.

[0060] PCL scaffolds were fabricated using 3D printing: internal infill angle 0° / -90°, external infill angle 45° / -45°, printing speed 1000 mm / min, layer height 0.2 mm, infill rate 27%, resulting in PCL scaffolds with a diameter of 5 mm and a height of 1 mm, which were then sterilized and dried for later use.

[0061] A PCL scaffold was immersed in CPSH hydrogel to form a uniform coating on the scaffold surface and within its pores, resulting in a CPSH@PCL composite scaffold; without the addition of Cu 2+ The sample obtained by treating with FE3 solution is denoted as P@PCL ( Figure 2 The resulting composite scaffold surface is blue-green, indicating that Cu... 2+ Successfully introduced and evenly distributed.

[0062] (3) Structural and physicochemical characterization of CPSH and CPSH@PCL: Transmission electron microscopy (TEM) observations revealed that the interior of CPSH consists of a three-dimensional porous network composed of uniformly interwoven nanofibers. Figure 3 Scanning electron microscopy (SEM) confirmed that a uniform hydrogel coating formed on the CPSH@PCL surface and was tightly bonded to the scaffold. Figure 4 ).

[0063] Water contact angle tests showed that the CPSH coating reduced the PCL surface contact angle from 101° to 26°, significantly improving the hydrophilicity of the scaffold. Rheological tests indicated that the storage modulus G′ was consistently higher than the loss modulus G″ in the range of 0.1–10 rad / s, and both were greater than 1000 Pa, demonstrating that the hydrogel possesses stable solid-like properties. Figure 5 ).

[0064] Zeta potential testing showed that the FE3 polypeptide was negatively charged (~ -50 mV), and Cu 2+ Positively charged (~ +10 mV). After self-assembly, the CPSH potential increased to -14.55 mV, indicating coordination binding. Circular dichroism spectroscopy showed that both FE3 and CPSH exhibited β-sheet structures.

[0065] Fourier transform infrared (FT-IR) spectroscopy showed that, compared to the FE3 peptide, the carboxyl vibration peak shift and the disappearance of the C–OH absorption peak in CPSH confirmed that Cu 2+ Forms a coordination cross-linking network with the carboxyl group of the polypeptide ( Figure 6 ).

[0066] Release experiments at pH 6.2 and 7.4 showed that Cu in the composite scaffold... 2+ It exhibits a biphasic release behavior, initially rapid and then sustained, lasting up to 216 hours. Figure 7 ).

[0067] Matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF) was used to detect [M-3H+Cu] 2+ ] - Isocomplex ion peaks ( Figure 8 This further verifies coordination formation.

[0068] (4) In vitro antibacterial properties of CPSH@PCL: According to the national standard GB / T 16886.12—XXXX / ISO 10993-12:2021, CPSH (0.1 g / mL) was soaked in sterile nutrient broth (LB) medium and incubated at 37℃ for 72 hours before the extract was collected; PCL and P@PCL extracts were prepared in the same way.

[0069] Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were selected as representative strains. The bacterial culture was treated and then adjusted to a concentration of 1×10⁻⁶. 6 CFU / mL was added to different material extracts and cultured for 12 hours, after which OD was measured. 600 Plate counts were performed. Results showed that almost no colonies formed in the CPSH@PCL group, while a large number of colonies appeared in the PCL, P@PCL, and blank control group (BLK). Figure 9 ) OD600 The results showed that CPSH@PCL inhibited both bacteria by more than 99% (p<0.0001).

[0070] MTT assay further confirmed that CPSH@PCL significantly reduced bacterial metabolic activity (>82%), while PCL and P@PCL had no significant antibacterial effect.

[0071] The above results indicate that CPSH@PCL possesses significant broad-spectrum antibacterial activity, and its effect mainly originates from the Cu in the hydrogel. 2+ The continuous release.

[0072] Biofilm formation inhibition experiment: Bacteria were prepared at a concentration of approximately 1*10 6 CFU / mL suspension was added in 100 μL to 96-well plates (three parallel wells per group) and cultured for 72 hours, with fresh LB liquid medium added after 36 hours. After culture, the culture medium was discarded, and the plates were gently washed three times with 0.9% sodium chloride solution to remove unattached bacteria. The plates were then fixed with methanol for 10 min, air-dried, and stained with 50 μL of 0.5% crystal violet solution for 30 min, followed by washing three times with deionized water. The dye was dissolved in 33% acetic acid, and the absorbance was measured at 570 nm to characterize the biofilm amount. CPSH@PCL almost completely inhibited biofilm attachment of both strains. Figure 10 ), OD after dye dissolution 570 The value decreased significantly.

[0073] Mature biofilm removal assay: Bacteria were cultured for 72 hours to form a mature biofilm as described above. After discarding the culture medium, 100 μL of different material extracts were added to each well for 12 hours. OD was then measured following the crystal violet staining procedure described above. 570 The value was used to evaluate the biofilm removal effect. CPSH@PCL also showed excellent disruptive ability: the matrix was extensively removed ( Figure 11 ), quantitative OD 570 Analysis showed a significant reduction in residual biofilm biomass. This indicates that the material released Cu... 2+ It can inhibit the initial adhesion of bacteria and their penetration and destruction of the biofilm matrix, thereby achieving efficient biofilm inhibition and removal.

[0074] CPSH@PCL synergistic effect, through Cu 2+ CPSH continuously disrupts cell membranes, providing potent broad-spectrum antibacterial and antibiofilm activity; simultaneously, the superhydrophilic CPSH coating further reduces bacterial adhesion. This dual mechanism of action makes CPSH@PCL an ideal platform for treating infectious bone defects and other complex orthopedic infections.

[0075] (5) Evaluation of the in vitro antioxidant properties of CPSH@PCL: According to the national standard GB / T 16886.12—XXXX / ISO 10993-12:2021, the scaffolds of each group were immersed in cell culture medium at a ratio of 0.1 g / mL and incubated at 37 ℃ and 5% CO2 for 72 hours. After centrifugation, the extract was collected for later use. The PCL group was used as a control.

[0076] When CPSH@PCL was placed in an H2O2 solution, a large number of bubbles were rapidly generated on its surface, while PCL showed no significant change. This indicates that the material possesses catalase-like activity, capable of decomposing H2O2 to generate water and oxygen. TMB colorimetric results further confirm its ability to continuously consume exogenous H2O2. Figure 12 ).

[0077] Oxidative stress was induced in BMSCs using H2O2 (final concentration 400 μM), followed by incubation with extracts from each group for 12 hours, and DCFH-DA fluorescence detection was performed. The results showed that the intracellular ROS level in the CPSH@PCL group was significantly reduced, indicating a significant protective effect against oxidative damage.

[0078] Further RT-qPCR analysis revealed that CPSH@PCL significantly upregulated the expression of antioxidant-related genes: SIRT1 expression increased by approximately 5-fold, and SOD2 and CAT expression increased by approximately 7-fold.

[0079] In summary, CPSH@PCL can directly catalyze the removal of ROS and activate intracellular antioxidant pathways, thereby improving cell tolerance and promoting the repair of infected bone defects.

[0080] (6) Biocompatibility evaluation of CPSH@PCL: Whole blood was diluted with PBS to prepare a 5% red blood cell suspension. Deionized water (positive control), physiological saline (negative control), and scaffold extracts from each group were added separately. After incubation at 37 °C for 1, 3, and 5 hours, the supernatant was collected by centrifugation, and the absorbance was measured at 540 nm to calculate the hemolysis rate. The results showed that the hemolysis rate of each scaffold group was significantly lower than the 5% limit specified in ISO 10993-4.

[0081] Bone marrow mesenchymal stem cells (BMSCs) and osteoblasts (OBs) were cultured using scaffold extract, and their viability / death staining, migration, and proliferation were evaluated. Viability / death staining results showed that viable cells predominated in all groups, and there was no significant difference in CPSH@PCL levels between the control group and the control group.

[0082] Scratch assays showed that CPSH@PCL significantly promoted the migration of BMSCs and OBs at 12 and 24 hours, with a more significant promoting effect on OBs in the early stages. Figure 13 ).

[0083] The CCK-8 results showed that CPSH@PCL supported continuous cell proliferation for 5 days.

[0084] In summary, CPSH@PCL exhibits low hemolytic activity and low cytotoxicity, and can promote cell migration and proliferation, demonstrating good biocompatibility and tissue repair potential.

[0085] (7) In vitro osteogenic properties of CPSH@PCL: BMSCs were cultured using scaffold extract and differentiated using osteogenic induction medium. Alkaline phosphatase (ALP) and Alizarin Red (ARS) staining were performed after 7 and 14 days of culture. Results showed that ALP activity and mineralized nodule formation were significantly higher in the CPSH@PCL group than in the control group, indicating that it can promote early osteoogenesis and later matrix mineralization.

[0086] RT-qPCR results showed that ( Figure 14 ), CPSH@PCL significantly increased ALP, RUNX2, OPN and OCN Expression of osteogenic-related genes; expression of glycolysis-related genes HIF-1α, GLUT1, HK2, PDK1 and LDHA The expression levels were significantly increased, indicating that the material can promote osteogenic differentiation through HIF-1α-mediated glycolytic metabolic reprogramming.

[0087] In summary, CPSH@PCL promotes osteogenic differentiation in vitro through a dual mechanism of osteogenic gene activation and metabolic regulation.

[0088] (8) In vivo antibacterial and osteogenic properties of CPSH@PCL: Twenty-four SD rats were randomly divided into three groups: PCL group, P@PCL group, and CPSH@PCL group (n = 8). An infectious, critical-size skull defect model was established: a 5 mm diameter full-thickness bone defect was prepared in the skull, inoculated with methicillin-resistant Staphylococcus aureus (MRSA), and then a corresponding scaffold was implanted. The rats were observed for 4 weeks post-surgery.

[0089] In vivo antibacterial evaluation was performed one week post-surgery. Skull samples were cultured in PBS with shaking for 12 hours, then spread for further culture. Results showed almost no colonies in the CPSH@PCL group, while the control group exhibited abundant colony growth, indicating that the scaffold possesses significant in vivo antibacterial activity. Figure 15 ).

[0090] Micro-CT analysis was performed 4 weeks post-surgery. Results showed that the CPSH@PCL group exhibited the most significant new bone formation. Figure 16 The bone regeneration rate was approximately 40%, and bone mineral density (BMD), bone volume fraction (BV / TV), and trabecular bone number (Tb.N) were all significantly improved.

[0091] Histological analysis showed that the CPSH@PCL group had a significantly reduced inflammatory response, significantly increased collagen deposition and new bone formation, and strong positive OCN immunostaining, indicating the formation of mature bone tissue.

[0092] In summary, CPSH@PCL achieved sustained antibacterial activity and significantly promoted bone regeneration in an infected bone defect model.

[0093] Example 2 (1) Obtaining FE3 and FE4 peptides: Ac-(FE)3-NH2 and Ac-(FE)4-NH2, hereinafter referred to as FE3 and FE4 respectively, were purchased externally, and their quality was determined by mass spectrometry and high performance liquid chromatography.

[0094] (2) FE3-Mn 2+ and FE4-Mn 4+ Preparation: Mn 2+ Solution preparation: Weigh manganese chloride (MnCl2), dissolve it in deionized water, and prepare a solution with a mass concentration of 20 mg / mL (equivalent to 0.36 mol / L). 2+ Aqueous solution, for later use.

[0095] FE3-Mn 2+ Preparation of hydrogel: The FE3 solution obtained in (1) was mixed with the Mn solution prepared above. 2+ Solution according to FE3 polypeptide: Mn 2+ The FE3 and MnCl2 solutions were mixed in a 1:1.5 molar ratio and thoroughly mixed to form a milky white hydrogel. The concentration of FE3 was 22 mg / mL and the concentration of MnCl2 was 4 mg / mL. This gel is denoted as FE3-MnCl2. 2+ .

[0096] FE4-Mn 4+ Preparation of hydrogel: The FE4 solution obtained in (1) was mixed with the Mn solution prepared above. 2+ Solution according to FE4 polypeptide: Mn 2+ The mixture was prepared by mixing in a 1:2 molar ratio. The resulting system was initially a light yellow fluid, containing 29 mg / mL FE4 and 6 mg / mL MnCl2. The system was then aged at 4 °C for 6 days. After inverting the system for 30 minutes, it showed no flowability, forming a yellowish-brown gel. Further aging at 4 °C for 10 days resulted in a darker brown gel that remained non-flowable even after inverting for 30 minutes, yielding a stable gel. Figure 17 ), denoted as FE4-Mn 4+ .

[0097] (3) FE3-Mn2+ and FE4-Mn 4+ Structural and physicochemical characterization: Zeta potential measurement: FE3-Mn 2+ Hydrogel and FE4-Mn 4+ The hydrogels were diluted 50-fold with pure water, and separate manganese chloride solutions and FE3 and FE4 peptide solutions with the same concentrations as the diluted samples were prepared. The manganese chloride solution and FE3-Mn were measured using a nanoparticle size / potential analyzer. 2+ FE4-Mn 4+ The Zeta potentials of FE3 and FE4 are shown in the figure ( Figure 18 As shown in the figure.

[0098] X-ray photoelectron spectroscopy (XPS) determination of Mn valence state composition and evolution: After the sample was rapidly frozen at -80 ℃ and freeze-dried, XPS was used to determine the valence state composition and evolution of FE3-Mn. 2+ The Mn 2p system was tested (prepared at 1 day and 10 days) and the FE4 system (prepared at 1 day for solution and 10 days for gel), and the Mn 2p was tested. 3 / 2 Peak fitting of the region to estimate Mn 2+ / Mn 4+ The ratio. The results show that: FE3-Mn 2+ With Mn 2+ Mainly, 1 day Mn 2+ Mn accounted for 97.67% and 10 days. 2+ 69.18% ( Figure 19 ); FE4 system with Mn 4+ The component is dominant, and Mn is prepared in 1 day. 4+ Mn accounted for 14.23% and 10 days. 4+ 75.69% ( Figure 20 ). For FE4-Mn 4+ Samples were prepared and tested at 1 month and 6 months, Mn 4+ The proportions were 76.15% and 76.58% respectively. Figure 21 ).

[0099] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS): FE3-Mn was detected using MALDI-TOF MS. 2+ [FE3-2H+ Mn] exists 2+ +e] - Characteristic ion peaks; FE4-Mn 4+ [FE4-4H+Mn] exists 4+ +e] - and [FE4-4H+Mn 2+ +Na + ] -Waiting for signal ( Figure 22 ).

[0100] Fourier transform infrared (FT-IR) results are shown below Figure 23 .

[0101] Morphology (TEM / SEM): The morphology of the samples was observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). TEM revealed a continuous network of interwoven nanofibers; SEM showed that the samples were stacked in a sheet / wrinkle-like manner and contained a porous structure. Figure 24 ).

[0102] Rheological testing: In the range of ω = 0.1-10 rad / s, both hydrogels exhibited G' > G (tan δ < 1), and G' was within 10 rad / s. 4 Above the Pa level; the composite viscosity η* decreases with increasing frequency ( Figure 25 Overall, FE3–Mn 2 + G' and η* are higher than FE4-Mn 4+ .

[0103] Mn 2+ Release assay (EDTA complexometric titration): The release of Mn in the medium under pH 6.4 and pH 7.3 conditions was determined using EDTA complexometric titration. 2+ The content was determined, and the cumulative release rate was calculated. Results showed that: FE3-Mn 2+ The cumulative release rate was 62.4% on day 14 at pH 6.4 and 42.9% at pH 7.3. FE4-Mn was determined by XPS. 4+ Mn 2+ Approximately 24.31%, obtained by titration of Mn 2+ The release amount was converted to obtain FE4-Mn 4+ The release rates on day 14 were approximately 79.4% and 51.0% at pH 6.4 and pH 7.3, respectively. Figure 26 ), and FE4-Mn 4+ The overall release rate is higher than that of FE3-Mn 2+ .

[0104] (4) FE3-Mn 2+ and FE4-Mn 4+ In vitro antitumor activity: Cytotoxicity (MTT): 4T1 and DC2.4 cells were seeded at 10,000 cells / well in 96-well plates and cultured for 24 hours. After adhesion, different treatment materials were added to achieve a manganese ion concentration of 4 μg / mL in the culture medium, and the cells were cultured for another 24 hours. Cell viability was assessed using the MTT assay (final concentration 0.5 mg / mL, incubation for 4 hours). After dissolving in DMSO, absorbance was read at 570 nm to calculate cell viability. Results showed that in DC2.4 cells, the cell viability after 24 hours of treatment with FE3 and FE4 was approximately 92%; the survival rate of the MnCl2 group was approximately 43%; and the survival rate of the FE3-MnCl2 group was... 2+ and FE4-Mn 4+ The survival rate after treatment was approximately 80%, which was significantly different from the MnCl2 group (p<0.0001). In 4T1 cells, the survival rate after FE3 and FE4 treatment was approximately 81%; the survival rate of the MnCl2 group was 42.33%; and the survival rate of the FE3-Mn group was... 2+ and FE4-Mn 4+ The survival rates of the two groups were approximately 64% and 70%, respectively, which were significantly different from those of the MnCl2 group (p<0.0001). Figure 27 ).

[0105] Cell migration (scratch assay): 4T1 cells were seeded at 60,000 cells / well in 6-well plates and cultured to approximately 90% confluence before scratching. FE3-Mn was then added. 2+ or FE4-Mn 4+ (Manganese ion concentration 4 μg / mL) Scratch changes were recorded and migration rates were calculated at 0, 6, 12, and 24 hours. Results showed that the scratches in the polypeptide (FE3, FE4) groups gradually closed within 24 hours; while those in the FE3-Mn group... 2+ and FE4-Mn 4+ The scratches increased over time, with 24-hour migration rates of -47.74% and -22.52%, respectively.

[0106] Hydrogen peroxide reaction / scavenging capacity (oxygen production phenomenon and TMB method): This involves the reaction of FE3-Mn... 2+ and FE4-Mn 4+ Bubble generation was observed after each hydrogel was mixed with hydrogen peroxide solution, and the H2O2 scavenging ability was quantitatively assessed at 450 nm using the TMB colorimetric method. Results showed that both manganese-based hydrogels produced significant bubbles after reacting with H2O2; FE4-Mn 4+ The gas production rate is higher than that of FE3-Mn 2+ Pure polypeptides showed virtually no bubbles when reacting with H2O2. Quantitative results showed that both manganese-based hydrogels possessed H2O2 scavenging ability, with the most significant scavenging effect observed in MnCl2 and FE3-Mn... 2+ The differences were p<0.0001 and p<0.001, respectively. Figure 28).

[0107] Intracellular ROS detection (DCFH-DA): 4T1 and RAW264.7 cells were seeded at 40,000 cells / well in 24-well plates. After adhesion, FE3-Mn was added. 2+ and FE4-Mn 4+ Treatment with 4 μg / mL manganese ions for 24 hours. Staining with DCFH-DA probe (final concentration 20 μM) followed by fluorescence microscopy observation. Results showed that in 4T1 cells, the green fluorescence of FE3 and FE4 groups was weaker; FE3-Mn... 2+ and FE4-Mn 4+ The fluorescence of the group was enhanced, and FE4-Mn 4+ The strongest fluorescence was observed in all groups in RAW264.7 cells. In contrast, the fluorescence in all groups was weaker.

[0108] HMGB1 immunofluorescence: 4T1 and DC2.4 cells were added to FE3-Mn under the conditions described above. 2+ and FE4-Mn 4+ Treatment with 4 μg / mL manganese ions for 24 hours, followed by staining with anti-HMGB1 antibody and fluorescent secondary antibody and microscopic observation. Results showed that the HMGB1 signal in groups FE3 and FE4 highly overlapped with the cell nucleus; FE4-Mn 4+ In the treatment group, HMGB1 was transferred from the nucleus to the cytoplasm.

[0109] GSH consumption: The GSH consumption of the material was determined using the DTNB method: the absorbance (412 nm) was measured after the material was mixed with a GSH standard solution and reacted for 5 min. Results showed that FE4-Mn 4+ Within 5 minutes, 84.74% of GSH was consumed, approximately FE3-Mn. 2+ 3.28 times.

[0110] Immune cell activation / polarization (flow cytometry): (1) DC2.4 cell maturation: DC2.4 cells were seeded at 50,000 cells / well in a 24-well plate and FE3-Mn was added. 2+ or FE4-Mn 4+ Treatment with 4 μg / mL manganese ions for 24 hours, followed by flow cytometry analysis of CD80 / CD86 (triple replicates per group). Results showed no significant difference between the peptide group and the blank control; FE3-Mn 2+ The CD80 Mean value of the control group increased by 161.6% compared with the control group (p<0.0001); FE4-Mn 4+ The mean values ​​of CD80 and CD86 in the group increased by 74.9% (p<0.01) and 13.45% (p<0.0001), respectively. Figure 29 , 30 ).

[0111] RAW264.7 macrophage polarization: RAW264.7 cells were seeded at 50,000 cells / well in 24-well plates, and FE3-Mn was added. 2+ or FE4-Mn 4+ Treatment with manganese ion concentration of 4 μg / mL for 24 hours was performed, and CD86 (M1), CD206, and CD163 (M2) were detected by flow cytometry (each group was repeated 3 times). Results showed that the CD86 mean values ​​in groups FE3 and FE4 were close to those in the blank control (p>0.05); FE3-Mn 2+ and FE4-Mn 4+ The CD86 mean in the control group was increased by 24.73% and 32.37% respectively (p<0.001). No significant uniform increase was observed in the M2 biomarker overall. Figures 31-33 ).

[0112] (5) FE3-Mn 2+ and FE4-Mn 4+ In vivo antitumor activity: To observe the local reaction of manganese-based hydrogels after in vivo injection, FE3-Mn 2+ FE4-Mn 4+ Subcutaneous injection was administered to healthy mice (n=3). One mouse was dissected daily for 1–3 days. A yellow, mucous-like local reaction was observed near the injection site.

[0113] To evaluate the in vivo antitumor effect of manganese-based hydrogels, a mouse subcutaneous 4T1 tumor model was established. The tumor volume was measured when it grew to approximately 120 mm. 3 Animals were randomly assigned to groups of 6 (n=100 mL) and administered 100 mL of the drug per tumor via subcutaneous injection on days 0, 2, and 4, respectively. 2+ or FE4-Mn 4+ The mice were continuously monitored until the endpoint was reached. During the experiment, the body weight of the mice in each group remained stable with no significant differences.

[0114] like Figure 34 As shown, the tumor volume in the saline group continued to increase over time; FE3-Mn 2+ and FE4-Mn 4+ Tumor growth in the group was significantly slowed. Endpoint statistics showed that at day 22, FE3-Mn... 2+ and FE4-Mn 4+ The tumor growth inhibition rates (TGI) were 71.09% and 89.86%, respectively. Figure 35 ).

[0115] To analyze changes in immune cells in tumor tissue, flow cytometry was used to detect the endpoint tumor tissue. Results showed that the material treatment group showed CD11b... +F4 / 80 + Increased proportion of myeloid / macrophage-related populations: approximately 53.7% in the saline group, FE3-Mn 2+ Group approximately 79.0%, FE4-Mn 4+ Group approximately 81.7% ( p <0.001)( Figure 36 ).

[0116] Given the tendency of 4T1 to metastasize to the lungs, H&E staining was performed on the lung tissue for observation. Dense areas of lesion-like cells were observed in the lung tissue of the saline group; FE3-Mn 2+ and FE4-Mn 4+ The alveolar structure of the group was relatively intact, and no obvious lesion-like areas were observed. Figure 37 ).

[0117] Multichannel immunofluorescence detection (CD20, CD4, CD8, iNOS) was performed on tumor tissue. In the saline group, immune cell signals showed a dispersed distribution; in the material-treated group, enhanced immune cell infiltration and localized aggregation / layered distribution were observed. Figure 38 Manual counting results show: FE4-Mn 4+ Group CD4 + CD8 + The CD20 infiltration densities were 642, 584, and 292 cells / mm², respectively. -2 Compared to FE3-Mn 2+ The groups showed increases of 6.9 times, 0.4 times, and 1.9 times, respectively. iNOS was visible in both material groups. + Cell infiltration, FE3-Mn 2+ 665 cells / mm -2 ;FE4-Mn 4+ 423 cells / mm -2 .

[0118] To assess changes in the expression of tumor-related genes, RT-qPCR analysis was performed on the endpoint tumor tissue. Figure 39 The results show that FE4-Mn 4+ Group STING pathway-related genes IFN-β1 , CXCL10 Significantly upward ( p <0.05), and accompanied by IFN-γ and IL-2 Adjust ( p <0.05). Furthermore, TNF-α The differences between groups were not significant; IL-4 Upregulated. Among oxidative stress-related genes, SOD2 and CAT In FE4-Mn 4+ The group was significantly upregulated. GPX4 No significant changes were observed, and accompanied by HIF1-α Differences in expression.

[0119] Example 3 (1) Obtaining FE3 polypeptide: Ac-(FE)3-NH2 was purchased externally, and its quality was determined by mass spectrometry and high performance liquid chromatography. It is referred to as FE3 below.

[0120] (2) FE3-Zn 2+ FE3-Ca 2+ FE3-Sr 2+ and FE3-Ce 4+ Preparation: Zn 2+ Solution preparation: Weigh zinc chloride (ZnCl2), dissolve it in deionized water, and prepare a Zn solution with a mass concentration of 20 mg / mL (equivalent to 0.30 mol / L). 2+ Aqueous solution, for later use.

[0121] Ca 2+ Solution preparation: Weigh calcium chloride (CaCl2), dissolve it in deionized water, and prepare a CaCl2 solution with a mass concentration of 20 mg / mL (equivalent to 0.50 mol / L). 2+ Aqueous solution, for later use.

[0122] Sr 2+ Solution preparation: Weigh strontium chloride (SrCl2), dissolve it in deionized water, and prepare a SrCl2 solution with a mass concentration of 20 mg / mL (equivalent to 0.23 mol / L). 2+ Aqueous solution, for later use.

[0123] Ce 4+ Solution preparation: Weigh cerium sulfate (Ce(SO4)2), dissolve it in deionized water, and prepare a Ce(SO4)2 solution with a mass concentration of 50 mg / mL (equivalent to 0.36 mol / L). 4+ Aqueous solution, for later use.

[0124] FE3-Zn 2+ Preparation of hydrogel: The FE3 solution obtained in (1) was mixed with the Zn prepared above. 2+ Solution according to FE3 polypeptide: Zn 2+ The FE3 and ZnCl2 solutions were mixed in a 1:1.5 molar ratio and thoroughly mixed to form a milky white hydrogel with a FE3 concentration of 22 mg / mL and a ZnCl2 concentration of 5.11 mg / mL, denoted as FE3-ZnCl2. 2+ .

[0125] FE3-Ca 2+Preparation of hydrogel: The FE3 solution obtained in (1) was mixed with the Ca solution prepared above. 2+ Solution according to FE3 polypeptide: Ca 2+ The FE3 and CaCl2 solutions were mixed in a 1:1.5 molar ratio. After thorough mixing, a milky white hydrogel was formed, with an FE3 concentration of 22 mg / mL and a CaCl2 concentration of 4.16 mg / mL, denoted as FE3-CaCl2. 2+ .

[0126] FE3-Sr 2+ Preparation of hydrogel: The FE3 solution obtained in (1) was mixed with the Sr prepared above. 2+ Solution according to FE3 polypeptide: Sr 2+ The FE3 and SrCl2 solutions were mixed in a 1:1.5 molar ratio and thoroughly mixed to form a milky white hydrogel with a FE3 concentration of 22 mg / mL and a SrCl2 concentration of 5.94 mg / mL, denoted as FE3-SrCl2. 2+ .

[0127] FE4-Ce 4+ Preparation of hydrogel: The FE3 solution obtained in (1) was mixed with the Ce2 solution prepared above. 4+ Solution according to FE3 polypeptide: Ce 4+ The FE3 and MnCl2 solutions were mixed in a 1:1.5 molar ratio and thoroughly mixed to form a milky white hydrogel with a concentration of 22 mg / mL FE3 and 12.46 mg / mL MnCl2, denoted as FE4-Ce. 4+ .

Claims

1. A metal-peptide-based hydrogel scaffold, characterized in that, This includes hydrogels formed by coordination of peptides with metal ions, wherein the peptide is Ac-(FX). n -NH2, where n is a natural number, Ac represents a carboxyl group, NH2 represents an amino group, (FX) n This represents n repeating units of FX, where F is a phenylalanine residue and X is selected from glutamic acid residues or aspartic acid residues.

2. The metal-peptide-based hydrogel scaffold according to claim 1, characterized in that, The metal ions are selected from Cu. 2+ Mn 2+ Mn 4+ Zn 2+ Ca 2+ 、Sr 2+ or Ce 4+ .

3. The metal-peptide-based hydrogel scaffold according to claim 1, characterized in that, n is 3 or 4.

4. The metal-peptide-based hydrogel scaffold according to any one of claims 1-3, characterized in that, The hydrogel scaffold includes a polymer scaffold, and the hydrogel is attached to the surface of the scaffold.

5. The metal-peptide-based hydrogel scaffold according to claim 4, characterized in that, The polymer scaffold was formed by 3D printing.

6. The metal-peptide-based hydrogel scaffold according to claim 4, characterized in that, The polymer scaffold is PCL.

7. The method for preparing a metal-peptide-based hydrogel scaffold according to claim 1, as described in any one of claims 1-6, is characterized in that, include: The polypeptide and the metal ions are mixed in solution and allowed to stand to self-assemble into the hydrogel.

8. The preparation method according to claim 7, characterized in that, The pH of the solution is 6.8 to 7.

2.

9. The preparation method according to claim 7, characterized in that, The preparation method further includes: impregnating the hydrogel with the polymer scaffold.

10. The application of the metal-peptide-based hydrogel scaffold according to any one of claims 1-6 in the preparation of antibacterial materials, bone defect repair materials or antitumor materials.

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