Biomacromolecule slow-release metal-polyphenol compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses a biomacromolecule slow-release metal-polyphenol compound as well as a preparation method and application thereof. The compound comprises metal ions, aromatic alkyl amine polyphenol and a biological macromolecular active drug, wherein the aromatic alkyl amine polyphenol is a compound with a structural general formula as shown in a formula (I), a formula (II), a formula (III) or a formula (IV) or a pharmaceutically acceptable salt of the compound. The compound is formed by self-assembly of metal ions, polyphenol and active drugs under a water phase condition, efficient entrapment of biological macromolecular drugs can be achieved under a mild condition, the drug loading capacity is 1%-95%, and the entrapment efficiency is 1%-100%. The compound can realize controllable release of the loaded biomacromolecular drug, and presents a long-term and continuous slow-release drug delivery effect, so that the stability and bioavailability of the drug in vivo are effectively improved, the drug delivery frequency and potential adverse reaction are reduced, and the compound has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a metal-polyphenol complex for sustained release of biological macromolecules, its preparation method, and its application. Background Technology

[0002] Biopeptides, proteins, and other macromolecular drugs, due to their high targeting, strong bioactivity, and low toxicity, have shown great potential in cutting-edge medical fields such as cancer treatment, metabolic disease management, and immune modulation. The biological function of these drugs is highly dependent on their precise and complex high-order spatial structures. However, these structures are extremely sensitive to the external environment, leading to easy degradation, aggregation, or inactivation in vivo. They generally suffer from short half-lives, require frequent dosing, and have low bioavailability, severely limiting their clinical translation and application. To overcome these shortcomings, drug delivery systems, especially sustained-release technologies that enable continuous drug delivery, have become a key research direction. Currently, commonly used strategies mainly include polyethylene glycol (PEG) modification and encapsulation with biodegradable polymer microspheres. PEG can form an "invisible" barrier on the drug surface through covalent bonding, prolonging its blood circulation time; polymer microspheres achieve slow drug release through physical encapsulation. However, these traditional technologies still face challenges such as low drug loading efficiency, with many nanosystems having a loading rate of less than 30%, resulting in large dosages, potential toxicity of excipients, and high production costs; harsh preparation conditions, often involving organic solvents, high temperatures, or severe shear forces, which can easily damage the natural conformation and activity of biomolecules; and uncontrollable release behavior, with severe initial burst release phenomena and release kinetics that are difficult to precisely control, affecting the safety and effectiveness of treatment.

[0003] In recent years, metal-polyphenol networks (MPNs) formed by the self-assembly of metal ions and polyphenolic ligands through coordination have faced challenges due to the relatively fixed structure and chemical properties of natural polyphenols and their limited variety. This makes it difficult to systematically and precisely control their coordination patterns with metal ions, network crosslinking density, and interactions with proteins. Consequently, the constructed carriers often fail to simultaneously achieve high drug loading and long-term sustained release. Unlike existing nanocarriers using natural polyphenols such as tannins, synthetic polyphenols possess tunable structural features; however, there are currently no reports on complexes synergistically constructed with aromatic alkylamine polyphenols and metal ions for protein delivery. Summary of the Invention

[0004] The present invention aims to provide a metal-polyphenol complex for sustained release of biological macromolecules, its preparation method and application. The metal-polyphenol complex has a high active drug loading rate and activity, and endows the system with controllable drug release behavior, so as to achieve long-term, sustained-release drug delivery effect, thereby effectively improving the in vivo stability and bioavailability of the drug.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A metal-polyphenol complex, the complex comprising a metal ion, an aromatic alkylamine polyphenol, and a bioactive macromolecular drug; Wherein, the aromatic alkylamine polyphenol is a compound having a general structural formula as shown in formula (I), (II), (III), or (IV), or a pharmaceutically acceptable salt thereof: ; Wherein, R1 is a benzene ring with -H or -OH at positions 2, 3, 4, 5, and 6, and at least two adjacent positions are simultaneously -OH; R2 represents the benzene ring with positions 2, 3, 4, 5, and 6 each being -H, -OH, -OR3, or -CN, where R3 is an alkyl group; and n is an integer from 1 to 100.

[0006] Preferably, the bioactive macromolecular drug is a water-soluble polypeptide or protein, including insulin, growth hormone, calcitonin, salmon calcitonin, teriparatide, liraglutide, dulaglutide, smegglutide, teduglutide, exenatide, beraglutide, antigepigide, lixisenatide, loxenatide, duraglutide, semaglutide, abiglutide, lixisenatide, leuprorelin, prorelin, gonadorelin, goserilin, buserorelin, sermorelin, nafarelin, histaminerelin, lanreotide, octreotide, somatostatin, terlipressin, atosiban; thymosin, thymopentin, thymosin, bone peptide, sarcosin, glutathione. Peptides, mannan peptides, Yunzhi glycopeptides, ginseng glycopeptides; hemoglobin, hemochloroglobin, serum albumin, bovine serum albumin, ovalbumin, ovalbumin, whey protein, β-lactoglobulin, lactoferrin, conalbumin, globulin, myosin, sarcoplasmic protein, keratin, casein, mucin, mucin, ferritin, transferrin, collagen, immunoglobulin G, cytochrome C, fibrinogen; trypsin, pepsin, pancreatic enzymes, papain, figase, chymotrypsin, histamine enzyme, thrombin, anterior pituitary troponin, thyrotropin-releasing hormone, parathyroid hormone, fibrinolytic enzyme, glucose oxygen Horseradish peroxidase, hyaluronidase, asparaginase, streptokinase, streptokinase, lysozyme, urokinase, reteplase, amylase, cellulase, collagenase, prothrombin kinase, pancreatic deoxyribonuclease; enfuviride, nasitide, bradykinin, enkephalin, dulaglutide, cortisone, epitubide, acimetidine, desmopotine, veracitide, bivalirudin, vasopressin, phenyltyrosine, sicalide, pentagastrin, secretin, vasoactive intestinal peptide, follicle-stimulating hormone, gonadotropin, prolactin, edotrexate, pentotriol, procarbamate, phenobarbital, sargastin; interferon Interferon-α, interferon-β, interferon-γ, anaprolactin, dinesiac interleukin, bone morphogenetic protein; bacitracin, brevicin, colistin, polymyxin sulfate, polymyxin, teicoplanin, vancomycin, norvancomycin, tervacin, daptomycin, glatiramer, glatiramer acetate, mirvastatin, nesiritide, carfilzomib, bortezomib, micafungin, anidofungin, caspofungin; botulinum toxin, coagulation factor VIII, coagulation factor IX, aprotinin, hirudin, ziconopeptide, enfuviride, thymopentin, angiotensin, atosiban, trachotoxin, bone peptide, any one or a mixture of several of these.

[0007] Preferably, the metal ion is selected from Ba. 2+ 、Sr 2+ Mg 2+ Ca 2+ Fe 2+ Mn 2+ Cu 2+ Zn 2+Co 2+ Ni 2+ Fe 3 + Al 3+ V 3+ Cr 3+ Zr 4+ Ti 4+ Ce 4+ Any one or a mixture of several of them.

[0008] The present invention also provides a method for preparing the aforementioned metal-polyphenol complex, comprising the following steps: S1. Dissolve aromatic alkylamine polyphenols in solvent I to obtain a polyphenol solution; S2. Dissolve the bioactive drug in deionized water to obtain an active drug solution; S3. Dissolve the metal cation salt in solvent II to obtain a metal cation solution; S4. Mix the polyphenol solution from S1, the active drug solution from S2, and the metal cation solution from S3 with solvent III to carry out a coordination reaction. After the reaction is complete, wash with deionized water and centrifuge to obtain the metal-polyphenol complex.

[0009] Preferably, in S1, the solvent I is water or an aqueous solution containing an alkali.

[0010] Preferably, in S3, the metal cation salt is selected from any one or a mixture of several of BaCl2, SrCl2, MgCl2, CaCl2, MnSO4, CuCl2, FeCl2, ZnSO4, Co(NO3)2, FeCl3, AlCl3, VCl3, CrCl3, EuCl3, GdCl3, ZrCl3, TiCl3, Bi(NO3)3, and (NH4)2Ce(NO3)2.

[0011] Preferably, in S3, solvent II is one of water, PBS buffer, HEPES buffer, Tris-HCl buffer, citrate buffer, or MES buffer.

[0012] Preferably, in S4, solvent III is one of PBS buffer, HEPES buffer, MOPS buffer, Tris-HCl buffer, bicine buffer, imidazole buffer, citrate buffer or MES buffer, and the pH value of solvent III is 7.4~10.5; the molar ratio of the metal cation to the aromatic alkylamine polyphenol is (100:1)~(1:100).

[0013] Preferably, the molar ratio of the metal cation to the aromatic alkylamine polyphenol is (10:1) to (0.5:1).

[0014] Preferably, in S4, the centrifugation speed is 15000 rpm and the centrifugation time is 10 min.

[0015] The present invention also provides the use of the metal-polyphenol complex as described in the preparation of medicaments for treating metabolic diseases, autoimmune diseases, infectious diseases or tumors.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a metal-polyphenol complex for sustained release of biomolecules, its preparation method, and its applications. Through coordination-driven self-assembly, it achieves efficient loading and controlled release of peptide and protein drugs. The nanoparticles are formed by the instantaneous cross-linking of metal ions and aromatic alkylamine polyphenols in a mild aqueous environment via coordination. The abundant catechol hydroxyl functional groups in the aromatic alkylamine polyphenol molecules can generate hydrogen bonds and van der Waals forces with various amino acid residues (such as amino and carboxyl groups) on the protein surface. Simultaneously, these functional groups can co-construct a dense and stable coordination network with metal ions, thereby achieving efficient loading of protein molecules and firmly fixing them within the nanoparticles. By precisely designing the molecular structure and types of aromatic alkylamine polyphenols, the intermolecular interactions between different components of the nanoparticles can be controlled. A rational intermolecular interaction network ensures both high loading rate and activity of the active drug and gives the system controllable drug release behavior, achieving long-term, sustained-release drug delivery effects. This effectively improves the in vivo stability and bioavailability of the drug, making it particularly suitable for the continuous treatment of chronic diseases.

[0017] This invention employs a green and mild reaction system, eliminating the need for toxic or volatile organic solvents. The entire reaction process is controllable and environmentally friendly. Furthermore, this method does not alter the amino acid sequence or chemical backbone structure of biomolecules, thus maximizing the preservation of their natural conformation and biological activity. The preparation method is easy to implement and highly controllable; by precisely controlling reaction parameters and synthesis conditions, nanoparticles with ideal size, morphology, and composition can be obtained. The highly controllable preparation technology results in products with uniform size and good reproducibility, facilitating large-scale production and quality control.

[0018] This metal-polyphenol complex has broad application prospects. It is not only suitable for transdermal drug delivery to promote tissue repair and regeneration, but also for intra-articular, intraperitoneal and systemic intravenous drug delivery. It can achieve efficient encapsulation and slow release of functional biomolecules, thus providing new treatment options for metabolic diseases, autoimmune diseases, infectious diseases and malignant tumors, and showing significant advantages in the continuous treatment of chronic diseases.

[0019] The complex described in this invention constructs a stable structure through controllable metal-polyphenol coordination, achieving efficient drug delivery and sustained release without altering the native conformation of the drug molecule. This technical solution possesses good universality and scalability, providing a new material basis and implementation pathway for the long-acting sustained release of biopharmaceutical drugs such as peptides and proteins.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1 The hydrated particle size distribution of bovine serum albumin-iron-polyphenol nanoparticles prepared in Example 4; Figure 2 Zeta potential diagram of bovine serum albumin-iron-polyphenol nanoparticles prepared in Example 4; Figure 3 The drug loading of the lysozyme-iron-polyphenol nanoparticles prepared in Example 1, the glucose oxidase-iron-polyphenol nanoparticles prepared in Example 3, and the bovine serum albumin-iron-polyphenol nanoparticles prepared in Example 4 (n=3). Figure 4 The in vitro release curve of the bovine serum albumin-iron-polyphenol nanoparticles prepared in Example 4; Figure 5 The results show the determination of the secondary structure of lysozyme and natural lysozyme bacteria released from the lysozyme-iron-polyphenol nanoparticles in Example 1. Figure 6 The image shows the results of the cell compatibility assessment experiment. Figure 7 This is a diagram showing the results of a cell hemolytic experiment. Figure 8 The results of changes in wound area after different treatments in diabetic mice used to establish a Staphylococcus aureus infection wound model. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] Source of experimental materials: In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0025] Example 1 This embodiment discloses a microfluidic preparation of lysozyme nanoparticles loaded with highly loaded polypeptide and protein drug complexes and iron-polyphenols.

[0026] S1. Dissolve N-[2-(3,4-dihydroxyphenyl)ethyl]-benzamide (polyphenol T1) in deionized water to obtain a polyphenol solution with a final concentration of 5 mg / mL; S2. Dissolve 1g of lysozyme in 50mL of deionized water to obtain a lysozyme solution; S3. Dissolve 1g of FeCl3·6H2O in 25mL of HEPES buffer (pH 8.5) to obtain a metal cation solution; S4. Using a syringe, draw 10 mL of the polyphenol solution with a final concentration of 5 mg / mL from S1, and 10 mL of the lysozyme solution from S2 and the metal cation solution from S3, respectively. Fix the syringes onto the microinjection pumps and mix them using a microfluidic device. After preparing the receiving device, click the start button on all microinjection pumps to start mixing. After mixing, wash with deionized water and centrifuge at 15000 rpm for 10 min to obtain lysozyme-iron-polyphenol nanoparticles.

[0027] Example 2 This embodiment discloses a method for the batch preparation of lysozyme nanoparticles loaded with highly loaded polypeptide and protein drug complexes and iron-polyphenols. The difference between this embodiment and Example 1 is that the three-phase solution is mixed by stirring.

[0028] S1. Dissolve N-[2-(3,4-dihydroxyphenyl)ethyl]-benzamide (polyphenol T1) in deionized water to obtain a polyphenol solution with a final concentration of 5 mg / mL; S2. Dissolve 1g of lysozyme in 50mL of deionized water to obtain a lysozyme solution; S3. Dissolve 1g of FeCl3·6H2O in 25mL of deionized water to obtain a metal cation solution; S4. Add the polyphenol solution with a final concentration of 5 mg / mL from S1, the lysozyme solution from S2, and the metal cation solution from S3 to HEPES buffer (pH 8.5) and shake to mix for 3 h. After the reaction is complete, wash with deionized water and centrifuge at 15000 rpm for 10 min to obtain lysozyme-iron-polyphenol nanoparticles.

[0029] Example 3 This embodiment discloses a method for the batch preparation of glucose oxidase nanoparticles loaded with iron-polyphenols, which are highly loaded with peptide and protein drug complexes.

[0030] S1. Dissolve N-[2-(3,4-dihydroxyphenyl)ethyl]-benzamide (polyphenol T1) in deionized water to obtain a polyphenol solution with a final concentration of 5 mg / mL; S2. Dissolve 1g of glucose oxidase in 50mL of deionized water to obtain a glucose oxidase solution; S3. Dissolve 1g of FeCl3·6H2O in 25mL of deionized water to obtain a metal cation solution; S4. The polyphenol solution with a final concentration of 5 mg / mL in S1, the glucose oxidase solution in S2, and the metal cation solution in S3 were added to HEPES buffer (pH 8.5) and shaken for 3 h. After the reaction was completed, the mixture was washed with deionized water and centrifuged at 15000 rpm for 10 min to obtain glucose oxidase-iron-polyphenol nanoparticles.

[0031] Example 4 This embodiment discloses a microfluidic preparation of bovine serum albumin nanoparticles loaded with iron-polyphenols, which are highly loaded with peptide and protein drug complexes.

[0032] S1. Dissolve N-[2-(3,4-dihydroxyphenyl)ethyl]-benzamide (polyphenol T1) in deionized water to obtain a polyphenol solution with a final concentration of 5 mg / mL; S2. Dissolve 1g of bovine serum albumin in 50mL of deionized water to obtain a bovine serum albumin solution; S3. Dissolve 1g of FeCl3·6H2O in 25mL of HEPES buffer (pH 8.5) to obtain a metal cation solution; S4. Using a syringe, draw 10 mL of the polyphenol solution with a final concentration of 5 mg / mL from S1, and separately draw 10 mL of the bovine serum albumin solution from S2 and 10 mL of the metal cation solution from S3. Fix the syringe on the microinjection pump and mix using a microfluidic device. After preparing the receiving device, click the start button on all microinjection pumps to start mixing. After mixing, wash with deionized water and centrifuge at 15000 rpm for 10 min to obtain bovine serum albumin-iron-polyphenol nanoparticles.

[0033] Example 5 This embodiment discloses a method for the batch preparation of bovine serum albumin nanoparticles loaded with iron-polyphenols, which are highly loaded with polypeptide and protein drug complexes. The difference between this embodiment and Example 4 is that the three-phase solution is mixed by stirring.

[0034] S1. Dissolve N-[2-(3,4-dihydroxyphenyl)ethyl]-benzamide (polyphenol T1) in deionized water to obtain a polyphenol solution with a final concentration of 5 mg / mL; S2. Dissolve 1g of bovine serum albumin in 50mL of deionized water to obtain a bovine serum albumin solution; S3. Dissolve 1g of FeCl3·6H2O in 25mL of deionized water to obtain a metal cation solution; S4. Add the polyphenol solution with a final concentration of 5 mg / mL from S1, the bovine serum albumin solution from S2, and the metal cation solution from S3 to HEPES buffer (pH 8.5) and shake to mix for 3 h. After the reaction is complete, wash with deionized water and centrifuge at 15000 rpm for 10 min to obtain bovine serum albumin-iron-polyphenol nanoparticles.

[0035] Example 6 This embodiment discloses a method for preparing bovine serum albumin nanoparticles loaded with iron-polyphenols, which are highly loaded with polypeptide and protein drug complexes. The difference between this embodiment and Example 5 is the different polyphenol structure.

[0036] S1. Dissolve N,N'-bis[2-(3,4-dihydroxyphenyl)ethyl]terephthalamide (polyphenol T2) in deionized water to obtain a polyphenol solution with a final concentration of 5 mg / mL; S2. Dissolve 1g of bovine serum albumin in 50mL of deionized water to obtain a bovine serum albumin solution; S3. Dissolve 1g of FeCl3·6H2O in 25mL of deionized water to obtain a metal cation solution; S4. Add the polyphenol solution with a final concentration of 5 mg / mL from S1, the bovine serum albumin solution from S2, and the metal cation solution from S3 to HEPES buffer (pH 8.5) and shake to mix for 3 h. After the reaction is complete, wash with deionized water and centrifuge at 15000 rpm for 10 min to obtain bovine serum albumin-iron-polyphenol nanoparticles.

[0037] Comparative Example 1 This comparative example discloses a microfluidic preparation of a highly loaded polypeptide and protein drug complex and iron-polyphenol nanoparticles.

[0038] S1. Dissolve N-[2-(3,4-dihydroxyphenyl)ethyl]-benzamide (polyphenol T1) in deionized water to obtain a polyphenol solution with a final concentration of 5 mg / mL; S2. Dissolve 1g of FeCl3·6H2O in 25mL of HEPES buffer (pH 8.5) to obtain a metal cation solution; S3. Using syringes, draw 10 mL of the polyphenol solution with a final concentration of 5 mg / mL from S1 and 10 mL of the metal cation solution from S2, respectively. Fix the syringes onto the microinjection pumps and mix them using a microfluidic device. After preparing the receiving device, click the start button on all microinjection pumps to start mixing. After mixing, wash with deionized water and centrifuge at 15000 rpm for 10 min to obtain iron-polyphenol nanoparticles.

[0039] Comparative Example 2 This comparative example discloses a microfluidic preparation of lysozyme nanoparticles loaded with highly loaded polypeptide and protein drug complexes and iron-tannic acid.

[0040] S1. Dissolve natural polyphenol tannic acid (Shanghai Dibai Biotechnology Co., Ltd.) in deionized water to obtain a tannic acid solution with a final concentration of 5 mg / mL; S2. Dissolve 1g of lysozyme in 50mL of deionized water to obtain a lysozyme solution; S3. Dissolve 1g of FeCl3·6H2O in 25mL of HEPES buffer (pH 8.5) to obtain a metal cation solution; S4. Using a syringe, draw 10 mL of the tannic acid solution from S1 with a final concentration of 5 mg / mL. Also using a syringe, draw 10 mL of the lysozyme solution from S2 and 10 mL of the metal cation solution from S3. Fix the syringes onto the microinjection pumps respectively. Mix using a microfluidic device. After preparing the receiving device, click the start button on all microinjection pumps to begin mixing. After mixing, wash with deionized water and centrifuge at 15000 rpm for 10 min to obtain lysozyme-iron-tannic acid nanoparticles.

[0041] The effectiveness of the above embodiments was verified: 1. The bovine serum albumin-iron-polyphenol nanoparticles provided in Example 4 were characterized by measuring their hydrated particle size and Zeta potential. The specific experimental procedure for determining the hydrated particle size is as follows: The above nanoparticles were added to deionized water and ultrasonically dispersed to obtain a dispersion. The hydrated particle size and particle size distribution were measured using a dynamic light scattering (DLS) instrument. Each sample was measured at least three times, and the average value was taken as the final result. The results are as follows: Figure 1 As shown.

[0042] The zeta potential of the bovine serum albumin-iron-polyphenol nanoparticles was determined by electrophoretic light scattering, and the results are as follows: Figure 2 As shown.

[0043] Depend on Figure 1 It can be seen that the particle size distribution of the bovine serum albumin-iron-polyphenol nanoparticles provided in Example 4 ranges from 100 to 500 nm.

[0044] Depend on Figure 2 It can be seen that the potential of bovine serum albumin-iron-polyphenol nanoparticles is approximately -3.5mV, and they carry a negative charge.

[0045] 2. The drug loading of the lysozyme-iron-polyphenol nanoparticles prepared in Example 1, the glucose oxidase-iron-polyphenol nanoparticles prepared in Example 3, and the bovine serum albumin-iron-polyphenol nanoparticles prepared in Example 4 was determined. The specific experimental procedure is as follows: the supernatant after centrifugation was collected, and the protein concentration in the supernatant was analyzed by HPLC. Based on the total amount of protein initially added and the content of free protein detected in the supernatant, the amount of protein loaded in the nanoparticles was calculated.

[0046] ; The results are as follows Figure 3 As shown.

[0047] Depend on Figure 3 It can be seen that the drug loading of the obtained lysozyme-iron-polyphenol nanoparticles is 61.2%, the drug loading of the obtained glucose oxidase-iron-polyphenol nanoparticles is 93.2%, and the drug loading of the obtained bovine serum albumin-iron-polyphenol nanoparticles is 84.8%.

[0048] 3. The in vitro release amount of bovine serum albumin-iron-polyphenol nanoparticles in Example 4 was determined. The specific experimental procedure is as follows: The bovine serum albumin-iron-polyphenol nanoparticles prepared in Example 4 were dispersed in phosphate buffer at 37°C. At different time points, the supernatant was collected by centrifugation, and an equal volume of fresh phosphate buffer was added. This process was repeated for eight consecutive days. The cumulative release amount at each time point was determined by HPLC analysis. The results are as follows: Figure 4 As shown.

[0049] Depend on Figure 4 It is known that bovine serum albumin-iron-polyphenol nanoparticles exhibit a continuous and slow release behavior under in vitro conditions, and the release process can be stably maintained for 8 days, effectively avoiding burst release.

[0050] 4. The secondary structures of lysozyme and natural lysozyme bacteria released from the lysozyme-iron-polyphenol nanoparticles in Example 1 were determined by circular dichroism (CD) spectroscopy. All circular dichroism data are expressed as mean residue ellipticity. The results are as follows: Figure 5 As shown.

[0051] Depend on Figure 5 It is evident that the conformational structure of lysozyme remained unchanged after being encapsulated in lysozyme-iron-polyphenol nanoparticles, and the natural lysozyme and the released lysozyme exhibited similar CD spectra. The molar ellipticity ratio between the negative bands at 208 nm and 222 nm indicates that the biological activity of this protein was well preserved during the assembly and release of the nanoparticles.

[0052] 5. Cell compatibility assessment experiment, the specific experimental protocol is as follows: Normal mouse embryonic fibroblasts were distributed at a density of 1 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 1000 mg / mL in 96-well plates and cultured at 37°C and 5% CO2 until cell adhesion occurred. Then, different concentrations (0, 0.125, 0.25, 0.5, 0.75, 1 mg / mL) of lysozyme, iron-polyphenol nanoparticles (Comparative Example 1), and the lysozyme-iron-polyphenol nanoparticle complex (Example 1) were added to the culture medium, and cultured for another 24 hours. Afterward, CCK-8 solution was added to each well, and incubation was continued for 2 hours. The absorbance of each well at 450 nm was measured using a microplate reader. The results are shown below. Figure 6 As shown.

[0053] Depend on Figure 6 It can be seen that even when the material concentration is increased to 1 mg / mL, the relative survival rate of the three groups of treated cells remains above 90%. Therefore, lysozyme, iron-polyphenol nanoparticles and lysozyme-iron-polyphenol nanoparticles have good in vitro cell compatibility.

[0054] 6. In the hemolytic activity assay, the specific experimental protocol was as follows: Fresh whole blood was collected, centrifuged, and the red blood cells were resuspended in phosphate buffer to prepare a homogeneous red blood cell suspension. The red blood cell suspension was then incubated with different concentrations of lysozyme-iron-polyphenol from Example 1 at 37°C for 3 hours. After incubation, the samples were centrifuged, and the absorbance of the supernatant at 540 nm was measured to assess the amount of hemoglobin released and calculate the hemolysis rate. The results are as follows: Figure 7 As shown.

[0055] Depend on Figure 7 It can be seen that even at the highest test concentration of 1 mg / mL, the hemolysis rate of the material is still less than 5%, indicating that the nanoparticles have good blood compatibility and biocompatibility.

[0056] 7. Animal experiments, the specific experimental protocol is as follows: To verify the therapeutic potential of lysozyme-iron-polyphenol (LYZ@Fe-T1) nanoparticles from Example 1 in infectious diabetic wounds, a Staphylococcus aureus infection wound model was established on the backs of streptozotocin-induced diabetic mice. A full-thickness skin excision wound with a diameter of 10 mm was created on the back of each mouse. On day 0, the wounds were treated with PBS (Control), lysozyme (LYZ), iron-polyphenol nanoparticles (Fe-T1) from Comparative Example 1, lysozyme-iron-tannic acid (LYZ@Fe-TA) from Comparative Example 2, or lysozyme-iron-polyphenol (LYZ@Fe-T1) nanoparticles from Example 1, respectively. The wounds were photographed on days 0, 3, 7, 10, and 14, and the changes in wound area were tracked. The results are as follows: Figure 8 As shown.

[0057] Depend on Figure 8 It can be seen that, compared with the blank group, the wound shrinkage of the LYZ, Fe-T1 and LYZ@Fe-TA groups was limited, and there were still obvious unhealed areas in the wound; while the LYZ@Fe-T1 treatment group had the smallest wound area and the healing speed was significantly better than the other groups.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A metal-polyphenol complex for sustained release of biomacromolecules, characterized in that, The complex comprises metal ions, aromatic alkylamine polyphenols, and bioactive macromolecules; Wherein, the aromatic alkylamine polyphenol is a compound having a general structural formula as shown in formula (I), (II), (III), or (IV), or a pharmaceutically acceptable salt thereof: ； Wherein, R1 is a benzene ring with -H or -OH at positions 2, 3, 4, 5, and 6, and at least two adjacent positions are simultaneously -OH; R2 represents the benzene ring with positions 2, 3, 4, 5, and 6 each being -H, -OH, -OR3, or -CN, where R3 is an alkyl group; and n is an integer from 1 to 100.

2. The metal-polyphenol complex according to claim 1, characterized in that, The bioactive macromolecular drugs are water-soluble polypeptides or proteins, including insulin, growth hormone, calcitonin, salmon calcitonin, teriparatide, liraglutide, dulaglutide, smegglutide, teduglutide, exenatide, beraglutide, antigepigide, liximab, loxenatide, duraglutide, semaglutide, abiglutide, liximab, leuprorelin, prorelin, gonadorelin, goserilin, buserorelin, sermorelin, nafarelin, histaminerelin, lanreotide, octreotide, somatostatin, terlipressin, atosiban; thymosin, thymopentin, thymosin, bone peptide, sarcosin, glutathione, and glycosides. Glycopeptides, Yunzhi glycopeptides, ginseng glycopeptides; hemoglobin, hemochloroglobin, serum albumin, bovine serum albumin, ovalbumin, ovalbumin, whey protein, β-lactoglobulin, lactoferrin, conalbumin, globulin, myosin, sarcoplasmic protein, keratin, casein, mucin, mucin, ferritin, transferrin, collagen, immunoglobulin G, cytochrome C, fibrinogen; trypsin, pepsin, pancreatic enzymes, papain, figase, chymotrypsin, histamine enzyme, thrombin, anterior pituitary troponin, thyrotropin-releasing hormone, parathyroid hormone, fibrinolytic enzyme, glucose oxidase Horseradish peroxidase, hyaluronidase, asparaginase, streptokinase, streptokinase, lysozyme, urokinase, reteplase, amylase, cellulase, collagenase, prothrombin kinase, pancreatic deoxyribonuclease; enfuviride, nasitide, bradykinin, enkephalin, dulaglutide, cortisone, epitubide, acimetidine, desmopotine, veracitide, bivalirudin, vasopressin, phenyltyrosine, sicalide, pentagastrin, secretin, vasoactive intestinal peptide, follicle-stimulating hormone, gonadotropin, prolactin, edotrexate, pentotrieptide, procarbamate, phenobarbital, sargastin; interferon -α, interferon-β, interferon-γ, anaerobicin, dinesiac interleukin, bone morphogenetic protein; bacitracin, brevicin, colistin, polymyxin sulfate, polymyxin, teicoplanin, vancomycin, norvancomycin, tervacin, daptomycin, glatiramer, glatiramer acetate, mirvaminide, nesiritide, carfilzomib, bortezomib, micafungin, anidofungin, caspofungin; botulinum toxin, coagulation factor VIII, coagulation factor IX, aprotinin, hirudin, ziconopeptide, enfuviride, thymopentin, angiotensin, atosiban, trachotoxin, bone peptide, any one or a mixture of several of these.

3. The metal-polyphenol complex according to claim 1, characterized in that, The metal ions are selected from Ba. 2+ 、Sr 2+ Mg 2+ Ca 2+ Fe 2+ Mn 2+ Cu 2+ Zn 2+ Co 2+ Ni 2+ Fe 3+ Al 3+ V 3+ Cr 3+ Zr 4+ Ti 4+ Ce 4+ Any one or a mixture of several of them.

4. A method for preparing a metal-polyphenol complex as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Dissolve aromatic alkylamine polyphenols in solvent I to obtain a polyphenol solution; S2. Dissolve the bioactive drug in deionized water to obtain an active drug solution; S3. Dissolve the metal cation salt in solvent II to obtain a metal cation solution; S4. Mix the polyphenol solution from S1, the active drug solution from S2, and the metal cation solution from S3 with solvent III to carry out a coordination reaction. After the reaction is complete, wash with deionized water and centrifuge to obtain the metal-polyphenol complex.

5. The method according to claim 4, characterized in that, In S1, solvent I is water or an aqueous solution containing alkali.

6. The method according to claim 4, characterized in that, In S3, solvent II is one of water, PBS buffer, HEPES buffer, Tris-HCl buffer, citrate buffer, or MES buffer.

7. The method according to claim 4, characterized in that, In S4, solvent III is one of PBS buffer, HEPES buffer, MOPS buffer, Tris-HCl buffer, bicine buffer, imidazole buffer, citrate buffer or MES buffer, and the pH value of solvent III is 7.4~10.5; the molar ratio of the metal cation to the aromatic alkylamine polyphenol is (100:1)~(1:100).

8. The method according to claim 4, characterized in that, In S4, the centrifugation speed is 15000 rpm and the centrifugation time is 10 min.

9. The use of the metal-polyphenol complex according to any one of claims 1-3 in the preparation of a medicament for treating metabolic diseases, autoimmune diseases, infectious diseases or tumors.

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