A polypeptide polyphenol nanogel and a coating manufacturing method and application thereof

Abstract

The application discloses a kind of polypeptide polyphenol nanogel and its coating manufacturing method, application, belong to the functional coating field of medical instrument surface or local administration carrier, raw material includes polypeptide, polyphenol component, boric acid and its derivative, oxidizing agent and other auxiliary reagent, the structure of the polypeptide is AAS-SAA type, including A peptide and B peptide coupled by disulfide bond at C terminal, the A peptide is MMP response type peptide;The polyphenol component contains catechol structure;The oxidizing agent is sodium periodate solution.Form dynamic reversible disulfide bond, dynamic reversible schiff base bond, dynamic reversible catechol-boric acid ester bond, the synergistic cooperation of three kinds of reversible dynamic bond, and introduce cutting peptide to MMP response in polypeptide part, realize to MMP and ROS response " double gate control ", with accurate selectivity.

Description

Technical Field

[0001] This invention belongs to the field of functional coatings for medical device surfaces or local drug delivery carriers, specifically relating to a polypeptide polyphenol nanogel and its coating preparation method and application. Background Technology

[0002] Currently, common technical approaches for functional coatings used on the surfaces of medical devices or as local drug delivery carriers include self-adhesive coatings based on polyphenols such as dopamine / tannic acid, and hydrogel films relying on small molecule crosslinking agents such as glutaraldehyde. However, existing coating components are often single-stimulus-responsive (pH or temperature) controlled-release systems, resulting in poor selectivity. In microenvironments such as tumors and inflammation, complex characteristics such as elevated matrix metalloproteinases (MMPs), enrichment of reactive oxygen species (ROS), and mild acidification occur. Most existing polyphenol or hydrogel coatings can only respond to one of these signals, leading to insufficient release precision and spatiotemporal selectivity. Furthermore, due to the influence of signal response mechanisms, most drug delivery systems release too quickly initially, making it difficult to achieve on-demand delivery based on "release after lesion signal triggering," often resulting in "burst release."

[0003] Secondly, existing coating crosslinking methods are often rigid or irreversible. For example, traditional glutaraldehyde or epoxy coatings are prone to embrittlement and difficulty in self-repair due to irreversible crosslinking, and may also lead to cytotoxic residues. Furthermore, coatings connected by single hydrogen bonds or π-π interactions are prone to detachment under blood flow or dynamic scouring, and have insufficient durability under fluid shear or body fluid environments.

[0004] Therefore, there is an urgent need for a smart coating for implantable or interventional medical devices, postoperative cavities, or tumor local precision drug delivery systems that can achieve dual signal gating and has properties such as adhesion and self-healing. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying polypeptide polyphenol nanogels and their coatings.

[0006] The objective of this invention is achieved through the following technical solution: The first aspect of this invention is to provide a polypeptide-polyphenol nanogel, comprising a polypeptide, a polyphenol component, boric acid or a derivative thereof, an oxidant, and other auxiliary reagents. The polypeptide has an AAS-SAA structure, comprising an A peptide and a B peptide coupled at the C-terminus via disulfide bonds, wherein the A peptide is an MMP-responsive peptide. The polyphenol component contains a catechol structure. The polypeptide accounts for 15-45 wt% of the solids in the raw materials. The total amine equivalent (A) of the polypeptide is... e The equivalent C of the catechol structure that can react with the polyphenol component e The ratio is A e ∶C e=0.6~1.2∶1; the molar ratio of the reactive catechol structure in the polyphenol component to the boric acid or its derivative is 1.0~2.5∶1; the oxidant is sodium periodate solution.

[0007] This application first designs an AAS-SAA type polypeptide, comprising peptide A and peptide B. Both short peptides contain a cysteine ​​residue (Cys) at their C-terminus, forming a disulfide bond coupling, thus constituting the AAS-SAA type polypeptide of this application. Peptide A is selected as an MMP-responsive peptide, capable of responding promptly to a microenvironment with high MMP expression, such as through specific cleavage of peptide A to achieve drug release. Peptide A and peptide B are coupled via a disulfide bond through the C-terminal Cys residue. After peptide A responds to the MMP-high expression environment, such as by releasing a drug, the presence of the dynamically reversible disulfide bond (-SS-) allows peptide A and peptide B to reconnect, achieving self-repair of the coating. Peptide B can be optionally selected as a short peptide with other functions or responses, such as a TAT-penetrating peptide, to achieve a multifunctional complex within the polypeptide.

[0008] Secondly, in conjunction with the polyphenol component, a catechol structure capable of being oxidized to a quinone group is incorporated. This allows the quinone group to covalently couple with the amino groups in the peptide through Schiff base reactions and Michael addition. Compared to traditional hydrogen bonds, this stronger linkage effectively prevents the peptide from detaching from the coating. Furthermore, the quinone group can covalently react with most active groups on the substrate surface, such as hydroxyl, amino, and thiol groups, forming a covalent bond between the coating and the substrate. This significantly enhances the overall adhesion of the coating, preventing detachment from blood vessels or tumor sites. More importantly, the Schiff base bond (-CH=N-) formed by the quinone group exhibits a dynamic response to the environment. For example, it breaks in the acidic microenvironment of a tumor and re-condenses under neutral or alkaline conditions, enabling intelligent drug delivery. Sodium periodate (NaIO4) solution is chosen as the oxidant for oxidizing the catechol structure, as it has high selectivity for the catechol structure and operates under mild conditions, without damaging the activity of other components in the coating.

[0009] Simultaneously, the dehydration condensation of boric acid or its derivatives with the remaining unoxidized catechin structure to form a reversible catechin-boronic acid ester bond can specifically respond to ROS, specifically reactive oxygen species such as ·OH and O2. - The reaction involves the addition of substances such as H2O2. ROS first oxidizes the catechol fragments in catechol borate esters, disrupting the stable structure of the five-membered ring, then triggering the hydrolytic breakage of the borate ester bonds, dissolving intra-chain / side ring crosslinks, and increasing porosity and permeability. Subsequently, localized catechols are further oxidized by ROS to quinones, breaking the metal-phenol coordination and reversibly rearranging with water / amine, resulting in dynamic pore reconstruction. Finally, MnO2 nanosheets are added as an auxiliary reagent to amplify the response to ROS: MnO2 + H2O2 → O2↑ + Mn 2+The consumption of H2O2 reduces non-specific oxidation, generates micro- and nano-sized O2 bubbles, produces transient stress, opens micropores, and allows a small amount of Mn to enter. 2+ It also participates in secondary metal-phenol coordination, providing a self-repair pathway. Through these actions, the catechol-boronic acid ester bond achieves precise dissociation in ROS-high expression environments (such as the tumor microenvironment) to meet the targeted release requirements of drug delivery coatings. Specifically, boric acid is chosen for two reasons: firstly, it reacts well with the catechol structure; the five-membered ring structure of the catechol-boronic acid ester forms steric hindrance, effectively "shielding" some other structures from contact with ROS, and the electron-deficient boron atom enhances ROS selectivity; secondly, the catechol-boronic acid ester bond is also dynamically reversible. During coating preparation, a stable five-membered borononic acid ester bond is formed, resulting in a dense coating structure. However, in a ROS-high expression microenvironment, the bond undergoes hydrolysis and breakage, releasing the drug. Subsequently, when the environment is suitable, such as when ROS expression is reduced or in a neutral environment, the boronic acid ester bond can reform, preventing excessive drug release and completing the coating's self-repair.

[0010] In summary, this invention designs a coating containing three main components: polypeptides, polyphenols, and boric acid. This coating forms three reversible dynamic bonds: dynamic reversible disulfide bonds, dynamic reversible Schiff base bonds, and dynamic reversible catechol-boronic acid ester bonds. The synergistic cooperation of these three reversible dynamic bonds, along with the introduction of MMP-responsive cleaving peptides into the polypeptide portion, achieves "dual gate control" for MMP and ROS responses. Simultaneously, it also possesses both coating adhesion and self-healing capabilities.

[0011] Furthermore, by controlling the proportion of peptides, the ratio of amino groups to reactive catechol structures, and the ratio of catechol structures to boric acid structures in the raw materials, the formation of cross-linking networks and the generation of dynamic bonds can be controlled. In the tumor microenvironment, if ROS appears in large quantities first, it will lead to the destruction of the cross-linking structure and premature delamination of the coating. If MMPs are inhibited by plasma inhibitors, the "MMP gate" cannot open. Therefore, by controlling the proportion of peptides and the molar ratio between the reacting structures, "Gate 1" (MMP) is located at a more topologically critical site, such as an interchain bridge, while "Gate 2" (ROS) is mainly placed in weak intrachain cross-links, allowing the raw materials to exert a better synergistic effect. Specifically, the total amine equivalent A... e With reactive catechol equivalent C e Satisfy: A e ∶C e =0.60~1.20∶1.00. Wherein, total amine equivalent A e Determined by the OPA method, the concentration of reactive amines (mM) in the system is based on the N-terminus and Lys side chain of the peptide; the reactive catechol equivalent (C) is also measured. e The concentration (mM) of catechol sites in the system that can be selectively oxidized to quinones is reflected by the catechol sites in DHBA / TA.

[0012] When Ae ∶C e When the ratio is approximately 0.6:1, i.e., amine is insufficient and quinone is excessive, amine acts as the rate-limiting node. The amine-quinone Schiff base / Michael bond preferentially occurs at the intersections of the polypeptide backbone with the highest spatial accessibility, especially at the two amine / N-terminus of AAS-SAA, forming interchain bridges / backbone nodes. These nodes are most sensitive to network integrity. Simultaneously, due to the excess quinone, the residual catechols at the ends of polyphenolic components such as DHBA / TA tend to be captured by boric acid or its derivatives via 1,2-diol, forming catechol-boron ester inner / side rings (mostly distributed within the chain segment and side chain). These weak intra-chain crosslinks contribute little to the overall load-bearing capacity but are extremely sensitive to ROS oxidation / hydrolysis. Secondly, when metal-phenol coordination is involved, such as Fe... 3+ At (0.2~0.3mM), it acts as a reconfigurable "secondary network" located in the polyphenol-rich region, placed far from the main chain nodes. In this case, the MMP gate (A peptide cleavage) is naturally located at the "inter-chain master node / bridge site" (cleavage = network de-ordering), while the ROS gate (boron ester + catechol oxidation) is mainly located at the "intra-chain weak crosslinks / side loops" (disconnection = instantaneous pore expansion, flux increase). When A... e ∶C e When the ratio is approximately 1.2:1, i.e., amine is in excess and quinone is deficient, the Schiff base / Michael bond density increases and occurs more frequently on the chain segment surface and at branching sites. The overall network is more flexible, and significant relaxation occurs immediately after MMP cleavage. At this point, the borate ester ratio is relatively low but still acts as a secondary gate opener, achieving the criteria of "MMP first, then ROS" or "opening with any triggering part, significant opening with dual triggering." Therefore, A... e ∶C e Within a window of 0.60 to 1.20:1, the system exhibits a self-consistent spatial division of labor between "strong interchain nodes (amine-quinone)" and "weak intrachain crosslinks (catechol-boronic acid ester / metal-phenol)," thereby anchoring MMP and ROS at their optimal destruction sites and achieving a series-parallel synergy of the gates: MMP preferentially destroys topologically critical nodes, ROS rapidly removes weak crosslinks and expands pores, and the release exhibits a step-amplification when the two signals are superimposed, while leakage is minimized under either single signal.

[0013] Preferably, the sequence of the A peptide is any one of: GPQGIAGQC (SEQ ID NO:1) (classic MMP-2 / 9 cleavage peptide), GPLGIAGQC (SEQ ID NO:2), PVGLIGRC (SEQ ID NO:3), PLGLWAGGC (SEQ ID NO:4) (adding GG to reduce steric hindrance), GPLGVRGC (SEQ ID NO:5), GPQGIWGQC (SEQ ID NO:6), GPLGIAGKC (SEQ ID NO:7) (introducing Lys amine near the C-terminus) or VPLSLYSGC (SEQ ID NO:8) (MMP-14 oriented).

[0014] AAS-SAA type peptides typically have an amino acid sequence length of 4-16 aa. As an MMP-responsive peptide, peptide A can have 1-2 Gly or Ser atoms added before the C-terminus as flexible spacers to reduce steric hindrance and increase the cleavage rate, in addition to the C-terminus being Cys for easy disulfide bond formation. Placing Gly or Gly-Ser spacers on both sides of the disulfide bridge between peptide A and peptide B can enhance enzyme exposure.

[0015] Preferably, the sequence of the B peptide is any one of the following: RKKRRQRRRC (SEQ ID NO:9) (TAT peptide), YGRKKRRQRC (SEQ ID NO:10) (short TAT peptide), GRGDSPGGC (SEQ ID NO:11) (RGD fibronectin targeting), CREKAGGC (SEQ ID NO:12) (thrombus / fibrin affinity), GNGRAHAGC (SEQ ID NO:13) (NGR / CD13 targeting linearization), RPARPARPGC (SEQ ID NO:14) (CendR / NRP-1 related permeation), KHHKHKHKC (SEQ ID NO:15) (His–Lys enrichment, pH / zinc response), or SKIKKPGGC (SEQ ID NO:16) (mildly enhances amines, maintains short sequence).

[0016] The B peptide coupled to the A peptide can be a peptide with various functions such as targeting, membrane penetration, and electrostatic enrichment, such as the TAT penetrating peptide. When increasing amine density is required, Lys can be preferentially introduced into the sequence of either the A or B peptide; while for stronger targeting, the series containing RGD / CREKA / NGR is preferred.

[0017] Preferably, the polyphenolic component is a combination of tannic acid and 3,4-dihydroxybenzaldehyde or dopamine.

[0018] When the polyphenolic component contains 3,4-dihydroxybenzaldehyde (DHBA), the quinone group generated can be oxidized in the presence of trace amounts of oxidants without destroying the aldehyde group. The aldehyde group can then undergo dehydration condensation with the amino group of the polypeptide to form additional Schiff base bonds, forming a synergistic effect with other corresponding mechanisms, such as enhancing the pH response. Simultaneously, tannic acid (TA) contains hydrogen bonds for metal coordination, enabling it to coordinate with metal ions to form a stable metal-phenol cross-linking network and improve biocompatibility.

[0019] It is also understood that, in addition to the preferred tannic acid and 3,4-dihydroxybenzaldehyde, the polyphenols in this application can also be other polyphenols with a catechol structure, such as the common dopamine.

[0020] Preferably, the boric acid or its derivative is 4-formylphenylboronic acid.

[0021] Boric acid or its derivative, 4-formylphenylboronic acid (FPBA), is preferred. It forms a reversible catechol-boronic ester bond with the catechol structure, providing additional aldehyde groups to participate in the Schiff base reaction. FPBA can simultaneously introduce ROS-reversible boronic esters and pH-reversible Schiff bases, increasing the density of reversible crosslinking sites without increasing toxicity, reducing initial leakage, and improving the signal resolution of the dual-gate system. Specifically, FPBA possesses a -CHO aldehyde group, which forms -C=N- with primary amines at pH 7.4~8.5, providing a reversible covalent anchor. Simultaneously, it accelerates hydrolysis at low pH (tumor / inflammation), facilitating on-demand release and self-repair reconstruction. Secondly, it forms a "site-protection" effect: FPBA first forms a reversible borate ester with catechol, limiting the opportunity for catechol to participate in amination, reducing early irreversible cross-linking, and inhibiting initial burst release. As ROS increases, the borate ester is oxidized / hydrolyzed, delaying the opening of secondary cross-linking / channels. Finally, the FPBA-catechin five-membered ring forms a fixed coordination geometry, making it easier to form a dense "secondary network" when coordinating with metal ions. After dissociation under ROS, the network relaxation increases, achieving a reversible "on-off" process.

[0022] Preferably, other auxiliary reagents in the raw materials include: nanozymes, wherein the nanozymes are MnO2 nanosheets, accounting for 0.2 to 2.0 wt% of the solid mass fraction in the raw materials.

[0023] Common nanozymes include manganese dioxide and iron oxide, but this application primarily uses manganese dioxide (MnO2) nanosheets. MnO2 nanozymes can alleviate tumor hypoxia and enhance the synergistic effect of some therapies (chemotherapy / immunotherapy). Common MnO2 nanosheets have a side length of 50-200 nm and a thickness of 2-10 nm, with preferred mass ratios provided. In the presence of H2O2, they can better catalyze the oxidation of catechol structures to quinones, thereby improving the sensitivity to ROS responses. Simultaneously, they can serve as a nanoskeleton to enhance structural stability. Introducing MnO2 nanosheets into the ROS-responsive part acts as an additional "gateway" for ROS responses. They are often sensitive to ·OH / H2O2, but are not falsely triggered at low ROS levels, exhibiting high selectivity.

[0024] Preferably, other auxiliary reagents in the raw materials include: a coordinating metal ion solution, wherein the concentration of the coordinating metal ions is 0.1~5.0 mM, and the coordinating metal ions include: Fe 3+ Zn 2+ or Ca 2+ Any one of them.

[0025] Based on the presence of tannic acid in polyphenols, the coordinateable hydrogen bonds of tannic acid can be used to selectively form a metal-phenol coordination secondary network with a certain concentration of metal ion solution. This can improve the toughness and density of the coating structure and enhance its resistance to body fluid erosion through coordination reconstruction.

[0026] A second aspect of the present invention provides a method for preparing a coating of polypeptide polyphenol nanogel, which involves using the aforementioned nanogel as a coating material and coating it onto a substrate to form a coating, comprising the following steps: S1 Ingredients: At room temperature, take 1-5 mg / mL of the AAS-SAA type polypeptide, 0.5-2 mg / mL of tannic acid, 1-5 mM of 3,4-dihydroxybenzaldehyde, and 1-3 mM of 4-formylphenylboronic acid, and add them sequentially to a 10 mM phosphate buffer solution with a pH of 7.4-8.5. Stir for 8-15 min to obtain a pre-prepared solution. If necessary, small molecules in the pre-prepared solution can be removed by ultrafiltration or dialysis. Adjust the pH to weakly alkaline (pH 7-9) using the phosphate buffer solution to ensure that tannic acid and other components are oxidized to quinone groups without destroying the bioactive components such as polypeptides and nanozymes.

[0027] S2 Oxidative Crosslinking: Add 0.05-0.50 mM NaIO4 solution dropwise to the pre-crosslinking solution, wherein the molar ratio of NaIO4 to the hydroxyl groups of the catechol structure in the pre-prepared solution is 0.02-0.08. After reacting for 15-30 min, add MnO2 nanosheets, with an endpoint concentration of 0.01-0.05 mg / mL, and stir for another 8-12 min to obtain a nanogel. The endpoint concentration here is calculated based on the total volume of the system after the reaction (i.e., the total volume of "pre-prepared solution + NaIO4 solution + MnO2 dispersion"). For ease of implementation, a concentration of 0.1-0.5 mg / mL is usually prepared first. -1 The amount of MnO2 stock solution to be added is calculated based on the total volume.

[0028] S3 Film Formation and Fixation: The pretreated substrate is immersed in the nanogel solution at 25~37℃ for 10~30 min. After removal, it is rinsed with phosphate buffer and then immersed in a 0.5~2mM coordination metal ion solution with pH=7.2~7.6 for 2~5 min to achieve dense metal-phenol coordination. The nanogel immersion and coordination metal ion solution immersion are repeated 1~5 times, each time to make the film thickness 100~170nm, so that the final total film thickness is 0.5~1μm. After rinsing, it is dried at 37℃ for 0.5~2h to obtain the polypeptide polyphenol nanogel coating on the surface of the substrate.

[0029] To facilitate coating, the prepared nanogel is usually diluted into a solution by adding a certain amount of buffer solution as needed, such as adding phosphate buffer solution, while providing a suitable pH environment for the gel.

[0030] The coating fabrication method of this application includes steps S1-S2 to first form a nanogel, followed by step S3 to fix it onto the substrate surface by impregnation to form a film. The uncoated nanogel has a particle size of 80-180 nm (PDI < 0.25) and a zeta potential of +10 to +25 mV. In step S3, after impregnating the nanogel to form the coating, the film thickness (QCM-D / ellipsometry) varies with the number of times the substrate is impregnated into the nanogel and metal ion solution. The film thickness after a single impregnation is 30-150 nm, and the coating film thickness after multiple impregnation cycles can reach 0.5-3 µm, which can effectively perform the coating function. Adhesion ≥4B (cross-cut adhesion, wet state ≥3B) is achieved on metal or polymer substrates through polyphenol adhesion and metal-phenol coordination.

[0031] Optionally, drug loading and end-capping can be performed during the preparation of the coating gel. For example, hydrophobic / hydrophilic drugs (such as DOX, paclitaxel, vancomycin) can be co-loaded in step S1, or loaded in step S3 by impregnation-adsorption. Unreacted quinones can be lightly end-capped with Tris-HCl (10~20mM, pH=7.4, 5min). The process conditions are: all-aqueous phase, pH=7.0~8.8, temperature 20~37℃, and total time ≤2h.

[0032] Preferably, the substrate pretreatment process in S3 includes pre-impregnating the substrate with tannic acid at a concentration of 0.5-2 mg / mL and a pH of 7.5-8.5 for 2-10 minutes. Additionally, for low surface energy polymers such as PTFE / ePTFE and medical silicone rubber, oxygen plasma activation for 30-120 seconds can be performed before pre-impregnation to improve the adhesion of the substrate.

[0033] The gel material of this application is suitable for coating various substrates, such as stainless steel, Ti alloys, Co-Cr, polyurethane (PU), polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), and medical silicone rubber. Substrate pretreatment is typically required. Metal substrates are ultrasonically cleaned (acetone:ethanol:water = 1:1:1) and dried with N2; polymer substrates are cleaned with oxygen plasma for 30-120 seconds. Preferably, tannic acid pre-impregnation can be used as an underlayer to initiate adhesion.

[0034] A third aspect of the present invention is to provide an application of a polypeptide polyphenol nanogel, wherein the polypeptide polyphenol nanogel is coated on the surface of an anticancer local drug delivery carrier, an anti-infection catheter, or a postoperative anti-recurrence cavity adhesive membrane.

[0035] The beneficial effects of this invention are: (1) This invention designs a coating gel containing three main components: polypeptide, polyphenol, and boric acid. It forms dynamic reversible disulfide bonds, dynamic reversible Schiff base bonds, and dynamic reversible catechol-boronic acid ester bonds. The three reversible dynamic bonds work together synergistically. In addition, a cleavage peptide that responds to MMP is introduced into the polypeptide part to achieve "dual gate control" of MMP and ROS response. It has precise selectivity, significantly reduces spontaneous release in healthy tissue (low MMP and ROS), and triggers step release at lesion sites (high MMP and ROS). The pharmacokinetic time-space window is narrower and more accurate.

[0036] (2) The AAS-SAA type polypeptide of the present invention can be compounded with a variety of short peptides with different functions to achieve functional compounding and has a stable expansion effect. The sequences of short peptides A and B can be replaced with RGD, NGR, CREKA, Angiopep-2, etc. in the range of 4 to 16aa to achieve different disease targeting and penetration requirements.

[0037] (3) The polyphenol component of the present invention forms catechol borate ester with boric acid, which acts as a second gate strongly correlated with ROS. While MMP cuts and destroys the main chain nodes, ROS induces borate ester decomposition and catechol oxidation. Significant release can only be achieved when the two signals are satisfied simultaneously or sequentially, which significantly reduces nonspecific leakage.

[0038] (4) This invention utilizes multiple dynamic bonds to achieve self-healing of the coating. Through the triple reversible network of disulfide bond exchange, metal-phenol coordination reconstruction, and reversible borate ester bonds, cracks or defects that may appear in the coating can be re-crosslinked in situ under wet conditions. The flux can be partially restored after scratches, and it can adapt to the blood flow environment. At the same time, polyphenol adhesion and metal coordination endow the coating with the ability to form films in situ on metals and inert polymers. It can achieve an adhesion of ≥3B without a primer. The coating has both adhesion and self-healing capabilities, giving full play to the synergistic effect between raw materials.

[0039] (5) The coating of the present invention is synthesized under conditions of all-aqueous phase, room temperature 37°C, and mild pH, without the need for toxic crosslinking agents, has better biocompatibility, and is suitable for various substrates and large-scale impregnation and spraying. By adjusting the ratio of amine / quinone and catechol / boric acid, the number of metal ion densification cycles, etc., the synergistic release effect is significantly amplified and the membrane mechanics and density are appropriately adjusted. Attached Figure Description

[0040] Figure 1 This relates to the dual-gate chemical mechanism of the polypeptide-polyphenol nanogel coating of the present invention.

[0041] Figure 2 This is the cumulative release curve of the coating in Example 3 of the present invention.

[0042] Figure 3 This is a graph showing the scratch width recovery rate test results of the coating in Embodiment 3 of the present invention.

[0043] Figure 4 This is a graph showing the relationship between the number of immersion cycles and the coating thickness in Example 3 of the present invention.

[0044] Figure 5 This is a diagram showing the wet shear adhesion test results of the substrate surface in Embodiment 3 of the present invention.

[0045] Figure 6 The figure shows the ROS removal capability test results of the coating in Example 3 of the present invention.

[0046] Figure 7 This is a diagram showing the compatibility test results between the coating and cells in Example 3 of the present invention.

[0047] Figure 8 This is a diagram showing the hemolysis experiment results of Example 3 of the present invention. Detailed Implementation

[0048] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] See Figure 1 The present invention provides a technical solution, comprising the following steps: S1 Ingredients: At room temperature, take 1~5 mg / mL of the AAS-SAA type polypeptide, 0.5~2 mg / mL of tannic acid, 1~5 mM of 3,4-dihydroxybenzaldehyde, and 1~3 mM of 4-formylphenylboronic acid, and add them sequentially to a phosphate buffer solution with a pH of 7.4~8.5 and a concentration of 10 mM. Stir for 8~15 min to obtain a pre-prepared solution. If necessary, small molecules in the pre-prepared solution can be removed by ultrafiltration or dialysis.

[0050] S2 Oxidative Crosslinking: Add 0.05~0.50mM NaIO4 solution dropwise to the pre-prepared solution, wherein the molar ratio of NaIO4 to the hydroxyl groups of the catechol structure in the pre-prepared solution is 0.02~0.08. After reacting for 15~30min, add MnO2 nanosheets, with an endpoint concentration of 0.01~0.05mg / mL, and stir for another 8~12min to obtain nanogel.

[0051] S3 Film Formation and Fixation: The pretreated substrate is immersed in the nanogel at 25~37℃ for 10~30 min. After removal, it is rinsed with phosphate buffer and then immersed in a 0.5~2mM coordination metal ion solution with pH=7.2~7.6 for 2~5 min to achieve dense metal-phenol coordination. The nanogel immersion and coordination metal ion solution immersion are repeated 1~5 times, each time to make the film thickness 100~170nm, so that the final total film thickness is 0.5~1μm. After rinsing, it is dried at 37℃ for 0.5~2h to obtain the polypeptide polyphenol nanogel coating on the surface of the substrate.

[0052] Example 1

[0053] The implementation process of this embodiment follows the specific implementation method, controlling the target synthesis ratio as: total amine:quinone = 2.00:3.33 ≈ 0.60:1, to construct a nanogel coating with high cross-linking density and a relatively "hard" texture, exhibiting good elution resistance. The specific raw material formulation and related process conditions are as follows: Peptide A is: GPQGIAGQC (SEQ ID NO:1) (8aa, MMP-2 / 9 substrate).

[0054] The B peptide is: YGRKKRRQRRRGGC (SEQ ID NO:17) (13aa, TAT core + GGC).

[0055] The AAS–SAA type polypeptide is: GPQGIAGQC-CGGRRRQRRKKRGY (SEQ ID NO:18); concentration: 0.50mM, approximately 125~150mg per 100mL.

[0056] Polyphenol component: Dopamine (DA): 3.33mM, approximately 60~65mg per 100mL (calculated as dopamine hydrochloride DA•HCl).

[0057] Boric acid: FPBA: 15~23mg per 100mL (≈1.0~1.5mM).

[0058] Oxidizing agent NaIO4 solution: 0.15~0.30mM (approximately 3.2~6.4mg per 100mL) (trace oxidation, satisfying NaIO4:catechol hydroxyl group = 0.02~0.08).

[0059] Metal coordination ion solution: FeCl3: 0.25 mM, approximately 6.8 mg per 100 mL (calculated as FeCl3•6H2O); MnO2 nanosheet: 50 μg·mL -1 5mg per 100mL.

[0060] S1 Ingredients: Under room temperature conditions, take the above AAS-SAA type peptide, DA, and 4-formylphenylboronic acid, and add them sequentially to a phosphate buffer solution with pH=8.5 and a concentration of 10mM. Stir for 8~15min to obtain a pre-prepared solution, wherein DA and NaIO4 are pre-mixed and pre-oxidized for 2~3min.

[0061] S2 Oxidative Crosslinking: Add NaIO4 solution dropwise to the above pre-prepared solution, react for 15-30 min, then add MnO2 nanosheets, with an endpoint concentration of 0.01-0.05 mg / mL, and stir for another 8-12 min to obtain nanogel.

[0062] S3 Film Formation and Fixation: The pretreated substrate is immersed in the above nanogel at 25~37℃ for 10~30 min. After removal, it is rinsed with phosphate buffer and then immersed in a 0.5~2mM coordination metal ion solution with pH=7.4 for 2~5 min to achieve dense metal-phenol coordination. The nanogel immersion and coordination metal ion solution immersion are repeated 3 times, each time with a film thickness of 140~170nm, so that the final total film thickness is about 0.8μm. After rinsing, it is dried at 37℃ for 0.5~2h to obtain a polypeptide polyphenol nanogel coating on the surface of the above substrate.

[0063] Test analysis showed that the coating's D50 was approximately 110–140 nm, and ζ was approximately +15–+25 mV; the initial burst release of the coating was <20% (24 h, pH 7.4); the release accelerated within 1–2 h when MMP and ROS were present simultaneously; and the release was further accelerated within 50 ng / mL of MMP-2. -1 The cumulative release of H2O2 (100 μM) in the presence of A+H2O2 (100 μM) over 24 hours was approximately 70%; the hemolysis rate was <3%. e ∶C e The concentration ratio was controlled at 0.6–0.7:1, FPBA at 1.0–1.5 mM, and MnO2 at 0.02–0.05 mg / mL. -1 Under these conditions, the coating can achieve low initial leakage, efficient release triggered by dual gates, and good blood compatibility.

[0064] Example 2

[0065] The implementation process of this embodiment refers to the specific implementation method. The target synthesis ratio is controlled as follows: total amine: quinone = 2.00: 1.67 ≈ 1.20: 1. The amine ratio is relatively high, the residual free amine ratio is relatively high, the coating is "softer" and more compatible with loading drugs or nucleic acids.

[0066] The specific raw material formula and proportions, as well as the relevant process conditions, are as follows: Peptide A is: GPLGIAGKC (SEQ ID NO:7) (1 Lys is introduced into peptide A to increase the amine site, 9aa), N-terminal 1 amine + Lys × 1 = 2 amines.

[0067] The B peptide is: RKKRRQRRRC (SEQ ID NO:9) (9aa), N-terminal 1 amine + Lys×2 = 3 amines.

[0068] The AAS–SAA type polypeptide is: GPLGIAGKC-CRRRQRRKKR (SEQ ID NO:19); concentration: 0.40mM, approximately 90~110mg per 100mL.

[0069] Polyphenol component: Dopamine (DA): 1.67mM, approximately 30~33mg per 100mL (calculated as DA•HCl).

[0070] Boric acid: FPBA: 15~23mg per 100mL (≈1.0~1.5mM).

[0071] Oxidizing agent NaIO4: 0.08~0.20mM (approximately 1.7~4.3mg per 100mL) The process conditions for other raw materials, auxiliary reagents, substrates, and manufacturing steps were kept the same as in Example 1. The FeCl3 solution concentration was reduced to 0.20 mM to avoid the coating gel being too hard. The growth thickness of each immersion cycle was approximately 100~130 nm.

[0072] Tests and analysis showed that the coating had a voltage rating of ζ≈+10~+18mV; it could partially open the gate when MMP or ROS were present alone, and the opening was significantly accelerated when both were present; it had a faster self-healing speed; the hemolysis rate was <2%; and other properties remained good.

[0073] Example 3

[0074] The implementation process of this embodiment refers to the specific implementation method. The specific raw material formula ratio and related process conditions are as follows: Peptide A is: GPQGIAGQC (SEQ ID NO:1) (MMP substrate).

[0075] The B peptide is: RKKRRQRRRC (SEQ ID NO:9) (CPP / positive charge enrichment).

[0076] AAS–SAA type peptide: GPQGIAGQC-CRRRQRRKKR (SEQ ID NO:20); concentration: 0.50mM, 100mg.

[0077] Polyphenol component: TA100mg + DHBA = 30mg (≈2.2mM).

[0078] Boric acid: FPBA: 20mg (≈1.3mM).

[0079] Oxidizing agent NaIO4 solution: 0.1 mM.

[0080] Metal coordination ion solution: Fe 3+ 0.20~0.30mM; MnO2 nanosheets: 20~50μg·mL -1 .

[0081] Substrate: 316L stainless steel sheet, for local drug delivery to the tumor.

[0082] S1 Ingredients: Under room temperature conditions, take the above AAS-SAA type peptide, tannic acid, 3,4-dihydroxybenzaldehyde, and 4-formylphenylboronic acid, and add them sequentially to a phosphate buffer solution with pH=8.5 and a concentration of 10mM. Stir for 12 min to obtain the pre-prepared solution.

[0083] S2 Oxidative Crosslinking: Add 0.08 mM NaIO4 solution dropwise to the above pre-crosslinking solution, react for 20 min, then add MnO2 nanosheets. The final concentration is 0.02~0.05 mg / mL. Stir for another 8~12 min to obtain nanogel.

[0084] S3 Film Formation and Fixation: After plasma cleaning for 60 seconds, 316L sheets were immersed in the above nanogel solution at 25-37°C for 10-30 minutes. After removal, they were rinsed with phosphate buffer and then immersed in a 0.5-2 mM, pH 7.4 coordination metal ion solution for 2-5 minutes to achieve dense metal-phenol coordination. This nanogel immersion and coordination metal ion solution immersion were repeated 5 times, each time maintaining a film thickness of 140-170 nm, resulting in a final total film thickness of approximately 0.9 μm. After rinsing, the film was dried at 37°C for 0.5-2 hours to obtain a polypeptide-polyphenol nanogel coating on the substrate surface. Doxorubicin (DOX, 0.5 mg / mL, 30 minutes) was loaded into the coating, and Tris was used for 15 mM capping for 5 minutes.

[0085] Tests and analysis showed that the initial release of the coating was <20% (24h, pH 7.4), and the cumulative release increased to approximately 70% over 24h when MMP-2 (50ng / mL) and H2O2 (100µM) coexisted; Raman / XPS confirmed the presence of quinone-amine / metal-phenol; and the self-healing recovery rate after scratch-immersion cycles was >70%. In A... e ∶C e The concentration ratio was controlled at 0.6–0.7:1, FPBA at 1.0–1.5 mM, and MnO2 at 0.02–0.05 mg / mL. -1 Under these conditions, the coating achieves a dual-gate synergy where strong nodes are preferentially cut off by MMP and weak intrachain crosslinks are rapidly released by ROS. It also exhibits low initial leakage, rapid trigger release, significant self-repair, long-lasting adhesion, and compatibility with blood / cells.

[0086] Specific test results are as follows: Figures 2-8 As shown, Figure 2 The cumulative release curve of the coating in Example 3 is obtained from... Figure 2 It can be seen that the coating release increases dramatically when both gates are in use, proving the synergistic effect of MMP cutting off strong nodes and ROS unblocking weak crosslinks. No such effect was observed in the control / single gate mode. Figure 3 This is a graph showing the scratch width recovery rate test results of the coating in Example 3. Figure 3 It can be seen that the scratch recovery rate is >70% within 6~8 hours, verifying the reversible reconstruction channels provided by FPBA Schiff base / boronic acid ester and metal-phenol. Figure 4 This is a graph showing the relationship between the number of immersion cycles and the coating thickness in Example 3. Figure 4 It can be seen that the film thickness is nearly linear with the number of cycles, proving that the process is controllable and scalable. Figure 5 This is a diagram showing the wet shear adhesion test results of the substrate surface in Example 3. Figure 5 It can be seen that the high wet shear adhesion is maintained even after the action of MMP+ROS, indicating that the bottom anchoring is reliable. Figure 6 This is a graph showing the ROS removal capability test results of the coating in Example 3. Figure 6 It can be seen that the coating has a significant ability to remove H2O2 / •OH, and MnO2 is further enhanced, thus balancing the protection of the structure and the triggering of the response. Figure 7 This is a diagram showing the compatibility test results of the coating and cells in Example 3. Figure 7 It can be seen that the survival rate of L929 and HUVEC is ≥80~95%, indicating good cell compatibility. Figure 8 This is a diagram showing the hemolysis test results of the coating in Example 3. Figure 8 It can be seen that the hemolysis rate is <3%, which is below the 5% threshold, indicating that the blood compatibility meets the standard.

[0087] Example 4

[0088] The implementation process of this embodiment refers to the specific implementation method. The specific raw material formula ratio and related process conditions are as follows: Peptide A is: GPLGIAGQC (SEQ ID NO:2) (MMP substrate variant). The B peptide is: KHHKHKHKC (SEQ ID NO:15) (His–Lys enrichment, which is beneficial for antibacterial and pH / metal coordination) AAS–SAA type peptide: GPLGIAGQC-CKHKHKHHK (SEQ ID NO:21); Concentration: 0.35~0.50mM (in 50mL solution, M w (Approximately 3.0 kDa), feed intake 52~75 mg.

[0089] Polyphenol component: TA 40mg + DHBA = 10mg.

[0090] FPBA: 10mg.

[0091] Oxidizing agent NaIO4 solution: 0.05 mM.

[0092] Ca 2+ 1mM; MnO2 nanosheets 0.5mg.

[0093] Substrate: PU catheter.

[0094] The process conditions for S1 to S2 are the same as those in Example 3.

[0095] S3 Film Formation and Fixation: The pretreated substrate was immersed in the above nanogel solution at 25-37℃ for 10-30 min. After removal, it was rinsed with phosphate buffer and then immersed in a 0.5-2 mM, pH 7.4 coordination metal ion solution for 2-5 min to achieve dense metal-phenol coordination. The nanogel immersion and coordination metal ion solution immersion were repeated 5 times, each time with a film thickness of 140-170 nm, resulting in a final total film thickness of approximately 0.9 μm. After rinsing, it was dried at 37℃ for 0.5-2 h to obtain a polypeptide polyphenol nanogel coating on the surface of the substrate. Vancomycin 1 mg·mL⁻¹ was loaded into the coating. -1 .

[0096] Tests and analysis showed that when ROS levels increased in a bacterial polysaccharide / extracellular matrix environment, the coating rapidly opened its outer channels, resulting in a >2 log decrease in viable bacterial count over 24 hours. After 7 days of static water immersion, the adhesion remained at ≥3 B. The coating demonstrated effective anti-infection properties and stable long-term adhesion, confirming that dual-gate control is equally effective in microbial ROS scenarios.

[0097] Comparative Example 1

[0098] The specific implementation process of Comparative Example 1 is consistent with that of Example 3, except that MnO2 nanosheets are not added.

[0099] Tests and analysis revealed that the lack of H2O2→O2 bubbles / local stress and Mn 2+ The secondary coordination amplification effect reduces the sensitivity of the ROS gate, resulting in insufficient drug release in the early stage and inadequate flux in the later stage.

[0100] Comparative Example 2

[0101] The specific implementation process of Comparative Example 2 is consistent with that of Example 3, except that FPBA is not used.

[0102] Test analysis revealed that the lack of catechol-boronic acid esters and the additional Schiff base's "occupation-delay-reversibility" effect significantly reduced weak intra-chain crosslinks, leading to early leakage without triggering, and a decrease in ROS trigger amplitude. The dual-gate control was changed to a single-gate control, resulting in decreased selectivity and an increase in initial bursts.

[0103] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A polypeptide polyphenol nanogel, characterized in that, The raw materials include polypeptides, polyphenolic components, 4-formylphenylboronic acid, oxidants, and nanozymes. The structure of the polypeptides is AAS-SAA type, including A peptide and B peptide coupled at the C-terminus by disulfide bonds. The A peptide is an MMP-responsive peptide, and the B peptide is a peptide with targeting, membrane-penetrating, or electrostatic enrichment functions. The polyphenol component contains a catechin structure; the total amine equivalent A in the polypeptide is... e The equivalent C of the catechol structure that can react with the polyphenol component e The ratio is A e ∶C e =0.6~1.2∶1, the molar ratio of the reactive catechol structure to the 4-formylphenylboronic acid in the polyphenol component is 1.0~2.5∶1; the polyphenol component is tannic acid and 3,4-dihydroxybenzaldehyde; The oxidant is sodium periodate solution; the nanozyme is MnO2 nanosheets; The preparation method of the polypeptide polyphenol nanogel includes the following steps: S1 Ingredients: At room temperature, take 1~5 mg / mL of the AAS-SAA type polypeptide, 0.5~2 mg / mL of the tannic acid, 1~5 mM of the 3,4-dihydroxybenzaldehyde and 1~3 mM of the 4-formylphenylboronic acid, and add them sequentially to a phosphate buffer solution with a pH of 7.4~8.5 and a concentration of 10 mM. Stir for 8~15 min to obtain a pre-prepared solution; S2 Oxidative Crosslinking: Add 0.05~0.50mM sodium periodate solution dropwise to the pre-prepared solution, wherein the molar ratio of NaIO4 to the catechol hydroxyl groups in the pre-prepared solution is 0.02~0.

08. After reacting for 15~30min, add the MnO2 nanosheets, with an endpoint concentration of 0.01~0.05mg / mL, and stir for another 8~12min to obtain the polypeptide polyphenol nanogel.

2. The polypeptide polyphenol nanogel according to claim 1, characterized in that, The sequence of the A peptide is any one of GPQGIAGQC, GPLGIAGQC, PVGLIGRC, PLGLWAGGC, GPLGVRGC, GPQGIWGQC, GPLGIAGKC, or VPLSLYSGC.

3. The polypeptide polyphenol nanogel according to claim 1, characterized in that, The sequence of the B peptide is any one of: RKKRRQRRRC, YGRKKRRQRC, GRGDSPGGC, CREKAGGC, GNGRAHAGC, RPARPARPGC, KHHKHKHKC, or SKIKKPGGC.

4. The polypeptide polyphenol nanogel according to claim 1, characterized in that, The raw materials also include: a coordinating metal ion solution, wherein the concentration of the coordinating metal ions is 0.1~5.0 mM, and the coordinating metal ions include: Fe 3+ Zn 2+ or Ca 2+ Any one of them.

5. A method for preparing a coating of polypeptide polyphenol nanogel as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S3 Film Formation and Fixation: The pretreated substrate is immersed in the solution of the polypeptide polyphenol nanogel at 25~37℃ for 10~30 min. After removal, it is rinsed with phosphate buffer and then immersed in a 0.5~2mM coordination metal ion solution with pH=7.2~7.6 for 2~5 min. The nanogel immersion and coordination metal ion solution immersion are repeated 1~5 times, each time with a film thickness of 100~170nm, so that the final total film thickness is 0.5~1μm. After rinsing, it is dried at 37℃ for 0.5~2h to obtain the polypeptide polyphenol nanogel coating on the surface of the substrate.

6. The method for preparing a coating of polypeptide polyphenol nanogel according to claim 5, characterized in that, The pretreatment process of the substrate in S3 includes pre-soaking the substrate in tannic acid at a concentration of 0.5-2 mg / mL and a pH of 7.5-8.5 for 2-10 minutes.

7. An application of a polypeptide polyphenol nanogel, characterized in that, A coating is formed by coating the surface of a polypeptide polyphenol nanogel as described in any one of claims 1 to 4 onto the surface of an anticancer local drug delivery carrier, an anti-infection catheter, or a postoperative anti-recurrence cavity patch.

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