A dual-module cascade signal amplification hydrogel system and its application in tissue regeneration

Abstract

The application relates to the technical field of hydrogels, and discloses a double-module cascade signal amplification hydrogel system and application thereof in tissue regeneration, which comprises a first module, a second module and a hydrogel three-dimensional support; the first module comprises a capture unit and a signal conversion unit, the second module comprises a signal receiver and a cascade enzymatic reaction network, and the first module and the second module are both distributed in the interior or surface of the hydrogel support. The double-module cascade signal amplification hydrogel system constructed by the application realizes intelligent regulation and control of the whole process of damage microenvironment signal sensing-signal conversion-exponential amplification-time sequence effect output, can accurately identify and capture weak endogenous damage signals in the body at the level of picomole to nanomole, amplifies the signals through double-enzyme cascade reaction, converts the signals into strong effect signals which can directly mediate tissue repair, and solves the core problems of insufficient signal intensity, uncontrollable release and low treatment efficiency of traditional regeneration materials.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and more specifically, to a dual-module cascaded signal amplification hydrogel system and its application in tissue regeneration. Background Technology

[0002] Tissue regeneration and repair after injury is a highly ordered, multi-cellular, and multi-factor temporally regulated complex biological process. From skin wound healing, bone defect repair, and cartilage remodeling to the functional regeneration of solid organs such as myocardium, nerves, and liver, all depend on the precise coordination of a series of events within the injury microenvironment, including signal initiation, signal transduction, cell recruitment, matrix synthesis, and tissue remodeling.

[0003] Under physiological conditions, endogenous tissue regeneration is initiated by initial damage signals released at the site of injury. These signals are collectively referred to as damage-associated molecular patterns (DAMPs), which mainly include high-mobility group box 1 (HMGB1), adenosine triphosphate (ATP), heat shock proteins, hyaluronic acid fragments, collagen terminal peptides, ROS signals, and intracellularly released proteases. When tissue integrity is compromised, these signals are rapidly released, activating pattern recognition receptors on the surfaces of local immune cells and stromal cells, initiating a repair cascade of reactions including inflammation clearance, stem cell recruitment, angiogenesis, and stromal reconstruction, propelling the tissue from a damaged state back to homeostasis. During this process, the intensity, duration, and spatiotemporal distribution of the endogenous signals directly determine the regeneration efficiency.

[0004] However, in pathological microenvironments such as large-area trauma, chronic infection, diabetes, osteoporosis, aging, and ischemia-hypoxia, endogenous repair signals generally suffer from insufficient intensity, extremely short half-life, susceptibility to enzymatic degradation, easy dilution by tissue fluid, and limited diffusion range. To compensate for the lack of endogenous repair signals, traditional tissue engineering and regenerative medicine strategies often employ direct loading and passive sustained release of exogenous bioactive factors. For example, collagen scaffolds loaded with bone morphogenetic protein-2 (BMP-2) have been approved by the FDA for spinal fusion; hydrogels loaded with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been used to promote angiogenesis and wound healing; and systems loaded with insulin-like growth factor (IGF-1) and transforming growth factor (TGF-β) have been used for cartilage and tendon regeneration. While these strategies have improved repair efficacy to some extent, they still suffer from a series of inherent drawbacks that severely limit their clinical translation and safe application: First, the burst release effect is significant, with growth factors being released in large quantities within the first few hours to days, resulting in local concentrations far exceeding physiological levels, which can easily induce serious complications such as ectopic osteoblastic formation, hemangioma, excessive inflammation, and even tumor proliferation; Second, the stability is extremely poor, as protein factors rapidly denature and become inactive at physiological temperatures, pH levels, and in the presence of enzymes, resulting in a very short effective window that is difficult to match the tissue regeneration cycle of several weeks or even months; Third, the safety risks are prominent, with high-dose, long-term use of exogenous growth factors posing potential tumorigenicity, immunogenicity, and dose-dependent toxic side effects; Fourth, the lack of adaptive regulation capability, as traditional sustained-release systems are typically in an "open-loop" mode, only able to release passively, linearly, and continuously, unable to sense the microenvironment state, respond to endogenous signals, start and stop on demand, dynamically adjust the output intensity, or capture, amplify, and enhance weak endogenous signals.

[0005] In recent years, environmentally responsive hydrogels have been extensively studied, including intelligent delivery systems that respond to pH, redox, enzymes, temperature, and glucose. These systems can achieve controlled release of active substances under specific stimuli, improving the blind release capability of traditional sustained-release systems to some extent. However, existing responsive systems still have significant limitations: most can only achieve single-trigger, linear release, or simple on / off release, lacking a nonlinear amplification relationship between signal output and input, and unable to convert weak physiological signals at the picomolar to nanomolar level into sufficiently strong therapeutic signals; at the same time, the systems have limited functionality, failing to cover the multi-stage and multi-type signal requirements of early tissue regeneration (anti-inflammatory, mid-stage angiogenesis, late-stage differentiation, and matrix remodeling), and lack modular, replaceable, and scalable platform designs.

[0006] In the fields of bioelectronics, molecular biology, and synthetic biology, signal amplification technologies are highly mature, such as enzyme-catalyzed cascade reactions, polymerase chain reactions, and immune signal amplification, which can achieve high gain and high specificity output of weak signals. However, in the field of biomedical materials, hydrogel systems that can sense weak endogenous signals in situ within the damaged microenvironment, achieve autonomous and continuous signal amplification through multi-level enzyme-catalyzed cascades, output customized therapeutic signals according to the regeneration stage sequence, and simultaneously possess good biocompatibility and tissue adaptability have not yet been reported, and no mature products have been launched.

[0007] Furthermore, tissue regeneration is a highly time-dependent dynamic process: the early stage focuses on suppressing excessive inflammation, clearing DAMPs, and protecting cells; the middle stage focuses on recruiting stem cells, promoting angiogenesis, and building a nutrient supply network; and the later stage focuses on promoting directed cell differentiation, matrix synthesis and mineralization, and achieving functional reconstruction. The types, concentrations, and modes of action of the required effector signals differ significantly at different stages, and hydrogels with single components, single functions, and single release modes are insufficient to meet the regeneration needs throughout the entire cycle. If a modular, cascadeable, replaceable, and scalable universal hydrogel platform can be constructed, it can flexibly adapt to the regeneration needs of different tissues, different stages, and different pathological microenvironments by changing the signal recognition module and effect output module. This would greatly overcome the current technological bottlenecks and provide a disruptive solution for refractory tissue damage.

[0008] Therefore, providing a dual-module cascaded signal amplification hydrogel system and its application in tissue regeneration has important practical significance. Summary of the Invention

[0009] In view of this, the present invention proposes a dual-module cascaded signal amplification hydrogel system and its application in tissue regeneration, aiming to solve at least one of the problems in the current background art.

[0010] This invention proposes a dual-module cascaded signal amplification hydrogel system, the system comprising a first module, a second module, and a hydrogel three-dimensional scaffold; The first module includes a capture unit and a signal conversion unit. The capture unit is capable of specifically identifying and binding to at least one endogenous initial signal molecule in the damaged microenvironment, and the signal conversion unit releases the initial signal molecule in response to the binding event. The second module includes a signal receiver and a cascaded enzyme reaction network. The signal receiver specifically recognizes the primary signal molecule, and the cascaded enzyme reaction network consists of at least two tandem enzyme reactions, which can convert the presence of the primary signal molecule into an exponentially amplified output of the effector molecule. Both the first module and the second module are distributed inside or on the surface of the hydrogel scaffold.

[0011] Preferably, the capture unit is selected from antibodies, nucleic acid aptamers, extracellular domains of receptor proteins, lectins, or DAMPs-binding proteins; The endogenous initial signaling molecules are selected from high-mobility group box 1, heat shock protein, ATP, free DNA, S100 protein, IL-1β, IL-6, TNF-α, TGF-β1 or exosome membrane markers.

[0012] Preferably, the signal conversion unit includes a pre-embedded signal molecule precursor, a breakable linker arm, and a limiting protease or chemical breakage trigger. The breakable linker includes a matrix metalloproteinase recognition sequence, a thrombin recognition sequence, a disulfide bond, or a thioacetate bond. The primary signaling molecule is selected from glucose oxidase, horseradish peroxidase, alkaline phosphatase, pyrophosphatase, nitric oxide synthase, calcium ion chelator, or chemotactic peptide.

[0013] Preferably, the cascaded enzyme reaction network is selected from any of the following systems: (a) A glucose oxidase and horseradish peroxidase cascade system, using glucose and oxygen as substrates, to generate hydrogen peroxide or oxidative cross-linking products. (b) The alkaline phosphatase and phosphorylated polypeptide substrate cascade system generates phosphate ions and calcium phosphate mineralization precursors; (c) A cascade system of tyrosinase and polyphenol substrates generates an oxidatively cross-linked polyphenol network; (d) Pyruvate oxidase and peroxidase cascade system; The cascade reaction amplifies the initial signal by a factor of 100 to 10,000 within 30 minutes to 72 hours.

[0014] Preferably, the hydrogel three-dimensional scaffold is composed of one or more of the following: methacrylamide gelatin, methacrylamide hyaluronic acid, thiol-modified sodium alginate, polyethylene glycol diacrylate, chitosan, polypeptide RADA16, or copolymers thereof. The hydrogel is an interpenetrating network or porous structure with a pore size of 20-200 micrometers, a compressive modulus of 1-50 kPa, and a degradation half-life that can be controlled within the range of 7-90 days by the degree of cross-linking or enzyme-sensitive cross-linked peptides.

[0015] Preferably, the first module and the second module are co-located inside the same hydrogel through physical embedding, chemical bonding or phase separation, and the transmission of the output signal from the first module to the second module is mainly mediated by diffusion, with the spatial interaction distance controlled within 50 micrometers to prevent signal dilution.

[0016] This invention provides a method for preparing the dual-module cascaded signal amplification hydrogel system described above, comprising the following steps: (1) Prepare a functionalized hydrogel prepolymer, and fix the capture unit onto the prepolymer by covalent bonding or specific non-covalent bonding to obtain the first module precursor solution; (2) The signal conversion unit, the breakable connecting arm and the pre-embedded signal molecule precursor are added to the first module precursor solution, mixed thoroughly and homogenized to remove bubbles, to obtain the hydrogel precursor system loaded with the first module. (3) Mix the enzyme components, substrate components, signal receivers and stabilizers of the cascade enzyme reaction network to prepare the second module premix; (4) The hydrogel precursor system of the first module and the premixed liquid of the second module are mixed evenly at a volume ratio of 1:0.5-1:5, and then formed by photocrosslinking, ionic crosslinking, Schiff base crosslinking or thermal induction crosslinking to obtain a dual-module co-positioning hydrogel. (5) The obtained hydrogel is washed, shaped, and aseptically freeze-dried or wet-method aseptically preserved to obtain the dual-module cascaded signal amplification hydrogel system.

[0017] The present invention also provides an application of the system described above in the preparation of a drug that promotes bone tissue regeneration, wherein the cascade enzyme reaction network outputs phosphate ions to form in-situ hydroxyapatite with calcium ions; and the initial signal is HMGB1 or TGF-β1 released in the bone defect microenvironment.

[0018] The present invention also provides the application of the system described above in the preparation of drugs that promote the healing of chronic skin ulcers, repair of peripheral nerve damage, or angiogenesis after myocardial infarction. The cascade enzyme reaction network is selected from the GOx / HRP system for generating antibacterial and angiogenic hydrogen peroxide and reactive oxygen species, or selected from the ALP / phosphorylated peptide system for generating a biomimetic mineralization layer of the basement membrane.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention constructs a dual-module cascaded signal amplification hydrogel system, achieving intelligent control of the entire process from signal perception of the damaged microenvironment to signal conversion, exponential amplification, and time-sequential effect output. It can accurately identify and capture weak endogenous damage signals at the picomolar to nanomolar level in vivo, amplifying them 100-10000 times through a dual-enzyme cascade reaction, transforming them into a strong signal that can directly mediate tissue repair. This solves the core problems of insufficient signal intensity, uncontrollable release, and low treatment efficiency of traditional regenerative materials. Compared with traditional scaffolds that rely on exogenous growth factors, this system does not require high doses of active proteins, avoiding burst release, inactivation, ectopic proliferation, and potential tumorigenic risks. While improving repair efficacy, it significantly enhances biosafety and clinical stability.

[0020] This invention employs a modular design, allowing for flexible adaptation to different tissue regeneration needs, such as skin ulcers, bone defects, nerve injuries, and myocardial repair, by replacing the capture unit, signal conversion unit, and cascaded enzymatic network. It possesses high versatility and scalability. Furthermore, the hydrogel scaffold used in the system has precisely controllable pore size, mechanical strength, and degradation rate, achieving a high degree of matching with the host tissue microenvironment. This provides an ideal three-dimensional space for cell adhesion, proliferation, and differentiation, realizing efficient synergy between signal amplification and tissue engineering scaffold structure. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0022] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] This invention provides a dual-module cascaded signal amplification hydrogel system, the system comprising a first module, a second module, and a hydrogel three-dimensional scaffold; The first module includes a capture unit and a signal conversion unit. The capture unit is capable of specifically identifying and binding to at least one endogenous initial signal molecule in the damaged microenvironment, and the signal conversion unit releases the initial signal molecule in response to the binding event. In this invention, the capture unit is selected from antibodies, nucleic acid aptamers, extracellular domains of receptor proteins, lectins, or DAMPs-binding proteins. The endogenous initial signaling molecules are selected from high-mobility group box 1, heat shock protein, ATP, free DNA, S100 protein, IL-1β, IL-6, TNF-α, TGF-β1 or exosome membrane markers.

[0027] In this invention, the signal conversion unit includes a pre-embedded signal molecule precursor, a breakable linker arm, and a limiting protease or chemical breakage trigger. The breakable linker includes a matrix metalloproteinase recognition sequence, a thrombin recognition sequence, a disulfide bond, or a thioacetate bond. The primary signaling molecule is selected from glucose oxidase, horseradish peroxidase, alkaline phosphatase, pyrophosphatase, nitric oxide synthase, calcium ion chelator, or chemotactic peptide.

[0028] The first module of this invention is the damage signal sensing unit and signal conversion hub of the system, undertaking the key functions of specifically recognizing damage microenvironment signals and initiating cascade amplification reactions. It consists of two parts: a capture unit and a signal conversion unit. Specifically, the capture unit, as the core of signal recognition, is composed of antibodies, nucleic acid aptamers, extracellular domains of receptor proteins, lectins, or DAMPs-binding proteins. It can accurately identify and specifically bind to endogenous initial signal molecules released after tissue damage, including high-mobility group box 1 (HMP-B1), heat shock proteins, ATP, free DNA, S100 protein, IL-1β, IL-6, TNF-α, TGF-β1, and exosome membrane markers, etc., achieving high specificity and high sensitivity sensing of the damage state and avoiding interference from non-specific signals. The signal conversion unit consists of a pre-embedded signal molecule precursor, a breakable linker arm, and a terminating protease or chemical breakage trigger. The breakable linker arm contains responsive structures such as matrix metalloproteinase recognition sequences, thrombin recognition sequences, disulfide bonds, or thioketal bonds. When the capture unit binds to the initial signal molecule, it triggers the activation of the terminating protease or a chemical breakage response, causing the breakable linker arm to specifically break, thereby releasing the pre-embedded primary signal molecule, including glucose oxidase, horseradish peroxidase, alkaline phosphatase, pyrophosphatase, nitric oxide synthase, calcium ion chelators, or chemotactic peptides, completing the conversion from biological damage signal to enzymatic trigger signal, providing the initiation signal for the cascade reaction of the second module.

[0029] The second module includes a signal receiver and a cascaded enzyme reaction network. The signal receiver specifically recognizes the primary signal molecule, and the cascaded enzyme reaction network consists of at least two tandem enzyme reactions, which can convert the presence of the primary signal molecule into an exponentially amplified output of the effector molecule. In this invention, the cascaded enzyme reaction network is selected from any of the following systems: (a) A glucose oxidase and horseradish peroxidase cascade system, using glucose and oxygen as substrates, to generate hydrogen peroxide or oxidative cross-linking products. (b) The alkaline phosphatase and phosphorylated polypeptide substrate cascade system generates phosphate ions and calcium phosphate mineralization precursors; (c) A cascade system of tyrosinase and polyphenol substrates generates an oxidatively cross-linked polyphenol network; (d) Pyruvate oxidase and peroxidase cascade system; The cascade reaction amplifies the initial signal by a factor of 100 to 10,000 within 30 minutes to 72 hours.

[0030] The second module of this invention is the system's signal receiving and exponential amplification execution unit, responsible for converting the weak primary signal released by the first module into a high-intensity effect signal that can directly mediate tissue regeneration. It consists of a signal receiver and a cascaded enzymatic reaction network. The signal receiver possesses molecular recognition specificity, accurately binding to the primary signal molecules released by the first module and rapidly activating the cascaded enzymatic reaction network, avoiding signal delay or false activation. The cascaded enzymatic reaction network consists of at least two tandem enzymatic reactions, capable of exponentially amplifying the primary signal by 100 to 10,000 times, completing signal amplification and effect output within 30 minutes to 72 hours, meeting the signal intensity requirements of different stages of tissue regeneration. Its specific amplification system and functions include: The glucose oxidase and horseradish peroxidase cascade system uses glucose and oxygen as substrates to generate hydrogen peroxide and oxidative cross-linking products, achieving antibacterial, anti-inflammatory, and angiogenesis-promoting effects. The alkaline phosphatase and phosphorylated peptide substrate cascade system generates phosphate ions and calcium phosphate mineralization precursors, which can combine with calcium ions to form in-situ hydroxyapatite, mediating bone tissue regeneration and biomimetic basement membrane mineralization. The tyrosinase and polyphenol substrate cascade system generates an oxidatively cross-linked polyphenol network, strengthening the hydrogel scaffold structure and promoting tissue matrix remodeling. The pyruvate oxidase and peroxidase cascade system generates reactive oxygen species effector molecules, regulating the inflammatory microenvironment and promoting damage repair. Through these cascade amplification effects, the second module transforms weak trigger signals into strong therapeutic signals, ultimately achieving adaptive, high-gain tissue regeneration regulation.

[0031] Both the first module and the second module are distributed inside or on the surface of the hydrogel scaffold.

[0032] In this invention, the hydrogel three-dimensional scaffold is composed of one or more of the following: methacrylamide gelatin, methacrylamide hyaluronic acid, mercapto-modified sodium alginate, polyethylene glycol diacrylate, chitosan, polypeptide RADA16, or copolymers thereof. The hydrogel is an interpenetrating network or porous structure with a pore size of 20-200 micrometers, a compressive modulus of 1-50 kPa, and a degradation half-life that can be controlled within the range of 7-90 days by the degree of cross-linking or enzyme-sensitive cross-linked peptides.

[0033] The hydrogel three-dimensional scaffold of this invention is the core carrier and physical support platform of a dual-module cascaded signal amplification hydrogel system, providing a stable three-dimensional microenvironment and structural foundation for the entire signal sensing, conversion, amplification, and tissue regeneration process. This scaffold is composed of biocompatible materials such as methacrylamide gelatin, methacrylamide hyaluronic acid, mercapto-modified sodium alginate, polyethylene glycol diacrylate, chitosan, peptide RADA16, or its copolymers. It possesses an interpenetrating network or porous structure with a pore size controlled between 20 and 200 micrometers and a compressive modulus between 1 and 50 kPa. The degradation half-life can be precisely controlled between 7 and 90 days through cross-linking degree or enzyme-sensitive cross-linked peptides to match the regeneration cycles of different tissues such as bone, skin, nerves, and myocardium. Its core functions are reflected in the following aspects: Firstly, it provides fixed sites for the first and second modules, achieving co-localization of the two modules through physical embedding, chemical bonding, or phase separation, avoiding diffusion and dilution of signal molecules, while controlling the signal transmission distance from the first module to the second module within 50 micrometers to ensure signal transmission efficiency. Secondly, it provides a biomimetic extracellular matrix microenvironment for host cell adhesion, migration, proliferation, and differentiation, supporting the morphological construction of tissue regeneration. In addition, its porous structure and degradable properties can also achieve efficient exchange of nutrients and metabolic waste, gradually degrading as tissue regeneration progresses, reserving space for the deposition of new tissue matrix. It can also be combined with a third functional layer such as an anti-inflammatory factor sustained-release layer, a chemokine gradient layer, and an antimicrobial peptide loading layer to expand the system's multifunctionality.

[0034] In this invention, the first module and the second module are co-located inside the same hydrogel through physical embedding, chemical bonding or phase separation, and the transmission of the output signal from the first module to the second module is mainly mediated by diffusion, with the spatial interaction distance controlled within 50 micrometers to prevent signal dilution.

[0035] This invention provides a method for preparing the dual-module cascaded signal amplification hydrogel system described above, comprising the following steps: (1) Prepare a functionalized hydrogel prepolymer, and fix the capture unit onto the prepolymer by covalent bonding or specific non-covalent bonding to obtain the first module precursor solution; Specifically, the preferred method involves preparing a biocompatible hydrogel prepolymer solution for constructing a three-dimensional scaffold. One or more blended materials selected from methacrylamide gelatin, methacrylamide hyaluronic acid, mercapto-modified sodium alginate, polyethylene glycol diacrylate, chitosan, or peptide RADA16 are dissolved in sterile phosphate-buffered saline (PBS), deionized water, or HEPES buffer under ice bath conditions to prepare a homogeneous prepolymer solution with a mass fraction of 5%–20%. Subsequently, capture units (antibodies, nucleic acid aptamers, extracellular domains of receptor proteins, lectins, or DAMPs-binding proteins) are added to the prepolymer solution. The capture units are stably immobilized on the polymer chain through covalent bonding (EDC / NHS activated carboxyl-amino coupling, Michael addition, Schiff base reaction) or specific non-covalent binding (such as biotin-streptavidin interaction, heparin-growth factor binding, hydrophobic interactions). The capture unit with a final concentration of 10 ng / mL to 100 μg / mL was fixed onto the prepolymer at a mass ratio of 1:500 to 1:5000 by covalent coupling or specific non-covalent binding of EDC / NHS, wherein the molar ratio of EDC:NHS:prepolymer carboxyl groups was controlled at 1.2 to 2.0:1.0:1.0. The reaction was carried out at 4°C in the dark for 2 to 12 hours to obtain the first module precursor solution.

[0036] (2) The signal conversion unit, the breakable connecting arm and the pre-embedded signal molecule precursor are added to the first module precursor solution, mixed thoroughly and homogenized to remove bubbles, to obtain the hydrogel precursor system loaded with the first module. Specifically, in the preferred embodiment, the functional components required for the signal conversion unit, including the pre-embedded signal molecule precursor, the cleavable linker arm, the site-limiting protease, or the chemical cleavage trigger, are sequentially added to the first module precursor solution obtained in step (1). The molar ratio of the cleavable linker arm to the pre-embedded signal molecule precursor is 1.0~1.5:1.0, and the molar ratio of the site-limiting protease to the cleavable linker arm is 1:50~1:200. The signal conversion unit, the cleavable linker arm, and the pre-embedded signal molecule precursor are mixed, and then added to the first module precursor solution at a volume ratio of 1:5~1:20 between the signal conversion mixture and the first module precursor solution. The final concentration of the pre-embedded signal molecule precursor is controlled to be 5U / mL~200U / mL (enzymes) or 10μM~500μM (peptides). After thorough mixing, vacuum homogenization and degassing are performed to obtain a hydrogel precursor system with a solid content of 8wt%~25wt% loaded with the first module. The precursors for the pre-embedded signal molecules include primary signal molecules such as glucose oxidase, horseradish peroxidase, alkaline phosphatase, pyrophosphatase, and chemotactic peptides; the breakable linker uses responsive structures such as matrix metalloproteinase (MMP) recognition sequences, thrombin recognition sequences, disulfide bonds, or thioketal bonds; and the limiting protease is selected according to the characteristics of the damaged microenvironment.

[0037] (3) Mix the enzyme components, substrate components, signal receivers and stabilizers of the cascade enzyme reaction network to prepare the second module premix; Specifically, a second module functional premix is ​​prepared separately to achieve signal reception and exponential amplification. The dual / multi-enzyme components corresponding to the cascade enzyme reaction network, the specific substrate, the signal receiver, and the enzyme stabilizer are thoroughly mixed in the following proportions: the molar ratio of the dual enzyme components is controlled at 1:1 to 1:5, with a final concentration of 1 U / mL to 100 U / mL; the molar ratio of the substrate to the corresponding enzyme is 50 to 500:1; the molar ratio of the signal receiver to the primary signal molecule is 1:1 to 1:10; and the stabilizer is added at 2 vol% to 10 vol% glycerol, 1 wt% to 5 wt% sucrose / trehalose, and 0.1 wt% to 1 wt% BSA. The mixture is gently mixed for 15 to 30 minutes under aseptic conditions in an ice bath to obtain a second module premix with a solid content of 2 wt% to 10 wt%. If a glucose oxidase / horseradish peroxidase (GOx / HRP) cascade system is used, then GOx, HRP, glucose solution and oxygen saturation system are added; if an alkaline phosphatase / phosphopeptide (ALP / phosphopeptide) cascade system is used, then ALP, phosphorylated peptide substrate and calcium ion source are added; at the same time, a signal receiver is added to specifically recognize the primary signal released by the first module.

[0038] (4) The hydrogel precursor system of the first module and the premixed liquid of the second module are mixed evenly at a volume ratio of 1:0.5 to 1:5, and then formed by photocrosslinking, ionic crosslinking, Schiff base crosslinking or thermal induction crosslinking to obtain a dual-module co-positioning hydrogel. Specifically, the preferred method is to precisely mix the first module hydrogel precursor system from step (2) with the second module premix from step (3) at a volume ratio of 1:0.5-1:5, and then mix them for 10-30 minutes in an ice bath with low-speed stirring to achieve a uniform co-positioning distribution of the first and second modules, while ensuring that the signal transmission distance is controlled within 50 micrometers to prevent signal diffusion and dilution. After mixing, the composite precursor solution is injected into a custom mold, and the corresponding crosslinking method is selected according to the material system. Photocrosslinking: Add 0.1%~1% (w / v) photoinitiator, at 365 nm and 10~50 mW / cm 2 Irradiate under ultraviolet light for 5-30 minutes; Ionic crosslinking: Adding Ca 2+ Ba 2+ Divalent cation solutions trigger cross-linking of polysaccharides such as sodium alginate; Schiff base crosslinking: The amino group reacts in situ with the aldehyde group to form a dynamic covalent bond; Thermally induced cross-linking: Low temperature and mild heating trigger the self-assembly of peptides or temperature-sensitive polymers.

[0039] After cross-linking, a three-dimensional porous co-positioned hydrogel with uniformly distributed dual modules is obtained.

[0040] (5) The obtained hydrogel is washed, shaped, and aseptically freeze-dried or wet-method aseptically preserved to obtain the dual-module cascaded signal amplification hydrogel system.

[0041] Specifically, the preferred method is to wash the obtained hydrogel 3-5 times with sterile PBS or HEPES buffer at a volume ratio of 10:1 to 50:1 to remove unreacted monomers, residual initiators, free enzymes, and unbound components, thus avoiding cytotoxicity. The hydrogel is then precisely shaped according to clinical application requirements (e.g., sheet-like, columnar, porous sponge-like, or irregular defect-matching morphology), and subsequently preserved using either sterile lyophilization or wet sterile methods. Aseptic freeze-drying: in 40℃~ After pre-freezing at 80℃, the sponge is freeze-dried under vacuum to prepare a dry sponge support, which is convenient for long-term storage and transportation at room temperature / low temperature. Wet sterile preservation: Immerse in sterile buffer containing a small amount of antibacterial agent and seal for preservation to maintain the three-dimensional network of hydrogel and enzyme activity.

[0042] The final result is a dual-module cascaded signal amplification hydrogel system that can sense damage signals, amplify them in a cascaded manner, and regulate tissue regeneration in a time sequence.

[0043] The present invention also provides an application of the system described above in the preparation of a drug that promotes bone tissue regeneration, wherein the cascade enzyme reaction network outputs phosphate ions to form in-situ hydroxyapatite with calcium ions; and the initial signal is HMGB1 or TGF-β1 released in the bone defect microenvironment.

[0044] The present invention also provides the application of the system described above in the preparation of drugs that promote the healing of chronic skin ulcers, repair of peripheral nerve damage, or angiogenesis after myocardial infarction. The cascade enzyme reaction network is selected from the GOx / HRP system for generating antibacterial and angiogenic hydrogen peroxide and reactive oxygen species, or selected from the ALP / phosphorylated peptide system for generating a biomimetic mineralization layer of the basement membrane.

[0045] Example 1 (1) To prepare a functionalized hydrogel prepolymer, a 10 wt% methacrylamide gelatin prepolymer solution was prepared using sterile phosphate buffer as solvent. HMGB1 nucleic acid aptamer with a final concentration of 500 ng / mL was covalently coupled to the prepolymer at a mass ratio of 1:2000 with the prepolymer via EDC / NHS, wherein the molar ratio of EDC:NHS:prepolymer carboxyl groups was controlled to be 1.5:1.0:1.0. The reaction was carried out at 4°C in the dark for 6 h to obtain the first module precursor solution.

[0046] (2) The signal conversion unit, the cleavable linker and the pre-embedded signal molecule precursor were mixed at a molar ratio of 1.2:1.0 for the cleavable linker arm (MMP enzyme-cleaved peptide) and 1:100 for the pre-embedded signal molecule precursor (glucose oxidase). The mixture was then added to the first module precursor solution at a volume ratio of 1:10 for the signal conversion mixture and the first module precursor solution. The final concentration of glucose oxidase was controlled to be 20 U / mL. After thorough mixing, the mixture was vacuum homogenized and degassed to obtain a hydrogel precursor system with a solid content of 12 wt% loaded with the first module.

[0047] (3) Mix glucose oxidase, horseradish peroxidase, glucose substrate, signal receiver and stabilizer in the cascade enzyme reaction network. The molar ratio of the two enzyme components is controlled at 1:2 and the final concentration is 10U / mL. The molar ratio of substrate to corresponding enzyme is 200:1. The molar ratio of signal receiver to primary signal molecule is 1:5. The amount of stabilizer added is 5 vol% glycerol, 2 wt% trehalose and 0.5 wt% BSA. Gently mix for 20 min under aseptic conditions in an ice bath to prepare the second module premix with a solid content of 5 wt%.

[0048] (4) Mix the hydrogel precursor system of the first module and the premixed liquid of the second module at a volume ratio of 1:1 and stir at low speed in an ice bath for 15 min; add 0.5 wt% of photoinitiator of the total system and cross-link and form a solid content of 10 wt% by irradiation with 365 nm ultraviolet light for 10 min.

[0049] (5) The obtained hydrogel was washed four times with sterile PBS at a volume ratio of 20:1 and shaped into a circular sheet. 2 wt% trehalose freeze-drying protectant was added, and the dry scaffold was obtained by pre-freezing at -80℃ and vacuum freeze-drying. Finally, a dual-module cascaded signal amplification hydrogel system was obtained.

[0050] Example 2 (1) Preparation of functionalized hydrogel prepolymer: A solution of 8 wt% methacrylamide hyaluronic acid prepolymer was prepared using sterile HEPES buffer as solvent; TNF-α antibody with a final concentration of 1 μg / mL was added at a mass ratio of 1:1000 to the prepolymer via biotin. Streptavidin was non-covalently immobilized on the prepolymer and reacted at 4°C in the dark for 4 hours to obtain the first module precursor solution.

[0051] (2) The signal conversion unit, the breakable linker and the pre-embedded signal molecule precursor were mixed at a molar ratio of 1.0:1.0 between the breakable linker (disulfide bond) and the pre-embedded signal molecule precursor (alkaline phosphatase) and a molar ratio of 1:150 between the limiting reducing agent and the breakable linker. The mixture was then added to the first module precursor solution at a volume ratio of 1:15 between the signal conversion mixture and the first module precursor solution. The final concentration of alkaline phosphatase was controlled to be 15 U / mL. After thorough mixing, the mixture was vacuum homogenized and degassed to obtain a hydrogel precursor system with a solid content of 10 wt% loaded with the first module.

[0052] (3) Mix alkaline phosphatase, phosphorylated polypeptide substrate, calcium ion source, signal receiver and stabilizer in the cascade enzyme reaction network. The molar ratio of enzyme to substrate is 1:300, the molar ratio of signal receiver to primary signal molecule is 1:3, and the amount of stabilizer added is 3wt% sucrose and 0.3wt% BSA. Gently mix for 25 min under aseptic conditions in an ice bath to prepare the second module premix with a solid content of 4wt%.

[0053] (4) The hydrogel precursor system loaded with the first module and the premixed liquid of the second module are mixed evenly at a volume ratio of 1:2 and stirred at low speed in an ice bath for 20 min; CaCl2 solution with a molar ratio of 1.5:1 to hyaluronic acid unit is added for ionic cross-linking to obtain a dual-module co-positioning hydrogel with a solid content of 8wt%.

[0054] (5) The obtained hydrogel was washed three times with sterile HEPES buffer at a volume ratio of 30:1 and shaped into a column. It was then soaked in antibacterial PBS at a volume ratio of 10:1 and sealed for wet storage to finally obtain a dual-module cascaded signal amplification hydrogel system.

[0055] Application Example 1 (1) The dual-module cascaded signal amplification hydrogel system prepared in Example 1 was used with HMGB1 as the endogenous initial signal. The signal was amplified through the glucose oxidase / horseradish peroxidase (GOx / HRP) cascade reaction, and hydrogen peroxide was generated in situ. It was used for adaptive repair of chronic skin ulcers in diabetic rats and had anti-inflammatory, antibacterial and angiogenesis-promoting functions.

[0056] (2) Experimental animals and model construction SPF-grade SD diabetic rats, 8 weeks old and weighing 200±20g, were selected and acclimatized for 3 days. Anesthetized with sodium pentobarbital via intraperitoneal injection, the back was shaved and disinfected with iodine. A full-thickness skin defect ulcer model, extending to the fascia layer, was established using a 10mm diameter punch. After successful modeling, the rats were randomly divided into 3 groups of 6 rats each. Model control group: The wound was not treated in any way, only bandaged with sterile gauze; Conventional hydrogel group: Implantation of an equal volume of blank GelMA hydrogel scaffold; Example 1 group: Implantation of the dual-module cascaded signal amplification hydrogel system prepared in Example 1.

[0057] (3) Application and administration method The hydrogel scaffold was trimmed into a circular piece that perfectly matched the wound surface and tightly adhered to the ulcer base. It was then secured and bandaged with sterile medical tape and gauze. The dressing and scaffold were changed every two days post-surgery, and the patient was observed for 14 consecutive days. The patient had free access to food and water and was kept normally.

[0058] (4) Performance testing methods Wound healing status: The wound was photographed on days 0, 3, 7, and 14 postoperatively, and the wound healing rate was calculated using ImageJ software.

[0059] Signal amplification efficiency: Wound tissue was taken on the 7th day after surgery. HMGB1 content was detected by ELISA, and H2O2 concentration was detected by chemiluminescence. The signal amplification factor was calculated.

[0060] Histological observation: Samples were taken 14 days postoperatively, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with HE and Masson staining to observe granulation tissue growth, collagen deposition, and inflammatory infiltration.

[0061] Angiogenesis evaluation: Immunofluorescence staining was used to detect positive expression of CD31 and α-SMA, and the density of new blood vessels was statistically analyzed.

[0062] Antibacterial effect: The number of Staphylococcus aureus and Escherichia coli colonies was determined by plate counting method from the wound exudate, and the bacterial clearance rate was calculated.

[0063] (5) The test results are shown in Table 1. Table 1 Test Results As shown in Table 1, the system in Example 1 can accurately identify HMGB1 signals in the microenvironment of diabetic ulcers. Through a cascade of enzymatic reactions, it can amplify the signal by more than 1,000 times, significantly improve the wound healing speed, promote granulation tissue proliferation and angiogenesis, and efficiently remove wound bacteria. It has significant advantages in the repair of diabetic chronic ulcers.

[0064] Application Example 2 (1) Using the dual-module cascaded signal amplification hydrogel system prepared in Example 2, with TGF β1 is an endogenous initial signal that generates phosphate in situ through an alkaline phosphatase / phosphopeptide (ALP / phosphopeptide) cascade reaction, which combines with calcium ions to form hydroxyapatite, and is used for adaptive mineralization and regeneration of critical bone defects in rat skulls.

[0065] (2) Experimental animals and model construction SPF-grade SD rats, 8 weeks old and weighing 220±20g, were selected and acclimatized for 3 days. After anesthesia with sodium pentobarbital, the head was prepared and disinfected, the skin was incised along the midsagittal line, the periosteum was separated, and a circular critical bone defect model was prepared in the parietal bone using a 5 mm diameter cranial drill, avoiding damage to the dura mater. After modeling, the rats were randomly divided into 3 groups, with 6 rats in each group. Blank control group: No material was filled into the bone defect; Pure hyaluronic acid hydrogel group: implantation of blank hyaluronic acid hydrogel scaffolds; Example 2 group: Implantation of the dual-module cascaded signal amplification hydrogel system prepared in Example 2.

[0066] (3) Application and implantation method The hydrogel scaffold was shaped into a cylinder with a diameter of 5 mm and a thickness of 2 mm and tightly embedded into the skull defect area, ensuring that the scaffold fits perfectly against the edge of the bone defect. Subsequently, the periosteum, muscle and skin were sutured layer by layer, disinfected with iodine, and the child was fed normally with free access to food and water.

[0067] (4) Performance testing methods Micro CT scan: Samples were taken at 4 and 8 weeks post-surgery for microscopic examination. CT scans and 3D reconstructions were used to calculate bone volume fraction (BV / TV) and trabecular bone thickness (Tb.Th).

[0068] Mineralization signal amplification efficiency: The amount of calcium and phosphorus deposited in the defect area was determined by ICP method and compared with the initial TGF. Calculate the mineralization amplification factor based on the β1 signal intensity.

[0069] Histology and immunohistochemistry: Tissue samples were taken 8 weeks postoperatively, fixed and decalcified, sectioned, and observed for HE staining and Masson Goldner staining. Immunohistochemistry was used to detect positive expression of OCN and Runx2, and the positive area rate was calculated.

[0070] (5) The test results are shown in Table 2. Table 2 Test Results Based on Table 2, it can be seen that the system in Example 2 can respond to TGF in the bone defect microenvironment. β1 signaling enables efficient in-situ mineralization through a cascade of enzymatic reactions, significantly increasing the amount and maturity of new bone formation and promoting bone defect repair, thus showing promising application prospects in the field of bone tissue engineering.

[0071] 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 it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A dual-module cascaded signal amplification hydrogel system, characterized in that, The system includes a first module, a second module, and a hydrogel three-dimensional scaffold; The first module includes a capture unit and a signal conversion unit. The capture unit is capable of specifically identifying and binding to at least one endogenous initial signal molecule in the damaged microenvironment, and the signal conversion unit releases the primary signal molecule in response to binding. The second module includes a signal receiver and a cascaded enzyme reaction network. The signal receiver specifically recognizes the primary signal molecule, and the cascaded enzyme reaction network consists of at least two tandem enzyme reactions, which can convert the presence of the primary signal molecule into an exponentially amplified output of the effector molecule. Both the first module and the second module are distributed inside or on the surface of the hydrogel scaffold.

2. The dual-module cascaded signal amplification hydrogel system according to claim 1, characterized in that, The capture unit is selected from antibodies, nucleic acid aptamers, extracellular domains of receptor proteins, lectins, or DAMPs-binding proteins.

3. The dual-module cascaded signal amplification hydrogel system according to claim 1, characterized in that, The endogenous initial signaling molecules are selected from high-mobility group box 1, heat shock protein, ATP, free DNA, S100 protein, IL-1β, IL-6, TNF-α, TGF-β1 or exosome membrane markers.

4. The dual-module cascaded signal amplification hydrogel system according to claim 1, characterized in that, The signal conversion unit includes a pre-embedded signal molecule precursor, a breakable linker arm, and a site-limiting protease or chemical breakage trigger. The breakable linker includes a matrix metalloproteinase recognition sequence, a thrombin recognition sequence, a disulfide bond, or a thioacetate bond. The primary signaling molecule is selected from glucose oxidase, horseradish peroxidase, alkaline phosphatase, pyrophosphatase, nitric oxide synthase, calcium ion chelator, or chemotactic peptide.

5. The dual-module cascaded signal amplification hydrogel system according to claim 1, characterized in that, The cascaded enzyme reaction network is selected from any of the following systems: (a) A glucose oxidase and horseradish peroxidase cascade system, using glucose and oxygen as substrates, to generate hydrogen peroxide or oxidative cross-linking products. (b) The alkaline phosphatase and phosphorylated polypeptide substrate cascade system generates phosphate ions and calcium phosphate mineralization precursors; (c) A cascade system of tyrosinase and polyphenol substrates generates an oxidatively cross-linked polyphenol network; (d) Pyruvate oxidase and peroxidase cascade system; The cascade reaction amplifies the initial signal by a factor of 100 to 10,000 within 30 minutes to 72 hours.

6. The dual-module cascaded signal amplification hydrogel system according to claim 1, characterized in that, The hydrogel three-dimensional scaffold is composed of one or more of the following: methacrylamide gelatin, methacrylamide hyaluronic acid, mercapto-modified sodium alginate, polyethylene glycol diacrylate, chitosan, polypeptide RADA16, or copolymers thereof. The hydrogel is an interpenetrating network or porous structure with a pore size of 20-200 micrometers, a compressive modulus of 1-50 kPa, and a degradation half-life that can be controlled within the range of 7-90 days by the degree of cross-linking or enzyme-sensitive cross-linked peptides.

7. The dual-module cascaded signal amplification hydrogel system according to claim 1, characterized in that, The first module and the second module are co-located inside the same hydrogel through physical embedding, chemical bonding or phase separation. The transmission of the output signal from the first module to the second module is mainly mediated by diffusion, and the spatial interaction distance is controlled within 50 micrometers to prevent signal dilution.

8. A method for preparing the dual-module cascaded signal amplification hydrogel system according to any one of claims 1-7, characterized in that, Includes the following steps: (1) Prepare a functionalized hydrogel prepolymer, and fix the capture unit onto the prepolymer by covalent bonding or specific non-covalent bonding to obtain the first module precursor solution; (2) The signal conversion unit, the breakable connecting arm and the pre-embedded signal molecule precursor are added to the first module precursor solution, mixed thoroughly and homogenized to remove bubbles, to obtain the hydrogel precursor system loaded with the first module. (3) Mix the enzyme components, substrate components, signal receivers and stabilizers of the cascade enzyme reaction network to prepare the second module premix; (4) The hydrogel precursor system of the first module and the premixed liquid of the second module are mixed evenly at a volume ratio of 1:0.5 to 1:5, and then formed by photocrosslinking, ionic crosslinking, Schiff base crosslinking or thermal induction crosslinking to obtain a dual-module co-positioning hydrogel. (5) The obtained hydrogel is washed, shaped, and aseptically freeze-dried or wet-method aseptically preserved to obtain the dual-module cascaded signal amplification hydrogel system.

9. The use of the system according to claim 1 in the preparation of a medicament for promoting bone tissue regeneration, characterized in that, The cascaded enzyme reaction network outputs phosphate ions to form in-situ hydroxyapatite with calcium ions; the initial signal is HMGB1 or TGF-β1 released in the bone defect microenvironment.

10. The use of the system of claim 1 in the preparation of a medicament for promoting the healing of chronic skin ulcers, the repair of peripheral nerve injuries, or angiogenesis after myocardial infarction, characterized in that, The cascaded enzyme reaction network is selected from the GOx / HRP system for generating antibacterial and angiogenesis-promoting hydrogen peroxide and reactive oxygen species, or from the ALP / phosphorylated peptide system for generating a biomimetic mineralization layer of the basement membrane.

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