Mechanically adaptive hydrogel for removing apoptotic cells and inhibiting tissue fibrosis, and preparation method and application thereof

Abstract

The application discloses a kind of for removing apoptotic cell, inhibiting the mechanical adaptive hydrogel of tissue fibrosis and preparation method and application thereof, and the hydrogel includes interpenetrating network polymer matrix and dynamic nanocomposite embedded in matrix.The hydrogel of the application provides structural support with AAm as the first crosslinking network, CSMA is given softness and high porosity connectivity as the second network, realizes high bearing and self-adaptive deformation to external pulling.The CP@ZV as nanonode, through the network stability of multiple-point non-covalent interaction is enhanced and the pulling energy generated by pre-stretching is dissipated.After pre-stretching attachment, nanonode cooperatively forms reverse mechanical buffer, effectively reduces the abnormal pulling stress perceived by edge cell, inhibits mechanical signal sustained activation, so as to restore macrophage cytophagic-mediated apoptotic cell removal function.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a mechanically adaptive hydrogel for clearing apoptotic cells and inhibiting tissue fibrosis, as well as its preparation method and application. Background Technology

[0002] Fibrotic scarring is a major obstacle to the repair of active wounds, and its occurrence is closely related to aberrant mechanical stress. Sustained mechanical load can activate mechanosensitive channels (such as Piezo1), leading to calcium signaling imbalance and ROS accumulation, triggering apoptosis and related inflammatory responses. In the skin injury microenvironment, apoptotic cells not only disrupt vascular homeostasis but also release pro-inflammatory and pro-fibrotic factors, thereby driving fibroblast overactivation and collagen deposition. Timely clearance of apoptotic cells is crucial for terminating the inflammatory response and halting fibrosis. During normal repair, macrophages promptly clear apoptotic cells and cell debris through cytotoxicity, thereby limiting the spread of pro-fibrotic signals and promoting inflammation resolution. However, aberrant mechanical environments (such as sustained traction or excessive compression) inhibit cytoskeleton rearrangement and phagocytic receptor aggregation, while perturbing YAP / TAZ signaling, thus weakening macrophage phagocytic capacity. Impaired cytotoxicity leads to apoptotic cell retention and sustained amplification of inflammation, creating conditions for fibrosis progression. Therefore, precise regulation of the mechanical environment of active wounds may be an important means of intervening in scar formation.

[0003] Hydrogel patches are widely used in wound repair due to their similarity to soft tissue mechanical properties and high plasticity. In recent years, "mechanical self-adaptation" has been considered a key property for regulating the mechanical microenvironment; that is, materials can actively regulate stress distribution through controllable deformation and energy dissipation, reducing abnormal mechanical stimulation perceived by cells and thus avoiding abnormal activation of mechanical signals. Within this framework, pre-stretched hydrogels can dissipate and disperse tensile stress, forming a "reverse mechanical buffer" at the wound edge, transferring locally excessive tensile energy to the interior of the hydrogel network, thereby mitigating the direct effects of pathological stress on cells. However, traditional hydrogels still face challenges in balancing mechanical self-adaptation and mechanical stability: while high cross-linking density enhances stability, it also increases rigidity and reduces energy dissipation, while limiting pore connectivity and material transport, which is detrimental to cell infiltration and microenvironment remodeling. The introduction of interpenetrating networks (IPNs) and nanofillers provides a solution for constructing hydrogels that combine mechanical adaptability and structural stability. Through segment interpenetration and multi-point non-covalent interactions, a hierarchical and well-connected network is formed, which achieves stress dispersion and energy dissipation while maintaining low stiffness and high ductility. This allows the hydrogel to continuously play a mechanical buffering role in dynamic mechanical environments and provides a more compliant mechanical interface for cells.

[0004] Based on this, developing a mechanically adaptive smart hydrogel that can regulate the abnormal local mechanical environment of the wound, inhibit the continuous activation of mechanical signals, and thereby restore the function of macrophage burial-mediated apoptotic cell clearance is of great significance for promoting scarless functional tissue regeneration and is also a key problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a mechanically adaptive hydrogel for clearing apoptotic cells and inhibiting tissue fibrosis, as well as its preparation method and applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide a mechanically adaptive hydrogel for clearing apoptotic cells and inhibiting tissue fibrosis, the hydrogel comprising an interpenetrating network polymer matrix and a dynamic nanocomposite material embedded in the matrix; The nanocomposite material comprises cellulose nanocrystals, a polydopamine layer coated on the surface of the cellulose nanocrystals, and drug-loaded metal-organic framework particles loaded through the polydopamine layer. The interpenetrating network polymer matrix comprises a first crosslinked network and a second crosslinked network; The first crosslinking network is a polymer network based on acrylamide monomers; The second crosslinking network is a polymer network based on methacrylamide chitosan.

[0007] Furthermore, the metal-organic framework is ZIF-8.

[0008] Furthermore, the drug loaded in the metal-organic framework is verteporfen.

[0009] The second aspect is to provide a method for preparing the aforementioned mechanically adaptive hydrogel, including the following steps: S1, Preparation of nanocomposite material: Polydopamine is polymerized on the surface of cellulose nanocrystals to form a coating layer, resulting in CNC@PDA; Drug is loaded into metal-organic framework particles to obtain drug-loaded particles; The drug-loaded particles are mixed with CNC@PDA to attach the drug-loaded particles to the surface of CNC@PDA, resulting in nanocomposite material; S2, Constructing the first network hydrogel: Mix the nanocomposite material, acrylamide monomer, crosslinking agent and first photoinitiator, and carry out the first photocrosslinking reaction to form the first crosslinked network hydrogel; S3, Constructing an interpenetrating network hydrogel: Immerse the first cross-linked network hydrogel in a second network precursor solution containing methacrylamide chitosan and a second photoinitiator, allowing the second network precursor solution to penetrate into the interior of the first cross-linked network hydrogel and then undergo a second photocrosslinking reaction to form an interpenetrating network structure hydrogel.

[0010] Further, in S1, dopamine hydrochloride is oxidatively polymerized in the dispersion of the cellulose nanocrystals in the presence of a Tris buffer solution at pH 8.5, thereby forming a polydopamine coating layer on the surface of the cellulose nanocrystals.

[0011] Furthermore, the drug is verteporfen, and the metal-organic framework is ZIF-8.

[0012] Furthermore, S2 specifically includes: Acrylamide monomer, crosslinking agent N,N'-methylenebisacrylamide, and a first photoinitiator are added to a nanocomposite dispersion with a concentration of 1-5 mg / mL; the concentration of the acrylamide monomer is 100-500 mg / mL, the mass ratio of the crosslinking agent to the acrylamide monomer is (0.1-1):100, and the mass ratio of the first photoinitiator to the acrylamide monomer is (0.1-0.5):100; a first photocrosslinking reaction is carried out to form a first crosslinked network hydrogel.

[0013] Furthermore, S3 specifically includes: Methacrylamide chitosan was dissolved in an aqueous solution of glacial acetic acid with a concentration of 0.05-0.1M, and a second photoinitiator was added to prepare a second network precursor solution with a methacrylamide chitosan concentration of 0.5-2 mg / mL. The first cross-linked network hydrogel was immersed in the second network precursor solution, and the second network precursor solution was allowed to penetrate into the interior of the first cross-linked network hydrogel to carry out a second photocross-linking reaction, forming an interpenetrating network structure hydrogel.

[0014] Furthermore, both the first photoinitiator and the second photoinitiator are lithium (2,4,6-trimethylbenzoyl) phosphate salts.

[0015] The third aspect is to provide the application of the aforementioned mechanically adaptive hydrogel in the preparation of products for the prevention or treatment of tissue fibrosis scars.

[0016] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: This invention combines interpenetrating networks and dynamic nanonodes to synergistically construct a mechanically adaptive hydrogel (Gel / VP) to regulate the abnormal mechanical environment of the wound site. Specifically, the hydrogel uses AAm as the first cross-linking network to provide structural support, and CSMA as the second network to impart flexibility and high porosity connectivity, thereby simultaneously achieving high load-bearing capacity and adaptive deformation in response to external traction. Meanwhile, CNC@PDA@ZIF8 (CP@ZV) nanonodes enhance network stability and dissipate the tensile energy generated by pre-stretching through multi-point non-covalent interactions. After pre-stretching and attachment, the nanonodes synergistically dissipate tensile energy, forming a controllable reverse mechanical buffer at the wound interface, effectively reducing the abnormal tensile stress sensed by marginal cells, inhibiting the continuous activation of mechanical signals, and thus restoring the macrophage burial-mediated apoptotic cell clearance function. Functional validation shows that this hydrogel significantly improves macrophage phagocytic efficiency in vitro, inhibits α-SMA myofibroblast aggregation in vivo, and achieves orderly collagen reconstruction and scar limitation. Attached Figure Description

[0017] Figure 1 In the table, A shows the Live / Dead staining results after Yoda1 (1–5 μM) treatment; B shows the protein levels of Piezo1 and YAP1 detected by Western blot; C shows the intracellular Ca2+ levels detected by Fluo-4 AM and DCFH-DA staining, respectively. 2+ ROS level; DF is a representative image of immunofluorescence staining for fibrosis markers (COL-1, α-SMA, and COL-3); G–I is the quantitative analysis of fluorescence intensity; J–L is the mRNA expression level of fibrosis markers; M is the expression of fibrosis and anti-fibrosis protein (TGF-β3) detected by Western blot; Actin is the internal control.

[0018] Figure 2 In the diagram, A represents TEM and SEM morphology images and elemental distribution maps; B represents SEM microstructure images and elemental distribution maps of the hydrogel; C represents XRD patterns; D represents swelling behavior curves of the hydrogel; E represents a schematic diagram of tensile testing of the interpenetrating network hydrogel; F represents tensile stress-strain curves of the hydrogel; and G represents tensile modulus statistics.

[0019] Figure 3In the image, A shows DCFH-DA fluorescence staining used to detect intracellular reactive oxygen species in the Control, Yoda1, VP, and Gel / VP treatment groups; B shows Western blot analysis of Piezo1 and YAP1 protein levels; C shows flow cytometry results of changes in macrophage phagocytic activity; D shows fluorescence microscopy imaging of macrophage morphology and intracellular phagocytic product distribution after phagocytosis of apoptotic cells, with a zoom image below showing magnified details; EF shows the phagocytic rate obtained through flow cytometry and statistical analysis of fluorescence staining; G shows immunofluorescence staining of macrophage CD86 and CD206; and HI shows quantitative analysis of immunofluorescence intensity.

[0020] Figure 4 In the diagram, A is the PCA distribution map; B is the differential gene volcano plot; C is the GO enrichment bar chart; D is the Top 50 differential gene expression heatmap; E is the KEGG pathway bubble chart; and F is the GSEA analysis enrichment curve.

[0021] Figure 5 In the image, AC represents the immunofluorescence imaging of fibrosis markers (COL-1, α-SMA, COL-3); DF represents the quantitative analysis of immunofluorescence intensity; GJ represents the mRNA expression of fibrosis markers and inflammatory markers (IL-1β); K represents the protein expression of fibrosis and anti-fibrosis (TGF-β3) markers analyzed by Western blot; and Actin is the internal control.

[0022] Figure 6 In the diagram, A is a schematic diagram of the wound healing process; B is a representative image of the wound appearance of the control group, Gel group, and Gel / VP group on days 3, 7, and 14; CD is a schematic diagram and quantitative analysis of the relative area of ​​the wound; E is the percentage of collagen fiber area based on Masson staining analysis; and F is a histological image of H&E staining and Masson trichrome staining.

[0023] Figure 7 In the images, A shows Sirius Red staining indicating collagen deposition in wound tissue; B shows YAP immunofluorescence indicating activation of mechanotransmission; C shows iNOS / Arg1 staining indicating macrophage phenotypic transformation; D shows α-SMA / TGF-β3 staining indicating myofibroblast activity; E shows quantitative analysis of fluorescence intensity of each marker; and F shows toxicological assessment of major organs. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0025] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0026] Example 1: Piezo1-mediated Ca 2+ Influx induces apoptosis, which in turn triggers scar formation. To investigate the effects of mechanical stimulation on cell function and scar formation in active wounds, the Piezo1 channel agonist Yoda1 was used to simulate biomechanical stimulation. The induction processes of calcium ion influx, apoptosis, and fibroblast activation in scar formation were systematically analyzed.

[0027] 1.1 Experimental Methods 1.1.1 Cell Culture and Treatment Human umbilical vein endothelial cells (HUVECs), rat fibroblasts (F208), and macrophages (RAW264.7) were used in experiments. HUVECs and F208 cells were cultured in Gibco medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. RAW264.7 cells were cultured in RPMI-1640 medium. All cells were incubated at 37°C in a 5% CO2 incubator until they reached the logarithmic growth phase before experimental treatment. Biomechanical stimulation was simulated using the Piezo1 selective agonist Yoda1 (HY-18723, MedChemExpress). Yoda1 solutions were prepared at different concentrations (1, 3, and 5 μM) and used for subsequent experiments after 24 hours of treatment.

[0028] 1.1.2 Cytotoxicity Detection Cytotoxicity was assessed using the Live / Dead staining method (C2015M, Beyotime). Calcein AM and PI dye were diluted 1:1000 with detection buffer to prepare working solutions. Cells were incubated in this staining working solution for 30 minutes, and cell viability was observed using a fluorescence microscope (Nikon, Japan).

[0029] 1.1.3 Ca²⁺ Concentration Detection Changes in intracellular calcium ion concentration were detected using a Fluo-4 AM fluorescent staining kit (S1060, Beyotime). Cells were incubated with Fluo-4 AM dye at 37°C for 30 minutes, and unbound dye was washed away with PBS after staining. The fluorescence intensity of the cells was observed under a microscope; the fluorescence signal reflected the intracellular calcium concentration. 2+ The concentration was determined, and finally, the fluorescence intensity was quantitatively analyzed using ImageJ software.

[0030] 1.1.4 ROS Level Detection Intracellular ROS levels were detected using a DCFH-DA reactive oxygen species staining kit (S0033S, Beyotime). After Yoda1 treatment, cells were co-incubated with diluted DCFH-DA dye for 30 minutes, followed by washing away unbound fluorescent probes with pre-warmed serum-free medium. Fluorescence signals were detected using a fluorescence microscope (Nikon, Japan). Fluorescence intensity was positively correlated with the amount of intracellular ROS, thus assessing intracellular oxidative stress levels.

[0031] 1.1.5 RNA extraction and gene expression analysis Real-time quantitative PCR (qRT-PCR) was used to quantitatively analyze the mRNA expression levels of specific genes. First, total RNA was extracted using TRIzol reagent, and then reverse transcribed into cDNA using a reverse transcription kit. Subsequently, qRT-PCR was performed using ColorSYBR Green qPCR Master Mix. GAPDH was used as an internal control gene, and its relative expression level was calculated using the 2-ΔΔCT method. All kits were purchased from EZBioscience; specific primer sequences (Sangon, Shanghai) are shown in Table 1.

[0032] Table 1

[0033] 1.1.6 Co-culture model of apoptotic endothelial cells and fibroblasts First, HUVECs were treated with Yoda1 to induce apoptosis, and then apoptotic cells were collected and co-cultured with fibroblasts for 24 hours. After co-culture, the expression of COL-1, COL-3, and α-SMA in fibroblasts was analyzed by immunofluorescence staining and qRT-PCR. Furthermore, the expression of fibrosis-related proteins was analyzed using Western blot. Total cellular proteins were extracted using RIPA lysis buffer (PC101, YARN), separated by SDS-PAGE electrophoresis, and transferred to PVDF membranes. After blocking with 5% skim milk powder, the membranes were incubated with specific antibodies, including COL-1, COL-3, α-SMA, TGF-β3, Piezo1, and YAP1. After initial incubation, the membranes were washed and incubated with appropriate secondary antibodies (all antibodies purchased from Proteintech). After washing, the target protein bands were visualized using ECL chemiluminescence immunoassay (SQ201, YARN).

[0034] 1.1.7 Data Analysis All experimental data were statistically analyzed using GraphPad Prism software, and results are expressed as mean ± standard deviation (SD). Differences between groups were compared using one-way ANOVA or t-tests, with a significance threshold of p < 0.05. Significance levels in the figures are indicated by symbols: “*” indicates a significant difference (0.01 ≤ p < 0.05), “**” indicates a highly significant difference (0.001 ≤ p < 0.01), “***” indicates an extremely significant difference (0.0001 ≤ p < 0.001), and “****” indicates an extremely high significance difference (p < 0.0001).

[0035] 1.2 Results and Discussion 1.2.1 Yoda1 induces endothelial cell apoptosis, Ca²⁺ influx, and upregulation of the Piezo1 / YAP1 pathway. Increased Yoda1 concentration was accompanied by a significant increase in the proportion of dead cells, with a significant increase in the number of dead cells in the 5 μM treatment group. Figure 1 (A) This suggests that high concentrations of Yoda1 are toxic and can induce apoptosis or necrosis. Under Yoda1 stimulation, intracellular Ca²⁺ concentration and ROS levels in endothelial cells significantly increased (…). Figure 1 The expression of Piezo1 and its downstream transcription factor YAP1 was significantly upregulated (C), showing a dose-dependent increasing trend, indicating that cellular Ca²⁺ channels were activated and in a state of stress. Subsequently, the experiment found that the expression of Piezo1 and its downstream transcription factor YAP1 was significantly upregulated (C). Figure 1 (Figure B) verified that Yoda1 activates Ca²⁺ channels, thereby initiating the Piezo1-YAP1-mediated mechanoresponsive pathway. These results are consistent with existing literature, which reports that mechanical stress induces Ca²⁺ overload and ROS accumulation, which synergistically lead to apoptosis. It can be inferred that Yoda1, in simulating mechanical stimulation, promotes endothelial cell apoptosis, thus reproducing the pathological mechanism of "apoptotic cell activation and fibrosis" in the wound microenvironment.

[0036] 1.2.2 Apoptotic endothelial cells induce fibroblasts to transform into scar phenotypes. By constructing a co-culture model of apoptotic endothelial cells (ACs) and fibroblasts, it was verified that apoptotic cells significantly induced the transformation of fibroblasts into a scar phenotype, as evidenced by a significant upregulation of COL-1, COL-3, and α-SMA expression. Figure 1 In DL (low-density lipoprotein), mRNA expression was also significantly increased. Western blot results further confirmed that apoptotic cells led to a significant upregulation of scar-related markers ( ). Figure 1 (M). This phenomenon may be related to the activation of fibroblasts through paracrine mechanisms by pro-inflammatory and pro-fibrotic factors released by ACs, leading to their differentiation into myofibroblasts, increasing collagen deposition and contractile capacity, and ultimately promoting scar formation.

[0037] Example 2: Preparation and characterization of stress-buffering hydrogel dressing (Gel / VP) 2.1 Materials Vertepofen (VP), bleomycin (BLM), cellulose nanocrystals (CNC), and glacial acetic acid were purchased from Maclean's; ZIF-8 was purchased from Xianfeng Nano; dopamine hydrochloride (PDA) was purchased from Yuanye Biotechnology; lithium (2,4,6-trimethylbenzoyl)phosphate (LAP) was purchased from Huaxia Siyin; acrylamide (AAm), N,N'-methylenebisacrylamide (Bis), and methacryloxychitosan (CSMA) were purchased from Sigma-Aldrich. Other conventional chemical reagents were purchased from Sigma-Aldrich or Maclean's and used directly without further purification.

[0038] 2.2 Preparation of CNC@PDA 4 mL of 10 wt% cellulose nanocrystal (CNC) suspension was added to 36 mL of 10 mM Tris (pH=8.5) buffer solution and thoroughly dispersed by vortexing and sonication. Then, 200 mg of dopamine hydrochloride was added, and sonication was used to promote dissolution. The resulting mixture was placed on a constant-speed shaker and reacted under light-protected conditions (covered with aluminum foil) for 6-12 h. During this process, dopamine was gradually oxidized to ortho-quinone and underwent intermolecular condensation to generate highly adhesive polydopamine (PDA), which spontaneously deposited onto the CNC surface. The system turning dark brown indicated that PDA polymerization was essentially complete, thus yielding the CNC@PDA product. During this process, the reaction vessel must be kept partially sealed to ensure dissolved oxygen exchange required for the dopamine self-polymerization reaction.

[0039] 2.3 Preparation of CNC@PDA@ZIF-8 (hereinafter referred to as CP@ZV) composite material Pre-dissolved verteporfin (VP) solution was slowly added to ZIF-8 dispersion and thoroughly mixed under light-protected conditions to promote the diffusion and adsorption of VP molecules into the ZIF-8 channels, thereby completing its encapsulation and fixation within the framework to obtain ZIF-8@VP nanoparticles.

[0040] Next, 20 mg of ZIF-8@VP nanoparticles were weighed and ultrasonically dispersed in 20 mL of the prepared CNC@PDA solution, allowing ZIF-8 and PDA to adhere and be fixed through surface catechol-metal interactions. The mixture was reacted on a shaker for 4-6 hours, during which time the container remained partially uncovered. After the reaction was complete, the CP@ZV nanocomposite material was obtained.

[0041] 2.4 Construction of AAm / CP@ZV single-network hydrogel 1 g of AAm, 5 mg of Bis, and 3 mg of photoinitiator LAP were added to 10 mL of CP@ZV solution and thoroughly mixed by sonication. The resulting precursor solution was then transferred to a hydrogel mold for photo-initiated crosslinking. LAP generated free radicals that initiated the polymerization of AAm, which formed a three-dimensional covalent crosslinked structure through Bis. Simultaneously, inorganic nanostructures were solidified within the network through hydrogen bonding, coordination, and physical entanglement, ultimately yielding an AAm / CP@ZV single-network hydrogel.

[0042] 2.5 Construction of CSMA / AAm / CP@ZV interpenetrating hydrogel 10 mg of methacrylamide chitosan (CSMA) was weighed and dissolved in 10 mL of deionized water containing 0.05–0.1 M glacial acetic acid (pH ≈ 4). After dissolution, the solution was filtered through a 0.45 μm filter to remove undissolved particles. Then, 3 mg of LAP was added to the clear solution and ultrasonically dispersed to obtain the second network precursor solution, CSMA. The prepared AAM monolayer hydrogel was immersed in the CSMA solution. Under the full swelling effect of the hydrogel network, CSMA molecules could penetrate into its internal pores. After secondary photocrosslinking, the methacrylamide groups of CSMA formed a second independent covalently crosslinked network under photoinitiation, which spatially interpenetrated with the original AAM network and produced a synergistic effect through hydrogen bonding and physical entanglement, thus forming a CSMA / AAm / CP@ZV hydrogel (Interpenetrating network, hereinafter referred to as Gel / VP) with an interpenetrating network structure.

[0043] 2.6 Material Characterization 2.6.1 Physicochemical Structure The porosity and microstructure of the material were characterized using scanning electron microscopy (SEM, Hitachi SU8600) and transmission electron microscopy (TEM, FEI Talos F200S). The elemental composition and spatial distribution of the samples were analyzed using energy-dispersive X-ray spectroscopy (EDS, Oxford Ultimax 40) and mapping mode analysis to confirm the distribution characteristics of elements such as C, N, O, and Zn. The crystal structure of the samples was analyzed using powder X-ray diffraction (XRD, Bruker D8 AA25) to determine the formation and phase composition of the composite structure.

[0044] After the hydrogel was frozen and fractured by liquid nitrogen and sputtered with gold, its internal skeletal morphology was observed using SEM. At the same time, elemental mapping was performed on the target area to verify the uniform embedding and dispersion of the composite components in the three-dimensional network of the hydrogel.

[0045] SEM, TEM, and XRD analyses characterized the physicochemical structures of ZIF-8, CNC@PDA, and CP@ZV. The results showed that ZIF-8 exhibited a polyhedral structure with well-defined interfaces. Figure 2 In the middle (A), the signals of C, N, and Zn elements are highly consistent, and typical diffraction peaks appear at 14.8°, 16.4°, and 22.6°. Figure 2 The presence of C indicates that the crystal structure is complete and the chemical composition is uniform. In contrast, CNC@PDA exhibits a continuous fibrous network, with a rougher fiber surface after PDA coating and a significantly enhanced distribution of C, N, and O elements, proving successful PDA deposition. For CP@ZV, the fiber surface is loaded with a large number of particles, and the Zn element highly overlaps with the fiber skeleton, while the characteristic peaks of ZIF-8 are still clearly visible, indicating that ZIF-8 has achieved in-situ attachment on the fiber surface, forming a crystal structure with smaller particle size and higher dispersion.

[0046] Based on the above composite nano-units, in the structural characterization of single-network and interpenetrating-network hydrogels, the elemental signals of C, N, O, and Zn are uniformly distributed in the pore walls and framework region, indicating that the composite nano-units are successfully embedded and stably exist in the hydrogel network structure. Figure 2 (B)

[0047] 2.6.2 Swelling properties The swelling behavior was assessed by immersing the pre-fabricated hydrogel in deionized water, removing it at different time intervals, absorbing the surface moisture, and then weighing it. The swelling ratio (SR) was calculated using a formula, where W0 is the initial mass and Wt is the mass at time t.

[0048]

[0049] The two differ significantly in their microstructure and swelling behavior. Figure 2(D). Specifically, the Singlenetwork hydrogel exhibits a dense structure with small pores, stabilizing after rapid initial water absorption, with an equilibrium swelling ratio of 97.63% ± 1.44%, effectively reducing the excessive swelling (>200%) of AAm hydrogels. In contrast, the Interpenetrating Network hydrogel displays a more interconnected macroporous structure. Thanks to the strong hydrophilicity of CSMA, the interpenetrating network achieves a higher degree of hydration and a more open pore structure during construction. Furthermore, the Interpenetrating Network hydrogel has an equilibrium swelling ratio of only 58.27% ± 2.46%, because the interpenetration of the two networks creates more chain segment constraints, suppressing swelling; simultaneously, the multi-point anchoring of rigid ZIF-8 and flexible CNC fibers further enhances the stability of the framework, thereby reducing volume expansion.

[0050] 2.6.3 Tensile mechanical properties Tensile tests were performed on rectangular hydrogels measuring 20 mm in length, 10 mm in width, and 2 mm in thickness using an Instron 5542 universal testing machine (equipped with a 100 N sensor) at a loading rate of 2 mm / min. At least three parallel samples were tested in each group. Mechanical parameters were recorded, and tensile stress-strain curves were plotted to calculate Young's modulus.

[0051] During macroscopic stretching ( Figure 2 The interpenetrating network hydrogel exhibits excellent elasticity, maintaining continuous deformation without local rupture even under high strain conditions. Further research revealed that the single network exhibits stress fluctuations during the intermediate strain stage. Figure 2 In the case of F), the stress curve of the interpenetrating network always increases smoothly, and the Young's modulus can reach 8.42±1.56 kPa. Figure 2 The stress at break (G) was not significantly different from that of the single network (15.52±8.79 kPa). Furthermore, the interpenetrating network exhibited a higher elongation at break (100%), indicating more stable stress transfer and superior toughness throughout the tensile process.

[0052] This "high modulus-high ductility" characteristic stems from the graded contribution of the dual-network structure at different strain stages: on the one hand, the chain segments inside the interpenetrating network are more hydrated, and the second network is more flexible, enabling the material to exhibit greater chain segment mobility and compliance within a small strain range; on the other hand, as the strain increases, the synergistic load-bearing capacity of the CP@ZV multi-point anchoring structure and the interpenetrating network is gradually activated, providing stronger structural support for the material, enabling it to effectively disperse the load and maintain overall stability under large deformation, thereby achieving a balance between flexibility, elasticity, and mechanical buffering capacity.

[0053] Example 3: Validation of Gel / VP's Inhibition of Mechanical Stress-Related Fibrosis at the Cellular Level 3.1 Experimental Methods 3.1.1 Macrophage function detection and immunophenotypic analysis In the assay to detect the phagocytic function of macrophages, macrophages were first co-cultured with the experimental material for 24 hours, followed by treatment with Yoda1 for 24 hours, and finally co-incubated with apoptotic endothelial cells for 4 hours. During this process, macrophages were labeled with calcein (C2015M, Beyotime), and apoptotic endothelial cells were labeled with Dil (D3911, Invitrogen). The phagocytic capacity of macrophages was quantitatively analyzed by flow cytometry. Simultaneously, the nuclei and cytoskeleton of macrophages were stained, and the cell burial effect was observed using a confocal microscope (SP8, Leica, Germany) to evaluate the impact of Yoda1 treatment and experimental material intervention on macrophage function.

[0054] Furthermore, immunofluorescence staining was used to observe the polarization of macrophages in the immune environment. After co-culturing the materials and cells and treating with Yoda1, the cells were first fixed with 4% paraformaldehyde, followed by permeabilization and blocking. The cells were incubated overnight with primary antibodies CD86 and CD206, and then incubated with the corresponding fluorescently labeled secondary antibodies. The cell nuclei were labeled with DAPI, and the cytoskeleton was labeled with Actin. The expression of macrophage M1 and M2 markers was observed using a confocal microscope (SP8, Leica, Germany), and the fluorescence signal intensity was quantitatively analyzed using ImageJ software.

[0055] 3.1.2 RNA-seq transcriptome analysis To further investigate the effects of Gel / VP on the macrophage transcriptome, high-throughput RNA-seq was performed on the macrophage transcriptomes of both the Gel / VP-treated and control groups. First, total RNA was extracted from cells using TRIzol reagent, and high-throughput transcriptome sequencing was performed using the Illumina HiSeq platform. Principal component analysis (PCA) was used to assess gene expression differences between the groups and to screen for differentially expressed genes. Subsequently, GO and KEGG enrichment analyses were used to reveal the underlying biological processes and signaling pathways.

[0056] 3.1.3 Fibrosis Model and Intervention Effect Evaluation The inhibitory effect of Gel / VP composite material on fibrosis was investigated using an indirect co-culture system. First, fibroblasts were treated with 2 μM BLM for 24 hours to establish a fibrosis pathological model. Then, macrophages were subjected to Yoda1 stimulation, apoptotic cell uptake, and Gel / VP intervention, respectively. The specific groupings were as follows: the group receiving Yoda1 stimulation and apoptotic cell uptake was named the RAW / Acs group; the group receiving Yoda1 stimulation and material intervention alone was named the Gel / VP group; and the group receiving Yoda1 stimulation, apoptotic cell uptake, and material intervention simultaneously was named the RAGV group. Next, conditioned medium from each macrophage treatment group (RAW / Acs, Gel / VP, RAGV) was collected and co-cultured with BLM-induced activated fibroblasts for 24 hours (Control, BLM, RAW / Acs, Gel / VP, RAGV). After intervention, immunofluorescence staining was used to observe the effect of secretions from each group on the expression of fibrosis markers (COL-1, COL-3, α-SMA). Furthermore, the expression of genes and proteins related to fibrosis (COL-1, COL-3, α-SMA), anti-fibrosis (TGF-β3), and inflammation (IL-1β) was further detected by qRT-PCR and Western blot methods.

[0057] 3.1.4 Data Analysis All experimental data were statistically analyzed using GraphPad Prism software, and results are expressed as mean ± standard deviation (SD). Differences between groups were compared using one-way ANOVA or t-tests, with a significance threshold of p < 0.05. Significance levels in the figures are indicated by symbols: “*” indicates a significant difference (0.01 ≤ p < 0.05), “**” indicates a highly significant difference (0.001 ≤ p < 0.01), “***” indicates an extremely significant difference (0.0001 ≤ p < 0.001), and “****” indicates an extremely high significance difference (p < 0.0001).

[0058] 3.2 Results and Discussion 3.2.1 Gel / VP synergistically activates macrophage phagocytic function and remodels the immune microenvironment Previous studies have demonstrated that Yoda1 induces Ca²⁺ influx and ROS accumulation in endothelial cells by activating Piezo1 channels, thereby triggering apoptosis, a phenomenon consistent with existing literature. However, if apoptotic cells are not promptly eliminated, they will drive fibroblasts to transform into a scar phenotype, keeping the local microenvironment in a state of persistent inflammation and fibrosis. Therefore, regulating Piezo1-related pathological signals, enhancing phagocytic clearance capacity, and remodeling the immune microenvironment are important strategies for achieving scarless repair.

[0059] After material preparation and characterization, their regulatory effects on the Piezo1-YAP1 pathway and inflammatory environment were investigated. Results showed that both VP and Gel / VP effectively inhibited Yoda1-induced ROS accumulation, with the Gel / VP group exhibiting the most significant inhibitory effect. Figure 3 (A). Furthermore, Western blot analysis showed that Yoda1 upregulated the expression of Piezo1 and its downstream YAP1, while VP and Gel / VP reversed this trend (…). Figure 3 The results (B) suggest that it can intervene in the sustained activation of mechanosensitive signals and alleviate cellular stress and inflammatory responses. This effect mainly stems from the uniform anchoring of CP@ZV in the interpenetrating network of Gel / VP, which makes the local micromechanical environment more stable, thereby weakening the abnormal activation of Piezo1 by external mechanical stimulation; the pore-released VP of ZIF-8@VP can sustainably intervene in YAP1-related pathological signals; at the same time, PDA binding to metal ions can inhibit the generation of ROS and the scavenging of free radicals.

[0060] Based on the close link between Piezo1 overactivation and fibrosis, the effects of abnormal mechanical stimulation on macrophage function were assessed using flow cytometry and confocal imaging. Figure 3 (In CF). Specifically, Yoda1 treatment significantly inhibited the phagocytic capacity of macrophages, reducing the phagocytic rate from 22.50±3.87% and 12.66±3.09% to 7.50±2.38% and 1.71±0.62%. However, intervention with VP and Gel / VP effectively restored this function, with the Gel / VP composite showing a more significant effect, increasing the phagocytic rate to 64.50±7.19% and 41.72±6.16%. This improvement allows macrophages to fully utilize their function of clearing apoptotic cells and cell debris, thereby limiting the spread of pro-fibrotic signals and promoting inflammation resolution.

[0061] Further research revealed that abnormal mechanical stimulation not only weakens phagocytic function but also drives it towards an inflammatory immune state, and even exacerbates local tissue damage and fibrosis. Immunofluorescence staining results showed ( Figure 3Yoda1 (containing G, H, and I) induces macrophage polarization towards the pro-inflammatory M1 type (CD86) and inhibits the anti-inflammatory repair M2 type (CD206). Notably, intervention with VP and Gel / VP effectively reversed this polarization trend and promoted CD206 expression, with Gel / VP showing the most significant immunomodulatory effect. This advantage can be attributed to the interpenetrating network structure of Gel / VP providing excellent mechanical buffering capacity and highly hydrophilic surface properties, reducing non-specific protein adsorption, thereby reducing macrophage immune recognition of materials and alleviating inflammatory stimulation caused by mechanical stress. At the same time, the sustained release of VP in the ZIF-8 porous structure avoids cellular stress caused by a sudden increase in drug concentration, and continuously and gently regulates the YAP-related signaling pathway, thereby stabilizing macrophage function and promoting its transformation into a repair-oriented macrophage.

[0062] In summary, the gel / VP hydrogel, based on the excellent mechanical buffering and hydrophilic regulation capabilities provided by its interpenetrating network structure, as well as the sustained regulation of YAP signaling and oxidative stress by gel contraction and VP sustained release, significantly enhances the ability of macrophages to clear apoptotic cells and inflammatory factors, and promotes their transformation into repair-oriented cells. Ultimately, it achieves the remodeling of the immune microenvironment and provides an effective intervention strategy for inflammation control and tissue repair.

[0063] 3.2.2 Transcriptome Mechanism of Gel / VP-Mediated Macrophage Functional Remodeling RNA-seq analysis was used to explore the molecular basis of Gel / VP's improvement of macrophage function. The results showed significant separation at the transcriptional level between the Gel / VP treatment group and the control group, identifying 302 upregulated genes and 113 downregulated genes, suggesting that Gel / VP clearly remodeled the overall expression status and function of macrophages. Figure 4 The Top 50 differentially expressed gene heatmap showed that genes related to calcium ion channels (Cacna1a, Cbr2), redox reactions (Ms4a6c), cytoskeleton dynamics (Gtpbp2, Syne2), and the YAP pathway (Angptl6, Atp6v0a1) all underwent significant changes. Figure 4 The differentially expressed genes (D) were highly consistent with the roles of Gel / VP in mechanical stress buffering, ROS regulation, and cell motility improvement. GO and KEGG enrichment results indicated that these differentially expressed genes were mainly involved in key immune processes such as phagocytosis regulation, calcium channel activity, and inflammatory responses. Figure 4 (E). GSEA further showed that Gel / VP significantly upregulated functional groups such as "Cytoskeletal motor activity," "Actin-based cell projections," and "GTPase regulator activity," while inhibiting stress pathways related to calcium overload. Figure 4 The presence of β-F (in the middle F) suggests that Gel / VP restores macrophage phagocytic activity by modulating YAP signaling and mechanical stress. In summary, Gel / VP reveals its potential mechanism in immune regulation and antifibrotic therapy by reshaping the transcriptional profile of macrophages.

[0064] 3.2.3 Gel / VP remodels macrophage secretions to drive transcellular antifibrotic regulation The above results confirm that Gel / VP can enhance the phagocytic capacity of macrophages and induce their M2 polarization, thereby reshaping the immune microenvironment with anti-inflammatory and repair characteristics. To verify its transmission mechanism in anti-fibrosis, an indirect co-culture system was used for further investigation. First, macrophages were treated with Yoda1 stimulation, apoptotic cell uptake, and Gel / VP intervention, respectively. Then, the conditioned medium of each treatment group was collected to intervene in bleomycin (BLM)-induced activated fibroblasts to evaluate its effect on fibrosis-related phenotypes.

[0065] BLM treatment significantly upregulated the expression of fibrosis marker proteins COL-1, COL-3, and α-SMA, indicating that fibroblasts were in a highly activated state. Figure 5 (AC). However, after stimulation with conditioned medium in the RAW / ACs group, the activation level was alleviated, and the regulatory effect of the Gel / VP group was more obvious. Particularly noteworthy was the RAGV group (supernatant obtained after macrophages phagocytose apoptotic cells and receive Gel / VP intervention), which showed the most significant inhibition of COL-1, COL-3, and α-SMA expression. Quantitative fluorescence (QF) Figure 5 DF), mRNA level ( Figure 5 (GJ) and Western blot ( Figure 5 The results from the study consistently showed that RAGV treatment significantly inhibited the expression of fibrotic factors and pro-inflammatory factors (IL-1β), accompanied by the upregulation of anti-fibrotic factor (TGF-β3), indicating that this strategy not only has anti-inflammatory capabilities but may also activate tissue repair signaling pathways.

[0066] The formation of this anti-fibrotic effect likely depends on the reprogramming of macrophage functional profiles by Gel / VP. First, the material effectively alleviates oxidative stress by slowly releasing VP, maintaining macrophage homeostasis and inducing M2 polarization, thereby producing restorative cytokines. Second, the Gel / VP material itself possesses excellent mechanical buffering and hydrophilic regulation properties, with low protein adsorption and low inflammatory stimulation on its surface, helping to reduce the stress response of macrophages to environmental stimuli and remodel their secretions. Finally, it is noteworthy that macrophages in RAGV undergo two phases: apoptotic cell clearance and Gel / VP hydrogel intervention. This dual regulation may endow them with higher immune tolerance and pro-repair properties, prompting them to secrete more anti-fibrotic factors such as TGF-β3 and reduce the release of pro-inflammatory factors such as IL-1β. This allows for effective delivery to fibroblasts via conditioned culture medium, achieving a cascaded fibrosis-inhibiting effect at the "immune-mesenchymal" level, demonstrating cross-cellular level anti-fibrotic potential.

[0067] Example 4: Gel / VP accelerates repair and inhibits scar formation in a rat skin trauma model. 4.1 Experimental Methods All animal experiments were approved by the Animal Research Committee of Tongren Hospital Affiliated to Shanghai Jiao Tong University (Approval No.: A2025-062-01). Male SD rats aged 6-8 weeks were anesthetized under isoflurane inhalation. Subsequently, the dorsal skin was shaved and strictly disinfected, and standardized circular full-thickness skin defects were prepared using a disposable 16mm diameter punch. PBS, Gel, and Gel / VP materials were then injected into the wound using a 1mL syringe. The wound was covered with a transparent dressing to reduce the risk of contamination and infection, and the dressing was changed every 48 hours. Wound healing was recorded using a digital camera on postoperative days 7 and 14, and the animals were euthanized at the corresponding time points. The regenerated skin tissue was completely removed, laid flat on filter paper to maintain its natural shape, and fixed in 4% paraformaldehyde for 24 hours.

[0068] Fixed tissues were routinely dehydrated, paraffin-embedded, and cut into 5μm serial sections. H&E staining, Masson staining, and Sirius red staining were performed to assess epithelial regeneration, collagen fiber arrangement and deposition patterns, and the degree of fibrotic remodeling at different postoperative time points. Subsequently, immunofluorescence staining was performed on tissue samples: iNOS / Arg1 was used to evaluate local inflammation and macrophage polarization; α-SMA / TGF-β3 was used to analyze fibroblast activation and scar formation-related signals; and YAP was used to detect mechanobiological cellular responses and tissue regeneration activity.

[0069] 4.2 Results and Discussion Further histological evaluation was conducted to assess the repair effect of Gel / VP in a rat skin trauma model. Figure 6 (A). Optical observation shows ( Figure 6 In the middle BD group, the Gel / VP group showed significant reduction in wound area within week 1 and almost complete closure (>95%) within week 2; the healing speed of the Gel group was also significantly faster than that of the control group, but overall it was still weaker than that of the Gel / VP group, with more noticeable residual wounds. HE, Masson, and Sirius Red staining results showed that ( Figure 6 EG, Figure 7 In the Gel / VP group (A), more continuous new epithelium was formed in the first week, with less inflammatory infiltration and a more compact and orderly arrangement of collagen fibers. By the second week, its dermal structure was closer to that of normal skin. Although the Gel group also showed some degree of tissue reconstruction, it was weaker than the Gel / VP group in terms of epithelial integrity, fiber arrangement and inflammation control.

[0070] Gel / VP hydrogel significantly inhibited YAP activation in the wound area ( Figure 7 (B), while promoting the transformation of macrophages from pro-inflammatory to reparative types ( Figure 7 In the middle C group, iNOS expression was decreased and Arg1 expression was increased. While the Gel group exhibited the same regulatory trend, the magnitude was limited. Figure 7 In addition, the number of α-SMA-positive myofibroblasts was significantly reduced in the Gel / VP group, while maintaining a higher level of TGF-β3 (E). Figure 7 Gel (or Gel / VP) treatments were beneficial in reducing scarring and promoting more normal ECM remodeling. Further toxicological evaluation showed that the major organs (heart, liver, spleen, lungs, and kidneys) of rats treated with Gel or Gel / VP maintained normal histological structure, comparable to the control group. Figure 7 No detectable toxicity was found in the samples from the F sample, confirming the biocompatibility of both materials.

[0071] Overall, both gel and gel / VP have the ability to promote tissue repair, but gel / VP performs better. Gel, with its moisturizing properties, elasticity, and porous structure, provides a moist, breathable, and relatively uniform stress environment for the wound. Simultaneously, it utilizes the opposing mechanical forces of gel contraction and wound stretching to inhibit Piezo1-YAP pathway activation, thereby buffering external mechanical stress and supporting epithelialization and early collagen remodeling. In contrast, gel / VP, with the addition of VP, possesses even stronger mechanical buffering capacity and more sustained drug release, maintaining a more stable mechanical and immune microenvironment during wound healing. This more effectively inhibits YAP activation, promotes the transformation of macrophages into repair-oriented cells, and reduces the degree of fibrosis.

[0072] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A mechanically adaptive hydrogel for eliminating apoptotic cells and inhibiting tissue fibrosis, characterized in that, The hydrogel comprises an interpenetrating network polymer matrix and a dynamic nanocomposite material embedded in the matrix; The dynamic nanocomposite material comprises cellulose nanocrystals, a polydopamine layer coated on the surface of the cellulose nanocrystals, and drug-loaded metal-organic framework particles loaded through the polydopamine layer. The interpenetrating network polymer matrix comprises a first crosslinked network and a second crosslinked network; The first crosslinking network is a polymer network based on acrylamide monomers; The second crosslinking network is a polymer network based on methacrylamide chitosan.

2. The mechanically adaptive hydrogel for clearing apoptotic cells and inhibiting tissue fibrosis according to claim 1, characterized in that, The metal-organic framework is ZIF-8.

3. The mechanically adaptive hydrogel for clearing apoptotic cells and inhibiting tissue fibrosis according to claim 1, characterized in that, The drug loaded in the metal-organic framework is verteporfen.

4. A method for preparing a mechanically adaptive hydrogel as described in any one of claims 1-3, characterized in that, Includes the following steps: S1, Preparation of nanocomposite material: Polydopamine is polymerized on the surface of cellulose nanocrystals to form a coating layer, resulting in CNC@PDA; Drug is loaded into metal-organic framework particles to obtain drug-loaded particles; The drug-loaded particles are mixed with CNC@PDA to attach the drug-loaded particles to the surface of CNC@PDA, resulting in nanocomposite material; S2, Constructing the first network hydrogel: Mix the nanocomposite material, acrylamide monomer, crosslinking agent and first photoinitiator, and carry out the first photocrosslinking reaction to form the first crosslinked network hydrogel; S3, Constructing an interpenetrating network hydrogel: Immerse the first cross-linked network hydrogel in a second network precursor solution containing methacrylamide chitosan and a second photoinitiator, allowing the second network precursor solution to penetrate into the interior of the first cross-linked network hydrogel and then undergo a second photocrosslinking reaction to form an interpenetrating network structure hydrogel.

5. The preparation method according to claim 4, characterized in that, In step S1, dopamine hydrochloride is oxidatively polymerized in the dispersion of the cellulose nanocrystals in the presence of a Tris buffer solution at pH 8.5, thereby forming a polydopamine coating layer on the surface of the cellulose nanocrystals.

6. The preparation method according to claim 4, characterized in that, The drug is vertepofen, and the metal-organic framework is ZIF-8.

7. The preparation method according to claim 4, characterized in that, S2 specifically includes: Acrylamide monomer, crosslinking agent N,N'-methylenebisacrylamide, and a first photoinitiator are added to a nanocomposite dispersion with a concentration of 1-5 mg / mL; the concentration of the acrylamide monomer is 100-500 mg / mL, the mass ratio of the crosslinking agent to the acrylamide monomer is (0.1-1):100, and the mass ratio of the first photoinitiator to the acrylamide monomer is (0.1-0.5):100; a first photocrosslinking reaction is carried out to form a first crosslinked network hydrogel.

8. The preparation method according to claim 4, characterized in that, S3 specifically includes: Methacrylamide chitosan was dissolved in an aqueous solution of glacial acetic acid with a concentration of 0.05-0.1M, and a second photoinitiator was added to prepare a second network precursor solution with a methacrylamide chitosan concentration of 0.5-2 mg / mL. The first cross-linked network hydrogel was immersed in the second network precursor solution, and the second network precursor solution was allowed to penetrate into the interior of the first cross-linked network hydrogel to carry out a second photocross-linking reaction, forming an interpenetrating network structure hydrogel.

9. The preparation method according to claim 4, characterized in that, Both the first photoinitiator and the second photoinitiator are lithium (2,4,6-trimethylbenzoyl) phosphate salts.

10. The use of the mechanically adaptive hydrogel according to any one of claims 1-3 in the preparation of products for the prevention or treatment of tissue fibrosis scars.

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