Density-driven all-bio-based Janus hydrogel and preparation method and application thereof

Abstract

The application discloses a kind of full biological Janus hydrogel based on density driving in the field of biological medicine and its preparation method and application, the present application utilizes the gravity stability brought by the density difference of N-acryloyl aspartic acid precursor solution and methacrylated gelatin precursor solution, by sequential pouring, combined with one-step photopolymerization process, successfully prepared a kind of adherent-adhesion type full biological Janus hydrogel with excellent mechanical properties, the Janus hydrogel can withstand large size strain stretching without delamination, also has other physical and chemical asymmetric characteristics, such as gradient degradation, asymmetric hydrophilicity and asymmetric swelling, can adapt to complex gastric environment and gastric perforation repair cycle, realize strong adhesion while preventing postoperative adhesion, provide an effective way for the design of advanced surgical sealant.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a density-driven, fully bio-based Janus hydrogel, its preparation method, and its applications. Background Technology

[0002] Gastric perforation (GP) is a life-threatening emergency that can lead to peritonitis and systemic inflammation. Traditional surgical sutures often result in additional tissue damage; therefore, biocompatible material adhesion is an important approach to achieving gastric wall sealing and remodeling. Poly(N-acryloyl aspartic acid) PAASP, derived from natural amino acids, is an excellent material for repairing gastrointestinal wounds due to its high density of dicarboxyl groups, which can anchor tissues through hydrogen bonds. Literature reports that PAASP-35 weight percentage hydrogel has high tensile strength (73 kPa), ultra-high elongation (1700% tensile strain), and strong adhesion to porcine skin (120 kPa). However, this high adhesion presents a challenge in preventing postoperative adhesions.

[0003] Janus hydrogels are materials with asymmetric functions on both sides, providing an effective strategy for resolving this contradiction. Pu's research team coated one side of PAASP hydrogel with FeCl3-impregnated filter paper, using Fe... 3+ Coordination selectively inhibits the adhesion of catechols; however, metal ion cross-linking systems often induce chronic inflammatory responses and local oxidative stress, which may hinder natural gastric regeneration.

[0004] Gelatin possesses a natural RGD structure, and its tissue adaptability can help repair gastric perforations. Under normal circumstances, it is relatively fragile. Modified methacrylamide gelatin (GelMA) exhibits excellent thermal stability and high storage modulus after photoinitiated polymerization. However, compared with PAASP, its adhesion properties are significantly weaker.

[0005] Tang's research team prepared Janus hydrogels by utilizing the thermal response phase change properties of GelMA. By using surfaces at different temperatures, such as 65 ℃ and 25 ℃, the stretching and folding of GelMA units were triggered, thereby achieving the switching between adhesive and non-adhesive states. However, compared with actively designed chemical barriers, its physical isolation effect is weaker. Wen's research team prepared a Janus hydrogel by coating a genipin-crosslinked polylysine adhesive layer on a prepolymerized non-adhesive GelMA layer. Its disadvantage is that the process steps are complicated and the chemical crosslinking reduces the degradation performance.

[0006] Wang's research team prepared a Janus hydrogel by shielding the hydrophilic carboxyl groups on the top layer through the self-assembly of hydrophobic lauryl methacrylate, causing it to aggregate at the bottom. Liu's research team achieved a one-pot Janus hydrogel preparation by using light / heat to realize the spontaneous gradient distribution of chitosan and polyethylene. This natural layering method shows significant advantages in the preparation of asymmetric hydrogels. PAASP and GelMA are both bio-based polymers. If the natural layering of PAASP and GelMA can be achieved by utilizing the differences in their specific physical properties, the resulting Janus hydrogel can not only avoid the inflammatory response caused by metal-modified PAASP, but also promote the repair of gastric perforation wounds. Summary of the Invention

[0007] The purpose of this invention is to solve the above-mentioned problems by providing a density-driven, fully bio-based Janus hydrogel, its preparation method, and its applications, especially for the sealing treatment of gastric perforation.

[0008] The present invention achieves the above objectives through the following technical solutions: As a first aspect of the present invention, a method for preparing a density-driven, fully bio-based Janus hydrogel is provided. The method comprises: utilizing the density difference between the N-acryloyl aspartic acid precursor solution and the methacrylated gelatin precursor solution, achieving gravity-stabilized stratification through sequential casting, and combining this with a one-step photopolymerization process to prepare the fully bio-based Janus hydrogel; wherein, sequential casting refers to casting the N-acryloyl aspartic acid precursor solution first and then casting the methacrylated gelatin precursor solution.

[0009] As a further optimization of the present invention, the N-acryloyl aspartic acid precursor solution is prepared by dissolving N-acryloyl aspartic acid in deionized water and adding a photoinitiator; the mass-volume concentration of the N-acryloyl aspartic acid precursor solution is 40-45%, w / v.

[0010] As a further optimization of the present invention, the methacrylamide gelatin precursor solution is prepared by transferring Laponite nanoclay dispersed in deionized water into a methacrylamide gelatin solution containing a photoinitiator. The degree of methacrylation of the methacrylated gelatin is 15-45%; the mass-volume concentration of the methacrylated gelatin in the methacrylated gelatin precursor solution is 5-10% (w / v); and the mass-volume concentration of the Laponite nanoclay in the methacrylated gelatin precursor solution is 0.65-0.70% (w / v).

[0011] As a further optimization of the present invention, an N-acryloyl aspartic acid precursor solution is first injected into a mold, and then a methacrylamide gelatin precursor solution is poured onto its surface. The mold after pouring is placed under ultraviolet light for irradiation, and the fully bio-based Janus hydrogel can be obtained by one-step photopolymerization.

[0012] As a further optimization of the present invention, the volume ratio of the N-acryloyl aspartic acid precursor solution to the methacrylated gelatin precursor solution is 5-7:1.

[0013] As a second aspect of the present invention, a fully bio-based Janus hydrogel prepared by any of the above preparation methods is also provided. The fully bio-based Janus hydrogel has an adhesion-anti-adhesion bilayer structure, wherein a poly(N-acryloyl aspartic acid) adhesion layer is formed by an N-acryloyl aspartic acid precursor solution under the initiation of a photoinitiator, and a methacryloyl gelatin anti-adhesion layer is formed by methacryloyl gelatin and Laponite nanoclay under the initiation of a photoinitiator. The two layers form a seamless interface through interfacial covalent bonding and bridging effect of Laponite nanoclay.

[0014] As a third aspect of the present invention, the application of density-driven, fully bio-based Janus hydrogel as described in any of the foregoing claims in the preparation of medical anti-adhesion patches is also provided.

[0015] As a further optimization of the present invention, the application site of the all-biobased Janus hydrogel is any one of the following: stomach, liver, kidney, intestines or skin tissue.

[0016] As a fourth aspect of the invention, the application of a density-driven, fully bio-based Janus hydrogel as described in any of the foregoing claims in the preparation of a gastric perforation repair sealant is also provided.

[0017] As a further optimization of the present invention, the all-bio-based Janus hydrogel is applied as a surgical sealant to the gastric perforation wound. The bicarboxyl groups of the poly(N-acryloyl aspartic acid) adhesion layer mediate hydrogen bonding and strongly adhere to the gastric tissue, forming an active expanding embolism to seal the gastric perforation wound. The hydrophilicity of the methacryloyl gelatin anti-adhesion layer is used to construct a dense hydration layer to achieve postoperative anti-adhesion.

[0018] The beneficial effects of this invention are as follows: This invention utilizes the gravitational stability resulting from density differences to successfully prepare an adhesion-anti-adhesion type fully bio-based Janus hydrogel with excellent mechanical properties through sequential casting combined with a one-step photopolymerization process. The structure consists of a poly(N-acryloyl aspartic acid) adhesion layer (PA layer) and a methacrylamide gelatin anti-adhesion layer (G layer), exhibiting a tensile strain of 1529.7% and a tensile stress of 18 kPa. The addition of Laponite nanoclay bridges the interface, preventing delamination. Combined with covalent bonds formed through interfacial penetration, this hydrogel achieves strong adhesion to gastric tissue, with a burst pressure of 135 mmHg and a peel strength of 216.3 ± 3.2 N / m, and remains stable even after long-term immersion in phosphate-buffered saline (PBS) or simulated gastric juice (SGF). Further testing revealed that, in addition to the difference in adhesion between the two sides, the Janus hydrogel also possesses several asymmetries that are beneficial for the repair of gastric perforations. For example, the PA layer has a higher degree of swelling than the G layer, which helps to form an active, expanding embolus; the G layer has better hydrophilicity than the PA layer, which can construct a dense hydration layer to prevent postoperative adhesion; the gradient degradation characteristics of the PA and G layers match the wound healing cycle, etc. More importantly, in experiments on the repair of gastric perforations in mice and the prevention of postoperative adhesions, this all-bio-based material avoids the chronic inflammation problems that may be caused by common metal ion-based adhesive shielding systems, providing an effective approach for the design of advanced surgical sealants. Attached Figure Description

[0019] Figure 1 The synthesis of N-acryloyl aspartic acid (AASP) provided by this invention is shown in the figure. Figure a shows the synthetic route of AASP, figure b shows the H NMR spectrum, and figure c shows the FTIR spectrum. Figure 2 The structural characterization of GelMA provided by the present invention is shown in the figure. Figure a is the H NMR spectrum of GelMA; Figure b is the FTIR spectrum of GelMA; Figure c is the degree of methacrylation (DM) of different GelMAs determined by H NMR and 2,4,6-trinitrobenzenesulfonic acid (TNBS) determination method. Figure 3 The effect of light intensity on the tensile strength of PAASP hydrogel is provided in this invention; Figure 4This invention provides a schematic diagram of the preparation of PA-GX / Y / Z Janus hydrogel. Figure a shows the hydrogel prepared by sequential casting with AASP and GelMA under UV light induction, along with photographs of the hydrogel and interface layer. Figure b is a schematic diagram of the microstructure of the Janus hydrogel, which includes an anti-adhesion layer, an interface layer, and an adhesion layer. Figure c shows photographs of the Janus hydrogel in a bent or twisted state, and its adhesion effect on a movable finger joint. Figure d shows the adhesion of the dicarboxyl groups to tissue via hydrogen bonds formed by amino and carboxyl groups. Figure 5 This invention provides the preparation of hydrogels under different pouring sequences; Figure 6 SEM characterization of the PA-GX / Y / Z Janus hydrogel provided by the present invention; Figure a shows the pore size characterization of different G layers and PA layers; Figure b shows the SEM image of the interface cross section of the PA-G-15 / 5 / 7 Janus hydrogel; Figures cj show the SEM images of the PA layer and different G layers, respectively. Figure 7 Mechanical property characterization of the PA-GX / Y / Z Janus hydrogel provided by this invention; Figure af shows the PA / G layer volume ratio, C G The influence of DM on mechanical properties, and the corresponding elastic modulus and toughness; Figure 8 The mechanical properties of the hydrogels provided by this invention are compared and shown in physical images; Figure a shows a comparison of the properties of different PA-GX / Y / Z Janus hydrogels; Figure b shows a photograph of PA-G-15 / 5 / 7 Janus hydrogels attached to a continuously expanding balloon. Figure 9 The compressive properties of the PA-G-15 / 5 / 7 Janus hydrogel provided by this invention are characterized. In the figure, a) shows the compressive stress-strain curves of the G-15 / 5 / 7 layer, the PA layer, and the PA-G-15 / 5 / 7 Janus hydrogel; b) shows a photograph of the recovery of the PA-G-15 / 5 / 7 Janus hydrogel after compression; c) shows the compressive stress-strain curves of the PA-GX / Y / Z Janus hydrogel in three consecutive compression cycles. Figure 10 The effect of Laponite on the mechanical properties of PA-G-15 / 5 / 7 hydrogel provided by this invention; Figure 11 Degradation characteristics of the PA layer, G-15 / 5 / 7 layer and PA-G-15 / 5 / 7 Janus hydrogel provided by the present invention in PBS; Figure 12The contact angle properties of the PA-GX / Y / Z Janus hydrogel provided by this invention are characterized; in the figure, a is the contact angle image of different surfaces; b is the contact angle value of the PA-GX / Y / Z Janus hydrogel. Figure 13 The swelling properties of the PA-GX / Y / Z Janus hydrogel provided by this invention are characterized. In the figure, Figure a shows the asymmetric swelling behavior and adhesion stability of the PA-G-15 / 5 / 7 Janus hydrogel after being immersed in PBS and SGF for 60 minutes; Figure b shows the asymmetric swelling behavior curve of the PA-G-15 / 5 / 7 Janus hydrogel. Figure 14 A schematic diagram of the experimental apparatus for testing burst pressure and peel strength provided by the present invention; in the figure, a is a schematic diagram of the experimental apparatus for testing burst pressure; b is a schematic diagram of the experimental apparatus for testing peel strength. Figure 15 The adhesion properties of the PA-GX / Y / Z Janus hydrogel provided by the present invention are characterized; in the figure, figure a shows the burst pressure of different PA-GX / Y / Z Janus hydrogels; figure b shows the burst pressure of PA-G-15 / 5 / 7 Janus hydrogels on different organs. Figure 16 The peel strength of the PA-GX / Y / Z Janus hydrogel provided by this invention is characterized; in the figure, a shows the peel strength of the PA layer on different PA-GX / Y / Z Janus hydrogels; b shows the peel strength between the PA layer and the G layer in the PA-G-15 / 5 / 7 Janus hydrogel; cd shows the peel strength of the PA layer in the PA-G-15 / 5 / 7 Janus hydrogel after soaking in PBS or SGF for different times. Figure 17 The relative cell viability of PA-G-15 / 5 / 7 Janus hydrogel at 450 nm wavelength, as detected by CCK-8 assay, provided by this invention; Figure 18 Characterization of the postoperative anti-adhesion performance of PA-GX / Y / Z Janus hydrogel provided by the present invention; Figure a shows a mouse gastric perforation model treated with Janus hydrogel patch and surgical suture; Figure b shows normal gastric tissue and gastric perforation sections collected from mice treated with Janus hydrogel or surgical suture on the 7th day after surgery, stained with hematoxylin and eosin. Figure 19 The elongation at break and burst pressure properties of the PA-G-15 / 5 / 7 Janus hydrogel prepared for this invention are compared with those of other gel products reported in the literature. Detailed Implementation

[0020] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] The present invention achieves the following technical solution through at least one embodiment: This invention provides a method for preparing density-driven, fully bio-based Janus hydrogels, comprising the following steps: Preparation of N-acryloyl aspartic acid precursor solution: N-acryloyl aspartic acid was dissolved in deionized water and a photoinitiator was added to prepare the precursor solution. The mass-volume concentration of the N-acryloyl aspartic acid precursor solution was 40-45% w / v. The photoinitiator was preferably Irgacure 2959, and the mass-volume concentration of the photoinitiator in the N-acryloyl aspartic acid precursor solution was 0.46-0.51% w / v. Preparation of methacrylated gelatin precursor solution: Laponite nanoclay dispersed in deionized water was transferred to a methacrylated gelatin solution containing a photoinitiator to prepare the precursor solution. The degree of methacrylation of the methacrylated gelatin was 15-45%; the mass-volume concentration of the methacrylated gelatin in the precursor solution was 5-10% (w / v); the mass-volume concentration of the Laponite nanoclay in the precursor solution was 0.65-0.70% (w / v); and the photoinitiator was further preferably LAP, with a mass-volume concentration of 0.18-0.22% in the precursor solution. The prepared N-acryloyl aspartic acid precursor solution was first injected into a mold, and then a methacrylated gelatin precursor solution was poured onto its surface. Finally, the mold was placed under ultraviolet light for irradiation, and the fully bio-based Janus hydrogel was obtained through one-step photopolymerization. The volume ratio of the N-acryloyl aspartic acid precursor solution to the methacrylated gelatin precursor solution was 5-7:1. The ultraviolet irradiation process parameters were: ultraviolet wavelength of 365 nm and ultraviolet light intensity of 6-30 mW / cm². 2 .

[0022] I. Materials and Reagents Aspartic acid was purchased from Aladdin Biochemical Co., Ltd. (Shanghai, China); Laponite (Lapo, chemical formula Si8Mg) 5.45 Li 0.4 O 24 Na 0.7Purchased from BYK Chemical AG (Germany); The porcine skin gelatin was purchased from Sigma-Aldrich Reagents Ltd. (Shanghai, China). Photoinitiators: Lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP) and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959), purchased from Maclean Biochemical Co., Ltd. (Shanghai, China); N-Acryloylaspartic acid (AASP) was synthesized by the reaction of acryloyl chloride with aspartic acid. The synthetic reaction formula and characterization results are shown below. Figure 1 ; Methacrylamide gelatin (GelMA) was prepared by reacting pigskin gelatin with methacrylic anhydride. The characterization results of products with different degrees of methacrylation (DM of 15, 30, and 45) (denoted as GelMA-15, GelMA-30, and GelMA-45) are shown in the figure. Figure 2 ; Unless otherwise specified, all other reagents are commercially available chemicals and should be used directly without further purification; II. Methods Unless otherwise specified, the experimental methods described in the following examples can be performed using conventional methods.

[0023] 1. One-step synthesis of PA-GX / Y / Z Janus hydrogel Figure 2 A schematic diagram of the preparation of PA-GX / Y / Z Janus hydrogel is provided; the specific preparation steps are as follows: (1) At room temperature, AASP(C PA AASP solution was prepared by dissolving 43% of the sample in deionized water and adding the photoinitiator Irgacure 2959 (0.51% w / v). (2) The Laponite dispersion in deionized water was transferred to a GelMA-15 solution containing photoinitiator LAP (dissolved at 40°C) to make the final concentrations of Laponite, LAP and GelMA in the GelMA-15 solution 0.67%, 0.20% and 5% (w / v) respectively, and then stained with methylene blue to obtain the GelMA solution; (3) Two precursor solutions of different concentrations were injected into the mold in the following order: first, concentrated AASP solution was injected, then GelMA solution was injected, with a volume ratio of 7:1 (AASP:GelMA). After being irradiated with ultraviolet light at a wavelength of 365 nm for 30 minutes at room temperature and pressure, Janus hydrogel with a blue-green upper layer (denoted as G layer) and a transparent lower layer (denoted as PA layer) was formed, which was named PA-G-15 / 5 / 7. Using a similar preparation method, a series of PA-GX / Y / Z hydrogels were synthesized, where X, Y, and Z represent the degree of methacrylation (DM, 15%, 30%, or 45%), GelMA concentration (C), etc. G The raw material formulations of these hydrogels are summarized in Table 1. (5-10% w / v) and the volume ratio of PA / G layers (5:1, 6:1 or 7:1).

[0024] In addition, monolayer PA hydrogels and monolayer G hydrogels were prepared as comparisons. The preparation method of monolayer PA hydrogel is as follows: at room temperature, AASP(C PA A concentrated AASP solution was prepared by dissolving 43% (w / v) in deionized water and adding Irgacure 2959 (0.51% w / v) as a photoinitiator. The solution was then irradiated with ultraviolet light at a wavelength of 365 nm for 30 minutes to obtain a monolayer PA hydrogel. The ultraviolet irradiation parameters were 6 mW / cm². 2 The LG-3535 ultraviolet light equipment, with AC 90-240V, 50 Hz, and 18 W, was purchased from Shenzhen Lianhuicheng Technology Co., Ltd.

[0025] The preparation method of monolayer G hydrogel is as follows: following the above preparation method of PA-GX / Y / Z, without casting the PA layer, the monolayer G hydrogel is prepared.

[0026] Previous studies have investigated different light intensities (6, 16, 30 mW / cm²). 2 The effect of ) on the tensile strength of monolayer PAASP hydrogel, such as Figure 3 As shown; therefore, the ultraviolet irradiation process parameters used in this study are 6 mW / cm². 2 The LG-3535 ultraviolet light equipment, with an AC output of 90-240 V, 50 Hz, and a power of 18 W, was purchased from Shenzhen Lianhuicheng Technology Co., Ltd.

[0027] Figure 4 The Janus hydrogel with a blue-green top layer and a colorless bottom layer was prepared by sequential casting of AASP and GelMA under UV light, with only about 1 mm of color halo remaining at the interface. Figure 4 b shows a schematic diagram of the microstructure of Janus hydrogel with an anti-adhesion layer, an interface layer, and an adhesion layer; Figure 4 c shows photos of Janus hydrogel in a bent or twisted state, as well as its adhesion effect on active finger joints; Figure 4 d shows that the dicarboxyl group adheres to the tissue through hydrogen bonds formed by the amino and carboxyl groups.

[0028] Figure 5 This indicates that the order of addition of the precursor solution is crucial for the successful preparation of Janus hydrogels; reversing the order leads to severe mixing of the two layers, which can be explained by Rayleigh-Taylor instability in hydrodynamics. Simple measurements show that a 43% w / v AASP solution (ρ≈1.15 g / cm³) is suitable for this purpose. 3 The density of the solution was significantly higher than that of the 5% w / v GelMA-15 solution (ρ≈1.02 g / cm³). 3 When the high-density AASP solution occupies the lower layer, the system is in a low gravitational potential energy state; the interface remains flat and stable, with only limited molecular diffusion. At the same time, despite the low concentration, the macromolecule GelMA still has a certain viscosity at room temperature, and can adhere to the surface of the AASP solution for a short time without significant mixing. However, when the high-density AASP solution is injected into the GelMA solution, the gravity-induced density gradient triggers a finger flow effect, causing the AASP solution to penetrate downwards. This leads to convective mixing of the two components before photopolymerization begins. Therefore, the strategy of AASP followed by GelMA is adopted. While maintaining the hierarchical structure at the macroscopic level, the AASP monomers and GelMA molecular chains can undergo microscale diffusion and interpenetration at the contact interface. The subsequent photopolymerization triggers in-situ crosslinking of monomers on both sides of the interface. At the same time, the molecular chains that diffuse to the opposite region are permanently locked at the interface through covalent bonds, forming an interpenetrating polymer network (IPN).

[0029] Table 1. Raw material formulation of Janus hydrogel PA-GX / Y / Z ; Note: a: C PA a: AASP solution concentration; b: X, degree of methacrylation (DM) in GelMA solution; c: C G d: concentration of GelMA solution; Z: volume ratio of PA / G layer; e: C Lapo The concentration of Laponite in the GelMA solution.

[0030] Hydrogel performance characterization Unless otherwise stated in the figure captions, all performance characterization experiments were repeated three times; error bars represent standard deviations (sd) unless otherwise stated; statistical significance was assessed using one-way ANOVA; *p<0.05, **p<0.01, ***p<0.001.

[0031] 2.1 Scanning Electron Microscopy (SEM) Analysis To observe the bonding state at the hydrogel interface, the longitudinal cross-section of the lyophilized hydrogel was analyzed using a ZEISS GeminiSEM 300 field emission scanning electron microscope. The results are as follows: Figure 6 As shown: The pore size characterization data of different G layers (G-15 / 5 / 5, G-15 / 5 / 6, G-15 / 5 / 7, G-15 / 7.5 / 7, G-15 / 10 / 7, G-30 / 5 / 7, G-45 / 5 / 7) and PA layers are summarized in Figure 6 a; Figure 6 b shows that the PA-G-15 / 5 / 7 Janus hydrogel has two regions with significantly different pore sizes: the upper G layer exhibits a loosely interconnected microporous structure with a pore size of approximately ~53.8±2.4 μm. Figure 6 e); the lower PA layer exhibits a dense microporous structure, with the pore size significantly reduced to ~18.6±7.1 μm ( Figure 6 f); Most importantly, the continuous and seamless transition between the PA layer and the G layer strongly indicates the presence of molecular diffusion and physicochemical cross-linking at the interface.

[0032] Figure 6 ce、 Figure 6 gj demonstrated the influence of X, Y, and Z parameters on the microstructure of the G layer. All samples exhibited a well-defined, interconnected porous network structure. As the Z value was adjusted from 5 to 7, the pore size of the upper G layer decreased slightly (from 56.4 ± 2.0 μm to 53.8 ± 2.4 μm, see [reference]). Figure 6 The ce), which may be attributed to the improved surface heat transfer efficiency due to the thinning of the layer during freeze-drying; when the Y value increases from 5 to 10, the average pore size decreases from 53.8±2.4 μm to 29.8±0.8 μm ( Figure 6 e Figure 6 In solutions with higher concentrations (gh), a denser network structure is formed; as the X value increases from 15 to 45, the pore size significantly decreases to 19.9 ± 1.2 μm (gh). Figure 6 e Figure 6 This is due to the denser three-dimensional network formed by the higher group density (ij), which is caused by the higher group density.

[0033] 2.2 Mechanical property testing Tensile tests were conducted using an electronic universal testing machine (Lugong, STD 50, China) at a constant beam moving speed of 20 mm / min; rectangular specimens with dimensions of 30 mm × 8 mm × 2 mm were used; stress (σ) was calculated from load capacity (F) and cross-sectional area (S); tensile strain (ε) was the elongation at break; the elastic modulus was taken from the average slope of the 10% to 20% tensile strain range in the stress-strain curve; and toughness was the area under the curve.

[0034] Compression tests were conducted using an electronic universal testing machine (MTS, CMT 6103, China) at a loading rate of 1 mm / min; cylindrical specimens (Φ5 mm × 4.6 mm) were loaded to the preset 95% strain value; for three-cycle compression tests (without any holding time), the target strain value was set to 80%.

[0035] The results are as follows Figure 7 , Figure 8 , Figure 9 As shown: Figure 7 Compared to PA-G-15 / 5 / 6 and PA-G-15 / 5 / 5, PA-G-15 / 5 / 7 Janus hydrogel exhibits superior tensile properties, achieving a tensile strain of 1529.7% and a tensile strength of approximately 18 kPa. The thinner G layer can more effectively function as a sacrificial layer, reducing energy loss while maintaining the load-bearing function of the PA layer. Figure 7 b further demonstrates that, compared to PA-G-15 / 5 / 6 and PA-G-15 / 5 / 5, the PA-G-15 / 5 / 7 Janus hydrogel exhibits the highest elastic modulus (~10.4 kPa) and superior toughness (~0.16 MJ / m). 3 ); Figure 7 cf displays different C G Hydrogels with different DM values ​​all exhibit similar phenomena, with PA-G-15 / 5 / 7 Janus hydrogel consistently demonstrating excellent toughness. Notably, compared to monolayer PA hydrogels, Janus hydrogels show reduced tensile stress and strain, consistent with the high storage modulus of the G layer. This effect can be mitigated by minimizing the G layer concentration, degree of substitution, and volume. Considering the isolation effectiveness of the G layer, the currently most readily prepared optimal formulation is PA-G-15 / 5 / 7 Janus hydrogel. Comprehensive radar chart analysis (see) Figure 8 a; Figure 8 b demonstrates that even when the balloon is rapidly inflated, the two layers remain strongly bonded and do not separate.

[0036] Figure 9 a presents the compression analysis results of PA-G-15 / 5 / 7 Janus hydrogel and its upper and lower monolayers. Although the PA monolayer hydrogel maintained the highest compressive strength, the Janus hydrogel achieved a compressive strength of 2.82 MPa, which significantly exceeded the strength value of the G monolayer hydrogel.

[0037] Figure 9b shows photographs of the manual compression-recovery test. To further evaluate the mechanical stability under repeated compression, an instantaneous three-cycle compression test was performed on the PA-G-15 / 5 / 7 hydrogel. Figure 9 c) The gradual increase in peak compressive strength at the same strain level indicates that PA-G-15 / 5 / 7 Janus hydrogel has excellent damage tolerance.

[0038] Laponite (Lapo) is a synthetic silicate clay. In this invention, it is incorporated into a GelMA solution. Positively charged amino acid residues on the GelMA chain (such as lysine and arginine [Lys / Arg+]) can adsorb onto the oppositely charged Lapo surface. When a stable GelMA-Lapo physical network is cast onto a pre-cast AASP solution, Lapo diffuses downwards, forming hydrogen bonds with carboxyl groups on the AASP through its surface hydroxyl groups. This multi-level non-covalent interaction across interfaces is crucial for preventing the Janus structure from peeling off in complex environments. Figure 4 b).

[0039] Experiments showed that the addition of 0.67% (w / v) Laponite significantly improved the tensile properties of the resulting Janus hydrogel, increasing the tensile strain from 1360% to 1529.7% and the stress from 10.3 kPa to 17.8 kPa. Figure 10 ).

[0040] 2.3 Characterization of Asymmetric Properties (1) Degradation performance test Monolayer PA hydrogels (PA layer), monolayer G hydrogels (G-15 / 5 / 7 layer), and PA-G-15 / 5 / 7 Janus hydrogels were stored at 4 °C for 24 h, and then lyophilized to obtain their initial dry weight. Each lyophilized gel sample was placed in a 24-well plate containing 2 mL of PBS solution and incubated at 37 °C for 3 weeks. The PBS solution was changed every 3 days, and the incubated samples were lyophilized and weighed on days 1, 7, 14, and 21 to obtain the in vitro degradation rate.

[0041] Figure 11The degradation characteristics of the PA layer, G-15 / 5 / 7 layer, and PA-G-15 / 5 / 7 Janus hydrogel in phosphate-buffered saline (PBS) were demonstrated. The G-15 / 5 / 7 layer showed a degradation rate of only 13.6 ± 1.3% within 7 days, effectively maintaining the physical barrier function and preventing external adhesion. This result is consistent with the repair time of gastric perforation in mice, with the gastric tissue wound essentially healed by 7 days. The degradation rate of the G-15 / 5 / 7 layer exceeded 50% at 14 days, reaching approximately 76.5 ± 2.7% by 21 days. This rapid degradation characteristic significantly reduced the risk of prolonged foreign body reaction. In contrast, the PA layer maintained approximately 90% of its mass within the same time period, providing a long-lasting mechanical scaffold for the 21-day medical repair cycle.

[0042] (2) Contact angle test The Janus hydrogel strips (3 cm × 0.8 cm) prepared above were fixed on the adjustable lifting platform of a contact angle micrometer (CHD-JCJ180A-1); droplets were added through a microsyringe, and images were acquired at fixed time intervals. The results are as follows. Figure 12 a, Figure 12 As shown in b: The surface hydrophilicity of the G-15 / 5 / 7 layer and the PA layer was quantified by contact angle measurement. As the X and Y values ​​increased or the Z value decreased, the contact angle gradually increased, indicating that the denser GelMA cross-linked structure enhances hydrophobicity. However, for the PA-G-5 / 5 / 7 Janus hydrogel, the G-15 / 5 / 7 layer exhibited excellent hydrophilicity, with a contact angle of 52.8°, which was significantly lower than that of the PA layer (64.0°). This allowed it to form a strong wetting layer and effectively prevent postoperative organ adhesion.

[0043] (3) Swelling performance test PA-G-15 / 5 / 7 Janus hydrogels, with or without attachment to pigskin, were immersed in phosphate-buffered saline (PBS) or simulated gastric fluid (SGF), respectively. Photographs were taken at predetermined time points (within 60 minutes), and their horizontal dimensions were measured. Dynamic swelling behavior was monitored, and their dimensional stability and long-term tissue adhesion were assessed. Results are as follows: Figure 13 a, Figure 13 As shown in b: Figure 13 a demonstrates that, driven by water adsorption and polymer network relaxation, the PA-G-15 / 5 / 7 Janus hydrogel exhibits spontaneous volume expansion over time in both media, resulting in an increase in horizontal dimension. The hydrogel attached to pigskin, however, shows relatively mild lateral overexpansion, further demonstrating its strong adhesion. Figure 13b shows a magnified view of the interface after the hydrogel was attached to pigskin and soaked for a long time. Regardless of whether the hydrogel was in a free state or attached state, its swelling rate in SGF was lower than that in PBS, which may be attributed to the limitation of water molecule adsorption by the acidic environment. Surprisingly, with the extension of soaking time, wrinkles of different degrees appeared on the surface of G-15 / 5 / 7 layer, which is obviously due to the relatively high swelling rate of PA layer. This asymmetric swelling property is expected to achieve active swelling plug function while maintaining the same external dimensions, thereby more effectively sealing irregular gastric perforations.

[0044] 2.5 Adhesion performance test Take fresh pig skin or pig organs and carefully scrape off the surface fat with a scraper; then immerse them in phosphate-buffered saline (PBS) at 37 °C for 1 hour to remove residual lipids on the surface. The burst pressure test was performed using a custom-designed measuring device. The test procedure was as follows: A 2 mm incision was made on the surface of a 4 cm × 4 cm porcine stomach tissue sample using a scalpel; the incision was sealed with hydrogel (Φ10 mm × 4 mm) and then fixed to the testing device connected to the injection pump (see schematic diagram of the testing device). Figure 14 (as shown in a); rupture pressure is defined as the maximum pressure value before slow pressurization; similar tests were performed on pig liver, kidney, intestines, and skin tissue.

[0045] For the peel test, the hydrogel was sandwiched between two pieces of pigskin of the same size (3 × 0.8 cm), with tears at the edges of the pigskin; the unmeasured surfaces were firmly glued to the pigskin with super glue; the schematic diagram of the test setup and the sample clamping method are as follows. Figure 14 As shown in b; in addition, the test samples were soaked in phosphate-buffered saline (PBS) and artificial gastric juice (SGF) for different times, but the test protocol was the same.

[0046] The results of the burst pressure test are as follows Figure 15 As shown, Figure 15 a shows that the burst pressure of PA-G-15 / 5 / 7 Janus hydrogel in the stomach reached 135.0±2.7 mmHg, which is significantly higher than that of fibrin glue (52.0±1.7 mmHg) and monolayer PA hydrogel (105.8±2.1 mmHg). This significant improvement is a key performance indicator for clinical hemostatic sealants. Furthermore, with concentration C G With the increase of DM value, the burst pressure increases synchronously, which proves the excellent storage modulus and efficient stress transfer capability of the GelMA network. Similar measurements on other porcine organs and tissues all exceeded 100 mmHg. Figure 15b); On the other hand, the rigidity of the G-15 / 5 / 7 layer restricts the viscoelastic energy dissipation at the peeling front, resulting in a decrease in the peel strength of the PA layer with increasing G-15 / 5 / 7 layer volume, concentration, and DM. Figure 16 a) Bidirectional peel strength tests were conducted using PA-G-15 / 5 / 7 Janus hydrogel as a representative example. The results confirmed its distinctly different adhesive properties on both sides: the PA layer was 216.3±3.2 N / m, and the G-15 / 5 / 7 layer was 6.2±0.8 N / m. Figure 16 b) Figure 16 c and Figure 16 Figure d shows the change in adhesion strength of PA-G-15 / 5 / 7 Janus hydrogel after soaking in PBS and SGF for a specific time. The values ​​decreased to 158.12 N / m and 170 N / m, respectively, indicating that the material has excellent adhesion properties and potential for practical biomedical applications.

[0047] 2.7 Postoperative anti-adhesion performance test Prior to in vivo experiments, studies had confirmed that PA-G-15 / 5 / 7 Janus hydrogel possesses excellent biocompatibility. Figure 17 Then, the postoperative anti-adhesion performance of PA-G-15 / 5 / 7 Janus hydrogel was tested. The specific steps are as follows: Six mice were randomly divided into two groups. A standardized gastric incision was made after abdominal incision to establish a mouse model of gastric perforation. The grouping was as follows: The hydrogel group was applied to the incision site with Φ8 mm PA-G-15 / 5 / 7 Janus hydrogel dressing; The incision was closed using standard surgical sutures. Seven days later, the healing of the gastric wound was assessed, and tissue samples were collected for H&E staining pathological examination.

[0048] Figure 18 The study compared the wound healing of the control group and the experimental group at 0 and 7 days post-surgery. Anatomical observation at 7 days showed that the suture group had poor healing, with gastric fluid leakage and a rough, uneven surface. In contrast, the wound covered by the hydrogel group was almost completely healed, and the stomach returned to its normal anatomical contour. Its surface was smooth and without adhesion, and significant vascular redistribution was visible at the repair site. It can be inferred that the hydrogel not only provides an effective physical barrier but also actively promotes local angiogenesis and tissue regeneration.

[0049] Figure 18 b shows the histological analysis of the healing quality of the two treatment regimens using H&E staining; Figure 18b clearly shows the standard anatomical structure of the blank group (normal gastric tissue). The orange arrow points to the mucosa, which contains neatly arranged single-layer columnar epithelium; the red arrow points to the lamina propria, which is densely filled with gastric glands; the blue arrow points to the submucosa, which contains loose connective tissue; and the green arrow points to the muscularis propria, which contains thick smooth muscle and a continuous serosa. The sutured group shows complete rupture with extensive disordered granulation tissue, necrotic debris, and immature fibrous tissue. In contrast, the images of the hydrogel group show significantly improved regeneration and are very similar to the normal gastric wall. Existing literature reports Fe 3+ It can prevent PAASP from adhering at the site of gastric perforation; however, dense fibrous capsules and inflammatory zones are often observed at the interface, which may be due to oxidative stress induced by metal ions or significant stiffness mismatch. The results above show that the hydrogel prepared by the present invention does not have this problem, and the postoperative anti-adhesion and regeneration effects are more excellent.

[0050] 2.8 Performance Comparison with Existing Technologies The PA-G-15 / 5 / 7 Janus hydrogel prepared in this invention was compared with other gel products reported in the literature in terms of elongation at break and burst pressure performance. The prior art literature is summarized in Table 2.

[0051] Table 2. Summary of Existing Technical Documents ; Note: For the tissues used in the burst pressure test, references 1 and 7-8 used pig intestines; reference 2 used pig lungs; references 3-5 used pig skin; reference 6 used dura mater; and this invention and references 9-10 both used pig stomach.

[0052] The results are as follows Figure 19 As shown, PA-G-15 / 5 / 7 Janus hydrogel exhibits excellent performance in both mechanical and medical sealing aspects.

[0053] III. Conclusion This invention successfully prepared a fully bio-based Janus hydrogel for gastric perforation repair using a one-step photo-irradiation process by leveraging the density difference between high-concentration AASP and low-concentration GelMA. This structure forms a seamless interface through covalent bonding with Laponite, enabling it to withstand large-scale mechanical stretching without delamination. The Janus structure also possesses other physicochemical asymmetric properties, such as gradient degradation, asymmetric hydrophilicity, and asymmetric swelling, perfectly adapting to the complex gastric environment and aligning with the gastric perforation repair cycle. More importantly, this fully bio-based Janus hydrogel avoids the risk of biological inflammatory reactions that may be triggered by traditional metal ion impregnation methods. This strategy allows for further screening of more suitable bio-based materials, thereby achieving simultaneous improvements in adhesion, sealing performance, and mechanical properties.

[0054] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a density-driven, fully bio-based Janus hydrogel, characterized in that, The preparation method is as follows: taking advantage of the density difference between the N-acryloyl aspartic acid precursor solution and the methacrylated gelatin precursor solution, gravity-stabilized stratification is achieved through sequential casting, and the fully bio-based Janus hydrogel is prepared by combining a one-step photopolymerization process; wherein, sequential casting means casting the N-acryloyl aspartic acid precursor solution first and then casting the methacrylated gelatin precursor solution.

2. The method for preparing a density-driven, fully bio-based Janus hydrogel according to claim 1, characterized in that, The N-acryloyl aspartic acid precursor solution is prepared by dissolving N-acryloyl aspartic acid in deionized water and adding a photoinitiator; the mass-volume concentration of the N-acryloyl aspartic acid precursor solution is 40-45%, w / v.

3. The method for preparing a density-driven, fully bio-based Janus hydrogel according to claim 1, characterized in that, The methacrylamide gelatin precursor solution is prepared by transferring Laponite nanoclay dispersed in deionized water into a methacrylamide gelatin solution containing a photoinitiator. The degree of methacrylation of the methacrylated gelatin is 15-45%; the mass-volume concentration of the methacrylated gelatin in the methacrylated gelatin precursor solution is 5-10% (w / v); and the mass-volume concentration of the Laponite nanoclay in the methacrylated gelatin precursor solution is 0.65-0.70% (w / v).

4. The method for preparing a density-driven, fully bio-based Janus hydrogel according to claim 1, characterized in that, The N-acryloyl aspartic acid precursor solution is first injected into a mold, and then the methacryloyl gelatin precursor solution is poured onto its surface. The mold after pouring is placed under ultraviolet light for irradiation, and the fully bio-based Janus hydrogel can be obtained by one-step photopolymerization.

5. A method for preparing a density-driven, fully bio-based Janus hydrogel according to claim 1 or 4, characterized in that, The volume ratio of the N-acryloyl aspartic acid precursor solution to the methacryloyl gelatin precursor solution is 5-7:

1.

6. A fully bio-based Janus hydrogel prepared using the preparation method according to any one of claims 1-5, characterized in that, The fully bio-based Janus hydrogel has an adhesion-anti-adhesion bilayer structure. The adhesion layer is formed by N-acryloyl aspartic acid precursor solution under the initiation of photoinitiator, and the anti-adhesion layer is formed by methacryloyl gelatin and Laponite nanoclay under the initiation of photoinitiator. The two layers form a seamless interface through interfacial covalent bonding and bridging effect of Laponite nanoclay.

7. The application of the fully bio-based Janus hydrogel as described in claim 6 in the preparation of medical anti-adhesion patches.

8. The application according to claim 7, characterized in that, The fully bio-based Janus hydrogel can be applied to any one of the following tissues: stomach, liver, kidney, intestines, or skin.

9. The use of the fully bio-based Janus hydrogel as described in claim 6 in the preparation of a gastric perforation repair sealant.

10. The application according to claim 9, characterized in that, The fully bio-based Janus hydrogel was applied as a surgical sealant to the gastric perforation wound. The bicarboxyl groups of the poly(N-acryloyl aspartic acid) adhesion layer mediated hydrogen bonding to strongly adhere to the gastric tissue, forming an active expanding embolism to seal the gastric perforation wound. The hydrophilicity of the methacryloyl gelatin anti-adhesion layer was used to construct a dense hydration layer to prevent postoperative adhesion.

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