Sprayable dual-network hydrogel microsphere adhesive, method of making and use thereof

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses a sprayable double-network hydrogel microsphere adhesive as well as a preparation method and application thereof. The preparation method comprises the following steps: liquid paraffin and sorbitan monooleate, polyoxyethylene sorbitan monolaurate are taken as oil phases to form an emulsion; the emulsion is fully mixed with petroleum ether, centrifugal separation is performed on the gel hydrogel microspheres, and freeze-drying is performed on the gel hydrogel microspheres for storage in the dark; preparation of ropivacaine liposomes; ultrapure water is added into the lipid membrane to obtain a primary liposome suspension, the hydrogel microspheres are used to adsorb the liposomes, and the obtained double-network hydrogel microspheres are collected. The sprayable double-network hydrogel microsphere adhesive as well as the preparation method and application thereof can be used to prepare hydrogel microspheres which can quickly and uniformly cover irregular wounds, and the liposomes carrying ropivacaine can provide sustained analgesic release, so that the problems of maintaining sustained adhesion in a blood-saturated wound and prolonging pain relief are simultaneously solved.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, and in particular to a sprayable dual-network hydrogel microsphere adhesive, its preparation method, and its application. Background Technology

[0002] Wound closure is a critical goal of surgical and trauma care, essential for preventing infection, promoting tissue regeneration, and restoring physiological function. Deep, irregular bleeding wounds present particularly challenging clinical problems due to their ubiquity in daily life and the inherent complexity of their treatment. These wounds exhibit several major complications, including surgical inaccessibility, which hinders the effective placement of sutures in tortuous cavities to stop ongoing bleeding from the vascular system; impaired interfacial adhesion under moist conditions; and chronic neuropathic pain exacerbated by mechanical stress on injured nerves.

[0003] Traditional methods, such as needle and suture suturing and skin staplers, remain the gold standard for wound closure due to their reliability. However, these methods not only show limited effectiveness for deep, irregular bleeding wounds but also have significant limitations, including mechanical damage to surrounding tissues, the risk of infection, and the potential for inducing inflammation and scarring. Furthermore, sutures require skilled application and can cause discomfort during removal, while staples can lead to tissue ischemia and delayed healing. Adhesives, such as adhesive patches and bioadhesives, have emerged as a promising solution, unlike sutures, as they can distribute pressure evenly across the wound, reducing localized pressure and promoting faster healing. However, this solution is still insufficient. On the one hand, wound dressings / patches cannot conform to irregular contours, creating gaps that allow bacterial invasion and exudate buildup. Moreover, they quickly fail to adhere in a blood-filled environment. On the other hand, bioadhesives (such as fibrin glue and cyanoacrylate) exhibit limited penetration beyond the surface of the bleeding site due to premature polymerization upon contact with blood, a phenomenon known as the "self-sealing effect," which allows deeper bleeding to go uncontrolled. Furthermore, their brittle interfaces often break under physiological stress.

[0004] Adhesive powders offer unique advantages by filling complex wound structures through particle flow and rapid liquid absorption. Unlike patches, they can conformally adapt to 3D defects while concentrating clotting factors through superabsorption, surpassing biological adhesives in hemostasis speed. Particle size and inhomogeneity severely impair their performance in bleeding wounds, as inhomogeneity leads to low absorbency and uneven distribution under pulsating pressure creates weaknesses that make them prone to adhesive failure. However, most milled powders exhibit uncontrollable size variations, directly compromising adhesive stability in bloody environments. Therefore, there is a need to develop adhesive powders with uniform particle size.

[0005] Meanwhile, existing solutions neglect pain management during the extended healing period, thus prolonging recovery time. Therefore, the mismatch between the duration of analgesia and the wound healing period remains a challenge for achieving long-term pain control. Further research is needed to develop more effective and sustained-release formulations that can provide adequate pain relief throughout the wound healing process.

[0006] To overcome these dual challenges, particularly achieving strong wet adhesion in bleeding environments while providing long-term analgesia, a sprayable dual-network hydrogel microsphere adhesive system has been developed, offering a comprehensive solution for improving postoperative recovery and patient comfort. Summary of the Invention

[0007] The purpose of this invention is to provide a sprayable dual-network hydrogel microsphere adhesive, its preparation method, and its application. The prepared sprayable hydrogel microspheres can quickly and consistently cover geometrically irregular wounds that cannot be covered by conventional dressings, while the embedded liposomes carrying ropivacaine provide sustained analgesic release. By simultaneously addressing the issues of maintaining continuous adhesion in blood-saturated wounds and prolonging pain relief, this invention addresses a key unmet need in trauma and surgical recovery, providing a comprehensive solution for improving postoperative recovery and patient comfort.

[0008] To achieve the above objectives, the present invention provides a method for preparing a sprayable dual-network hydrogel microsphere adhesive, comprising the following steps: Step 1, Preparation of the aqueous phase of the hydrogel microsphere precursor: Dissolve polyvinyl alcohol in water, and then add poly(ethylene glycol) diacrylate, photoinitiator, N-hydroxysuccinimide acrylate and acrylic acid in sequence to form a prepolymer aqueous solution. Place at 55°C for at least 30 min to avoid bubbles. Step 2: Using liquid paraffin as the oil phase, add dehydrated sorbitan monooleate and polyoxyethylene dehydrated sorbitan monolaurate, stir and heat to 55°C, gradually add the aqueous phase solution to the oil phase, and continuously irradiate with ultraviolet light to solidify the water droplets in the emulsion to form hydrogel microspheres. Step 3: Mix the hydrogel microspheres thoroughly with petroleum ether and separate the phases at ambient temperature for 1 hour. Decant the supernatant, centrifuge to separate the hydrogel microspheres, and purify them by three consecutive washing cycles with a gradient series of ethanol / deionized water mixture. Collect the purified hydrogel microspheres, freeze-dry them for 24 hours, and store them in the dark. Step 4, Preparation of ropivacaine liposomes: Lecithin choline, cholesterol, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)-2000 are mixed evenly, free ropivacaine base is added to the mixture, and the mixture is evaporated under reduced pressure to form a thin lipid film; Step 5: Add ultrapure water to the lipid membrane, mix and sonicate to obtain a primary liposome suspension. Mix the hydrogel microspheres with the liposome suspension and incubate for 1 hour under continuous orbital oscillation. Collect the obtained double-network hydrogel microspheres by centrifugation, freeze-dry and store in a nitrogen-purged bottle at -20°C.

[0009] Preferably, in step 1, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate or α-ketoglutaric acid.

[0010] Preferably, in step 2, after the emulsion is formed, the system is continuously irradiated with a 365nm ultraviolet lamp for 30-60 minutes under continuous stirring. Before terminating the stirring process, the structural integrity of the hydrogel microspheres is verified by microscopic observation.

[0011] Preferably, in step 3, the volume ratio of ethanol to deionized water in the gradient series ethanol / deionized water mixture is: 50:50 for the first wash, 75:25 for the second wash, and anhydrous ethanol for the third wash. Each wash includes vortex mixing for 30 seconds and centrifugation under the same parameters.

[0012] Preferably, in step 5, a thermally stable liposome extruder is used to extrude the liposome suspension through a polycarbonate membrane with continuous pore size to obtain a liposome suspension with uniform particle size distribution.

[0013] Preferably, in step 5, the hydrogel microspheres and liposome suspension are incubated at a ratio of 10 mL: 10 mg under continuous orbital oscillation for 1 hour.

[0014] Preferably, in step 5, the obtained dual-network hydrogel microspheres are collected by centrifugation and washed twice with deionized water to remove unbound liposomes, thus purifying the dual-network hydrogel microspheres.

[0015] The present invention also provides a sprayable dual-network hydrogel microsphere adhesive, which is prepared by the above-described preparation method.

[0016] The application of sprayable dual-network hydrogel microsphere adhesives can be used to prepare wound dressings that promote surgical wound healing and provide sustained pain relief.

[0017] The advantages and beneficial effects of the above-mentioned sprayable dual-network hydrogel microsphere adhesive, its preparation method, and its application are as follows: 1. This invention develops a dual-network hydrogel microsphere adhesive system composed of polyvinyl alcohol (PVA), polyethylene glycol diacrylate (PEGDA) and acrylic acid (AA), which is functionalized with N-hydroxysuccinimide acrylate (AAC-NHS) to achieve strong adhesion to wet tissues.

[0018] 2. The introduction of ropivacaine liposomes (Rop-Lipo) in this invention achieves sustained local analgesia, addressing the dual challenges of wound closure and postoperative pain management. These hydrogel microspheres exhibit excellent mechanical properties, rapid swelling, high liquid absorption, and superior tissue adhesion, exceeding clinical standards in overlap shear, T-peel, and tensile tests.

[0019] 3. In vivo evaluations of this invention demonstrated rapid and effective hemostasis in a rat liver defect model, as well as accelerated wound healing and reduced inflammation in dorsal and plantar incision models. Sustained release of ropivacaine from the hydrogel microspheres significantly prolonged analgesia (≥168 hours), reduced nociceptive neuronal activation, and inhibited glial cell responses in the dorsal horn of the spinal cord. Biocompatibility assessments confirmed minimal cytotoxicity and systemic safety. These multifunctional hydrogel microspheres represent a promising advance in enhancing postoperative recovery, combining reliable wound adhesion with prolonged local pain relief to improve postoperative outcomes. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the components of hydrogel microspheres; Figure 2 In the image, a represents the UV curing process of the PVA / AAC precursor, b represents a representative microscopic image of the PNP hydrogel microspheres (scale bar: 500 μm), c represents a representative fluorescence image and microscopic image of the AMCA-labeled PR hydrogel microspheres (scale bar: 500 μm, upper right microscopic image), and d represents representative SEM images of the PNP and hydrogel microspheres (scale bars: 50 μm and 100 μm). Figure 3 In Figure a, rheological properties are shown: frequency scanning tests reveal the storage modulus (G′) and loss modulus (G″) of the hydrogel microspheres (n=3). Figure b shows the Fourier transform infrared spectroscopy confirming the successful addition of ropivacaine liposomes. Figure c shows the drug release curves of PR hydrogel microspheres, PNP hydrogel microspheres mixed with ropivacaine, and PNP microspheres mixed with ropivacaine hydrochloride solution (n=3). Figure d shows the particle size distribution of the hydrogel microspheres measured by dynamic light scattering (DLS) (n=3). Figure 4 Figure a shows a schematic diagram of the spraying of dual-network hydrogel microspheres, and figure b shows the expansion behavior of PNP and PR hydrogel microspheres over time (n=3). Figure 5 In Figure 1, a represents the mechanical property curve and quantitative analysis of ASTM F2205-05 (lap shear strength), b represents the mechanical property curve and quantitative analysis of ASTM F2256-05 (peel test), and c represents the mechanical property curve and quantitative analysis of ASTM F2258-05 (tensile strength) (n=8). Figure 6Image a shows a photographic demonstration of two pigskin substrates adhered to by PNP hydrogel microspheres, maintaining a weight of 300 (contact area 2.5 cm²). 2 b) shows photographs of PNP hydrogel microspheres adhering to the surfaces of multiple organs (heart, spleen, kidney, lung, muscle, and skin), with yellow dashed squares indicating incision locations. c) shows intraoperative photographs documenting the hemostatic performance of gauze (Gauze control), cyanoacrylate (CA clinical reference), porcine fibrin adhesive (Fibrin biological control), and PNP / PR hydrogel microspheres in a rat liver defect model. Figure 7 In the table, 'a' represents the amount of bleeding, and 'b' represents the corresponding quantification of the time required to achieve hemostasis (n=3). Figure 8 These are representative live / dead cell staining images of the present invention; Figure 9 In the middle section, a represents HUVECs treated with PNP / PR hydrogel microspheres, ropivacaine nanoparticles (Rop), and a control group; b represents the CCK-8 survival rate of NIH3T3 cells (n=4); c represents the quantitative analysis of the PI-positive cell ratio in each HUVEC group; d represents the quantitative analysis of the PI-positive cell ratio in each NIH3T3 group (n=4); and e represents the hemolysis detection of PNP / PR hydrogel microspheres cultured with rabbit erythrocytes using PBS (negative control) and 1% Triton X-100 (positive control) (n=4). Representative photographs of centrifuged samples show hemoglobin release. Figure 10 Image a shows representative photographs of wound healing in a rat dorsal incision model on days 1, 4, 7, 10, and 14 post-surgery. Treatment methods included control, stitch, fibrin adhesive, cyanoacrylate (CA), PNP, and PR hydrogel microspheres. Image b shows hematoxylin and eosin (H&E) staining of the incision site on days 7 and 14 post-surgery, with the incision area highlighted in yellow boxes (scale bar = 500µm). Image c shows Masson trichrome staining of the incision site on days 7 and 14 post-surgery (scale bar = 500µm). Figure 11 In Figure a, we present a quantitative analysis of the change in wound width over time; in Figure b, we present a quantitative analysis of collagen content based on Masson's trichrome staining on postoperative days 7 and 14; and in Figure c, we present a quantitative analysis of the mean fluorescence intensity (MFI) of TNF-α and IL-6 based on immunofluorescence staining on postoperative days 7 and 14. Figure 12 Immunofluorescence staining of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was performed on days 7 and 14 postoperatively (scale bar = 500m). Figure 13 In the middle section, a shows actual photos of the incision sites on postoperative days 1, 4, and 7 for different treatment groups (control group, stitch group, PNP group, PNP+Rop group, and PR group), and b shows the typical trajectory of the open field test for each group on days 1, 4, and 7. Figure 14 For the quantitative analysis of behavioral tests, a is the mechanical withdrawal threshold (MWT), b is the claw withdrawal latency (PWL), and c is the distance traveled in the open field test. Figure 15 Image a shows immunofluorescence staining of DRGc-Fos and TRPV1 (scale bar: 100 μm); image b shows quantitative analysis of ATF3 immunofluorescence intensity; image c shows immunofluorescence staining of GFAP and Iba-1 in the dorsal horn of the spinal cord, including magnified and merged images (scale bars: 50 μm and 10 μm). Figure 16 In the table, a represents the quantitative analysis of c-Fos immunofluorescence intensity and the proportion of c-Fos / TRPV1 co-expression in c-Fos-positive cells; b represents the immunofluorescence staining of ATF3 in DRG (scale bar: 100μm); c represents the quantitative analysis of ATF3 immunofluorescence intensity; d represents the quantitative analysis of GFAP immunofluorescence intensity; and e represents the quantitative analysis of Iba-1 immunofluorescence intensity. Figure 17 Infrared spectrum of PNP hydrogel microspheres; Figure 18 The effect of different UV curing times on the final formation of hydrogel microspheres is shown, where a is 10 min, b is 30 min, and c is 60 min. Figure 19 Quantitative analysis of the swelling rate of PNP hydrogel microspheres (n=3); Figure 20 DLS for ropivacaine liposomes; Figure 21 In section a, representative mechanical curves and quantitative analysis of lap shear strength are presented; in section b, representative mechanical curves and quantitative analysis of T-peel strength are presented; and in section c, representative mechanical curves and quantitative analysis of tensile strength are presented (n=8). p<0.05, p<0.01, p<0.001, p<0.0001); Figure 22 In the figure, a represents the histological analysis after the foot thoracotomy experiment in SD rats, and b represents the hematological parameters. Figure 23 Skin trunk reflex response in a rat dorsal incision model (n=6); Figure 24 Histological analysis of foot incision wound healing in SD rats. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. Unless otherwise defined, the reagents, equipment, and other materials used in this invention are all commercially available.

[0022] Example 1 A method for preparing a sprayable double-network hydrogel microsphere adhesive for medical wounds includes the following steps: Step 1, Preparation of the aqueous precursor phase for hydrogel microspheres: Accurately weigh a predetermined mass of PVA (polyvinyl alcohol, degree of polymerization 1700, 99% hydrolyzed, Aladdin) and dissolve it in deionized water to obtain a 7 wt% homogeneous solution. At 55°C, add the following components sequentially to the PVA solution: 0.05 wt% poly(ethylene glycol) diacrylate (Sigma-Aldrich's PEGDA), 0.2 wt% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 97%, TCI) as a photoinitiator, 0.3 wt% N-hydroxysuccinimide acrylate (AAC-NHS, BOCSciences), and 35 wt% acrylic acid (AA, 99%, Aladdin). Homogenize the mixture for 5 minutes using vortex mixing (vortex generator 500 rpm) to ensure uniform distribution of all components, forming a transparent aqueous solution of the prepolymer. Incubate the solution at 55°C for at least 30 minutes to avoid air bubbles.

[0023] Step 2: Liquid paraffin (95% v / v) was placed in a beaker as the oil phase, followed by the addition of 4.75% v / v sorbitan monooleate (Span 80) and 0.25% v / v polyoxyethylene (20) sorbitan monolaurate (Tween 20, Signa). The mixture was thoroughly homogenized and heated to 55°C under continuous mechanical stirring. Subsequently, an aqueous solution (10% by volume) was gradually added dropwise to the oil phase containing Span 80 and Tween 20 at a stirring speed of 1000 rpm. The resulting water-in-oil (W / O) emulsion was mechanically stirred for 1 hour to ensure stability. After the emulsion formed, the system was continuously irradiated with a 365 nm UV lamp for 30–60 minutes under continuous stirring to achieve complete crosslinking and curing of the hydrogel microspheres. Before terminating the stirring process, the structural integrity of the hydrogel microspheres was verified by microscopic observation.

[0024] Step 3: The emulsion was thoroughly mixed with petroleum ether (analytical grade, ≥99.5% purity, Aladdin) and phase-separated at ambient temperature for 1 hour. The supernatant was decanted, and the hydrogel microspheres were separated by centrifugation at 5,000 rpm (equivalent to 3,000 × g, rotor radius 8 cm) for 5 minutes using a refrigerated centrifuge. Subsequent purification consisted of three consecutive wash cycles in a gradient series with an ethanol / deionized water mixture: initially 50:50 (v / v), then 75:25 (v / v), and finally anhydrous ethanol (HPLC grade, ≥99.9% Aladdin). Each wash step included vortex mixing (30 seconds) and centrifugation at the same parameters to ensure complete removal of oil phase residues and emulsifiers. The purified hydrogel microspheres (PNP hydrogel microspheres) were collected and freeze-dried in a freeze dryer (condenser temperature: -50°C, vacuum: <10 Pa) for 24 hours. The final product was stored in a light-protected container at 4°C to minimize photodegradation.

[0025] Step 4, Preparation of Ropivacaine Liposomes: The liposome formulation was prepared using a modified thin-film hydration method. 7 mg of lecithinylcholine (EPC, >99% purity), 1 mg of cholesterol (CHOL, >99% purity), 0.2 mg of 1,2-dicalpalmitoyl-sn-glycerol-3-phosphate choline (DPPC, >99% purity), and 0.2 mg of 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)-2000 (DSPE-polyethylene glycol)] were added to the lipid mixture. Ropivacaine (Rop, >99% purity) free base (3 mg, equivalent to a final concentration of 0.1% w / v) was then added. The organic solvent was evaporated under reduced pressure at 50°C for 30 minutes using a rotary evaporator to form a thin lipid film.

[0026] Step 5: 3 mL of ultrapure water (preheated to 50 °C) was added to the lipid membrane, followed by vortex mixing (1 min) and probe sonication using an ultrasonic instrument (30 s, 20 kHz, 150 W) to obtain a primary liposome suspension. To obtain a uniform particle size distribution, the suspension was extruded through a polycarbonate membrane with continuous pores using a thermally stable liposome extruder. The final ropivacaine-loaded liposomes (Rop-Lipo) were stored at 4 °C for subsequent characterization.

[0027] PNP hydrogel microspheres (10 mg) and liposome suspension were incubated at a ratio of 10 mL:10 mg (v / w) under continuous orbital oscillation (150 rpm, 25 °C) for 1 hour to promote swelling-mediated loading. This process allows the hydrogel microspheres to encapsulate liposomes through porous structure absorption during polymer matrix swelling.

[0028] The obtained PR hydrogel microspheres (double-network hydrogel microspheres) were collected by centrifugation (3000×g, 10 min, 4 °C) and washed twice with deionized water to remove unbound liposomes. The purified hydrogel microspheres (PR hydrogel microspheres) were lyophilized under optimized conditions using a laboratory-scale freeze dryer: initial drying for 24 h at a condenser temperature of -50 °C and a vacuum pressure <10 Pa, followed by a secondary drying at 25 °C for 6 h. The final PR hydrogel microspheres exhibited a porous morphology and were stored at -20 °C in nitrogen-purged vials until further use.

[0029] Verification Example 1. Characterization of PNP hydrogel microspheres and PR hydrogel microspheres.

[0030] The microstructure and morphology of various lyophilized hydrogel microspheres were examined using scanning electron microscopy (SEM). Fourier transform infrared spectroscopy (FT-IR) was used to analyze the structure and functional groups of the materials. The rheological properties of the hydrogel microspheres were analyzed using rotational rheometer (TA).

[0031] 2. Ropivacaine in vitro release curve.

[0032] PNP hydrogel microspheres (100 mg) were loaded with ropivacaine hydrochloride, ropivacaine base, and PR (equivalent to 10 mg ropivacaine) in dialysis bags (molecular weight cutoff 14 kDa, MWCO). The dialysis bags were immersed in 20 mL of phosphate-buffered saline (PBS, pH 7.4) containing 0.02% (w / v) Tween-80 and incubated with shaking at 37 °C. At predetermined time intervals, 1 mL of release medium was collected and replaced with an equal volume of fresh PBS to maintain the bath conditions. The cumulative release of ropivacaine (Rop) was quantified by UV-Vis spectrophotometry based on a pre-established calibration curve.

[0033] 3. Mechanical experimental analysis of PNP hydrogel microspheres and PR hydrogel microspheres.

[0034] The tissue adhesion strength of hydrogel microspheres was evaluated using pigskin samples through lap shear tests, 180° peel tests, and tensile tests, according to ASTM standards F2255-05, F2256-05, and F2258-05, respectively. Fresh pigskin samples (25 × 25 mm, 1 mm thick) were kept hydrated throughout the experiments. For the lap shear analysis (ASTM F2255-05), samples coated with hydrogel microspheres were subjected to tensile loading at a speed of 5 mm / min using a universal testing machine (Tinius Olsen H10KS) with an overlap width of 1.0 ± 0.2 mm. The 180° peel resistance (ASTM F2256-05) was measured by controlling the separation of the adhesive paper strip at a crosshead speed of 300 mm / min. Tensile adhesion properties (ASTM F2258-05) were determined by measuring force-displacement curves under uniaxial tension at 10 mm / min.

[0035] The in vitro adhesion properties of PNP hydrogel microspheres were systematically evaluated on freshly excised porcine tissues (liver, kidney, spleen, lung, and heart) under simulated physiological conditions. Tissue samples were pretreated in phosphate-buffered saline (PBS, pH 7.4) at 37°C for 30 min to maintain hydration. Linear incisions (20 mm long) were made using a sterile scalpel perpendicular to the tissue surface, following a standardized protocol. Approximately appropriate amounts of PNP hydrogel microspheres were adhered to each cross-section of the incision, followed by manual pressure application for 120 seconds using a sterile latex glove to ensure uniform interfacial contact. Adhesion integrity was assessed by visual observation 20 min after application under standardized lighting conditions.

[0036] 4. Biosafety testing.

[0037] 1×10 5Cells (NIH3T3 and HUVECs, respectively) were incubated with hydrogel microspheres in Dulbecco's modified Eagle medium (DMEM) for 48 hours. NIH3T3 and HUVECs cells were irradiated with calcein AM / PI (2×10⁶ m; 2×10⁶ m) at 37°C for 30 minutes, and images were obtained using fluorescence microscopy. Cell viability was assessed using a Cell Counting Kit-8 (CCK8). The control group was set at 100% viability, where cells were cultured without hydrogel microspheres. For the hemolysis assay, fresh anticoagulated rabbit blood (4 mL, dissolved in anticoagulated rabbit blood, Hongquan Biotechnology) was centrifuged at 2,000 rpm for 5 minutes to separate red blood cells. The supernatant was discarded, and the precipitated red blood cells (RBCs) were washed three times with physiological saline (0.9% NaCl) and then resuspended in saline to prepare a 5% (v / v) RBC suspension. For testing, 0.2 g of each hydrogel microsphere sample was mixed with 1 mL of RBC suspension and 4 mL of PBS in test tubes (experimental group). The positive control was replaced with 0.1% (v / v) Triton X-100 instead of PBS, and the blank control was replaced with PBS instead of the hydrogel microspheres. All tubes were incubated in a shaking water bath (150 rpm). After incubation, the mixture was centrifuged for 5 minutes, and the absorbance A of the supernatant was measured at 545 nm using a UV-Vis spectrophotometer. The hemolysis rate (%) was calculated as follows:

[0038]

[0039] A sample A represents the absorbance of the sample. negetive control Absorbance of blank control, A positive control The absorbance is for the positive control.

[0040] The sample, negative control (PBS), and positive control (Triton X-100) represent the absorbance values ​​of each group.

[0041] Prior to euthanasia, 2 mL of whole blood was collected from each rat via cardiac puncture using EDTA-K2 anticoagulant tubes for analysis of hematological parameters (white blood cell count, red blood cell count, hemoglobin, platelet count, etc.) and serum biochemical parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CRE), etc.). After euthanasia, major organs (heart, lungs, liver, spleen, and kidneys) were harvested and subjected to H&E staining for histological evaluation.

[0042] 5. Rat liver hemostasis experimental model.

[0043] For the rat liver resection model, male SD rats (6 to 8 weeks old, 180 to 200 g) were purchased and randomly divided into five groups (n=3 per group). First, heparin (3000 U / kg) was injected intraperitoneally 30 minutes before surgery for anticoagulation. Animals were anesthetized with sodium pentobarbital and fixed to a manipulation board. The liver of the rat was exposed through an abdominal incision. The liver was partially resected 1 cm from the liver margin. For active hepatic bleeding, porcine fibrin adhesive (Fibrin), cyanoacrylate (CA), PNP hydrogel microspheres, and PR hydrogel microspheres were applied. The control group received no treatment. Hemostasis time was recorded. If hemostasis was not observed within 10 minutes, the rat was euthanized to reduce animal pain. In separate experiments, blood was collected using filter paper within 3 minutes post-surgery. The filter paper was then weighed, and the blood loss was calculated.

[0044] 6. Animal research.

[0045] All animal experiments were approved by the Animal Protection Committee of Shanghai Jiao Tong University (A2024430-001). Adult male Sprague-Dawley (SD) rats weighing 250-300g were purchased from Vital River Animal Technology Co., Ltd. and placed in ventilated cages with a 12-hour light-dark cycle.

[0046] 7. Rat dorsal incision experimental model.

[0047] Male Sprague-Dawley rats (6–8 weeks old, ~210 g) were used to demonstrate the application of PNP hydrogel microspheres in wound closure. After anesthesia with sodium pentobarbital, the rats' backs were shaved and disinfected with povidone-iodine. A 2 cm full-thickness skin incision was made along the midline of the back of each rat using a scalpel. PNP and PR hydrogel microspheres were injected into the incision and then held in place with fingers for 2 minutes. For comparison, porcine fibrin adhesive was applied to the incision and held in place with fingers for 2 minutes. Cyanoacrylate glue (CA n-butyl-2-cyanoacrylate) was applied to the wound surface according to the manufacturer's protocol to form a polymer film. Conventional sutures were also used for comparison. Rats were euthanized at 7 and 14 days post-surgery. Skin around the wound was collected for histological evaluation and immunohistochemical staining. Three animals were used for each treatment at each time point. Histological images were obtained using an inverted fluorescence microscope or a confocal laser scanning microscope (CLSM). Using ImageJ software, wound area and collagen deposition levels were analyzed based on H&E and Masson trichrome staining images, respectively. Immunofluorescence intensities of IL-6 and TNF-α (inflammatory factors) were quantified using ImageJ software.

[0048] The local analgesic effect of PR hydrogel microspheres was also evaluated using a rat dorsal needle acupuncture test. Following dorsal incision surgery, mechanosensitive hyperalgesia was assessed using a 26G von Frey filament (UgoBasile) attached to an 18G needle. Each rat received six consecutive stimulations (each lasting 1–2 seconds, spaced 10 minutes apart), applied vertically to the surgical site. Cutaneous muscle withdrawal reflex (CTMR), defined as rapid abdominal muscle contraction or aversive movement upon stimulation, was recorded and analyzed for the number of CTMRs in each group of rats.

[0049] 8. Experimental model of rat foot incision.

[0050] The analgesic effect of PR hydrogel microspheres was evaluated using a plantar incision model. Male Sprague-Dawley rats (weighing 200-250g) were randomly divided into different groups (n=6 per group). Under isoflurane anesthesia (induction: 3% in 100% O2; maintenance: 1.5-2%), the right hind paw was disinfected with iodine. A 1cm longitudinal incision was made through the skin and underlying fascia, starting 0.5cm from the heel and extending towards the toes. The plantar muscles were exposed using eye clamps, and longitudinal myotomy was performed while preserving the muscle origin and insertion. Hemostasis was achieved by gentle squeezing before therapeutic application. After hemostasis, the incisions were treated with different materials, and the rats were divided into 6 groups: blank group, no treatment; incision group, foot incision was performed, no further treatment; suture group, the wound was sutured with 5-0 nylon suture after incision; PNP group, PNP hydrogel microspheres (50 mg, particle size ~200 μm) were applied to the wound after incorporation; PNP+Rop group, ropivacaine (Rop, 0.2% w / w) powder was mixed with PNP hydrogel microspheres and applied to the wound after incision; PR group, PR double network hydrogel microsphere powder was applied to seal the wound after incision.

[0051] 9. Behavioral analysis of a rat foot incision model.

[0052] Behavioral analysis of rats after plantar incision modeling was performed using the von Frey test, Hargreaves test, and open field test (OFT) to evaluate the analgesic effect of PR hydrogel microspheres. In the von Frey test, rats were acclimatized for 15–30 minutes in a transparent acrylic chamber (20 × 20 × 40 cm) with a wire mesh floor (0.2 × 0.2 cm aperture). The test began when the animals did not exhibit exploratory / defecation behavior. The calibration probe of an electric von Frey analgesic device (model 2393 series, Shanghai Yuyan Instrument Co., Ltd.) was applied vertically to the plantar surface near the paw incision. Each probe was stimulated 3 times at intervals greater than 1 minute, gradually increasing to the rat's body weight. Two or more obvious pain-related behaviors, such as paw withdrawal, were observed and considered as a valid pain response. The previously recorded g value was designated as the mechanical pain threshold. Baseline measurements were obtained from the control group, and the mechanical pain threshold and corresponding changes in different groups of rats were recorded at the corresponding time intervals.

[0053] In the Hargreaves test, the thermal sensitivity of the hind paws in rats was assessed using the Planter test (37570, UGO Basilé Ltd.). Two days prior to testing and on each test day, rats were acclimatized to the testing environment and equipment for 30 minutes. Each rat was placed in a single-person plexiglass chamber on a glass platform, with the temperature maintained at 30°C. The thermal sensitivity of the hind paws was assessed by directing a light beam at the plantar surface and recording the paw withdrawal latency (PWL). The light intensity was adjusted to produce a typical PWL of 10–14 seconds in blank rats, with automatic cutoff at 20 seconds to prevent tissue damage. If no withdrawal occurred within 20 seconds, a 20-second PWL was recorded. The average PWL of three readings at intervals ≥5 minutes for each paw was calculated and analyzed.

[0054] In the open field test, the focus was on assessing the spontaneous movement activity of the rats. Following surgery and drug administration, the rats were placed in the center of an open arena measuring 120cm × 120cm × 60cm and allowed to freely explore the arena for at least 30 minutes. The trajectory and total distance traveled were recorded using a camera connected to an intelligent video computerized tracking system (XR-XZ301, Shanghai Xinruan Information Technology Co., Ltd.).

[0055] 10. Analysis of the signaling pathways from the lower limbs to the spinal cord.

[0056] Rats were anesthetized with inhaled isoflurane, then perfused with 20 mL of saline, followed by 15 mL of 4% paraformaldehyde. The lumbosacral spinal cord segments (L4-L6) and corresponding dorsal root ganglia (DRGs) were dissected and post-fixed in 4% PFA at 4°C for 24 hours. After post-fixation, the segments were dehydrated using a sucrose gradient before being cut into 20 μm sections. ATF3, TRPV1, and c-Fos immunostaining were used to detect neuronal activation in DRGs, while Iba-1 and GFAP staining were used to identify microglia and astrocytes. ImageJ software was used for quantitative analysis of neuronal astrocyte and microglia activation, and mean fluorescence intensity was assessed to determine activation levels.

[0057] 11. Statistical analysis.

[0058] All analyses were performed using GraphpadPrism software version 9.5.0. Data are expressed as mean with standard deviation. Data were subjected to one-way ANOVA. The following symbols are used to indicate statistical significance: *p<0.05, **p<0.01, **p<0.001, ****p<0.0001.

[0059] 12. Results Analysis.

[0060] (1) Preparation and characterization of PNP hydrogel microspheres and PR hydrogel microspheres.

[0061] Polyvinyl alcohol / polyethylene glycol diacrylate / N-hydroxysuccinimide acrylate (PNP) hydrogel microspheres were successfully synthesized using a combined emulsification and photocrosslinking method. Figure 1 Polyvinyl alcohol (PVA) serves as a flexible framework, forming a three-dimensional network through hydrogen bonds between hydroxyl groups. Photocrosslinking between PEGDA, AA, and AAC-NHS forms a secondary network, enhancing the PVA structure through free radical polymerization of diacrylate and acrylate groups under 365 nm illumination. Fourier transform infrared spectroscopy (FTIR) confirmed the covalent bonds formed between PVA hydroxyl groups and PEGDA acrylate at 1700 cm⁻¹. Figure 17 AAC-NHS not only increases crosslinking density, but its NHS esters can also mediate tissue adhesion through amide bonds with collagen amino groups. Brief hydrogen bonds between the AA carboxyl groups and tissue hydroxyl groups provide the initial adhesive contact, while stable amide bonds ensure prolonged adhesion maintenance. The hydrogel microsphere precursor solution exhibits rapid UV-induced gelation properties under UV irradiation, as demonstrated by its crosslinking ability within 30 seconds. Figure 2(a) To quantitatively evaluate the viscoelasticity of photocurable PNP hydrogel microspheres, dynamic oscillatory rheological measurements were performed. The results showed the dominant elastic behavior, with a storage modulus (G') of 2535±12 Pa, significantly exceeding the loss modulus (G”) of 153±5 Pa (G' / G”≈16.6). Figure 3 In section a), it is shown that a robust and elastic network structure has been formed.

[0062] PNP hydrogel microspheres were prepared using a photocrosslinking method combined with emulsion technology, with PNP hydrogel microsphere precursors as the aqueous phase. The aqueous phase consisted of a hydrogel microsphere precursor solution containing lithium phenyl (2,4,6-trimethylbenzoyl)phosphinic acid (LAP) as a photoinitiator, while the oil phase consisted of liquid paraffin stabilized with surfactants Span 80 and Tween 20. Notably, the dynamic properties of the emulsion system required extended irradiation time compared to static hydrogel microsphere curing. Different UV exposure times were tested to optimize the hydrogel microsphere preparation method. The results showed that insufficient UV irradiation (<10 min) led to incomplete polymerization, resulting in structural collapse and a flocculent morphology after freeze-drying. Conversely, excessive irradiation (>60 min) caused over-crosslinking, aggregation of hydrogel microspheres, and precipitation due to interfacial instability. Figure 18 To optimize the uniformity and dispersibility of the hydrogel microspheres, the procedure includes: (1) initial high-speed emulsification (1000 rpm, 60 min) to obtain monodisperse droplets; (2) reduced agitation (500 rpm) during 30 min of UV curing to minimize shear-induced aggregation; and (3) post-curing low-speed mixing (500 rpm, 5 min) to ensure residual monomer consumption and structural stability. The hydrogel microspheres produced by this method exhibit narrow particle size distribution, spherical integrity, and excellent dispersion stability, which has been confirmed by morphological analysis after freeze-drying. Figure 2 (b)

[0063] SEM images show that the PNP hydrogel microspheres exhibit a well-defined porous surface structure. Figure 2 (middle d, left), which gives it high absorption capacity. PNP hydrogel microspheres exhibit excellent swelling properties, with a swelling rate of 945±5% (middle d, left). Figure 19 In this case, Rop-Lipo prepared by rotary evaporation film deposition method has an average hydrodynamic diameter of 167.4 ± 2.1 nm (PDI = 0.18). Figure 20 Ropivacaine-loaded liposomes (Rop-Lipo) can be readily loaded into PNP hydrogel microspheres via absorption. Meanwhile, the incorporation of Ropivacaine-loaded liposomes had no observable effect on the formation of these morphological features. Figure 2 (middle d, right).

[0064] The 7-amino-4-methyl-3-coumarinacetic acid (AMCA) labeling of Rop resulted in blue fluorescence in response to the amino groups on the Rop, and successful loading of the Rop could be confirmed by fluorescence imaging of the hydrogel microspheres. Figure 2 (c) Furthermore, FTIR spectroscopy analysis revealed characteristic absorption bands in the drug-loaded hydrogel microspheres (PR hydrogel microspheres), which were absent in the blank carrier. Figure 3 b): 1538cm 1 (NH tape) and 2922cm⁻ 1 (CH alkane stretching). Perfect spectral matching between the loaded hydrogel microspheres and pure ropivacaine confirmed successful encapsulation without chemical degradation. Notably, the sustained release profile of ropivacaine from the PR hydrogel microspheres was significantly improved by employing a two-stage delivery strategy using lipid vesicles and a hydrogel microsphere network. In vitro release experiments showed that the drug release duration of the PR system was extended to 240 hours, exhibiting a significant prolongation compared to PNP hydrogel microspheres loaded with hydrochloride (~12 hours) or free ropivacaine base (~24 hours). Figure 3 (c)

[0065] Particle size distribution analysis confirmed comparable size characteristics between the two formulations. The average diameter of the PNP and PR hydrogel microspheres was approximately 191.9 ± 8.7 μm. Figure 3 In the middle (d), over 80% of the hydrogel microspheres are distributed in the range of 148–228 μm, a range reported to have better liquid absorption than other particle sizes. Furthermore, thanks to their suitable diameter and uniform dispersion, our PNP and PR hydrogel microspheres can form a fine mist when sprayed through an atomizer bottle. Figure 4 (a)

[0066] Hydration property assessment showed minimal differences between formulations, due to the swelling capacity of PNP hydrogel microspheres being 919.76±20.58%, PR hydrogel microspheres being 912.95±37.59%, and water retention rates of 614.61±56.51% and 582.09±72.08%, respectively. Figure 4 (b)

[0067] (2) PNP hydrogel microspheres and PR hydrogel microspheres have strong adhesion and excellent ability to seal in vivo bleeding points.

[0068] The adhesive properties of ropivacaine-loaded PNP hydrogel microspheres (PR hydrogel microspheres) were rigorously evaluated using standardized mechanical characterization methods according to ASTM F2255-05 (lap shear strength), ASTM F2256-05 (T-peel strength), and ASTM F2258-05 (tensile strength). The adhesive properties of PR hydrogel microspheres with lipid-encapsulated ropivacaine at concentrations of 1%, 2.5%, 5%, and 10% were compared with those of widely used clinical adhesive fibrin sealants. The results showed that the adhesive strength decreased in a concentration-dependent manner. Figure 21 PR-MS containing 10% ropivacaine showed no statistically significant difference in adhesive strength compared to porcine fibrin adhesive (p>0.05). Conversely, hydrogel microspheres containing 1% ropivacaine exhibited the highest mechanical strength and significantly stronger adhesion compared to porcine fibrin (p<0.05). Based on the established clinical concentration range of 0.5–1% ropivacaine for regional nerve block, the 1% ropivacaine-loaded PR-MS formulation, which maintains optimal adhesive performance while delivering the therapeutic agent, was selected for further experimental characterization and animal studies.

[0069] Both types of hydrogel microspheres exhibited significantly stronger tissue adhesion than the commercial tissue adhesive porcine fibrin adhesive (p<0.0001). In the overlap shear test ( Figure 5 In the second test (a), PNP and PR reached maximum adhesion strengths of 134.46 kPa and 129.96 kPa, respectively, significantly higher than porcine fibrin's 30.06 kPa. Similarly, in the T-peel test (… Figure 5 Figure b) shows that the adhesion energy of PNP is 125.34 J / m, PR is 111.04 J / m, and porcine fibrin is 39.52 J / m. Tensile test ( Figure 5 c) further confirmed that PNP (213.79 kPa) and PR (207.36 kPa) were superior to porcine fibrin (85.47 kPa; n=8). Although the maximum tensile strength of PR was slightly lower than that of PNP, this difference was not statistically significant. Notably, PNP hydrogel microspheres adhered to both porcine skin samples successfully lifted a weight of 300 g. Figure 6 (a) Adhesion experiments on isolated organs further confirmed these findings. After standardized incisions were made on isolated porcine heart, liver, kidney, lung, muscle, and skin tissues, PNP hydrogel microspheres were uniformly applied to the wound sites using a powder sprayer. After manual pressure for 20 seconds, complete closure of visceral organ defects and effective adhesion between muscle and skin tissues were observed. Figure 6 (b)

[0070] To verify the superior tissue adhesion and rapid occlusion capabilities of PNP hydrogel microspheres, an in vivo hemostatic efficacy evaluation was conducted using a rat liver defect model. Figure 6 c and Figure 7 (a, b) Standardized full-thickness wounds were created on the exposed liver surface of live Sprague-Dawley (SD) rats, followed by wound closure using gauze, cyanoacrylate, porcine fibrin adhesive, PNP hydrogel microspheres, and PR hydrogel microspheres. Hemostasis time and bleeding volume were quantitatively recorded. Results showed that both the PNP and PR groups achieved hemostasis within 20 seconds, with significantly less blood loss than 50 mg, superior to the gauze control group (p<0.01) and commercial porcine fibrin adhesive (p<0.05). Although cyanoacrylate showed similar hemostasis time and blood loss to PNP, its exothermic polymerization during hydration raised the local temperature above 80°C, causing thermal damage to liver tissue. Furthermore, the fragile membrane formed by cyanoacrylate ruptured under respiratory motion and hemodynamic pressure, posing a risk of secondary hemorrhage or embolism. The physical barrier further hindered the hepatocyte migration and regenerative microenvironment. These findings demonstrate that PNP and PR hydrogel microspheres rapidly absorb tissue fluid upon contact with bleeding wounds, establishing a stable adhesive physical barrier to accelerate coagulation. Their excellent in vivo sealing properties highlight their potential as emergency materials for wound control.

[0071] (3) Evaluation of the biocompatibility of PRs.

[0072] Biocompatibility is a fundamental requirement for the clinical application of hydrogel microsphere dressings. Here, the biocompatibility of the adhesive hydrogel microspheres of this invention was investigated using in vitro and in vivo methods.

[0073] To assess the cell compatibility of hydrogel microspheres, we evaluated cell viability by co-incubating extracts of various hydrogel microspheres with NIH-3T3 fibroblasts and human umbilical vein endothelial cells (HUVECs). Cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay after 24 hours. Figure 9 As shown in Figures a and b, the viability of NIH-3T3 cells and HUVECs in the PNP group was not significantly different from that in the control group, indicating that cytotoxicity was negligible. Slightly lower cell viability was observed in the Rop group, which may be attributed to the cytotoxic effect of high concentrations of ropivacaine. Notably, cell viability in the PR group was significantly higher than that in the ropivacaine-only group (p=0.0003), which may be due to the more complex cross-linked structure of the dual-network hydrogel microspheres (PR) and the encapsulation within liposomes enhancing overall stability, jointly slowing the release of ropivacaine and thus reducing cytotoxicity. Live / dead cell staining results ( Figure 9 (c) and (d) confirmed the CCK-8 results, indicating that most cells in the PR group appeared healthy, with intact morphology and characteristic spindle shape. Figure 8Only a minimal number of dead cells were observed. Considering the direct contact between the binder powder and blood, we further evaluated the blood compatibility of the hydrogel microspheres. After incubation with rabbit blood, their effects on erythrocytes were assessed using the following methods: hemolysis (erythrocyte rupture) or aggregation. Triton X-100 and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. Absorbance at 545 nm was measured by UV-Vis spectrophotometry after 1 hour of incubation. Figure 9 As shown in Figure e, compared with the blank control, neither PNP nor PR hydrogel microspheres induced significant hemolysis, and the hemolysis rate in all groups was less than 3%, indicating excellent blood compatibility.

[0074] For in vivo studies, we closely monitored the mental state and weight changes of rats for 7 days following the physical intervention. We found that rats in each group were in good mental condition and their weight steadily increased. Comprehensive histological examination of the heart, liver, spleen, lungs, and kidneys using H&E staining confirmed the absence of acute toxicological effects. Figure 22 (a) Myocardial fibers are arranged in a cylindrical branching network with centrally located nuclei and abundant capillaries in the interstitium. The liver lobe structure is intact, with hepatocytes arranged radially, and the nuclei are round and uniform with pale perinuclear staining. The spleen has clear red and white pulp, with deeply stained lymphocytes in the white pulp. The red pulp is rich in erythrocytes, appearing red, and contains splenic cords and sinusoids. The vesicle structure is intact, the alveolar walls are a single layer of squamous epithelium, the interstitial masses are uniform, and there is no congestion. The glomeruli are clearly structured, and the renal tubular epithelial cells are neatly arranged without interstitial fibrosis. All observed structures and cell morphologies are consistent with the structure and histology of normal, healthy organs. Serological indicators of the heart (CK-MB, LDH), liver (AST, ALT), and kidneys (Cr, BUN) in each group are within the normal range. Figure 22 (b)

[0075] (4) Application of PR hydrogel microspheres in rat dorsal incision model.

[0076] PNP and PR hydrogel microspheres were formed by the hydration of their hydrogel microspheres and evaluated as wound spray bandages in SD rats with dorsal incisions. The SD rats were randomly assigned to six groups (n=6): untreated control, cyanoacrylate (CA), porcine fibrin adhesive, suture, PNP, and PR. Macroscopic assessment revealed different healing patterns. Figure 10 In step a), quantitative analysis confirmed that, compared with the control group, wound closure was significantly accelerated in both the PNP and PR groups (p<0.0001). Figure 10 (b) By day 14, complete epithelial formation was achieved in the PNP and PR groups, while persistent defects remained in the control group. Histological evaluation by H&E staining ( Figure 11(a) On day 7, the control group showed extensive non-epithelialized areas and inflammation, while the PNP, PR, and Stitch groups showed reduced epithelial defects, although Stitch showed exacerbated inflammation due to suture-induced foreign body reaction. By day 14, although CA and porcine fibrin achieved epithelial formation, partial epidermal-dermal separation indicated fragile neoepithelial tissue, while the PNP and PR groups showed robust epidermal-dermal integration with preserved skin appendages. Masson's trichrome staining further showed that collagen deposition was denser in the PNP and PR groups on day 7. Figure 10 c and Figure 11 (b) The cells were more organized, indicating enhanced extracellular matrix remodeling.

[0077] Furthermore, inflammatory profiling using TNF-α and IL-6 quantification showed that on day 7, cytokine levels in the PNP and PR groups were significantly lower than in other groups (p<0.01). Figure 12 and Figure 11 (c) By day 14, IL-6 levels continued to decrease, indicating not only excellent biocompatibility but also a positive regulatory effect on the inflammatory response. Notably, preliminary assessment of the analgesic effect using the cutaneous trunk muscle reflex (CTMR) test showed that PR hydrogel microsphere treatment of the incision provided transient pain suppression, which returned to baseline levels by day 5. Mechanosensitive inhibition persisted for approximately 4 days after application. Figure 23 This supports its multifunctional therapeutic properties.

[0078] (5) Application of PR hydrogel microspheres in rat foot incision model.

[0079] To further investigate the analgesic mechanism of the composite material, we established a rat paw incision model. Pain levels and motor activity were assessed using the von Frey filament (mechanical withdrawal threshold of the mean squared tipping point), the hot plate test (pulling-out latency, PWL), and the open field test (OFT). Macroscopic assessment (…) Figure 13 a) and H&E staining ( Figure 24 This revealed comparable wound closure processes in both the hydrogel microsphere group and the suture group, both significantly faster than the control group. The PNP group and the suture group exhibited similar pain and movement curves. Figure 13 b and Figure 14(a, b, c) During the initial 72 hours post-incision, decreased tolerance to PWL and MWT, as well as reduced OFT activity, were observed due to wound pain. As the wound healed, the rats gradually adapted to their environment. However, compared to the control group, the incision group consistently maintained lower PWL, MWT, and OFT levels. Due to analgesic effects, the PR and PNP+Rop (ropivacaine) groups showed MWT values ​​and motor levels comparable to the control group in the initial 12 hours, even exceeding the tolerance of the control rats in the hot plate test. After 24 hours, as ropivacaine in the PNP+Rop group was absorbed and metabolized, its PWL and MWT values ​​rapidly decreased to levels close to those of the PNP group, subsequently following curves similar to those of other control groups, reflecting the effect of hind paw pain on sensorimotor function after cessation of analgesia. Conversely, due to the sustained release of the drug from hydrogel microspheres and liposomes, the PR group maintained high pain tolerance in the initial 96 hours, gradually approaching the control group as healing progressed. Throughout the 168-hour observation period, the rats maintained a high level of locomotor activity, with OFT (open field test) values ​​comparable to the control group. This indicates enhanced exploratory drive and reduced pain interference in the experimental cohort. These results demonstrate that encapsulation in hydrogel microspheres and liposomes can prolong and locally release ropivacaine, providing sustained analgesia and reducing post-incision pain in the hind paws of rats.

[0080] (6) PR inhibits the activation of pain-related peripheral neurons.

[0081] To elucidate the analgesic mechanism of PR, samples were collected from the L4 / L5 dorsal root ganglion (DRG) and dorsal horn of the spinal cord 7 days post-intervention to assess c-Fos expression in sensory neurons. Cells co-expressing c-Fos and TRPV1 indicated activation of sensory neurons. Figure 15 As shown in Figure a, on day 7, based on the highest c-Fos expression, the Stitch, PNP, and PNP+Rop groups exhibited similar high sensory neuronal activation. Conversely, compared to the control group, the PR group showed significantly lower c-Fos expression, only slightly higher than the blank group, which was not statistically significant (p=0.7876). Figure 16 (a) Conversely, compared with the suture group and the PR group, the proportion of double-positive cells (c-Fos+TRPV1+ / TRPV1+) among all TRPV1-positive cells in the PNP group was significantly higher (p<0.01). Figure 16 (b) The incision group showed the highest proportion of double-positive cells, but this was not statistically different from the PNP and suture groups. The proportion of double-positive cells in the PNP+Rop group was slightly reduced due to the incorporation of Rop. However, it still showed a significantly higher level than the PR group, due to the rapid release of Rop without liposomes from the PNP hydrogel microsphere network (approximately 100% within 24 hours).

[0082] ATF3, a core stress transcription factor in the DRG, has a dual function of promoting regeneration and regulating pain, and is normally expressed at low levels in the DRG of normal rats. Its expression is significantly increased in response to noxious stimuli. Figure 15 b and Figure 16 In the study, compared with the blank control group (no-damage control group), all experimental groups showed significantly increased ATF3 expression. Notably, a statistically significant difference in ATF3 expression was observed between the PR group and the PNP+Rop group (p=0.0026)—both of which showed analgesic effects—indicating that PR intervention has excellent long-lasting analgesic effects.

[0083] Glial cells in the dorsal horn of the spinal cord play a crucial role in neural development and maintenance by providing protection, support, nutrition, and scavenging harmful substances. Furthermore, neuronal signaling within the glial region of the dorsal horn facilitates the transmission of information in the nervous system, particularly nociceptive information. Expression of GFAP (glial fibrillary acidic protein) and Iba-1 (ionized calcium-binding adapter molecule 1) in the dorsal horn of the spinal cord marks the activation of astrocytes and microglia, respectively. Glial cells in the dorsal horn play a vital role in neural development and maintenance by providing protection, support, nutrition, and scavenging harmful substances. Figure 15 As shown in Figure c, compared with the blank group, both the control group and the PNP group showed significantly increased expression levels of GFAP and Iba-1 (p<0.0001). Compared with the cut group, the PNP+Rop group showed a slight downregulation of GFAP (p<0.0001). Figure 16 (d) and Iba-1 (p=0.0001); Figure 16 (e) Facial expression. However, compared with the PNP+Rop group, the PR group showed significantly reduced expression of GFAP (p=0.0138) and Iba-1 (p=0.0363). Consistent with previous studies, our findings suggest that PR alleviates incision pain by persistently inhibiting c-Fos expression in the DRG and suppressing glial cell activation in the dorsal horn of the spinal cord.

[0084] This invention develops a dual-network hydrogel microsphere adhesive system composed of polyvinyl alcohol (PVA), polyethylene glycol diacrylate (PEGDA), and acrylic acid (AA), functionalized with N-hydroxysuccinimide acrylate (AAC-NHS) to achieve strong adhesion to wet tissues. The introduction of ropivacaine liposomes (Rop-Lipo) enables sustained local analgesia, addressing the dual challenges of wound closure and postoperative pain management. The hydrogel microspheres exhibit excellent mechanical properties, rapid swelling, high liquid absorption, and superior tissue adhesion, exceeding clinical standards such as fibrin glue and cyanoacrylate in overlap shear, T-peel, and tensile tests. In vivo evaluation demonstrated rapid and effective hemostasis in a rat liver defect model, as well as accelerated wound healing and reduced inflammation in dorsal and plantar incision models. Sustained release of ropivacaine from the hydrogel microspheres significantly prolonged analgesia (≥168 hours), reduced nociceptive neuronal activation, and inhibited glial cell responses in the dorsal horn of the spinal cord. Biocompatibility assessment confirmed minimal cytotoxicity and systemic safety. These multifunctional hydrogel microspheres represent a promising advance in enhanced postoperative recovery (ERAS), combining reliable wound adhesion with prolonged local pain relief to improve postoperative outcomes.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a sprayable dual-network hydrogel microsphere adhesive, characterized in that, The steps include the following: Step 1, Preparation of the aqueous phase of the hydrogel microsphere precursor: Dissolve polyvinyl alcohol in water, and then add poly(ethylene glycol) diacrylate, photoinitiator, N-hydroxysuccinimide acrylate and acrylic acid in sequence to form a prepolymer aqueous solution. Place at 55°C for at least 30 min to avoid bubbles. Step 2: Using liquid paraffin as the oil phase, add dehydrated sorbitan monooleate and polyoxyethylene dehydrated sorbitan monolaurate, stir and heat to 55°C, gradually add the aqueous phase solution to the oil phase, and continuously irradiate with ultraviolet light to solidify the water droplets in the emulsion to form hydrogel microspheres. Step 3: Mix the hydrogel microspheres thoroughly with petroleum ether and allow them to separate at ambient temperature for 1 hour. Decant the supernatant, centrifuge to separate the hydrogel microspheres, and perform three consecutive washing cycles for purification. Collect the purified hydrogel microspheres, freeze-dry them for 24 hours, and store them in the dark. The first wash uses a mixture of ethanol and deionized water with a volume ratio of 50:50, the second wash uses a mixture of ethanol and deionized water with a volume ratio of 75:25, and the third wash uses anhydrous ethanol. Each wash includes vortex mixing for 30 seconds and centrifugation under the same parameters. Step 4, Preparation of ropivacaine liposomes: Lecithin choline, cholesterol, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)-2000 are mixed evenly, free ropivacaine base is added to the mixture, and the mixture is evaporated under reduced pressure to form a thin lipid film; Step 5: Add ultrapure water to the lipid membrane, mix and sonicate to obtain a primary liposome suspension. Mix the hydrogel microspheres with the liposome suspension and incubate for 1 hour under continuous orbital oscillation. Collect the obtained double-network hydrogel microspheres by centrifugation, freeze-dry and store in a nitrogen-purged bottle at -20°C.

2. The method for preparing the sprayable dual-network hydrogel microsphere adhesive according to claim 1, characterized in that: In step 1, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate or α-ketoglutaric acid.

3. The method for preparing the sprayable dual-network hydrogel microsphere adhesive according to claim 1, characterized in that: In step 2, after the emulsion is formed, the system is continuously irradiated with a 365nm ultraviolet lamp for 30-60 minutes under continuous stirring. Before stopping the stirring process, the structural integrity of the hydrogel microspheres is verified by microscopic observation.

4. The method for preparing the sprayable dual-network hydrogel microsphere adhesive according to claim 1, characterized in that: In step 5, a thermally stable liposome extruder is used to extrude the liposome suspension through a polycarbonate membrane with continuous pore size to obtain a liposome suspension with uniform particle size distribution.

5. The method for preparing the sprayable dual-network hydrogel microsphere adhesive according to claim 1, characterized in that: In step 5, the hydrogel microspheres and liposome suspension were incubated for 1 hour under continuous orbital oscillation at a ratio of 10 mL: 10 mg.

6. The method for preparing the sprayable dual-network hydrogel microsphere adhesive according to claim 1, characterized in that: In step 5, the obtained dual-network hydrogel microspheres are collected by centrifugation and washed twice with deionized water to remove unbound liposomes, thus purifying the dual-network hydrogel microspheres.

7. A sprayable dual-network hydrogel microsphere adhesive, characterized in that: It is prepared by the preparation method described in any one of claims 1-6.

8. The application of the sprayable dual-network hydrogel microsphere adhesive according to claim 7, characterized in that: It is used in the preparation of wound dressings that promote surgical wound healing and relieve persistent pain.

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