Sprayable double-network hydrogel microsphere adhesive as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses a sprayable double-network hydrogel microsphere adhesive and a preparation method and application thereof.The preparation method comprises the following steps that liquid paraffin, sorbitan monooleate and polyoxyethylene sorbitan monolaurate serve as oil phases, and an emulsion is formed; fully mixing the emulsion with petroleum ether, centrifugally separating gel microspheres, freeze-drying, and storing in a dark place; preparing a ropivacaine liposome; and adding ultrapure water into the lipid membrane to obtain a primary liposome suspension, adsorbing the liposome by using the hydrogel microspheres, and collecting the obtained dual-network hydrogel microspheres. According to the sprayable double-network hydrogel microsphere adhesive as well as the preparation method and the application thereof, prepared hydrogel microspheres can quickly and consistently cover irregular wounds, and liposome loaded with ropivacaine provides continuous analgesic release, so that the painful wound healing effect is improved. By simultaneously solving the problems of maintaining continuous adhesion and prolonging pain relief in blood-saturated wounds.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical biomaterials, in particular to a sprayable double network hydrogel microsphere adhesive as well as a preparation method and application thereof. BACKGROUND

[0002] Wound closure is a key objective of surgical and trauma care, essential to prevent infection, promote tissue regeneration, and restore physiological function. Due to its ubiquity in daily life and inherent therapeutic complexity, deep, irregularly bleeding wounds pose particularly arduous clinical challenges. These wounds exhibit several major complications, including inaccessibility to surgical procedures, which hinders the effective placement of sutures to stop the vasculature from continuous bleeding in tortuous cavities, impaired interfacial adhesion under wet conditions, and exacerbated long-term neuropathic pain due to mechanical stress on injured nerves.

[0003] Traditional methods, such as needle and thread suturing and skin staplers, remain the gold standard for wound closure due to their reliability. However, these methods not only show limited effectiveness on deep, irregularly bleeding wounds, but also have significant limitations, including mechanical damage to surrounding tissues, risk of infection, and potential to induce inflammation and scarring. Moreover, suturing requires skilled application and can cause discomfort during removal, while steel staples cause tissue ischemia and delayed healing. Adhesives, such as adhesive tapes and biological adhesives, have emerged as a promising solution, unlike sutures, which can distribute pressure evenly over the wound, reduce local pressure, and promote faster healing. However, this solution is still insufficient. On the one hand, wound dressings / tapes cannot conform to irregular contours, creating gaps that allow bacterial invasion and exudate accumulation. Moreover, they quickly lose adhesion in blood-filled environments. On the other hand, biological adhesives (e.g., fibrin glue, cyanoacrylate) exhibit limited penetration beyond the surface layer of the bleeding site due to premature polymerization upon blood contact, known as the "self-sealing effect", which leaves deeper bleeding uncontrolled. Furthermore, their brittle interfaces frequently break under physiological pressure.

[0004] Adhesive powders fill complex wound structures through particle flow and rapid liquid absorption, providing unique advantages. Unlike tapes, they can conformally adapt to 3D defects while concentrating clotting factors through a superabsorption effect, outperforming biological adhesives in hemostatic speed. Particle size and heterogeneity severely impair their performance in bleeding wounds, as heterogeneous particle sizes result in low and uneven distribution of liquid absorption under pulsatile pressure, creating weak points prone to adhesion failure. However, most milled powders exhibit uncontrollable size variations, which directly compromise adhesion stability in blood environments. In this context, there is a need to develop adhesive powders with uniform sizes.

[0005] At the same time, the existing solutions ignore the pain management during the prolonged healing period, thus prolonging the recovery time. Therefore, the mismatch between the duration of analgesia and the wound healing period remains a challenge to achieve 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, especially to achieve firm wet adhesion in a bleeding environment while achieving long-term analgesia, a sprayable dual-network hydrogel microsphere adhesive system is developed, which provides a comprehensive solution to improve postoperative recovery and patient comfort. SUMMARY

[0007] The purpose of the present application is to provide a sprayable dual-network hydrogel microsphere adhesive and its preparation method and application. The prepared sprayable hydrogel microsphere form can quickly and uniformly cover the geometrically irregular wounds that cannot be covered by conventional dressings, while the embedded ropivacaine-loaded liposomes provide sustained analgesic release. By simultaneously addressing the need for sustained adhesion in blood-saturated wounds and prolonged pain relief, the unmet key needs in wound and surgical recovery are addressed, providing a comprehensive solution to improve postoperative recovery and patient comfort.

[0008] To achieve the above-mentioned purpose, the present application provides a preparation method of a sprayable dual-network hydrogel microsphere adhesive, comprising the following steps: Step 1, preparation of hydrogel microsphere precursor aqueous phase: dissolve polyvinyl alcohol in water, then add poly(ethylene glycol) diacrylate, photoinitiator, N-hydroxysuccinimidyl acrylate, and acrylic acid in sequence to form a pre-polymer aqueous solution, and place it at 55℃ for at least 30 min to avoid bubbles; Step 2, add sorbitan monooleate and polyoxyethylene sorbitan monolaurate to liquid paraffin as oil phase, stir and heat to 55℃, gradually add the aqueous phase solution to the oil phase, and continuously irradiate the water droplets in the emulsion with ultraviolet light while stirring to solidify, forming hydrogel microspheres; Step 3, mix the hydrogel microspheres with petroleum ether and phase separate at ambient temperature for 1 hour, decant the supernatant, centrifuge the hydrogel microspheres, purify them with three consecutive washing cycles with a gradient series of ethanol / deionized water mixture, collect the purified hydrogel microspheres, and freeze-dry for 24 hours, store in the dark; Step 4, preparation of ropivacaine liposomes: uniformly mix lecithin, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000, add ropivacaine free base to the mixture, and evaporate under reduced pressure to form a thin lipid film; Step 5, ultrapure water was added to the lipid film, mixed and sonicated to obtain a primary liposome suspension, the hydrogel microspheres were mixed with the liposome suspension, incubated for 1 hour under continuous orbital shaking, the resulting double network hydrogel microspheres were collected by centrifugation, freeze-dried and stored at -20℃ in nitrogen-purged bottles.

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

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

[0011] Preferably, in step 3, the volume ratio of ethanol to deionized water in the gradient series of ethanol / deionized water mixture is: 50:50 for the first washing, 75:25 for the second washing, and no water for the third washing, each washing includes vortex mixing for 30 s and centrifugation under the same parameters.

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

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

[0014] Preferably, in step 5, the resulting double network hydrogel microspheres are collected by centrifugation and washed twice with deionized water to remove unbound liposomes, and the double network hydrogel microspheres are purified.

[0015] The present application also provides a sprayable double network hydrogel microsphere adhesive prepared by the above preparation method.

[0016] The application of the sprayable double network hydrogel microsphere adhesive is used in the preparation of wound dressings to promote surgical wound healing and sustained pain relief.

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

[0018] 2. The introduction of ropivacaine liposomes (Rop-Lipo) in the present application enables sustained local analgesia, addressing the dual challenge of wound closure and postoperative pain management. The hydrogel microspheres exhibit superior mechanical properties, fast swelling, high liquid uptake, and excellent tissue adhesion, surpassing clinical standards in lap shear, T-peel, and tensile tests.

[0019] 3. In vivo evaluation of the present application 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. The sustained release of ropivacaine from the hydrogel microspheres significantly prolonged analgesia time (≥ 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. This multifunctional hydrogel microsphere represents a promising advancement in enhancing postoperative recovery, combining reliable wound adhesion with prolonged local pain relief to improve postoperative outcomes. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a schematic diagram of the hydrogel microsphere components; Figure 2 in which a is the UV curing process of the PVA / AAC precursor, b is a representative micrograph of PNP hydrogel microspheres (scale bar: 500 microns), c is a representative fluorescence image and microscope image of PR hydrogel microspheres labeled with AMCA (scale bar: 500 microns, upper right microscope image), d is a representative SEM image of PNP and hydrogel microspheres (scale bar: 50 microns and 100 microns); Figure 3 in which a is the rheological properties: frequency sweep test showing the storage modulus (G') and loss modulus (G") of the hydrogel microspheres (n = 3), b is the Fourier transform infrared spectroscopy confirming the successful incorporation of ropivacaine liposomes, c is the drug release profile of PR hydrogel microspheres, PNP hydrogel microspheres mixed with ropivacaine monomer, and PNP microspheres mixed with ropivacaine hydrochloride solution (n = 3), d is the particle size distribution of the hydrogel microspheres measured by dynamic light scattering (DLS) (n = 3); Figure 4 in which a is a schematic diagram of the dual-network hydrogel microsphere spraying, b is the swelling behavior of PNP and PR hydrogel microspheres over time (n = 3); Figure 5 in which a is the mechanical property curves and quantitative analysis of ASTM F2205-05 (lap shear strength), b is the mechanical property curves and quantitative analysis of ASTM F2256-05 (peel test), c is the mechanical property curves and quantitative analysis of ASTM F2258-05 (tensile strength) (n = 8); Figure 6Figure 1. a is a photograph demonstrating the adherence of PNP hydrogel microspheres to two pieces of porcine skin substrates, maintaining a weight of 300 (contact area 2.5 cm 2 ), b is a photograph documenting the adherence of PNP hydrogel microspheres to the surface of multiple organs (heart, spleen, kidney, lung, muscle, and skin), the yellow dashed square indicates the incision location, c is an intraoperative photograph documenting the hemostatic performance of Gauze (control), cyanoacrylate (CA clinical reference), porcine fibrin glue (fibrin biological control), and PNP / PR hydrogel microspheres in a rat liver defect model; Figure 7 Figure 1. a is a photograph demonstrating the adherence of PNP hydrogel microspheres to two pieces of porcine skin substrates, maintaining a weight of 300 (contact area 2.5 cm Figure 8 Figure 2. Representative live / dead staining images of the present application; Figure 9 Figure 2. Representative live / dead staining images of the present application; a is HUVECs treated with PNP / PR hydrogel microspheres, Ropivacaine nanoparticles (Rop), and control groups, b is CCK-8 viability of NIH3T3 cells (n=4), c is quantitative analysis of PI positive cell ratio of HUVECs in each group, d is quantitative analysis of PI positive cell ratio of NIH3T3s in each group (n=4), e is hemolysis detection of PNP / PR hydrogel microspheres incubated with rabbit red blood cells using PBS (negative control) and 1% Triton X-100 (positive control) (n=4), representative photographs of centrifuged samples showing hemoglobin release; Figure 10 Figure 3. a is representative photographs of wound healing in a dorsal incision model of rats at 1st, 4th, 7th, 10th, and 14th day post-surgery, treatment methods include control, stitch, porcine fibrin glue (fibrin), cyanoacrylate (CA), PNP, and PR hydrogel microspheres, b is hematoxylin and eosin (H&E) staining of incision sites at 7th and 14th day post-surgery, yellow square highlights the incision area (scale bar = 500 pm), c is Masson’s trichrome staining of incision sites at 7th and 14th day post-surgery (scale bar = 500 pm); Figure 11 Figure 3. a is representative photographs of wound healing in a dorsal incision model of rats at 1st, 4th, 7th, 10th, and 14th day post-surgery, treatment methods include control, stitch, porcine fibrin glue (fibrin), cyanoacrylate (CA), PNP, and PR hydrogel microspheres, b is quantitative analysis of collagen content based on Masson’s trichrome staining at 7th and 14th day post-surgery, c is quantitative analysis of mean fluorescence intensity (MFI) of TNF-a and IL-6 based on immunofluorescence staining at 7th and 14th day post-surgery; Figure 12 Figure 4. Immunofluorescence staining of tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) at 7th and 14th day post-surgery (scale bar = 500 m); Figure 13 a Actual photographs of the incision site at day 1, 4 and 7 post- surgery for different treatment groups (control, stitch, PNP, PNP+Rop and PR groups), b Typical trajectories of the open field test for each group at day 1, 4 and 7; Figure 14 Quantitative analysis of the behavioral tests, where a is the mechanical withdrawal threshold (MWT), b is the paw withdrawal latency (PWL), c is the distance traveled in the open field test; Figure 15 a Immunofluorescence staining of DRG c-Fos and TRPV1 (scale bar: 100 pm), b Quantitative analysis of ATF3 immunofluorescence intensity, c Immunofluorescence staining of spinal dorsal horn GFAP and Iba-1, including magnified images and merged images (scale bars: 50 pm and 10 pm); Figure 16 a Quantitative analysis of c-Fos immunofluorescence intensity and the proportion of c-Fos / TRPV1 co-expression in c-Fos positive cells, b Immunofluorescence staining of ATF3 in DRG (scale bar: 100 pm), c Quantitative analysis of ATF3 immunofluorescence intensity, d Quantitative analysis of GFAP immunofluorescence intensity, e Quantitative analysis of Iba-1 immunofluorescence intensity; Figure 17 Infrared spectrum of PNP hydrogel microspheres; Figure 18 Effect of different UV curing time on the formation of final hydrogel microspheres, where a is 10 min, b is 30 min, c is 60 min; Figure 19 Quantitative analysis of the swelling rate of PNP hydrogel microspheres (n = 3); Figure 20 DLS of ropivacaine liposomes; Figure 21 a Representative mechanical curves and quantitative analysis of strength for lap shear strength, b Representative mechanical curves and quantitative analysis of strength for T-peel strength, c Representative mechanical curves and quantitative analysis of strength for tensile strength (n = 8, p < 0.05, p < 0.01, p < 0.001, p < 0.0001); Figure 22 a Histological analysis of the plantar incision experiment in SD rats, b Hematological parameters; Figure 23 Skin trunk reflex response in a dorsal incision model in rats (n=6); Figure 24 Histological analysis of the healing of a plantar incision wound in SD rats. DETAILED DESCRIPTION

[0021] The technical solutions of the present application are further described below by means of the accompanying drawings and examples. Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those with ordinary skills in the art to which the present application pertains. Unless otherwise defined, the reagents, devices, and the like materials used in the present application are all obtained from conventional commercial sources.

[0022] Example 1 The preparation method of the medical wound sprayable double-network hydrogel microsphere adhesive comprises the following steps: Step 1, preparation of a hydrogel microsphere precursor aqueous phase: accurately weigh a predetermined mass of PVA (polyvinyl alcohol, degree of polymerization 1700, 99% hydrolysis, Aladdin) and dissolve it in deionized water to obtain a uniform solution of 7 wt%. At 55°C, the following components are sequentially added to the PVA solution: 0.05 wt% poly(ethylene glycol) diacrylate (PEGDA from Sigma-Aldrich), 0.2 wt% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 97%, TCI) as a photoinitiator, 0.3 wt% acrylic acid N-hydroxysuccinimide ester (AAC-NHS, BOC Sciences), and 35 wt% acrylic acid (AA, 99%, Aladdin). The mixture is homogenized by vortex mixing (vortex generator 500 rpm) for 5 minutes to ensure uniform distribution of all components, forming a transparent pre-polymer aqueous solution. The mixed solution is placed at 55°C for at least 30 minutes to avoid air bubbles.

[0023] Step 2, liquid paraffin (95% v / v) is 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 is thoroughly homogenized and heated to 55°C under continuous mechanical stirring. Subsequently, the aqueous phase solution (10% by volume) is 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 is mechanically stirred for 1 hour to ensure stability. After the emulsion is formed, the system is continuously irradiated with a 365 nm UV lamp for 30-60 minutes under continuous stirring to achieve complete cross-linking and solidification of the hydrogel microspheres. The structural integrity of the hydrogel microspheres is verified by microscopic observation before the stirring process is terminated.

[0024] Step 3, The emulsion was thoroughly mixed with petroleum ether (analytical pure, purity > 99.5%, Aladdin) and phase separated at ambient temperature for 1 hour. The supernatant was decanted and the hydrogel microspheres were isolated using a refrigerated centrifuge at 5,000 rpm (equivalent to 3,000 x g with a rotor radius of 8 cm) for 5 minutes. Subsequent purification involved three consecutive washing cycles with a gradient series of ethanol / deionized water mixtures: initially 50:50 (v / v), followed by 75:25 (v / v), and finally absolute ethanol (HPLC grade, > 99.9%, Aladdin). Each washing step involved 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 lyophilized in a freeze-dryer (condenser temperature: -50 °C, vacuum: < 10 Pa) for 24 hours. The final product was stored in light-protected containers at a temperature of 4 °C to minimize photodegradation.

[0025] Step 4, Preparation of Ropivacaine liposomes: Liposome formulation was prepared using a modified film hydration method. 7 mg of egg phosphatidylcholine (EPC, > 99% purity), 1 mg of cholesterol (CHOL, > 99% purity), 0.2 mg of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, > 99% purity), and 0.2 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[ammonium (polyethylene glycol)-2000 (DSPE-polyethylene glycol) were subsequently added to the lipid mixture with Ropivacaine (Rop, > 99% purity) free base (3 mg, equivalent to 0.1% w / v final concentration). The organic solvent was evaporated using a rotary evaporator at 50 °C under reduced pressure for 30 minutes to form a thin lipid film.

[0026] Step 5, 3 mL of ultrapure water (preheated to 50 °C) was added to the lipid film, followed by vortex mixing (1 minute) and probe sonication using an ultrasonic apparatus (30 seconds, 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 a continuous pore size using a thermally stable liposome extruder. The final Ropivacaine-loaded liposomes (Rop-Lipo) were stored at 4 °C for subsequent characterization.

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

[0028] The resulting PR hydrogel microspheres (double network hydrogel microspheres) were collected by centrifugation (3000 x g, 10 min, 4 °C) and washed twice with deionized water to remove unbound liposomes. The purified hydrogel microspheres (PR hydrogel microspheres) were freeze-dried using a lab-scale freeze-dryer under optimized conditions: primary drying at -50 °C condenser temperature for 24 h under vacuum pressure < 10 Pa, followed by 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 freeze-dried hydrogel microspheres were examined using a scanning electron microscope (SEM). Fourier-transform infrared spectroscopy (FT-IR) was used for structural and functional group analysis of the materials. The rheological properties of the hydrogel microspheres were analyzed with a rotational rheometer (TA).

[0031] 2. In vitro release profile of ropivacaine.

[0032] PNP hydrogel microspheres (100 mg) were loaded with ropivacaine hydrochloride, ropivacaine base, and PR (equivalent to 10 mg of ropivacaine) in dialysis bags (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 at 37 °C with shaking. At predetermined time intervals, 1 mL of release medium was collected and replaced with an equal volume of fresh PBS to maintain sink conditions. The cumulative release of ropivacaine (Rop) was quantified by ultraviolet-visible (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 the hydrogel microspheres was evaluated by lap shear test, 180° peel test and tensile test using pigskin samples according to ASTM standards F2255-05, F2256-05 and F2258-05, respectively. Throughout the experiments, fresh pigskin samples (25 x 25 mm, 1 mm thick) were kept under hydrated conditions. For lap shear analysis (ASTM F2255-05), a tensile loading was applied to the hydrogel microspheres coated samples using a universal testing machine (Tinius Olsen H10KS) with 1.0 ± 0.2 mm overlap width at a speed of 5 mm / min. The 180° peel resistance (ASTM F2256-05) was measured by controlling the separation of the adhesive tissue paper strips at a crosshead speed of 300 mm / min. The tensile adhesive performance (ASTM F2258-05) was determined by measuring the force-displacement curve under uniaxial tension at 10 mm / min.

[0035] The in vitro adhesion performance of PNP hydrogel microspheres was systematically evaluated on freshly excised porcine tissues (liver, kidney, spleen, lung and heart) under simulated physiological conditions. The tissue samples were pre-treated 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 blade perpendicular to the tissue surface following a standardized protocol. An appropriate amount of PNP hydrogel microspheres was adhered to each cross-section of the incision, which was then manually pressurized using sterile latex gloves for 120 s to ensure uniform interfacial contact. The adhesion integrity was evaluated by naked eye observation 20 min post-application under standardized lighting conditions.

[0036] 4. Biosafety testing.

[0037] 1 x 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 stained by adding calcein AM / PI (2 x 106m; 2 x 106m) and irradiated (37 °C, 30 min) for fluorescence microscopy. Cell Counting Kit-8 (CCK8) was used to detect cell viability. The viability of the control group was set to 100%, in which the cells were cultured without hydrogel microspheres. For hemolysis test, fresh anticoagulant rabbit blood (4 mL, dissolved anticoagulant rabbit blood, Macro Gen Biotech Co., Ltd.) was centrifuged at 2,000 rpm for 5 min to separate red blood cells. The supernatant was discarded, and the precipitated red blood cells (RBC) were washed three times with normal 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 a test tube (experimental group). The positive control used 0.1% (v / v) Triton X-100 instead of PBS, and the blank control used PBS instead of hydrogel microspheres. All test tubes were incubated in a shaking water bath (150 rpm). After incubation, the mixture was centrifuged for 5 min, and the absorbance A of the supernatant was measured at 545 nm using a UV-visible spectrophotometer. The hemolysis rate (%) was calculated as follows:

[0038]

[0039] A sample A is the absorbance of the sample negetive control A is the absorbance of the blank control positive control A is the absorbance of the positive control.

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

[0041] Before euthanasia, 2 mL of whole blood was collected from each rat using an EDTA-K2 anticoagulant tube by cardiac puncture for analysis of hematological parameters (white blood cell count WBC, red blood cell count RBC, hemoglobin HGB, platelet count PLT, etc.) and serum biochemical profile (alanine aminotransferase ALT, aspartate aminotransferase AST, urea nitrogen BUN, creatinine CRE, etc.). After euthanasia, the main organs (heart, lung, liver, spleen, and kidney) were harvested and subjected to H&E staining for histomorphological evaluation.

[0042] 5. Rat liver hemostasis experiment model.

[0043] For the rat liver incision model, male SD rats (6-8 weeks old, 180-200 g) were purchased and randomly divided into five groups (n=3 for each group). First, the rats were anticoagulated with heparin (3000 U / kg) by intraperitoneal injection 30 minutes before the operation. The animals were anesthetized with sodium pentobarbital and fixed on the operation board. The rat liver was exposed through an abdominal incision. The liver was partially resected at a distance of 1 cm from the liver margin. Porcine fibrin glue (Fibrin), cyanoacrylate (CA), PNP hydrogel microspheres, and PR hydrogel microspheres were applied when the liver was actively bleeding. The control group was not treated. The hemostatic time was recorded. If hemostasis was not observed within 10 minutes, the rats were euthanized to alleviate animal pain. In a separate experiment, blood was collected with filter paper within 3 minutes after the operation. Then, the filter paper was weighed, and the amount of blood loss was calculated.

[0044] 6. Animal study.

[0045] All animal experiments were approved by the Animal Protection Committee of Shanghai Jiao Tong University (A2024430-001). Adult male 250-300 g Sprague-Dawley (SD) rats were purchased from Vantianlihua Animal Technology Co., Ltd. and placed in a ventilated cage 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 were shaved on the back and disinfected with povidone-iodine. A 2 cm full-thickness skin incision was made on the midline of the back of each rat with a scalpel. PNP hydrogel microspheres and PR hydrogel microspheres were injected into the incision, and then clamped with fingers for 2 minutes. For comparison, porcine fibrin glue was applied to the incision, and then clamped with fingers for 2 minutes. According to the manufacturer's protocol, cyanoacrylate glue (CA n-butyl-2-cyanoacrylate) was applied to the wound surface to form a polymer film. For comparison, traditional sutures were also used. The rats were euthanized 7 and 14 days after the operation. The 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 with an inverted fluorescence microscope or a confocal laser scanning microscope (CLSM). The wound area and collagen deposition level were analyzed based on H&E and Masson trichrome staining images, respectively, using ImageJ software. The immunofluorescence intensity of IL-6 and TNF-a (inflammatory factors) was quantified using ImageJ software.

[0048] The local analgesic effect of PR hydrogel microspheres was also evaluated using the rat dorsal incision pinprick test. Mechanical hyperalgesia was assessed using 26G von Frey filaments (Ugo Basile) attached to 18G needles after dorsal incision surgery. Each rat received six consecutive stimulations (1-2 seconds each, 10 minutes apart) applied perpendicularly to the surgical site. Cutaneous tail muscle reflex (CTMR), defined as a rapid abdominal muscle contraction or aversive movement upon stimulation, was recorded and analyzed for the number of CTMRs for each group of rats.

[0049] 8. Rat plantar incision model.

[0050] The analgesic effect of PR hydrogel microspheres was evaluated using the plantar incision model. Male Sprague-Dawley rats (body weight 200-250 g) were randomly divided into different groups (n = 6 for each group). Under isoflurane anesthesia (induction: 3% in 100% 02; maintenance: 1.5-2%), the right hind paw was disinfected with iodine. A 1-cm longitudinal incision was made through the skin and underlying fascia, starting 0.5 cm proximal to the heel and extending toward the toes. The plantar muscle was exposed using eye forceps, and a longitudinal myotomy was performed, while the muscle origin and insertion were preserved. Hemostasis was achieved by gentle compression before treatment application. After hemostasis, the incision was treated with different materials, and the rats were divided into 6 groups: blank group, no treatment; incision group, plantar incision, no further treatment; suture group, after incision, the wound was sutured with 5-0 nylon suture; PNP group, after incorporation, PNP hydrogel microspheres (50 mg, particle size ~ 200 μm) were applied to the wound; PNP+Rop group, after incision, only Rop (0.2% w / w) powder mixed with PNP hydrogel microspheres was used to adhere the wound; PR group, after incision, PR double network hydrogel microsphere powder was applied to seal the wound.

[0051] 9. Animal behavior analysis of rat plantar incision model.

[0052] Behavioral analysis was performed on rats after plantar incision modeling using 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 acclimated in a transparent acrylic chamber (20 x 20 x 40 cm) with a wire mesh floor (0.2 x 0.2 cm aperture) for 15-30 minutes. When the animals showed no exploratory / defecation behavior, the test began. The calibrated probe of the electric von Frey analgesia instrument (Model 2393 series, Shanghai Yuyan Instrument Co., Ltd.) was applied vertically to the plantar surface near the incision of the rat paw. Each probe was stimulated 3 times with more than 1 minute interval, gradually increasing to the body weight of the rat. Two or more times of obvious pain-related behaviors such as paw withdrawal were observed as valid pain response. The previously recorded gram value was designated as the mechanical pain threshold. The baseline measurement was obtained from the blank group, and the mechanical pain threshold and the corresponding change of different groups of rats were recorded within the corresponding time period.

[0053] In the Hargreaves test, the thermal sensitivity of the rat hind paw was evaluated by the Planter test (37570, UGO Basile Ltd.). Two days before the test and on each test day, rats were acclimated to the test environment and equipment for 30 minutes. Each rat was placed in a single glass platform in an organic glass chamber, and the temperature was maintained at 30°C. The thermal sensitivity of the hind paw was evaluated by directing the 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, and was automatically cut off at 20 seconds to prevent tissue damage. If no withdrawal occurred within 20 seconds, a PWL of 20 seconds was recorded. The average PWL of 3 readings with an interval of ≥5 minutes for each paw was calculated for analysis.

[0054] In the open field test, the spontaneous motor activity of rats was evaluated. After surgery and drug administration, rats were placed in the center of a 120 cm x 120 cm x 60 cm open arena 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 a smart video computerized tracking system (XR-XZ301, Shanghai Xinsuo Information Technology Co., Ltd.).

[0055] 10. Analysis of signal pathways of pain transmission from lower limbs to 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 2In the middle a). To quantitatively evaluate the viscoelasticity of photocured PNP hydrogel microspheres, dynamic oscillatory rheometry was performed. The results showed a predominantly 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 the middle a), indicating the formation of a robust elastic network structure.

[0062] PNP hydrogel microspheres were prepared by photo-crosslinking method combined with emulsion technique using PNP hydrogel microsphere precursors as the aqueous phase. The aqueous phase included a hydrogel microsphere precursor solution containing lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP) as a photoinitiator, while the oil phase consisted of liquid paraffin stabilized with surfactants Span 80 and Tween 20. It is worth noting that the dynamic nature of the emulsion system requires prolonged irradiation time compared to the static hydrogel microsphere solidification. To optimize the preparation method of hydrogel microspheres, different UV exposure times were performed. The results showed that insufficient UV irradiation (< 10 minutes) led to incomplete polymerization, resulting in structural collapse and flocculated morphology after lyophilization. On the contrary, excessive irradiation (> 60 minutes) would lead to over-crosslinking, aggregation of hydrogel microspheres, and precipitation due to interface instability Figure 18 To optimize the uniformity and dispersibility of hydrogel microspheres, the protocol included: (1) initial high-speed emulsification (1000 rpm for 60 minutes) to obtain monodispersed droplets; (2) reducing agitation (500 rpm) during the 30-minute UV curing process to minimize shear-induced coalescence; (3) post-curing low-speed mixing (500 rpm for 5 minutes) to ensure residual monomer consumption and structural stability. This method produced hydrogel microspheres with a narrow particle size distribution, spherical integrity, and excellent dispersion stability, which was confirmed by the morphology analysis after lyophilization Figure 2 In the middle b).

[0063] SEM images showed that PNP hydrogel microspheres exhibited a well-defined porous surface structure Figure 2 In the middle d, left), which enabled them to have high absorbent capacity, PNP hydrogel microspheres showed excellent swelling properties with a swelling ratio of 945 ± 5% Figure 19 In this case, Rop-Lipo with an average hydrodynamic diameter of 167.4 ± 2.1 nm (PDI = 0.18) Figure 20 could be easily loaded in PNP hydrogel microspheres by absorption. At the same time, the incorporation of ropivacaine-loaded liposomes (Rop-Lipo) had no observable effect on the formation of these morphological features Figure 2 In the middle d, right).

[0064] Rop reacted with the amino group on Rop was labeled with 7-amino-4-methyl-3-coumarin acetic acid (AMCA) which emits blue fluorescence, and the successful loading of Rop can be confirmed by the fluorescence image of the hydrogel microspheres Figure 2 c). In addition, FTIR spectroscopic analysis showed characteristic absorption bands of drug-loaded hydrogel microspheres (PR hydrogel microspheres) that were not present in the blank carrier Figure 3 b): 1538 cm⁻ 1 (N-H band) and 2922 cm⁻ 1 (C-H alkane stretch). The complete spectral match between the loaded hydrogel microspheres and pure ropivacaine confirmed successful encapsulation without chemical degradation. Notably, the sustained release profile of ropivacaine from PR hydrogel microspheres was significantly improved by employing a two-stage delivery strategy with lipid vesicles and hydrogel microsphere networks. 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 ropivacaine free base (~24 hours) Figure 3 c).

[0065] Particle size distribution analysis demonstrated comparable size characteristics between the two formulations. The average diameter of PNP and PR hydrogel microspheres was approximately 191.9 ± 8.7 microns Figure 3 d), with over 80% of the hydrogel microsphere particles distributed in the range of 148-228 μm, which was reported to have better wicking rates than other particle sizes. Furthermore, with appropriate diameters and uniform dispersity, our PNP and PR hydrogel microspheres could form fine dust mists when sprayed through an atomizer bottle Figure 4 a).

[0066] Hydration property evaluation showed minimal differences between the formulations, with the swelling capacity of PNP hydrogel microspheres being 919.76 ± 20.58%, that of PR hydrogel microspheres being 912.95 ± 37.59%, and the water retention rates being 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 in vivo bleeding point sealing ability.

[0068] The adhesion properties of PNP hydrogel microspheres loaded with ropivacaine (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), ASTM F2258-05 (tensile strength). The adhesion properties of PR incorporating liposome-encapsulated ropivacaine at concentrations of 1%, 2.5%, 5%, and 10% were compared to the widely used clinical adhesive fibrin sealant, and the results showed a concentration-dependent decrease in adhesion strength Figure 21 ). PR-MS containing 10% ropivacaine showed no statistically significant difference in adhesion strength compared to porcine fibrin sealant (p > 0.05). In contrast, hydrogel microspheres containing 1% ropivacaine had the highest mechanical strength compared to porcine fibrin, showing significantly stronger adhesion (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 adhesion properties while delivering the therapeutic agent, was selected for further experimental characterization and animal studies.

[0069] Both hydrogel microspheres exhibited significantly stronger tissue adhesion than the commercial tissue adhesive, porcine fibrin sealant (p < 0.0001). In lap shear tests Figure 5 a), PNP and PR achieved maximum adhesion strengths of 134.46 kPa and 129.96 kPa, respectively, significantly higher than the 30.06 kPa of porcine fibrin. Similarly, T-peel tests Figure 5 b) showed adhesion energies of 125.34 J / m for PNP, 111.04 J / m for PR, and 39.52 J / m for porcine fibrin. Tensile tests Figure 5 c) further confirmed the superiority of PNP (213.79 kPa) and PR (207.36 kPa) over 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 two porcine skin samples successfully lifted a 300g weight Figure 6 a). These findings were further confirmed in ex vivo organ adhesion tests. After creating standardized incisions 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 internal organ defects and effective adhesion between muscle and skin tissues were observed Figure 6 b).

[0070] To validate the exceptional tissue adhesion and rapid occlusion capabilities of PNP hydrogel microspheres, in vivo hemostatic efficacy evaluations were performed using a rat liver defect model Figure 6 c and d). The results showed that PNP hydrogel microspheres were able to completely stop bleeding within 5 minutes of application, demonstrating superior hemostatic efficacy compared to porcine fibrin sealant (p < 0.05).Figure 7 In vivo a, b). Standardized full-thickness wounds were created on the exposed liver surface of live Sprague-Dawley (SD) rats, followed by application of Gauze, cyanoacrylate, porcine fibrin glue, PNP hydrogel microspheres, and PR hydrogel microspheres for wound closure. Hemostatic time and blood loss were quantitatively recorded. The results showed that both PNP and PR groups achieved hemostasis within 20 seconds with significantly less blood loss than 50 mg, outperforming the gauze control group (p < 0.01) and commercial porcine fibrin glue (p < 0.05). Although cyanoacrylate showed similar hemostatic time and blood loss as PNP, its exothermic polymerization upon hydration increased the local temperature to over 80 °C, causing thermal injury to the liver tissue. Moreover, the brittle film formed by cyanoacrylate ruptured under respiratory motion and hemodynamic pressure, posing a risk of secondary bleeding or embolism. The physical barrier further hinders hepatocyte migration and regenerative microenvironment. These findings indicate that PNP and PR hydrogel microspheres rapidly absorb interstitial fluid upon contact with a bleeding wound, establishing a stable adherent physical barrier to accelerate coagulation. The superior closure performance in vivo highlights their potential as an emergency material for controlling traumatic wound.

[0071] (3) Biocompatibility evaluation of PRs.

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

[0073] To evaluate the cell compatibility of the hydrogel microspheres, we assessed cell viability by co-incubating the extracts of various hydrogel microspheres with NIH-3T3 fibroblasts and human umbilical vein endothelial cells (HUVECs). After 24 hours, cell survival was determined using a cell counting kit-8 (CCK-8) assay. As shown in Figure 9 As shown in Figs. 8a and 8b, there was no significant difference in the viability of NIH-3T3 cells and HUVECs in the PNP group compared to the control group, indicating that the cytotoxicity can be negligible. A slightly lower cell survival was observed in the Rop group, which can be attributed to the cytotoxic effect of high concentrations of ropivacaine. Notably, the cell viability in the PR group was significantly higher than that in the group with ropivacaine only (p = 0.0003), which can be due to the more complex cross-linked structure of the double-network hydrogel microspheres (PR) and the encapsulation in liposomes that enhance the overall stability, collectively slowing down the release of ropivacaine and thus reducing cytotoxicity. The live / dead cell staining results Figure 9 Figs. 8c and 8d confirm the CCK-8 results, showing that most cells in the PR group appear healthy with intact morphology and characteristic spindle shape Figure 8), while only minimal dead cells were observed. Considering the direct contact of the adhesive powder with blood, we further evaluated the hemocompatibility of the hydrogel microspheres. After incubation with rabbit blood, their effects on red blood cells were evaluated by assessing hemolysis (rupture of red blood cells) or aggregation. Triton X-100 and phosphate buffered saline (PBS) were used as positive and negative controls, respectively, and the absorbance at 545 nm was measured by UV-visible spectrophotometry after 1 h of incubation. As shown in Fig. 5a, both PNP and PR hydrogel microspheres did not induce significant hemolysis compared to the blank control, with a hemolysis rate of less than 3% for all groups, indicating excellent hemocompatibility. Figure 9

[0074] For in vivo experiments, we closely monitored the mental state and body weight changes of rats within 7 days after physical intervention, and found that the mental state of rats in each group was good and the body weight increased steadily. Comprehensive histological examination of H&E staining of heart, liver, spleen, lung and kidney confirmed the absence of acute toxicological effects Figure 22 a). The myocardial fibers were arranged in a cylindrical branching network with the nucleus in the center and the interstitial body rich in capillaries. The liver lobule structure was intact, and the hepatocytes were arranged in a radial pattern with round and uniform nuclei and perinuclear light staining. The spleen had clear red and white pulp, and the white pulp lymphocytes were deeply stained. The red pulp was rich in red blood cells and red in color, with splenic cord and splenic sinus structure. The vesicle structure was intact, the alveolar wall was single-layer flat epithelium, the interstitial group was uniform, and there was no congestion. The glomerular structure was clear, and the renal tubular epithelial cells were arranged in order without interstitial fibrosis. All observed structures and cell morphology were consistent with normal, healthy organ structure and histology. The serum indexes of heart (CK-MB, LDH), liver (AST, ALT) and kidney (Cr, BUN) of each group were 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 were evaluated as wound spray bandages in SD rats with dorsal incisions, which were randomly divided into six groups (n=6): untreated control (control), cyanoacrylate (CA), porcine fibrin adhesive (Fibrin), suture (Stitch), PNP and PR. Macroscopic evaluation revealed different healing patterns Figure 10 a), and quantitative analysis confirmed that the wound closure of PNP and PR groups was significantly accelerated compared with the control group (p<0.0001 Figure 10 b); by day 14, complete epithelialization was achieved in the PNP and PR groups, while persistent defects still existed in the control group. Histological evaluation by H&E staining Figure 11 ​In summary, a) at day 7, extensive areas of non-epithelialization and inflammation were observed in the control group, while PNP, PR and Stitch groups showed reduced epithelial defects, although Stitch showed exacerbated inflammation due to foreign body reaction induced by suturing. By day 14, while CA and porcine fibrin achieved epithelization, partial epidermal-dermal separation indicated a fragile newly formed epithelium, while PNP and PR groups showed robust epidermal-dermal integration with preserved skin appendages. Trichrome Masson staining further showed that PNP and PR groups had denser collagen deposition at day 7 Figure 10 In summary, c and Figure 11 b) were more organized, indicating enhanced extracellular matrix remodeling.

[0077] Furthermore, the inflammatory profile quantified by TNF-a and IL-6 showed that at day 7, cytokine levels were significantly lower in PNP and PR groups than in other groups (p<0.01 Figure 12 and Figure 11 In summary, c), by day 14, IL-6 continued to decrease, indicating not only excellent biocompatibility but also a positive regulatory effect on the inflammatory response. Notably, preliminary assessment of analgesic effect by cutaneous tail-flick muscle reflex (CTMR) test showed that PNP hydrogel microspheres-treated incisions had transient inhibition of pain, with mechanical hyperalgesia inhibition lasting approximately 4 days after application before returning to baseline levels at day 5 Figure 23 ), supporting its multifunctional therapeutic properties.

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

[0079] To further investigate the analgesic mechanism of the composite material, we established a rat plantar incision model. Von Frey filament (mechanical withdrawal threshold of MWT), hot plate test (paw withdrawal latency, PWL), and open field test (OFT) were used to assess the level of pain and motor activity. Macroscopic evaluation Figure 13 a) and H&E staining Figure 24 revealed comparable wound closure processes in the hydrogel microspheres group and the suture group, both of which were significantly faster than the control group. PNP and suture groups showed similar pain and motor curves Figure 13 b and Figure 14In the first 72 hours after incision, a decrease in tolerance to PWL and MWT and a decrease in OFT activity were observed due to the painful stimulus of the wound. As the wound healed, the rats gradually adapted to the environment. However, the incision groups always maintained lower levels of PWL, MWT and OFT compared to the blank group. The PR and PNP+Rop (ropivacaine) groups showed MWT values and motor levels comparable to the blank control group in the first 12 hours due to the analgesic effect, even exceeding the tolerance of the blank rats in the hot plate test. After 24 hours, when the 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 a curve similar to the other control groups, reflecting the effect of hind paw pain on sensorimotor function after stopping analgesia. Conversely, the PR group maintained high pain tolerance in the first 96 hours due to the sustained release of the drug from the hydrogel microspheres and liposomes, gradually approaching the blank group as healing progressed. Throughout the 168-hour observation period, rats maintained high levels of motor activity, with OFT (open field test) values comparable to the control groups. This indicates an increase in exploratory drive and a decrease in pain interference in the experimental cohort. These results indicate that encapsulation in hydrogel microspheres and liposomes enables prolonged, local release of ropivacaine, providing sustained analgesia and reducing post-incision pain in the rat hind paw.

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

[0081] To elucidate the analgesic mechanism of PR, L4 / L5 dorsal root ganglion (DRG) and spinal cord dorsal horn samples were collected 7 days after intervention to assess c-Fos expression in sensory neurons. Cells co-expressing c-Fos and TRPV1 indicate activation of sensory neurons. As shown in Figure 6a, on day 7, the Stitch, PNP and PNP+Rop groups showed similar high sensory neuron activation according to the highest expression of c-Fos. Conversely, the PR group exhibited significantly lower c-Fos expression compared to the control groups, only slightly higher than the blank group, with no statistical significance (p=0.7876 Figure 15 Figure 6a). Conversely, the proportion of double-positive cells (c-Fos+TRPV1+ / TRPV1+) was significantly higher in the PNP group compared to the Stitch and PR groups (p<0.01 Figure 16 Figure 6a). Conversely, the proportion of double-positive cells (c-Fos+TRPV1+ / TRPV1+) was significantly higher in the PNP group compared to the Stitch and PR groups (p<0.01 Figure 16 Figure 6a). Conversely, the proportion of double-positive cells (c-Fos+TRPV1+ / TRPV1+) was significantly higher in the PNP group compared to the Stitch and PR groups (p<0.01

[0082] ATF3, a core stress transcription factor in DRG, has a dual function of promoting regeneration and modulating pain, and is normally expressed at low levels in normal rat DRG. Its expression is significantly increased in response to noxious stimuli. In Figure 15 b and Figure 16 In c, all experimental groups showed a significant increase in ATF3 expression compared to the blank control group (sham control group). 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 - suggesting that the PR intervention has superior long-lasting analgesic effects.

[0083] Glial cells in the dorsal horn of the spinal cord play an important role in neural development and maintenance by providing protection, support, nutrition, and removal of harmful substances. In addition, neuronal signaling within the glial zone of the dorsal horn of the spinal cord facilitates information transmission in the nervous system, especially noxious 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, which play a crucial role in neural development and maintenance by providing protection, support, nutrition, and removal of harmful substances. As shown in Figure 15 In c, both the control and PNP groups showed a significant increase in the expression levels of GFAP and Iba-1 compared to the blank group (p<0.0001). 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) expression. However, the PR group showed a significant decrease in the expression of GFAP (p=0.0138) and Iba-1 (p=0.0363) compared to the PNP+Rop group. Consistent with previous studies, our findings suggest that PR reduces incisional pain by continuously suppressing c-Fos expression in DRG and inhibiting glial cell activation in the dorsal horn of the spinal cord.

[0084] The present application develops a dual-network hydrogel microsphere adhesive system consisting of polyvinyl alcohol (PVA), polyethylene glycol diacrylate (PEGDA) and acrylic acid (AA), functionalized with acrylic acid N-hydroxysuccinimide ester (AAC-NHS) to achieve secure adhesion to wet tissues. The introduction of ropivacaine liposomes (Rop-Lipo) enables sustained local analgesia, addressing the dual challenge of wound closure and postoperative pain management. The hydrogel microspheres exhibit superior mechanical properties, rapid swelling, high liquid uptake and excellent tissue adhesion, outperforming clinical standards such as fibrin glue and cyanoacrylate in lap shear, T-peel and tensile tests. In vivo evaluation demonstrates 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. The sustained release of ropivacaine from the hydrogel microspheres significantly prolongs analgesia time (≥168 hours), reduces nociceptive neuronal activation, and suppresses glial cell responses in the spinal cord dorsal horn. Biocompatibility evaluation confirms minimal cytotoxicity and systemic safety. This multifunctional hydrogel microsphere represents a promising advancement in enhanced recovery after surgery (ERAS), combining reliable wound adhesion with prolonged local pain relief to improve postoperative outcomes.

[0085] Finally, it should be noted that the above examples are only intended to illustrate the technical solutions of the present application and not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for preparing a sprayable dual-network hydrogel microsphere adhesive, characterized in that, Includes 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.

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 3, the volume ratio of ethanol to deionized water in the gradient series ethanol / deionized water mixture is as follows: 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.

5. 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.

6. 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.

7. 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.

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

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

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