Antibacterial hemp nanocellulose self-gelling hemostatic powder and preparation method thereof

Abstract

The present application relates to the technical field of medicine, in particular to a kind of antibacterial hemp nanocellulose self-gelling hemostatic powder and preparation method.The specific technical scheme is as follows: a kind of preparation method of antibacterial hemp nanocellulose self-gelling hemostatic powder, polyethyleneimine solution is mixed with hemp nanocellulose suspension, then mixed with polyacrylic acid solution, the mixed solution is quickly placed in liquid nitrogen and frozen, then vacuum freeze-dried, grinded into powder to obtain antibacterial hemp nanocellulose self-gelling hemostatic powder.The hemostatic powder not only has antibacterial and analgesic functions, but also has rapid self-gelation ability, can adapt to irregular wound surface and quickly achieve hemostasis.The overall preparation process is simple, low in cost, suitable for various medical scenarios, convenient to use, and has good popularization and application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an antibacterial hemp nanocellulose self-gelling hemostatic powder and its preparation method. Background Technology

[0002] Self-gelling hemostatic powders are materials that can rapidly absorb water and form a gel in situ at the wound site upon contact with bodily fluids (such as blood and tissue fluid). They are widely used in hemostasis, wound dressings, flexible wearable devices, and drug delivery systems. For example, there are reports of preparing self-gelling hemostatic powders using natural or synthetic polymers such as gelatin, carboxymethyl chitosan, and sodium alginate. These materials typically form hydrogel structures based on physical cross-linking or chemical reactions. However, in practical applications to achieve rapid and stable hemostasis, traditional hemostatic powders still face many problems, such as the inability to form stable scabs, easy detachment, and the potential risk of thrombosis; some materials form gels slowly after contact with liquids, failing to meet the "golden minute" hemostatic window, which refers to the first few minutes after trauma as the high-risk period for hemorrhagic death, and the first hour after trauma is known as the "golden hour," during which timely and effective treatment can significantly improve survival rates; the gel has weak strength and poor adhesion to the wound surface, making it easy to detach due to tissue movement; traditional gel materials themselves do not have antibacterial properties, easily leading to wound infection; some polymer cross-linking agents may have potential toxicity; and they are difficult to apply effectively to deep or irregular wounds.

[0003] To address the aforementioned issues, this invention provides an antibacterial hemp nanocellulose self-gelling hemostatic powder and its preparation method. The prepared self-gelling hemostatic powder, with its excellent tissue adhesion, can firmly adhere to the wound surface, effectively reducing the risk of detachment and improving hemostasis efficiency. Simultaneously, the self-gelling hemostatic powder rapidly absorbs blood and tissue fluid at the wound site, forming a hydrogel in situ at the wound, which not only quickly stops bleeding but also reduces the risk of infection and provides a protective barrier for the wound. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an antibacterial hemp nanocellulose self-gelling hemostatic powder and its preparation method. This invention uses nanocellulose as a framework, combining it with polyethyleneimine and polyacrylic acid to achieve rapid gelation and construct a mechanically stable gel network. The material possesses excellent natural antibacterial capabilities due to the natural cannabidiol (CBD) component contained within hemp fibers. Thanks to the ultra-high specific surface area and flowable powder morphology of nanocellulose, it can adapt to various complex and irregular wounds. All raw materials are derived from natural systems, exhibiting both excellent biocompatibility and low toxicity.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention discloses a method for preparing antibacterial hemp nanocellulose self-gelling hemostatic powder. The method involves mixing a polyethyleneimine solution with a hemp nanocellulose suspension, then mixing the mixture with a polyacrylic acid solution. The resulting solution is then rapidly placed in liquid nitrogen for freezing, followed by vacuum freeze-drying and grinding into powder to obtain the antibacterial hemp nanocellulose self-gelling hemostatic powder.

[0006] Preferably, the mass ratio of polyethyleneimine to polyacrylic acid is 2-8:8-2, and the amount of hemp nanocellulose added is 0.1wt% to 0.5wt% of the mass of the polyethyleneimine solution.

[0007] Preferably, the liquid nitrogen freezing time is 5 to 10 minutes, the vacuum freeze-drying pressure is 8 to 10 Pa, and the temperature is -60 to -45°C.

[0008] Preferably, the molecular weight of the polyethyleneimine is 70,000 and the molecular weight of the polyacrylic acid is 240,000.

[0009] Preferably, the concentrations of the polyethyleneimine solution and the polyacrylic acid solution are both 10 wt%.

[0010] Preferably, the preparation process of the hemp nanocellulose suspension is as follows: hemp nanocellulose is dispersed in deionized water, sheared at 12000 r / min for 5-10 min, and then subjected to 5 high-pressure homogenization cycles at 60 MPa; subsequently, it is centrifuged at 8500 r / min for 10 min to obtain the hemp nanocellulose suspension.

[0011] Preferably, the concentration of the hemp nanocellulose in deionized water is 10. -2 g / mL.

[0012] Correspondingly, an antibacterial hemp nanocellulose self-gelling hemostatic powder prepared by the preparation method described above.

[0013] The present invention has the following beneficial effects: 1. Polyethyleneimine and polyacrylic acid interact physically (e.g., through hydrogen bonding and electrostatic interactions). The addition of hemp nanocellulose not only stabilizes the mechanical properties of the gelled material but also provides some antibacterial activity. Compared to traditional hydrogels, hemp-modified nanocellulose self-gels exhibit lower toxicity, making them an ideal novel hemostatic material.

[0014] 2. The preparation process of this invention is simple and the operation process is clear, making it suitable for large-scale production; secondly, the raw materials are readily available and the overall cost is low, which is conducive to its promotion and application; in addition, the obtained product has the functions of rapid hemostasis, good biocompatibility and antibacterial function, and has broad clinical application prospects. Attached Figure Description

[0015] Figure 1 The infrared spectrum of the PEI / PAA / CNF prepared in Example 1; Figure 2 Scanning electron microscopy cross-sectional morphology of PEI / PAA composite gels with different hemp nanocellulose addition amounts; Figure 3 A photograph of the PEI / PAA / CNF composite gel undergoing an inverted adhesion and load-bearing test to demonstrate its inverted adhesion and load-bearing capacity. Figure 4 The effect of hemp nanocellulose content on gel swelling properties; Figure 5 Stress-strain curves of PEI / PAA composite gels with different amounts of hemp nanocellulose added; Figure 6 The images show the antibacterial effects of PEI / PAA and PEI / PAA / large CNF composite gel on Staphylococcus aureus and Escherichia coli in plate coating. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0018] This invention provides a method for preparing antibacterial hemp nanocellulose self-gelling hemostatic powder. The method involves mixing a polyethyleneimine solution with a hemp nanocellulose suspension, then mixing the mixture with a polyacrylic acid solution. The resulting solution is rapidly frozen in liquid nitrogen for 5–10 minutes, followed by vacuum freeze-drying at a pressure of 8–10 Pa, a temperature of -60 to -45°C, and a time of 24–48 hours. After freeze-drying, the powder is ground to obtain the antibacterial hemp nanocellulose self-gelling hemostatic powder. The mass ratio of polyethyleneimine (PEI) to polyacrylic acid (PAA) is 2–8:8–2, and the amount of hemp nanocellulose added is 0.1 wt%–0.5 wt% of the mass of the polyethyleneimine solution. The molecular weight of the polyethyleneimine is 70,000 (Mw 70,000), and the molecular weight of the polyacrylic acid is 240,000 (Mw 240,000). The concentrations of the polyethyleneimine solution and the polyacrylic acid solution are both 10 wt%.

[0019] Furthermore, a hemp nanocellulose suspension was prepared by treating hemp bast fibers with inorganic acid hydrolysis and oxidative modification. The specific preparation process is as follows: (1) The hemp phloem was cut into small, uniform segments, repeatedly rinsed and rubbed with tap water, and then dried at room temperature to constant weight. The segments were then pulverized using a plant pulverizer and sieved through a 60-mesh sieve. The resulting sample was placed in deionized water and boiled for 2 hours to remove water-soluble impurities from the phloem; Soxhlet extraction was performed using benzene-ethanol (V... 苯 :V 乙醇 The 2:1 mixture solution was refluxed at 90℃ for 3 hours to remove wax, fat and other substances from the raw material. The resulting solid was transferred to a 5% NaOH solution and reacted at 90℃ for 3 hours to remove hemicellulose. To further remove lignin, the treated raw material was immersed in a 0.15mol / L NaClO2 solution, the pH was adjusted to 3-4 using glacial acetic acid, and the mixture was magnetically stirred in a 75℃ constant temperature water bath for 4 hours. After washing until neutral and drying, the purified hemp cellulose was obtained.

[0020] (2) 6.00±0.05 g of hemp cellulose was dispersed in 600 mL of deionized water. After soaking and stirring until the fibers swelled, 0.025 mmol / g of 2,2,6,6-tetramethylpiperidine-1-oxygen radical (TEMPO) and 0.5 mmol / g of sodium bromide were added to the resulting cellulose suspension, followed by the addition of 10 mmol / g of NaClO to initiate TEMPO-mediated oxidation at room temperature. The pH of the mixture was adjusted to between 10.2 and 11.5 by adding 0.5 mol / L NaOH dropwise, and the mixture was stirred continuously for 3 h at a temperature of 25 °C. After the oxidation reaction was completed, excess ethanol was added immediately to terminate the reaction. The mixture was then thoroughly washed with deionized water and dialyzed using a dialysis bag with a molecular weight cutoff of 14000 Da. The resulting sample was stored at 4 °C for analysis.

[0021] (3) The sample obtained in step (2) was placed in a vacuum freeze dryer and freeze-dried under conditions of 8-10 Pa pressure and -90-80℃ temperature to obtain solid oxidized hemp cellulose. The obtained solid oxidized hemp cellulose was added to deionized water in a certain proportion to prepare a solution with a concentration of 10 -2A cellulose dispersion of g / mL was prepared; then, an IKA T25 homogenizer was used at 12000 rpm for 5–10 min to fully disperse and mechanically disintegrate the solid oxidized hemp cellulose; subsequently, an HPH-L2 high-pressure homogenizer was used for 5 complete homogenization cycles at 60 MPa, with each cycle consisting of the entire dispersion passing through the homogenization system once. The unfibrillated or partially fibrillated precipitate was then separated by centrifugation at 8500 rpm for 10 min. The final hemp nanocellulose suspension was transferred to a sealed container and stored at 4°C, with a concentration of 1%.

[0022] This invention provides an antibacterial hemp nanocellulose self-gelling hemostatic powder prepared by the aforementioned preparation method.

[0023] The present invention will be further described below with reference to specific embodiments.

[0024] Unless otherwise specified, all "%" in the following examples refer to mass percentages.

[0025] Example 1 Take 1g of 50% polyethyleneimine (PEI), add 3g of 1% hemp nanocellulose suspension and 1g of deionized water, and mix thoroughly on a magnetic stirring table. Separately, take 2g of 25% polyacrylic acid (PAA), add 3g of deionized water, and mix thoroughly on a magnetic stirring table. Then, rapidly mix the two solutions and immerse them in liquid nitrogen for 10 minutes, then place them in a vacuum freeze dryer and freeze-dry at 10Pa and -50℃ for 48 hours. Finally, remove the dried freeze-dried material and grind it into a uniform powder using a ball mill to obtain the target product—antibacterial hemp nanocellulose self-gelling hemostatic powder, namely PEI / PAA / hemp nanocellulose composite gel (i.e., PEI / PAA / CNF, abbreviated as PPC), in which the amount of hemp nanocellulose added is 0.3wt%.

[0026] Comparative Example 1 Take 1g of 50% polyethyleneimine, add 1g of 1% hemp nanocellulose suspension and 3g of deionized water, and mix thoroughly on a magnetic stirring table. Separately, take 2g of 25% polyacrylic acid, add 3g of deionized water, and mix thoroughly on a magnetic stirring table. Then, rapidly mix the two solutions and immerse them in liquid nitrogen for 10 minutes, followed by freeze-drying in a vacuum freeze dryer for 48 hours. After drying, remove the sample, grind it into powder using a ball mill, and obtain the final composite material. The amount of hemp nanocellulose added is 0.1wt%.

[0027] Comparative Example 2 1 g of 50% polyethyleneimine was added to 4 g of 1% hemp nanocellulose suspension and mixed thoroughly on a magnetic stirring table. Separately, 2 g of 25% polyacrylic acid was added to 1 g of 1% hemp nanocellulose suspension and 2 g of deionized water and mixed thoroughly on a magnetic stirring table. The two solutions were rapidly mixed and immersed in liquid nitrogen for 10 min, then freeze-dried in a vacuum freeze dryer for 48 h. After drying, the sample was removed and ground into powder using a ball mill to obtain the final composite material. The amount of hemp nanocellulose added was 0.5 wt%.

[0028] The test methods and results are as follows: Figure 1 The infrared spectrum of the PEI / PAA / CNF prepared in Example 1; 1. Testing instruments and conditions Fourier transform infrared spectroscopy was used for testing. Approximately 1–2 mg of PEI / PAA gel powder sample, dried to constant weight, was mixed with dried potassium bromide (KBr) powder at a mass ratio of approximately 1:100. The mixture was then thoroughly ground in an agate mortar under infrared lamp irradiation until the particle size of the mixed powder was less than 2 μm. The uniformly ground powder was placed in a tableting mold, and a hydraulic press was used to apply pressure at 8000–15000 kg / cm². 2 Apply pressure and maintain it for at least 1 minute to compress a transparent sheet approximately 13 mm in diameter and 0.5 mm thick. Use a pure KBr pellet as the background blank and set the scanning range to 4000–400 cm⁻¹. -1 The number of scans was 32, and the resolution was 4 cm. -1 The entire sample preparation and testing process was conducted in a dry environment to avoid interference from moisture in the air on the test results.

[0029] Preparation of PEI / PAA gel: 1 g of 50% polyethyleneimine (PEI) was added to 4 g of deionized water and mixed thoroughly on a magnetic stirring table. Separately, 2 g of 25% polyacrylic acid (PAA) was added to 3 g of deionized water and mixed thoroughly on a magnetic stirring table. The two solutions were then rapidly mixed and immersed in liquid nitrogen for 10 min, followed by lyophilization at -50°C and 10 Pa for 48 h. Finally, the lyophilized material was removed and ground into a uniform powder using a ball mill to obtain the product, namely PEI / PAA gel powder.

[0030] 2. Results Analysis Comparison of the infrared spectra of PEI, PAA, and PEI / PAA gel reveals that the PEI / PAA gel retains the characteristic absorption peaks of both PEI and PAA, without exhibiting characteristic absorption peaks corresponding to new covalent bonds such as amide bonds, indicating that no significant covalent chemical reaction occurs between PEI and PAA. However, compared to pure PEI and pure PAA, some characteristic absorption peaks in the PEI / PAA gel show significant shifts. This is mainly manifested in the characteristic absorption peaks of amino groups (-NH2 / -NH-) in PEI, and the C=O stretching vibration peak and OH stretching vibration peak of carboxyl groups (-COOH) in PAA, both showing varying degrees of shift. These changes are primarily attributed to the strong electrostatic interaction or ion association (-COO-) between the amino groups on the PEI molecular chain and the carboxyl groups on the PAA molecular chain. - ··· + H3N-), which in turn alters the electron cloud density and chemical microenvironment surrounding the relevant functional groups.

[0031] Further comparison of the infrared spectra of PEI / PAA gel and PEI / PAA / CNF composite gel (PPC) revealed that no new characteristic absorption peaks appeared in the composite gel, indicating that the introduction of hemp nanocellulose did not undergo a significant covalent chemical reaction with PEI or PAA. However, compared with PEI / PAA gel, the composite gel exhibited significant differences in some characteristic wavelength bands after the addition of hemp nanocellulose. Specifically, at approximately 3400 cm⁻¹... -1 The absorption peaks attributable to the stretching vibration of the hydroxyl group (-OH) broadened significantly and shifted. At the same time, the absorption peaks attributable to the stretching vibrations of the amino group (NH) and the carboxyl group (C=O) also showed varying degrees of peak position shift and peak shape change.

[0032] The spectral differences observed only in the hemp nanocellulose composite system are mainly due to the presence of numerous polar groups, especially abundant hydroxyl groups (-OH), in the hemp nanocellulose molecular chain. In the composite system, these hydroxyl groups can interact with the amino (-NH2 / -NH-), carboxyl (-COOH), and carboxylate (-COO-) groups in the PEI / PAA gel network. - Strong intermolecular hydrogen bonds are formed, such as -OH···N- and -OH···O=C-. Simultaneously, the hydroxyl groups in hemp nanocellulose may also form synergistic electrostatic-hydrogen bond couplings with ionic groups in the system. These multiple physical interactions alter the force constants of chemical bonds such as NH, C=O, and OH in the PEI / PAA network and their chemical microenvironment, resulting in significant changes in the position, intensity, and width of the relevant infrared characteristic absorption peaks compared to pure PEI / PAA gel.

[0033] The above results demonstrate that hemp nanocellulose has been successfully introduced into the PEI / PAA gel system, forming strong physical interactions with the polymer matrix through hydrogen bonding and physical entanglement, rather than binding to PEI or PAA through covalent grafting. These physical interactions coexist synergistically with the electrostatic interactions between PEI and PAA, jointly constructing a physical cross-linking network within the composite gel.

[0034] Figure 2 Scanning electron microscopy cross-sectional morphology of PEI / PAA composite gels with different hemp nanocellulose addition amounts; 1. Testing instruments and conditions The microstructure of the samples was observed using a field emission scanning electron microscope (FET). During testing, the operating voltage was set to 5–10 kV, the working distance to 8–15 mm, and images were acquired using a secondary electron detector (SE2) or an InLens detector. Before observation, the samples underwent gold sputtering to improve conductivity and reduce the charging effect. The gold sputtering conditions were: vacuum degree 8–10 Pa, sputtering current 6–8 mA, and sputtering time 2–4 min.

[0035] 2. Sample Preparation PEI / PAA gel and PEI / PAA / hemp nanocellulose composite gel samples with different amounts of hemp nanocellulose were cut into block samples with a size of approximately 5 mm × 5 mm × 3 mm. The samples were placed in pure water and sealed to swell until mass equilibrium was reached, i.e., weighed every 2 hours until the difference between two adjacent weighing results did not exceed 1%, to ensure that the pores in the gel network were fully expanded.

[0036] After swelling equilibrium is reached, the sample is transferred to liquid nitrogen for rapid freezing for approximately 10 minutes to quickly freeze the hydrogel and reduce the damage to the pore structure caused by ice crystal formation. The freeze-hardened sample is then subjected to brittle fracture using pre-cooled tweezers or a blade to expose the fresh fracture surface. Subsequently, the fracture-side-up sample is rapidly transferred to a freeze dryer and freeze-dried at -80 to -50°C and a vacuum level below 20 Pa for 24 to 48 hours to allow sufficient sublimation and removal of moisture. The dried sample is then fixed to the sample stage with conductive adhesive, fracture-side-up, and sputter-coated with gold before use.

[0037] 3. Testing Procedures The prepared sample was loaded into the scanning electron microscope sample chamber and evacuated to a vacuum level better than 1×10⁻⁶. -4 Pa. The microstructure of the sample cross-section was observed at different magnifications, including ×100, ×500, ×1000, ×5000, and ×10000, and images were collected from representative areas. At least three different areas were observed for each sample group, and no fewer than three images were collected for each area.

[0038] Image analysis software was used to measure the pore size of the gel in the acquired images. At least 100 pores were randomly measured for each sample, and the average pore size and pore size distribution of the sample were calculated accordingly.

[0039] 4. Results Analysis Scanning electron microscopy (SEM) results showed that the PEI / PAA gel and the composite gels with different amounts of hemp nanocellulose exhibited a typical three-dimensional porous network structure. With increasing hemp nanocellulose content, no significant structural changes were observed in the cross-sectional morphology of the composite gels. Pore size statistics further indicated that there was no significant difference in the average pore size of the composite gels with different hemp nanocellulose contents, suggesting that the introduction of hemp nanocellulose did not significantly affect the overall network pore structure of the PEI / PAA gel.

[0040] These results corroborate the infrared spectroscopy analysis. Hemp nanocellulose was mainly introduced into the gel system through hydrogen bonding and physical entanglement, without altering the basic cross-linked network framework of the PEI / PAA gel. Therefore, the gel pore size did not change significantly with the amount of hemp nanocellulose added. Furthermore, no local structural distortions due to significant agglomeration of hemp nanocellulose were observed in the scanning electron microscope images, indicating that the hemp nanocellulose has good dispersibility in the gel matrix, and the prepared composite gel maintained good structural uniformity overall.

[0041] Figure 3 A photograph of the PEI / PAA / CNF composite gel undergoing an inverted adhesion and load-bearing test to demonstrate its inverted adhesion and load-bearing capacity. 1. Sample preparation A 25mm × 75mm glass slide was used as the support substrate. A small amount of lyophilized PEI / PAA / CNF composite gel powder was uniformly adhered to the surface of one end of the slide. Fresh pig back skin was taken, and after removing subcutaneous fat and the epidermis, it was cut into rectangular specimens measuring 20mm × 40mm and immersed in phosphate-buffered saline (PBS) for 30 minutes to rehydrate. After rehydration, the pig skin specimens were removed and laid flat in a petri dish with the dermis facing upwards for later use.

[0042] 2. Test Procedure Holding a glass slide coated with the lyophilized composite gel powder, flip the slide so that the powdered side faces down and suspend it vertically about 5 mm above the pigskin specimen. Then slowly lower the slide until the gel surface contacts the dermal layer of the pigskin, and record the time corresponding to the contact and adhesion process.

[0043] After the gel adheres to the pigskin, the glass slide is slowly lifted and inverted. By observing whether the pigskin specimen falls off, it can be determined whether a stable adhesion has been formed between the gel and the pigskin.

[0044] 3. Test Results Timing results showed that the composite gel on the glass slide contacted the pigskin surface at 8.83 s and achieved stable adhesion at 10.04 s. Therefore, the time required from gel contact with the pigskin surface to the formation of a stable adhesion that prevents the pigskin specimen from detaching when inverted is approximately 1.21 s.

[0045] Within approximately 1 second of contact between the gel and pigskin, the resulting adhesive system was able to lift the pigskin specimen. The mass of the pigskin specimen exceeded 100 times the mass of the gel itself on the glass slide, indicating that the composite gel can form a strong tissue adhesion in a relatively short time.

[0046] 4. Results Analysis The above results indicate that the PEI / PAA / CNF composite gel of the present invention has a rapid adhesion response after contact with porcine skin tissue, can establish effective and stable interfacial adhesion within about 1 second, and can withstand a load far exceeding the mass of the gel itself, indicating that the composite gel has good tissue adhesion performance.

[0047] This rapid adhesion property helps to shorten the fixation time of materials on tissue surfaces, giving it excellent immediate fixation capabilities and ease of operation in applications such as tissue wound closure, rapid fixation of tissue surfaces, and wearable device attachment.

[0048] Figure 4 The effect of hemp nanocellulose content on gel swelling properties; 1. Materials and Instruments: Prepare the freeze-dried gel sample to be tested, phosphate buffered saline (PBS, pH 7.4), petri dishes or centrifuge tubes, precision analytical balance (accuracy 0.1 mg), and filter paper.

[0049] 2. Sample preparation: Freeze-dry the gel sample to be tested to constant weight to obtain a dry gel. Record its weight as Wd.

[0050] 3. Testing Procedures 1) Immersion swelling: Completely immerse the known weight (Wd) of the dry gel sample in a sufficient amount of swelling medium, phosphate-buffered saline (PBS). Sufficient medium should be provided to ensure the sample remains submerged throughout the swelling process.

[0051] 2) Setting conditions: Place the entire system under constant temperature (25℃) conditions to allow the sample to begin to swell.

[0052] 3) Reaching Equilibrium: Allow the sample to swell fully in the medium until equilibrium is reached. For most hydrogels, 24 hours is sufficient to reach swelling equilibrium. During this period, if swelling kinetics need to be studied, samples can be taken out and weighed at specific time points of 0.5, 5, 10, 15, 20, 25 min…24 h.

[0053] 4) Remove surface moisture: After the predetermined time point, use tweezers to remove the swollen gel and immediately use filter paper to gently absorb excess moisture from its surface. Be careful not to squeeze the gel, as this may squeeze out the absorbed moisture and affect the results.

[0054] 5) Weighing and recording: Quickly weigh the swollen gel after the surface moisture has been absorbed, and record its weight as Ws.

[0055] 6) Repeat the operation: Place the weighed gel back into the swelling medium to continue swelling. Repeat steps 4) and 5) until the mass of two consecutive weighings no longer changes (i.e., swelling equilibrium is reached). The weight at which equilibrium is reached is the final Ws.

[0056] 4. Test Results: The 0.5 wt% formulation exhibits the lowest swelling peak and relatively slow subsequent degradation due to excessive cellulose aggregation and overly dense cross-linking. The 0.1 wt% formulation, on the other hand, suffers from insufficient cross-linking, resulting in weak network strength and excessively rapid degradation. The 0.3 wt% formulation, however, achieves the highest swelling peak (strongest water absorption capacity) at 0.5 min and the slowest weight loss rate after the peak (most structurally stable), thus balancing high swelling rate and low degradation rate, making it the optimal formulation. In short, the 0.3 wt% formulation achieves the best balance between "water absorption and swelling" and "anti-disintegration."

[0057] Figure 5 Stress-strain curves of PEI / PAA composite gels with different amounts of hemp nanocellulose added; 1. Experimental Materials and Pretreatment Take fresh or frozen pig back skin, thaw it, remove subcutaneous fat and epidermis, and cut it into strips 25±0.1 mm wide and 100±0.5 mm long. Immerse the pig skin strips in phosphate-buffered saline (PBS) for 30 min to fully rehydrate them. Before testing, use filter paper to absorb excess moisture from the surface of the strips to ensure that there are no free water droplets on the bonding surface.

[0058] 2. Instruments and Equipment The test was conducted using a universal material testing machine equipped with a 10-100N range sensor and a pneumatic push clamp. The load indication error of the testing machine did not exceed ±0.5%.

[0059] 3. Testing Procedures (1) Overlapping bonding: Take two pretreated pigskin specimens, and evenly coat the gel powder onto the bonding end of one of the pigskin specimens. The width of the coating area is 25 mm and the length is 10 mm, that is, the bonding area is 250 mm². 2 Align the adhesive ends of another piece of pigskin specimen with the gel layer surface to form an overlapping shear structure. Apply a compressive force of 5–10 N and hold for 30 seconds to remove air bubbles from the adhesive interface and ensure a uniform gel layer thickness.

[0060] (2) Curing treatment: Place the overlapped pigskin-gel composite sample in an environment with a temperature of 25±2℃ and a relative humidity of 50±5% for static curing; or, depending on the characteristics of the gel material, use ultraviolet light irradiation or other methods to fully crosslink and cure it.

[0061] (3) Tensile loading: Install the cured specimen in the upper and lower clamps of the universal testing machine, adjust the clamp position so that the longitudinal axis of the pigskin specimen is parallel to the tensile direction, and ensure that the clamp does not clamp the gel bonded area. Stretch at a constant speed of 2 to 10 mm / min, preferably 5 mm / min, until the bonded interface is completely destroyed, and record the maximum destructive load during the test.

[0062] 4. Test Results and Analysis Depend on Figure 5 It can be seen that the amount of hemp nanocellulose added has a certain influence on the mechanical properties of the PEI / PAA composite gel. When the amount of hemp nanocellulose added is 0.1wt%, the tensile strength of the composite gel reaches its maximum value; when the amount added increases to 0.3wt%, the elongation at break of the composite gel reaches its maximum value, while its tensile strength remains at a high level.

[0063] Based on the comprehensive evaluation results of the composite gel, including its tensile mechanical properties, swelling properties, microstructure uniformity, and interfacial interactions reflected by infrared spectroscopy, it is evident that when the addition amount is 0.3 wt%, the composite gel exhibits excellent comprehensive performance in terms of fracture strength, flexibility, and ductility, with a suitable elongation at break. Simultaneously, the internal network structure maintains good uniformity, and the interfacial interactions are strong. Although the elongation at break is slightly higher when the addition amount is 0.1 wt%, its fracture strength and swelling properties decrease, and a certain degree of agglomeration occurs in the microstructure, weakening the interfacial bonding force. Therefore, this invention preferably uses a hemp nanocellulose addition amount of 0.3 wt% as the preferred formulation.

[0064] Figure 6 The image shows the antibacterial effect of hemp nanocellulose PEI / PAA composite gel on Staphylococcus aureus and Escherichia coli in plate coating. 1. Experimental materials and preparation of bacterial suspension Gram-positive Staphylococcus aureus (ATCC 6538) and Gram-negative Escherichia coli (ATCC 8099) were used as indicator strains. The test strains were inoculated into nutrient broth and cultured at 37°C with shaking for 24 hours to obtain activated bacterial suspensions. The fresh bacterial suspensions were then serially diluted with sterile phosphate-buffered saline (PBS), and the absorbance was measured using a microplate reader. Based on a pre-established relationship between absorbance and viable cell concentration, the bacterial suspension concentration was adjusted to 1.33 × 10⁻⁶. 6 CFU / mL, for later use.

[0065] 2. Sample grouping and co-cultivation The experiment group, material control group, and blank control group were set up. The experiment group used PEI / PAA / CNF composite gel powder with a hemp nanocellulose content of 0.3wt%; the material control group used pure PEI / PAA gel powder without hemp nanocellulose; the blank control group only added bacterial suspension and did not add any gel sample.

[0066] Equal masses of powder samples from the experimental group and the material control group were weighed and placed in sterile conical flasks. 5 mL of a 1.33 × 10⁻⁶ solution was added to each flask. 6 The bacterial suspension at CFU / mL was thoroughly shaken to mix. An equal volume of bacterial suspension was added to the blank control group. Subsequently, each Erlenmeyer flask was placed in a 37°C constant temperature shaker and co-cultured at 120 rpm for 12 h.

[0067] 3. Coating and Cultivation After co-culturing, take bacterial suspensions from each group and perform serial 10-fold dilutions using sterile PBS. The dilution factor is determined based on the actual bacterial suspension concentration and can range from 10 to 10. -1 ~10 -9 Take 100 μL of each dilution of bacterial culture and spread it evenly on the surface of the solidified nutrient agar plate.

[0068] After coating, place the plate face up and let it stand for 10-15 minutes until the bacterial culture is fully absorbed by the culture medium. Then, invert the plate and incubate it in a 37°C constant temperature incubator for 24 hours.

[0069] 4. Colony count and inhibition rate calculation After incubation, plates with colony counts ranging from 30 to 300 were selected for counting. Three parallel plates were prepared for each group, and the average colony count was taken. The antibacterial rate of the composite gel relative to the pure PEI / PAA gel was calculated according to formula (I): Inhibition rate (%) = [(N0 - N1) / N0] × 100% (I) Wherein, N0 is the material control group, i.e., the average viable count of the pure PEI / PAA gel powder group; N1 is the experimental group, i.e., the average viable count of the PEI / PAA / CNF composite gel powder group. The blank control group is used to verify the normal growth of the test bacteria under the set culture conditions.

[0070] When converting colony concentration, the dilution factor and the actual coating volume should also be considered to convert the colony count result into the viable bacteria concentration in the bacterial solution, with the unit being CFU / mL.

[0071] 5. Test Results and Analysis Colony counting results showed that the initial bacterial concentration was 1.0 × 10⁻⁶. 6 Under the same co-culture conditions and CFU / mL, a large number of dense colonies grew on the culture plates of the blank control group and the pure PEI / PAA gel powder group, while the number of colonies on the PEI / PAA / CNF composite gel powder group was significantly reduced.

[0072] Calculations showed that, compared with pure PEI / PAA gel without hemp nanocellulose, the PEI / PAA / CNF composite gel powder had an inhibition rate of 96.0% and 96.8% against Staphylococcus aureus and Escherichia coli, respectively, with an inhibition rate of over 90% against both tested bacteria, indicating that the composite gel powder has a good antibacterial effect.

[0073] The above results indicate that the introduction of hemp nanocellulose can improve the antibacterial properties of the PEI / PAA gel system. This is presumably because hemp nanocellulose has a large specific surface area and contains abundant polar groups such as hydroxyl groups, which helps improve the composite gel network structure and increase the contact between the material and the bacterial surface. Simultaneously, hemp nanocellulose may promote the uniform distribution and effective exposure of nitrogen-containing groups in PEI within the composite system, making it easier for them to interact electrostatically with the negatively charged bacterial cell surface, thereby affecting cell membrane integrity and normal physiological function. Furthermore, the adsorption of moisture and nutrients from the bacterial growth environment by the composite gel may also inhibit bacterial growth and proliferation to some extent.

[0074] In summary, the PEI / PAA / CNF composite gel powder of this invention exhibits good antibacterial effects against two representative strains of Staphylococcus aureus and Escherichia coli, and has the potential to be applied to medical antibacterial dressings and related biomedical materials.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an antibacterial hemp nanocellulose self-gelling hemostatic powder, characterized in that: The solution of polyethyleneimine was mixed with hemp nanocellulose suspension and then mixed with polyacrylic acid solution. The mixed solution was quickly placed in liquid nitrogen and frozen, then vacuum freeze-dried and ground into powder to obtain antibacterial hemp nanocellulose self-gelling hemostatic powder.

2. The preparation method according to claim 1, characterized in that: The mass ratio of polyethyleneimine to polyacrylic acid is 2-8:8-2, and the amount of hemp nanocellulose added is 0.1wt% to 0.5wt% of the mass of the polyethyleneimine solution.

3. The preparation method according to claim 1 or 2, characterized in that: The liquid nitrogen freezing time is 5 to 10 minutes, the vacuum freeze-drying pressure is 8 to 10 Pa, and the temperature is -60 to -45℃.

4. The preparation method according to claim 1, characterized in that: The molecular weight of the polyethyleneimine is 70,000, and the molecular weight of the polyacrylic acid is 240,000.

5. The preparation method according to claim 1, characterized in that: The concentrations of the polyethyleneimine solution and the polyacrylic acid solution are both 10 wt%.

6. The preparation method according to claim 1, characterized in that: The preparation process of the hemp nanocellulose suspension is as follows: hemp nanocellulose is dispersed in deionized water, sheared at 12000 r / min for 5-10 min, and then subjected to 5 high-pressure homogenization cycles at 60 MPa; subsequently, it is centrifuged at 8500 r / min for 10 min to obtain the hemp nanocellulose suspension.

7. The preparation method according to claim 6, characterized in that: The concentration of the hemp nanocellulose in deionized water is 10. -2 g / mL.

8. An antibacterial hemp nanocellulose self-gelling hemostatic powder prepared by the preparation method according to any one of claims 1-7.

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