Ultra-fast self-gelling self-expanding self-propelling high-adhesion procoagulant hemostatic powder for non-compressible hemorrhage hemostasis in pigs

Domain: neuroscience
Published: 2026-01-29
Editorial outcome: minor_revision

Abstract
Non-compressible hemorrhage remains the chief cause of battlefield mortality and civilian trauma death. Here, we propose a hemostatic strategy based on ultra-fast self-gelling self-expanding powder of polyacrylic acid (PAA), polyethyleneimine (PEI) and foaming agent, which achieves non-compressible hemorrhage hemostasis through the multiple synergistic effects of expansion plugging, self-gelling adhesion and activating the coagulation factors. The hemostatic powder integrates rapid physical crosslinking with spontaneous gas foaming, exhibits a fast gelation rate, high expansion ratio, strong tissue adhesion, and activation of red blood cells, platelets, and fibrin. The optimized formulation (PP/PT5-TXA30) achieves superior hemostatic performance compared to commercial powders in rat liver volumetric defect, femoral artery and vein transection, as well as complete transection of the subclavian artery and vein in rabbits. Notably, in a lethal porcine model of complete subclavian artery and vein transection, PP/PT5-TXA30 achieves superior performance of non-compressible hemorrhage compared to gauze and XStat™. Additionally, PP/PT5-TXA30 accelerates full-thickness skin wound healing. This work demonstrates a strategy based on ultra-fast self-gelling and self-expanding mechanisms for developing hemostatic materials for non-compressible hemorrhage.

Introduction
Uncontrollable hemorrhage—defined as bleeding at amputation sites or junctional areas (e.g., groin) that cannot be managed through manual or mechanical compression due to trauma in battlefield or civilian accidents—is a leading cause of preventable death1–4. Such non-compressible hemorrhage represents 90% of potentially survivable fatalities among military personnel and constitutes 30–40% of trauma-related deaths in civilian populations. Consequently, it stands as the leading cause of mortality in emergency care settings5,6. Traditional hemostatic materials, such as gauze7, sponges8,9, and glues10, typically require manual or physical pressure to achieve hemostasis, but their efficacy is often limited in these scenarios11,12. Therefore, the development of hemostatic strategies for non-compressible hemorrhage, aimed at reducing both blood loss and time to hemostasis, is urgently needed to significantly lower trauma-related mortality in both battlefield and civilian emergency settings13.

Given that non-compressible hemorrhage typically occurs in deep wounds and internal organs, often involving profuse bleeding accompanied by irregular wound geometries and rupture of deep-seated arteries and veins, manual or physical compression is not feasible, necessitating emergency and pre-hospital hemostatic intervention14. Researchers have developed a variety of hemostatic materials for non-compressible hemorrhage based on physical, chemical, and biological mechanisms1,15, including sponges/foaming agents6,8, sealants/adhesives11, and platelet mimetics16. Among these, expandable hemostatic materials have been widely recognized as the most promising translational approach due to their wound-adaptive capacity and immediate physical blockage effect, enabling rapid control of non-compressible hemorrhage17,18. Existing expandable hemostatic materials can be classified based on their expansion mechanisms into the following categories: (1) Shape memory sponge-based expandable hemostatic materials (e.g., XStat™, cryogels). The U.S. military utilizes XStat™, a commercial injectable system loaded with compressed cellulose sponges. These sponges rapidly swell to occupy the cavity and generate localized pressure in deep, non-compressible wound tracts6,14,19, thereby achieving hemostasis. However, it lacks biochemical procoagulant activity, and its relatively low blood absorption capacity and compressive strength may prolong hemostasis time and increase blood loss. To address these limitations, we previously reported a series of injectable cryogels that achieve effective hemostasis in non-compressible hemorrhage through the synergistic effects of biochemical procoagulation and expansion-induced sealing20–25. Shape memory sponge-based hemostatic materials are easy to sterilize and convenient for storage. Nonetheless, they may be unable to access concealed bleeding sites or treat complex wounds with narrow grooves or voids, cannot cope with internal or diffuse bleeding in the abdominal cavity, and excessive expansion may tear the wound, resulting in secondary hemorrhage. To better adapt to wound characteristics and avoid excessive compression of the wound, a second category emerged: (2) Fluid foaming-type expandable hemostatic materials (e.g., ResQFoam, expandable hemostatic hydrogels). Upon injection into the wound site, ResQFoam generates foam through contact with blood, which exerts pressure at the bleeding site to achieve hemostasis. However, the complex administration procedure of ResQFoam, along with its operational intricacies that necessitate trained personnel, poses a significant risk of misadministration, and the risk can lead to severe complications or even death, thereby limiting its application in emergency care1,19. To simplify the application process, our team engineered an injectable, self-propelling hydrogel adhesive with inherent procoagulant properties and fast-setting capability17. This hydrogel adhesive exhibits a fast expansion rate, high self-expanding capacity, and strong tissue adhesion, enabling effective control of complex bleeding while minimizing excessive compression on surrounding tissues. Nevertheless, its relatively low mechanical strength and liquid form—characterized by poor sterilization compatibility (low irradiation tolerance), as well as limited storage stability and portability—restrict its practical use in emergency settings. Therefore, retaining the high hemostatic efficiency of fluid foaming-type expandable materials while overcoming the challenges of sterilization and mechanical weakness associated with liquid formulations has become a major bottleneck in the development of hemostatic material design targeting non-compressible hemorrhage.

In this study, we develop an ultra-fast self-gelling, self-expanding, self-propelling, highly adhesive, procoagulant portable hemostatic powder by integrating the moisture-induced self-gelling property of powders with self-expanding and self-propelling capabilities. Dopamine-modified montmorillonite (PDA-MMT) is incorporated into polyethyleneimine/polyacrylic acid powder (PP/PT), which possesses ultra-fast self-gelling and wet adhesion properties, to enhance its adhesive performance and hemostatic efficacy. Furthermore, a gas-generating strategy based on protonated tranexamic acid (TXA-NH +) and sodium carbonate (Na CO ) is introduced into the self-gelling hemostatic powder system to confer self-expanding and self-propelling behaviors, while synergistically accelerating coagulation. We systematically characterize the blood absorption and gelation capacity, mechanical properties, tissue adhesiveness, antibacterial activity, biocompatibility, and volumetric expansion performance of the hemostatic powder. The hemostatic efficacy in non-compressible hemorrhage is evaluated using rat models of liver volumetric defect, cardiac puncture, and femoral artery/vein transection, as well as lethal rabbit and porcine models involving complete transection of the subclavian artery and vein. In addition, the powder’s ability to achieve sutureless closure of full-thickness skin incisions and promote wound healing is assessed. The results demonstrate that the self-gelling expandable hemostatic powder not only possesses portability, storage stability, and ease of use, but also exhibits high wet tissue adhesion, rapid self-expanding and self-propelling properties, and superior antibacterial and procoagulant activities. It effectively controls bleeding from surface wounds, irregularly shaped deep cavity injuries, and non-visible hemorrhage sites, and further accelerates skin wound healing.

Results
We prepared a hemostatic powder with ultra-fast self-gelling, self-expanding, and self-propelling properties, which triggers rapid gelation upon contact with blood and exhibits good adhesion and coagulation-promoting properties. This hemostatic powder can be used to control irregular, non-compressible hemorrhage (Fig. 1). First, montmorillonite (MMT) was modified with hydrochloric acid to enhance its dispersion in water, allowing dopamine to self-assemble on the MMT to obtain dopamine-modified MMT (PDA-MMT). Thermogravimetric analysis (TGA) results showed that PDA was successfully coated on MMT, with a dopamine coating ratio of approximately 2.51% (Supplementary Fig. 1a). After PDA coating, the Zeta potential of MMT decreased from −14.3±1.3mV to −25.6±0.2mV (Supplementary Fig. 1b). Then, PDA-MMT was incorporated into polyethyleneimine (PEI) and polyacrylic acid (PAA), which exhibit ultra-fast self-gelling and wet adhesion, to enhance the adhesion and hemostatic properties. After freeze-drying and grinding, PEI/PAA/PDA-MMT powder (abbreviated as PP/PT) was obtained. In the preparation of PP/PT powder, the mass ratio of PEI to PAA was maintained at 1:1, and the amount of PDA-MMT in the PP/PT powder was varied to enhance the adhesion strength of PP/PT. The specific feed ratio is shown in Supplementary Table 1. The introduction of catechol groups enhances the adhesion properties of the powder17,26. However, as the content of PDA-MMT increased, the adhesion strength of the powder initially increased and then decreased. This is due to a change in the cohesion between the polymers12,27. The PP/PT with 5 wt% PDA-MMT exhibited the optimal adhesion strength (>40kPa) (Supplementary Fig. 2). Therefore, in subsequent experiments, we used the self-gelling hemostatic powder with 5wt% PDA-MMT content, named PP/PT5. To verify the successful preparation of the material, we characterized the product using SEM, EDS, and FT-IR spectroscopy. Material morphology was examined via SEM, and the successful synthesis of PDA-MMT and PP/PT was confirmed by EDS (Supplementary Fig. 3 and Supplementary Fig. 4). The molecular structure of the materials was systematically characterized using FT-IR, as shown in Supplementary Fig. 5. In the spectrum of PDA-MMT composite, a significant N–H in-plane bending vibration absorption peak was observed at 1603cm−1, which confirmed that dopamine was successfully surface-modified onto the montmorillonite matrix. Further comparison of the spectral features revealed that the characteristic absorption peak of the carboxyl group (–COOH) in pure PAA appeared at 1696cm−1, while the vibration peaks of primary/secondary amines (–NH /–NH) in PEI were observed in the 1585cm−1 region. Notably, when constructing the PP/PT5 composite system, the aforementioned characteristic peaks shifted to 1632cm−1 and 1541cm−1, respectively, and no new characteristic peaks were observed. This spectroscopic evidence indicates that the crosslinking interactions between PEI, PAA, and PDA-MMT in the system primarily arise from physical bonding mechanisms such as hydrogen bonding and electrostatic interactions, rather than the formation of covalent bonds12,27. The crosslinking interactions between PEI, PAA, and PDA-MMT in the system primarily arise from physical bonding mechanisms such as hydrogen bonding and electrostatic interactions, rather than the formation of covalent bonds27. This conclusion provides key spectroscopic evidence for the non-covalent crosslinking nature of the composite material.

To enhance the material’s expandable properties, a foaming agent was introduced into the system to obtain PP/PT5-TXAn; the specific feed ratios are provided in Supplementary Table 2. The protonated tranexamic acid (TXA-NH +) and sodium carbonate (Na CO ) rapidly react to continuously release CO₂ gas bubbles, causing the system to expand and providing self-propelling capabilities. After the foaming reaction, TXA-NH + remains in the system as tranexamic acid, which can further promote hemostasis by inhibiting plasminogen activation, thereby enhancing the material’s hemostatic performance6,28. Next, the content of the foaming agent was optimized by evaluating the mechanical properties, expansion performance, and hemostatic capability of the hemostatic powder to identify the most optimal expandable self-gelling hemostatic powder, PP/PT5-TXA30. The key steps in the preparation of PP/PT5-TXA30 hemostatic powder are shown in Fig. 2a. Experimental verification demonstrated that the self-gelling, expandable, self-propelling hemostatic powder rapidly gelled upon contact with blood, while gas production as a propellant caused the system to expand, physically sealing the wound and promoting coagulation through biochemical synergistic effects. Consequently, the material achieves exceptional hemorrhage control in a lethal, non-compressible pig model.

Methods
Materials
Polyethyleneimine (PEI, molecular weight ≈70,000Da, 50wt% aqueous solution) and tranexamic acid (TXA-NH ) were purchased from Aladdin (Shanghai, China). Polyacrylic acid (PAA, molecular weight ≈240,000Da, 25wt% aqueous solution) was obtained from J&K Scientific (Beijing, China). Sodium carbonate (Na CO ), Montmorillonite (MMT), and dopamine (DA) were purchased from Macklin. All reagents were used as received without further purification. Antibody TNF-a (ab 1793) was purchased from commercial sources (Abcam, UK), and antibody CD31 (AF6191) was purchased from commercial sources (Affinity, UK). L929 (BNCC100314) cell line was purchased from Shangcheng Beinachuanglian Biotechnology Co., LTD.

Synthesis of dopamine-modified montmorillonite (PDA-MMT, PT)
Dopamine-modified montmorillonite (PDA-MMT, abbreviated as PT) was prepared according to an established protocol29. Briefly, 5g of montmorillonite was treated with 100mL of 3M HCl under stirring at 45°C for 8h. The mixture was subsequently centrifuged and washed repeatedly (5–6 cycles) until the supernatant tested negative for chloride ions using silver nitrate. The resulting solid was collected and dried overnight at 50°C. In parallel, a Tris buffer solution (150mL) was adjusted to pH 8.5 with HCl, followed by the addition of 1.5g of dopamine under stirring until complete dissolution. The acid-treated montmorillonite was then introduced into this dopamine solution and stirred at room temperature for 24h. After reaction, the product was isolated by centrifugation, washed thoroughly with water (3–4 times) until the supernatant became clear, and finally lyophilized for further use.

Synthesis of organic acid protonated tranexamic acid (TXA-NH +)
The preparation of protonated tranexamic acid (TXA-NH +) using organic acids was carried out according to protocols described in earlier studies40. Concentrated HCl was added to 0.5M TXA-NH until the pH reached 4.3, and then solid TXA-NH + was collected after freeze-drying.

Preparation of polyethyleneimine/polyacrylic acid/dopamine-modified montmorillonite (PEI/PAA/PT, PP/PTn) powder
Dopamine-modified montmorillonite (PT) was mixed with polyethyleneimine (PEI, 10wt%) and polyacrylic acid (PAA, 10wt%) at various ratios, followed by lyophilization and grinding to obtain PP/PTn powders. Specifically, we added PT to an equal volume of 10wt% PEI (Mn=70,000) aqueous solution and 10wt% PAA (Mn=240,000) aqueous solution at 1%, 3%, 5%, 10%, and 20% by mass and mixed them uniformly. The mixture was immediately immersed in liquid nitrogen for about 15min without pouring out any liquid, and then freeze-dried to remove water. Finally, the dried solid was ground with a mortar and pestle to obtain PP/PTn powder, and the detailed content of each component is shown in Supplementary Table 1. In addition, we mixed 10wt% PEI (Mn=70,000) and 10wt% PAA (Mn=240,000) in a volume ratio of 5:5. Immerse in liquid nitrogen, freeze-dry, and grind to obtain PP powder.

Preparation of self-gelling self-expanding high-adhesion procoagulant hemostatic PP/PT5-TXAn powder
Optimized Polyethyleneimine/polyacrylic acid/dopamine-modified montmorillonite (PP/PT5), protonated tranexamic acid (TXA-NH ⁺), and sodium carbonate (Na CO ) were mixed in different ratios, followed by drying and storage to obtain the PP/PT5-TXAn hemostatic powder. TXA-NH + and Na CO act as foaming agents in the system, generating CO gas upon contact with liquids. PP/PT5, TXA-NH +, and Na CO were mixed in the proportions shown in Supplementary Table 2 to obtain a fast self-gelling, self-expanding, high-adhesion hemostatic powder PP/PT5-TXAn.

Morphology, elemental composition, and FT-IR spectra of the powder
The morphology of freeze-dried MMT, PT, PEI/PAA (PP), and PP/PT powders was observed by using a field emission scanning electron microscope (FEI Quanta FEG 250). The surface of the MMT, PT, PEI/PAA (PP), and PP/PT powders was sprayed with gold before observation. In addition, the elemental compositions of MMT, PT, and PP/PT5 were analyzed using energy dispersive X-ray spectroscopy (EDS) equipped in the SEM by mapping measurements.
FT-IR spectra of PT, PEI, PP/PT, and PAA were recorded in the range of 4000–400cm−1 by using a Nicolet 6700 FT-IR spectrometer (Thermo Scientific Instrument).

Blood uptake ratio test
Chitosan HP, PP, PP/PT5, and PP/PT5-TXA30 powders were weighed and recorded as m , and then immersed in anticoagulated blood water at 37°C for 10s, respectively. Then, the samples were dried, and their weight were recorded as m. Blood uptake ratio was calculated as follows:
Blood uptake ratio = (m - m )/m × 100% (1)

Swelling ratio test
The powders were immersed in PBS at room temperature to transform into hydrogels. And then, immersed in PBS buffer (0.01 M, pH=7.4) and placed on a shaker at 37°C. The shaking speed was set as 100 rpm. When reaching the preset time interval, the hydrogel was taken out of the bottle, and its surface water was removed using filter paper. Then the hydrogel was weighed. Measurements were repeated three times for each group. The swelling ratio is calculated using the following formula:
Swelling ratio (%) = (M - m)/m × 100% (2)
where M and m represent the wet weight after reaching the equilibrium swelling state and the initial weight of the samples, respectively.

Mechanical properties tests
The rheological property of the powder expanding to the maximum volume was performed using a TA rheometer (DHR-2), and the testing data was collected by TRIOS Installer (version 5.1.1.46572) provided by TA Instruments. Before the test, the powder was mixed with PBS at room temperature to form the hydrogel. The expanded hydrogels were cut into discs with dimeter of 20mm and a thickness of 1.5mm. Before the collection of the data, the hydrogel disc was placed between 20mm parallel plates. Oscillation-frequency test with 1% constant strain and the frequency varying from 1 to 100 rad/s at 37°C was used to evaluate the stiffness of the expanded hydrogels.
The compression strength of the expanded hydrogels was tested by using a TA rheometer (DHR-2), and the testing data was collected by TRIOS Installer (version 5.1.1.46572) provided by TA Instruments. Before the test, the powder was mixed with PBS into a cylindrical tube with a diameter of 10mm at room temperature to form the hydrogel. After being expanded to the maximum volume, the hydrogel was taken out and cut into a cylindrical shape with a diameter of 10mm and a height of 10mm, and then placed between parallel plates with a diameter of 20mm and a gap of 10mm. The maximum compression strain was set to 80%, and the compression speed was set to 100μm/s.
Tensile tests were performed on the dumbbell-shaped samples with the custom clamps of the Instron Materials Test system (MTS Criterion 43, MTS Criterion) at a cross-head speed of 5mm/min, and the testing data was collected by Testworks 4 (version 4.12D) provided by MTS Systems Corporation (MTS). The tensile strain was defined as the change in length relative to the initial gauge length. Measurements were repeated three times for each group.

Volumetric expansion test
1g of PP/PT5-TXAn with varying foaming agent content was injected into an equal volume of heparinized whole blood to study its blood absorption, self-gelling, and expansion properties. The volumetric expansion performance of powders was determined by calculating the volume expansion ratio and recording the volume expansion time17. In brief, 2mL of blood was put into a container, and then 1g of powder was mixed with the blood. The end of the mixing was regarded as the start time. The timing was stopped when the hydrogel expanded to the maximum volume. The initial volume, volume after expansion, and volume expansion time of the hydrogels were recorded. Each powder was tested in triplicate. The calculation formulas of the volume expansion ratio are as follows:
Expansion ratio (%) = Vt/Vs × 100% (3)
Where Vt and Vs represent the preset time volume and initial volume, respectively.

Simulation calculation
A fluid-structure coupling module in ANSYS Workbench 2020 R1 was used to simulate the expansion process of the hydrogel precursor41. A rotational geometry model of the wound cavity, with a shape similar to an actual wound, was established. The component outside of the wound cavity was assigned as soft tissues with the Mooney-Rivlin 2 model, with the material constant C10=0.347MPa, C01=0.0352MPa, the density of 1048kgm−3, and the incompressibility parameter D1=0.00134MPa−1. A mass-flow inlet with a normal mass-flow rate of 0.05kg/s was used at the bottom boundary of the wound cavity section to simulate the continuously generated bubbles. The laminar flow module was employed. The fluid materials were set as carbon dioxide with 1.7878kgm−3 and 1.37×10−5kg·m−1·s−1. The large deflection was available to simulate the geometric nonlinear characteristics of the soft tissue. A fixed constraint was added at the inlet of the cavity, while a displacement constraint was added on the upper and lower surfaces of the soft tissue. We focused on the changes from 0s to 8s, with a step size of 0.5s.

Tissue adhesion test
The tissue adhesion strength of the powders was evaluated by using porcine skin tissue via a lap shear test. The porcine skin tissue was cut into 10mm × 30mm rectangular shapes. Then, the powder was put on the porcine skin, and PBS buffer was added in situ at room temperature to form a hydrogel. The contact area of the two tissues was kept at 10mm × 10mm. Then the samples were placed at 37°C for 1h under the condition of maintaining high humidity. Then, the samples were lap shear-tested to failure on an Instron Materials Test system (MTS Criterion 43, MTS Criterion) equipped with a 50N load cell by using a cross-head speed of 5mm/min at room temperature, and the testing data was collected by Testworks 4 (version 4.12D) provided by MTS Systems Corporation (MTS). All measurements were repeated six times for all groups.

Bursting pressure test
In order to test the performance of the powder as tissue sealant, the burst pressure test was conducted by referring to the previous work with some modifications39. In brief, the fresh porcine skin tissues were cut into rectangles (20 × 20mm), and then a 2-mm-diameter hole was punched in the center of the skin tissue. After that, the skin tissue was fixed on a hole (diameter: 5mm) in the upper surface of a custom-made cubic box, and the powder was put onto the center of the skin tissue, and PBS was added in situ at room temperature to form the hydrogel. A digital pressure gauge (MIK-Y290, Asmik, China) and a syringe pump (20mL) were connected to the cubic box, and the cubic box and the tubes were filled with PBS before the test. During the test, PBS was injected into the apparatus gently, and the burst pressure of the whole process was recorded. The test was repeated for 3 times.

Antibacterial activity of the powder
The in vitro antibacterial activity of hydrogels against Methicillin-Resistant Staphylococcus Aureus (MRSA) and Escherichia Coli (E. coli) was evaluated using the surface contact antibacterial test by referring to the reported method with modifications24,49,51. 50mg powders were placed into 48-well plates. After 60min of UV radiation, 10μL of bacterial suspension (107 CFU/mL) was added onto the surface of the powders, and the inoculated hydrogels were incubated at 37°C for 2h. After the incubation, 990µL of PBS was added to the well to resuspend any bacterial survivors. 10μL of bacterial suspension (107 CFU/mL) suspended in 990µL of PBS served as the negative control. After incubating for 18–24h at 37°C, the colony-forming units on the agar plate were counted. Each sample was repeated three times. The bacterial killing ratio of the powder is calculated using the following formulas:
Bacterial killing ratio (%) = (Nc - Np)/Nc × 100% (4)
Where Nc and Np represent the count of bacteria in the control group and the count of bacteria in the powder group, respectively.

Hemocompatibility, cytotoxicity, and in vivo host response of self-gelling hemostatic powder
The biosafety of PP/PT5-TXAn powders was evaluated by hemocompatibility, cytocompatibility, and in vivo biocompatibility tests.
The hemolytic activity of the powders on blood cells was evaluated by evaluating the hemolysis ratio of the rat blood cells, as we reported previously21,25,48,52. The rat blood was centrifuged at 200×g for 10min to obtain blood cells. After removing the supernatant, the resulting blood cells were washed with PBS buffer and then diluted to a final concentration of 5% (v/v) blood cell suspension. 0.5mL of powder after gelling was placed into a 24-well plate, and then 600µL of PBS buffer and 600µL of blood cell suspension were added. The 24-well plate was incubated at 37°C with a shaking speed of 100 rpm for 1h. After that, the blood cell suspension was transferred to a tube and centrifuged at 200xg for 10min. 100µL of the supernatant was added to the 96-well plate, and the absorbance of the solution was read at 540nm using a microplate reader. 0.1% of Triton X-100 was set as a positive control, while PBS buffer was set as the negative control. Each group was repeated 6 times. The hemolysis ratio was calculated using the following formula:
Hemolysis ratio (%) = [(A - A )/(A - A )] × 100% (5)
Where A, A, and A represent the absorbance values of samples, positive control, and PBS, respectively. The cytotoxicity of the powders was evaluated by a contact test method39,53. For the contacting test method, the freeze-dried hydrogels were first cut into disks with a diameter of 8mm and a height of 1mm, and then sterilized by immersing them into 75% alcohol. The complete growth medium was Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1.0×105 U/L penicillin (Hyclone), and 100mg/L streptomycin (Hyclone). L929 cells were seeded into a 48-well plate with a cell density of 7500 cells/well. After being cultivated for one day, the sample disks equilibrated in the culture medium were added into each well, and the culture medium level was kept slightly lower than the sample’s upper surface to ensure the fine contact between the cells and the samples. After culturing for 24h, the cell viability under the powders was tested by Live/Dead Viability/Cytotoxicity Kit assay. After culturing for 1 day, respectively, the cell viability under the powders was tested by alamarBlue® assay. 20µL of alamarBlue® reagent in 200µL culture medium was added to each well after removing the sample disks and medium. After incubating at 37°C for 4h, 100µL of the medium in each well was transferred to a 96-well black plate (Costar). Fluorescence was read by a microplate reader (Ex. 560nm; Em. 600nm). Each group was repeated 6 times. Cell adhesion and viability were observed under an inverted fluorescence microscope (IX53, Olympus). The cell viability was calculated using the following formula:
Cell viability (%) = [(A - A )/(A - A )] × 100% (6)
Where A is the fluorescence value of the experimental group, A is the fluorescence value of the positive control group, and A is the fluorescence value of the blank background (10µL of alamarBlue® in 90µL of complete growth medium).
The in vivo biocompatibility of the powder was verified by a subcutaneous implantation test17. All the samples were cut into the same shape and size (diameter of 8mm and height of 2mm), sterilized with 75% ethanol, and rinsed in phosphate-buffered saline (PBS) overnight. Four symmetrical small incisions were created on the back of each rat (female, Sprague Dawley, 180–210g). The prepared samples were placed into the incisions, and then the skin was closed. After 7 days and 28 days, the rats were sacrificed, and the testing samples were excised with the adjacent tissues. The obtained material-tissue samples were embedded in paraffin, sectioned, and mounted onto slides. The acute inflammatory response and chronic inflammatory response were measured by both hematoxylin and eosin (H&E) staining. The stained slides were observed and analyzed by a microscope. Each group was repeated 3 times.

Dynamic whole-blood-clotting test
The whole-blood-clotting ability of the powders was evaluated by referring to the reference with some modifications23. Gelatin sponge, Combat Gauze, and commercial powder were set as the control groups. The powders or control groups were placed into polypropylene tubes and then prewarmed to 37°C. 10μL of 0.1M CaCl solution was mixed with 190μL of citrated rat whole blood, and then 50μL of the recalcified whole blood was immediately added to the powder or control surface. The tubes were incubated at 37°C for 30s, 60s, 90s, and 120s, respectively. After the incubation, the blood cells that were not trapped in the clot were hemolyzed with 10mL of DI water, and the absorbance of the resulting hemoglobin solution was measured at 540nm. The absorbance of 50μL of recalcified whole blood in 10mL DI water was used as the reference value (positive control). Each sample was repeated six times. The blood-clotting index (BCI) was calculated using the following equation:
BCI (%) = (I - I )/(I - I ) × 100% (7)
where I, I , and I represent the absorbance of samples, positive control, and DI water, respectively.

Blood cell adhesion and platelet aggregation test
The blood cell adhesion and platelet aggregation tests were conducted referring to references17,20,23. In brief, put the powder, gelatin sponge, and Combat Gauze (same weight) into 24-well plates. For the blood cell adhesion test, the samples were immersed in PBS for 1h at 37°C. Following that, the whole blood with ACD anticoagulant solution (volume ratio: 6:1) was added dropwise onto the sample and then incubated for 5min at 37°C. For the platelet aggregation test, platelet-rich plasma (PRP) was separated from the whole blood with ACD anticoagulant solution by centrifugation of blood at 500×g for 15min. The PRP was then added dropwise onto the sample and incubated for 1h at 37°C. All samples were then washed with PBS solution three times to remove the physically adhered blood cells, and then fixed with 2.5% glutaraldehyde for another 2h. After that, blood cells were dehydrated using 50%, 60%, 70%, 80%, 90%, and 100% ethanol solutions with a time interval of 10min. Finally, the samples were dried and observed using SEM.

Plasma recalcification time test
Platelet-rich plasma (PRP) was separated from the whole blood with ACD anticoagulant solution by centrifugation of blood at 500×g for 15min. 10mg of different powder samples were weighed in a glass bottle, then 90μL PRP and 10mL CaCl solution were added to it, and the coagulation time was recorded as PRT. Each sample was repeated three times54.

In vivo hemostatic performance of self-gelling hemostatic powder
Hemostatic performance in non-compressible hemorrhage was analyzed using rat liver volumetric defect and complete femoral artery laceration hemorrhage models10,17,20,39. The hemostatic effect of the material on surface non-compressible hemorrhage was assessed in a rat cardiac puncture hemorrhage model and a tail amputation hemorrhage model12,17,24,38,55.

Rat liver volumetric defect bleeding model. For the rat liver non-compressible hemorrhage model, the rats (SD rats, weight 250–300g, male) were randomly and equally divided into 5 groups. The animals were anesthetized and fixed on a surgical corkboard. The liver of the rat was exposed by abdominal incision, and serous fluid around the liver was carefully removed to prevent inaccuracies in the estimation of the blood weight obtained by the hemostatic samples. Then, a 6mm wide and 3mm deep narrow wound was created. Immediately after wiping off the blood using gauze, the Chitosan HP, PP powder, PP/PT5 powder, and PP/PT5-TXA30 powder were applied on the site of the lesion. The group without hemostatic materials was used as the blank control group. The hemostatic time and blood loss were recorded. Each group contains 5 rats.

Rat femoral artery and vein complete transection bleeding model. For the rat femoral artery complete transection bleeding model, the rats (SD rat, 250–300g, male) were anesthetized and fixed on a surgical corkboard. A midline skin incision approximately 1–1.5 cm long was made adjacent to the femoral vessels, and the femoral artery was then exposed. The femoral artery of the rats was cut off with surgical scissors, and the bleeding blood was quickly wiped using gauze. The commercial powder (Chitosan HP), PP powder, PP/PT5 powder, and PP/PT5-TXA30 powder were immediately applied to the bleeding site, respectively. During the hemostasis, the blood loss, hemostatic time, and dead time were recorded. No treatment after bleeding served as the control group. Each group contains 5 rats.

Rat heart puncture hemostasis model. For the rat heart puncture hemostasis model, the rats (SD rat, 250–300g, male) were anesthetized and fixed on a surgical corkboard. A midline skin incision of about 2–3 cm long was made near the chest, and then the heart was exposed. The rat heart was punctured with a needle, then the commercial powder (Chitosan HP) and PP/PT5-TXA30 powder were immediately applied to the bleeding site, respectively. During the hemostasis, the blood loss, hemostatic time, and dead time were recorded. No treatment after bleeding served as the control group.

Rat tail truncation model. For the rat tail truncation model, the rats (SD rat, 250–300g, male) were anesthetized and fixed on a surgical corkboard. Cut off with scissors about a third of the length of the tail, then treated immediately with the commercial powder (Chitosan HP) and PP/PT5-TXA30 powder, respectively. During the hemostasis, the blood loss and hemostatic time were recorded. No treatment after bleeding served as the control group.

Hemostatic effect of self-gelling hemostatic powder on non-compressible hemorrhage from complete transection of the rabbit subclavian artery/vein
Following previously reported methods25, the hemostatic effect of the powder was evaluated on non-compressible hemorrhage by performing hemostasis on fully transected rabbit subclavian arteries and veins. The hemostatic powder was syringe-loaded for injection, with an untreated group and a commercial chitosan-based hemostatic powder group as controls. New Zealand White rabbits (weight of about 2kg, male) were anesthetized and fixed on the surgical corkboard. A cut-down was performed to expose the right femoral artery and femoral vein. Subsequent transection of the femoral artery and vein using the scissors resulted in a hemorrhage model with an invisible bleeding site. The commercial powder (Chitosan HP) and PP/PT5-TXA30 powder were injected into the bleeding site through a skin incision. No treatment after bleeding served as the control group. During the hemostatic process, the weighed gauze was used to absorb the flowing blood. The hemostatic time and blood loss were recorded accordingly. Any additional bloodshed from the wound site was recorded as blood loss. If there is no blood flow from the wound site, the hemostasis time is 0min. Each group contains 5 rabbits.

Hemostatic effect of self-gelling hemostatic powder on non-compressible hemorrhage from complete transection of pig subclavian artery/vein
The efficiency of the hemostatic powder in non-compressible hemorrhage was evaluated by complete transection of the subclavian artery and vein in pig17,25,56. Male Cross-bred Yorkshire pigs (n=3 per group) were fasted for ≥12h (water permitted) prior to surgery. Anesthesia was induced via ear vein injection of 3% pentobarbital (1mL/kg). Following intubation, the animals were positioned dorsally on a surgical table. The right femoral artery and vein were cannulated to allow continuous monitoring of mean arterial pressure (MAP) and for fluid administration, respectively. Core temperature was maintained at 37–39°C using a rectal probe and heating pad.
The pigs were abducted, with the left front leg to allow surgical access to the subclavian vessels. A 4.5 cm incision was made parallel to the sternum, over the cranial superficial pectoralis muscle. After dividing the pectoral muscles, the subclavian artery, vein, and brachial nerve plexus were exposed, and a 5 cm section of the subclavian artery and vein was then dissected free from surrounding tissues. The potential wound cavity volume was pre-measured by saline filling. Topical 2% lidocaine was applied to induce vasodilation. A 10-min stabilization period commenced, requiring a stable MAP > 65mmHg, core temperature of 37–39°C, and an arterial diameter > 6mm. At the midaxillary line, the subclavian artery, vein, and nerve plexus were completely transected with surgical scissors. A 30-s free-bleeding period was allowed. Pretreatment blood loss was calculated as the sum of (1) blood aspirated during free bleeding and initial application, and (2) the pre-measured cavity volume (accounting for pooled blood). Animals were randomized to receive either the injectable hemostatic powder or standard gauze immediately after free bleeding. For the hemostatic powder group, the material was applied to fill the cavity without subsequent manual compression. In the gauze group, the cavity was packed followed by 3min of mild manual pressure. The time to fill the cavity was recorded as the application time. MAP was recorded at application and at 15-min intervals thereafter. Resuscitation with prewarmed Lactated Ringer’s solution (37°C) was administered to maintain MAP ≥ 65mmHg. 1h observation period followed the 5min application window. Five minutes after the start of the 1h observation, the wound site was inspected for signs of bleeding. Any additional bloodshed from the wound site was collected and recorded as post-treatment blood loss. The 1h observation phase commenced immediately following the completion of manual compression (for gauze) or upon injection of the hemostatic powder. Prospectively identified primary endpoints include hemostasis (defined as no blood exiting the wound cavity) at 4min, hemostasis at 60min, and survival at 60min (survival defined by MAP > 60mmHg). The secondary endpoints, defined a priori, comprised the mean arterial pressure (MAP) recorded at the study’s conclusion, total blood loss measured following intervention, and the time required to administer the treatment.

Wound healing effect of the self-gelling hemostatic powder on full-thickness skin incisions on the back of rats
The wound healing experiment was conducted by referring to our previous report50. The animal experiments were approved by the institutional review board of Xi’an Jiaotong University. All rats were randomly and equally divided into 4 groups: TegadermTM film, glue, suture, and powder groups. Each group contained 6 rats. The rats were anesthetized, and then the dorsal region of the mouse above the tail but below the back was shaved for further surgery. Two full-thickness wounds with a length of 20mm were made on each side of the mouse midline. Subsequently, different groups of samples were applied to the wound site. For wound area monitoring, on the 0th, 3rd, 5th, 7th, and 14th day, the rats were anesthetized, and then the wound area was photographed. And the tensile strength of the wound was tested by the stretching machine.
To evaluate the epidermal regeneration and inflammation in the wound area, the collected samples on the 7th and 14th day were fixed with 4% paraformaldehyde for 1h, embedded in paraffin, and then cross-sectioned into 4μm thickness slices. The obtained slices were then stained with Hematoxylin-Eosin (Beyotime, China) and Masson. All slices were analyzed and photographed by a microscope (IX53, Olympus, Japan). Meanwhile, TNF-α immunofluorescence staining was performed to observe the inflammatory response of the wounds in each group. The angiogenesis of wounds in each group was observed by CD31 immunofluorescence staining.

Statistical analysis
Results express continuous variables as mean ± standard deviation (SD). Following ≥3 experimental replicates per condition. Statistical analyses included Two-tailed Student’s t-tests for two-group comparisons and One-way ANOVA plus Tukey’s post-hoc testing for multi-group analyses (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Statistical significance was set at p<0.05. OriginPro (version 2025b) was used to analyze all the statistical data; Images were analyzed by ImageJ (version 1.53k).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript innovatively proposed a new non-compressible hemorrhage hemostasis strategy based on ultrafast self-gelling self-expanding powder. The hemostatic powder integrates rapid physical crosslinking with spontaneous gas foaming, exhibits a fast gelation rate, high expansion ratio, outstanding antibacterial activity, and strong tissue adhesion, facilitating its application in the management of non-compressible hemorrhage. The ultrafast self-gelling self-expanding powder can achieve non-compressible hemorrhage hemostasis through the multiple synergistic effects of expansion plugging, self-gelling adhesion and activating the coagulation factors. This is an innovative and meaningful work in the field of non-compressible hemorrhage hemostasis. However, there still existed some issues in this article as following. Therefore, I recommend it to accept in Nature Communications after minor revision.\n1. How to prepare the hemostatic powder? The representative photographs of key steps in the preparation of the hemostatic powder should be added to Figure 2.\n2. Please standardize abbreviations. For instance, “PP/PT5-TXA30” should be fully defined at first mention in Abstract.\n3. The diagram in Figure 1 is not clearly labeled. It is recommended to add arrows or text descriptions to more intuitively demonstrate the hemostatic mechanism of the powder.\n4. Some experimental methods are described briefly, pointing to the SI (Supplementary Information) section, which hinders readers’ ability to read and obtain key information. the key steps need to be briefly summarized in the main text. For example, key reaction conditions or ratios can be added in “2.2 Synthesis of Hemostatic Powder Precursor Materials.”\n5. Some statistical annotations are missing or inconsistent (e.g., significance markers in certain figures). Please review and ensure consistency throughout. Such as Figure 3b.\n6. It is recommended to polish the language of the manuscript, especially the introduction and discussion. Some sentences are long and can be split appropriately to improve readability. For example, “Given that non-compressible hemorrhage typically occurs in deep wounds and internal organs—often involving profuse bleeding accompanied by irregular wound geometries and rupture of deep-seated arteries and veins—manual or physical compression is not feasible, necessitating emergency and pre-hospital hemostatic intervention.”\n7. “2.3 Synthesis of Polyethyleneimine/Polyacrylic Acid/Dopamine-Modified….”, and 2.4,and 3.1. the word “Synthesis” should be replaced by “preparation”\n8. Line 215, the “control deep” should be “control group”?\n9.How to measure the “Mechanical properties of the Hemostatic Powder”, form section of 3.2, authors measured the Mechanical properties of powder-derived hydrogels rather than that of Powder. For uniaxial compression tests, the size of gelled powders should be given.\n10, How about the biocompatibility of PP/PT5-TXA30, suggesting to add discussion about the biocompatibility of PP/PT5-TXA30", "reviewer_2": "In this manuscript, the authors report an ultrafast, self-gelling, self-expanding hemostatic powder based on polyacrylic acid, polyethyleneimine, and a foaming agent. This hemostatic powder achieves non-compressible hemorrhage hemostasis by the synergistic effects of expansion and occlusion, self-gelling and adhesion, and activation of coagulation factors. The authors fully characterized the physicochemical properties and procoagulant activity and the excellent in vivo hemostasis property of the hemostatic powder in different animal models including pigs. While the manuscript demonstrates high novelty and clinical research value, several issues need to be minor revised and clarified before acceptance in Nature Communications. These are outlined below.\n1. Why doesn't the powder swelling rate at 0 min start at 0? Is there a difference in the swelling behavior of the powder in PBS buffer versus blood?\n2. It is recommended to zoom in on representative areas of red blood cells, platelets, and fibrin networks to further observe changes in cell morphology.\n3. The powder has been proven to be effective in treating a variety of incompressible bleeding in the body. From a clinical perspective, the hemostatic material needs to be removed after hemostasis. Can the gel formed by this powder be successfully removed after surgery?\n4. In the volume expansion ratio test results of Figure 3b, there is no analysis of significant differences between each group. Please complete the p-value analysis.\n5. There are many reports on the gel formation of hemostatic powder. What are the advantages of this manuscript compared with other reports? It would be best if it could be compared with the currently reported hemostatic powders in terms of mechanical properties, adhesion strength and other aspects.\n6. The new expandable hemostatic powder reported in the manuscript has good in vivo hemostatic performance. What improvements or problems this material needs to face before clinical translation? This part can be appropriately discussed and supplemented in the conclusion of the manuscript.", "reviewer_3": "In the manuscript titled “Ultra-fast Self-gelling Self-expanding Self-propelling High-adhesion Procoagulant Hemostatic Powder for Non-compressible hemorrhage Hemostasis in Pigs,” a hemostatic powder with self-gelling , self-expanding, and self-propelling properties was developed. The incorporation of protonated amino acids (TXA-NH₃⁺) and sodium carbonate (Na₂CO₃) as foaming agents enabled rapid gelation, volumetric expansion, and enhanced hemostatic efficacy. This work demonstrates the excellent hemostatic performance of developed biomaterials across multiple animal models (rats, rabbits, pigs). the presented findings are comprehensive and significant. Reviewer has the following comments:\n1. The current introduction provides a somewhat lengthy description of the background and limitations of existing materials for uncontrollable bleeding. It could be condensed to more quickly introduce the design philosophy and innovative aspects of this work.\n2. The conclusion section is currently rather weak and reads merely as a summary of results. To strengthen the manuscript, the authors should significantly revise this section to: Briefly reiterate the core scientific rationale behind the material's success (i.e., the synergy between physical and biochemical hemostasis) rather than just restating its properties.Key limitations include the small sample size in the porcine model (n=3), the use of healthy animals which do not mimic the physiologic derangements of severe trauma (e.g., coagulopathy, hypothermia, acidosis), and the lack of long-term biocompatibility and degradation data.\n3. Dopamine (DA)-modified MMT is crucial for adhesion, but what is the grafting rate of DA onto MMT? How does the zeta potential of MMT change after grafting? It is suggested to provide related chemical characterizations.\n4. Does the pressure generated by material expansion within a sealed cavity pose a risk of compressive injury to adjacent critical structures such as nerves? It is recommended to discuss or provide related data.\n5. The text mentions that hydrogels have “poor sterilization compatibility.” What sterilization method is used for this powder? (Gamma irradiation? Ethylene oxide?) It is necessary to specify and demonstrate that this sterilization method does not damage the material's chemical structure and functionality.\n6. The paper highlights the advantages of powder formulations in terms of storage stability and portability. It is suggested to provide stability data to support this core advantage.\n7. The authors claim to achieve “ultra-fast” gelation within 2 seconds. However, this key parameter is supported only by qualitative video evidence (Supplementary Video S1) and lacks quantitative rheological data—particularly sweep experiments demonstrating the evolution of G' and G'' over time and the precise gelation point.\nSome minor comments are listed below:\n1. In Figure 2, the legend for panel 2h states that the data is \"mean ± SD\", but there are no error bars in the figure. Please add error bars.\n2. Regarding Figure 7e, the figure currently lacks significant markers for intergroup comparisons (such as asterisks). Given that the text description mentions a strong significant effect (p < 0.001), these markers must be added to the figure for an intuitive interpretation of the statistical results."}, "author_response": "Response to the reviewers’ comments\nWe greatly thank you for these professional and constructive comments about our\nmanuscript. These comments are valuable and helpful for revising and improving our\nmanuscript. Following the editor and reviewers’ comments and suggestions, we have\nmade the following explanations to make our results convincing.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript innovatively proposed a new non-compressible hemorrhage\nhemostasis strategy based on ultrafast self-gelling self-expanding powder. The\nhemostatic powder integrates rapid physical crosslinking with spontaneous gas foaming,\nexhibits a fast gelation rate, high expansion ratio, outstanding antibacterial activity, and\nstrong tissue adhesion, facilitating its application in the management of non-\ncompressible hemorrhage. The ultrafast self-gelling self-expanding powder can achieve\nnon-compressible hemorrhage hemostasis through the multiple synergistic effects of\nexpansion plugging, self-gelling adhesion and activating the coagulation factors. This\nis an innovative and meaningful work in the field of non-compressible hemorrhage\nhemostasis. However, there still existed some issues in this article as following.\nTherefore, I recommend it to accept in Nature Communications after minor revision.\n1. How to prepare the hemostatic powder? The representative photographs of key steps\nin the preparation of the hemostatic powder should be added to Figure 2.\nResponse: Thanks for your constructive question and suggestion. First, the preparation\nmethod of hemostatic powder is as follows: Dopamine-modified montmorillonite was\nmixed with polyethyleneimine (10 wt%) and polyacrylic acid (10 wt%) at various ratios,\nP\nA\nG\nE\n\\\nfollowed by lyophilization and grinding to obtain PP/PTn powders. Subsequently, the\noptimized PP/PT5 powder was mixed with protonated tranexamic acid (TXA-NH +)\n3\nand sodium carbonate (Na CO ) to obtain the final PP/PT5-TXAn hemostatic powder.\n2 3\nWe have added the above brief preparation procedure of the PP/PT5-TXAn hemostatic\npowder to the manuscript. Secondly, by following your advice, we have added\nrepresentative photographs of key steps in the preparation of hemostatic powder to\nFigure 2a, and the representative pictures added are as follows:\nFigure 2a. The representative photographs of key steps in the preparation of the\nPP/PT5-TXA30 hemostatic powder.\n2. Please standardize abbreviations. For instance, “PP/PT5-TXA30” should be fully\ndefined at first mention in Abstract.\nResponse: Thanks for your reminding. By following your suggestions, we have\ncarefully checked the entire article and tried our best to standardize all abbreviations.\n3. The diagram in Figure 1 is not clearly labeled. It is recommended to add arrows or\ntext descriptions to more intuitively demonstrate the hemostatic mechanism of the\npowder.\nResponse: Thanks for your constructive suggestion. By following your advice, we have\nadded labels to correspond the hemostatic stages with the hemostatic mechanism of the\nhemostatic powder in Figure 1 to more intuitively demonstrate the hemostatic\nP\nA\nG\nE\n\\\nmechanism of the powder.\n4. Some experimental methods are described briefly, pointing to the SI (Supplementary\nInformation) section, which hinders readers’ ability to read and obtain key information.\nthe key steps need to be briefly summarized in the main text. For example, key reaction\nconditions or ratios can be added in “2.2 Synthesis of Hemostatic Powder Precursor\nMaterials.”\nResponse: Thanks for your nice comment. We have carefully considered your\nsuggestions and have briefly summarized the experimental methods of the following\nimportant parts in the main text to facilitate readers to read and obtain key information:\n2.2 Synthesis of Hemostatic Powder Precursor Materials;\n2.6 Evaluation of the Expansion Properties of Self-gelling Hemostatic Powder;\n2.12 Hemostatic Effect of Self-gelling Hemostatic Powder on Non-compressible\nHemorrhage from Complete Transection of Pig Subclavian Artery/Vein\n5. Some statistical annotations are missing or inconsistent (e.g., significance markers in\ncertain figures). Please review and ensure consistency throughout. Such as Figure 3b.\nResponse: Thanks for your important reminding. We carefully checked the statistical\nfigures throughout the paper to ensure that the statistical annotations were complete and\nconsistent. Moreover, Figure 3b shows the experimental results of the volume\nexpansion ratio of PP/PT5-TXA30 hemostatic powder over time upon contact with\nanticoagulated blood, and each column shows the volume expansion ratio of the\nhemostatic powder at different time points, so there is no comparison between groups.\n6. It is recommended to polish the language of the manuscript, especially the\nintroduction and discussion. Some sentences are long and can be split appropriately to\nimprove readability. For example, “Given that non-compressible hemorrhage typically\nP\nA\nG\nE\n\\\noccurs in deep wounds and internal organs—often involving profuse bleeding\naccompanied by irregular wound geometries and rupture of deep-seated arteries and\nveins—manual or physical compression is not feasible, necessitating emergency and\npre-hospital hemostatic intervention.”\nResponse: Thanks for your constructive advice. By following your suggestion, we have\npolished the language of the entire article, especially simplifying and splitting the long\nsentences you suggested to improve the reading convenience of readers.\n7. “2.3 Synthesis of Polyethyleneimine/Polyacrylic Acid/Dopamine-Modified….”, and\n2.4, and 3.1. the word “Synthesis” should be replaced by “preparation”\nResponse: Thanks for your nice advice. Your suggestion is very pertinent. Compared\nwith “synthesis”, “preparation” is more suitable to describe the preparation process of\nhemostatic powder. We have changed “synthesis” to “preparation” in the relevant parts\nof the whole paper.\n8. Line 215, the “control deep” should be “control group”?\nResponse: Thanks for your reminding. We have changed “control deep” to “control\ngroup” in line 215 and carefully checked the whole article and tried our best to correct\nthe errors in the article.\n9.How to measure the “Mechanical properties of the Hemostatic Powder”, form section\nof 3.2, authors measured the Mechanical properties of powder-derived hydrogels rather\nthan that of Powder. For uniaxial compression tests, the size of gelled powders should\nbe given.\nResponse: Thanks for your important reminding. In Section 3.2, we measured the\nmechanical properties of the hydrogel formed by the self-gelation of the hemostatic\npowder, not the mechanical properties of the hemostatic powder. In the uniaxial\ncompression test, the hydrogel was cylindrical with a diameter of 10 mm and a height\nof 10 mm, and we have added this information to the main text.\n10, How about the biocompatibility of PP/PT5-TXA30, suggesting to add discussion\nabout the biocompatibility of PP/PT5-TXA30.\nResponse: Thanks for your constructive suggestion. The biocompatibility of the\nhemostatic powder was tested by hemolysis test, in vitro cytocompatibility test and in\nvivo subcutaneous implantation test in rats. Firstly, we evaluated the blood\ncompatibility of the powder by testing its hemolysis ratio. The experimental results\nshowed that the hemolysis ratio of the PP/PT5-TXA30 hemostatic powder was less than\n5%, showing good blood compatibility. Secondly, the cytocompatibility of the PP/PT5-\nTXA30 hemostatic powder was evaluated using L929 fibroblasts co-cultured for 24 h\nwith disc-shaped preformed gel powders (20 mg) under a contact mode assay, and\nPP/PT5-TXA30 hemostatic powder groups exhibited cell viability above 95%\ncompared to the TCP control group, demonstrating good cell compatibility. Finally, to\nfurther evaluate the biocompatibility of the materials, preformed PP/PT5-TXA30\nhemostatic powders were subcutaneously implanted into SD rats, H&E staining\nrevealed that PP/PT5-TXA30 hemostatic powders induced relatively mild acute and\nchronic inflammatory responses at both 7- and 28-days post-implantation. Collectively,\nthese findings demonstrate that the PP/PT5-TXA30 hemostatic powder exhibits good\nhemocompatibility, in vitro cytocompatibility, and in vivo biocompatibility, and has the\npotential to serve as an effective hemostatic material. We have added the above\ndiscussion on the biocompatibility of hemostatic powder to Section 3.5 of the main text,\nand the modifications are highlighted in red font in the manuscript.\nReviewer #2 (Remarks to the Author):\nIn this manuscript, the authors report an ultrafast, self-gelling, self-expanding\nP\nA\nG\nE\n\\\nhemostatic powder based on polyacrylic acid, polyethyleneimine, and a foaming agent.\nThis hemostatic powder achieves non-compressible hemorrhage hemostasis by the\nsynergistic effects of expansion and occlusion, self-gelling and adhesion, and activation\nof coagulation factors. The authors fully characterized the physicochemical properties\nand procoagulant activity and the excellent in vivo hemostasis property of the\nhemostatic powder in different animal models including pigs. While the manuscript\ndemonstrates high novelty and clinical research value, several issues need to be minor\nrevised and clarified before acceptance in Nature Communications. These are outlined\nbelow.\n1. Why doesn't the powder swelling rate at 0 min start at 0? Is there a difference in the\nswelling behavior of the powder in PBS buffer versus blood?\nResponse: Thanks for your nice questions. Firstly, the swelling test process of\nhemostatic powder is as follows: the powders were immersed in PBS at room\ntemperature to transform into powder-derived hydrogel. And then, immersed in PBS\nbuffer (0.01 M, pH = 7.4) and placed on a shaker at 37℃ to further evaluate the swelling\nproperties of the powder-derived hydrogel. Therefore, at 0 min, the swelling ratio is the\nweight of the powder-derived hydrogel divided by the original weight of the powder,\nso the swelling ratio is not 0. Secondly, by following your question, we added swelling\nexperiments of PP/PT5-TXA30 hemostatic powder in blood to evaluate the difference\nin its swelling behavior in PBS buffer and blood. As shown in Figure S6, the swelling\nratio of the powder-derived hydrogel at different time points in blood was not\nsignificantly different from that in PBS buffer. The hydrogel's low swelling in blood\nensures the stability of its mechanical properties during hemostasis and subsequent\nremoval.\nP\nA\nG\nE\n\\\n) PP/PT5-TXA30 in PBS buffer\n%\n200\n(\no\ni\nt\na\nr 160\ng\nn\ni\nl\nl\ne\nw 120\nS\n80\n0 20 40 60 80 100120140160180200\nTime (min)\nFigure S6. Swelling ratios curves of PP/PT5-TXA30 hemostatic powder-derived\nhydrogel in PBS buffer and blood (n=3).\n2. It is recommended to zoom in on representative areas of red blood cells, platelets,\nand fibrin networks to further observe changes in cell morphology.\nResponse: Thanks for your suggestion. By following your suggestion, in Figure 5, we\nused yellow arrows to indicate representative areas of red blood cells, platelets, and\nfibrin networks related to coagulation to further observe changes in cell morphology\nand confirm the procoagulant function of hemostatic powder.\n3. The powder has been proven to be effective in treating a variety of incompressible\nbleeding in the body. From a clinical perspective, the hemostatic material needs to be\nremoved after hemostasis. Can the gel formed by this powder be successfully removed\nafter surgery?\nResponse: Thank you for your constructive question. As you asked, the removal of\nhemostatic materials is crucial for their clinical application. Therefore, during material\ndesign, we enhanced the postoperative removability of the hemostatic powder through\nthe following strategies: (1) the hemostatic powder was combined into a whole through\nP\nA\nG\nE\n\\\nself-gelation, which improved its removal convenience; (2) we screened a high-strength\nself-gelling system based on high molecular weight PEI/PAA to enhance the\nmechanical strength of the expanded hydrogel, which further ensured the efficient\nremoval of the hemostatic powder after hemostasis. Based on the above material design,\nas shown in the Figure S18, the PP/PT5-TXA30 hemostatic powder has demonstrated\nthe ability to be efficiently removed after stopping bleeding in the Lethal porcine model\nof complete subclavian artery and vein transection.\nFigure S18. Demonstration of hemostasis and post-hemostasis removal of the PP/PT5-\nTXA30 hemostatic powder in a rabbit the Lethal porcine model of complete subclavian\nartery and vein transection.\n4. In the volume expansion ratio test results of Figure 3b, there is no analysis of\nsignificant differences between each group. Please complete the p-value analysis.\nResponse: Thanks for your important reminding. We carefully checked the statistical\nfigures throughout the paper to ensure that the statistical annotations were complete and\nconsistent. Moreover, Figure 3b shows the experimental results of the volume\nexpansion ratio of PP/PT5-TXA30 hemostatic powder over time upon contact with\nanticoagulated blood, and each column shows the volume expansion ratio of the\nhemostatic powder at different time points, so there is no comparison between groups.\nP\nA\nG\nE\n\\\n5. There are many reports on the gel formation of hemostatic powder. What are the\nadvantages of this manuscript compared with other reports? It would be best if it could\nbe compared with the currently reported hemostatic powders in terms of mechanical\nproperties, adhesion strength and other aspects.\nResponse: Thanks for your constructive suggestion. Compared with the gel formation\nof hemostatic powder in previous reports, we introduced a foaming component into the\nhemostatic powder for the first time, giving the hemostatic powder the ability to expand\na expand\nafter gel formation, and thus proposed a new hemostatic strategy based on ultrafast self-\ngelling and self-expanding powder. This expansion ability gives the powder-derived\nhydrogel better physical wound sealing and self-propelling capabilities, making it more\nsuitable for non-compressible hemorrhage. Moreover, the hemostatic powder-derived\nhydrogel designed in this work also showed obvious advantages in comprehensive\nproperties such as mechanical properties, adhesion properties, self-healing properties,\nanti-swelling and burst pressure. The newly added discussion can be found in the\nResults and Discussion section of the manuscript.\n6. The new expandable hemostatic powder reported in the manuscript has good in vivo\nhemostatic performance. What improvements or problems this material needs to face\nbefore clinical translation? This part can be appropriately discussed and supplemented\nin the conclusion of the manuscript.\nResponse: Thanks for your nice question. As you mentioned, the hemostatic powder\ndesigned in this work requires numerous refinements and explorations before clinical\ntranslation, such as optimizing the removal of the hemostatic powder after hemostasis\nand achieving large-scale production. Based on your suggestions, we have discussed\nthe potential translational potential and bottlenecks of the hemostatic powder in the\nconclusion section of this article. We will continue to optimize the performance of the\nhemostatic powder and strive to promote its clinical translation.\nP\nA\nG\nE\n\\\nReviewer #3 (Remarks to the Author):\nIn the manuscript titled “Ultra-fast Self-gelling Self-expanding Self-propelling High-\nadhesion Procoagulant Hemostatic Powder for Non-compressible hemorrhage\nHemostasis in Pigs,” a hemostatic powder with self-gelling, self-expanding, and self-\npropelling properties was developed. The incorporation of protonated amino acids\n(TXA-NH₃⁺) and sodium carbonate (Na₂CO₃) as foaming agents enabled rapid gelation,\nvolumetric expansion, and enhanced hemostatic efficacy. This work demonstrates the\nexcellent hemostatic performance of developed biomaterials across multiple animal\nmodels (rats, rabbits, pigs). the presented findings are comprehensive and significant.\nReviewer has the following comments:\n1. The current introduction provides a somewhat lengthy description of the background\nand limitations of existing materials for uncontrollable bleeding. It could be condensed\nto more quickly introduce the design philosophy and innovative aspects of this work.\nResponse: Thanks for your constructive suggestion. By following your advice, we have\ncondensed the description of the background and limitations of existing materials for\nuncontrollable bleeding in introduction part. Moreover, we have brought forward and\nexpanded the design philosophy and innovative aspects of this work so that readers can\nobtain the highlights and effective information of the article earlier.\n2. The conclusion section is currently rather weak and reads merely as a summary of\nresults. To strengthen the manuscript, the authors should significantly revise this section\nto: Briefly reiterate the core scientific rationale behind the material's success (i.e., the\nsynergy between physical and biochemical hemostasis) rather than just restating its\nproperties. Key limitations include the small sample size in the porcine model (n=3),\nthe use of healthy animals which do not mimic the physiologic derangements of severe\ntrauma (e.g., coagulopathy, hypothermia, acidosis), and the lack of long-term\nbiocompatibility and degradation data.\nP\nA\nG\nE\n\\\nResponse: Thanks for your constructive comments. By following your suggestions, In\nthe conclusion section, we added a discussion of the core scientific rationale behind the\nmaterial's success to enhance the completeness of the manuscript. For example, (1) in\nthe material design section, we added a discussion of the principle of rapid self-gelation\nof PEI/PAA hemostatic powder; (2) in the pro-coagulant section, we added a discussion\nof the possible intrinsic mechanism of the material's pro-coagulant effect; (3) in the\nbiocompatibility section, we added a discussion of the hemocompatibility, in vitro\ncytocompatibility, and in vivo biocompatibility of hemostatic powders, and the\nmodifications are highlighted in red font in the manuscript.\nRegarding the key limitations you raised:\n(1) As for the selection of sample size in the porcine model: First, we referenced\nseveral high-level literatures that used a porcine artery rupture model to evaluate the\nhemostatic performance of materials for our sample size1-5. Second, when the sample\nsize was 3, our designed hemostatic powder showed highly significant differences (p <\n0.001) from the gauze control group in multiple dimensions such as hemostasis time\nand blood loss, so we selected a sample size of 3 in in the porcine model. Moreover,\nbased on your suggestion, we will increase the sample size of large animals (such as\nporcine model) in the clinical translation experiments of this hemostatic powder and\nthe subsequent related work of our group.\n(2) As for the use of healthy animals which do not mimic the physiologic derangements\nof severe trauma (e.g., coagulopathy, hypothermia, acidosis): In the clinical\ntransformation experiments of this hemostatic powder and the subsequent related work\nof our group, we will add animal bleeding models under pathological conditions such\nas coagulation disorders, hypothermia, and acidosis (such as coagulation disorder fatal\nporcine subclavian arterial/venous complete transection non-compressible hemorrhage\nmodel) to evaluate the hemostatic performance of the hemostatic material for non-\ncompressible hemorrhage.\n(3) As for the lack of long-term biocompatibility and degradation data: We have\nevaluated the short-term and long-term biocompatibility of the PP/PT5-TXA30\nP\nA\nG\nE\n\\\nhemostatic powder through in vivo implantation experiments. H&E staining revealed\nthat PP/PT5-TXA30 hemostatic powders induced relatively mild acute and chronic\ninflammatory responses at both 7- and 28-days post-implantation, these findings\ndemonstrate that the PP/PT5-TXA30 hemostatic powder exhibits good in vivo\nbiocompatibility and holds potential as an effective hemostatic material.\n3. Dopamine (DA)-modified MMT is crucial for adhesion, but what is the grafting rate\nof DA onto MMT? How does the zeta potential of MMT change after grafting? It is\nsuggested to provide related chemical characterizations.\nResponse: Thanks for your important questions. The grafting rate of PDA on MMT\nwas studied by thermogravimetric analysis. As seen from Figure S1a, the weight\ndecreased upon increasing the temperature from ambient temperature to 800 °C. The\nstage starts at approximately 170°C and ends at 660°C, corresponding to PDA\ndecomposition. After 80°C, the remaining weight was 84.42% and 81.91% for MMT\nand PDA-MMT, respectively, which indicates that PDA has been successfully grafted\nonto MMT, and the grafting ratio of dopamine is about 2.51%. Moreover, As shown in\nFigure S1b, after PDA grafting, the zeta potential of MMT decreased from -14.3±1.3\nmV to -25.6±0.2 mV.\nFigure S1. a) Thermalgravimetric analysis (TGA) curves of MMT and PDA-MMT. b)\nThe zeta potential of MMT and PDA-MMT.\nP\nA\nG\nE\n\\\n4. Does the pressure generated by material expansion within a sealed cavity pose a risk\nof compressive injury to adjacent critical structures such as nerves? It is recommended\nto discuss or provide related data.\nResponse: Thanks for your constructive question. As you mentioned, compressive\ninjury to adjacent critical structures is an important issue that should be considered in\nthe design of expandable hemostatic materials. Therefore, we further studied the\nexpansion behavior of self-expanding self-gelling hemostatic powder in the bionic\nwound cavity through simulation. The results showed that the expanding hydrogel\nexerts a maximum extrusion pressure of 35 kPa on the surrounding tissues, which is\nmuch lower than the breaking strength of skin (~10 MPa)6, blood vessels (~2 MPa)7\nand muscles (~100 kPa)8, , and also lower than the in-situ stress of surrounding tissues\non nerves (50 kPa)9. Therefore, it will not cause compressive injury to the surrounding\ntissues. Moreover, for non-compressible hemorrhage that occurs on the battlefield or in\ndaily emergency care, the most important thing is to stop the bleeding quickly to save\nthe patient's life.\n5. The text mentions that hydrogels have “poor sterilization compatibility.” What\nsterilization method is used for this powder? (Gamma irradiation? Ethylene oxide?) It\nis necessary to specify and demonstrate that this sterilization method does not damage\nthe material's chemical structure and functionality.\nResponse: Thanks for your nice question. As shown in the certificate of irradiation for\nthe PP/PT5-TXA30 hemostatic powder, the PP/PT5-TXA30 hemostatic powder was\nsterilized by electron beam irradiation (Electron Beam Sterilization Device: DZ type\n10MeV/20KW electron linear accelerator; irradiation intensity: 15.0 kGy). Furthermore,\nas shown in Figures S19a-d, we evaluated the self-gelling time, tissue adhesive strength,\nand expansion properties of the PP/PT5-TXA30 hemostatic powder before and after\nirradiation sterilization. The results showed that irradiation sterilization had no\nsignificant effect on the key properties of the powder-derived hydrogel.\nP\nA\nG\nE\n\\\nCertificate of Irradiation for the PP/PT5-TXA30 hemostatic powder:\nP\nA\nG\nE\n\\\nFigure S19. Evaluation of stability of hemostatic powder after irradiation sterilization.\na) Self-gelling time of PP/PT5-TXA30 hemostatic powder after irradiation sterilization\nby oscillatory time sweep. b) Comparison of the expansion properties of PP/PT5-\nTXA30 hemostatic powder before and after irradiation sterilization. c and d)\nComparison of the tissue adhesive strength of PP/PT5-TXA30 hemostatic powder\nbefore and after irradiation sterilization.\n6. The paper highlights the advantages of powder formulations in terms of storage\nstability and portability. It is suggested to provide stability data to support this core\nadvantage.\nResponse: Thanks for your suggestion. As shown in Figure S20, we evaluated the self-\ngelling time, expansion properties, and tissue adhesive strength of the PP/PT5-TXA30\nhemostatic powder before and after one-year of storage. The results showed that one-\nyear storage had no significant effect on the key properties of the powder-derived\nhydrogel, demonstrating the good storage stability of the PP/PT5-TXA30 hemostatic\npowder.\nP\nA\nG\nE\n\\\nFigure S20. Evaluation of storage stability of PP/PT5-TXA30 hemostatic powder. a)\nSelf-gelling time of PP/PT5-TXA30 hemostatic powder after one-year storage by\noscillatory time sweep. b) Comparison of the expansion properties of PP/PT5-TXA30\nhemostatic powder before and after one year of storage. c and d) Comparison of the\ntissue adhesive strength of PP/PT5-TXA30 hemostatic powder before and after one\nyear of storage.\n7. The authors claim to achieve “ultra-fast” gelation within 2 seconds. However, this\nkey parameter is supported only by qualitative video evidence (Supplementary Video\nS1) and lacks quantitative rheological data—particularly sweep experiments\ndemonstrating the evolution of G' and G'' over time and the precise gelation point.\nResponse: Thanks for your constructive suggestion. By following your advice, we\nquantitatively measured the gelation time of the PP/PT5 powder using a TA rheometer\n(Figure S8a). As shown in Figure S8b, the PP/PT5 powder self-gelled within 10 seconds.\nThe gelation time was slightly longer than the previous qualitative evidence\n(Supplementary Video S1), possibly because the disc-shaped powder took some time\nto absorb PBS buffer from the surrounding area.\nP\nA\nG\nE\n\\\nFigure S8. a) The schematic of using a TA rheometer to test the self-gelling time of the\nPP/PT5 powder. b) Self-gelling time determination of PP/PT5 powder by oscillatory\ntime sweep.\nSome minor comments are listed below:\n1. In Figure 2, the legend for panel 2h states that the data is \"mean ± SD\", but there are\nno error bars in the figure. Please add error bars.\nResponse: Thanks for your important reminding. First of all, the description \"Data are\npresented as mean ± SD (* p < 0.05, * * p < 0.01, * * * p < 0.001)\" in the last part of\nthe legend of Figure 2 is for all the small figures in Figure 2. We apologize for the\nconfusion. Secondly, Figure 2h is a representative tensile stress–strain curves of\nhydrogels derived from hemostatic powders, so there are no error bars in the Figure 2h.\nMoreover, we have carefully checked the whole article and tried our best to correct the\nerrors in the article.\n2. Regarding Figure 7e, the figure currently lacks significant markers for intergroup\ncomparisons (such as asterisks). Given that the text description mentions a strong\nsignificant effect (p < 0.001), these markers must be added to the figure for an intuitive\ninterpretation of the statistical results.\nResponse: Thanks for your important reminding. First, the strongly significant\ndifferences (p < 0.001) described in the discussion section of this manuscript refer to\nP\nA\nG\nE\n\\\nthe significant differences in the blood loss between the groups, not for the statistical\ndifferences in the mortality ratios of the rabbits in the groups. We apologize for the\nconfusion. Secondly, in Figure 7e, the mortality ratio of rabbits in each group = the\nnumber of dead rabbits/the total number of rabbits, so only a single data can be obtained\nin each group, and no significant difference between groups can be counted. Moreover,\nwe have carefully checked the whole article and tried our best to correct the errors in\nthe article.\nReferences\nmassive bleeding on anticoagulation therapy. Proceedings of the National\nAcademy of Sciences 121, e2316170121, doi:doi:10.1073/pnas.2316170121\n(2024).\nwith enhanced permeability and mechanical properties for non-compressible\nhemostasis in pigs. Nature Communications 15, 5460, doi:10.1038/s41467-024-\n49578-2 (2024).\nnanofabric. Nature Communications 16, 4910, doi:10.1038/s41467-025-60244-\nz (2025).\ncontrolled balance of hydrophilicity and hydrophobicity. Nature\nCommunications 13, 552, doi:10.1038/s41467-022-28209-8 (2022).\nwith Procoagulant Activity and Rapid Gelation for Lethal Massive Hemorrhage\nManagement. Advanced Materials 36, 2308701,\ndoi:https://doi.org/10.1002/adma.202308701 (2024).\nP\nA\nG\nE\n\\\ndoi:10.1038/ncomms7649 (2015).\nTissues, Materials and Biological Reactions (eds Mitsuo Niinomi, Takayuki\nNarushima, & Masaaki Nakai) 71-98 (Springer Berlin Heidelberg, 2015).\nA cadaveric study. Journal of the Mechanical Behavior of Biomedical Materials\n172, 107180, doi:https://doi.org/10.1016/j.jmbbm.2025.107180 (2025).\ndefined acute crush injuries. Journal of Orthopaedic Research 10, 657-664,\ndoi:https://doi.org/10.1002/jor.1100100508 (1992).\nP\nA\nG\nE\n\\"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors made careful revision in this revised manuscript and address my concerns.I have no other comments.", "reviewer_2": "The authors have addressed all my concerns, and this manuscript is recommended for acceptance.", "reviewer_3": "the revision has addressed the reviewer's comment well. reviewer has no further comments"}, "author_response": ""}]

Decision letter