Hemostatic powders are widely used in clinical and emergency settings, but typically present with low wet adhesion, cytotoxicity issues, and are not suitable for fatal incompressible bleeding. Decheng Wu/Chong Zhang, Hospital of Southern University of Science and Technology and Mileage Zhang, co-developed a novel gel-viscous powder (GAP), which integrates chitosan microspheres (CM), four-arm polyethylene glycol amine (Tetra-PEG-NH2) and four-arm polyethylene glycol succinimidyl succinate (Tetra-PEG-SS). When applied to the wound site, macroporous CM can quickly absorb the interfacial fluid, and at the same time, hydrated GAP becomes a hydrogel with stable and firm adhesion to wet tissue by covalent bonding. In vitro and in vivo results show that the optimized formulation of GAP has strong tissue adhesion, high burst pressure and enhanced blood clotting capacity, as well as excellent biocompatibility and on-demand removal performance. Compared with CM, commercial fibrin glue and Yunnan baiyao (YB), the hemostatic effect of rat liver, spleen and femoral artery injury models was significantly improved. GAP also stops severe bleeding from the pig's internal organs. In general, the proposed GAP has many advantages such as good biocompatibility, fast and effective hemostasis, low cost, and easy to use, and is a promising hemostatic agent for lethal incompressible hemostatic drugs. The study was published in Advanced Functional Materials as a paper titled "Gelable and Adhesive Powder for Lethal Non-Compressible Hemorrhage Control."
The study describes a novel gel-viscous powder (GAP) for the control of fatal incompressible bleeding. To achieve fast and effective hemostasis, GAP integrates three different components, including chitosan microspheres (CM), four-arm pegylated amine (Tetra-PEG-NH2), and four-arm polyethylene glycol succinimide succinate (Tetra-PEG-SS). CM's macroporous structure enables rapid removal of interfacial fluids, which is essential for immediate and robust wet adhesion in wet environments. Positively charged chitosan is also active in promoting blood clotting. GAP is designed to synergistically address these limitations by rapidly absorbing blood and promoting blood clotting while gelling in situ, forming a strong viscous hydrogel to seal bleeding injury. The biocompatibility of GAP was investigated by in vitro cytotoxicity and in vivo rat subcutaneous implantation assays. Compared with CM, commercial fibrin glue and Yunnan baiyao (YB), the gel significantly improved blood cell/platelet adhesion, clotting capacity, and hemostasis in rat liver, spleen, and femoral artery injury models. In addition, GAP-derived adhesive hydrogels can also be safely removed from tissue surfaces as required.
Programme 1. Design and mechanism of gel-viscous powders (GAP).
【Preparation and gelation capacity of GAP】
The improvement in the absorption capacity of CM liquid is mainly attributed to the improvement of porosity. CM made from 1.5 wt% chitosan concentration was chosen for this study due to the high porosity and liquid absorption capacity leading to rapid removal of adhesion of the interface fluid. The Tetra-PEG component plays a key role in GAP gelation and the gelation ability is enhanced at higher Tetra-PEG content. Thus, the formation of GAP-derived hydrogels upon hydration can be attributed to the efficient crosslinking reaction between the NHS ester of Tetra-PEG-SS and the primary amine group of Tetra-PEG-NH2.
Figure 1. Physical characteristics of CM and GAP
【Adhesion properties】
The torsion test was first used to evaluate the adhesion performance of GAP deposits on different wet pig tissues (skin, blood vessels, spleen, and heart). GAP-3-derived hydrogels can withstand deformations caused by various external forces, such as twisting, bending, scratching and water rinsing without peeling after 30 s of hydration, revealing the formation of stable and robust hydrogel-tissue bonding interfaces. The burst pressure was further tested using a homemade unit to evaluate the adhesive and sealing capabilities of the engineered powder. In the test powder, GAP-3 had a burst pressure ≈8.63 times higher than fibrin glue, and this value was also significantly greater than the normal systolic blood pressure (≈ 120 mmHg), supporting the formation of a firm adhesion and seal, which is especially necessary in the case of pressurized arterial bleeding.
After applying GAPs to wet tissue, the dehydrating nature and porous structure of CM enable rapid and efficient absorption of interfacial water, which is then triggered by covalent crosslinking of Tetra-PEG-SS with Tetra-PEG-NH2/CM to trigger in situ self-gel. At the same time, the removal of the interface water enables the formation of stable covalent bonds by a reaction between the terminal NHS ester and the amine/thiol group on the underlying tissue. Ultimately, GAP is transformed into a viscous hydrogel with a strong adhesion interface that is beneficial for keeping wounds sealed, maintaining a moisture environment, minimizing potential tissue damage, promoting wound healing, and more.
Figure 2. Adhesion properties of GAP on wet biological tissues
Trigger attribute removal
In the presence of the basic drug cysteamine (CA), Tetra-PEG hydrogels show accelerated dissolution by succinyl ester bond breakage. Gently wipe the GAP-induced adhesive layer on pig skin using a cotton swab dipped in cysteamine solution, PBS as a control. After 10 min of wiping with a PBS-soaked swab, a large amount of hydrogel remains and adheres tightly to the tissue. The adhesive hydrogel treated with CA showed significant disintegration within 5 minutes and was almost completely removed within 10 minutes with no signs of tissue damage, indicating promising clinical applications as removable wound sealants.
Figure 3. Adhesion mechanism and on-demand removal performance of GAP
【In vitro coagulation capacity】
The in vitro coagulation capacity of GAP is first assessed by examining the clotting time after co-incubation with whole blood. GAP was found to have excellent and effective clotting capacity. Further red blood cell and platelet adhesion assays were performed to better understand blood clotting mechanisms. The results showed that the porous structure of CM can also promote rapid absorption of whole blood and concentrated blood cells. More importantly, gelation on GAP contact with blood enables the enrichment of blood cells through physical retention and chemical interactions between Tetra-PEG-SS and the cell surface. Therefore, the excellent procoagulant ability of GAP-3 can be attributed to the synergistic effect of CS procoagulant activity, rapid and strong blood absorption and its self-gelling ability.
Figure 4. In vitro coagulation properties of GAP
【Biocompatibility and degradability】
The biocompatibility of hemostatic materials is a key prerequisite for their future clinical applications. First, hemolytic activity assays were carried out and their excellent hemocompatibility as blood contact materials was found. Then, in vitro cytocompatibility is assessed by CCK-8 and live/dead staining assays. The GAP group showed cell viability and morphology comparable to the control group. The results showed that the engineered composite powder had excellent cytocompatibility. In addition, it also has good in vitro degradation ability.
Figure 5. In vitro and in vivo biocompatibility of GAP
【Hemostasis performance in the body】
The liver and spleen are highly fragile organs and are considered the most commonly injured internal organs after abdominal trauma. The hemostatic sealing performance of the engineered rubber powder in vivo was first evaluated in a rat incompressible hepatosplenoid hemorrhage model. In short, GAP-3 reduced blood loss and hemostasis by 92% and 80% on average, significantly higher than 30-35% and 45-50% in the CM and YB groups.
The use of a rat femoral artery hemorrhage model further challenges the hemostatic ability of the binder powder. Arterial vessels are tranverted to induce bleeding. The application of gauze to the injured oral cavity failed to stop arterial bleeding, while GAP-3 quickly absorbed blood and formed a strong seal within 3 minutes to stop bleeding, and no secondary bleeding occurred during the observation period of 30 minutes.
Figure 6. In vivo hemostatic efficacy of GAP in a rat model
In order to further evaluate the hemostatic efficacy in vivo and demonstrate the potential application of this engineered powder, a preclinical functional evaluation was carried out in a pig internal organ bleeding model. After depositing GAP (≈2 g) on the wound under gentle pressure, the initial massive bleeding can be controlled within 3 minutes. The resulting hydrogel and blood clot complex remained stable, and no second bleeding occurred after water flushing and manual shaking of the liver and spleen. It is verified that the adhesive powder can provide a strong adhesive and efficient hemostatic seal for fatal and incompressible pig visceral injuries, showing high practical application potential.
Figure 7. In vivo hemostatic efficacy of GAP in pig models
【Summary】
The study describes a method for the treatment of fatal incompressible bleeding by integrating macroporous CM and Tetra-PEG-based components (Tetra-PEG-NH2/Tetra-PEG-SS). The resulting GAP can quickly absorb the interface fluid and promote blood clotting, while spontaneously forming a strong tissue adhesion hydrogel in situ, which synergistically achieves efficient hemostasis. The results show that the optimized formulation of GAP-3 has strong tissue adhesion strength, high burst pressure and enhanced coagulation performance, and has excellent biocompatibility in both in vitro and in vivo tests. In rat models of liver, spleen, and femoral artery injury, GAP-3 was superior to CM, commercial fibrin glue, and YB in hemostatic effects. What's more, GAP-3 can stop severe bleeding from pigs' internal organs. In addition, GAP can also be safely removed as needed without causing damage. Overall, comprehensive in vitro and in vivo data support the high potential of engineered GAP as a hemostatic agent for emergency applications.
However, the research still has a lot of room for improvement in future work. For example, when compared to injectable hydrogels or self-expanding porous materials, GAP may have difficulty entering deep bleeding sites such as gunshot or knife wounds to promote hemostasis. The hemostatic effect of GAP should be further validated using trauma models with more fatal bleeding. Additional long-term in vivo experiments are required prior to clinical practice to comprehensively examine the degradation, biotoxicity, and wound healing properties of GAP. Future work will also focus on a deeper understanding of in situ gels and adhesion mechanisms to guide the development of highly efficient hemostatic materials.
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Original link:
https://doi.org/10.1002/adfm.202305222