Antibacterial and rapidly absorbable hemostatic sponge by aldehyde modification of natural polysaccharide

Severe hemorrhage from trauma is a leading cause of preventable death, particularly in military, pre-hospital, and emergency settings where rapid control of bleeding and infection is crucial. Ideal hemostatic agents should achieve rapid hemostasis, provide antibacterial protection, be easy to remove, promote healing, be affordable, stable, and biocompatible. Polysaccharide-based hemostats (e.g., dextran, cellulose, alginate, chitosan, starch derivatives) are attractive due to bioactivity and safety but often suffer from limited hemostatic activity, contamination risk, short shelf life, insufficient coagulation and anti-infection capabilities, and slow in vivo absorption—limitations that hamper their effectiveness for massive bleeding. Prior enhancement strategies include optimizing physical structure, chemical modification, bioinspired designs, and composites, yet challenges remain: potential toxicity of derivatives, slow absorption, difficult removal, complex fabrication, and low yield. This study addresses the need for a high-yield, biocompatible, rapidly acting, antibacterial, and rapidly absorbable polysaccharide hemostat suitable for massive hemorrhage by designing an aldehyde-modified riclin sponge (AR), optimized at 50% theoretical oxidation (AR50).

The authors review polysaccharide hemostats used clinically or commercially (dextran, cellulose: Surgicel, BloodSTOP; alginate: Algosteril, KALTOSTAT; chitosan: HemCon, Celox, Chito SAM; starch: TraumaDEX). While these materials are safe and available, they often lack strong hemostatic activity and antibacterial function, and may be slow to absorb in vivo. Chemical modification approaches (acylation, carboxymethylation, phosphorylation, sulfation, alkylation) and physical structuring (porosity, channel design, size) have improved performance. Bionic designs and composites can add antibacterial and adhesion properties. However, reports of polysaccharide-based hemostats validated in lethal large-animal hemorrhage models remain limited, and issues such as toxicity of derivatives, slow degradation, and removal difficulties persist. Riclin (a succinoglycan from Agrobacterium sp. ZCC3656) offers high yield and biocompatibility, motivating its selection for aldehyde modification to enhance both active and passive hemostasis.

Materials and synthesis: Riclin polysaccharide solutions (2 wt.%) were oxidized with sodium periodate (NaIO4) at theoretical oxidation ratios of 25%, 50%, 75%, and 100% to generate aldehyde-modified riclin (AR25, AR50, AR75, AR100). The oxidation cleaves vicinal diols to dialdehydes; partial hemiacetal formation and ring opening occur. After 2 h oxidation and 24 h dialysis (MWCO 500–1000 Da), solutions were freeze-thawed repeatedly and freeze-dried to form sponges. Chemical characterization: Degree of oxidation was quantified by hydroxylamine hydrochloride titration (AR25 19.2%, AR50 39.4%, AR75 50.2%, AR100 68.4%). 1H-NMR (aldehyde signals 9.0–9.3 ppm; hemiacetal 5.0–6.5 ppm), FT-IR (C=O at ~1720 cm−1 increasing with oxidation), XPS (appearance of C=O at 289 eV; increased O content), UV-Vis (absorption at 228–230 nm), zeta potential (riclin −38.5 mV; AR25 −22.4 mV; AR50 −18.2 mV; AR75 −18.0 mV; AR100 16.0 mV), and XRD (amorphous broad peaks) confirmed aldehyde introduction and structural changes. Physical and mechanical characterization: SEM assessed porous microstructure; AR sponges showed quasi-honeycomb channels whose aperture decreased with oxidation. Concentration effects (1–3 wt.%) were evaluated; AR50 at 2 wt.% showed stable macrostructure with high porosity (~92.5%) and mean channel diameter ~91.5 μm, rapid wetting, robust shape memory, and suitable compressive properties. Cyclic compression-decompression (up to 80% strain) assessed elasticity; AR50 maintained recovery over cycles with ultimate compressive strengths ~19 kPa; other AR levels showed brittleness/damage at high strain. Liquid absorption: Liquid absorption ratio (LAR) and kinetics in normal saline (NS), simulated body fluid (SBF), and whole blood were measured over 60 s; absorption speed (initial slope) quantified. Hemostatic assays in vitro: Whole-blood clotting index (BCI) and whole-blood clotting time (WBCT) compared AR50 to commercial hemostats (Celox, gelatin sponge, Surgicel, gauze). Thromboelastography (TEG) performed without kaolin activator to assess clot initiation/rate/strength. Plasma pathway assays measured PT, APTT, and TT using platelet-poor plasma (PPP), and plasma clotting kinetics over 60 min. Cellular mechanisms: Erythrocyte adhesion quantified by hemoglobin readout after lysis; erythrocyte clotting kinetics over 20 min; SEM assessed RBC morphology and polyhedral transformation over time. Platelet adhesion quantified by LDH assay at 15/30/60 min; Ca2+ fluorescence (Fluo-4 AM) assessed activation; flow cytometry (CD41a/CD62p) measured activation; SEM visualized platelet morphology/activation. In vivo hemostasis: Massive bleeding models were established in rats, rabbits, and pigs. Key large-animal models included porcine hepatic laceration (cross incision), hepatic perforation (2 cm hole), and femoral artery scission/laceration in rabbits and pigs. AR50 sponge was compared to gauze, Surgicel, gelatin sponge, Celox, Chito SAM, and QCG (kaolin gauze). Outcomes: hemostasis time and total blood loss; histology examined clot/wound interface; SEM of sponge-blood interface after treatment. Antibacterial testing: In vitro bacteriostatic activity against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa via inhibition zones, growth kinetics (OD600, CFU plating), and live/dead staining (SYTO 9/PI). SEM and TEM assessed bacterial ultrastructural damage. Degradation and biosafety: In vitro degradation in SBF (25 °C) and DPBS (37 °C); pH- and temperature-dependent degradation profiles (pH 5–10 at 25 °C and 37 °C). In vivo absorption via subcutaneous implantation in rats with serial retrieval (days 0–25) to measure residual mass and histology (H&E). Hemolysis assay with human RBCs, cytocompatibility (NIH3T3 proliferation, ROS), and rabbit skin irritation tests. Ethics: Human blood obtained under institutional approval with informed consent; animal protocols approved by Bengbu Medical College ethics committee.

- Optimized material: AR50 (theoretical 50% oxidation; measured ~39% aldehyde) formed a quasi-honeycomb, high-porosity sponge with rapid wetting, robust shape memory, and suitable elasticity/rigidity balance. - Ultra-high absorption: Blood absorption capacity reached 59.4 g g−1; rapid liquid infiltration through channels enabled fast uptake and compression stabilization. - Rapid clotting in vitro: AR50 showed the strongest BCI performance among tested hemostats and the shortest whole-blood clotting time (~50 s), with SEM showing progressive platelet activation and RBC aggregation from 15–240 s. - Plasma pathways: Minimal effect on PT/APTT; TT decreased for AR50/AR75, indicating accelerated fibrin formation in the common pathway. Plasma clotting kinetics stabilized fastest with AR50 (~5 min), suggesting limited but present fibrin formation; primary hemostatic activity stemmed from blood cell interactions. - Erythrocyte targeting: Highest RBC adhesion efficiency within 15 min (AR50 96.74% vs Celox 7.93%, gelatin 39.37%, Surgicel 61.72%). RBCs rapidly contracted into tessellated polyhedra, forming tightly packed clusters with stable clots (low hemoglobin absorbance by 4 min). - Platelet targeting and activation: Platelet adhesion rate increased to ~94.33% at 60 min. AR50 elevated intracellular Ca2+ fluorescence versus other hemostats and increased CD41a/CD62p double-positive platelets to 32.7% (vs blank 10.7%, spongy riclin 12.6%), indicating active platelet activation in addition to physical adhesion. - Large-animal hemostasis superiority: • Porcine hepatic laceration: AR50 achieved hemostasis in 68 s with 2.6 g blood loss (vs Surgicel 262 s/7.9 g; Chito SAM 284 s/10.4 g; gauze 312 s/15.4 g). • Porcine hepatic perforation: 140 s and 4.9 g (vs Surgicel 242 s/11.3 g; Chito SAM 332 s/19.8 g; gauze 436 s/25.4 g). • Rabbit femoral artery scission: 54 s and 2.6 g (vs Surgicel 119 s/6.6 g; gelatin+gauze 184 s/14.7 g; Celox 216 s/14.8 g). • Porcine femoral artery laceration: 140 s and 4.2 g (vs Surgicel 242 s/11.3 g; QCG 171 s/19.5 g; Chito SAM 126 s/21.3 g), highlighting markedly reduced blood loss and competitive or superior times to most comparators. SEM of in vivo clots showed dense clusters of activated platelets interwoven with polyhedral RBCs at the contact surface; limited fibrin presence suggested the sponge channels partly substitute fibrin’s mechanical role. - Antibacterial efficacy: Clear inhibition zones (E. coli 5.1 mm; S. aureus 4.2 mm; P. aeruginosa 2.0 mm); live/dead staining and CFU reduction confirmed superior killing versus other hemostats. SEM/TEM showed membrane/cell wall disruption, leakage, and vacuolization. Antibacterial potency increased with aldehyde content; mechanism attributed to aldehyde reactions with bacterial cell wall peptidoglycan/proteins plus prolonged contact via sponge channels. - Rapid degradation/absorption: In SBF at 25 °C, AR sponges fully degraded within 24 h (AR50 ~16 h); in DPBS at 37 °C, complete degradation within 15–60 min. Degradation accelerated with higher pH and temperature. In vivo, AR50 lost ~72.3% mass by day 2 and was virtually absorbed by days 10–15 without impairing healing. - Biocompatibility and hemocompatibility: No significant inflammation in surrounding skin; no organ pathology; rabbit skin irritation absent; hemolysis rate <0.5%; NIH3T3 proliferation and ROS assays indicated minimal cytotoxicity.

The AR50 sponge addresses critical shortcomings of current polysaccharide hemostats by combining rapid, high-capacity absorption with a microarchitecture that targets and organizes blood cells at the bleeding interface. Its quasi-honeycomb channels and aldehyde-modified chemistry promote robust RBC adhesion and polyhedral deformation, while actively stimulating platelets (intracellular Ca2+ elevation, CD62p upregulation) to form tightly packed, mechanically resilient cell clusters. This cell-centric hemostatic mechanism reduces reliance on fibrin formation, which is advantageous in high-pressure arterial and visceral bleeding where rapid clot mechanical integrity is essential. The material’s capacity to conform and maintain structure under compression further stabilizes hemostasis. In large-animal models of lethal hemorrhage, AR50 consistently shortened hemostasis times and markedly reduced blood loss relative to standard hemostats, particularly in liver injuries and arterial lacerations, demonstrating translational potential for pre-hospital and battlefield use. Its aldehyde-enabled antibacterial action against both Gram-negative and Gram-positive bacteria supports infection control at the wound site, and the rapid, pH/temperature-responsive degradation enables easy removal or safe absorption, mitigating risks of secondary injury from debridement. Collectively, these properties align with the clinical need for a rapidly acting, broadly applicable, and safe hemostatic agent.

This work introduces an aldehyde-modified riclin sponge (AR50) with a unique quasi-honeycomb architecture that delivers rapid, robust hemostasis by targeting erythrocytes and platelets to form tightly packed cell clusters with minimal dependence on fibrin. AR50 shows ultra-high blood absorption, strong shape memory, appropriate tissue adhesion, broad-spectrum antibacterial activity via bacterial cell wall disruption, and rapid in vivo absorption with excellent biocompatibility. In lethal porcine and rabbit hemorrhage models, AR50 reduced hemostasis times and blood loss compared with most commercial hemostats. Given the scalable production of riclin and straightforward fabrication, AR50 is a promising candidate for emergency, pre-hospital, and battlefield hemostasis. Ongoing preclinical and clinical evaluations will further establish its efficacy and safety in humans. Future work should optimize aldehyde content for maximal efficacy with minimal cytotoxicity, explore long-term outcomes in varied wound environments, and investigate integration with adjunct therapies or delivery systems.

- Clinical translation: Human clinical data are not yet available; current evidence is from in vitro assays and animal models. - Antibacterial spectrum: Reduced efficacy against Pseudomonas aeruginosa compared to E. coli and S. aureus, likely due to lower outer membrane permeability. - Aldehyde content balance: Higher oxidation enhances antibacterial action and degradation rate but can increase brittleness and potential cytotoxicity; optimization is required. - Coagulation pathways: While PT/APTT were unaffected and TT decreased, detailed effects on individual coagulation factors and long-term hemostasis stability in coagulopathic conditions were not fully explored. - Performance variability: Some comparators (e.g., Chito SAM in porcine femoral artery time) showed faster hemostasis time but with higher blood loss; broader standardized head-to-head studies are needed across injury types and conditions.