Research Of Modified Chitosan Hemostatic Sponge Based On Plant Polysaccharides/antibacterial Components

Traumatic hemorrhage is one of the crucial reasons for military and civilian casualties.The blood transfusions after massive bleeding may lead to death due to coagulation disorders and complications such as multiple organ failure.Therefore,timely hemostasis is essential to reduce blood loss and improve personnel survival.In the process of hemostasis,besides massive blood loss,external bacteria can quickly enter the body or deep tissues through the open wounds and cause infections,which may even lead to final sepsis and death.Therefore,it is essential to develop fast and efficient hemostatic materials with antibacterial properties.As a hemostatic dressing,the hemostatic sponge is suitable for complex wound treatment with a uniform porous structure supporting cellular interactions and promoting cell proliferation with relatively soft and breathable properties.Simultaneously,the hemostatic sponge can absorb a large amount of water,concentrate red blood cells,enhance the adhesion of red blood cells and platelets,thus accelerating blood clotting.Chitosan(CS),as a natural animal-derived polysaccharide,is widely used in the preparation of hemostatic dressings.However,the pristine CS hemostatic sponge can hardly meet these requirements for complex wound treatments.Therefore,in this study,the deacetylation degree was improved by the deacetylation of commercially CS by concentrated Na OH solution under mild heating conditions.The oxidation of CNC using Na IO₄ produced dialdehyde cellulose(DAC).The physical and chemical methods were used to combine and conjugate CS with CNC/DAC in various mass ratios(1:2,1:1,2:1,4:1,6:1,8:1,10:1)to obtain a series of composite sponges.The characterization of raw materials and composite sponges were characterized by FTIR,NMR,SEM,stress-strain test,water absorption performance,stability performance,i.e.,to screen the optimal preparation methods and targeted samples.After deacetylation treatment,the degree of deacetylation of the resulting CS increased from 66.0%to 99.3%,indicating more exposure of reactive-NH₂ groups as reactive sites.Through selective oxidation,the obtained DAC bear-CHO sites provided the basis for the subsequent preparation of hemostatic sponges through Schiff base crosslinking reaction with CS.The mechanical properties,stability,and water absorption of the composite sponges prepared by such a method were significantly improved compared with CS,which was significantly better than the physical method.Especially 2CS-DAC(m _CS:m _DAC=2:1)could absorb 56.9±6.5 times of its own weight of water in 10 s.It had good resilience porous structures,and its mechanical properties were not significantly affected by repeated compression after50 times.Then,the above validated composite sponges were then systematically analyzed for in vitro coagulation,hemolytic rate,mice and rabbit animal trauma models,and cytotoxicity.The hemostatic sponges'formulation and preparation process with good hemostatic properties and biocompatibility were screened and determined,and their coagulation mechanism was also preliminarily explored.Both in vitro and in vivo hemostasis experiments showed that the use of chemical composite sponges could reduce blood loss and significantly shortened hemostasis time compared with CS,CNC,and Celox^TM.The2CS-DAC samples showed excellent hemostatic effects in both thromboelastography(TEG)coagulation index and hemostasis models of mouse tail vein and rabbit femoral artery amputation.The 2CS-DAC shortened the hemostatic time by 39.8%,38.4%,and34.2%,and reduced the bleeding amounts by 66.0%,75.8%,and 64.7%,compared with gauze,CS,and Celox^TM,respectively in mice tails tests.Based on the complete coverage of the wound,1g 2CS-DAC shorten the hemostasis time by 47.7%,6.7%and 1.6%,and reduced the bleeding amounts by 70.5%,44.0%and 7.1%;compared with 8.0g gauze,3.7g CS and 3.0g Celox^TM,respectively in femoral artery hemostasis.So,2CS-DAC can achieve similar or even better hemostatic effects with lower amounts.The secondary damage was eliminated during wound debridement due to its softness and easy peeling off the mice's tail.Simultaneously,the chemically prepared composite sponge could maintain similar low cytotoxicity and good hemocompatibility compared with CS,CNC,and Celox^TM.Preliminary analysis of the coagulation mechanism showed that the composite sponge could greatly adhere/activate red blood cells and platelets to form a solid clot through the endogenous coagulation pathway due to the efficient water absorption capacity of its porous sponge structure and the synergistic electrostatic interaction with blood.Thus such sponges could accelerate the coagulation process,improve coagulation efficiency and reduce blood loss effectively at the same time.The screened composite sponge was modified with Ag NPs and GTMAC by physical or chemical methods.Its comprehensive performance was evaluated by FTIR,XRD,SEM,WBCT,hemolytic rate,and antibacterial experiments.The antibacterial composite sponges Agx@2CS-DAC loaded with Ag NPs were prepared by dip-coating into Ag NO₃solution and Na BH₄ reduction.We also prepare QCS with different substitution degrees by GTMAC modification onto CS chains,and then continue to synthesize the 2QCS-DAC composite sponge which were also evaluated by FTIR,XRD,SEM,in vitro coagulation,hemolytic rate,and the antibacterial properties against two colonies of E.coli and S.aureus.Antibacterial modification of composite sponges:The composite sponges prepared by physical method still maintained the original porous structure and softness,and different forms of nanoparticles were visible on the surface.The Ag NPs contents of the resulting Ag@2CS-DAC gradually increased as the concentration of Ag NO₃ impregnation solution increases.The WBCT showed that the incorporation of Ag NPs did not affect its coagulation performance.Simultaneously,the bactericidal rate was>99.9%against E.coli and S.aureus(10⁵ CFU/m L)of Ag_2.5/5/10/20@2CS-DAC at0.4mg/m L.However,the in vitro measurement showed that the hemolytic rates of all Ag_x@2CS-DAC composite sponges were much higher than the safety limit of 5%,which may possibly be caused by the release of Ag NPs.In future experiments,we would modify the composite sponge with Ag NPs by adding the crosslinking agent such as Genipin and glutaraldehyde to adjust the release contents of Ag NPs,to obtain safe and reliable Ag-containing antibacterial hemostatic materials.In the chemical modification,with the increase of m _GTMAC:m _NH2(CS)from 1:1,2:1 to3:1(X:1),the resulting QCS_xsubstitution was 39.7±0.6%,54.9±2.2%and 71.9±2.1%,respectively.Meanwhile,the zeta potential of QCS_x in acetic acid solution and aqueous solution increased with the increase of substitution degree,especially the zeta potential of QCS_x in aqueous solution were 25.2±1.5 m V,43.8±2.6 m V,and 56.7±4.2 m V,which is much higher than the 7.4±1.6 m V of CS dispersion.This was because the successful quaternary ammonium modification converted CS from acid-soluble to water-soluble with higher positive charges.The three QCS_x were then reacted with DAC according to2:1(m:m)to prepare antibacterial composite sponges,which still had good porous structures under SEM observation.Meanwhile,in vitro coagulation experiments indicated that the hemostatic time of the three 2QCS_x-DACs decreased significantly following the substitution degrees.In particular,the clotting time of 2QCS₃-DAC improved 76.6%,59.8%,and 44.0%than blank,Celox^TM,and 2CS-DAC,respectively,which may be inseparable from a large amount of positive charge.At 12.8 mg/m L,the three 2QCS-DAC composite sponges'bactericidal rates could reach above 98%against S.aureus(10⁵ CFU/m L),among which the 2QCS₃-DAC showed the best antibacterial ability.A similar trend can also be concluded in the case of against E.coli.The hemolysis experiments showed that the hemolytic rates of all the three composite sponges were lower than 5%,indicating that the introduction of quaternary ammonium groups did not affect the good hemocompatibility.Different concentrations of 2QCS₃-DAC immersion solution were used to analyze the effect of cell proliferation on L929 cells.As the concentration of the immersion solution decreases from 0.1 mg/m L to 12.8 mg/m L,the inhibition rate of cell proliferation gradually increased.In future research,it is expected to seek a balance concentration with low cytotoxicity while achieving high efficiency to kill bacteria.In this study,the antimicrobial hemostatic sponge with excellent properties was investigated using CS and CNC as raw materials via Schiff base reaction,nano-Ag and GTMAC as antimicrobial agents.The optimal composite sponges reported herein were expected to serve as a safe hemostatic material for hemostatic applications in civilian and military wounds treatments.Moreover,it has a broad potencial application in injectable hemostatic materials for emergency treatment of deep and complex wounds due to its good resilience ability.