Yan Fang,Peiyuan Li,Weikang Zhou

Fujian Provincial Key Laboratory of Polymer Materials,College of Chemistry and Materials Science,Fujian Normal University,Fuzhou,Fujian Province,350007,China

Abstract:Rapid control of heavy hemorrhaging is critical to save the life of injured individuals.Herein,we developed a novel hemostat employing chitosan-based microspheres,which was prepared by sequential microemulsion of chitosan solution,sol-gel phase transition,and surface modification by 3,4-dihydroxyhydrocinnamic acid(HCA).HCA-modified chitosan microspheres(CSMS-HCA)displayed a porous structure,with a high specific surface area(~211.3 m2/g),accelerating their rapid absorption of blood and concentrating red cells and platelets.The CSMS-HCA exhibited much better in vitro and in vivo hemostatic efficacy than porous chitosan microspheres did.Such a rapid hemostat in the form of microspheres is highly effective in treating deep and irregular wounds,owing to easy accessibility to injured sites.

Keywords:chitosan;microspheres;hemostasis;wet-tissue adhesion

1 Introduction

Uncontrollable hemorrhage is a major factor for mortality from traumatic injuries,which frequently occurs in battlefields,civilian accidents,and in operating theatres[1].As reported,almost 30% of trauma deaths result from excessive bleeding,and nearly 50% of these deaths occur before emergency medics arrive[2-3].Therefore,timely and effective hemostasis is urgently required to treat life-threatening traumatic bleeding.

At present,numerous materials in various forms,such as gauze,sponges,hydrogels,and powders,have been delicately designed and developed for rapid and effective control of bleeding[4-9].Among them,powder hemostatic agents are the most popular,as they can be applied to wounds of any shape and depth,and can be used to suppress external hemorrhage that is not amenable to other bleeding control methods,including direct pressure applied to vessels,compression dressings,and bands[8].As one of the prime candidates,chitosan has been frequently applied to fabricate hemostatic powder,due to its abundant availability,inherent pharmacological properties,and beneficial biological characteristics such as biocompatibility, biodegradability, anti-infection,hemostatic activity,healing acceleration,non-toxicity,and low-immunogenicity[8,10-11].The hemostatic properties of chitosan are attributable to its cationic nature[12].During the initial hemostasis stage,adhesion,activation,and aggregation of platelets play critical roles.Because the surface membranes of erythrocytes and platelets are negatively charged,the positively charged chitosan electrostatically attracts erythrocytes and platelets,inducing them to enmesh and form a clot.Additionally,chitosan adsorbs fibrinogen and plasma proteins,enhancing platelet aggregation[13].Meanwhile,this mechanism does not depend on a patient's own clotting mechanisms and is therefore effective even in patients with coagulation disorders.Therefore,chitosan has been produced on a large scale and commercialized,primarily in the form of powder[8].However,chitosan powder is not suitable for controlling severe bleeding because it lacks wet tissue adhesion ability.When they were applied to severe bleeding,the powder was easily dispelled by outflowing blood,leading to poor hemostasis.

Recently,mussel-inspired tissue adhesive materials have attracted significant attention[14-16].3,4-dihydroxy-L-phenylalanine(L-DOPA),which was first found in the byssal threads of marine mussels and later in adhesion pads,is generated by tyrosine-mediated enzymatic conversion from tyrosine to L-DOPA[17].The catechol ortho-dihydroxyphenyl group,which is the side chain of L-DOPA,is responsible for mussel adhesion,and exhibits superior adhesiveness to various organic/inorganic surfaces[18].The catechol group may interact with substrates through hydrogen bonds,covalent bonds,hydrophobic interactions,metal coordination,covalent crosslinking,π-πstacking,and cationic-πinteractions.Therefore,the adhesive moieties of L-DOPA have frequently been introduced into various polymers,such as hyaluronic acid,alginate,heparin,poly(vinyl alcohol)(PVA),and poly(acrylic acid),to enhance their adhesive properties.Liao et al[19]found that the adhesion strength to porcine skin of a conductive hydrogel consisting of PVA,polydopamine(PDA),and carbon nanotube(CNT)was 5 kPa,while that of PDA-PAAm (PAAm=polyacrylamide)copolymer hydrogel to porcine skin could reach a maximum of 15 kPa when the dopamine/acrylamide(DA/AA)ratio was 0.8 wt%.Another DA-modifiedεpoly-L-lysine-polyethylene glycol-based hydrogel showed a much improved adhesion strength of 147 kPa to porcine skin[20].Hence,in this work,through biomimicking mussels,we sought to introduce catechol groups onto the chitosan powder to enhance its wet tissue adhesion properties.The physical and chemical structures of catechol-conjugated chitosan powder were systematically characterized.Itsin vitroblood coagulation behavior andin vivohemostasis performance involving non-compressible wounds were investigated.

2 Materials and methods

2.1 Materials

Chitosan powder(CSP,degree of deacetylation≥95%,viscosity 100-200 mPa·s)was purchased from Shanghai Aladdin Industrial Corporation,China.Tetraethyl orthosilicate (TEOS, AR),cetyltrimethylammonium bromide(CTAB,AR),acetic acid(CP),Span 80(CP),Tween 60(CP),ethanol(AR),NaOH(AR),petroleum ether(AR),3,4-dihydroxyhydrocinnamic acid(HCA),and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC)were supplied by Sinopharm Chemical Reagent Co.,Ltd.,China.Sprague-Dawley(SD)rats approximately 200-250 g in weight were bought from Shanghai Slack Experimental Animal Co.,Ltd.,China.

2.2 Preparation of chitosan microspheres

Specifically,1 g chitosan powder was dispersed in 99 mL distilled water.Then,1 mL acetic acid(AcOH)was added to obtain a 1%(w/V)chitosan solution.1 g NaOH was dissolved in 150 mL ethanol solution(water:ethanol=1∶14,V/V)and pre-cooled at-20℃as an inverse solution.The emulsifier(Tween 60∶Span 80=0.2∶4.8,w/w)was added to petroleum ether to form a continuous phase solution with a total emulsifier content of 5%(w/V).The dispersed phase was added dropwise to the continuous phase under magnetic stirring(1200 r/min)and emulsified at 40℃for 3 h to obtain a W/O emulsion.Then,it was poured into liquid nitrogen,followed by the addition of the precooled inverse solution to regenerate chitosan microspheres.The product was washed to neutral pH value with water and ethanol in sequence,and then dried at 50℃in a vacuum oven to obtain porous chitosan microspheres(CSMS).

2.3 Preparation of catechol-conjugated CSMS

HCA(0.8 equivalent to amines on chitosan)was added to the CSMS suspension.After EDC(1 equivalent to HCA)was dissolved in ethanol(50 mL),the solution was added dropwise to the chitosan/HCA aqueous solution.The coupling reaction proceeded for 1 h(pH value=4.5).To remove unreacted reagents,the solution was dialyzed against HCl(pH value=3-4)containing NaCl for 2 days using a regenerated cellulose membrane(molecular weight cutoff of 12000-15000).In addition,after further dialysis against HCl for 2 days,the solution was dialyzed against deionized water for 6 h.The final product was lyophilized,and the catechol-conjugated chitosan microspheres were coded as CSMS-HCA.

2.4 Characterization

The morphology of the specimens was observed using a scanning electron microscope(SEM,JSM-7500F,JEOL,Japan).All samples were coated with gold using an ion sputter coater at 30 mA for 90 s before SEM observations.The structures of CSMS and CSMS-HCA were characterized by Fourier transform infrared spectrometry(FT-IR)in KBr form(model 1600,Perkin Elmer Co.,USA).Thermal stability was analyzed using a thermogravimetric analyzer(TGA,TGA/SDTA851e,Switzerland).Microsphere diameters and surface pore sizes were measured using Smileview(version 2.0).Statistical average values were reported for the measurement of at least 100 microspheres at different locations.Excess phosphate-buffered saline(PBS)was removed using filter paper.The specific surface area was measured by nitrogen adsorption-desorption isotherm analysis on a BELSORP-mini II multi-station automatic surface area and porosity analyzer(MicrotracBEL Japan,Inc.).

The crystal structures of CSMS and CSMS-HCA were elucidated using X-ray diffraction(XRD)patterns with Cu Kαradiation(λ=0.15406 nm)at 40 kV and 30 mA,recorded in the 2θrange from 5° to 45°.The samples were ground into powders and dried in a vacuum oven at 60℃for 48 h.The crystallinity index(CrI)of chitosan samples was determined using Eq.(1).

whereI110is the maximum intensity(2θ=20°)of the(110)lattice diffraction,andIamis the intensity of amorphous diffraction at 2θ=16°of chitosan samples.

2.5 Blood cell and platelet adhesion

Samples were pre-warmed in PBS(pH value=7.4)at 37℃for 20 min,followed by removal of PBS.Then,fresh citrate-anticoagulated rat blood was slowly added dropwise onto the surface of the samples.The cells were incubated at 37℃for 1 h.Subsequently,the resultant samples were washed liberally with PBS(pH value=7.4)to remove unbound blood cells.The samples were then treated with 2.5% glutaraldehyde for 24 h.The samples were dehydrated with 10%,20%,30%,40%,50%,60%,70%,80%,90%,and 100%ethanol for 10 min.Finally,the resultant samples were dried at 37℃for 48 h,and then observed using SEM.

2.6 In vitro pro-coagulant activity

The animal care and study in this research followed the rules of the Research Animal Ethics Committee of Fujian Normal University.At the end of each animal experiment,rats were euthanized by exsanguination under anesthesia.A 2-mL disposable plastic tube with 10 mg sample was placed in a 37℃water bath.Fresh blood was obtained from rat heart with a medical vacuum collection tube containing sodium citrate anticoagulant(3.8%sodium citrate∶blood=1∶9,V/V),and was maintained at 37℃.One milliliter of the asobtained anticoagulated blood was added to the sample tube.Time was recorded immediately after addition of 100μL 0.1 mol/L CaCl2aqueous solution to the blood.The tube was tilted every 10 s to observe if the blood had gelatinized.The clotting time was recorded when the blood completely lost flowability.Blood without microspheres was used as a blank control.Each sample was repeated six times.

2.7 In vivo hemostasis

Hemostatic performance was evaluated in rat femoral artery and liver injury models.Forty-eight male rats were randomly divided into eight groups,and each group comprised six rats.Rats were anesthetized by injecting 1 mL 10% chloral hydrate into the peritoneal cavity.The liver was exposed using a scalpel and scissors.A severe bleeding wound with a length of 10 mm and depth of 5 mm was made to the liver.Samples(1 mg)were placed on the wound immediately and pressed.Blood loss and hemostatic time were recorded.For hemostasis in the rat femoral artery injury model,the left femoral artery was exposed using a scalpel and scissors.A severe arterial bleeding wound was created by completely cutting off the rat femoral artery.Samples(1 mg)were placed on the wound immediately and pressed.Blood loss and hemostatic time were recorded.The tested rats were euthanized with an overdose of chloral hydrate at the end of the experiment.

3 Results and discussion

3.1 Preparation and structure of CSMS and CSMSHCA

The fabrication process of CSMS-HCA is schematically presented in Fig.1.First,CSMS was prepared by microemulsion formation,together with the sol-gel method.Chitosan was dissolved in acetic acid to obtain a homogeneous and transparent solution.Tween 60/Span 80 used as an emulsifier was added to obtain a colloidal suspension.A mixture containing NaOH,water,and ethanol was employed as an inverse solution to cause the microspheres to aggregate into a new phase to form a gel.After freeze-drying,CSMS were obtained.To prepare catechol-conjugated microspheres,carboxylic acid-terminated catechol,i.e.,HCA was employed,which was conjugated with amine residues in the chitosan backbones via carbodiimide coupling.

The structures of CSMS and CSMS-HCA were systematically characterized by FT-IR,XRD,and TG.The FT-IR spectra of CSP,CSMS,and CSMS-HCA are displayed in Fig.2.The CSP spectrum displayed a series of characteristic bands at 3438,1671,and 1592 cm−1,which were assigned to the N—H stretching,amide I,and amide II stretching vibrations of chitosan,respectively.CSMS displayed a similar spectrum to that of CSP,suggesting that the chemical structure of chitosan was not destroyed by the"sol-gel"process.Compared with CSP and CSMS,a new peak at 1180 cm−1was ascribed to the C—O vibration of nonoxidized catechol that emerged in the CSMS-HCA spectrum,suggesting that HCA was successfully conjugated onto chitosan.

The XRD patterns of CSP,CSMS,and CSMS-HCA are presented in Fig.3.Three crystalline diffraction peaks were observed in the CPS,indexed as(020),(110),and(130)lattice diffraction of chitosan.However,only two reflection peaks,without(130),were observed in CSMS and CSMS-HCA.Furthermore,the calculatedCrIof CPS was only 42.95%,which was lower than that of CSMS(51.58%)and CSMS-HCA(52.23%).These results suggested that original chitosan chain packing was restructured,and chitosan chains were restructured to form more regular aggregate architecture during the"sol-gel"process,leading to a higherCrIvalue.It is noted that theCrIvalue of CSMS was similar to that of CSMS-HCA,indicating that the structure of CSMS was retained well during the chitosan-catechol preparation.

The TG and DTG thermograms of CSMS and CSMS-HCA(50℃-600℃)are presented in Fig.4.The thermal decomposition process of CSMS is divided into three stages:water evaporation,depolymerization,and residue decomposition & pyrrole ring breakage.The fastest pyrolysis temperature of CSMS-HCA was at approximately 295℃,which was comparable with that of CSMS(300℃).These observations suggested that CSMS-HCA is thermally stable,and suitable for hemostasis inclusion.

The surface structures of CSMS and CSMS-HCA were observed by SEM.As shown in Fig.5,CSMS had a size of approximately 350μm,with a porous structure from the surface to the interior of the microspheres.SEM visualization of CSMS-HCA demonstrated a rich interconnected interior pore structure similar to that of CSMS.Hence,the catecholconjugated process had little influence on the structure of CSMS,which is in accordance with XRD results.The porous structures of CSMS and CSMS-HCA were further characterized by nitrogen adsorption-desorption isotherm analyses(Fig.6).The corresponding physicochemical parameters,such as the BET specific surface area(as,BET),pore size,and pore volume,are listed in Table 1.As shown in Fig.6,the isotherms of CSMS and CSMS-HCA were both identified as type IV,which is a characteristic of mesoporous materials.The corresponding average pore diameters of CSMS and CSMS-HCA were both in the range of 17-19 nm(Table 1).The porosities of CSMS and CSMS-HCA were 46.64% and 47.48%,respectively.Furthermore,the BET specific surface areas of CSMS and CSMSHCA were 183.9 m2/g and 211.3 m2/g,respectively.Such a porous structure with a high specific surface area would facilitate microspheres to rapidly absorb water in blood,thus concentrating clotting factors needed for hemostasis.

3.2 In vitro blood coagulation behavior

The hemostatic efficiency of a material is related to its coagulation ability,which is indexed to blood coagulation time.As shown in Fig.7,the blood gelation time of CSMS was(124.2±3.9)s,which was shorter than that of the control(blank)group.It was noted that the blood gelation time was shortened to(118.6±1.9)s after modification of CSMS by HCA,suggesting that thein vitroblood pro-coagulation efficiency of CSMS-HCA was significantly better than that of CSMS.To investigate coagulation mechanisms involving CSMS-HCA,blood clotting on the tested hemostats was observed by SEM.As shown in Fig.8,many red blood cells(RBCs)and platelets adhered to the surfaces of the CSMS.However,as for CSMSHCA,there were not only numerous red blood cells and platelets,but also fibrins distributed across the surface.As reported,fibrin is a key parameter during the hemostatic process,as it can cross-link platelets and blood cells to form a three-dimensional network structure,leading to the formation of blood clots and thereby improving hemostasis.

Table 1 Physical parameters of CSMS and CSMS-HCA

3.3 In vivo hemostatic evaluation

Thein vivohemostatic performance of CSMS-HCA was evaluated using a rat liver injury model,and a rat femoral artery injury model.Commercial cotton gauze was used as a control.As shown in Fig.9(a),the hemostatic time of cotton gauze was up to(211.8±2.1)s and(109.0±2.0)s for the liver and femoral artery injury models,respectively.As for CSMS,the hemostatic time was shortened to(79.2±2.5)s and(68.0±2.0)s for the liver and femoral artery injury models,respectively.It was noted that CSMS-HCA exhibited superior hemostatic performance to CSMS.The hemostatic time was only(44.0±2.6)s and(52.2±1.6)s for the liver and femoral artery injury models,respectively.Digital images of the hepatic and femoral artery injury wounds covered with CSMS-HCA are shown in Fig.9(b)and Fig.9(c),respectively.When bleeding ceased,dark-red blood clots consisting of gelled blood and microspheres formed on the wounds.The outstanding hemostatic performance of CSMS-HCA is ascribed to the following factors:①the porous structure of CSMSHCA can absorb plasma rapidly to concentrate red blood cells and platelets to form RBC-platelet clots;②the catechol groups on the surface of the microspheres tightly adhered to tissue cells surrounding the wound by hydrogen bonding,and formed a closed system to prevent blood outflow,which caused platelets and erythrocytes to further accumulate and form a thrombus to quickly control blood loss;③catechol groups with a negative charge also activated coagulation factor XII of the"clotting cascade"to promote hemostasis.

4 Conclusions

Catechol-conjugated microspheres were successfully prepared by sequential microemulsion of chitosan solution,sol-gel phase transition,and surface modification by 3,4-dihydroxyhydrocinnamic acid(HCA).HCA-modified chitosan microspheres(CSMSHCA)displayed a porous structure with a high specific surface area(~211.3 m2/g),accelerating the rapid absorption of blood,and concentrating red blood cells and platelets.CSMS-HCA microspheres showed much betterin vitroandin vivohemostatic efficacy than porous chitosan microspheres(CSMS)did.Such a rapid hemostat in the form of microspheres may be highly effective in treating deep and irregular wounds,owing to ready access to injured sites.

Acknowledgments

This work was supported by the Natural Science Foundation of Fujian Province(2019J05059)and the Social Development of Instructive Program of Fujian Province(2017Y0025).