Construction Of Hemostatic-antibacterial Dual Functional Coatings Based On Self-assembly Nanohemostats And In Vitro And In Vivo Evaluations

Uncontrolled hemorrhage is the leading cause of traumatic death in warfare,accidents and disasters.In addition,owing to delayed hemostasis,massive bleeding in patients with coagulopathy is more difficult to control and can be life-threatening.Infection after injuries can seriously influence the prognosis of wounds,leading to increased burden on the wound care.Hence,there is an urgent need for advanced hemostatic materials with excellent hemostatic efficacy and superior antibacterial activity.Our group have previously reported a positively charged nanoparticle,abbreviated as PEI/UDCA nanoparticle,which was fabricated by ursodeoxycholic acid(UDCA)medicated self-assembly of polyethylenimine(PEI).PEI/UDCA nanoparticle can induce platelets activation and aggregation,thereby achieving rapid hemostasis in various bleeding models.Based on PEI/UDCA nanoparticle,herein we designed universal hemostatic-antibacterial dual-functional coatings which can achieve rapid hemostasis and infection prevention at the same time.Here,in situ oxidative polymerization of dopamine or tannic acid(TA)at alkaline pH value was conducted to prepare electronegative polydopamine(PDA)or TA coatings as the adhesive layer.Electrostatic force-induced deposition of positively charged PEI/UDCA nanoparticles(PUNPs)onto PDA or TA coatings gave rise to PDA-nanoparticles bilayer coatings(PDANPs)or TA-nanoparticles bilayer coatings(TANPs).PDANPs and TANPs coatings can induce platelets activation and aggregation,thereby minimizing blood loss in healthy and thrombocytopenic rodent animals after venipuncture.Apart from excellent hemostatic capacity,PDANPs and TANPs coatings also possess superior antibacterial activities against gram-positive and gram-negative bacteria.Methods1.Construction and characterization of PUNPsPUNPs(the weight ratio was 2:1 for UDCA: PEI)were fabricated by a dialysis method.The morphology of PUNPs was characterized through transmission electron microscopy observation(TEM).Nanoparticle size,size distribution,and zeta-potential were measured on Malvern Zetasizer Nano ZS instrument.2.Preparation and characterization of PDANPs needles and TANPs needlesBased on an immersive layer by layer assembly technology,PDANPs needles and TANPs needles were respectively prepared by successive immersion of cleaned needles in aqueous solution of dopamine(pH 8.5)and aqueous dispersion of PUNPs or aqueous solution of tannic acid(pH 7.8)and aqueous dispersion of PUNPs.Successful formation of PDANPs or TANPs coating on the surface of needles was confirmed by scanning electron microscopy(SEM),energy dispersive spectroscopy(EDS),and in vivo imaging system(IVIS).3.Effect of PDANPs and TANPs coatings on platelets activation and aggregationPlatelet rich plasma(PRP)was prepared according to Landsbergs method.Platelets labeled with FITC-anti-CD61 antibody were time-lapse photographed by confocal laser scanning microscopy(CLSM)during incubation with PDANPs or TANPs coatings to observe what happened on platelets.Colocalization of PUNPs and platelets on PDANPs or TANPs coatings was also determined by CLSM.Platelets activation induced by PDANPs or TANPs coatings was confirmed using CLSM by labeling activated platelets with PE-anti-CD62 P antibody.Platelets aggregation and morphological alternations on the coatings were further confirmed using SEM.4.In vitro thromboplastic behavior of PDANPs and TANPs coatings.Thrombocytopenic blood was prepared in vitro as previously reported.Fresh sodium citrate anticoagulant blood and thrombocytopenic blood were placed on PDANPs or TANPs coatings respectively for 40 min.The clots formed on PDANPs or TANPs coatings were assessed by SEM.5.Hemostatic capacity of PDANPs and TANPs needles in healthy rodent animals.Blood loss and bleeding times were quantified to assess hemostatic efficacy of PDANPs and TANPs needles after the rat femoral vein injection,the rat saphenous vein vertical penetration,and the mice tail vein injection.Bare needles served as a control.Femoral vein acupunctured with PDANPs or TANPs needles was excised and treated for SEM observation on the clots at the puncture site.Needles used in the mice tail vein injection were fixed,dehydrated,and observed using SEM to investigate platelets activa tion and aggregation on the PDANPs or TANPs needles.To demonstrate whether the coatings shed during venipuncture,IVIS was used to measure the fluorescence signals at the puncture site after the rat femoral vein and the mice tail vein injection with PDANPs-Cy5 and TANPs-Cy5 needles.And then the rat femoral vein was isolated and sectioned for CLSM observation.6.Hemostatic effects of PDANPs and TANPs needles in thrombocytopenic rats.Thrombocytopenia was induced by intraperitoneal injection of 20 mg/kg busulfan in rats.PDANPs and TANPs needles were employed to puncture or penetrate through the femoral vein and the saphenous vein of thrombocytopenic rats.After the needles were r emoved,blood loss and time to achieve hemostasis were measured to assess the hemostatic capability of PDANPs and TANPs needles.7.Antibacterial activities of PDANPs and TANPs coatings.Staphylococcus aureus(gram-positive)and Acinetobacter baumannii(gram-negative)were used to evaluate antibacterial activities of PDANPs and TANPs coatings.After incubation with PDANPs and TANPs coatings for 24 h,bacterial viability was determined by colony count assay and live/dead bacterial cell staining assay.SEM o bservation was also carried out to image morphological alternations of bacteria attached on PDANPs and TANPs coatings.8.In vivo biosafety evaluation of PDANPs and TANPs needles.Femoral vein of Sprague Dawley rats was punctured with PDANPs and TANPs needles and conventionally reared for 14 days after hemostasis.The body weight was daily monitored.After 14 days,rats were sacrificed and blood samples were drawn for hemat ological analysis.Major organs were isolated for histopathological section stained by H&E.Results1.Based on a dialysis method,self-assembly of UDCA and PEI at weight ratio of 2:1 generated well-shaped microsphere.The average size of PUNPs was 209±2 nm with centralized size distribution profile.PUNPs had a positive δ-potential of 67.3±0.6 m V.The existence of PUNPs on the surface of PDANPs and TANPs needles was confirmed using SEM,EDS,and IVIS which demonstrated the successful deposition of PUNPs onto the needles with the help of electronegative PDA and TA adhesive layer based on layer by layer assembly strategy.2.Real-time monitoring by CLSM showed that the number of platelets aggregations on PDANPs or TANPs coatings increased over time.In addition,during time-lapse imaging,platelets contraction was observed as well.Platelets activation induced by the coatings was confirmed by colocalization of CD62P(activated platelets)with CD61(all platelets).The colocalization of PUNPs with platelets on the coatings could be clearly observed,indicating that platelets aggregation and activation were induced by PUNPs on the coatings.Platelets aggregation and morphological changes resulting from platelets activ ation were further affirmed by imaging with SEM.Blood clots,composed of platelets,red blood cells,and f ibrin network,were observed on the coatings by SEM,indicating that the coatings exhibited potent procoagulant ability whether incubated with sodium citrate-anticoagulated blood or thrombocytopenic blood.3.To evaluate the hemostatic capability in vivo,the rat femoral vein,saphenous vein,and the mice tail vein were separately punctured using PDANPs and TANPs needles.Compared with bare needles,needles bearing PDANPs or TANPs coatings caused much less blood loss or even no bleeding.Platelets aggregation,morphological alternations,and small clots were observed on the surface of PDANPs needles used in mice tail vein injection,which revealed that PDANPs coating can activate platelets and accelerate blood clotting in seconds.The results of in vivo imaging and CLSM observation elucidated that there were the residual PDANPs or TANPs coating at the acupuncture site to promote coagulation continuously after the needles were removed.4.To demonstrate PDANPs and TANPs needles remain efficient in thrombocytopenia models,the needles were employed to perform femoral vein injection and saphenous vein vertically penetration in busulfan-induced thrombocytopenic rats.It’s encouraging that there were apparent decrease in blood loss and bleeding times,indicating that PDANPs and TANPs needles maintained their hemostatic capacities with no reduction in efficacy when the platelet count decreased to a half or even one-tenth of the normal level.5.No bacteria colony was observed on agar plate onto which the bacteria incubated with PDANPs or TANPs coatings were seeded,indicating that PDANPs and TANPs coatings efficiently eradicated S.aureus and A.baumannii in solution.CLSM images showed that almost all of the bacteria attached on the PDANPs or TANPs coatings were stained by SYTO 9 and PI meanwhile which revealed that very few bacteria on the coatings survived.After incubation with PDANPs and TANPs coatings,the cellular morphologies of S.aureus and A.baumannii observed by SEM exhibited collapsed shape with coarse and fracted cell walls.These results demonstrated the remarkable antibacterial activity against S.aureus(gram-positive)and A.baumannii(gram-negative)of PDANPs and TANPs coatings.6.The potential adverse effects were measured after the rat femoral vein injection with PDANPs and TANPs needles.Weight gain and organ index of major organs didn’t show any aberrance compared to control group.Hematological parameters,biochemical markers relevant to liver and kindey functions,and coagulation function were all in the normal range as well.Conclusions1.Based on an immersive layer by layer assembly strategy,we prepared PDANPs a nd TANPs hemostatic needles respectively.PDA coating and TA coating with negative charge were separately formed onto the surface of needles(26 G)by the self-polymerization of dopamine and tannic acid under the alkaline condition.Positively charged PUNPs were assembled onto PDA or TA needles through electrostatic interaction.2.PUNPs on the PDANPs and TANPs coatings can induce platelets activation and a ggregation,thereby promoting clot formation and achieving rapid hemostasis.It’s worth mentioning that PDANPs and TANPs coatings are able to potentiate thrombocytopenic blood coagulation as well.3.Blood loss and bleeding time can be minimized after venipuncture with PDANPs and TANPs needles in healthy and busulfan-induced thrombocytopenic rodent animals.Short-term safety study revealed that PDANPs and TANPs needles are safe enough for clinical application.4.Apart from hemostatic capacity,PDANPs and TANPs coatings also possess su perior antibacterial activities against Staphylococcus aureus(gram-positive)and Acinetobacter baumannii(gram-negative)which perfectly meet clinical needs for hemostatic dressings.