In order to achieve satisfactory hemostatic efficiency, the selection of proper components for preparation of the powdery hemostatic agent is critical. In this study, we chose sodium alginate and chitosan as base materials, not only because they have good properties, such as biocompatibility and biodegradation, but also because they both have certain hemostatic abilities with different hemostatic mechanisms. Moreover, chitosan is a natural cationic polysaccharide polymer; the cationic nature and high charge density of chitosan in solution allows it to form stable ionic complexes with multivalent water-soluble anionic polymers under mild physiological conditions [22
]. Alginate is an anionic polysaccharide polymer that can form complexes with polycations such as calcium, chitosan, and polylysine [27
The chitosan-alginate microparticles can be prepared using the gelation method or by emulsification and cross-linking technology. The driving force for the microparticle formation in the gelation method is the decrease in solubility. The mutual neutralization between oppositely charged alginate and chitosan decreases the solubility of the entire system. On the other hand, the guluronic acid moieties of alginate interact with calcium ions to form so-called “egg-box” structures, in which two adjacent macromolecules are linked by means of a physical bond [29
]. However, in emulsification and cross-linking, the formation of chitosan-alginate microspheres is mainly caused by the differences in their solubility in organic solvents and aqueous solutions.
Due to the porous structure, which can retain a significant amount of water within the pores, the water uptake ability of the microparticles prepared in this study was much better than that of the microparticles prepared by the gelation method.
The cytotoxicity of the microspheres was evaluated in L929 mouse fibroblast cells. As the main cellular components of connective tissues, fibroblast cells are widely used in cytotoxicity studies of biomaterials [30
]. In order to avoid confounding factors resulting from the physical trauma of the microspheres, the extract dilution method was chosen over the direct exposure method [31
]. We think that the inhibition of the extract on the cell proliferation during the initial period may be due to the leakage of micro-amounts of organic compounds which remained in the microsphere. However, owing to the promotion of cell proliferation by chitosan [32
], with the increase of culture time, the influence of micro-amounts organic compounds was counteracted, and the cells recovered gradually. Thus, the relative cell proliferation rate reached 100% in later periods of cell culture.
The use of chitosan and sodium alginate carriers as drug delivery systems has gained wide interest [33
]. Generally, drug release from carrier is affected by many factors, such as the properties of materials used in the carrier, preparation method of the carrier, and pH values and ionic strength of the release media. First, the drug-loaded particles were prepared by dissolved tranexamic acid in the chitosan/alginate colloidal solution. Then, the mixture was added drop-wise to petroleum, and with the formation of the particles, tranexamic acid was encapsulated in the particles. Interestingly, we found that most of the tranexamic acid was removed during the subsequent purification process. Therefore, we initially prepared porous composite particles with chitosan and sodium alginate, and then using physical absorption, the tranexamic acid was loaded on the composite particle.
Because the particles come in contact mainly with blood when applied to wounds, and the composition of blood can affect the release of tranexamic acid, we carried out the drug release tests in both PBS and anticoagulated blood plasma. The initial burst release clearly revealed that the tranexamic acid was absorbed mainly on the surface of the microspheres, and the porous structure of the particles facilitated the release of tranexamic acid. However, the burst release of tranexamic acid was exactly what we expected, because high doses of tranexamic acid are required in direct hemostatic application. Owing to the fact that the pH value of the PBS solution used in this study was almost equal to that of anticoagulated blood plasma (7.4), but the ionic strength of both systems was different, we attributed the diversity of tranexamic acid release in both systems to the ionic strength and the influence of proteins in the plasma. However, identifying the detailed mechanism behind this will require further research.
Flashclot is developed by the Honghua Pharmaceutical Co. Ltd and the Fourth Military Medical University. Because its hemostatic performance is similar to Quickclot, Flashclot is also defined as quick-acting styptic powder [36
]. Flashclot is made of zeolite, which has a porous structure. In this paper, Flashclot was used as the control to evaluate the hemostatic performance of alginate/chitosan microspheres. As mentioned previously, the alginate/chitosan microparticles also have many small pores on their surfaces. When applied to wounds, the micropores can quickly absorb the water from blood and cause the aggregation of platelets, blood coagulation factors, fibrins, red blood cells, and blood clotting factors to the surface of particles to form a blood clot, and to seal the crevasse of blood vessels. In this study, unlike Flashclot, the alginate/chitosan microspheres did not generate heat when absorbing water from blood and did not result in reinjury of the treated liver.
In addition to the water absorbing ability of the micropores, the positive charge on the surface of chitosan can attract red blood cells and platelets and enhance the adhesion and aggregation of platelets, which results in the formation of a cell embolus or thrombus and promotes blood clotting. In turn, the increase of the local concentration of platelets, blood coagulation factors, and fibrin initiate and enhance the internal clotting mechanism [37
In addition to the activation of the blood clotting system, it is well known that when the tissues of the endomembrane are exposed after the cutting of blood vessels, the fibrinolytic system is also activated. Unfortunately, the constructed thrombus can be dissolved over time. Tranexamic acid is a local hemostatic agent, which can inhibit the degradation of fibrin by inhibiting plasminogen activation which forms fibrinolytic enzymes. Thus, in this study, as the tranexamic acid-loaded composite particles were used on the wound, the porous composite particles enhanced the formation of the thrombus. In addition, the tranexamic acid released from the particles inhibited the degradation of the thrombus. Due to the synergistic effects of several hemostatic mechanisms, the tranexamic acid loaded composite particles exhibited an improved hemostatic efficiency.