| Blood transfusion systems have greatly benefited to human health and welfare for treatment of severe anemia, trauma and major surgeries induced hemorrhagic shock. Nevertheless, some problems such as infection, blood type mismatching, immunological response and short shelf life still exist. It is of great importance to launch substantial efforts to develop a blood substitute.The blood substitutes can be divided into two kinds: perfluorocarbon-based oxygen carriers(PFBOC) and hemoglobin-based oxygen carriers( HBOCs). Published animal studies and clinical trials carried out in a perioperative setting have demonstrated that HBOCs take effect for a short time during and after surgery, no improvement in mortality and an increased incidence of adverse reactions. Well-designed artificial oxygen carriers that meet requisite criteria are expected to be realized eventually. Encapsulation of Hb is one idea to shield the toxicities of molecular Hbs. However, intrinsic issues of encapsulated Hbs must be resolved: tissue oxygenation, blood circulation time, blood pressure elevation and heme stability. So this study aims to prepare a novel kind of HBOCs to resolve these problems.Objective: Layer-by-Layer Self-Assembly (LbL) offers a new method for surface modification of materials. To prepare a novel artificial O2 carrier based on Layer-by-Layer Self-Assembly and to investigate whether this kind of nanoparticles had kept heme stability and prolonged blood circulation time for further study.Methods: Anionic Hb-PLGA-PSS-NPs consisted of polymer blends of poly(lactide-co-glycolide) (PLGA) and poly(styrene-co-4-styrene-sulfonate) (PSS) were prepared and investigated. Based on the core of Hb-PLGA-PSS-NPs, shells composed of biocompatible polymers, including chitosan and dextran sulfate sodium salt (Dextran), were introduced by LbL technique. The LbL process was confirmed by the detection of dynamic light scattering (DLS), zeta-potential, scanning electron microscope (TEM). And release behavior and in vitro oxygen carrying activity of Hb-PLGA-PSS-NPs and polymeric nanoparticles coated by 6 layers were evaluated respectively.Result 1 The obtained Hb-PLGA-PSS-NPs were approximately spherical in shape with average size of 226.8±23.4nm and Zeta-potential of -68.62mV. Under optimized preparation condition, the encapsulation efficiency of Hb could achieve 99.3%, and the loading efficiency of nanoparticles was 28.6% when the nanoparticles were preserved at 4℃. Besides, in vitro release behavior manifested that Hb was sustained released. It was also confirmed that the biological activity of Hb after encapsulation. P50 of Hb-PLGA-PSS-NPs was 30.2 mmHg, and the hill index was 2.134, which did not seem to be greatly affected by the encapsulation procedure. Result 2 Results show that the average diameter of the nanoparticles coated by 6 layers was 287 nm. In-vitro release behavior showed that Hb was released more slowly as coated layers increased. This result has significant point in prolonging blood circulation time. And P50 of Hb-PLGA-PSS-NPs was 29 mmHg, and the hill index was 2.036, which did not seem to be greatly affected by the encapsulation procedure.Conclusion: Obtained results indicate that this kind of polymeric nanoparticles based on LbL have nice oxygen-carrying capability, and sustained-releasing of Hb have positive point in prolonging blood circulation time. This study can provide new methods and nice study data for further study.
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