Reducing Oxidative Toxicity Of Hemoglobin-based Oxygen Carriers By Polydopamine Modification

Reducing Oxidative Toxicity of Hemoglobin-based Oxygen Carriers by Polydopamine ModificationBlood transfusion maintains normal physiologic functions and can help to save lives.It is applied widely in surgical procedures,natural disasters,and battlefield wounds.However,the risks of cross-matching and severe shortage of blood seriously affect the availability of blood transfusion.The limitations of blood have driven the development of red blood cell(RBC)substitutes because they have long shelf-lives and low risk of virus transmission without the requirement for blood-type matching.Until now,there have been two main types of RBC substitutes: perfluorocarbon emulsions and hemoglobin-based oxygen carriers(HBOCs).The former have not been investigated thoroughly because of their complicated preparation procedures and adverse reactions.HBOCs have attracted considerable attention and a large volume of research has been carried out on them.Since the 1950 s,various approaches have been adopted to improve HBOCs to prevent renal clearance and hypertension,such as crosslinking,surface modification and encapsulation.Various HBOCs have been investigated clinically.South Africa and Russia approved bovine Hb crosslinked with glutaraldehyde(HBOC-201)manufactured by Biopure Corporation(Cambridge,MA,USA)for patients with acute anemia.In addition,Poly Heme(Northfield Laboratories,Evanston,IL,USA),a human Hb product polymerized with glutaraldehyde,and Hem Assist(Baxter Healthcare Corporation,Deerfield,IL,USA),a diaspirin-crosslinked Hb,have been investigated clinically.HBOCs carry a low clinical risk,increase the oxygen supply and aid treatment of battlefield trauma,but their development is always challenging.Severe adverse effects have been documented in recent clinical trials,such as hypertension,myocardial infarction,gastrointestinal discomfort and acute renal failure.Hence,the clinical application of HBOCs has been not approved by the US Food and Drug Administration.These adverse effects have hindered the further development and licensing of HBOCs.The oxidative side reactions of Hb are the most frequently reported effects in the overall toxicology of HBOCs.The oxidative toxicity of HBOCs has been illustrated in two main aspects.First,Hb in its reduced state can be auto-oxidized to generate methemoglobin(Met Hb)because of the lack of a reductase system compared with RBCs.In addition,the formation of free radicals increases the multiple secondary injuries caused by massive,rapid blood transfusions during the oxidative process of HBOOCs,such as ischemia–reperfusion injury.Hence,strategies for synthetic HBOCs involve the formidable challenges of maintaining oxygen-carrying capacity while minimizing Met Hb formation and reducing oxidative toxicity with the ability to scavenge free radicals.To resolve these problems,studies have been carried out to crosslink or add antioxidant enzymes against the oxidative side reactions of HBOCs.However,complicated procedures and toxic reagents promote oxidative side reactions and affect the quality of HBOCs during crosslinking or polymerization.It is,therefore,essential to develop simple strategies to synthesize HBOCs with antioxidant properties to lower the risk of Hb auto-oxidation and oxidative damage.In 2007,Lee and colleagues found dopamine(DA)to be prone to oxidative auto-polymerization in alkaline solutions(p H >7.5)and could be attached strongly to substrate surfaces.Polydopamine(PDA)is a novel coating material that not only protects the underlying materials from external erosion but also confers new functionalities to them.PDA has become a rapidly growing area of research in biologic and biomedical sciences.PDA has shown great promise for HBOCs applications because it:(1)can be attached strongly to virtually all types of substrate surfaces in an efficient and simple process without the need for organic reagents;(2)can act as an antioxidant to prevent Hb auto-oxidation and reduce oxidative damage;(3)has excellent hemocompatibility and biocompatibility.Therefore,study of PDA application in HBOCs should focus on reduction of Hb auto-oxidation,and scavenging of free radicals,which could reduce oxidative damage.Three main scientific problems must be solved.First,the method to synthesize HBOCs with PDA modification should be investigated in details,such as the starting concentration of DA and p H value.Changes in the components on the Hb surface are important for illustrating the mechanics of PDA modification.Second,the hemocompatibility and cytotoxicity of PDA-modified HBOCs should be demonstrated.Third,the antioxidant properties of PDA-modified HBOCs,such as the prevention of Hb auto-oxidation and reduction of oxidative damage,should be determined.Chapter I: Preparation and physicochemical properties of PDA-modified nanoparticlesPDA modification should be taken into account for HBOCs development.PDA-modified Hb nanoparticles have been designed and synthesized.(1)The effect of parameters on fabrication of Hb-PDA nanoparticles was investigated,such as the concentration of DA monomer,type of buffer,and p H of the reaction solution.To prepare PDA-modified HBOCs,48.8 mg of Hb was incubated with 9.76 mg of DA in Tris-HCl buffer(10 m M,p H 8.5)with slight stirring for 210 min at 4°C.(2)The change of surface moieties showed that catechol groups have crucial roles in the adhesion of PDA on the Hb surface.(3)The results of UV–vis spectrophotometry,Fourier transform infrared spectroscopy and circular-dichroism revealed that the preparation process had little influence on the secondary structure of Hb.(4)Hb-PDA nanoparticles showed good dispersion in 0.9% Na Cl and phosphate-buffered saline(PBS)according their size at 7 days.Chapter II: Preparation and physicochemical properties of PDA-modified microparticles/nanoparticlesHb-MPs particles were fabricated by co-precipitation of manganese chloride and sodium carbonate.Human albumin located on the surface of Hb-MPs prevented agglomeration.Then,Hb on the surface was crosslinked with GA and the template dissolved in ethylenediamine tetra-acetic acid.Afterwards,PDA was introduced to modify Hb-MPs by self-polymerization of monomers of dopamine hydrochloride.(1)It was shown the Hb-MPs-PDA micro/nanoparticles were ellipsoid and the average diameter of the particles was ?750 nm as observed by scanning electron microscopy and transmission electron microscopy.(2)An increasing initial concentration of DA increased the amount of PDA on Hb-PDA nanoparticles surface.(3)Hb-MPs-PDA micro/nanoparticles were stable and did not form aggregates in PBS,0.9% Na Cl,human blood plasma,or neurobasal cell culture medium.Chapter III: Hemocompatibility and cytotoxicity of PDA-modified HBOCsPDA-modified HBOCs with good blood compatibility and low cytotoxicity have been demonstrated.(1)PDA-modified HBOCs exhibited excellent hemocompatibility.It was shown that Hb-PDA nanoparticles had no effect on coagulation.PDA-modified HBOCs did not induce the aggregation or hemolysis of RBCs.PDA-modified HBOCs had very little influence on platelet aggregation.(2)Endothelial and A549 cells were selected to determine the cytotoxicity of PDA-modified HBOCs which did not affect the viability of endothelial cells.(3)Hippocampal cells and cerebral cortex cells were selected to determine the cytotoxicity of Hb-MPs-PDA micro/nanoparticles which show little neurotoxicity.Chapter IV: Oxidative protection of PDA-modified HBOCsReduction of PDA modification for oxidative toxicity of HBOCs has been investigated.(1)To determine the amount of oxygenated-Hb and Met Hb in PDA-modified HBOCs,it was shown that PDA modification decreased the auto-oxidation of Hb and maintained the capacity to carry oxygen.The partial pressure of oxygen due to Hb-PDA nanoparticles increased by 10% compared with that of Hb.Compared with Hb micro/nanoparticles,the partial pressure of oxygen due to different Hb-MPs-PDA1 micro/nanoparticles increased by 26%.(2)PDA-modified HBOCs had excellent antioxidant capacity because they scavenged various free radicals,such as DPPH?ABTS+ and H2O2.(3)Effective suppression of generation of ROS due to PDA modification was noted.PDA-modified HBOCs helped to relieve H2O2-induced oxidative damage,especially for mitochondria.These results suggested that PDA-modified HBOCs could be used to construct novel oxygen carriers with the capacity to reduce oxidative side reactions.In summary,PDA modification should be taken into account for HBOCs development.The effect of parameters on PDA modification was investigated to synthesize PDA-modified HBOCs with different sizes and construction.Catechol groups had crucial roles in PDA adhesion on the surface of Hb.PDA modification provides a novel strategy to design and prepare HBOCs.PDA-modified HBOCs exhibited excellent hemocompatibility and low cytotoxici ty.PDA modification is believed to prevent Hb auto-oxidation and maintain the capacity to carry oxygen.Also,PDA-modified HBOCs help to relieve oxidative damage.These results demonstrated that PDA modification benefited the improvement of HBOCs against oxidative toxicity.