| Since the discovery of blood type antigen by Landsteinter in 1900, allogeneicblood transfusion has developed into a routine clinical practice; it has contributed tohuman health and welfare. Infections diseases such as hepatitis viruses, HIV andother potential emerging infective agents have become widespread social problems,immunological responses such as anaphylaxis and graft-versus-host disease andcontingencies of blood type incompatibility further limit the utility of blood products.As a result, the requisites for artificial oxygen carriers that we developed should benot only effectiveness for tissue oxygenation, but also have these characteristics asfollowing, (1) universal biocompatibility like human blood (no need to type andcrossmatch); (2) perfect ability of transporting oxygen to the tissue; (3) maintenanceof arterial blood pressure, pH, ion balance, and colloidal osmotic pressure, etc.; (4) noinfection, no pathogens, and no immune response, etc.; (5) stability for long-termstorage (e.g. over 6 months) at room temperature for stockpiling for any emergency;(6) long half-time circulation in vivo and no renal toxicity; (7) rapid metabolizationand prompt elimination in vivo; (8) availability at a reasonable cost and easy ofadministering.There are two general categories of oxygen carriers: perflurocarbons (PFCs) andhemoglobin-based oxygen carriers (HBOCs). PFCs are synthetic linear or cycliccompounds made of fluorine atoms replacing hydrogen atoms along an 8-10 moleculecarbon backbone. PFCs have good ability of oxygen dissolution so that they are likelyto have perfect oxygen transfusion to the tissue in vivo. But PFCs perform a long halflifecirculation and are difficult to eliminate from the body,generally speaking,theycan stay up to 30-60 hours in the circulation of blood. Furthermore, PFCs can lead to some side effects, such as a temporary decrease in platelet count and flu-likesymptoms (myalgia and fever). The second category, hemoglobin-based oxygencarriers, are very promising, because their raw materials are hemoglobin from the redblood cells. They can also been divided into three generations according to theirdevelopment. First-generation blood substitutes are simply an oxygen-carrier basedon the use of modified Hb without the presence of RBC enzymes or the RBCmembrane. They include surface modification of hemoglobin, intramolecular crosslinkinghemoglobin, polymerized hemoglobin, and genetic recombinant hemoglobin.Second-generation blood substitutes are to use nanotechnology to assemblehemoglobin with superoxide dismutase and catalase into nanodimension PolyHb-SOD-CAT. They can play potential role as a protective therapeutic agent in clinicalsituation of ischemia and oxidative stress in stroke, myocardial infarction, sustainedsevere hemorrhagic shock, organ transplantation and in cardiopulmonary bypass.Obviously, in the first and the second generations, hemoglobin molecules contactwith the blood directly, so high purity of hemoglobin are needed. What's more, theyeasily lost their efficacy via autoxidation or being attacked by the immune system.And they usually have short half-life in vivo because of their exposure in the bloodcirculation. So the researchers all over the world have a high regard to thephysiological significance of cellular structure of red blood cells:(1) a decreased highcolloidal osmotic pressure; (2) prevention of the removal of Hb from bloodcirculation; (3) prevention of direct contact of toxic Hb molecules and the endotheliallining; (4) retardation of reactions with endogenous NO and CO; (5) preservation ofthe chemical environment in cells, such as the concentration of phosphates (2, 3-DPG,ATP, etc.) and other electtolytes; (6) RBCs are the major component that rendersblood as non-Newtonian and viscous, which is necessary to pressurize the peripheralartery for homogeneous blood distribution and for the maintenance of bloodcirculation; (7) the RBC cellular structure retards O2-release in comparison toacellular Hb solutions, thereby retaining O2 to peripheral tissues where O2 is required.For those reasons, the optimal structure of Hb-based O2 carriers might be to mimic the RBC cellular structure. So the third generation blood substitutes are born at theright moment. Third-generation blood substitutes is liposome-encapsulated Hb (LEH)which are closer to RBCs because they contain Hb and all of the enzymes of RBCs.Some of the characteristics of Hb-vesicles as a transfusion alternative are below: (1)Human Hb is purified completely via pasteurization at 60℃and ultrafiltration; theblood type antigens and pathogens are eliminated for utmost safety; (2) encapsulationof concentrated Hb solution (>35g/dL), like RBCs; (3) prevention of plateletactivation; (4) PEG modification for dispersion stability for long-term storage and inthe bloodstream; (5) the cellular structure, which resembles that of RBCs, shields allside effects of Hb molecules, such as scavenging NO and CO; (6) the particle size(250nm) is appropriate for sterilization, circulation persistence and biodistribution; (7)HbV do not show COP, additiona of a plasma substitute solution such as recombinantalbumin is effective to regulate COP.Rencently, a biodegradable polymer, polylactide(PLA), was more and morestudied as the HbV matrix due to its favorable properties. Firstly, it is perfectlybiodegradable and biocompatible, it can be finally degraded into CO2 and H2O invivo without any side effects. Secondly, the structure of PLA-based material can beeasily tailored to meet demands of different applications. For example, PEG segmentcan be attached to the end of PLA to form an amphiphilic structure, which can selfassembleinto micelles in aqueous solution and widely used in many biomedicalfields. Functional groups are also introduced into the backbone of PLA bycopolymerization of LA and other functionalized monomers. Through thesefunctional groups, many bioactive molecules can be conjugated to the biodegradablepolymer and endow it with certain bioactivities. Thirdly, compared with traditionalHbV matrixes, PLA-based materials are superior in mechanical properties, whichlead to easier manufacturing, better stability and less cost.Main results of the work are listed as follows:(1) MPC, 5-methyl-5-alkynyloxycarbonyl-1,3-dioxan-2-ketone, a new type ofaliphatic polyester bearing an alkynyl group was prepared. 1H-NMR analysis investigated the microstructure of the polymer. Then, a triblock copolymer, PEGMPC-PLA, was prepared by ring-opening copolymerization of L-lactide (LA), MPCand polyethylene glycol (PEG) with diethyl zinc (ZnEt2) as initiator. Themicrostructure of the triblock copolymer was investigated by 1H-NMR and 13C-NMRanalysis. DSC results indicated that the triblock copolymer displayed a single glasstransitiontemperature (Tg), which was indicative of a block copolymer instead ofrandom copolymer. The adhesion and spreading of ECV-304 cells on the copolymerwere better than that on PLA films. Therefore, this biodegradable amphiphilic blockcopolymer is expected to be used as a biomaterial for drug delivery and tissueengineering. And then, the triblock polymer PEG-MPC-PLA was self-assembled intocore-shell nanomicelles in aqueous solution. DLS, ESEM, AFM, and fluorescencespectra indicated the microstructure and morphology of the nanomicelles. Cellexperiments showed that these novel nanomicelles had impoved adhesion,proliferation and perfect biocompatibility behavior of VERO cells on the polymerfilms.(2) Bovine hemoglobin was conjugated to the core of the nanomicelles via clickchemistry. DLS, ESEM, TEM, and AFM indicated the microstructure andmorphology of hemoglobin conjugated nanomicelles and the nanomicellesrespectively, and the differences between them are remarkable. Then, FITC-BSA as acontrol, the confocal laser scanning microscope (CLSM) image indicates the efficacyof conjugation between BSA and micelles, which and also demonstrate the wellconjugation between hemoglobin and the micelles, because the two proteins, BSAand Hb, are alike in structure and composition. At last, by UV, the hemoglobinconjugated nanomicelles showed the ability to carry oxygen with the half-life beingabout 18 hours, predicting the applications as a new artificial oxygen carrier in thefuture. |
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