RedBloodCellsSubstitutes– FutureVision – António Filipe SousaNº64427 MBioNano

Introduction The purpose of this paper is to determine a valid application of vesicles for encapsulating a concentrated human Hb, and diferent kinds of enzymes, in order to obtain a fully operational and self functional artificial red bloood cell. The resulting Hb-vesicle (HbV) can serve as an O2 carrier with the ability comparable to red blood cells (RBC). Some advantages of obtaining Hb-based O2carrieres (HBOCs) are:• Absence of blood-type antigens and transmission of known and unknown blood-borne disease;• Long Term stability and storage;• Low toxicity and prompt metabolism, even after massive infusion;[2]• Physichochemical properties that are adjustable to resemble those of human blood and• Reasonable production expense and cost performance. [1]

Introduction Liposome-encapsulated hemoglobin (LH) was chosen for this paper as a promising artificial oxygen (O2) carrier. The capsular structure of LH is favorable for preserving the chemical environment for Hb and reducing its side effects. [3][1] The main purpose is to mimic the double layer membrane of the biological red blood cells. So, we want to fabricate a artificial membrane with optimal Amounts of Protein (49%), Lipid(43%) and Carbohydrate (8%)

The method chosen for the preparation of the artificial red blood cellssuspension would be the follow at sterile conditions:• Human Hb purified from outdated, donated blood. Preparation of polyHb based on crosslinkingpolyHb with superoxydedismutase and catalase.PreparationMethod• At the same time, metHbreductase, and other different kinds of enzymes can be included with the PolyHb. • Preparation of polyHb with pyridoxal 5’-phosphate as an allostericeffector (increases oxygen affinity).• The lipid bilayer was composed of a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, and 1,5-O-dihexadecyl-N-succynyl-L-glutamate (DHSG) and 1,2- distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG5000. [4]• HbVswere suspended in a physiological salt solution, sterilized using filters (pore size, 0.45 um). [4]• Parameters of the HbV are as follows: particle diameter, 250 nm and oxygen affinity (P50), 30 Torr. [4]

MoleculeTransportation:Sodium-potassiumpump,Calciumpump,Aminoacids,Glucose.PreparationMethod2 Moleculesoflactateand 2 of ATP, bytheEmbden-Meyerhofway.Enzymesrequired:Hexokinase,Phosphofructokinase,PiruvateKinase,NAD coenzyme.Anti-Oxydantprotection:Superoxydedismutase,Superoxydecatalase,NADPH coenzyme,Otherslike, metahemoglobinreductase, G6P, GSH andVitamin E.

Is a very important parameter to consider using HbV as an oxygen carrier. Since the estimated diameter of the smallest blood capillaries in the human body is 4–7 um, the particle sizes should optimally be below 4 um to avoid embolism. ParticlesSizeParameteres for using HbV(a) Thetransmittanceelectron micrograph of rat blood 1 day after infusion of HbV. Many HbV particles are visible in the red circles. (b) Flow patterns of the mixture of HbV and RBC suspended in recombinant human serum albumin in a narrow tube (centerline flow velocity: 1 mm/s. The RBCs tend to flow in the centerline, whereas HbV particles are dispersed homogeneously ina suspensionmedium.[1]

ParticlesSize

With this paper we want to mimic the RBC O2 affinity, so we expect to obtain a curve of this kind:Oxygen_carryingCapacityHbVshould act as a particle in the blood and not as a solute; the colloid osmotic pressure of the HbV suspension should be nearly zero. The rheological property of an artificial oxygen carrier is important because the infusion amount should be considerably large, which might affect the blood viscosity and hemodynamics. The measurement of the O2-release rate of the HbV must be similar to the RBCs. Parameteres for using HbV

Oxygen_carryingCapacity

HbVparticles are recognizedas foreign materials and finally captured mainly by the reticuloendothelialsystem. These are outstandingcharacteristicsin comparison with molecular Hb that shows a shorter circulation time because it is filtered through the kidneysIn a series of safety studies of HbV, it has been clarified that the cellular structure and the size of the HbV are advantageous for maintaining a steady blood circulation withoutvasoconstrictionandhypertension.• Physiological Capacity of the Reticuloendothelial System for the Degradation of Hemoglobin Vesicles Parameteres for using HbVNextwewillseetheefectsofHbVinthe RES ofrats, during a studyby, Hiromiet al.Note: DRI=DailyRepeatedInfusion[5]

Theresultswere:Parameteres for using HbVNumbersofRBC’sshowedmoderatechanges.DilutionofBlood, andhypervolemia.Slighthypertensioninthefirstdayafter DRI.[5]

Advantages of artificial O2 carriers including HbVare the absence of blood-type antigens and infectious viruses, along with stability for a long-term storage for any emergency that might overwhelm the RBC transfusioncapacity.Thepossibilityofobtaining a fullyindependentHbV, withnotonlyhemoglobin, butalsowithalltheenzymesnecessary for a regular oxygenflowbetwenbloodandtissues, isbecoming a possibilityand a areaofinvestigationwithmuchinterest.Thefuture?Conclusions …No more bloodrespiratorydeseases!!…Super athelitcs!!…Only 5-6 inspirationsper minute!!

[1] ­H. Sakai, et al, (2007). Hemoglobin-vesicles as artificial oxygen carriers: present situation and future visions. Journal of Internal Medicine, 263; 4–15.[2] Chang, Thomas Ming Swi(2006)'Evolution of Artificial Cells Using Nanobiotechnology of Hemoglobin Based RBCBlood Substitute as an Example',Artificial Cells, Blood Substitutes, and Biotechnology,34:6,551 — 566[3]Matsumoto T, et al. Model analysis of local oxygen delivery with liposome-encapsulated hemoglobin. Med Eng Phys (2008), doi:10.1016/j.medengphy.2008.08.005[4] Hiromi Sakai, et al (2004). Metabolism of hemoglobin-vesicles (artificial oxygen carriers) and their influence on organ functions in a rat model. Biomaterials 25, 4317–4325.[5] Hiromi Sakai, et al (2004). Physiological Capacity of the Reticuloendothelial System for the Degradation of Hemoglobin Vesicles (Artificial Oxygen Carriers) after Massive Intravenous Doses by Daily Repeated Infusions for 14 Days. TheJournalofPharmacologyand Experimental Therapeutics, 311:874–884.Bibliography[6] Jian Zhao, et al (2006). Preparation of hemoglobin-loaded nano-sized particles with porous structure as oxygen carriers. Biomaterials 28, 1414–1422.[7] Robert M. Winslow (1999). New Transfusion Strategies: Red Cell Substitutes. Annu. Rev. Med. 50:337.53