CA1253801A - Substitute for human blood and method of making the same - Google Patents

Abstract

ABSTRACT
The method of this invention yields a two phase liquid aqueous system which replicates the two phase heterogeneous physicochemical system of naturally occurring whole human blood. The method produces a composition of matter which comprises a whole blood substitute when polymerized hemoglobin ox pyridoxylated-polymerized hemoglobin, and preferably, other specific additives are incorporated. When said blood substitute is processed further, a microencapsulated composition with time release characteristics that can transport oxygen is produced. The invention overcomes the obstacles that have prevented the use of modified hemoglobins like pyridoxylated-polymerized hemoglobin in the preparation of oxygen transport systems.

Description

The invention concerns an improved composition useful as a substitute for human blood and as an oxygen transport system, and methods of preparation.
In response to an evident need, a number of 5 oxygen transporting solutions have recently been developed.
Each reflects a different approach. At this point of development, most of these preparations exhibit either manufacturing or clinical difficulties. In some in-stances, both problems are present.
PerEluorocarbon based compositions were among the earliest of these oxygen ~ransport solutions.
While such compositions possess oxygen carrying capability, difficulties in dwell time, in administering the prepara-tion and the suspicion of a toxic po-tential has raised 15 serious questions regardiny the safety and utility of this product.
The eEJ~ort to replicate erythrocytes through the deve:lopment o~ liposomes containing stroma free hemc)globin represent~ a second approach. tRef. Djorejevichr 20 L: Miller,~ Lipid Encapsulated Hemoglobin as a Synthetic Erythrocyte, Fed. Proc. 1977, 36:567)~ The evidence to date suggests that in this approach, un-desireable and erratic effects are known to follow when the hemoglobin of the composition attaches to the 25 exterior of the liposome during the process of manu-facture or leaks from the encapsulating liposome after the product is introduced into the circulation of the recip:ient. :~n cither event, ,Eree helrloglob:i,rl is liherat-ed into the c:irculation oE the recipient. I'he possible consecluences of this event are well known to clinicians andothers sk:illed in the art.
A third approach to the development of an oxygen transportincJ fluid is based upon efforts to modify the hemoglobin molecule through the process of pyridoxylation and polymerization. See: DeVenuto, F. and Zegna, A. Prepara-tion and Evalua-tion of -the 10 Pyridoxalated-Polymerized l-lemoglobin Molecule. Surg.
Res. 35-205, 212 (1933).
At least two major clifficulties, appear to be associated with solutions containing modified hemoglobin.
The firs-t involves the problem of oxygen release; the 15 second is the loss of the composition from the vascular space. The problem of the toxicity of this composition has apparently not been explored. These difficulties raise doubts about the utility of presently known oxygen transporting solutions based on modified hemo-20 globin.
The fourth approach to the developmen-t of an oxygen -transporting solution is based upon -the a~plicants' inventions disc:Losed in U.S. Patent Nos. ~,3~3,797 and ~,~39,~2~. Tl-le process lnvolvecl in thls approach 25 rnakes us~ oE a two phase lic,luid aqueous hQterocJelleolls sy~telll ancl yie:ld.; rl suhs,t:itute for humall ~]oocl.
'I'he here:ill disclosed invention represents a sign:iEicant sclentific ,advance in that through its use oE a coacervate system the problems associated with 30 unmodified solutions, polymerized hemoglobins and pyridoxy-lated-pol,ymerized hemoglobins, are avoided.
The manufacturing sequence of this invention is designed: (1) to yield a coacervate sys-tem which can serve as a whol,e blood substi-tute ~ith the incorporati,on 35 oE polymerized hemoglobin or pyridoxylated hemoglobin.

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and (2) to provide, if desired, a form oE microencapsu~lated hemoglobin wi-th an approxima-te equivalence to the cytoplasm o-F erythrocytes or paeked red cells. The oxygen carrying solu~ion herein described can also restore and maintain normal oncotic pressure when infused into the circulatory system.
For purposes of this application, -the applican-ts draw a distinction be-tween synthetie blood products disclosed in U. S. Patent Nos. 4,343,797 and 4,439,424, and oxygen transport solutions. For the most part the oxygen release eurve of the latter differs signi-ficantly from that of the prior art.
It is an object of this invention to provide a composition of matter whieh can serve as a whole blood substitute. It is another object to provide a conven-ient method for the manufaeture of these compositions based on -the process of coacerva-tion. It is a further object to provide a composition that has physiologieal charaeteristics equivalent to those of paeked red eells, and fur-tller a convenient method of preparing -this composition. Moreover, this i.nven-tion provides a cornposition w.ith charaeteris-t:ics sim:ilar to the cyto-plasm o:E erythrocytes an~ a method of produe1.ng the S ~
:Ln par:t:icu:Lar, the :invent:ion provkles a .;ynthet:i.c whol.e b:Lood substitute, consisting essentially oE a non-tox:ic two phase ].iquid system, both said phases being aqueous, one of sai.d phases heing a relatively non-polar coacervate phase having physiological and physicoehemical properties substantially equivalent to erythrocytes, the o-ther of said phases being a relatively polar liquid aqueous phase ha.ving physiological and physicochemical properties substan-tially equivalent to blood plasma, said relatively non-polar coacervate phase being insoluble in and in equilibrium wi-th said relatively po].ar liquid aqueous phaset and incorporated in-to said two phase system a hemoglobin componen-t selected from polymerized hemoglobin, pyridoxylated-polymerized hemoglobin and mixtures thereof; said two phase liquid system having physiological and physi.co-chemical proper-ties essentially similar to those of whole bloodO
Moreover, the invention provides a me-thod of preparing a composition of matter which is useful as a blood substitute, said method characterized by the steps of (a) combining albumin and phospholipid in water; (b) thoroughly mixing the compositions; (c) storing said mixture undisturbed until the composition of step (a) separates into two layers, one above the other, the lower layer being a substantially non-polar coacervate phase, and the upper layer being an equilibrium water phase; ~d) continuing the separation process until no increase in the volume of the coacervate phase can be observed; (e) centrifuging the composition until inspection reveals a clear demarcati.on of the two phases; (f) separati.ng the two phases; and (g) addiny a hemoglobin cornponent selected from polymerized hemoglobin, pyr:idoxylated-polymerized hemoglobin and mixtures the:reof, to the coacervate phase.
'rh;.'; :inv~ntion compr:ises compositions of matter and methods by means of wh:ich -they can be prepared.
The claimed inventions comprise a combination of endoyenous componen-ts and water which yields a two phase heteroyeneous physicochemical coacervate system similar to that of human blood. This coacervate system is the basis of -the herein disclosed discovery.
In practice, any appropriate non toxic coacerva-te system can be used to manufac-ture -the products oE this inven-tion, and further, any endoyenous bioloyical surface active ayent or derivative thereof, such as albumin, lecithin, gelatin, etc., can be used to prepare a coacerva-te sys-tem appropria-te -Eor the method ~3~

and product of this lnvention~ Appropria-te non--toxic exogenous cornponents, i.e. acacia gel, can also be used to prepare sui-table coacervate systems.
The method of making -this invention begins with the preparation of a two phase aqueous liquid system, also referred to as a coacerva-te sys-tem. When the prep-aration of -the coacervate sys-tem is completed, i-t will consist of two phaseso (l) an internal suspension, relatively non-polar phase, commonly referred to as the coacervate phase; and (2) an associated, relatively polar ex-ternal suspension or equilibrium phase. Both phases are in equilibrium with and insoluble in each other. The coacervate phase of this two phase system can comprise from about 0.5% to 99.5% by volume of -the system; correspondingly, the associated equilibrium phase can comprise from about 0.5% to 99.56 by volume of the system. The preferred proportions are 50%
volume to volume of each of these components. The hemoglobin component is incorpora-ted into the two phase system.
The coacervate phase of the claimed coacerva-te sys-tem possesses physioloyical properties equi.valent to -the cytoplasm of erythrocytes or packecl red cells.
~'. '.-,uc'h, thiC, phclsc~ oE l:he coacervate system has siy-n:i.E:icarlt o~yclen transport capabil:ity. The equilibriurnphase possesses physiological and physiochemical prop-erties equivalent -to -the plasma of human blood.
The claimed two phase aqueous liquid system, (i.e. coacervate system) incorporating specific hemoglobin components func-tions as a whole blood substitute and may be drawn off during the process of manufacture. When componen-ts such as appropria-te proteins, electrolytes, sterols, any of several available forms of hemoglobin and an oxygen releasing entity are added -to -the coacervate sys-tem, the sys-tem achieves a biochemical equivalence approaching -that of human blood. If a composition wi-th properties similar to the cytoplasrn of erythrocytes or of packed red cells is desired, the disclosed coacervate system and additives are subjected to warming and/or other procedures. In such process the end product is microencapsulated hemoglobin.
Enzymes, nutrients and drugs are among the additives tha-tmay be incorporated in the preparation of the blood substitute. The polymerized hemoglobin used in the invention is based on human and other forms of mammalian blood, i.e., bovine blood, etc. The human source is preferred.
When modified hemoglobin, i.e., the pyridoxy-lated-polymerized form, is incorporated in the claimed preparation, -the problems presently associated with such forms of hemoglobin, i.e. oxygen re- 7 /
/

lease, at normal o~yyen tens;.ons and loss of hemoglobin solution from -the vascular system, are eliminated.
This is enhanced through the addition of an oxygen releasing molecule, such as di-phospho-glycerate to the 5 coacervate system in the course o~ the manufacturing process. As used in this invention, di-phospho-gly-cerate acts to release oxygen from hemoglobin precisely as it does in -the body. Small quantities oE urea may also be added, if desired, during the preparation of 10 this composition to further the release of oxygen from the said composition.
Loss o~ the oxygen transport solution from the vascular space is prevented in this invention by two factors: (1) by emulsifying the preparation wherein 15 the resulting emulsified droplets in which the hemo-globin is contained are manufactured to a size of ap-proximately 7 microns, i.e., the size of normally oc-curring erythrocytes. Emulsified droplets of this size permit oxygena-tion of tissues, prevent escape of the 20 solution from the circulation and allow en-try of the hemoglobln bearing droplets into the microcircula-tion.
The method of preparation, however, provides for the droplet size, if desire~, to vary from 100 millimicrons to 15 micronC3.
ZS I.o~-;s o:~ the solutiorl Erom the vascular space :i..s also preverlted :i.n th:is invention throucJh the ef-Eects of electrical charyes present Oll the surface of drople-ts of the :Einished product and on the surfaces of arterial and venous branches of the circulatory system.
30 Thus, the surfaces of blood vessels are negatively charged; the electrical charge on the surface of the emuLsified drople-ts of the claimed composition is also negative. The resultant repulsant effect serves to pre-vent t.he loss of the solution from the circulatory s~stem.
In the finished product, the composition may by comprised of emulslfied droplets of the same size or of any cornbination of sizes, depending upon the in- F
tended useO Thus, in a ~iven version of this invention a preponderance of emulsified droplets of a size smaller 5 than 0.6 microns may be indicated, example: when it is desired that the claimed composition penetrate in-farcted area(s) in the vascular system.
While the disclosed invention indicates that .
equal proportions of albumin and lecithin are preferred 10 in the preparation of the claimed composition, it is possible to produce coacervate systems using unequal proportions of albumin and lecithin. In the case of such usage, however, the resultiny coacervate system may not have the optimal yield of the coacervate phase.
15 However, the coacervate phase of such systems may pos-sess other desireable characteristics known to those skilled in the art, e.g., oxygen transport.
The claimed invention also contains a pro-cess in the manufacturing procedure which yields de-20 rivative compositions. One of these is the physio-logical equivalent of the cytoplasm of erythrocytes.
When hemoglohin is added to this preparation, the equivalent of packed red cells is produced. The deriva-tive preparations can be subjected to microencapsulEI-~5 ti.on procedures and to ~ heating step. The heating step wi.ll act to harden the surface of the coacervate phase droplets of -the composition to any desired degree.
This results in compositions with sustained release characteristics. If desired, achemical process using 30 non-toxic members of the aldehyde group may be used.
The heating procedure is preferred.
With the exception of the inventors' contri-bution to the prior art,-the scientific literature con-tains no reference to a two phase heterogeneous physico-35 chemical system which permits the effective incorpora~

tion of modified forms of hemoglobin and further, which can serve as a useful substitute for human blood. In addition, there is no literature reference to a composi-tion which possesses physico-chemical properties that 5 approximate those of the cytoplasm of erythrocytes or of packed red cells.
In order to explain the claimed invention, the follo~ing is a general example of a preferred method s of preparation. Specific examples appear in the fol~
lO lowing section of this disclosure.
In the preparation of the disclosed composition, the component ingredients are prepared and combined un-der sterile conditions. All water used in the manu- -facturing process must be sterile and pyrogen free. -The preparation of the appropriate coacervate system constitutes the first step of the method neces~
sary to produce the product of this invention. The preferred ingredients of this step are albumin and a suitable phospholipid. In this method lecithin is pre-20 ferred. However, other phospholipids known to those skilled in the art such as cephalin, isolecithin, sphingomyolin, phosphatidyl serine, phosphatidic acid, phosphatidyl irlo.sl-tal, phosphatidyl choline may also be us ~
~n Ihe preferred method, equal weight to volume proportions of albumin ancl lecithin are added to an amoun~ of sterile water that will yield lO0 mls. of aqueous solution. The mixture is then thorouyhly mixed by vortex mixer. The preferred proportions for each 30 component, i.e. albumin and lecithin, are 3% weight to ~~
volume. Unequal proportions of albumin and lecithin can yield a coacervate system. However, this method is not preferred.
In the preferred method of preparation any 35 quantity of albumin and lecithin can be used, provided ~r~

-- 10 ~ '~
the requirement of the proportions of the inyredients is observed and quantity of water used is adjusted ac-cordingly .
Follcwing thorough mixing the solution is 5 stored in suitable containers. In the preferred method, the solution is stored undisturbed until the ma~imum yield of the coacervate phase oE the coacervate system has been achieved. Maximum yield is the point at which no significant increase in the volume of the coacer-10 vate phase can be observed. This determination can bemacle by direct visual inspection or other suitable means~ As is kr,own to those skilled in the art, longer -periods of storage produce greater yields of the coacer-vate phase. ~-~
The storage step may take place at tempera-tures ranging from freezing point to about 4 degrees C.
and up to room temperature or higher. In the preferred method, storage takes place at a temperature of from about 4 degrees to 10 degrees C.
When it is observed that the maximum yield of the coacervate phase has been achieved, the coacervate system is centriEuged un-til observation indicates that a clear divls:i.on exists at the interEace of the two phage~s c~ tlle coacervate system. :[f an oxygen trans-25 pork SO Lut i.OIl i.S desired, the system is emulsified, Ihe particle size of which may range from 100 milli-m:icrons to 10 microns. The composition is placed in re-frigerated storage until needed. If the manufacturing objective is to produce a synthetic blood, the fol-30 lowing steps are initiated. -The two phases are then separated by means of a separatory funnel. The equilibrium phase i5 set aside for subsequent recombination with the coacervate phase. Any of the previously preferred to forms of hemo-35 globin is then mixed into the coacervate phase in an 3~

amoun-t that will produce lift sustaininy oxygen -tens,ions in the ~inished produc-t. Suitable forms of the hemoglobin component are selected from polymerized hemoylobin, pyridoxylated-polymerized hemoglobin and rnixtures -thereof.
In this disclosure pyridoxylated-polymerized hemoglobin is preferred. It is noted that the source of the hemoglobin component may be human or bovine.
After this step, any oxygen liberatlng en-tity, such as di-phospho-glycera-te is added, and mixed into the coacervate phase. The amount added may range from 1~ or less to 6~ or more weight to volume. In this disclosure 4% weight to volume of di-phospho-glycerate is preferred.
The next step consists of recombining the equilibrium phase wi-th the coacervate phase which now includes the addi-tives described above. This is fol-lowed by a step in which the preparation is emulsified and an electrolyte is added. Any of the electroly-tes known to those skilled in the art, i.e. sodium chloride, potassium chlorlde, macJnesium chloride, or calcium chlor:icle may he used. The purpose oE this adclition is l:o rende~r the comr?osilion i-;otorl:ic with llurllarl blood.
Sodiuln chloriclc Ls the preEerr~d elec~rolyte and is adcled in that qualltity that will produce the desired ~5 isotonicity. At this point, if desired, 1 mg per cent of urea my be added. This componen-t can act to facili-tate the release of oxygen from the hemoglobin present in the claimed composition. If desired, a sterol from the following group is added: chlosterol, ergosterol, 7-dehydrocholestero].,C~sitosterol, /~sitosterol, ~
si-tosterol, campesterol or mixtures thereof. Choles-terol is preferred. 0.1 -to 10 rng. per cent of choles terol may be added ~o the preparation to improve -the stability of the composition. The preferred amount of cholesterol added to the composition is 1 my. per cent.
Following this step the p~l of the preparation is adjusted to 7.4 to 7.5 by the drop by drop addition of either HCl or sodium bicarbonate, depending upon the 5 pH of the preparation. Any other suitable non toxic acidifying or alkalizing agent may be used in place of hydrochloric acid or sodium bicarbonate, however, the agents named are preferred. t Upon completion of this step, the compo-lQ sition is again emulsified using either a colloid mill, sonification or other emulsifying technique known to those skilled in the art. This step produces emul-sified droplets which contain the hemoglobin component.
The droplets can range in size from less than 100 milli-15 microns to 15 microns and above; the preferred size is x that of normal erythrocytes. However, the invention pro- -vides for the possibility that specific medical treat-ments may require that the size of the droplets be of smaller dimensions. If desired, enzymes, nutrients and 20 drugs may be added to the coacervate phase of the com-position or to the composition at this stage of manu-facture.
If the manufacturing objective is to produce a composition that has the physiological properties of L
25 erythroc~tes or packed red celLs, the first step of that proccss consists of warming the preparation described imrnediately ahove. This step is accomplished by warminy the preparation in a water bath or controlled oven to a temperature ranying from 15 to 50C for from 20 seconds 3~ to 3 hours in order to produce a cross li.nking of the albumin and lecithin of the composition. The effect of this process is a hardening of the surface of the emul-sified droplets. The degree of hardness obtained is a func-tion of the duration and temperature of the warming 35 step. Thus, subjecting the composition for relatively shorter periods of time to higher temperatures will r~

yield approximately the same degree of hardening of the emulsified droplet surfaces as subjecting the compo-sition to lower temperatures for relatively longer periods of time. In point of fact, a spectrum of de-S grees of surface hardness is possible at this point ofmanufacture by varying the variables of time and tem-perature. r' In this invention the degree of structuringor hardening of the surface of the emulsified droplets 10 can range from fluid-like to semi-solid, i.e. gel-like to rigid. When the desired degree of surface hardness of the emulsified droplets has been achieved, the droplets are filtered from the emulsion. The fil-trate is discarded. The droplets are removed from the 15 filter bed, washed thoroughly with normal saline or other suitable solution and then dried by any of the con-ventional methods.
If desired, dif~ering proportions of the dried preparation with differing degrees of shell ~o hardness may be co~lbined, during the process of re-constituting the preparation with normal saline or other suitable solutions. ~lternatively, droplets of the same degree of surface hardness may be usecl :in the process of reconstitution. :Cn either formulation, the 25 composition will po~sess special oxygerl release proper-tics, and wilL be capable oE prompt, sustained and/or prolonged effect~
~ ross linking may also be achieved through chemical means known to those skilled in the art, i.e., 30 through the use of gluteraldehyde, etc. The method of heating is preferred in this invention. If it is de-sired to produce a product that has physiological prop-erties similar to the cytoplasm of erythrocytes, the pro-cedure described above is followed except that the hemo-35 globin component is omitted.
When the manufacturing steps are completed, ~ 53~

- lA -the products, i.e., the oxygen transport solution, the blood substitute, or either of the derivative compo-sitions,can be transfused into the circulatory system, where the individually described functions will be 5 carried out: transport of physiological gases, restora-tion of blood pressure, transport of drugs and enzyme systems etc. Alternatively, each composition can be stored, preferably at from 4 to 10 degrees C until need-ed. If the composition is to be infused into a human lO or animal following refrigerated storage, it should be warmed to body temperature (37 degrees C) before in-fusion.
SPECIFIC EXAMP~ES
Examples of how the claimed composition(s) of 15 matter may be prepared follow:

5% weight to volume proportions of albumin and lecithin are added to an amount of sterile water that will yield lO0 mls of aqueous solution. The mixture is then thoroughly mixed by vortex mlxer.
Following ~horough mix:ing, the solution is ~kored undis~urbed Imti:L the maximum yield of the co-acervate phasc oE the coacervate system has been achieved.
The storage step takes place at ~ de~rees C.
When it is observed that the maximum yield of the coacervate phase has been achieved, the coacervate system is centri~uged until observation indicates that a clear division exists at the inter-Eace of the two phases of the coacer~ate system. The two phases are 30 then separated by means of a separatory funnel. The equilibrium phase is set aside for subsequent recombina~
tion with the coacervate phase. 15 grams of pyridoxa-lated-polymerized hemoglobin are then dispersed into the coacervate phase. After this step~ ~% weight to volume 35 di phospho-glycerate is added and mixed into the co-B~

acer~ate phase.
The next step consists of recombining theequilibrium phase and the coacervate which contains the above named cornponents and emulsifying the preparation, 5 and adding that quantity of soclium chloride as will render the composition isotonic with human blood. At this point, 1 mg. per cent of urea is added. 1 mg. of cholesterol is added as the following step. The compo-sition is then mixed vigorously until all additives are 10 dispersed.
Following this step, the pl~ of the preparation is adjusted to 7.~ to 7.5 by the drop by drop addition of ~ither HCl or sodium bicarbonate, depending upon the pH of the preparation at this stage of manuacture.
lS Upon cornpletion of this step, the composition is again emulsiEied using a col:Loid mill. 'L'he resulting emulsified droplets which contain the hemoglobin compc)rl-ent are preparecl to be 7 microns in size~
13~MPI,~, 2 _, _ ._ .. . _ . .
200 m]s o~ 5~j solllt;orl of a:lbumirl is addecl to 200 mls. of a 3% solution of lecithin and mixed thoroughly.
The remaining steps o~ the procedure follow those of Example 1.
EX~MPLE 3 The procedure of Example 1 is followed ex-cep-t that the urea adding step is omitted.

The procedure of Example 1 is followed ex-cept that the cholesterol addillg step:isomitted.

200 mls. of a 5% solution of albumin is added to 200 mls. of a 7~ solution of lecithln and mixed thoroughly. The remaining steps of the procedure fol-low Example 1.

200 mls. oE a 3% solution of albumin isthoroughly mixed with 200 mls of a 3% solu-tion of iso-lecithin. The solution is then stored undisturbed at 4 degrees C for 24 hours. The remaining s-teps of the procedure follow Example 1.

, The procedure of Example 1 is followed ex-cept that the steps involving the addition of cholesterol 15 and urea are omit-ted.

The procedure oE Example 1 is Eollowed to completion. The resulting composition is then sub-jectecl to a warrll:irlcJ step. Thls consists o,f placing the ~ solution :i,n a wcLte~r bath al, 25 declrees C Eor five min-ul:es. ~t ttlc erl~'l of tllis per:iod the cIrople-ts of the c~ompositi.on ar~ iltered from the emulsion, and washed thoroughly with normal saline solution and dried by conventional means. 100 mls of normal saline solution 25 are aclded to -the product resulting from this pxocess thereby reconstituting a composition, the physio-logical properties of which are equivalent to the cyto-plasm of packed red cells.

The procedure of Example 8 is followe~ ex-cept that the warming stage is carried out at 30 degrees C
Eor 1 minu-te.

'EXAMP:LE 10 The procedure of Example 1 is followed ex-cept that 2~ weight to volume of di-phospho-glycerate is used.
EX~MPLE 11 The procedure of Example 1 is followed ex-cept that 1 mg. per cent of ergosterol is used in place of cholesterol.

The procedure of Example 1 is followed ex-cept that the emulsified droplets in the finished pro-duct are prepared to be 100 millimicrons in size.

The procedure follows Example 1 except that lS after the emulsification step,essential amino acids such as L-lysine, L-tryptophan, L-his-tidine, L-phenyl~
alanine, L-leucine, L-isoleucine, L~hreonine, L-~aline, L-orgine, and L-methionine can be added in the amounts and mixtures as indicated by the needs of -the individual situa-tion-EXAMPLE 1~
The procedure follows Example 1 except -tha-t hemoglobin and di-phospho-cJlyc:erate components are omitLe-:l lrom the manuEacturincJ process. This examp:le pr.orduces a compo,:ition wh:ich approximates the physio-I.ogical properties of the cytoplasm of erythrocytes.

Claims (39)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A synthetic whole blood substitute, consisting essentially of a non-toxic two phase liquid system, both said phases being aqueous, one of said phases being a relatively non-polar coacervate phase having physiologi-cal and physicochemical properties substantially equiva-lent to erythrocytes, the other of said phases being a relatively polar liquid aqueous phase having physiological and physicochemical properties substantially equivalent to blood plasma, said relatively non-polar coacervate phase being insoluble in and in equilibrium with said relatively polar liquid aqueous phase, and incorporated into said two phase system a hemoglobin component selected from polymerized hemoglobin, pyridoxylated-polymerized hemoglobin and mixtures thereof; said two phase liquid system having physiological and physico-chemical properties essentially similar to those of whole blood.

2. A synthetic whole blood substitute according to claim 1, wherein the relatively non-polar coacervate phase comprises from .05% to 99.5% by volume, of the two phase liquid system.

3. A synthetic whole blood substitute according to claim 1, wherein the phases are emulsified, whereby said relatively non-polar coacervate phase is in the form of coacervate droplets suspended in said relatively polar liquid aqueous phase.

4. A synthetic whole blood substitute according to claim 3, wherein said droplets are essentially of size within the range from 100 millimicrons to 10 microns.

5. A synthetic whole blood substitute according to claim 1, wherein the pH of the two phase liquid system is in the range of from 7.35 to 7.45.

6. A synthetic whole blood substitute according to claim 1, wherein the two aqueous phases comprise a protein or protein derivatives with surface active properties, an electrolyte, a surface active agent and water.

7. A synthetic whole blood substitute according to claim 6, wherein the protein or protein derivative is selected from albumin, gelatin or modified fluid gelatin.

8. A synthetic whole blood substitute according to claim 6, wherein the electrolyte is selected from sodium chloride, magnesium chloride, calcium chloride, potassium chloride and mixtures thereof.

9. A synthetic whole blood substitute according to claim 6, wherein the surface active agent is a phos-pholipid or a derivative thereof.

10. A synthetic whole blood substitute according to claim 9, wherein the phospholipid is selected from lecithin, cephalin, isolecithin, sphingomyelin, phospha-tidyl serine, phosphatidic acid, phosphatidyl inositol, phosphatidyl choline or mixtures thereof.

11. A synthetic whole blood substitute according to claim 1, wherein one or both of the aqueous phases include urea, electrolytes, hemoglobin, di-phosphoglycerate, sterols, or mixtures or combinations thereof.

12. A synthetic whole blood substitute according to claim 11, wherein the hemoglobin is pyridoxylated-polymerized hemoglobin.

13. A synthetic whole blood substitute according to claim 11, including di-phospho-glycerate.

14. A composition according to claim 11, wherein the sterol is selected from cholesterol, ergosterol, 7-dehydrocholesterol, .alpha. sitosterol, .beta. sitosterol, .gamma. sitosterol or campesterol, or mixtures thereof.

15. A composition according to claim 11, wherein the electrolytes are selected from NaCl, KCl, MgCl, CaCl2 or mixtures thereof.

16. A method of preparing a composition of matter which is useful as a blood substitute, said method characterized by the steps of (a) combining albumin and phospholipid in water; (b) thoroughly mixing the components; (c) storing said mixture undisturbed until the composition of step (a) separates into two layers, one above the other, the lower layer being a substan-tially non-polar coacervate phase, and the upper layer being an equilibrium water phase; (d) continuing the separation process until no increase in the volume of the coacervate phase can be observed; (e) centrifuging the composition until inspection reveals a clear demarcation of the two phases; (f) separating the two phases; and (g) adding a hemoglobin component selected from polymerized hemoglobin, pyridoxylated-polymerized hemoglobin and mixtures thereof, to the coacervate phase.

17. The method of claim 16, wherein the phospholipid is selected from lecithin, cephalin, iso-lecithin, sphingomyelin, phosphatidyl serine, phosphatidic acid, phosphatidyl inositol, phosphatidyl choline, or mixtures thereof.

18. The method of claim 16, wherein the hemoglobin is pyridoxilated-polymerized hemoglobin.

19. The method of claim 18, including the step of adding from 0.5% to 10% weight to volume of di-phospho-glycerate to the coacervate phase, after the addition of pyridoxylated-polymerized hemoglobin.

20. The method of claim 19, including the further step of combining the equilibrium phase of the coacervate system and the associated coacervate phase now containing said additives.

21. The method of claim 20, including the further step of emulsifying the composition.

22. The method of claim 21, including the further step of adding an electrolyte in an amount that will render the isotonicity of the preparation equal to that of human blood.

23. The method of claim 22, wherein the electrolyte is selected from sodium chloride, potassium chloride, calcium chloride, magnesium chloride, or mixtures thereof.

24. The method of claim 23, including the further step of adding 0.1 to 1 mg. urea.

25. The method of claim 24, including the step of adding a sterol.

26. The method of claim 25, wherein the sterol is selected from cholesterol, ergosterol, 7-dehydrochole-sterol, .alpha. sisterol, .beta. sitosterol, .gamma. systerol, campesterol, and mixtures thereof.

27. The method of claim 25, including the step of adding from 0.1 to 10 mg. percent cholesterol.

28. The method of 27, including the step of adjusting the pH of the preparation to 7.35 to 7.4 by the dropwise addition of either hydrochloride acid or sodium chloride.

29. The method of claim 28, including the further step of emulsifying the composition after said pH adjustment.

30. The method of claim 29, wherein the particles of said emulsion range from 100 millimicrons to 10 microns.

31. The method of claim 21, wherein the emulsified composition is subjected to a process to harden the surfaces of the emulsified droplets contained within said emulsion.

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32. The method of claim 31, wherein the hardening process is based upon either a physical or a chemical procedure.

33. The method of claim 32, wherein the physical process is comprised of a warming step.

34. The method of claim 33, wherein the composition is subjected to a warming procedure in which the composition is placed in a water bath, the temperature of which is from 15°C to 50°C.

35. The method of claim 34, wherein the warming period is from 20 seconds to 3 hours.

36. The method of claim 31, wherein the surface hardened droplets are filtered from the composition.

37. The method of claim 36, wherein the emulsified droplets containing the hemoglobin component are washed thoroughly.

38. The method of claim 37, wherein the composition is reconstituted by the addition of any physiologically suitable solution.

39. The method of claim 38, wherein the solution is normal saline solution.