Preparation of synthetic frythrocytes

A sterile preparation of synthetic erythrocytes consisting of hemoglobin fraction encapsulated within water-immiscible amphiphylic membranes provides a total hemoglobin of at least about 12 gm percent at a hematocrit of 50%. A lipid composition is prepared and dispersed by agitation in a sterile, stroma-free 30-45 gram percent hemoglobin fraction. The dispersion is pressurized to between about 400 to about 900 kg/cm.sup.2, and the pressure is substantially instantaneously released by passing the mixture to a lower pressure region through an orifice having an area of between about 0.1 and about 10.0 mm.sup.2, thereby forming the synthetic erythrocytes. The preparation is filtered through a filter which passes particles having a diameter less than about 0.22 micron to eliminate any monocellular organisms that may be present and to eliminate larger synthetic erythrocytes. The synthetic erythrocyte preparation, if dried under vacuum to remove the major portion of the water from the encapsulated hemoglobin fraction, substantially transforms the erythrocytes but retains the integrity of the encapsulating lipid composition membranes. In the dried form, the sterile erythrocytes are storable for extended periods.

BACKGROUND OF THE INVENTION 
Erythrocytes are the red cells of blood which serve the biological function 
of transporting respiratory gases. In nature, the walls of the red cells 
are membranes which contain many different kinds of proteins and lipid 
materials. Oxygen passes through the erythrocyte walls and is exchanged 
for carbon dioxide which the erythrocytes carry away from the tissue. 
It has long been common in the practice of medicine to take blood from a 
donor and transfuse this into the blood circulatory system of a patient 
who is deficient in hemoglobin. There are, however, difficulties in the 
preparation of blood for transfusion and substantial difficulties in 
maintaining adequate reserves of whole blood and/or blood components for 
transfusion. 
One difficulty is that the natural erythrocytes in the blood of animals and 
humans deteriorate relatively soon after the blood is drawn, and present 
regulations require that the blood must be used for human transfusion 
within 21 days after it is drawn. Another serious inconvenience is that 
the blood of the donor must be typed and transfusions generally made into 
subjects whose blood is of the same type as that of the donor. Both of 
these disadvantages are due to the presence of proteins which are 
contained within the membranes of the natural erythrocytes. 
U.S. Pat. No. 4,133,874 discloses a process in which a lipid in an organic 
solvent is spun to form a film on the interior walls of a container, and 
this film allowed to dry. Stroma-free hemoglobin is added, and by the use 
of ultrasound, hemoglobin is encapsulated within lipid composition 
membranes to form synthetic erythrocytes. 
The '874 patent teaches that synthetic erythrocytes having hemoglobin 
solution encapsulated in lipid composition membranes can be used to 
transport respiratory gases in warm blooded animals; however, one with 
knowledge of the function of erythrocytes would recognize that 
improvements over the preparations described in the '874 patent might 
greatly enhance their utility. 
The sonification method in the '874 patent is useful for producing 
synthetic erythrocytes under laboratory conditions but is not readily 
adaptable to mass production techniques. Importantly, the sonification 
method presents obstacles to providing and maintaining sterility of the 
preparation. 
The most concentrated hemoglobin fraction encapsulated in the disclosure of 
the '874 patent is 22 gram percent hemoglobin, i.e., about two-thirds the 
concentration of hemoglobin within the erythrocytes of healthy humans. 
Using this concentration of hemoglobin, the preparation at a 50 percent 
hematocrit (slightly greater than normal whole blood) is necessarily less 
than 12 percent, and accounting for the synthetic erythrocyte membranes 
and the void volume between packed cells, the total hemoglobin at 50 
percent hematocrit would not be more than about 9 gram percent. This 
compares quite unfavorably with the total hemoglobin of about 15 gram 
percent found in normal human blood. 
The synthetic erythrocytes formed by the sonification process described in 
the '874 patent have a range of diameters of from about 0.1 microns to 
about 10 microns. The upper end of this size range is generally unsuitable 
for transfusion into warm blooded animals, being too large to fit through 
capillaries (human erythrocytes have a diameter of about 7 microns). 
Synthetic erythrocytes should be somewhat smaller than natural 
erythrocytes because synthetic erythrocytes are less flexible and do not 
pass as easily through the constricted capillaries. Several advantages 
accrue by providing synthetic erythrocytes within a narrow size range at 
the lower end of the size range described in the '874 patent. 
An essential attribute of a synthetic erythrocyte preparation for 
transfusion into animals, and particularly humans, is that the preparation 
be sterile. The introduction of a synthetic erythrocyte preparation 
represents a dilution of infection-resistant agents normally present 
within blood, including antibodies produced by lymphocytes. In any case, a 
synthetic erythrocyte preparation should not introduce infectious agents. 
Synthetic erythrocyte preparations cannot be sterilized by heat or any 
other sterilization method which would denature the hemoglobin or 
destabilize the synthetic erythrocyte membranes. 
An important projected use of synthetic erythrocytes preparations is to 
substitute for whole blood in remote locations where there is no readily 
available source of fresh blood. Whereas the shelf life of blood is about 
21 days under refrigeration, synthetic erythrocyte preparations may be 
stored for considerably longer periods. It would be desirable to have 
synthetic erythrocytes which may be stored substantially indefinitely even 
when not refrigerated. 
SUMMARY OF THE INVENTION 
The present invention provides a preparation of sterile synthetic 
erythrocytes having a total hemoglobin concentration of at least about 12 
gram percent at a hematocrit of 50 percent. A water-immiscible composition 
is prepared which is between about 60 and about 90 weight percent lipids, 
between about 10 and about 40 weight percent of a sterol and between 0 and 
about 10 weight percent of an agent that imparts a negative charge to the 
surface of the composition. A stroma-free hemoglobin fraction is separated 
from whole blood, and its hemoglobin concentration is adjusted to between 
about 30 and about 45 gram percent. The water-immiscible amphiphylic 
composition and hemoglobin fraction are mixed at a volume ratio of between 
about 1:3 and about 1:10, and the mixture is agitated to disperse globules 
of the water-immiscible composition less than about 1 mm in diameter 
within the hemoglobin fraction. The resultant dispersion is pressurized to 
a gage pressure of between about 450 kg/cm.sup.2 and about 900 
kg/cm.sup.2, and the pressurized dispersion is subjected to high shear 
conditions by passing it through an orifice having a cross-sectional area 
of between about 0.05 mm.sup.2 and about 10.0 mm.sup.2 to substantially 
instantaneously release the pressure, thereby forming a preparation of 
synthetic erythrocytes in which hemoglobin fraction is encapsulated within 
outer membranes of the lipid composition. The preparation is filtered 
through a filter having a pore size that passes particles having a maximum 
diameter of below about 0.22 micron.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Herein the term "synthetic erythrocyte" is used to refer to a tiny spheroid 
in which hemoglobin fraction is encapsulated in a lipid composition 
membrane. 
The term "lipid" refers to various materials that are soluble in non-polar 
solvents, including fats, waxes, phosphatides, cerebrosides, and related 
and derived compounds which constitute the principle structural components 
of the living cell. The lipids used herein may be obtained from a wide 
variety of sources. A lipid composition is selected which does not induce 
immunological responses in a host mammal. 
It is believed that lipid composition encapsulation of a hemoglobin 
fraction is a surface related effect in which polar moieties of the lipids 
face inward to the encapsulated hemoglobin fraction and outward to the 
free hemoglobin fraction while non-polar moieties aggregate centrally 
within the membrane. It is believed that lipid membranes formed by the 
processes of the invention are two molecules thick with non-polar ends of 
lipid molecules joined end-to-end. The lipid composition must have 
sufficient polar moieties to form the membrane, and it is preferred that 
the phospholipids comprise at least about 50% by weight of the lipid 
composition. Lecithin is a preferred lipid composition for forming the 
lipid membrane being high in phospholipid content and generally free of 
immune response-inducing agents. 
The lipid composition preferably incorporates a sterol, such as 
cholesterol, to enhance the mechanical strength of the synthetic cell 
membrane. The sterol is preferably present in amounts of between about 10% 
and about 40% by weight of the lipid composition. 
The lipid composition may incorporate an agent to adjust the charge of the 
lipid composition and thereby the charge of the synthetic cell surface. 
The surface of a natural erythrocyte is negatively charged, a feature 
which prevents aggregation of the cells. The zeta potential of a natural 
erythrocyte is about 18 millivolts, and that the zeta potential of the 
synthetic erythrocyte may be adjusted to between about 7 and about 23 
millivolts. Suitable agents for adjusting the charge include phosphatidic 
acid, dicetyl phosphate and pharmaceutically acceptable salts thereof. The 
charge adjusting agent may comprise from 0 up to about 10 weight percent 
of the lipid composition. 
The lipid composition is prepared by dissolving its various components in 
an organic solvent or a mixture of solvents, such as chloroform, 
chloroform-methanol mixture or dichloromethane, to produce a homogeneous 
mixture, and thereafter, the organic solvent is evaporated away. The 
organic solvent system is selected to destroy infectious agents present in 
the lipid compositions. It is desirable to substantially remove the 
solvent prior to admixture with hemoglobin lest the solvent denature the 
hemoglobin. The lipid composition has a paste-like consistency. 
Hemoglobin is obtained from washed red blood cells lysed by conventional 
means. The cells are washed by repeated suspensions in isotonic saline (or 
balanced salt solutions) followed by centrifugation to pack the cells. 
Mechanical methods, such as freezing and thawing the cells, ultrasonic 
disruption of the membranes hypotonic lysing, or high pressure (70-210 
kg/cm.sup.2) disruption are generally preferred to chemical membrane 
disruption methods so that the amount of hemoglobin denaturization is 
minimized. The cells may, however, be chemically lysed by toluene, a 
solvent which does not denature hemoglobin. 
In one method of separating the stroma from the hemoglobin of the lysate, 
the pH of the lysate is reduced from the physiological pH of about 7.4 to 
below about 5.0 where the stroma readily precipitates out by 
centrifugation. A clear, concentrated hemoglobin solution is obtained by 
filtration which removes remaining cell solids. Alternatively, stroma may 
be removed by ultrafiltration, e.g., through 100,000 pore size filter. 
The gas transport capacity of the synthetic erythrocyte preparation is a 
function of the total amount of encapsulated hemoglobin. It is desirable 
that the total amount of encapsulated hemoglobin per volume of synthetic 
erythrocyte preparation approach or even exceed the total amount of 
hemoglobin encapsulated in a similar volume of natural erythrocytes, and 
for purpose of this invention, a synthetic erythrocyte preparation should 
provide a total hemoglobin of at least about 12 gram percent at a 
hematocrit of 50 percent. The preferred size range of the erythrocytes in 
the preparation is between about 0.05 and about 0.2 micron in diameter. To 
achieve a total hemoglobin level approaching that of normal blood, it is 
preferred that the hemoglobin fraction used for encapsulation be at the 
high end of the hemoglobin concentration range (28-33 weight percent) 
found within natural erythrocytes and preferably even higher. Highly 
concentrated hemoglobin solutions, however, are quite viscous making them 
difficult to encapsulate, and a fifty gram percent hemoglobin fraction 
represents the most highly concentrated solution that can be generally be 
encapsulated. For purposes of the invention, it is preferred that the 
hemoglobin concentration of the hemoglobin fraction be between about 30 
and about 45 weight percent. 
Where a more highly concentrated hemoglobin fraction is desired than is 
obtainable from cell lysis, it is necessary to concentrate the hemoglobin. 
The hemoglobin may be concentrated by filtering the hemoglobin fraction 
with a filter having a pore size sufficient to pass water and other small 
molecules, e.g. about 10,000 MW filter, but retaining the larger 
molecules. The hemoglobin fraction might also be concentrated by 
evaporating water, e.g., through lyophilization; however, if the water is 
withdrawn, it is also desirable to remove a corresponding amount of 
electrolytes so that the encapsulated hemoglobin fraction is not unduely 
high in electrolyte concentration causing it to draw in water through 
osmosis, expanding and possibly lysing the lipid composition membrane. 
It is important that the hemoglobin be sterile lest the artifical 
erythrocytes introduce infection to the transfused animal. Excess heat, of 
course, would denature the hemoglobin. In above-mentioned U.S. Pat. No. 
4,133,874, bacteriostatic agents, such as gentamycin and tetracycline, 
were added to the hemoglobin solution. While such agents may be added, 
their use is preferably avoided because the product erythrocytes are 
intended to be generally acceptable to all recipients, and bacteriostatic 
agents may induce allergic response in certain patients. A preferred 
method of sterilizing the hemoglobin solution is by passing the solution 
through a membrane permeable by hemoglobin but impermeable to living 
cells. Such a membrane has a pore size which permits passage of particles 
less than about 0.22 microns in diameter. Suitable membranes for this 
purpose are sold by Nuclepore and Millipore. 
The lipid composition is dispersed in the hemoglobin solution by blending 
with a high speed mixer to break the lipid composition into globules 
having an average size less than about 1 mm in diameter. The ratio of 
lipid composition to hemoglobin fraction is not critical, the ratio 
affecting efficient utilization of the components more than erythrocyte 
production. However, the volume ratio of hemoglobin fraction to lipid 
composition should be greater than about 3:1 or else significant amounts 
of lipid spheroids will be formed having no encapsulated hemoglobin. On 
the other hand, a large excess of hemoglobin fraction is wasteful of 
hemoglobin and increases the required capacity of hemoglobin recycling 
systems. A preferred volume ratio of hemoglobin fraction to lipid 
composition is between about 5:1 and about 10:1. 
At the high hemoglobin concentrations used to form the synthetic 
erythrocytes and within the preferred volume ratio range of hemoglobin 
fraction to lipid compositions, quite viscous dispersions are formed. For 
example, a dispersion of 35 gm % hemoglobin solution mixed at a 10:1 
volume ratio with a lipid composition comprising 40.7 g lecithin, 20.7 g 
cholesterol, and 6.9 g dicetyl phosphate is found to have a viscosity of 
982 centipoise at 37.degree. C. and 1640 centipoise at 4.degree. C., as 
measured using a Brookfield cone plate viscometer with a CP-42 cone and 
cone angle of 1.565 degrees and shear rate of 1.15 sec.sup.-1. Dispersions 
in accordance with this invention have viscosities, as measured above, of 
from about 1500 to about 3200 centipoise at 4.degree. C. The actual 
viscosity of each dispersion depends on several factors including the 
concentration of the hemoglobin fraction, the specific lipid composition, 
the volume ratio of hemoglobin fraction and lipid composition and the size 
of the dispersed lipid composition particles. The sonification method used 
for hemoglobin encapsulation in the above-mentioned 4,133,874 patent would 
be generally ineffective for encapsulating hemoglobin dispersions having 
such high viscosities. 
In accordance with the present invention, synthetic erythrocytes are formed 
from a hemoglobin fraction-lipid composition dispersion by subjecting the 
dispersion to substantial pressures and substantially instantaneously 
releasing the pressure. 
According to one method of forming the erythrocytes, the dispersion is 
pressurized, e.g., by mechanical means, to between about 400kg/cm.sup.2 
and about 900 kg/cm.sup.2, and preferably between about 450kg/cm.sup.2 and 
about 700kg/cm.sup.2. The pressurized dispersion is then passed through a 
restricted orifice or nozzle at high velocity to a region of lower 
pressure, thereby subjecting the dispersion to substantial shear forces 
which results in creation of the synthetic erythrocytes having thin lipid 
membranes encapsulating hemoglobin fraction. 
Another method of pressurizing the lipid-hemoglobin dispersion is by 
introducting a pressurized inert gas, such as nitrogen, into the vessel 
containing the dispersion. When the pressure is substantially 
instantaneously released by passing the gas-infused mixture through a 
restricted orifice, synthetic erythrocytes are formed. 
The shear force to which the dispersion is subjected is an important factor 
in determining the size distribution of the synthetic erythrocytes of the 
preparation. The size distribution of the erythrocytes of the preparation 
depends on several factors including the pressure, the orifice area, and 
the viscosity of the dispersion. At the present time, there does not exist 
a precise correlation of these factors with synthetic erythrocytes size 
distribution; however, it is known that the higher the pressure, the 
larger the permissible size of the orifice that will produce the necessary 
shear force to obtain predominantly monolaminar synthetic erythrocytes. 
Generally, within the above-described pressure ranges, the orifice size 
should be between about 0.1 square millimeter and about 10 square 
millimeters. For a given lipid composition and a given hemoglobin 
fraction, the pressure and orifice size may be adjusted to obtain a 
desired size distribution. 
For purposes of this invention, it is preferred that about 80 percent of 
the erythrocytes produced have a diameter within a 0.05 to 0.2 micron 
range. The small synthetic erythrocytes in this size range have high 
surface-to-volume ratios that increase gas exchange through the membrane, 
and synthetic erythrocytes of this size range have less tendency to 
aggregate than larger synthetic erythrocytes. Using synthetic erythrocytes 
less than about 0.2 micron in diameter reduces or eliminates the tendency 
of the synthetic erythrocytes to lodge within the capillaries. This small 
size range is also highly desirable for perfusion of ischemic tissue. 
Although larger size synthetic erythrocytes might be expected to have 
greater hemoglobin fraction to lipid composition membrane volume ratios, 
it is believed that larger synthetic erythrocytes tend to have 
multilaminar membranes negating this apparent advantage. It is found 
experimentally that the greatest amount of hemoglobin encapsulation occurs 
in the 0.05-0.2 micron diameter range which corresponds closely to the 
size of the majority of cells produced by the methods of the present 
invention. Below about 0.05 microns, the small size tends to substantially 
decrease the hemoglobin to lipid membrane volume ratio. 
Previous teachings of lipid encapsulation of aqueous solution suggest that 
single membrane layer liposomes have a diameter range of between about 
0.02 and about 0.05 micron, whereas liposomes may be formed between 0.1 
and 10 microns having multilaminar membranes. Suprisingly and 
unexpectedly, using lipid compositions and hemoglobin solutions with the 
above-described parameters and processing them within the above-described 
pressure and orifice size ranges, the tendency is to form liposomes that 
are predominantly in the 0.05 to 0.2 micron diameter size range and which, 
based upon the measured total hemoglobin of the preparation, appear to be 
predominantly monolaminar. 
After the synthetic erythrocytes are prepared, they are again passed 
through a filter that does not permit passage of particles having 
diameters greater than 0.22 microns in order to remove any unicellular 
infectious agents which were not previously removed or destroyed or which 
might have been later introduced. This filtering process also removes the 
small percentage of larger synthetic erythrocytes. 
In accordance with an important aspect of the present invention, it is 
found that the synthetic erythrocytes may be dried under vacuum to remove 
a major portion of the water content and that when so dried, the 
configuration of the synthetic erythrocytes changes dramatically. The 
dried synthetic erythrocytes may be stored for greatly extended periods of 
time and reconstituted merely by adding water or an aqueous solution. 
Despite the encapsulation of highly colored hemoglobin, the synthetic 
erythrocytes cannot be clearly seen under an optical microscope, the lipid 
membranes diffusing light so that a clear image cannot be seen. However, 
when the synthetic erythrocytes are dried to where the water concentration 
in the encapsulated hemoglobin fraction is below about 50 weight percent, 
the surface conditions which created the synthetic erythrocytes are 
substantially altered, and the synthetic erythrocytes undergo a 
transition. The transformed synthetic erythrocytes are readily 
distinguishable from the original synthetic erythrocytes, being easily 
seen under an optical microscope appearing as red spheroids. Thus, 
suprisingly, the lipid membranes remain intact even though the conditions 
of surface interaction, under which the membranes were created, are 
radically changed. Furthermore, it is found that the dried spheroids are 
reconstitutable into their original form merely by adding aqueous 
solution. In dried form, the synthetic erythrocytes are highly resistant 
to degradation and may be stored for long periods of time. For long-term 
storage, it is preferred that the water content of the synthetic 
erythrocytes be reduced to below about 10% by weight of the hemoglobin 
fraction and more preferably to below about 1% by weight of the hemoglobin 
fraction. 
Referring now in greater detail to the schematic diagram (FIG. 1) 
representing one mode of practicing the invention, squares or blocks are 
used to represent steps or units of equipment. 
Beginning at the top of FIG. 1, a washing saline solution from a container 
10 and whole blood from container 9 are passed through a centrifuge 11 
used to separate plasma from whole blood. The blood may have been drawn 
from humans or from other mammals. The plasma is separated off, and the 
fraction containing the red blood cells, or erythrocytes, is passed to a 
cell lysing apparatus 12 where the natural erythrocytes are subjected to 
high pressure to rupture the cell membranes after which the membranes and 
any tissue solids are removed by ultrafiltration at unit 13a, leaving a 
stroma-free hemoglobin fraction. The ultrafiltration also removes 
particles any cellular infectious agents. A unit 13b is used to 
concentrate the hemoglobin fraction to between about 30 and about 45 gram 
percent. The sterile, stroma-free hemoglobin is held in a sealed 
receptacle 13c. 
Turning now to the left-hand side of FIG. 1, there is a mixing vessel 14 
into which a quantity of a lipid material is fed from a container 15. 
There is also added sterol, such as cholesterol, from a container 16, and 
a surface charge-adjusting agent, such as dicetyl phosphate, is added from 
a container 17 to give the mixture the desired electrical charge. A 9:1 
v/v chloroform-methanol solution is added from a container 18. The 
phospholipid, sterol, and dicetyl phosphate are dissolved in the solvent 
using a mixer 19. 
The resulting non-polar solution is fed into a mixing vessel 20 having an 
associated agitator 22a, a vacuum source 22b and heater 22c. The heater 
22c mildly heats the solution while the vacuum source 22b draws off all of 
the solvent leaving a sterile lipid composition. The lipid composition is 
cooled to about 10.degree. C., and hemoglobin solution from vessel 13c is 
introduced. 
As the hemoglobin solution is introduced, the agitator 22a is actuated 
breaking the lipid composition into globules which disperse within the 
hemoglobin solution. Agitation is continued until substantially all of the 
lipid composition is broken into globules less than about 1 mm in 
diameter. 
From the mixing vessel 20, the dispersion is passed to a compression 
chamber 23 having a piston 23a, and one or more orifices 25 having an 
associated valve 25a. The compression chamber 23 has associated cooling 
apparatus 23b to cool the dispersion to between about 20.degree. C. and 
0.degree. C. 
The orifice 25 may be of the type illustrated in either of FIG. 2, FIG. 3, 
or FIG. 4. The type 25 illustrated in FIG. 2 is an ordinary circular 
orifice in the wall of a vessel. The type 25' shown in FIG. 3 is like that 
shown in FIG. 2 but with rounded corners at the edge of the orifice which 
causes the orifice to resemble that which is found in a common nozzle. The 
type 25" shown in FIG. 4 is a variable orifice. 
The piston 23a is actuated until the dispersion is sufficiently 
pressurized. As stated above, the required pressure depends on the 
viscosity of the dispersion and the particular orifice size. After the 
dispersion is fully pressurized, the valve 25a is actuated opening the 
orifice 25, resulting in the dispersion being expelled from the 
compressing chamber 23 into a collecting receptacle 26. As the lipid 
globules pass through the orifice at high speed, they are subjected to 
very high shear forces, and the dispersion emerges from the orifice 25 in 
the form of synthetic erythrocytes having lipid composition membranes 
encapsulating hemoglobin solution, the synthetic erythrocytes being 
suspended in the remaining hemoglobin solution. 
The synthetic erythrocytes from receptacle 26 are preferably washed by 
addition of a balanced salt solution. The synthetic erythrocytes are 
filtered in a unit 28 to remove unencapsulated hemoglobin solution from 
the synthetic erythrocytes. Then the synthetic erythrocytes are passed 
through a second filter 28a to reassure removal of any bacteria and remove 
any oversize synthetic erythrocytes. The filtered synthetic erythrocytes 
may be used, as packed cells as constituted, for transfusion as a product, 
or may be dried in a vacuum unit 29 for storage as packed cells and use at 
a later date. 
The filtered synthetic erythrocytes are suspended in a vessel 37 in a 
plasma-like solution, such as balanced salt solution to which albumin is 
added. This suspension may be directly transfused as a whole blood 
substitute into a mammalian animal, or may be dried in a vacuum unit 38 
for storage as reconstitutable artificial blood. 
The diluted hemoglobin solution, obtained as a byproduct of washing the 
synthetic erythrocytes at the filtering unit 28, may also be passed under 
pressure through a filter 39 having a pore size, e.g., 1000 MW, that 
retains the hemoglobin but allows passage of the water and small dissolved 
molecules in order to concentrate the recovered hemoglobin. The 
concentrated stroma-free hemoglobin is reintroduced along with fresh 
stroma-free hemoglobin into the mixing vessel 20. 
In a modified process, the modified steps being represented in FIG. 1A, the 
compression chamber 23' is associated with a source 36 of inert gas, such 
as nitrogen. In this modified process, the nitrogen, or other inert gas, 
is pumped from source 36 into the compressing chamber 23' and much of the 
gas is absorbed into the lipid and water in hemoglobin fraction dispersion 
in the compression chamber 23'. When a valve 25a' is opened so that the 
mixture containing the absorbed gas emerges rapidly from the orifice 25' 
with a sudden drop in the applied pressure, synthetic erythrocytes are 
formed as before. 
Following are specific examples which described carrying out the invention: 
EXAMPLE 1 
A lipid composition is prepared by dissolving 40.7 grams of egg lecithin, 
20.7 grams of cholesterol and 6.9 grams of dicetyl phosphate in 200 ml of 
a 9:1 v/v chloroform-methanol mixture. In a mixing vessel equipped with a 
heater, a stirrer, and a vacuum, the solvent drawn off with heat and 
vacuum. 
A hemoglobin fraction is obtained by lysing washed, packed erythrocytes and 
removing the stroma. The hemolysate is determined to have 29 gm percent 
hemoglobin. 20 gm percent hemoglobin solution is obtained by diluting the 
hemolysate with a balanced salt solution. 30 and 40 gram percent 
hemoglobin solutions are obtained by exposing the hemolysate to a filter 
which allows passage of water and electrolytes but which retains the 
hemoglobin. The remaining steps of the process are performed three times, 
once with each concentration (20, 30 and 40%) of hemoglobin fraction. 
The hemoglobin fraction is passed through a filter that permits passage of 
particles having diameters of 0.22 microns or less, and 200 ml of filtered 
hemoglobin fraction is introduced into the mixing vessel. The agitator 
within the vessel is activated spinning a blade at 16,000 rpm for 15 
minutes to disperse the lipid composition as small globules within the 
hemoglobin fraction. 
The dispersion is transferred to a compression chamber having a piston to 
reduce the volume and a valved circular orifice 1 mm in diameter. The 
piston is mechanically driven, creating a pressure of 700 kg/cm.sup.2, and 
then the valve is opened allowing the dispersion to escape to a 
receptacle. 
The resulting preparation contains synthetic erythrocytes suspended in the 
remainder of the hemoglobin fraction. The preparation is filtered to 
remove non-encapsulated hemoglobin fraction and the preparation is washed 
to remove all remaining hemoglobin. At this point, a sample of synthetic 
erythrocytes is removed from the washed, filtered preparation, and the 
size of the synthetic erythrocytes determined by freeze fraction electron 
microscopy. The following data represents the size distribution of the 
synthetic erythrocytes prepared using the 20, 30, and 40 gram percent 
hemoglobin fractions. Cell Size distribution as determined by electrom 
microscopy. 
______________________________________ 
Concentration of Hemoglobin 
Fraction 20 gm % 30 gm % 40 gm % 
______________________________________ 
number of cells counted 
283 293 397 
Mean diameter (microns) 
.1802 .1316 .1221 
Standard deviation 
.0999 .0753 .0752 
Min. diameter .0074 .0074 .0074 
Max. diameter .7037 .5852 .5852 
percent below 0.22 microns 
79.5 93.6 97.9 
in size 
______________________________________ 
The preparation is passed through a filter that allows passage of particles 
of about 0.22 microns or less, removing any monocellular microorganism and 
larger synthetic erythrocytes. The total hemoglobin is determined for the 
preparation from each hemoglobin solution, and the total hemoglobin at a 
fifty percent hematocrit for the 20 gram percent fraction is 9 gram 
percent, for the 30 gram percent fraction preparation 14 and for the 40 
gram percent fraction 19. The total hemoglobin in each case corresponds 
closely to the theoretical amount of hemoglobin encapsulated if all of the 
synthetic erythrocytes have monolaminar membranes. 
This experiment demonstrates that a sterile synthetic erythrocyte 
preparation can be produced according to the present invention having a 
total hemoglobin within the range of natural erythrocytes. 
EXAMPLE 2 
A dispersion of lipid globules in a 30 gm percent hemoglobin fraction is 
produced as in Example 1. The dispersion is transferred to a compression 
chamber with an associated source of pressurized nitrogen and a 1 mm. 
diameter orifice. The chamber is communicated to the source of pressurized 
nitrogen to raise the pressure in the chamber to 450 kg/cm.sup.2. The 
valve is then opened to release the pressurized dispersion through the 
orifice. 
The resulting preparation is washed and filtered to remove larger 
erythrocytes and any infectious agents. The washed and filtered 
erythrocyte preparation has a 12 gram percent total hemoglobin 
concentration at a 50% hematocrit. 
EXAMPLE 3 
The synthetic erythrocytes prepared in Example 1 from the 30 gram percent 
hemoglobin fraction are packed and subjected to vacuum conditions at 
4.degree. C. until no further weight loss is detected. The water 
concentration is determined to be less than 1 weight percent of the 
encapsulated hemoglobin. The synthetic erythrocyte suspension is examined 
under a microscope before and after drying. No clear image of cells is 
produced in the wet preparation. Dried synthetic erythrocyte suspension 
appears as red spheroids. Thus, it is demonstrated that synthetic 
erythrocytes, produced in accordance with this invention, undergo a 
substantial transformation when they are dried. 
The dried synthetic erythrocyte preparation is reconstituted with balanced 
salt solution. A determination of the free hemoglobin in the reconstituted 
suspension shows that less than about 5 percent of the hemoglobin is freed 
during lyophylization and reconstitution. 
EXAMPLE 4 
The washed synthetic erythrocyte preparared in Example 1 from the 30 gm 
percent hemoglobin fraction is suspended in equal volume of Ringer's 
solution containing 5 volume percent albumin. 
40 ml of the resulting synthetic erythrocyte suspension is administered to 
a rat by a technique wherein an infusion pump is employed to effect 
simultaneous withdrawal of blood from the femoral artery and infusion of 
the synthetic erythrocyte suspension into the femoral vein. All 40 ml of 
suspension (approximately 250% of the rat's natural blood volume) is 
administered over a period of 3 hours. The rat survives the transfusion 
for more than 24 hours and eventually dies of bacterial infection (septic 
shock). 
Described in the foregoing description are certain embodiments of the 
invention, but it is understood that our invention may be embodied in 
various forms, and many changes may be made, all within the spirit of the 
invention. For example, the hemoglobin fraction with dispersed lipid 
composition may be passed under pressure through as orifice several times. 
Various features of the invention are set forth in the following claims.