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Description  |
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FIELD OF THE INVENTION
This invention relates to a process for preparing artificial red cells.
More particularly, this invention relates to process for preparing
artificial red cells of aqueous hemoglobin solution encapsulated in
membranes of polymerized hemoglobin. Still more particularly, this
invention relates to preparing artificial red cells comprising
microdroplets of aqueous hemoglobin solution encapsulated in membranes of
polymerized, cross-linked hemoglobin by forming microdroplets of
hemoglobin solution in oil and cross-linking the hemoglobin at the surface
of the microdroplets with a suitable cross-linking agent.
BACKGROUND OF THE INVENTION
The search for blood substitutes has been prompted by many serious
drawbacks to the use of donor blood. One of the most serious problems
relates to the fact that an adequate supply of compatible donor blood may
not be available at the time and place where it is needed. This would be a
particularly serious problem during periods of disaster or war when blood
is most needed. The incompatibility of different blood types and the
relatively short storage life of whole blood limits the practicability of
collecting and storing large amounts of whole blood. According to some
estimates, as much as 30% of human blood collected is not reinfused into
human patients. Although red blood cells have a relatively short storage
life which contributes to a large amount of wasted blood, the hemoglobin
contained in the red cells appears to be unaffected if stored under the
proper conditions. However, the red cell membranes tend to degrade in
storage. Further, the transmission of disease, especially hepatitis, is a
problem that makes physicians hesitant to transfuse whole blood or plasma
into patients under conditions that are not life-threatening to the
patient. Thus, there is a definite need for effective blood substitutes.
The primary function of a plasma expander or blood substitute is to
maintain an adequate circulation volume of a solution that is nontoxic to
the human body and which will transport oxygen throughout the body to
sustain life until the body can remanufacture a supply of natural blood.
Artificial blood substitutes have received considerable attention during
the last decade. The development of fluorochemical emulsions that
transport oxygen and carbon dioxide in the blood stream offered hope of a
viable blood substitute. Unfortunately, however, a number of problems have
limited the use of fluorochemical emulsions in human patients.
Aqueous hemoglobin solution is the main constituent of red blood cells and
is the blood's oxygen carrier. It has also been demonstrated to be safe
when used in human patients. However, its intravascular persistance and
oxygen-release characteristics have proven to be inadequate. Free
hemoglobin completely disappears from the blood stream in less than about
eight hours and its affinity for oxygen is much greater than that of an
equivalent amount of hemoglobin encapsulated within the natural
red-blood-cell membrane. This greater affinity makes liberation of oxygen
to the tissues much more difficult. It is the red-blood-cell membrane that
contains the antigenic material which causes problems when mismatching of
donor and recipient occurs. Problems of intravascular coagulation and
renal damage have been demonstrated to be caused by the stroma present in
such membranes and not the hemoglobin molecule itself. Thus, the use of
stroma-free hemoglobin solution (SFHS) as a blood substitute offers many
potential advantages regarding immunology, storage and bio-compatibility.
Also, since free hemoglobin is not toxic to the kidney, SFHS seems an
ideal starting material for a blood substitute.
A number of attempts have been made using standard encapsulated techniques
employing various polymeric materials such as celluloses, polystyrenes and
polyamides to create artificial red cells by encapsulating hemoglobin
therein. However, these standard encapsulation techniques have produced
red cells non-biodegradable by ordinary metabolism. Alternatively, U.S.
Pat. Nos. 4,001,401; 4,053,590; and 4,061,736 disclose blood substitutes
and plasma expanders comprising polymerized, cross-linked, stroma-free
hemoglobin in either the oxyhemoglobin or deoxyhemoglobin form, said
polymers having a molecular weight ranging from about 64,000 to 1,000,000.
These blood substitutes are prepared by cross-linking stroma-free
hemoglobin in bulk solution with a suitable cross-linking agent that is at
least bifunctional in nature. However, the hemoglobin polymers are not
artificial red cells, because they are merely polymeric molecules as such
and do not consist of a membrane encapsulating a fluid phase which can
reversibly combine with oxygen. In another attempt at creating artificial
red cells, Miller et al in U.S. Pat. No. 4,133,874 disclose forming
artificial red cells by encapsulating hemoglobin in liquid lipid materials
comprising phospholipids, and optionally cholesterol, to form cells
typically ranging from 0.1 to 10 microns in their greatest dimension. The
lipid material is said to form a continuous membrane around the hemoglobin
solution. However, phospholipids have a tendency to release their contents
into other cells in the body. Kitajima et al in U.S. Pat. No. 3,879,510
show reinforcing the naturally-occurring membrane around red blood cells
by reacting the membrane with an isocyanate such as toluene diisocyanate.
In reinforcing the naturally occurring membrane, red blood cells are
dispersed in a suitable isotonic or hypertonic saline solution to which is
added an oil-in-water emulsion of liquid polyisocyanate which reacts with
the membranes, thereby reinforcing same. However, it is the red blood cell
membranes which contain materials that cause short storage life, and
incompatibility problems between different blood types.
Thus, there is a need for artificial red cells comprising an encapsulated,
stroma-free hemoglobin solution of a suitable size, strength and
flexibility to permit same to be used effectively as blood substitutes.
SUMMARY OF THE INVENTION
The present invention relates to a process for preparing artificial red
cells comprising microdroplets of stroma-free, aqueous hemoglobin solution
encapsulated in membranes of cross-linked, polymerized hemoglobin, said
process comprising forming microdroplets of hemoglobin solution in a
continuous oil phase, cross-linking hemoglobins at the surface of the
microdroplets with a suitable cross-linking agent to form the artificial
red cells and recovering the cells. In one embodiment of the process of
this invention a liquid membrane technique employing a water-soluble
cross-linking agent is used to form the cells. In this process, a
stroma-free, aqueous hemoglobin is emulsified in oil, the emulsion is
suspended in an aqueous suspending phase containing the cross-linking
agent to form the red cells and the red cells are recovered. In another
embodiment of the process of this invention, microdroplets of the
hemoglobin solution are formed in an oil phase and an oil-soluble
cross-linking is added to the oil phase either before or after contacting
the oil phase with the hemoglobin solution and forming the dispersion of
microdroplets of hemoglobin solution in the oil phase.
It should be understood that the process of this invention extends to
encapsulating any cross-linkable proteinaceous material, in which case the
cell size may broadly range from about 0.1 to 100 microns.
As a further embodiment of this invention, the artificial red cells or
encapsulated proteinaceous material made flexible and readily deformable
in nature by contacting them with an aqueous solution with respect to
which the encapsulated material is hypotonic.
DETAILED DESCRIPTION OF THE INVENTION
One process for preparing the artificial red cells of this invention is by
the use of a liquid membrane encapsulation technique. In this method, the
stroma-free hemoglobin is emulsified in a surfactant-containing oil to
form discrete, red-cell-size microdroplets of hemoglobin in a continuous
oil phase. In general, this process comprises the steps of:
(a) emulsifying a stroma-free, aqueous hemoglobin solution in a
surfactant-containing oil to form an emulsion comprising discrete
microdroplets of said hemoglobin solution in a continuous phase of said
oil;
(b) forming, in an aqueous suspending phase, liquid membrane capsules
comprising globules of said emulsion formed in (a), said globules
comprising an aqueous interior phase of discrete microdroplets of
hemoglobin solution and an exterior oil phase immiscible with both said
aqueous interior phase and said suspending phase and wherein said
suspending phase contains a water soluble cross-linking agent capable of
polymerizing hemoglobin;
(c) forming artificial red cells suspended in said globules of oil phase by
maintaining said capsules in said aqueous suspending phase for a time
sufficient for said cross-linking agent to polymerize hemoglobin at the
surface of said hemoglobin solution microdroplets present in said capsule
to form an artificial membrane around each of said microdroplets of
hemoglobin solution;
(d) separating said suspending phase from said oil phase containing the
artificial red cells; and
(e) recovering the artificial red cells from the oil phase.
However, it should be noted that if the oil used to form the emulsion is a
lipid, such as a phospholipid, it may not be necessary to use a surfactant
in the oil. As a further note, if the hemoglobin is emulsified in a
phospholipid instead of in an oil/surfactant combination, the liquid
membrane capsules formed in step (b) could be regarded as being similar in
structure to certain types of liposomes.
The cross-linking agent may be in the suspending phase when the liquid
membrane capsules are formed or it may be added to same after said
capsules are formed. The amount of cross-linking agent added to the
suspending phase may be just enough to form an artificial membrane of
polymerized hemoglobin around each microdroplet of hemoglobin emulsified
in the oil globule or it may be present in amounts substantially in excess
of that required to form the artificial membranes. In the latter event the
reaction will be stopped or quenched after a suitable predetermined period
by separating the aqueous suspending phase from the oil phase containing
the artificial red cells, by diluting the aqueous suspending phase, by
adding a material which reacts with the remaining cross-linking agent, or
by a combination of these methods. It is believed that the cross-linking
agent dissolved in the oil diffuses to the interface between the oil and
the microdroplets of hemoglobin solution. As has hereinbefore been stated,
the cross-linking agent or agents must have at least two functional groups
capable of reacting with the amine groups of lysine residues which are
located at numerous sites of the hemoglobin molecule. The cross-linking
agent must form intermolecular cross-links, although some intramolecular
cross-links can be tolerated. The cross-linking of the hemoglobin at the
surface of each microdroplet forms a film or membrane of polymerized
hemoglobin that effectively encapsulates the stroma-free hemoglobin
solution, thereby forming artificial red cells.
Important to the process of this invention is separating the artificial red
cells from the oil phase in which they were formed. This may be done by
separating the suspension of artificial red cells in the oil phase from
the aqueous suspending phase and washing the suspension several times with
an appropriate solution such as isotonic saline solution to remove any
unreacted cross-linking agent. The washed suspension is then contacted
with a second aqueous phase in the presence of a second surfactant under
conditions of agitation to form, suspend and break globules of the
artificial red cell-containing oil phase in said second suspending phase.
Alternatively, the second surfactant can be added directly to the
artificial red cell-containing oil phase prior to its contact with the
suspending phase. However, better results appear to be obtained if the
second surfactant is added to the second aqueous suspending phase. In
practice it has been found that a saline solution is an effective second
aqueous suspending phase and that when the breaking emulsion is agitated
with a large quantity of the saline solution to which the second
surfactant has been added, the artificial red cells become suspended in
the saline suspending phase. At this point, the artificial red cells may
also require treatment with one or more reagents to deactivate unreacted
functional groups of the cross-linking agent or to modify the surface
characteristics of the membranes. The artificial red cells may be
recovered from the suspending solution by centrifugation, filtration,
decanting or any suitable separation technique and may then be either
freeze-dried or resuspended in a suitable solution.
Optionally, if the first suspending phase contained only enough
cross-linking agent to form the artificial red cells, it is not required
that a second suspending phase be employed to free the cells. In this
case, the second surfactant may be added to the first suspending phase to
free the red cells.
It is extremely important in the process of recovering the artificial red
cells from the oil that the surfactant employed with the second suspending
phase (or added to the first suspending phase if only enough cross-linking
agent was present therein to form the artificial red cells) have an HLB at
least 9, preferably at least 11 and more preferably at least 13. That is,
the surfactant added to the aqueous suspending phase which breaks up the
oil phase and frees the artificial red cells must be preferentially
soluble in the aqueous phase and not the oil. As is known in the art, this
preferential water solubility can readily be defined in terms of HLB. It
is believed that the second surfactant having an HLB of at least 9
destabilizes the suspension of artificial red cells in the oil thereby
releasing the red cells and forming a three-phase system comprising an
aqueous, surfactant-containing suspending phase, an oil phase and the
artificial red cells.
Yet another process for making the artificial red cells of this invention
comprises emulsifying a stroma-free aqueous hemoglobin solution in a
surfactant-containing oil to form an emulsion comprising microdroplets of
hemoglobin solution dispersed in a continuous phase of said oil wherein
said oil phase contains a cross-linking agent which polymerizes the
hemoglobin at the surface of said microdroplets to form artificial red
cells dispersed in said oil phase. In this process, the cross-linking
agent can either be initially present in the oil or can be added to the
oil after the hemoglobin microdroplets have been formed therein. In this
process, it is quite naturally necessary for the cross-linking agent to be
oil soluble. As was the case for the liquid membrane encapsulation
technique described above, just enough cross-linking agent can be added to
the oil to effectively cross-link the hemoglobin at the surface of the
emulsified microdroplets, or the cross-linking agent or agents can be
present in sufficient excess such that the reaction has to be quenced by
diluting the oil, by quickly separating the so-formed artificial red cells
from the oil, by adding to the oil a material which reacts with the
remaining cross-linking agent, by contacting the emulsion with an aqueous
suspension phase containing the second surfactant under conditions of
agitation to break the emulsion, or a combination of these methods.
The artificial red cells of this invention may also be formed by starting
with liposomes containing hemoglobin solution suspended in a suitable
suspending phase. In this case, the liposomes are contacted with a
suitable cross-linking agent to form artificial red cells therein using
the same procedure as with the liquid membrane encapsulating technique,
except that one would start with step (c) of said procedure after adding
the cross-linking agent to the suspending phase. The so-formed suspension
of artificial red cells in lipid may then be broken and the artificial
cells freed by contacting the suspension with an aqueous solution in the
presence of a surfactant having an HLB of at least 9 as hereinbefore set
forth.
The artificial red cells of stroma-free, aqueous hemoglobin solution
encapsulated in membranes of cross-linked, polymerized, stroma-free
hemoglobin produced by the process of this invention are permeable to
oxygen and impermeable to hemoglobin. These artificial red cells can be
prepared having diameters of less than 8 microns in their greatest
dimension, preferably less than 4 microns and are able to maintain their
individual integrity under conditions of flow with shear rates of up to
about 2.times.10.sup.5 sec.sup.-1. If desired, they may consist
essentially of a stroma-free, aqueous hemoglobin solution encapsulated in
cross-linked hemoglobin, thus consisting essentially of stroma-free
hemoglobin and minute amounts of cross-linking agent. Alternatively, other
ingredients such as drugs, nutrients, hormones, enzymes and antibodies may
be incorporated into the hemoglobin solution prior to the encapsulation
thereof. These artificial red cells may be prepared by emulsifying
stroma-free hemoglobin solution, as microdroplets, in an oil phase,
reacting a suitable cross-linking agent with the hemoglobin at the surface
of the microdroplets of emulsified hemoglobin solutions thereby forming a
suspension of artificial red cells in the oil and then recovering the
artificial red cells. It should be understood however, that the scope of
this invention also extends to encapsulating any cross-linkable
proteinaceous material, in which case the cell size may broadly range from
about 0.1 to 100 microns. Further, the artificial red cells or
microdroplets of encapsulated proteinaceous material of this invention may
be made flexible and easily deformable in nature simply by contacting them
with an aqueous solution to which the encapsulated hemoglobin solution or
solution of proteinaceous material is hypotonic. Thus, contacting same
with an aqueous solution with respect to which the hemoglobin or
proteinaceous material solution is hypotonic causes some of the water in
the encapsulated solution to osmotically diffuse through the membrane into
the aqueous solution which depletes the interior of the artificial red
cell or microdroplet of encapsulated proteinaceous material of a portion
of its contents, thus rendering the cell flexible and readily deformed.
Hemoglobin solution useful for the artificial red cells of this invention
is prepared by starting with red blood cells separated from freshly drawn
human blood, from outdated whole blood, packed red cells obtained from
human donor centers or from red blood cells obtained from animal blood
such as bovine blood. There are many known ways to prepare stroma-free
hemoglobin. In one particular method, whole blood is drawn into bottles
containing an anticoagulant, centrifuged and the supernatent plasma
withdrawn. Next, the resultant red cells are washed in about 1 to 4
volumes of cold, isotonic or hypertonic sodium chloride solution to form a
suspension of red cells which is then centrifuged and the supernatant
removed and discarded. The red cells are generally washed an additional
two to three times with the wash being discarded after each
centrifugation. Procedures for preparing stroma-free hemoglobin solution
(SFHS) involve hemolysis, centrifugation, filtration and, optionally,
dialysis. To obtain stroma-free hemoglobin, the red blood cells are first
lysed in about one to four volumes of cold water or other lysing solutions
such as hypotonic phosphate buffers or hypotonic saline. After lysing, the
red cell suspension is shaken and cold toluene is added at about 10-200
volume percent of the red cells, usually about 10-30 volume percent. This
mixture is then shaken for four to ten minutes and left standing at from
4.degree. C. to 6.degree. C. for 24 to 72 hours to produce a triphasic
mixture. A lower, clearer, red layer is isolated and centrifuged at about
40,000 to 50,000 g for at least 60 minutes at about 4.degree. C. to
6.degree. C. Then, the upper supernatant is separated and filtered through
a suitable filter such as a diatomaceous earth filter. If desired,
residual low molecular weight salts and metabolites may be removed from
the stroma-free hemoglobin by dialysis against standard or medically
acceptable buffers which are well known to those in the art. The method
used to prepare SFHS to demonstrate the instant invention is set forth in
Example 6 supra.
Suitable cross-linking agents include those which are at least bifunctional
and, for the case where one desires to form artificial red cells, those
which result in a cross-linked hemoglobin membrane which is biodegradale
in a mammalian body so that the cells can be eliminated from the body
after their function has been performed. The bi or poly-functional
cross-linking agents must have at least two functional groups which can be
the same or different. These groups must be capable of reacting with and
cross-linking the functional groups of the proteinaceous material which,
in the case of hemoglobin, are primarily amino groups. By amino groups is
meant the N-terminal alpha amino group of the hemoglobin chains and those
of the basic amino acid residues such as lysine and arginine. The
following are intended to be illustrative, but non-limiting examples of
various categories of suitable cross-linking agents.
The functional groups of the cross-linking agent can be covalently bonded
to each other or they can be separated by an aliphatic or by an aromatic
ring. Exemplary aromatic stabilized functional groups are azo and halo
activated with a nitro group. These include compounds having a
heterocyclic ring with reactive groups bonded to the ring. For example,
triazines of the formula:
##STR1##
wherein R.sub.1 is halogen including fluoro, chloro and bromo, and R.sub.2
is a nucleophilic substitute such as an aliphatic or aromatic group, a
halogen, a lower alkyl of 1 to 8 carbons, and amino. Cross-linking agents
embraced by this formula are 2-amino-4-,6-dichloro-s-triazine and
chloro-s-triazine. The cross-linking agents include aromatic stabilized
agents prepared by the diazotation of an aromatic diamine, for example,
benzidine and its derivatives with nitrous acid to yield
bis-diazobenzidines of the formula:
##STR2##
wherein R.sub.3 is a member selected from the group consisting of a
covalent bond, alkylene of 1 to 5 carbons, phenylene, ether, sulfone and
secamine, R.sub.4 is halogen or nitro. R.sub.5 is hydrogen, nitro, lower
alkyl of 1 to 8 carbons, sulfonate (SO.sub.3 H) and carboxylate, and
R.sub.6 is halogen, diazo (--N:N--), isocyanate (NCO), and isothiocyanate
(NCS). Representative agents embraced by the formula include
bisdiazobenzidine 2,2'-sulfonic acid,
4,4'-difluoro-3,3'-dinitrophenylsulfone and
diphenyl-4,4'-diisothiocyanate.
Cross-linking agents suitable for the invention include compounds of the
formula:
##STR3##
wherein R.sub.7 is halogen and R.sub.8 is nitro, or hydrogen with at least
one R.sub.8 a nitro, as represented by the commercially available
activated halogenated reagent 1,5-difluoro-2,4-dinitrobenzene.
Cross-linking agents suitable for the purpose of the invention also include
compounds of the formula (R.sub.9).sub.2 C=O wherein R.sub.9 is hydrogen
or halogen, and compounds of the formula R.sub.10 --(CH.sub.2).sub.n
--R.sub.10 wherein R.sub.10 is the same or different and n is 1 to 8. The
agents also include compounds having a functional group bound to an
aromatic moiety either directly or through an alkylene bridge of the
formula R.sub.10 --(CH.sub.2).sub.m --C.sub.6 H.sub.4 --(CH.sub.2).sub.m
--R.sub.10 wherein R.sub.10 is the same or different and m is 0 to 3.
Cross-linking agents include the compounds having the functional groups
bonded to a cycloalkyl as represented by the formula:
##STR4##
wherein R.sub.10 is the same or different, p is 0 to 4, and q is 1 to 4.
The cross-linking agents include compounds having functional groups bonded
to an aliphatic chain interrupted with a nonfunctional group or having
nonfunctional groups bonded to the chain as represented by compounds of
the formula R.sub.10 --(CH.sub.2).sub.x --R.sub.11 --(CH.sub.2).sub.x
--R.sub.10 wherein R.sub.10 is the same or different, R.sub.11 is selected
from the group consisting of an ether bridge, an divalent amine and a
sulfone, and x is an alkylene of 1 to 5 carbon atoms, which each x the
same or different. Representative of the functional group embraced by
R.sub.10 include isocyanate, vinyl, imine, isothiocyanate, isocyanide,
aldehyde, epoxide, chloroformate, thiochloroformate, and imido lower alkyl
ester, and thiolactones of the formula:
##STR5##
wherein a is 1 to 3. Also, R.sub.10 can be an activated group formed by
reacting the carboxylic acid with a thionyl halide or phosphorus halide,
or an activated group formed by reacting an amide or an alkyl ester of the
carboxylic acid with hydrazine and then with nitrous acid to yield the
corresponding activated group COR.sub.12 wherein R.sub.12 is halogen or
azide. The activated group can also be formed by reacting the carboxylic
acid with N,N'-carbonyl diimidazole or a carbodiimide of the formula
R.sub.13 --N.dbd.C.dbd.N--R.sub.13 wherein R.sub.13 is the same or
different and are a lower alkyl, a lower cycloalkyl, di(lower)alkyl amino
lower alkylene, and heterocyclic lower alkyl including morpholino ethyl.
R.sub.12 can also be a
##STR6##
lower alkyl, and a
##STR7##
wherein n is 1 or 2.
Exemplary commercially available cross-linking reagents embraced by the
above formula include divinyl sulfone, epichlorohydrin, butadiene
diepoxide, ethylene glycol diglycidyl ether, glycerol diglycidyl ether,
dimethyl suberimidate dihydrochloride, dimethyl malonimidate
dihydrochloride, and dimethyl adipimidate dihydrochloride.
Representative of compounds bearing a functional isocyanate or
isothiocyanate group are the compounds listed below. Additionally, the
isocyanates or isothiocyanates can be synthesized by reacting an alkyl or
aryl amine with phosgene or thiophosgene. The isocyanates used for
cross-linking are diisocyanates and they react with the free amino groups
of hemoglobin producing urea or thiourea cross-linked sites. Typical
compounds include diphenyl-4,4'-diisothiocyanate-2,2'-disulfonic acid,
toluene diisocyanate, toluene-2-isocyanate-4-isothiocyanate,
3-methoxydiphenylmethane-4-4'-diisocyanate, propylene diisocyanate,
butylene diisocyanate, and hexamethylene diisocyanate.
Exemplary of cross-linking agents having an aldehyde or dialdehyde
functionality include formaldehyde, paraformaldehyde, formaldehyde
activated ureas such as 1,3-bis(hydroxymethyl) urea,
N,N'-di(hydroxymethyl) imidazolidinone prepared from formaldehyde
condensation with a urea according to the formula CH.sub.2 O+R.sub.16
HN--NHR.sub.16 .fwdarw.HOCH.sub.2 NR.sub.16 --CO--NR.sub.16 --CH.sub.2 OH
wherein R.sub.16 is hydrogen, alkyl, aryl or heterocyclic ring. Other
dialdehyde cross-linking agents include dialdehydes of the formula
OCH--R.sub.17 --HCO wherein R.sub.17 is member selected from the group
consisting of a covalent bond and a straight or branched chain alkylene of
1 to 8 carbons. Dialdehydes embraced by the formula include glyoxal,
malonic dialdehyde, succinic dialdehyde, glutaraldehyde, adipaldehyde,
3-methyl glutaraldehyde, propyladipaldehyde, phthalic dialdehyde,
terephthaldehyde and malonic dialdehyde.
Other cross-linking agents include derivatives of carboxylic acids and
carboxylic acid residues of hemoglobin activated in situ to give a
reactive derivative of hemoglobin that will cross-link with the amines of
another hemoglobin. Typical carboxylic acids useful for this purpose have
the formula CO.sub.2 H(CH.sub.2).sub.n CO.sub.2 H, and {(CH.sub.2).sub.n
COOH}.sub.3 CH wherein n is 1 to 8. The carboxylic acids include citric,
malonic, adipic and succinic. Carboxylic acid activators include thionyl
chloride, carbodiimides, N-ethyl-5-phenyl-isoxazolium-3'-sulphonate
(Woodward's reagent K), N,N'-carbonyldiimidazole,
N-t-butyl-5-methylisoxazolium perchlorate (Woodward's reagent L),
1-ethyl-3-dimethyl aminopropylcarbodiimide,
1-cyclohexyl-3-(2-morpholino-ethyl) carbodiimide, metho-p-toluene
sulfonate. The cross-linking reaction using a carboxylic acid can be
represented by the equation
##STR8##
Other cross-linking groups that can be used are prepared from esters and
thioesters activated by strained thiolactones, hydroxysuccinimide esters,
halogenated carboxylic acid esters and imidates. The above functional
reagents or methods for preparing them are reported in Bull. Soc. Chem.
Fr., Vol. 12, pages 4615 to 4617, 1971; Biochemical Aspects of Reactions
on Solid Supports, by Stark, George R., Chapter 1, published by Academic
Press, 1971; Chemtech, pages 47 to 55, 1974; Rev. Pure and Appl. Chem.
Vol. 21, pages 83 to 113, 1971; and British Pat. No. 1,252,770.
The oil used in the process of this invention may be any oil such as
mineral (preferably paraffinic), refined vegetable refined animal oil,
etc. to which the surfactant is added. It is obvious of course that the
oil used in preparing the hemoglobin emulsion should be of a type which is
inert with respect to the hemoglobin, the surfactant used and the
cross-linking agent. It is also obvious that the oil should not contain
materials that are toxic to mammalian bodies and which will diffuse into
either the hemoglobin solution or into the artificial red cells. Some
examples of suitable oils which can be used as a oil for emulsifying the
hemoglobin solution include hydrocarbon oils that have been refined to
remove toxic ingredients and which have molecular weights up to 1000, such
as paraffins, iosparaffins, naphthalenes and non-polynuclear aromatics.
Particularly suitable are mineral oils which have been highly refined for
use in human ingestion. Additionally, oils or treated oils from animal or
vegetable sources may be used provided they meet the criteria set forth
above. Silicon fluids can be used. Also, lipids such as phospholipids may
be used as the oil. Oils used in the process of forming the artificial red
blood cells of this invention may have a viscosity broadly ranging from
between about 0.3 to about 1,000 centipoise at normal temperature. A
preferred range is from about 1 to 150 and particularly preferred in from
about 2 to 20 centipoise.
The oil-soluble surfactant must not dissolve in the hemoglobin or react
with the cross-linking agent. This surfactant must be oil-soluble and may
be present in the oil from about 0.01 wt. % up to about 50 wt. % and
preferably from about 0.5 to 5 wt. % of the oil. A wide variety of
surfactants may be used in the oil to emulsify the hemoglobin solution
including those described in "Surface Active Agents and Detergents" by
Schwartz, Perry and Bush, Inter-sciences Publishers, Inc., New York and in
"Surface Chemistry" by Osepow, Reinhold, New York, 1962, Chapter 8. Of
course not all the surfactants included in these publications meet the
non-toxic and inert or non-transferring criteria set forth above.
Surfactants that have been satisfactorily employed to make the artificial
red cells of this invention include Santone 10-10-0 which is a
decaglycerol decaoleate and is available from the Durkee Industrial Foods
Group of SCM Corporation and polyamine derivatives having the general
formula:
##STR9##
where n varies from 10 to 60, x varies from 3 to 10 and Y is selected from
the group consisting of hydrogen and oxygen-containing hydrocarbyl
radicals having up to 10 carbons. In particular, higher molecular weight
polyamines with the structure of:
##STR10##
wherein m is an integer of about 40 have been found to be particularly
effective. This latter compound was commercially available as ENJ-3029
from Exxon Chemical Company.
The invention will be more readily understood by reference to the examples
below.
PREFERRED EMBODIMENT
Example 1
Stroma-free hemoglobin solution (SFHS) was prepared from fresh whole blood
from slaughterhouse cattle (bovine blood) which was collected in
sterilized 500 ml polypropylene bottles containing 80 ml of an
anticoagulant-antibiotic solution comprising 0.73% of citric acid, 2.2% of
sodium citrate, 2.45% of glucose, 0.93% of Penicillin "6" USP Potassium
(Grant Island Biological Supply Company) and 0.735 g of Streptomycin
Sulfate (Grant Island). The blood was immediately refrigerated. Red cells
were isolated by centrifuging, washing four times by resuspending them in
1.6% saline solution, centrifuging again, and discarding the supernatant.
The packed red cells were then lysed (osmotically ruptured) by the
addition of an equal volume of distilled water. Then the stroma (lipids
and debris from the red-cell membrane) was removed, from the hemolysate by
extracting with cold toluene, centrifugating for 30 min at 4.degree. C.,
and filtering through membranes of progressively finer porosity down to
0.2 .mu.m to produce the SFHS.
An emulsion of the SFHS in oil was prepared in a mixer comprising a 120 ml
glass jar, a shaft-mounted, 3-blade, 3.75-cm-diameter, propeller-type
stirrer positioned 0.5 cm from the bottom of the jar, and a 0.32 cm OD dip
tube extended through a cap which covered the jar to within 0.5 cm of the
bottom of the jar. The distance between the dip tube and the propeller was
0.3 cm. The oil phase was an isoparaffinic mineral oil having a viscosity
of about 2 centipoise at 38.degree. C. (Exxon Isopar M) containing 4% of
an oil-solube polyamine surfactant (Exxon ENJ 3029). The SFHS was injected
through the dip tube over a period of 1 minute into an equal weight of oil
contained in the jar while stirred at 4000 rpm. Stirring was continued for
5 minutes to form red-cell-size microdroplets of SFHS in oil.
Liquid membrane capsules were formed in a second stirred-jar device
identical to the one used to form the emulsion. The suspending phase was
75 ml of an aqueous solution containing 0.4% of glutaraldehyde in
phosphate buffered saline (1% of sodium chloride and 0.74% of sodium
biphosphate). A 25-ml batch of the SFHS-in-oil emulsion was injected
through the dip tube into the suspending phase which was being stirred at
1,000 rpm. After 5 min the stirrer was stopped, the phases were allowed to
separate, and the suspending phase was removed through the dip tube. The
artificial red cells remained in the oil phase. To remove unreacted
glutaraldehyde, the oil phase was washed three times by the addition of 75
ml of phosphate buffer solution, agitation for 1 min, phase separation,
and withdrawal of the aqueous phase.
A water soluble surfactant was used to eject the artificial red cells from
the oil phase. Five grams of Renex 30 (polyoxyethylene (12) tridecyl ether
supplied by ICI Americas Inc.) were dissolved in 75 ml of saline solution
and added through the dip tube. The mixture was stirred for 3 min and
allowed to stand. Within 5 min most of the oil had risen to the top of the
jar and a dark band containing the artificial red cells appeared at the
bottom of the aqueous phase. The artifical red cells were withdrawn
through the dip tube, washed 3 times in saline to remove the surfactant,
centrifuged and decanted. No free hemoglobin was observed in the
supernatant.
To demonstrate the mechanical strength of the artifical red-cell membrane,
a portion of the artificial red cells was suspended in saline solution,
and the suspension was drawn into a 10 cc syringe. A 22 ga needle was
placed on the syringe and the contents were forced out over a period of 30
sec to produce a shear rate of 3.63.times.10.sup.4 sec. The ejected
suspension was centrifuged and examined for the presence of free
hemoglobin. There was no evidence of hemolysis. The artificial red cells
were resuspended and forced through the needle over a period of 6 sec to
produce a shear rate of 1.84.times.10.sup.5 sec. Again, there was no
evidence of hemolysis.
Example 2
This example demonstrates the use of an oil soluble cross-linking agent in
the preparation of artificial red cells. An aqueous hemoglobin solution
was prepared by the procedure described in Example 1. The hemoglobin
concentration of the solution was determined to be 18.4 g/dl by the
Cyanomethemoglobin Method (Henry, Clinical Chemistry, 242-3, 1969). A 45
ml portion of this hemoglobin solution was injected into an equal volume
of stirred Isopar M containing 4% of ENJ 3029 in the same stirred-jar
device, and stirring was continued at 4000 rpm for 5 minutes to form an
emulsion of hemoglobin solution in oil. In a second, identical stirred jar
device, 0.42 ml of toluene diisocyanate (TDI) was added to 75 ml of the
Isopar M containing 4% of ENJ 3029, and this mixture was stirred at 200
rpm while 20 ml of the above emulsion was injected into the
oil/surfactant/TDI solution. Stirring was continued for 5 minutes to allow
a membrane of cross-linked hemoglobin to form around each microdroplet of
hemoglobin in the emulsion. Then 0.5 g of a second surfactant, Renex 690
having an HLB of 13.5 (polyoxyethylene alkyl aryl ether from ICI Americas
Inc.), which is preferentially soluble in water, was dispersed in the
emulsion to break it. Also 20 ml of a 1.6% saline solution was stirred
into the breaking emulsion to provide an aqueous medium for the ejected
droplets. When the mixture of phases in the broken emulsion was separated
by centrifugation, the artificial red cells collected in the bottom of the
tube and no dissolved hemoglobin was observed in the aqueous phase. When
the artificial red cells were suspended in a 5% solution of carboxymethyl
cellulose, their shape changed from spherical to elipsoidal, indicating
flexibility of the membrane when water was removed osmotically.
Example 3
This example demonstrates that artificial red cells can pick up and release
oxygen. A hemoglobin solution prepared by the procedure described in
Example 1 was deoxygenated by bubbling a stream of nitrogen through it for
one hour to form a deoxyhemoglobin solution. The remainder of the
procedure for forming the artificial red cells was carried out in a
nitrogen atmosphere. The deoxyhemoglobin solution (19.5 g/dl) was
emulsified in an equal volume of Isopar M, containing 4% of ENJ 3029 at a
stirrer speed of 4000 rpm in the stirred jar device.
An aqueous suspending phase was prepared by adding 0.4% of glutaraldehyde
to a phosphate buffer solution (1% of sodium chloride and 0.7% of sodium
biphosphate). A 25 ml portion of the emulsion was injected into 75 ml of
the suspending phase, and the mixture was stirred for 5 min at 800 rpm.
The emulsion was washed three times with the phosphate buffer solution and
broken by adding 0.5 g of Renex 30 (HLB of 14.5) and 40 ml of water with
manual shaking. The broken emulsion was centrifuged to recover the
artificial red cells.
The artificial red cells thus formed were dispersed in phosphate buffer and
equilibrated with air. A 20-microliter sample of this dispe | | |
