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Description  |
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BACKGROUND OF THE INVENTION
Liposomes which can carry enzymes and be labeled with antigens or
antibodies are described in British Patent Application No. 8,103,282 dated
Feb. 3, 1981. Liposomes labeled with antigens at their external surface
and containing an enzyme entrapped in their internal volume are mixed with
cognate antibody, complement, enzyme substrate and a test sample to
determine whether or not the liposomes permit substrate access to the
entrapped enzyme. This determination is made by detecting enzymatic
activity in the presence of substrate after exposure to the test sample.
Such techniques are referred to as enzyme membrane immunoassay techniques
or liposome immunoassays.
A key component of the liposome immunoassay (LIA) is analyte functionalized
liposome, (i.e., liposome with covalently attached antigen, antibody, or
other substance of interest). Analytes can be introduced into the
liposomes during their formation by incorporation of specific quantities
of analyte derivatives of phosphatidylethanolamine (PEA) or other
phospholipids.
Previous methods of preparation of analyte-functionalized liposomes utilize
PEA-diester intermediates for direct coupling with the analyte or its
derivatives. Complicated products are generated and difficulties are
usually encountered in purifying and characterizing the target compound.
A method of preparing phospholipid-analyte derivatives utilizing a
phosphotriester intermediate would avoid some of the difficulties
encountered by the diester method. However, no triester method of forming
such compounds has previously been shown in the art.
Phosphotriester intermediates, protected with benzyl groups, have been
previously described, c.f., J. D. Billimoria and K. O. Lewis; J. Chem.
Soc., (C), 1404 (1968). Such described intermediates are used in the
preparation of phospholipids but are not applicable to preparation of
phospholipid-analyte derivatives, since deprotection of such benzyl ester
intermediates to the target analyte derivatized phospholipid is not
compatible with most analytes required for the technology of the liposome
immunoassay described above. Removal of the benzyl groups by anionic
fission with sodium iodide produces benzyl iodide. That by-product is not
only difficult to remove but is also a highly reactive alkylating reagent
capable of reacting with any nucleophilic group on the analyte.
Use of phosphotriester methodology for different applications in
phospholipid chemistry has been previously disclosed by J. H. van Boom.
[C. A. A. van Boeckel et. al., Tetrahedron, 37, 3751 (1981); J. J.
Oltvoort et. al., Recueil Trav. Chim. Pays-Bas, 101, 87 (1982); C. A. A.
van Boeckel and J. H. van Boom, Tetrahedron Letters No. 37, 3561 (1979);
C. A. A. van Boeckel and J. H. van Boom, Tetrahedron Letters, 21, 3705
(1980).] These methods disclose use of phosphotriester methodology for
synthesis of naturally occurring teichoic acid fragments and modified
glycophospholipids for elucidation of their function in membranes and for
reasearch investigation of their physiological properties.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method of preparing
analyte functionalized liposomes for use in liposome immunoassay methods
capable of adaptation for any of a large variety of analytes.
It is also an object of the present invention to provide a method of
preparing analyte derivatized phospholipids which affords greater ease of
synthesis, separation, and/or characterization of intermediates than is
possible in previous methods.
It is another object of the present invention to provide a method of
preparing analyte derivatized phospholipids which permits ease of
production of multi-gram quantities required for LIA techniques.
It is a further object of the present invention to provide a method of
preparing analyte derivatized phospholipids which employs common
intermediates useful for preparation of several different analyte
derivatized phospholipid compounds.
Another object of the present invention is to provide a method of preparing
ligand-functionalized liposomes for the subsequent attachment of an
analyte to the ligand group for use in LIA techniques.
A still further and specific object of the present invention is to provide
a penicillin-G- or thyroxine-functionalized liposome for use in LIA
detection of penicillin or thyroxine.
The present invention is an advance in the state of the art for preparation
of analyte derivatized phospholipids. These compounds are a key component
of the liposomes required for competitive inhibition assay by the LIA
technology. The method utilizes phosphotriester rather than previously
used phosphodiester intermediates for synthesis of these derivatives. The
intermediates are uncharged compounds which are often far simpler to
prepare, purify and characterize than charged diester intermediates.
Common intermediates can be used for preparation of several target
compounds.
In the present invention, use of phosphotriester methodology is directed
toward multigram preparation of analyte or ligand derivatized amphiphilic
compounds, specifically phospholipids. Incorporation of such derivatives
containing either analytes or ligands plus analytes into liposomes
provides the analyte tagged, enzyme laden liposomes required for use in
LIA techniques. The same analyte-tagged liposomes, without an enzyme core,
can also be used as immunogens as described by Schuster, et al. (J.
Immunol., 122, 900 (1979)) and Dancy, et al. (J. Immunol., 122, 638
(1979)).
Incorporation of ligand derivatives without attached analytes provides
ligand functionalized liposomes. These can be modified chemically by using
condensing or coupling reagents to attach analytes or macromolecules to
provide either antigen or antibody tagged, enzyme laden liposomes as
required for LIA. Preparation of the ligand-functionalized liposome using
the present method is more fully described below.
According to the present invention, a phospholipid is used, having the
general formula
##STR1##
where R.sup.1 and R.sup.2 can independently be H, OH, R", OR" or
##STR2##
(where R" is a saturated or unsaturated, branched or straight-chain alkyl
or alkylene group of 1 to 24 carbons), wherein at least one of R.sup.1 or
R.sup.2 is
##STR3##
or OR"; and where R.sup.3 is a side chain with a functional group capable
of bonding to the analyte or ligand desired to be attached. The above
molecule is amphiphilic, having both a lipophilic portion and a charged
portion. The phospholipid is first treated with a protecting group, to
form a compound having the structure
##STR4##
where X is a protecting group for R.sup.3. This compound is then treated
with an alcohol or activated alkyl halide to afford a phosphotriester
having the structure
##STR5##
where Y is a phosphotriester blocking group. Once the blocking group is
attached, the R.sup.3 -protecting group is removed and the desired analyte
is attached at the R.sup.3 site, forming a triester intermediate of the
formula
##STR6##
where A is the analyte desired to be attached. The triester and any
analyte blocking groups are then removed, leaving an
analyte-functionalized phospholipid of the formula
##STR7##
That compound is then used to prepare an enzyme-laden liposome with an
external covalently attached analyte functional group.
An alternative to producing a phospholipid with a directly attached analyte
is to produce a phospholipid on which the analyte is attached by a ligand
which acts as a "leash", or spacer, i.e., the ligand is a bifunctional
compound which enables attachment at one end to the desired analyte and
the other end to the phospholipid. To produce such a phospholipid
compound, the ligand is attached to the phospholipid in the same method as
previously described for attaching an analyte. Attachment of a ligand to
the phospholipid by the present method would produce the triester
intermediate
##STR8##
where L is a ligand. One of three approaches may be employed for
attachment of an analyte to the ligand to produce the desired
functionalized liposome. One approach involves deprotecting the above
intermediate and using the phosphodiester ligand intermediate to form a
ligand-functionalized liposome, and then attaching the analyte to the
ligand functional group. A second approach is to attach the analyte
directly to the ligand group of the above intermediate to form a compound
having the structure
##STR9##
followed by subsequent removal of the triester and any analyte blocking
group and use of the phospholipid-ligand-analyte derivative to form the
analyte-ligand functionalized liposome.
A third alternative approach to the formation of
phospholipid-ligand-analyte combinations is the initial formation of
ligand-analyte derivatives, subsequent coupling to the phospholipid,
deprotection, and liposome formation. The route chosen for preparation of
these ligand derivatives depends very much on the relative stability of
the phospholipid and the specific analyte under conditions required for
their formation.
This newly developed phosphotriester approach for synthesis of key
intermediates provides a rapid and convenient method for the large scale
preparation of phospholipid-analyte derivatives. Triester intermediates
are quite soluble in organic solvents, can be prepared in high yield, are
readily functionalized with various analytes or analyte derivatives, and
are easily purified, readily characterized and conveniently converted to
the targeted phospholipid-analytes. The methodology has a high degree of
flexibility in that triester and analyte blocking groups can be varied to
assure compatability with the chemistry of the analyte in question.
The simplicity of the triester methodology is in sharp contrast to the
traditional diester synthetic route for preparation of
phospholipid-analyte derivatives. Phospholipid diesters are marginally
soluble in organic solvents and sometimes react poorly to give a
multiplicity of products which can be extremely difficult to purify. In
many cases, they are not readily characterized and/or not readily amenable
to large scale synthesis as required for commercial production.
As was mentioned earlier herein, van Boom and others have described the use
of phosphotriester methodology for synthesis of naturally occurring
teichoic acid fragments and modified glycophospholipids. The synthesis of
these compounds involves sequential phosphorylation of a glycerol
derivative and either a second glycerol derivative or a gentiobiose
derivative to form a phosphotriester derivative of the natural product.
These protected intermediates are ultimately converted to target compounds
by removal of several protecting groups including a phosphotriester group.
The van Boom procedures differ in four ways from those presented herein:
(1) The herein-described phophotriester synthesis starts with an intact
preformed naturally occurring phopholipid or a synthetic analog. The
procedure described by van Boom uses totally synthetic intermediates.
(2) The herein-described procedures are utilized for synthesis of
phospholipid-analyte, or -ligand-analyte derivatives as required for use
in immunoassays. These are not naturally occurring compounds. The
derivatives described by van Boom are naturally occurring compounds or
their derivatives. They are being prepared for elucidation of their
function in membranes and for research investigation of their
physiological properties.
(3) Different numbers and types of phosphoester linkages are formed during
the two reaction procedures. In the presently described procedure, a
single phosphoester protecting group is introduced. It functions as a
protecting group to convert a charged, chemically reactive phosphodiester
into a neutral, chemically inert phosphotriester. This protecting group is
removed at a later stage, after conjugation of the phospholipid with the
appropriate analyte or ligand and analyte.
The van Boom procedure requires formation of two new phosphoester linkages
with two different molecules. These new linkages form a phospholipid
backbone which was not present prior to these reactions. These linkages
remain intact in the final product. The phosphoester protecting group
which is ultimately removed was incorporated as part of the
phosphorylating reagent.
(4) One could envision preparation of phospholipid-analyte or
-ligand-analyte derivatives by modification of the procedures suggested by
van Boom. However, such an approach would be impractical because of the
multiplicity of reaction steps involved, low yield in preparation of
1,2-di-o-benzylsn-glycerol, and anticipated low yield in subsequent
reactions.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
In the present invention, synthesis of triester intermediates and
conversion to the targeted phospholipid-analyte derivative is a five step
process, as outlined schematically in Table 1. A sixth and final step
involves incorporation of phospholipid-analyte or ligand-analyte
derivatives into analyte tagged, enzyme laden liposomes as required for
the LIA assay. An alternative final step involves incorporation of the
phospholipid-ligand derivative into the liposome with subsequent coupling
to the analyte to form the analyte tagged, enzyme-laden liposome as
required for the LIA assay.
In the preferred embodiment of the present method, the phospholipid
utilized is a phosphatidylethanolamine (PEA) compound of the formula
##STR10##
Table 2 shows the reaction sequence of the present method using PEA
wherein penicillin-G is the analyte and the final product is
penicillin-G-functionalized liposome.
Table 3 summarizes the phosphatidylethanolamine and liposome derivatives
prepared to demonstrate utility of the phosphotriester synthetic approach
for preparation of analyte-functionalized liposomes.
TABLE I
______________________________________
Preparative Procedures for Synthesis
of Analyte Functionalized Liposomes
______________________________________
##STR11##
##STR12##
______________________________________
Legend
##STR13##
##STR14##
R.sup.3 = CH.sub.2 CH.sub.2 NH.sub.2
X = R.sup.3 (amine) protecting group
.+-.Y = phosphotriester blocking group
L = ligand
A = analyte
.circle.L = liposome
TABLE II
______________________________________
Chemical Modifications of Phosphatidylethanolamine
for Preparation of Penicillin Functionalized Liposomes
______________________________________
##STR15##
##STR16##
##STR17##
##STR18##
##STR19##
##STR20##
______________________________________
LEGEND
Tr = (C.sub.6 H.sub.5).sub.3 C
QSCl = 8quinolinesulfonylchloride
NT = 3nitro-1,-2,-4-triazole
NPE = 2(4-nitrophenyl)ethanol
PG(phenacyl) = 2(4-bromophenyl)-2-oxoethyl benzyl penicillanate
.circle.L = liposome
##STR21##
TABLE III
______________________________________
Summary of phosphatidylethanolamine and liposome derivatives
prepared to demonstrate utility of the phosphotriester synthetic
approach for preparation of analyte functionalized liposomes.
X Y L A Example
______________________________________
Step 1
BOC -- -- -- 1
Tr -- -- -- 4
Step 2
BOC phenacyl -- -- 2
Tr phenacyl -- -- 6
Tr NPE -- -- 5
Step 3
-- phenacyl -- -- 3
-- NPE -- -- 7
Step 4
-- phenacyl -- PG(phenacyl)
8
-- NPE -- PG(phenacyl)
9
-- phenacyl suc -- 11
-- phenacyl suc T4 12
-- phenacyl suc-ED PG (phenacyl)
16
Step 5
-- -- -- PG 10
-- -- suc T4 13
-- -- suc-ED PG 17
Step 6
-- -- -- PG 14
-- -- suc T4 15
-- -- suc-ED PG 18
______________________________________
X = amine protecting group
Y = phosphotriester derivative
L = leash
A = analyte
PEA = B,.gamma.-dipalmitoylD,L-2-phosphatidylethanolamine
BOC = tbutylcarbonyl
Tr = trityl
phenacyl = 2(4-bromophenyl)-2-oxoethyl
NPE = 2(4-nitrophenyl)ethyl
PG(phenacyl) = 2(4-bromophenyl)-2-oxoethylester of penecillin
suc = succinate
T4 = tetraiodothyronine
3MP = 3mercaptopropionyl
EAC = .epsilon. aminocaproyl
4MB = 4mercaptobutyryl
ED = ethylenediamine
The first step in the synthesis involves selective blocking of the free
amine group of the PEA to keep it intact but inert during subsequent
reactions. The protecting groups used are standard for protection of
primary or secondary amines. The only requirement for their use is the
ability to be removed during the third synthetic step without concomitant
cleavage of the targeted PEA triester or other key functional groups.
Suitable protecting groups include acyl (e.g. trifluoroacetyl, phthaloyl,
benzoyl, etc.), alkyl (e.g. triphenylmethyl derivatives, phenacyl,
tetrahydropyranyl, etc.), urethanes (e.g. carbobenzoxy [CBZ],
t-butoxycarbonyl [t-BOC], fluorenylmethoxycarbonyl [FMOC], etc.) and other
(e.g. sulfonyl, silyl, etc.) residues which meet the above criteria.
Examples 1 and 4 below describe the use of t-BOC(t-butylcarbonyl) and
trityl (triphenylmethyl) blocking procedures respectively. Tritylation of
amine groups of PEA has been previously utilized for amine protection in
the synthesis of chemically defined 0-(1,2-diacyl-sn-glycero-3-phosphoryl)
ethanolamines. (See R. Aneja et al., Bio Chem. Biophys. Acta; 187, 579
(1969).
The second reaction step involves conversion of the partially protected,
but negatively charged, phosphodiester (from step one) to a fully
protected neutral phosphotriester or other derivative. The standard route
for preparation of these triesters is chemical activation of the
phosphodiester and subsequent coupling with an appropriate alcohol.
Activating reagents may include but are not limited to carbodiimides
(dicyclohexylcarbodiimide [DCC];
1-ethyl-3[3-dimethylamino-propyl]carbodiimide [EDC]), sulfonic acid
derivatives (2,4,6-triisopropylbenzene sulfonylchloride [TPSCl],
mesitylene sulfonyl-3-nitro-1,2,4-triazole [MSNT], 8-quinolinesulfonyl
chloride [QSCl]/3-nitro-1,2,4-triazole [NT]) and other ester forming
reagents (Lewis or other strong acids, thionyl chloride, acid anhydrides,
carbonyl diimidazoles, etc.). Alcohols used for triester synthesis may
include but are not limited to 2-(4-nitrophenyl)-ethyl (NPE),
trichloroethyl (TCE), tribromoethyl (TBE), cyanoethyl (CE), p-chlorophenyl
(PCP), 2,4-dichlorophenyl (DCP) and methyl alcohol. An alternative route
for triester formation is reaction of the PEA diester salt with an
activated alkyl halide (e.g. methyl iodide, phenacyl bromides, etc.).
Other methods for triester formation include reaction with appropriate
diazoalkanes (e.g. diazomethane), orthoesters (e.g. trimethyl
orthoformate) or sulfonic acid esters (e.g. dimethyl sulfate).
Example 5 describes conversion of partially protected PEA to the
nitrophenethyl ester via an activated phosphodiester intermediate.
Examples 2 and 6 describe an alternative esterification procedure using a
phenacyl halide.
Step three of the synthetic sequence involves removal of the amine
protecting group under conditions which leave the remainder of the PEA
triester intact. Conditions required for protecting-group cleavage vary
with the type of amine protecting group and the nature of the
phosphotriester. Examples 3 and 7 below describe two types of acid
cleavage required for removal of BOC and trityl groups, respectively.
Step four of the synthetic sequence involves chemical condensation of an
analyte or partially protected analyte derivative with the amine function
of the PEA. This is convenient when the analyte has a functional group
which can be used to form a covalent linkage with the PEA amine residue.
Examples 8 and 9 describe reaction of a protected penicillin derivative
with the amine group on two types of triester derivative.
An alternative for step four is introduction of a ligand group which can
serve either as a "leash" or as a means of replacing the reactive amine
group with a chemically different group. The ligand is normally introduced
by initial attachment to the phospholipid and subsequent coupling to the
analyte. The latter step can be carried out either prior to or subsequent
to incorporation of the phospholipid-ligand into the liposome.
Example 11 describes introduction of the succinate residue as a ligand and
a means of changing phospholipid functionality. Example 12 describes
coupling of the succinate residue to the analyte thyroxine for preparation
of protected PEA.+-.(phenacyl) suc-T4.
"PEA.+-.(phenacyl)-Suc", the product described in Example 11, has been
deprotected and incorporated directly into liposomes, giving succinate
functionalized liposomes. Prefunctionalization of liposomes with such
ligand derivatives of phospholipids, with the same or chemically-altered
reactive groups (e.g. amines, carboxylic acids, hydroxides, etc.) provide
a means, via cross-linking and coupling reagents (e.g. succinimidyl
3-[2-pyridyldithio]propionate [SPDP], 2-iminothiolane,
carbonyl-diimidazole, dicyclohexylcarbodiimide [DCC]), for coupling
analytes, and particularly larger molecules, macromolecules (e.g.
peptides, proteins, antibodies, enzymes, nucleic acids, etc.) or their
derivatives to the liposome surface. Such antigen- or antibody-tagged,
enzyme laden liposomes are immunospecifically caused to act on specific
enzyme substrates in the presence of cognate antigen or antibody and
active complement. Bifunctional crosslinking agents may also be coupled to
phospholipids, incorporated into liposomes, and coupled directly to
reactive analyte derivatives.
An alternative method for introduction of a ligand between phospholipid and
analyte is initial ligand attachment to the analyte with subsequent
coupling to the phospholipid component. Examples 16 and 17 describe
preparation of a ligand derivative of penicillin-G and its coupling to
PEA.
Ligands or spacers are preferably alkyl groups of 20 carbons or less,
branched or unbranched, saturated or unsaturated, which contain two
distinct functional groups consisting of carboxyl, amine, hydroxyl or
sulfhydryl residues, preferably at the opposite ends of the ligand. One
end of the ligand is designed for attachment to the phospholipid
preferably via a carboxyl group. The other end of the ligand is attached
to other ligands or to analytes. The reactive group at this portion of the
ligand depends on the chemical nature of the ligand or analyte to which it
is being coupled. The actual mechanism for attachment of ligands to
phospholipids, analytes or other ligands varies depending on the nature of
reaction components. Anhydrides, carbodiimides, sulfonic acid derivatives,
N-hydroxysuccinamides, 2-pyridyldithio propionates, thiolanes and other
means of activation or reactive group formation are used for introduction
of these ligands. In addition to using single-ligand leashes, two or more
ligands may be used as spacers between the liposome and the analyte.
Ligands for the previously described reactions may include but are not
limited to 6-aminocaproyl, succinoyl, lysyl, diaminoethyl, diaminohexyl,
hydroxyethyl, hydrazyl and polylysyl residues. Bifunctional crosslinking
agents including but not limited to succinimidyl
3-[2-pyridyldithio]propionate (SPDP) or 2-iminothiolane may also be used
to introduce such ligands as 3-mercaptopripionyl or 4-mercaptobutyryl or
other groups.
Step five of the synthetic sequence involves removal of the phosphotriester
and analyte blocking group under conditions which do not adversely effect
the analyte phospholipid linkage or any other function in the molecule.
Conditions for this deprotection will vary considerably and depend on the
nature of the phospholipid and analyte as well as the triester and analyte
protecting groups. Examples 10 and 13 describe preparation of fully
deprotected PEA-penicillin-G and "PEA-suc-T4" as required for preparation
of analyte functionalized liposomes.
The sixth and final step is the preparation of analyte tagged, enzyme laden
liposomes by one of several methods, as will be further discussed below.
Table 2 shows the reaction sequence wherein penicillin-G-functionalized
liposomes are produced by the present methods. Details are given in the
designated examples.
The term analyte is used herein to refer to those materials which are
capable of being coupled to the surface of immunoreactive liposomes, e.g.,
antigens, antibodies, circulating hormones, antibiotics and other
therapeutic drugs, and derivatives thereof, as further described below.
Antigens which can be tested for or used as labels for the liposomes in
accordance with this invention are numerous. There are a number of
antigens, the quantitation of which is of significance in clinical
diagnostics. Many of these are now assayed by radioisotopic methods.
Assays for these by the LIA method would be a considerable improvement
inasmuch as hazardous, unstable reagents are not employed.
Antigens and antigenic materials which are to be analyzed for LIA purposes
include any which by themselves or with other products will produce
antibodies cognate therefor and thus detectable by the immune reaction.
For example, digoxin is considered an antigen because it with another
material will produce antibodies such that the antibody to digoxin can be
used in a test with either the antibody or digoxin used as the label
depending upon whether one is testing for the digoxin or the cognate
antibody. Such materials as bovine serum albumin, key hole limpet
heomocyanin or other macromolecular carriers are covalently coupled to the
digoxin or other "antigen" in forming antibodies. Thus the word "antigen"
as used herein is meant to include all antigenic materials whether
antigenic by themselves or in combination with other materials to produce
cognate antibodies in animals such as humans, rabbits, goats, sheep,
guinea pigs, bovine species and other mammals.
The LIA method may be employed to detect and quantitate specific antibodies
directed agains various antigens. The presence as well as the amounts of
such antibodies may be taken as indicators of the potential of immunity to
various infectious disease, previous exposure to disease, or active
infection.
The present method may be utilized to produce analyte-functionalized
liposomes to be benefically applied to the detection and estimation of
circulating hormones as indicators of endocrine function by employing LIA
techniques. A partial listing of these hormones would include:
______________________________________
thyroid hormones
thyroxine and triidothyronine,
parathyroid hormone and
calcitonin.
pancreatic hormones
insulin, proinsulin, and glucagon
pituitary hormones
prolactin, adrenocorticotropic
hormone, tyrotropin, oxytocin
and vasopressin
uterine and placental
chorionic gonadetropin,
hormones placental lactogens, chorionic
thyrotropin and relaxin.
steriod hormones
Estradiol, Estrone, Estriol,
Testosterone and
Dihydrotestosterone.
growth factors Urogastrone, Nerve growth factor
and the somatomedins
______________________________________
The method may be usefully applied to the intracellular messengers, the
cyclic nucleotides and prostaglandins.
The present invention may also be used to prepare analyte-functionalized
liposomes to be applied to the LIA screening of circulating levels of
therapeutic drugs, e.g. the cardiac glycosides; digoxin, digitoxin,
anticonvulsants, diphenylhydantoin, mesantoin, phenobarbital, and
mephobarbital. Of particular interest are those drugs with a narrow
therapeutic index, i.e., a certain minimal circulating level is required
for therapeutic efficacy while a moderately higher level elicits toxic or
harmful reactions.
Other analytes applicable to the present method include antibiotics such as
penicillin, streptomycin, and tetracyclines, chlortetracycline,
oxytetracycline, and tetracycline, chloramphenicol, erythromycin,
caromycin, and polymykin B. The aminoglycoside antibiotics gentamycin,
amikacin, tobramycin, kanamycin and neomicin employed in the management of
aerobic Gram negative bacillary infections can be conveniently assayed by
LIA, and corresponding functionalized liposomes may be prepared by the
methods of the present invention.
Functionalized liposomes for LIA may also be applied to the detection and
estimation of drugs of abuse such as opiates--morphine, heroin, meperidine
and methadon; ergot alkaloids, such as lysergic acid diethylamide,
marijuana, barbiturates and cocaine and its derivatives. The corresponding
phospholipid-analyte derivatives for LIA detection of these substances may
be prepared by the present method.
The analytes of the present method may include antigens for LIA diagnosis
in environments which are less well-equipped and sophisticated than
diagnostic laboratories. For example, the LIA method can be applied to
screening food and environmental toxins. In food screening, important
antigens would be mycotoxins and natural toxicants. This involves such
major toxins as aflatoxins, ochratoxin, patulin, penicillic acid,
zearelonone; and tricothecene toxins, as well as toxic metabolites such as
ipomeamerone that occur naturally in foods. Beyond the natural toxicants
there are a wide variety of environmental contaminants, the presence of
which in foods even in trace amounts poses a significant threat to
mankind. These may be industrial byproducts or pesticides e.g.
polychlorinated biphenyls, chlorinated dibenzo-p-dioxins, chlorinated
dibenzofurans, heptachlorepoxide, dieldrin, and DDT
1,1'-2,2,2-Trichloroethylidene)-bis[3-chlorobenzene]; 1,1,1 trichloro-2,2
bis(p-chlorophenyl)ethane.
The analytes of the present method need not be restricted to small
molecules as it has been shown (Humphries and McConnell Proc. Nat. Acad.
Sci. 71, 1691-1694, 1974) that macromolecular antigens such as egg albumin
may be coupled to the surface of immunoreactive liposomes. Thus, the
present invention may also be applied to detection of macromolecular
antigens-plasma proteins, hepatitis associated antigens,
histocompatibility markers.
Small (J. Am. Oil Chem. Soc. 45, 108-117 [1968]) provides a classification
of lipids based upon their interaction with water) both in bulk and at the
surface. Class II lipids are defined as "insoluble, swelling amphiphilic
lipids." Class II lipids include: phosphatidylethanolamines, lecithins,
phosphatidyl inositol, sphingomyelin, cerebrosides, phosphatidic acid,
plasmalogens, phosphatidyl serine, cardiolipins, and certain plant
sulfolipids.
Some Class II lipids are particularly appropriate for the formation of
phospholipid-analyte or -ligand-analyte derivatives by the triester
technology. These include phosphatidylethanolamine compounds of the
formula:
##STR22##
where R.sup.1 and R.sup.2 can independently be H, OH, R", OR" or
##STR23##
(where R" is a saturated or unsaturated, branched or straight-chain alkyl
or alkylene group of 1 to 24 carbons), wherein at least one of R.sup.1 or
R.sup.2 is
##STR24##
or OR".
In these constructions ethanolamine can be substituted by any side chain
(P) containing preferably one but also two or more reactive functional
groups (Q, R, etc.). P is an inert side chain consisting of 1 to 10 carbon
atoms in branched or straight chain form, with saturated or unsaturated
linkages and Q, R, etc. are one or more of the combination of amine,
carboxyl, hydroxyl, sulfhydryl, ethylene oxide, or any other reactive
functional group useful for attachment of analytes or ligands. Particular
substitutions for ethanolamine would include N-methyl ethanolamine,
serine, and N-2-hydroxyethylalanine.
The liposomes of the present invention are sometimes called smectic
mesophases or synthetic vesicles. They are in fact dry lipid films
suspended in aqueous media as have been describe by Uemura, K. and Kinsky,
S. C. (1972) Biochemistry 11, 4085-4094. Liposomes are belived to consist
of lipid bilayers which separate an internal aqueous compartment from an
external aqueous media and are in fact prototypes of biological membranes.
The liposomes mimic the properties of biological membranes. As is known,
they can be made to contain either enzyme substrates or enzymes. For
purposes of the present invention, the liposomes contain an enzyme and
have an outer surface substantially free of the enzyme which outer surface
encloses the enzyme and is labeled with an antigen or its cognate antibody
depending upon the test to be carried out. Preferably if one is testing
for the antibody, the liposome will be labeled with that antibody while if
one is testing for the antigen, the liposome will be labeled with the
antigen.
In preparing liposomes, it is necessary that lipids--such as those of Class
II--which are insoluble in water be introduced into an aqueous
environment. This can be achieved by a variety of methods.
By one such known method, lipids are physically dispersed into an aqueous
solution. A dry thin film of lipids is formed on the interior surface of a
suitable vessel. The aqueous solution containing the substances to be
entrapped within the liposomes is then placed in the vessel in contact
with the lipid film. The lipid film is then dispersed into the aqueous
solution by vigorous agitation of the vessel (glass beads approximately
0.1 mm in diameter may be included in the vessel to accelerate this
dispersion). Also, dispersion of the lipid film may be enhanced by
sonication through immersion of the vessel in a bath type sonicator or by
immersing the probe of a sonifier into the aqueous solution. Excessive
sonication may inactivate enzyme and can produce very small liposomes.
Alternatively, the lipids may be dissolved in an aqueous solution
containing a detergent lipid of Class III A or B such as laurylsulfate or
sodium deoxycholate. The detergent is then removed (e.g. by dialysis), and
the liposome bilayers are formed. Enoch and Strittmatter (Proc. Nat. Acad.
Sci. 76, 145-149 (1979)) have described the preparation of 1000 A
diameter, single-bilayer liposomes using sodium doxycholate as the
detergent which is dialyzed.
Another known technique involves the addition of aqueous solution to a
mixture of lipid and a volatile organic solvent which solvent is
subsequently removed by evaporation at reduced pressure. Szoka and
Papahadjopoulos (Proc. Nat. Acad. Sci. 75, 4194-4198 [1978]) have
described preparation of liposomes with very large internal aqueous space
by means of evaporation of organic solvents diethyl ether or isopropyl
ether.
The physical and detergent dialysis methods are particularly appropriate to
the present invention, as these produce acceptably large vesicles and are
quite gentle, thus unlikely to | | |
