 |
Description  |
|
|
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to synthetic lipid vesicles and the method of their
manufacture, encapsulating biologically active materials and to their use.
2. Brief Description of the Prior Art
Prior to our invention, several methods have been available to make
synthetic liposomes, encapsulating biologically active materials. For
example, Robinson, Trans. Faraday Soc., 56:1260-1264 (1960) and
Papahadjopoulos et al. (Biochim. Biophys. Acta, 135, 639, 1967) described
a method of forming phospholipid dispersions from an ether-lipid-aqueous
two-phase system that involved the evaporation of the ether by bubbling
nitrogen through the mixture. There was no attempt to use this procedure
to entrap organic materials and the trapping efficiency was not
investigated in detail. A similar evaporation technique from a
chloroform-aqueous two-phase system was described by Chowhan et al., in
Biochim. Biophys. Acta, 266:320-342 (1972). This procedure also involved
the use of excess aqueous phase and the slow removal of the chloroform
phase in order to produce a uniform population of phospholipid vesicles.
There was no attempt to maximize captured aqueous material and no
investigation into the trapping efficiency of this procedure.
Bangham et al. in J. Mol. Biol., 12:238-252 (1965) described multilamellar
lipid vesicles which could be characterized as having a small trapping
volume, a low trapping efficiency (of 10 percent) and a confined aqueous
space (15 to 35 A).
The small unilamellar vesicles produced by ultrasonication, described
initially by D. Papahadjopoulos and N. Miller (Biochim. Biophys. Acta,
135:624-638 [1967]) and by many others since then, have very low capture
efficiencies and are unsuitable for encapsulating large macromolecules due
to their small aqueous compartment (250 A).
Lipid vesicles prepared by injection of the lipids in an organic phase into
an aqueous solution were described by Batzri and Korn (Biochim. Biophys.
Acta, 298:1015 [1973]) using ethanol and by Deamer and Bangham in Biochim.
Biophys. Acta, 443:629-634 (1976) using ether. These methods produce
unilamellar or paucilamellar vesicles but, again do not achieve high
efficiencies in encapsulation. In the case of the ethanol injection this
low efficiency is due to the large aqueous volume in which the ethanol is
dispersed, and the small size of the vesicles produced by the technique.
In the case of the ether injection technique, it is due to a combination
of the large volume of aqueous space the ether is injected into, the small
amounts of lipid employed in the method, and the manner in which the
vesicles form.
A preparation of large unilamellar vesicles has been described by
Papahadjopoulos et al. in Biochim. Biophys. Acta, 394:483-491 (1975) that
involves a unique calcium-induced structural change in the lipid vesicle,
but this technique is restricted to a single phospholipid
(phosphatidylserine) and also has a relatively low efficiency of
encapsulation due to the method of reconstitution of the vesicles.
Another lipid vesicle preparation has been described in German Pat. No.
2,532,317, which involves centrifugation of a lipid-water-ether emulsion
into an aqueous phase. The disadvantage of this technique is that high
speed centrifugation is required and a large amount of the lipid-aqueous
emulsion becomes trapped at the interface and does not enter the aqueous
phase. This reduces the percentage of material entrapment.
The U.S. Pat. No. 3,804,776 is noteworthy for its disclosure of a method
for producing oil and fat encapsulated amino acids or polypeptides by
dispersing powders of the desired material for encapsulation in a molten
mixture of the fat or oil and thereafter pouring the molten mixture into
water. The encapsulated material is contained within relatively large
droplets of lipid which restricts their use to oral administration to an
animal. The method is somewhat restrictive in that it apparently is
limited to encapsulation of powders, and the lipid does not form a
bilayer.
Finally, mention may be made of U.S. Pat. No. 4,016,100 which describes the
entrapment of certain pharmaceuticals in lipid vesicles by freezing the
aqueous phospholipid dispersion of pharmaceutical and lipid. The
pharmaceutical compounds disclosed for encapsulation by the reference
method generally exhibit a high partition coefficient into an organic
phase from water. Therefore it would be expected that the material for
encapsulation would penetrate into the phospholipid bilayers of the
product vesicles. Theoretically this would provide a high degree of
encapsulation but there remains an open question as to the
bio-availability of the total material encapsulated. It would also be
expected that relatively high rates of encapsulation would not be obtained
if the technique were applied to encapsulate pharmaceuticals which are of
a more polar nature and less likely to penetrate the vesicle bilayers.
By the method of our invention, oligolamellar lipid vesicles (synthetic
liposomes) may be constructed rapidly, conveniently, under mild
conditions, in high yields, and in such a manner that they incorporate a
high percentage of a wide variety of biologically active material
processed with them. Representative of material which may be encapsulated
by the method of the invention are pharmaceutically active compounds and
compositions thereof, carbohydrates, nucleotides, polynucleotides (both
naturally occurring and synthetic) pesticides, including fungicides,
insecticides, miticides, nematocides and mollusicides, water soluble
fertilizers and agricultural nutrients, peptides, proteins, enzymes,
viruses and the like. Many of these materials do not normally penetrate
the plasma membrane of cells and may be inactivated in circulation within
a living organism or by contact with tissue and organ cultures. In the
case of pesticides and agricultural nutrients or fertilizers they may be
removed from the area of application by rain or irrigation. Encapsulation
of such materials protects them from inactivation or removal, i.e.;
maintains bioavailability. Bacterial cells such as C. parvum and E. coli
and the like may also be encapsulated by the method of the invention for
protection and bioavailability.
The method of the invention may also be used to encapsulate cosmetic
preparations which may be usefully employed as described in U.S. Pat. No.
3,957,971.
SUMMARY OF THE INVENTION
The invention comprises a method of encapsulating biologically active
materials in synthetic, oligolamellar lipid vesicles, which comprises;
providing a mixture of a vesicle wall forming compound in organic solvent
and an aqueous mixture of the biologically active material to be
encapsulated, the ratio of organic phase to aqueous phase being that which
will produce an emulsion of the water-in-oil type;
forming a homogeneous emulsion of said mixture, of the character produced
by ultra-sonic radiation;
removing organic solvent from the emulsion, whereby a mixture is obtained
having a gel-like character; and
converting the gel-like mixture to synthetic, oligolamellar vesicles
encapsulating the biologically active material.
The invention also comprises the intermediate gel-like material, the
product synthetic lipid vesicles, their use and carrier compositions
including the synthetic vesicles as the active ingredient thereof.
The term "biologically active material" as used throughout the
specification and claims means a compound or composition which, when
present in an effective amount, reacts with and/or affects living cells
and organisms.
The term "synthetic, oligolamellar lipid vesicles (liposomes)" as used
herein means man-made lipid vesicles, created in the laboratory and
characterized in part by few or single bimolecular lipid layers forming
the vesicles walls.
The method of the invention is useful to make synthetic, oligolamellar or
unilamellar lipid vesicles which in turn are usefully employed in a wide
variety of processes. For example, the lipid vesicles prepared by the
method of the invention may be used to enhance the bioavailability of
medications, to enhance enzyme replacement, oral drug delivery, topical
drug delivery, for introducing genetic information into cells in vitro and
in vivo, for the production of vaccines, for the introduction of
recombinant deoxyribonucleic acid segments into microbial cells, or as
diagnostic reagents for clinical tests following release of entrapped
"reporter" molecules. The lipid vesicles produced by the method of the
invention may also be employed to encapsulate cosmetic preparations,
pesticides, compounds for sustained slow release to effect the growth of
plants and the like.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
In a broad sense, the method of the invention calls for the formation first
of "inverted micelles" in an organic phase and then the removal of the
organic phase. The system then spontaneously reverts to a bilayer-like
structure, with a large amount of aqueous phase encapsulated in large
oligolamellar vesicles. The advantage of this method is that it gives high
capture efficiencies of aqueous phase and provides large, stable vesicles.
Phospholipids are excellent molecules for the formation of the "inverted
micelles" and then the subsequent bilayer of the vesicles. More
specifically, the method of the invention is carried out as follows.
The first step in the method of the invention is to provide a mixture of a
lipid vesicle wall forming composition in organic solvent and an aqueous
mixture of the biologically active material to be encapsulated in the
vesicle. Vesicle wall forming compounds are generally well known as are
the methods of their preparation. For example, any number of phospholipids
or lipid compounds may be used to form the vesicle walls. Representative
of such wall forming compounds are; phosphatidylcholine (hereinafter
referred to as "PC"), both naturally occurring and synthetically prepared,
phosphatidic acid (hereinafter referred to as "PA"),
lysophosphatidylcholine, phosphatidylserine (hereinafter referred to as
"PS"), phosphatidylethanolamine (hereinafter referred to as "PE"),
sphingolipids, phosphatidyglycerol (hereinafter referred to as "PG"),
spingomyelin, cardiolipin, glycolipids, gangliosides, cerebrosides and the
like used either singularly or intermixed such as in soybean phospholipids
(Asolectin, Associated Concentrates). In addition, other lipids such as
steroids, cholesterol, aliphatic amines such as long chain aliphatic
amines and carboxylic acids, long chain sulfates and phosphates, dicetyl
phosphate, butylated hydroxytoluene, tocophenol, retinol, and isoprenoid
compounds may be intermixed with the phospholipid components to confer
certain desired and known properties on the formed vesicles. In addition,
synthetic phospholipids containing either altered aliphatic portions such
as hydroxyl groups, branched carbon chains, cycloderivatives, aromatic
derivatives, ethers, amides, polyunsaturated derivatives, halogenated
derivatives or altered hydrophillic portions containing carbohydrate,
glycol, phosphate, phosphonate, quaternary amine, sulfate, sulfonate,
carboxy, amine, sulfhydryl, imidazole groups and combinations of such
groups can be either substituted or intermixed with the above mentioned
phospholipids and used in the process of the invention. It will be
appreciated from the above that the chemical composition of the lipid
component of the vesicles prepared by the method of the invention may be
varied greatly without appreciable diminution of percentage capture
although the side of the vesicle may be affected by the lipid composition.
A convenient mixture we have used extensively and which is representative
of lipid mixtures advantageously used in the method of the invention is
composed of PS and PC, or PG and PC as identified above (advantageously at
a 1:4 molar ratio in each instance). The PC, PG, PA and PE, may be derived
from purified egg yolk. Saturated synthetic PC and PG, such as dipalmitoyl
may also be used. Other amphipathic lipids that may be used,
advantageously also at 1:4 molar ratios with PC, are gangliosides,
globosides, fatty acids, stearylamine, long chain alcohols, and the like.
The liposome wall forming composition may be initially provided dissolved
in any inert solvent that can be substantially removed from the lipid or
phospholipid compound when desired. Representative of such solvents are a
wide variety of ethers, esters, alcohols, ketones, hydrocarbons (aromatic
and aliphatic including fluorocarbons), and silicones in which an aqueous
phase does not have an appreciable solubility. The solvents may be used
either alone or in admixture. For each solvent or mixture of solvents
however, the optimal ratio of lipid, aqueous space, and solvent is
different and must be determined for each case by trial and error
techniques as will be appreciated by those skilled in the art. The term
"inert solvent" as used herein means a solvent for the lipid or
phospholipid, which will not interfere with or otherwise adversely affect
the desired course of the method of the invention.
The phospholipid or lipid along with any lipid-soluble additives, are
advantageously evaporated from their solvent on to the sides of a suitable
reaction vessel. The organic phase, in which the "reversed phase
evaporation vesicles" of the invention will be formed is then added to the
vessel, i.e.; an inert organic solvent for the lipids and phospholipids as
described above. With mixing, dissolution of the lipid component of the
vesicles to be formed, previously deposited on the vessel walls is
obtained. A number of inert organic solvents are preferred for forming the
organic phase according to the method of the invention, depending on the
following conditions of the method employed. For low temperature
conditions, i.e.; removal subsequently of the organic phase at relatively
low temperatures, we find diethyl ether most advantageous, although
chloroform, or tetrahydrofuran may also be used advantageously. For higher
temperature processing, isopropyl ether is a preferred inert organic
solvent, particularly for preparing lipid vesicles containing saturated
phospholipids as the lipid component. Following dissolution of the
phospholipid or lipid to form the organic phase, an aqueous phase is added
to obtain a heterogeneous 2-phase mixture. The aqueous phase contains in
dissolution/suspension the compounds or compositions to be encapsulated in
the synthetic lipid vesicles produced by the method of the invention.
Preferably the aqueous phase is buffered to a pH suitable to maintain
stability of the material for encapsulation. The ionic strength of the
aqueous phase has a bearing on the encapsulation efficiency obtained in
the method of the invention. As a general rule, the higher the ionic
strength of the aqueous phase, the lower the percentage of entrapment. For
example, with 15 mM sodium chloride present, one can encapsulate circa 60
percent of the aqueous phase, while with 500 mM sodium chloride present,
only about 20 percent of the aqueous phase may be encapsulated. Thus, to
maximize the encapsulation of macromolecules, a buffer of low ionic
strength (less than 0.3) is preferably employed. The encapsulation
efficiency is also dependent to some degree on the concentration of lipid
or phospholipid present in the 2-phase system. Preferably the proportion
of lipid or phospholipid component is within the range of from about 0.5
mg to about 50 mg/ml. of the inert organic solvent. Preferably the ratio
of organic phase to aqueous phase is within the range of from about 2:1 to
about 20:1 v/v, most preferably about 4:1 to form a water-in-oil emulsion.
The heterogeneous 2-phase mixture obtained as described above is then
emulsified to obtain an emulsion of the character produced by ultrasonic
radiation. Preferably this is accomplished with the use of a bath type
sonicator, or for large volume preparations in an industrial size
emulsifier. Generally, the 2-phase mixture is sonicated for about 3 to 5
minutes, or until either a clear 1-phase or a homogeneous emulsion forms.
This is achieved by simply placing the container vessel in the sonicating
bath at an optimal level. Emulsification may be carried out over a wide
range of temperatures, i.e.; from about -10.degree. to about 50.degree.
C., advantageously at a temperature of from 0.degree.-20.degree. C. The
optimum conditions under which emulsification is carried out depends upon
the solvent, phospholipid, and volume of aqueous phase used in the
preparation. It will be appreciated that trial and error techniques may be
used to determine the optimum conditions for emulsification. The emulsion
mixture is then treated to remove a substantial portion of the inert
organic solvent. This may be carried out conveniently by use of a rotary
evaporator, at a temperature of circa 20.degree. C. to 60.degree. C. and
under a reduced pressure, i.e.; under vacuum (10 mm to 50 mm Hg). The
temperature employed for evaporation of the organic solvent from the
emulsion depends on the boiling point of the particular organic solvent
used in the emulsion and the stability of the biologically active material
being encapsulated. During evaporation, the emulsion first becomes a
viscous gel, which is an intermediate product. The gel is stable and can
be stored in this state for short periods of time, up to a week (at
least), at 4.degree. C. under an inert atmosphere such as nitrogen gas. A
small amount of water or buffer can then be added to the gel and the
resulting mixture evaporated for an additional period (circa 15 minutes)
to help remove residual traces of organic solvent, and to speed the
conversion of the gel into a homobeneous-appearing, suspension of
oligolamellar lipid vesicles. The gel may be converted by agitation or by
dispersion in an aqueous media such as a buffer solution. The vesicles
obtained range in diameter from 2,000 to 4,000 angstroms (average). A
significant proportion of the drugs, chemicals, macromolecules, or other
compounds and biological materials for encapsulation contained in the
aqueous buffer is captured within the lipid vesicles (up to circa 60
percent, depending on the amount of lipid, volume of the aqueous phase,
ratio of the organic phase to aqueous phase to lipid, type of inert
organic solvent(s) and, type of lipid(s) used in the process). The
non-incorporated aqueous material may be removed if necessary by
appropriate and known techniques such as by repeated centrifugations,
column chromatography, ion exchange chromatography, dialysis and like
procedures. The lipid vesicles with their encapsulated contents can then
be suspended in any isotonic buffer for use. The vesicles may be
sterilized by passage through a 0.4 micron filter (nucleopore) when
sterility is desired.
Representative of materials and compounds that may be encapsulated by the
method of the invention include but are not limited to drugs such as
cytosine arabinoside, and its phosphorylated derivatives; chemicals such
as cyclic 3', 5' adenosine monophosphate, sucrose, antibiotics such as
penicillin and streptomycin, polypeptide hormones such as insulin,
oxytocin, and vasopressin, macromolecules such as dextran, proteins such
as albumin, ferritin, and immunoglobulin G; enzymes such as alkaline
phosphatase; nucleic acids such as polyadenylic acid (poly A), ribonucleic
acids, deoxyribonucleic acids, virus and bacteria such as C. parvum and
like materials.
Advantageously the method of the invention is carried out under an inert
atmosphere. The term "inert atmosphere" as used herein means a
non-oxidizing atmosphere such as an atmosphere of nitrogen gas, argon and
like inert gases.
It will be observed from the above that the method of our invention differs
from the prior art methods in several ways. For example, according to our
method the material to be encapsulated is added into the organic phase
with the lipid where it is totally encapsulated. Furthermore, the organic
phase is substantially removed before an excess of an aqueous phase is
added. The emulsification of the initial aqueous phase into the organic
phase, and the removal of the organic phase prior to the addition of any
excess aqueous phase is essential for high capture percentage in this
method and is a critical difference between the process of our invention
and all previous methods heretofore described. The method of the invention
produces large oligolamellar vesicles from many different lipids either
alone or in combinations. An advantage of the method of the invention is
that the evaporation of the organic phase is performed under mild
temperatures and vacuum to obviate the potential for inactivation of
sensitive molecules.
The following examples describe the manner and process of making and using
the invention and represent the best mode contemplated by the inventors,
but are not to be construed as limiting. In all of the procedures
described below, one can include 0.5 to 1 mole of a fluorescent
phospholipid analog such as NBD-PE (Avanti Biochemicals) with the lipid or
phospholipid component in order to be able to visually follow the
separation of the vesicles on a column.
EXAMPLE I
Encapsulation of Enzymes
A 50 ml round bottom flask with a long extension neck is fitted with a
20/40 fitting so as to conveniently couple to a flash evaporator. The
flask is also fitted for continuous purging with nitrogen gas. The flask
is charged with 10.mu. moles of phosphatidylglycerol, 40.mu. moles of
phosphatidylcholine and 50.mu. moles of cholesterol dissolved in
chloroform. With rotary evaporation, the solvent is evaporated leaving a
thin lipid layer on the inner walls of the flask. The flask is then purged
with nitrogen gas and 5 ml of diethyl ether added with stirring to
dissolve the lipids. Then 1.5 ml of an aqueous mixture of 10 mM sodium
chloride buffered to a pH of 7.4 with 4 mM of
histidine/2-{[tris(hydroxymethyl)methyl]amino} ethanesulfonic acid
(hereinafter referred to as "TES") and 10 mg/ml of alkaline phosphatase is
added to the flask to form a heterogeneous, 2-phase mixture. The mixture
is then emulsified by sonication for 5 minutes at 0.degree. C. in a
bath-type ultrasonic cleaner (model T-80 80-IRS, Laboratory Supplies,
Hicksville, New York). The resulting emulsion is then evaporated on a
rotary evaporator (Buchi) at a temperature of 25.degree. C. and under a
reduced pressure (circa 10-50 mm Hg) using a water aspirator until a
viscous gel results. This gel is an intermediate precursor of the
synthetic liposomes to be prepared. The gel is stable and may be stored
for periods of at least one week at a temperature of circa 4.degree. C.
under an inert atmosphere.
To the gel obtained above there is added 1.5 ml. of the sodium
chloride/histide/TES buffer described above and the flask is rotated
gently to obtain any aqueous suspension of the gel. The resulting mixture
is evaporated at 30.degree. C. under a pressure of circa 10-50 mm Hg for
an additional 15 minutes to obtain an opaque suspension of phospholipid
vesicles (synthetic liposomes) having mean diameters of 0.2 to 0.6
microns. Prolonged evaporation without addition of extra buffer will
result in similar suspensions. Examination under an electron microscope
shows the vesicles to be substantially oligolamellar vesicles.
The opaque suspension of vesicles is passed through a Bio-gel A 1.5 agarose
column to separate the oligolamellar vesicles from unencapsulated alkaline
phosphatase mixture. The percent of encapsulation is calculated to be 34
percent. The separated vesicles may be suspended in any isotonic buffer
and used as the starting material for an enzyme reagent.
Similarly, repeating the above procedure for Example 1 but replacing the
alkaline phosphatase as used therein with 10 mg/ml of L-asparaginase,
synthetic vesicles are obtained encapsulating the L-asparaginase The
vesicles are useful in inhibiting certain tumor growths in mammals; see
Chang, T., Nature 229, 117-118 (1971) for the technique of use.
Similarly, repeating the above procedure of Example 1, but replacing the
alkaline phosphatase as used therein with 10 mg/ml of various
glycosidases, synthetic vesicles are obtained encapsulating the enzymes.
The product vesicles are useful in enzyme replacement therapy; see "Enzyme
Therapy in Lysosomal Storage Diseases", Tager, Hooghwinkel and Daems;
North-Hullang Publ. Co., (1974) for the technique of use.
Similarly, repeating the above procedure of Example 1 but replacing the
alkaline phosphatase as used therein with 84 mg/ml of
1-.beta.-D-arabinofuranosylcytosine, synthetic vesicles are obtained
encapsulating the arabinofuranosylcytosine for use in inhibiting certain
tumor growths in mammals; see Mayhew et al., Cancer Research, 36:
4406-4411, December 1976 for the technique of use. In like manner,
nucleoside analogues of ara-C, methotrexate or like antimetabolites may be
encapsulated for use in inhibiting certain tumor growths in mammals.
Similarly, repeating the above procedure of Example 1 but replacing the
alkaline phosphatase as used therein with 0.6 mg/ml of actinomycin D, this
compound is encapsulated. Actinomycin D encapsulated by the above
described procedure may be administered to patients suffering from
responsive cancers, following the method of Gregoriadis et al., Lancet,
June 29, 1974, pages 1313-1316; see also D. Papahadjopoulos, et al.,
Cancer Research, 36: 2988-2994, September 1976.
Similarly, repeating the above procedure of Example 1 but replacing the
phosphatase as used therein with the sodium salt of heparin dissolved in
phosphate buffered saline, synthetic vesicles are obtained encapsulating
heparin. The vesicles may be suspended in buffer solution and administered
intraveneously to a mammal as a sustained release anticoagulant, to treat
responsive afflictions. Similarly drugs such as meglumine antimoniate can
be encapsulated by the same procedure of Example 1. Meglumine antimoniate
encapsulated by the above described procedure can be used in mammals
against parasitic organisms such as Leishmaniasis. Similarly, repeating
the above procedure of Example 1 but replacing the phosphatase as used
therein with metal chelating compounds such as ethylenediaminetetraacetic
acid (EDTA), penicillamine, and the like, synthetic vesicles are obtained
that can be used in treating patients suffering from metal poisonings,
metal storage problems, or certain anemias; see U.S. Pat. No. 4,016,290.
EXAMPLE 2
Encapsulation of Nucleic Acids
The round-bottom flask described in Example 1, supra. is charged with
10.mu. moles of phosphatidylglycerol, 40.mu. moles of phosphatidylcholine
and 50.mu. moles of cholesterol dissolved in chloroform. The charge is
evaporated on a rotary evaporator to deposit a thin lipid layer on the
inner walls of the flask. The flask is purged with nitrogen gas and 5 ml
of diethyl ether added to the flask with stirring to redissolve the
lipids. Then 1.5 ml. of an aqueous mixture of 10 mM sodium chloride
buffered to a pH of 7.4 with 4 mM of histidine/TES and 1 mg/ml of
ribonucleic acid (RNA) (either polyadenylic acid or 25S tetrahymena
ribosomal RNA). The resulting mixture is emulsified by sonication for 5
minutes at a temperature of 0.degree. C. in the bath-type ultrasonic
cleaner (Laboratory Supplies, supra). The emulsion is then evaporated at a
temperature of 0.degree. C. and under a pressure of 10-50 mm Hg on the
flash evaporator until a gel is formed. To the gel there is added 1.5 ml
of the sodium chloride/histidine/TES buffer solution described above and
evaporation is continued for an additional 15 minutes. The resulting
opaque suspension is of synthetic vesicles encapsulating the ribonucleic
acid. After standing at a temperature of 20.degree. C. for about 30
minutes, the suspension is centrifuged at 100,000 X G for 30 minutes. The
supernatent is then removed, leaving as sediment the synthetic,
encapsulating vesicles. The percentage of RNA encapsulation is calculated
to be 40 to 43 percent. The vesicles are useful for inserting the RNA
through cellular membranes.
Similarly, repeating the above procedure of Example 2, but replacing the
ribonucleic acid as used therein with the insect virus nucleopolyhedrosis
and 1 mg per ml para-aminobenzoic acid, synthetic vesicles are obtained
encapsulating this virus for use as a pesticide. The vesicle encapsulated
pesticide may be used to control a number of insect pests as detailed in
chapter 29 Viruses and Invertebrates, A. J. Gibbs Ed., North Holland
Publishing Company, by spraying an effective amount of the encapsulated
pesticide, in a water mixture, on sites of infestation or potential sites
of infestation. Methods of determining effective amounts for a given pest
are well known; see for example U.S. Pat. Nos. 3,474,170; 3,476,836; and
3,478,029. The applied vesicles will remain effective for controlling the
organism over extended periods of time.
Similarly, other polyribonucleotides such as polyinosinic-poly-cytosinic
acid, or other synthetic polynucleotides can be substituted for the
ribonucleic acid in the procedure of Example 2 to form synthetic lipid
vesicles, encapsulating these compounds for use as anti-viral agents. The
techniques of using such vesicles are well known.
Similarly, repeating the above procedure but replacing the ribonucleic acid
as used therein with deoxyribonucleic acid, synthetic vesicles are
obtained encapsulating the deoxyribonucleic acid. The product vesicles may
be used as a means to transfer genetic information to either encaryotic or
bacterial cells.
EXAMPLE 3
Encapsulation of Insulin
The round-bottom flask described in Example 1, supra., is charged with
50.mu. moles of dipalmitoyl phosphatidylcholine and 50.mu. moles of
cholesterol in chloroform. The charge is evaporated on a rotary evaporator
to deposit the lipid mixture on the inner walls of the flask. The flask is
then purged with nitrogen gas and 7.5 ml of isopropyl ether and 7.5 ml of
chloroform added with stirring to redissolve the lipids. To the solution
there is added 1.5 ml of an aqueous mixture of insulin (bovine, 20 mg/ml
in 7 M urea) in aqueous buffer (10 mM triethylamine hydrochloride; pH
7.9). The resulting mixture is then emulsified by sonication for 5 minutes
at a temperature of 45.degree. C. in a sonicator bath (Laboratory
Supplies, supra.). The emulsion is then evaporated at a temperature of
45.degree. C. and under a pressure of 10-50 mm Hg. on a flash evaporator
until a gel is formed. To the gel there is added 1.5 ml of the sodium
chloride/TES buffer previously described, with stirring. Evaporation is
then continued for an additional 15 minutes. The resulting opaque
suspension is allowed to stand for 30 minutes at a temperature of
45.degree. C. The suspension is then passed through a G-75 Sephadex column
at room temperature (circa 26.degree. C.) to separate synthetic lipid
vesicles, encapsulating the insulin solution. The percentage of insulin
encapsulation is calculated to be 34 percent. The vesicles may be
suspended in phosphate buffered saline and administered orally or
intramuscularly at conventional dosage levels to patients suffering from
insulin controlled diabetes. The above procedure can be repeated at lower
temperatures during sonication and evaporation (0.degree.-20.degree. C.)
if fluid phospholipids are used for the formation of vesicles instead of
dipalmitolylphosphatidylcholine.
Similarly, repeating the above procedure but replacing the insulin with
other water-soluble peptide hormones, such as vasopresin, somatostatin or
their synthetic derivatives and analogues, synthetic lipid vesicles are
obtained encapsulating such material, for use in therapeutic applications
to mammals suffering responsive diseases.
EXAMPLE 4
Encapsulation of Sulfoxone
The round-bottom flask described in Example 1, supra., is charged with
50.mu. moles of soybean lecithin (Asolectin, Associated Concentrates,
Woodside, New York) and 50.mu. moles .beta.-sitosterol in chloroform. The
mixture is evaporated on a rotary evaporator to deposit the lipid mixture
of the inner walls of the flask. The flask is then purged with nitrogen
gas and 5 ml of diethylether is added with stirring to redissolve the
lipids. To the solution is added 1.5 ml of an aqueous mixture of sulfoxone
sodium, (disodium{sulfonylbis (p-phenylenimino}dimethanesulfinite) 25
mg/ml, and sodium ascorbate 5 mg/ml. The resulting mixture is emulsified
by sonication for 5 minutes at a temperature of 0.degree. C. in a
sonicator, (Laboratory Supplies, supra). The emulsion is then evaporated
at a temperature of 20.degree. C. and under a pressure of 10-50 mm Hg on a
flash evaporator until a gel is formed. To the gel there is added 1.5 ml
of a 10 mM ammonium chloride solution with stirring. Evaporation is
continued for an additional 15 minutes. The encapsulated sulfoxone is
separated from unencapsulated sulfoxone solution by dialysis. The vesicles
so obtained may then be used as an antibacterial preparation for
applications to plants, by suspending them in buffer solution and spraying
the solution over the root zones of the plant in an effective amount to
act as an antibacterial.
Similarly, repeating the above procedure but replacing the sulfoxone sodium
and sodium ascorbate with 50 mg/ml streptomycin sulfate, synthetic lipid
vesicles are obtained encapsulating such material, for use as an
antibacterial preparation for applications to plants.
As mentioned above, the ionic strength of the aqueous mixture for
encapsulation is a determining factor for the degree of encapsulation
obtained in the method of the invention. As the ionic strength of the
aqueous mixture for encapsulation increases, there is a decrease in both
the percentage of encapsulation and in the volume of encapsulated aqueous
space per .mu. mole of phospholipid. High concentrations of sucrose
glycerol, urea and the like do not have the same effect as increasing the
concentration of ionic species in the mixture for encapsulation. This
effect is shown in the following Example 5.
EXAMPLE 5
The procedure of Example 1, supra. is repeated six times, but in each case
the alkaline phosphatase as used in Example 1 is replaced with 0.84 mg/ml
of 1-.beta.-D-arabinofuranosylcytosine (ara-C) and the proportion of
sodium chloride in each repetition is varied to vary the ionic strength of
the ara-C mixture for encapsulation. The proportion of sodium chloride
present and the resulting degree of encapsulation obtained are shown in
Table 1, below.
TABLE 1
______________________________________
Run Percentage
No. Moles NaCl Encapsulation
______________________________________
1 0.50 15.0
2 0.15 37.5
3 0.10 42.5
4 0.04 47.5
5 0.02 62.3
6 0.00 62.5
______________________________________
The lipid concentration in the two phase mixture for emulsification also
has a bearing on the percentage of encapsulation obtained by the method of
the invention. For example, the percentage of ara-C encapsulation
decreases with decreasing total lipid concentrations. However, the aqueous
volume encapsulated per mole of phospholipid increases.
An aqueous volume of about 11.2 liters per mole of phospholipid is
encapsulated when the lipid totals about 100.mu. moles per 5 ml solvent
and increases to about 22.5 liters per mole of phospholipid when the total
lipid is reduced to 20.mu. moles. The range of 20-100 .mu.M/5 ml is
preferred, above 100 .mu.M producing increased numbers of multi-lamellar
vesicles. Example 6 below illustrates the percentage of ara-C
encapsulation obtained under various lipid concentrations.
EXAMPLE 6
The procedure of Example 1, supra, is repeated five times, but in each case
the alkaline phosphatase as used therein is replaced with 84 mg/ml of
1-.beta.-D-arabinofuranosylcytosine (ara-C) and the total lipid
concentration is varied for each repetition (while maintaining the same
ratio of PG:PC:cholesterol). The total proportion of lipid used in each
repetition and the percent of encapsulation obtained is shown in Table 2,
below.
TABLE 2
______________________________________
Run Total Lipid Percent of
No. (.mu. Moles) Encapsulation
______________________________________
1 20 15
2 40 25
3 60 30
4 80 31.2
5 100 37.5
______________________________________
The nature of the lipid or lipids employed in the method of the invention
does not appear to be critical. With the exception of negatively charged
lipids when used alone such as PS, PG, cardiolipin or phosphatidic acid,
different lipid compositions at similar proportions of
lipid/solvent/buffer have only a moderate effect on the degree of
encapsulation obtained. Example 7, below, illustrates this conclusion.
EXAMPLE 7
The procedure of Example 1, supra, is repeated several times, except that
the alkaline phosphatase as used therein is replaced with 0.84 mg/ml of
ara-C or 0.01 M of sodium chloride in 1/10 Dulbecco's phosphate buffered
saline instead of TES and in each repetition the lipid component is
varied. In Run No. 4, 3.5 ml. of diethyl ether and 1.05 ml of the
Dulbecco's saline (PBS) was used; in Run No. 5, 7.5 ml. of isopropyl ether
with 7.5 ml. of chloroform was used in place of the diethyl ether and the
second evaporation was conducted at 45.degree. C.; in Run No. 6, a mixture
of 7.5 ml. isopropyl ether and 7.5 ml. ethanol was used in place of the
diethyl ether; in Runs No. 7 and 8, a mixture of 5 ml. of diethyl ether
with 1 ml. of methanol was used in place of the diethyl ether alone. The
lipids used, the lipid concentration employed and the percentage of
encapsulation achieved in each case is shown in Table 3, below.
TABLE 3
______________________________________
Percent
Run Total Lipid
Encapsulation
No. Lipid Composition
(.mu. Moles)
ara-C sodium
______________________________________
1 PG/PC/Chol* (1:4:5)
100 62 47.5
2 PG/PC (1:4) 50 -- 34.3
3 PS/PC/Chol (1:4:5)
100 61 --
4 Stam**/PC/Chol (1:4:3)
56 -- 44.6
5 DPPC 50 -- 40.6
6 Sphingomyelin 66 24 --
7 PG 50 -- 18.6
8 PS 50 -- 22
______________________________________
*Chol represents cholesterol
**Stam represents stearylamine
The vesicles prepared by the method of the invention function as carriers
for the encapsulated, biologically active material which remains
bioavailable. The vesicle walls are impermeable to the encapsulated
materials as shown in Example 8.
EXAMPLE 8
Three 10 ml. portions of the vesicles prepared in Example 5, run No. 5 were
separated and dialyzed overnight to remove unencapsulated material present
in the suspension of ara-C encapsulated vesicles. Each dialyzed portion
was then placed in a dialysis bag and dialyzed against three consecutive
10 ml. portions of Dulbecco's phosphate buffered saline for 1 hour each at
various temperatures. The percentage of ara-C diffusing through the
dialysis bag per hour was then observed. The observations, temperatures
employed and length of dialysis are shown in Table 4, below.
TABLE 4
______________________________________
Percentage of entrapped material released per hour.
Temperature .degree.C.
Time 10 20 37
______________________________________
1 Hour .376 .697 1.97
2 Hours .221 .613 1.69
3 Hours .195 .649 1.54
______________________________________
EXAMPLE 9
Encapsulation of Bacteria
The round bottom flask described in Example 1, supra. is charged with
50.mu. moles PG:PC (1:4) and 50 moles cholesterol dissolved in chloroform.
With rotary evaporation, the solvent is evaporated leaving a film of the
lipid mixture on the inner walls of the flask. The flask is then purged
with nitrogen gas and 5 ml of diethylether is added with stirring to
redissolve the lipids. To the solution is added 1.5 ml of an aqueous
suspension of the heat-killed bacterium C. parvum In Dulbecco's phosphate
buffered saline*. The resulting mixture is emulsified by sonification for
5 minutes at 0.degree. C. in a sonicator, (Laboratory supplies, supra.).
The emulsion is then evaporated at a temperature of 20.degree. C. and
under a reduced pressure of 10-50 mm Hg on a flash evaporator until a gel
is formed. To the gel is added 1.5 ml of Dulbecco's phosphate buffered
saline and the evaporation is continued for an additional 15 minutes. The
encapsulated C. parvum is separated from unencapsulated C. parvum by
centrifugation on a sucrose gradient. Thirty percent of the presented C.
parvum is encapsulated. The vesicles so obtained may be used as an
adjuvant for immunotherapy against certain cancers as detailed in Seminars
Oncol. 1:367-378, 1974.
*FOOTNOTE: Dulbecco's phosphate buffered saline (NaCl, 0.137 M;
NaHPO.sub.4, 8.2.times.10.sup.-3 M; KCl, 2.7.times.10.sup.-3 M; KH.sub.2
PO.sub.4, 1.9.times.10.sup.-3 M; MgCl.sub.2, 1.1.times.10.sup.-3 M;
CaCl.sub.2, 0.9.times.10.sup.-3 M).
Similarly repeating the above procedure for Example 9 but replacing the C.
parvum as used therein with other immunostimulants such as BCG, intact
cancerous cells, or purification fractions of these, synthetic vesicles
are obtained encapsulating such material. These vesicles would be useful
in immunotherapy when administered to a mammal suffering from a responsive
affliction.
The Example 9 also demonstrates a particular advantage of the method of the
invention in that it provides a means of not only encapsulating materials
in solution, but materials in suspension in an aqueous media as well. The
material for encapsulation may be dissolved in the aqueous phase if
soluble. Alternatively, the material may be finely divided and suspended
in the aqueous medium or it may be macromolecules in suspension such as in
the case of virus or cellular materials. It appears that if there is room
for the suspended material within the product vesicle, it can be
encapsulated by the method of the invention. This is an advantage of the
method of the invention since many of the prior art methods of
encapsulation will not be feasible for the encapsulation of other than
dissolved materials.
The product vesicles prepared by the method of the invention have been
demonstrated to encapsulate large volumes and relatively large materials
having biological activity. Depending on the nature of the encapsulated
materials, the vesicles have a very broad range of utility. For example,
with encapsulated antibiotics they may be used in-vivo and in-vitro to
treat antibiotic resistant pathogens, including the gram-negative E. coli,
H. influenzae, P. aerunginosa and the like. The theory has been expressed
that drug resistance developed by bacterial organisms is a result of
developing impermeability of the cell wall. Delivery of an appropriate
antibiotic to the organism is facilitated when effected through a lipid
vesicle means since the lipid vesicle seems to readily penetrate the
bacterial cell wall, releasing the encapsulated antibiotic within the
pathogen.
Similarly, as briefly described above, by encapsulation of anti-parasite
compounds, one can deliver the encapsulated material to the
reticuloendothelial system of a host mammal. The lipid vesicle can deliver
the medication efficiently to neutralize the parasite with minimal toxic
effect on the host mammal.
The encapsulation of viral and bacterial agents such as inactivated polio
virus or P. aeruginosa provides a vaccine which may be administered to a
mammal to give that mammal immunization against disease caused by the
agent, with a reduction of toxicity potential associated with such
vaccines which are not encapsulated by the method of the invention.
The method of the invention may be used to encapsulate deoxyribonucleic
acid (DNA) described above. Encapsulated DNA fragments can be inserted
into living cells, both plant and animal through the delivery vehicle of
the lipid vesicle. The advantage is in labor and time saving over present
methods which comprise the plasmid and splicing techniques. The vesicle
encapsulated DNA is protected from degradation by enzymes.
* * * * *
|
|
 |
|
 |
Description |
|
