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Description  |
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TECHNICAL FIELD
The present invention relates to the art of liposomal encapsulation. More
specifically, the present invention relates to an improved procedure for
producing large multilamellar lipid vesicles (MLV), which may be used to
encapsulate a biologically active material, particularly lipophilic
substances.
BACKGROUND ART
Liposomes or lipid vesicles are onion-like structures comprising a series
of bimolecular lipid layers spaced from one another by an aqueous
solution, the outermost layer being lipid. Liposomes have been
advantageously used to encapsulate biologically active materials for a
variety of uses. The prior art describes a number of techniques for
producing synthetic liposomes. Most of these techniques relate to the
formation of unilamellar vesicles. For example, U.S. Pat. No. 4,078,052 -
Papahadjopoulos describes a procedure for producing large unilamellar
vesicles (LUV). This particular procedure, however, is restricted to the
lipid phosphalidyserine which was found to uniquely form the intermediate
cochleate structure, apparently essential to the formation of the large
lipid vesicles, in the presence of calcium cations.
A variety of other techniques have also been disclosed for producing small
unilamellar vesicles (SUV). In one approach, a mixture of the lipid and an
aqueous solution of the material to be encapsulated is warmed and then
subjected to vigorous agitation and ultrasonic vibration. In another
approach, U.S. Pat. No. 4,089,801 - Schneider, a mixture of a lipid, an
aqueous solution of the material to be encapsulated, and a liquid which is
insoluble in water is subjected to ultrasonication, whereby aqueous
globules encased in a monomolecular lipid layer are formed dispersed in
the water-insoluble liquid. The lipid vesicles are then formed by
combining the first dispersion with a second aqueous fluid and then
subjecting the mixture to centrifugation, whereby the globules are forced
through the monomolecular lipid layer dividing the two phases, thereby
forming the bimolecular lipid layer characteristic of liposomes. In still
another approach, (O. Zumbuehl and H. G. Weder, Biochim. Biophys. Acta.,
640: 252-262, 1981), the lipids and additives are solubilized with
detergents by agitation or sonication, yielding defined mixed micelles.
The detergents are then removed by dialysis.
Two alternate methods for the preparation of small unilamellar vesicles
(SUV) that avoid the need for sonication are the ethanol injection
technique (S. Batzri and E. D. Korn, Biochim. Biophys. Acta 198:
1015-1019, 1973) and the ether-infusion technique (D. Deamer and A. D.
Bangham, Biochim. Biophys. Acta 443: 629-634, 1976). In these processes,
the organic solution of lipids is rapidly injected into a buffer solution
where it spontaneously forms liposomes.
A more recent method for preparing large unilamellar lipid vesicles (LUV)
is the reverse phase evaporation technique described in U.S. Pat. No.
4,235,871 - Papahadjopoulos. This technique consists of forming a
water-in-oil emulsion of (a) the lipids in an organic solvent and (b) the
substances to be encapsulated in an aqueous buffer solution. Removal of
the organic solvent under reduced pressure produces a mixture having a
gel-like character which can then be converted to the lipid vesicles by
agitation or by dispersion in an aqueous media.
U.S. Pat. No. 4,016,100 - Suzuki et al describes still another method of
entrapping certain biologically active materials in unilamellar lipid
vesicles by freezing an aqueous phospholipid dispersion of the
biologically active materials and lipids.
For a comprehensive review of methods for preparing liposomes refer to a
recent publication by Szoka and Papahadjopoulos (Ann. Rev. Biophys.
Bioeng. 9: 467-508, 1980).
Methods for producing multilamellar lipid vesicles (MLV), are described by
Bangham et al (J. Mol. Biol. 13: 238-252, 1965) and by Mezei and
Gulasekharam, (Life Sci., 26: 1473-1477, 1980). The lipids and lipophilic
substances are first dissolved in an organic solvent. The solvent is then
removed under reduced pressure by rotary evaporation. The lipid residue
forms a thin film on the wall of the container. Upon the addition of an
aqueous solution, generally containing electrolytes or hydrophilic
biologically active materials, large multilamellar lipsomes are formed.
Small unilamellar vesicles can be prepared by sonication of the large
multilamellar vesicles.
Most of these processes suffer from either low encapsulation efficiency or
limitations in the types of materials that can be encapsulated or both.
For example, most of these processes are limited to the encapsulation of
hydrophilic materials, and cannot efficiently accommodate the
encapsulation of lipophilic substances. Moreover, all of the currently
available procedures, except the ones described by Bangham et al and by
Mezei and Gulasekharam, are only suitable for the encapsulation of
biologically active materials in oligolamellar, or unilamellar liposomes.
It is an object of the present invention to provide a process for
encapsulating biologically active materials in large multilamellar lipid
vesicles.
It is another object of this invention to provide a method for
encapsulating biologically active materials that results in a significant
increase in the encapsulation efficiency thereof.
It is still another object of this invention to provide a method of
encapsulating biologically active materials in large multilamellar lipid
vesicles that is not limited with respect to the material to be
encapsulated and can efficiently accommodate both lipophilic and
hydrophilic substances.
It is a further object of this invention to provide a procedure for
encapsulating biologically active materials in a multilamellar lipid
vesicle that can be conducted on a larger scale relative to prior art
procedures.
DISCLOSURE OF THE INVENTION
These and other objects are met by the present invention which comprises a
process for producing large multilamellar lipid vesicles comprising the
steps of:
(a) providing a vessel partially filled with inert, solid contact masses;
(b) providing a lipid component dissolved in a suitable organic solvent
within the vessel;
(c) removing the organic solvent by evaporation so as to form a thin lipid
film on the inner wall of the vessel and on the surfaces of the contact
masses;
(d) thereafter adding an aqueous liquid to the vessel and agitating the
vessel to form an aqueous dispersion of lipid; and
(e) allowing the dispersion to stand essentially undisturbed for a time
sufficient for the multilamellar vesicles to form.
If desired, the aqueous dispersion of the large multilamellar lipid
vesicles can be further treated; for example, ultrasonication or
filtration can be used to reduce the size of the vesicles or to change
their structure to oligolamellar or unilamellar structures.
According to a known procedure, the multilamellar vesicles can be filtered
through a series of polycarbonate filters, having decreasing pore sizes,
so as to form the unilamellar vesicles.
It is specifically contemplated that hydrophilic and/or lipophilic
biologically active substances will be encapsulated within the vesicles. A
particularly advantageous consequence of the large-sized vesicles produced
by this invention is that the risk of percutaneous transfer of the
formulation is substantially reduced or eliminated. Therefore, this
invention is particularly useful for encapsulating lipid soluble
medicaments intended to produce local (i.e., topical) rather than systemic
action.
As used in the specification and claims, the terms "biologically active
material" or "biologically active substance" means a compound or
composition which, when present in an effective amount, produces an effect
in living cells or organisms.
DETAILED DESCRIPTION
As disclosed, this process differs from the technique proposed by Bangham
in that the lipid film forming step is conducted in a vessel partially
filled with inert, solid contact masses. This modification has a
significant and unexpected impact on the overall encapsulation procedure.
In particular, we have observed a significant increase in encapsulation
efficiency, especially in the encapsulation of lipophilic substances.
Significant variation is possible in the size, size distribution, shape and
composition of the contact masses. The principal characteristics of the
contact masses are: (1) that the contact masses be inert to the materials
used in the formulation, in other words there should be no unwanted
interaction between the contact masses and the lipid, lipophilic
substances, organic solvent or aqueous liquid employed, and (2) that the
contact masses be solid throughout the processing steps, in other words
the contact masses should not dissolve or disintegrate and should provide
an appropriate solid surface for supporting the thin lipid film. Prior
experimental testing has used glass beads or balls as the inert, solid
contact masses and these materials have proven to be particularly
suitable. It is also expected that metal balls, e.g., stainless steel and
synthetic substances, e.g., plastics, will also be suitable in appropriate
circumstances. While spherical contact masses are preferred, since they
provide the maximum surface area in a given volume and are easily
fluidized during the agitation step, other regular and irregular shapes
could also be used.
The size of the contact masses used in any application will depend upon the
scale of operation, the intensity of agitation and other factors that will
be apparent to one skilled in this art. As an example, it is normally
appropriate to use contact masses having a size such that the ratio of the
vessel volume to the volume of an individual contact mass is between 50
and 50,000. Generally, spherical contact masses will have a diameter
between 1.0 mm and 100 mm. It is also contemplated that the contact masses
could have a range or distribution of sizes. However, our test work has
shown that equally sized contact masses adequately satisfy the
requirements of the invention. The number of contact masses employed will
depend upon their shape and size, the size of the vessel, the volume of
organic solvent used and the quantity of lipid and lipophilic substances
dissolved. An appropriate number is used for increasing the surface area
during the evaporation step and increasing the total area of the thin
lipid film formed, but reserving sufficient volume within the vessel for
movement of the contact masses during the agitation step.
The lipid vesicles of the present invention can be produced from
phospholipids, neutral lipids, surfactants or any other related chemical
compounds having similar amphiphilic properties. As is well known, these
materials can be classified according to the formula A-B where A is a
hydrophilic, generally polar group, e.g., a carboxyl group, and B is a
hydrophobic, i.e., lipophilic, non-polar group, e.g., a long chain
aliphatic hydrocarbon group. Suitable lipids include phosphatidylcholines,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
lysophosphatidylcholine and phosphatidylglycerol. In addition, other
lipophilic additives may be used for selectively modifying the
characteristics of the lipid vesicle, e.g., the stability and permeability
of the vesicle membrance. Such other substances include stearylamine,
phosphatidic acid, dicetyl phosphate, tocopherol, cholesterol, and lanolin
extracts. From the foregoing, it should be appreciated that the
composition of the lipid component can be substantially varied without
significantly reducing the improvement in encapsulation efficiency
provided by the present invention, and other lipids, in addition to those
listed above, can be used as desired.
According to the present invention, the lipid component, together with any
other lipophilic substances including biologically active materials, is
initially dissolved in a suitable, generally non-polar, organic solvent.
The organic solvent must be capable of being substantially removed from
the lipid by evaporation and must not otherwise affect any of the
lipophilic substances included in the formulation. Representative solvents
include: ethers, esters, alcohols, ketones and various aromatic and
aliphatic hydrocarbons, including fluorocarbons. The solvents may be used
alone or in combination; for example, a 2:1 mixture of chloroform and
methanol has been found to be suitable. The organic solvent is removed by
evaporation, which can conveniently be accomplished by use of a rotary
evaporator at temperatures generally between 20.degree. and 60.degree. C.
and under a less-than-atmospheric pressure. As is well known, the
evaporative conditions will strongly depend upon the physical properties
of the organic solvent and the lipophilic materials used in the
formulation.
After the lipid film forming step, the lipids are hydrated with an aqueous
liquid to form an aqueous dispersion of lipid. The required agitation can
be accomplished by the rotation or translation, i.e., vibration, of the
vessel. An important feature of the present invention is that the presence
of the inert, solid contact masses within the vessel provides an increased
and consistent level of mechanical agitation, which enhances the formation
of uniformly sized lipid vesicles. As is well known, this hydration step
is conducted above the transition temperature of the lipid components.
The aqueous liquid may be pure water; but will generally be an aqueous
solution of an electrolyte or a biologically active material. For example,
an aqueous solution of sodium chloride or calcium chloride may be
imployed. Additionally, active substances including pharmaceuticals such
as, vitamins, hormones, enzymes, antibiotics and bactericides, and
cosmetics such as, dyestuffs, perfumes and humectants may be included.
While most of the prior art procedures are limited to encapsulating
hydrophilic materials, the present invention can also accomodate the
encapsulation of hydrophobic, i.e., lipophilic materials. Testing has
shown that lypophilic medicaments, e.g. progesterone, can be encapsulated
at high efficiencies.
In other words, the present invention can be advantageously employed to
encapsulate either hydrophilic or lipophilic substances or both. In the
case of lipophilic materials, the substances to be encapsulated are
co-dissolved with the lipids in the organic solvent prior to the lipid
film forming step; while as noted above hydrophilic substances are
conveniently added to the aqueous liquid used to disperse the lipid film.
After agitating the lipid-aqueous liquid mixture, the resulting dispersion
is then allowed to remain undisturbed for a time sufficient to allow the
lipid vesicles to form and mature. Generally, it will be sufficient to
allow the vessel to stand undisturbed at room temperature for
approximately one to two hours. The aqueous dispersion of the
multilamellar lipid vesicles can then be recovered from the vessel
containing the inert, solid contact masses. If desired, any
non-incorporated active substances can be removed from the dispersion
using known techniques such as repeated centrifugations, dialysis or
column chromatography. The lipid vesicles can then be resuspended in any
suitable electrolytic buffer for subsequent use.
Since the procedure described by Bangham is the only prior art process of
which we are aware for encapsulating lipophilic materials in large
multilamellar lipid vesicles, we conducted a series of experiments
directly comparing the process of the present invention with the Bangham
procedure. In particular, we compared the two procedures so as to
determine their relative effectiveness in encapsulating lipophilic
substances. The following examples will vividly demonstrate the
significant and unexpected improvement in the encapsulation efficiency of
lipophilic materials made possible by the present invention.
EXAMPLE I
In this example, multilamellar lipid vesicles are prepared using the
procedure of this invention (Method A) and the procedure disclosed by
Bangham (Method B). The materials used in preparing the lipid vesicles and
the amounts thereof are listed below in Table 1. A small amount of
progesterone labelled with Carbon 14 was mixed with a quantity of
non-radioactive progesterone to facilitate the determination of its
encapsulation efficiency.
TABLE 1
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DL alpha dipalmitoyl 22.2 mg
phosphatidyl choline (DPPC)
Cholesterol 5.0 mg
Progesterone 5.0 mg
(containing 0.5 uCi; .sup.14 C)
Calcium chloride solution (8 mM)
5.0 ml
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In accordance with the method of this invention, Method A, the DPPC,
cholesterol and progesterone were co-dissolved in a chloroform-methanol
solvent (2:1) in a 50 ml round bottom vessel. Glass beads, having a
diameter of 5 mm, were added to the vessel and the solvent was evaporated
under vacuum in a rotary evaporator, thereby leaving a thin lipid film on
the glass beads and on the vessel wall. A warm calcium chloride solution
at 65.degree. C. was then added to the vessel, and the mixture was
vigorously shaken for one minute. Afterwards, the vessel was further
agitated by rotating it in the rotary evaporator, without applying a
vacuum, at 65.degree. C. for 30 minutes. The resultant dispersion was
allowed to stand for one hour.
According to the Bangham procedure, Method B, the DPPC, cholesterol and
progesterone were similarly dissolved in a chloroform-methanol solvent
(2:1) within a 50 ml round bottom vessel. The vessel did not contain any
contact masses. The organic solvent was evaporated under vacuum in a
rotary evaporator until a smooth, dry lipid film was observed on the wall
of the vessel. A calcium chloride solution, heated to a temperature of
65.degree. C. was then added to the contents of the vessel and the mixture
was vigorously shaken in a 65.degree. C. water bath for 30 minutes. The
resultant dispersion was then allowed to stand for one hour.
After allowing the newly formed liposomal preparations to stand at room
temperature for one hour, small aliquots (approximately 10 .mu.l) of each
of the preparations were examined under a microscope with a magnification
of 475X using polarized light to verify the formation of the large
multilamellar vesicles. The remaining portions of the liposomal
preparations were filtered through polycarbonate filters having an 8 .mu.m
pore size. The filtrates were then centrifuged at 22,000 Xg for 15 minutes
at 20.degree.C. The supernatant was decanted and the centrifugate was
resuspended in 5 ml of 8 mM aqueous calcium chloride solution. This
procedure was repeated twice. The centrifugate separated from the final
step of centrifugation was resuspended in 5.0 ml of 8 mM aqueous calcium
chloride solution, and 10 .mu.l aliquots from each preparation were used
to calculate the encapsulation efficiency. The results are presented in
Example III.
EXAMPLE II
The procedure of Example I was repeated three additional times, but in each
case the formulation was changed to that listed in Tables 2, 3 and 4,
respectively. In formulating the liposomes from the substances listed in
Table 4, a 1000 ml vessel was substituted for the 50 ml vessel.
TABLE 2
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Phosphatidylcholine (purified)
22.2 mg
Cholesterol 5.0 mg
Progesterone (0.5 uCi; .sup.14 C)
5.0 mg
Calcium Chloride (8 mM) 5.0 ml
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TABLE 3
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Dipalmitoyl phosphatidylcholine
22.2 mg
Cholesterol 5.0 mg
Stearylamine 2.0 mg
Progesterone (0.5 uCi; .sup.14 C)
5.0 mg
Calcium Chloride (8 mM) 5.0 ml
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TABLE 4
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Dipalmitoyl phosphatidylcholine
888.0 mg
Cholesterol 200.0 mg
Progesterone (1.0 uCi: .sup.14 C)
200.0 mg
Calcium Chloride (8 mM) 200.0 ml
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EXAMPLE III
The progesterone encapsulation efficiencies, using the test procedures
described in Examples I and II, are listed in Table 5. As shown by these
results, the present invention (Method A) provides a substantial and
unexpected increase in the encapsulation efficiency of lipophilic
materials as compared with the prior art (Method B) available for
accomplishing the same result.
TABLE 5
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TABLE % of Encapsulation
FORMULATION Method A Method B
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1 77.0 7.8
2 83.0 6.1
3 87.0 10.0
4 85.0 4.5
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In addition to enchancing encapsulation efficiency, the present invention
also makes it possible to produce liposomes on a larger scale. The Bangham
method can only produce small batches of liposomes (e.g., 100-200 ml)
otherwise the encapsulation efficiency substantially decreases. The batch
size when using our invention, however, can be significantly increased
simply by increasing the surface area of the vessel and the inert, solid
contact masses. This result is evidenced by the encapsulation efficiency
data in Table 5 for the formulation of Table 4, in which a 1000 ml vessel
was substituted for the 50 ml vessel used in the prior tests. This vessel
also contained a larger amount of solid inert contact masses, providing
much greater surface area for the lipid film formation. Consequently, the
present invention makes the large scale manufacturing of liposomes
possible.
While preferred embodiments of this invention have been discussed herein,
those skilled in the art will appreciate that changes and modifications
may be made without departing from the spirit and scope of this invention,
as defined in and limited only by the scope of the appended claims.
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