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BACKGROUND OF THE INVENTION
This invention relates generally to vesicles and more particularly to
polymerizable, lipid vesicles and the method of administering therapeutic
agents therewith.
Used herein, "lipid vesicle" and "liposome" refer to a hollow, spherical
like structure having an interior which can encapsulate aqueous solvents
and their solutes; and having a shell or membrane composed of lipid
layers. "Lipid microsphere" refers to a solid lipid spherical like
structure such that any substance dissolved in the microsphere must
generally be lipid soluble and, hence, hydrophobic or amphiphilic.
Typical methods of treating disease involve intravascular injections of
fairly toxic therapeutic agents. To maximize the efficacy at the desired
location, the blood plasma concentration of the therapeutic agent must be
kept at a high level throughout the body. This approach produces
undesirable side effects since the therapeutic agent acts upon many in
vivo systems. Encapsulation of the therapeutic agent into lipid
microspheres and targeting the microspheres to a specific release site
within the body have been suggested to avoid many undesirable side effects
caused by high blood plasma concentrations. The therapeutic agent is
encapsulated into microspheres containing ferromagnetic embedded
particles. The microspheres are injected into the body upstream of the
target site, allowed to flow in the blood stream to the target site, and
immobilized by an external magnetic field. The therapeutic agent leaks
into the target site or is released by in vivo enzymatic action. This
produces a high concentration of the therapeutic agent at the target site
while keeping the overall concentration in the blood plasma low thereby
minimizing undesirable side effects on the rest of the body.
There are, however, problems and limitations with this technique: only fat
soluble therapeutic agents can be easily encapsulated in lipid
microspheres and, after injection, the microspheres may be physically or
chemically degraded before they reach the target site. Additionally, there
is no effective means to control the rate of release of the therapeutic
agent. Current methods rely on leakage of the therapeutic agent from the
microsphere or on in vivo enzymatic activity that degrades the microsphere
at an indeterminate rate.
U.S. Pat. No. 4,247,406 discloses a magnetically localizable, biodegradable
microspheres formed from an amino acid polymer matrix embedded with
magnetic particles. The microspheres were formed from matrix materials,
such as albumin, which could be attacked in vivo by proteolytic enzymes
thus releasing the microsphere's contents. To obtain slower release rates,
enzymatic activity was inhibited by crosslinking the matrix using known
hardening agents. No method of increasing the life time or stability of
the vesicles was disclosed. The release rate for the microspheres'
contents was dependent on the degree of crosslinking and the inherent
proteolytic enzyme activity in vivo.
U.S. Pat. No. 4,345,588 discloses a method for immobilizing therapeutic
agent carrying microsphere in a capillary bed using an 8000 gauss magnetic
field following injection of the microspheres into an upstream artery and
subsequent migration of the vesicles to the desired capillary bed. No
method for releasing the therapeutic agent is disclosed. The microsphere
used were formed from nonpolymerizable lipids using the techniques known
in prior art. The therapeutic agent is carried by amino acid polymers
which are hardened by denaturation of the protein or by hardening with
formaldehyde.
U.S. Pat. No. 3,474,779 describes a method for administering therapeutic
agents in which magnetic microspheres are intravascularly administered so
that they pass into a capillary bed where they are caught by an applied
magnetic field, and magnetically retained in the capillary bed until the
therapeutic agent contained in the microsphere is released.
Microcapsules containing magnetic particles are disclosed in U.S. Pat. No.
2,971,916. The microcapsules of up to 150 micrometers in diameter are
formed by coacervation, the capsules having walls of hardened organic
colloid material enclosing an oily liquid containing a dispersion of
magnetic powder.
U.S. Pat. No. 3,663,687 teaches the use of biodegradable microspheres for
intravascular administration of therapeutic agents. The microspheres are
dimensioned so that they will lodge in the capillaries where they can be
degraded by enzymatic action thus releasing the therapeutic agent. The
patent further teaches that the rate of release of the therepeutic agent
can be varied by cross-linking the protein material forming the
microspheres.
Lipids have been used to encapsulate therapeutic agents in an effort to
selectively administer them to target sites. Rahman, Proc. Soc. Exp. Biol.
Med., 146, 1173 (1974), studied the effect of liposome encapsulated
antinomycin D on tumors in mice and concluded that mean survival times for
those mice treated with encapsulated therapeutic agents increased.
Gregoriades, Biomedical and Biophysical Research Communications, 65:537
(1975), studied the possibility of holding liposomes to targeted cells
using liposomes containing anti-tumor drugs.
Sozka et al., Am. Rev. Biophys. Bioeng., 9:467-508 (1980), reviews the
known methods for producing and characterizing lipid vesicles. Dispersion,
sonication, detergent solubilization and dialysis, solvent injection,
reverse phase evaporation, extrusion, fusion and freeze/thaw techniques
are typical methods used to produce lipid vesicles. A vesicle with desired
size and stability characteristics can be produced using one or more of
the above techniques in combination with various separating procedures
such as column chromatography.
Thus, the prior art is lacking a rugged and stable means for transporting
therapeutic agents to in vivo targeted sites. The vehicles used are
subject to premature enzymatic attack and physical degradation.
Additionally, current methods rely on timely enzymatic activity or leakage
to release the encapsulated therapeutic agent at the targeted site.
Neither method permits a controlled release of the therapeutic agent at
the site.
SUMMARY OF INVENTION
It is, therefore, an object of this invention to provide lipid vesicles
that are stable in vivo to unintended physical and chemical disruption.
A further object of this invention is to provide vesicles with
polymerizable membranes.
A further object of this invention is to provide vesicles that can carry
aqueous solutions of therapeutic agents.
A further object of this invention is to provide vesicles that can be
immoblized at an in vivo target site by an external magnetic field.
A further object of this invention is to control the rate of release of an
encapsulated therapeutic agent at the target site by oscillating the
magnetic field.
These and other objects are achieved by encapsulating therapeutic agents
and ferromagnetic particles in a lipid vesicle formed using polymerizable
lipids which are subsequently polymerized to form a stable vesicle
resistant to chemical and physical attack. The vesicles are attracted to
the targeted site by an external magnetic field. Once localized at the
target site, an oscillating magnetic field is used to destabilize the
vesicle membrane resulting in controled release of the therapeutic agent
at the target site.
Other objects, advantages, and novel features of the present invention will
become apparent form the following detailed description of the invention.
DESCRIPTION OF THE INVENTION
The vesicles of this invention are characterized by a relatively
impermeable lipid membrane that completely defines an enclosed volume
which can contain solvents, solid particles, and solutes. In the preferred
method of producing the vesicles, the solvent is evaporated from a mixture
of lipids-polymerizable lipids in a rotary evaporator such that the lipids
coat the inside surface of the rotary evaporator. A solution containing
the therapeutic agent and the magnetic particles is added to the
evaporator. The lipids are dispersed by vortexing the resulting mixture to
get the lipids off the wall of the evaporator and into solution. The
mixture is placed in a ultra-filtration cell and forced through
polycarbonate membranes until vesicles having the desired size and
membrane characteristics are produced. The vesicles should not exceed 5
micrometers in diameter and preferably measure less than 3 micrometers
with an average of 1-2 micrometers. Also, the vesicles should have a
unilamellar membrane which can be made more or less permeable by the
motion of the encapsulated magnetic particles. The vesicles containing the
encapsulated material and separated from the nonencapsulated material by
column chromatography and polymerized using Ultraviolet Light.
The lipids used to form the vesicles are most commonly phospholipids,
single-chain amphiphiles, or lysophosphatides, phospholipids being the
preferred group. These include lipids with polymerizable moieties, the
preferred group being unsaturated phospholipids containing, for example,
diacetylene, butadiene, methacryloyl, or other similar moieties.
Particular examples include bis [1,2-(methacryloyloxy)
dodecanoyl]-L-alpha-phosphatidylcholine, a 16 carbon dimethacrylate
phospholipid, and
1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, a 23 carbon
diacetylene phospholipid. The lipids should have branched or linear,
unsubstituted aliphatic chains containing between eight and thirty carbon
atoms, the preferred group having between 14 and 24 carbon atoms.
Non-polymerizable lipids useful in practicing the invention include
phospholipids such as phosphatidylcholine, phosphatidylserine,
sphingomyelin or cardiolipin.
The magnetic particles which are to be encapsulated in the vesicle can be
any ferromagnetic substance, preferably mono-domain size magnets from
magnetic sensing bacteria, magnetites, ferrites or simply very fine iron
filings, particularly ferrite particles having a particle size not
exceeding 1000 angstroms, preferably between 100 and 500 angstroms.
Additives useful in preparing the vesicles include hydrophobic entities
such as cholesterol, dicetylphosphate, or short chain lysolecithins,
preferably cholesterol.
Surfactants useful in preparing the present invention include anionic
agents such as sodium dodecylsulfate, cationic surfactants such as
dialkyldimethylammonium hydroxide, and nonionic surfactants such as
polyoxyethylene sorbitan monooleate. Naturally occurring surfactants such
as lysolecithins are also useable. The precise nature of the surfacant is
not critical to the practice of the invention.
The vesicles can be made using dispersion, sonication, detergent
solubilization and dialysis, solvent injection, reverse phase evaporation,
French press extrusion through filters, fusion and freeze/thaw techniques,
preferably extrusion through polycarbonate filters. The method chosen to
form the vesicles can affect the size of the vesicles. With some methods
it may be necessary to use the extrusion process to reduce the size of
larger vesicles. Additionally, the size of the vesicles useful in the
present method can be controlled by taking only the fraction from the
chromatography column which contains the desired size range.
The invention having been generally described, the following examples are
given as particular embodiments of the invention and to demonstrate the
practice and advantages thereof. It is understood that the examples are
given by way of illustration and are not intended to limit the
specification or the claims to follow in any manner.
The following are specific examples of the preparation of
magnetically-localizable lipid vesicles according to the present
invention.
EXAMPLE I
The vesicles can be made by removing the solvent with a rotary evaporator
from a 75 micromole mixture of bis [1,2-(methacryloyloxy)
dodecanoyl]-L-alpha-phosphatidylcholine, cholesterol, and dicetylphosphate
(DCP) 5:4:1. 75 micromoles of the therapeutic agent and 110 mg of ferrite
should be added to the lipid, which is dispersed by vortexing. The mixture
is placed at 20.degree. C. in an Amicon model 12 ultrafiltration cell and
forced by argon pressure through polycarbonate membranes twice each
through membranes with 6000 angstrom, 4000 angstrom, and 2000 angstrom
pores. The resulting mixture of vesicles and non-encapsulated materials is
separated at 4.degree. C. on a column of Sepharose CL-6B. The purified
vesicles should be concentrated on an M.sub.r 100000 cut-off ultrafilter,
or by centrifugation at 5000 rev/min for 20 minutes.
EXAMPLE II
The procedure for preparing the vesicles is identical to that of Example I
except no dicetylphosphate or cholesterol is used.
EXAMPLE III
The vesicles are fomed by identical prodedure to Example I except that
dimyristoyl phosphatidylserine, a charged phospholipid is used instead of
dicetylphosphate. The ratio of the lipids are kept the same.
EXAMPLE IV
The procedure followed Example I except Dimyristoyl phosphatidylcholine
used in place of bis [1, 2-(methacryloyloxy) dodecanoyl]
L-alpha-phosphatidylcholine. The extrusion temperature is around
22.degree. C.
EXAMPLE V
Liposomes were prepared as in Example I except monodomain magnets from
magnetic sensing bacteria is the magnetic substance used.
In the present invention, the vesicles are formed using polymerizable
lipids which are subsequently polymerized by exposing the vesicles to
ultra-violet light. Using a Rayonet Photochemical Reactor Chamber (model
RPR-100), it takes between 5-30 minutes at a UV strength of about 25
watts. Alternatively, the vesicles can be formed from lipid/polymerizable
lipid mixtures so as to vary the permability of the vesicle membrane. Once
formed, the vesicles, containing the therapeutic agent and ferromagnetic
particles, can be injected upstream from the target site. The vesicles
migrate through the blood stream to the target area where they can be
immobilized by an 8000 gauss magnetic field. Once immobilized, the
vesicle's contents can be released by oscillating the magnetic field at a
rate sufficient to vibrate the embedded ferromagnetic particles. The total
contents of the vesicle can be released by oscillating the magnetic field
sufficiently to lyse the membrane. Alternatively, particularly with the
mixed lipid/polymerizable lipid vesicle, the contents can be released at a
controlled rate by varying the oscillation rate so as to destabilize the
membrane making it more permeable to the therapeutic agent but not so as
rupture the membrane.
The magnetic field can be oscillated at a rate between 10 and 1200 cycles
per second but a range between 500 and 1000 cycles per second is prefered.
The magnetic field can have any strength necessary to immobilize the
vesicles. A range between 5000 and 12000 Gauss is prefered with 7000 to
9000 Gauss being most preferred.
For example, vesicles containing oncolytic agents could be injected
intra-arterially upstream from a tumor, localized in the tumor by the
magnetic field, and disrupted by oscillating the magnetic field. The
toxicity of the oncolytic agents is, therefore, confined to the area where
the tumor is located.
Therapeutic agents which can be encapsulated in the vesicles include
hydrophillic materials such as vindesine sulfate, fluorouracil,
antinomycin D, and the like. Basically, any known oncolytic agent,
anti-inflamatory agent, anti-arthritic agent or similar agent which is
hydrophillic can be incorporated into the vesicles.
Obviously many modifications and variations of the present invention are
possible in light of the above teachings. It is therefore to be understood
that within the scope of the appended claims the invention may be
practiced otherwise than as specifically described.
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