U.S. patent application number 11/304248 was filed with the patent office on 2006-07-27 for lipid particles having asymmetric lipid coating and method of preparing same.
Invention is credited to Yuanpeng Zhang.
Application Number | 20060165770 11/304248 |
Document ID | / |
Family ID | 33162201 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060165770 |
Kind Code |
A1 |
Zhang; Yuanpeng |
July 27, 2006 |
Lipid particles having asymmetric lipid coating and method of
preparing same
Abstract
A method of preparing lipid particles having an asymmetric lipid
coating is described. The lipid composition of the outer lipid
coating of the particles varies from the inner to outer surfaces.
The asymmetric lipid particles are formed by preparing a lipid
composition containing a charged lipid and a therapeutic agent,
where the particles each have an outer lipid coating with an
external lipid leaflet and an internal lipid structure. The
particles are then incubated under conditions effective to remove
the charged lipid from the external lipid leaflet, thus rendering
the lipid coating asymmetric. The particles have the ability to
their regain surface charge via translocation of the lipids.
Inventors: |
Zhang; Yuanpeng; (Cupertino,
CA) |
Correspondence
Address: |
PHILIP S. JOHNSON;JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
33162201 |
Appl. No.: |
11/304248 |
Filed: |
December 13, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10814703 |
Mar 30, 2004 |
7005140 |
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11304248 |
Dec 13, 2005 |
|
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60519905 |
Nov 14, 2003 |
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60459305 |
Mar 31, 2003 |
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Current U.S.
Class: |
424/450 ;
435/458 |
Current CPC
Class: |
A61K 9/1277 20130101;
A61K 47/6911 20170801; A61K 9/1271 20130101; A61K 9/1272
20130101 |
Class at
Publication: |
424/450 ;
435/458 |
International
Class: |
A61K 9/127 20060101
A61K009/127; C12N 15/88 20060101 C12N015/88 |
Claims
1. A method of preparing lipid particles having an external lipid
coating, comprising: preparing lipid particles comprised of (i) a
charged lipid and (ii) a therapeutic agent, said particles each
having an external lipid leaflet; and incubating said particles
under conditions effective to remove said charged lipid from the
external lipid leaflet.
2. The method of claim 1, wherein said preparing is comprised of
preparing lipid particles composed of a lipid composition
containing at least one cationic lipid.
3. The method of claim 1, wherein said preparing comprises (i)
forming lipid vesicles composed of said lipid composition and (ii)
complexing said lipid vesicles with said therapeutic agent.
4. The method of claim 1, wherein said incubating comprises
incubating said lipid particles in a medium containing uncharged
lipid vesicles.
5. The method of claim 4, wherein said incubating further includes
adding to the medium a lipid-polymer-ligand conjugate.
6. The method of claim 1, wherein said lipid particles are
liposomes.
7. The method of claim 6, wherein said incubating further includes
adding to the medium a lipid derivatized with a hydrophilic
polymer.
8. The method of claim 7 wherein said adding is comprised of adding
a phospholipid derivatized with polyethyleneglycol.
9. The method of claim 1, wherein said incubating is at a
temperature of less than about 15.degree. C.
10. The method of claim 1, wherein said incubating is for a time of
greater than about 5 hours.
11. The method of claim 1, wherein said preparing is comprised of
preparing lipid particles having an entrapped therapeutic agent
selected from the group consisting of a charged drug, a protein, a
peptide, and a nucleic acid.
12. The method of claim 11, wherein said therapeutic agent is a
protein or peptide.
13. A composition, comprising lipid particles having a lipid
coating comprised of an outer lipid leaflet and an inner lipid
structure, said lipid coating formed of a lipid composition (i)
comprising a charged lipid and (ii) having a gel-crystalline phase
transition temperature, said lipid particles having no appreciable
charge at a temperature lower than said phase transition
temperature but having a measurable charge after incubation at a
temperature above said phase transition temperature.
14. The composition of claim 13, wherein said lipid composition
comprises a cationic lipid.
15. The composition of claim 13, wherein said lipid particles
further include a therapeutic agent having a charge.
16. The composition of claim 15, wherein said therapeutic agent is
a nucleic acid.
17. The composition of claim 15, wherein said lipid composition has
a phase transition of between about 34-38.degree. C.
18. The composition of claim 15, wherein said lipid particles are
liposomes.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 10/814,703 filed Mar. 30, 2004, which claim the benefit of U.S.
Provisional Application No. 60/459,305, filed Nov. 14, 2003 and
U.S. Provisional Application No. 60/459,305 filed Mar. 30, 2003.
All of these documents are incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a lipid particle
composition having an asymmetric lipid coating, for use in delivery
of therapeutic agents to a person, and more specifically, to a
cell.
BACKGROUND OF THE INVENTION
[0003] Lipid vesicles, or liposomes, have demonstrated utility for
delivering therapeutic agents and diagnostic agents to target
tissues and organs. Lipid vesicles have an aqueous interior
enclosed by one or more lipid bilayers, where the therapeutic agent
is entrapped in the aqueous interior spaces or within the lipid
bilayer. Thus, both water-soluble and water-insoluble drugs can be
transported by lipid vesicles within the aqueous spaces and the
lipid bilayer, respectively.
[0004] The action of many drugs involves their direct interaction
with sites inside the cell. For action, the drug must pass through
the cell membrane to reach the cytoplasm. Success in achieving
intracellular delivery of a liposome-entrapped agent has been
limited for a variety of reasons. One reason is that liposomes,
after systemic administration to the bloodstream, are rapidly
removed from circulation by the reticuloendothelial system. Another
reason is the inherent difficulty in delivering a molecule, in
particular a large and/or a charged molecule, into the cellular
cytoplasm and/or the nucleus.
[0005] The limitation of rapid uptake by the reticuloendothelial
system has largely been overcome by the addition of a hydrophilic
polymer surface coating on the liposomes to mask the vesicle from
recognition and uptake by the reticuloendothelial system. The
extended blood circulation lifetime of liposome having a coating of
polyethyleneglycol (PEG) polymer chains (U.S. Pat. No. 5,013,556)
allows for a greater opportunity for uptake by a cell.
[0006] Delivery of charged molecules intracellularly remains a
technical challenge. In particular, delivery of nucleic acids, both
DNA and RNA, has been challenging, due to the charge and size of
the molecules. Proteins, peptides, and charged drug compounds
involve the same technical hurdle of transport across a cell
membrane. One approach to delivery of negatively charged agents,
particularly nucleic acids fragments for gene therapy, has been to
complex the DNA or RNA with a cationic lipid. Electrostatic
interaction of the cationic lipid with the nucleic acid permits
formation of lipid-nucleic acid particles in a size range suitable
for in vivo administration. The positively charged cationic lipid
on the outer particle surfaces is beneficial for interaction with
negatively-charged cellular membranes, to promote fusion or uptake
of the lipid-nucleic acid particles into the cell.
[0007] However, the presence of the positive charge on the external
surface of lipid particles prepared with cationic lipids is
detrimental to the goal of achieving a long blood circulation
lifetime for widespread biodistribution. The charge on the
particles causes immediate binding with the tissue surfaces at or
near the site of administration, substantially limiting the
availability of particles for circulation and distribution to the
target site. It would be desirable to design a lipid vesicle
composition that is neutral upon administration to permit
biodistribution, yet that is charged after a period of time, i.e.,
after biodistribution of the particles, to permit interaction with
cell membranes for binding and intracellular delivery of the
entrapped agent.
SUMMARY OF THE INVENTION
[0008] Accordingly, it is an object of the invention to provide a
lipid particle composition that includes a charged lipid for
interaction with a charged therapeutic agent, yet which bears
minimal external surface charge after formation.
[0009] It is another object of the invention to provide a lipid
particle composition that includes a charged lipid and has minimal
external surface charge after particle formation, but that is
capable of developing a charge over time, such as during incubation
at physiological temperature.
[0010] In one aspect, the invention includes a method of preparing
lipid particles having an external lipid coating. The method
comprises preparing lipid particles composed of (i) a lipid
composition containing a charged lipid and (ii) a therapeutic
agent. The particles each have an outer lipid coating having an
external lipid leaflet and an internal lipid structure. The
particles are then incubated under conditions effective to remove
the charged lipid from the external lipid leaflet.
[0011] In one embodiment, the lipid particles are composed of a
lipid composition containing at least one cationic lipid.
[0012] In another embodiment, step of preparing comprises (i)
forming lipid vesicles composed of the lipid composition and (ii)
complexing the lipid vesicles with the therapeutic agent.
[0013] Incubation of the lipid particles, in one embodiment,
involves incubation in a medium containing uncharged lipid
vesicles. In another embodiment, a lipid-polymer-ligand conjugate
can be added to the incubation medium. In other embodiment, the
incubation medium can further include a lipid derivatized with a
hydrophilic polymer. An exemplary lipid derivatized with a
hydrophilic polymer is a phospholipid derivatized with
polyethyleneglycol.
[0014] In other embodiments, incubation of the particles is
conducted at a temperature of less than about 15.degree. C. and/or
for a time of greater than about 5 hours.
[0015] In one embodiment, the lipid particles are liposomes.
[0016] In still other embodiments, the lipid particles are prepared
to have an entrapped therapeutic agent selected from the group
consisting of a charged drug, a protein, a peptide, and a nucleic
acid.
[0017] In another aspect, the invention includes a composition
comprising lipid particles having a lipid coating comprised of an
outer lipid leaflet and an inner lipid structure. The lipid coating
is formed of a lipid composition (i) comprising a charged lipid and
(ii) having a gel-crystalline phase transition temperature, where
the lipid particles have little or no appreciable charge at a
temperature lower than the lipid composition's phase transition
temperature, but have a measurable charge after incubation at a
temperature above the phase transition temperature.
[0018] In one embodiment, the lipid composition has a phase
transition of between about 34-38.degree. C.
[0019] In yet another aspect, the invention includes a method of
preparing lipid particles having an asymmetric charged lipid
composition in its outer lipid coating prior to in vivo
administration. The method includes preparing lipid particles
comprised of (i) a lipid composition containing a charged lipid and
(ii) a therapeutic agent, where the particles each have an outer
lipid coating having an external lipid leaflet and an internal
lipid structure. The particles are incubated under conditions
effective to remove charged lipids from the external lipid
leaflet.
[0020] In one embodiment, the incubation is done by incubating at a
temperature of less than about 15.degree. C. In another embodiment,
the incubation period is for a time of greater than about 5 hours.
In another embodiment, the incubating medium is comprised of
neutral lipid vesicles.
[0021] These and other objects and features of the invention will
be more fully appreciated when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A-1E are illustrations of lipid particles described
herein;
[0023] FIG. 2A is a flow chart schematic showing the steps for
formation of lipid particles;
[0024] FIG. 2B is a schematic depiction of an exemplary lipid-DNA
particle at each formation step;
[0025] FIG. 3 is a graph of zeta potential, in mV, as a function of
cationic lipid concentration (DOTPA, mole percent) for cationic
liposomes;
[0026] FIG. 4A is a graph of zeta potential, in mV, of asymmetric
lipid particles comprised of DMTAP/DOPE/cholesterol/mPEG-DS
(50:24:24:2) as a function of incubation time, in hours, at
37.degree. C. in a buffer (diamonds) and in buffer containing
neutral lipid vesicles (triangles);
[0027] FIG. 4B is a graph of zeta potential, in mV, of asymmetric
lipid particles comprised of DMTAP/DOPE/cholesterol/mPEG-DS
(50:25.5:22.5:2) as a function of incubation time, in hours, at
37.degree. C., in a buffer (diamonds) and in buffer containing
neutral lipid vesicles (squares);
[0028] FIG. 5 is a graph of zeta potential, in mV, of asymmetric
lipid particles comprised of DMTAP/DOPE/cholesterol/mPEG-DS
(50:25.5:22.5:2) and stored at 4.degree. C. for two months, as a
function of incubation time, in hours, at 37.degree. C., in a
buffer (diamonds) and in buffer containing neutral lipid vesicles
(squares); and
[0029] FIG. 6 is a graph showing luciferase expression in cells, in
pg/mg protein, for six lipid particle formulations composed of
DMTAP/DOPE/cholesterol/mPEG-DS (50:25.5:22.5:2), where Formulations
1-3 are control particles with no asymmetric bilayer and
Formulations 4-6 are lipid particles having an asymmetric outer
bilayer, where all formulations were incubated at 37.degree. C.
prior to transfection for times of 0 hours (Formulations 1, 3), 48
hours (Formulations 2, 5) and 60 hours (Formulations 3, 6).
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0030] "Lipid particle" as used herein intends particles of any
shape or size that have at least one lipid bilayer. That is, the
term includes unilamellar, plurilamellar, and multilamellar
vesicles. In some particles, portions of the particle may be
unilamellar and other portions may be multilamellar. The particles
may be spherical, or may be more globular in shape. Included within
the term "lipid particle" are liposomes as well as complexes of
lipids with other particle components. The particle may have a
defined aqueous space, i.e., a liposome, or may have pockets or
regions of aqueous space(s), i.e., lipid complexes.
[0031] Abbreviations: DMTAP:
1,2-dimyristoyl-3-trimethylammoniumpropane; DOPE:
dioleoylphosphatidylethanolamine; PEG: polyethyleneglycol; DS:
distearoyl; mPEG-DS: methoxy(polyethyleneglycol)-distearoly.
II. Lipid Particles and Method of Preparation
[0032] In one aspect, the invention relates to a method for
preparing lipid particles that have an external lipid coating
having an outer surface and an inner lipid portion, and a
compositional gradient across the lipid coating extending between
the outer surface and inner lipid region. Such particles having a
compositional gradient across all or a portion of the lipid coating
have what is referred to as an asymmetric lipid composition, as
will now be illustrated in FIGS. 1A-1E, where idealized
illustrations of such lipid particles are shown. FIG. 1A shows a
unilamellar lipid particle 10 having a single outer lipid coating
12. A portion of the lipid coating is shown in exploded view to
better illustrate the arrangement of the lipids. The coating is
comprised of an external coating surface 14 and an internal coating
surface 16. As illustrated here, the coating takes the form of a
lipid bilayer; however this illustration is idealized and the lipid
coating may have a more complex arrangement of lipids. The external
coating surface corresponds to an outer lipid leaflet of the
coating, where the polar head groups of the lipids in the leaflet
are oriented for contact with the external bulk medium in which the
particles are suspended. The internal coating surface corresponds
to an inner lipid structure, which can be a lipid leaflet or can be
a more complex arrangement of lipids, where the polar head groups
of the lipids in the inner leaflet are oriented for contact within
the aqueous space of the particle. The outer lipid leaflet has a
low or minimal charge, by virtue of being comprised predominantly
of lipids bearing no positive (cationic) or negative (anionic)
charge, for example, neutral vesicle-forming lipids or other
neutral lipids. In a preferred embodiment, the outer lipid leaflet
is comprised of neutral or anionic lipids; that is, the outer lipid
leaflet contains little or no appreciable cationic charge due to
the presence of cationic lipids. The inner lipid structure is
comprised of charged lipids. This feature is illustrated in FIG. 1A
by the indication of positively charged lipids at 18, 20 in the
inner lipid structure that provide a positive charge on the
internal coating surface. For this lipid particle having a
unilamellar lipid coating, the difference in lipid composition
between the inner and outer lipid leaflets form what is referred to
herein as an asymmetric lipid coating. More specifically, the
difference in charged lipid composition across the thickness of the
outer lipid coating is referred to as an asymmetric outer lipid
coating.
[0033] FIGS. 1B-1C are illustrations of multilamellar lipid
vesicle, also prepared by the method to be described to yield an
asymmetric outer lipid bilayer or coating. With initial reference
to FIG. 1B, vesicle 24 has a plurality of nested, concentric lipid
coating layers, indicated as idealized bilayers 26, 28, 30. In
practice, the number of lipid bilayers may be many more than the
three illustrated in the drawing and may be more complex in lipid
arrangement than the simplified bilayer shown. A portion of the
lipid coat, comprised of lipid bilayers 26, 28, 30, is shown in
exploded view for ease of viewing the lipid arrangement. Lipid
coating 30 is the outermost lipid layer and is in contact with the
external medium in which the particles are suspended. Outer lipid
coating 30 has an external surface 32 and an internal surface 34,
where the external surface is defined by an outer lipid leaflet 36
and the internal surface defined by an inner lipid structure 38.
Outer lipid leaflet 36 is distinct in its lipid composition
relative to the lipid composition of inner lipid structure 38 in
that the outer lipid leaflet has fewer charged lipids, and more
preferably fewer cationic lipids. In contrast, the inner lipid
structure includes cationic lipids that result in a positive
surface charge along the internal surface 38, as indicated by the
plus symbols at 40, 42. Thus, in the multilamellar lipid particle
illustrated in FIG. 1B, the outer lipid coating is asymmetric with
respect to its lipid composition.
[0034] FIG. 1C shows a lipid particle 44 similar to lipid particle
24 of FIG. 1B, and like elements are identified by like numerical
identifies. Particle 44 includes a plurality of lipid layers, such
as layers 26, 28, 30. Outer lipid coating 30 has an external
surface 32 and an internal surface 34. In this embodiment, the
lipid composition of outer lipid coating 30 is relatively constant
between external surface 32 and internal surface 34, where the
outer lipid coating has minimal charged lipids. The lipid layer
internal from the outermost lipid coating 30, however, includes
charged lipids in its composition as indicated by the plus symbols
43, 45, 47. Thus, in this embodiment of the lipid particle, the
asymmetric lipid coating is with respect to the lipid composition
of the outer lipid coating 30 to the inner lipid structure or lipid
layers. That is, the lipid coating as a whole is asymmetric with
respect to its lipid composition as it extends from the outer
particle surface to the inner particle regions.
[0035] FIG. 1D shows another embodiment of a multilamellar lipid
particle 46, similar to that described in FIG. 1B. Particle 46,
however, includes a lipid derivatized with a hydrophilic polymer,
such as lipids 48, 50, 52, which are representative of the
derivatized lipids in each of the lipid bilayers, 54, 56, 58 shown
in the drawing. As will be discussed below, particles having a
polymer-derivatized lipid distributed across the particle's lipid
coat are formed by including the polymer-derivatized lipid in the
lipid mixture during particle formation. The outer lipid coating 58
in particle 46 has an asymmetric lipid composition due to the
absence of charged lipids in outer lipid leaflet 60 and the
presence of charged lipids, such as lipids 62, 64, in inner lipid
structure 66. Presence of charged lipids in the inner lipid leaflet
results in a charged inner lipid coating surface, as indicated by
the plus symbols along the inner coating surface.
[0036] The lipid particle in FIG. 1D includes an additional feature
that can optionally be included. The lipid particles can also be
prepared to include targeting ligands, such as ligands 70, 72, that
act as homing devices to bring the lipid particles to a desired
site for therapeutic action. Targeting ligands attached to lipid
particles are described, for example, in U.S. Pat. Nos. 5,891,468,
6,056,973 and 6,180,134, the disclosure of these patents with
respect to moieties suitable for targeting ligands, preparation of
lipid conjugates carrying the ligands, and preparation of lipid
particles comprising lipid-ligand conjugates is incorporated by
reference herein. Targeting ligands can be incorporated into lipid
particles after formation by incubating the pre-formed lipid
particles in a micellar solution of lipid-ligand or
lipid-linker-ligand conjugates. Targeting ligands can also be
incorporated into the lipid particles by including a lipid-ligand
conjugate in the lipid composition for particle formation.
[0037] Another exemplary lipid particle is illustrated in FIG. 1E.
As noted above, the illustrations in FIGS. 1A-1D are highly
idealized, showing spherical particles, with defined lipid layers
and defined aqueous spaces. In practice, the particles may be much
more complex in structure, as partially illustrated in FIG. 1E.
FIG. 1E shows a lipid-DNA particle 51 comprised of cationic lipids
(denoted by a solid head group, such as lipids 53, 55) and neutral
lipids (denoted by an open head group, such as lipids 57, 59). DNA
61 is disposed in the interior of the particle and via charge
interaction is coated with more cationic lipids than with neutral
lipids. The particle has a defined outer lipid leaflet 63 and an
internal lipid structure comprised of the lipid layers internal to
the outer lipid leaflet. The inner lipid structures thus include
the inner leaflet 65 opposing outer leaflet 63 and the inner
bilayers surrounding the DNA, such as bilayer 67, 69. Particle 51
has pockets of aqueous space, such as pocket 71, but does not have
the defined aqueous internal compartment common in conventional
liposomes.
[0038] A. Lipid Particle Preparation
[0039] As discussed above, particularly with respect to FIGS.
1B-1C, particles described herein have an asymmetric lipid coating
with respect to either a single lipid layer in the particle (e.g.,
FIG. 1B where the lipid composition of the outer lipid layer is
asymmetric) or with respect to the lipid coating as it extends from
the outer particle surface to inner particle regions (FIG. 1C).
Preparation of such asymmetric lipid particles will now be
described.
[0040] Lipid particles having an asymmetric outer lipid coating are
prepared according to a method illustrated in general terms in FIG.
2A, where a flow chart schematic of the basic steps involved in
formation of the lipid particles is provided. FIG. 2B provides a
schematic depiction of the nature of an exemplary lipid particle at
each step in formation. In broad terms, the first step is to
prepare lipid vesicles from a lipid composition that includes a
charged lipid, such as a cationic lipid. Next, the lipid vesicles
are mixed with a therapeutic agent, for complexation of the agent
with the charged lipid vesicles, thereby forming lipid particles.
An exemplary particle corresponding to this step is shown as
particle 80 in FIG. 2B. Particle 80 is comprised of cationic lipids
(denoted by a solid head group, such as lipids 82, 84) and neutral
lipids (denoted by an open head group, such as lipids 86, 88). DNA
90 is disposed in the interior of the particle and via charge
interaction is coated with more cationic lipids than with neutral
lipids.
[0041] With continuing reference to FIG. 2A, the lipid particles
are then incubated in a medium and under conditions that achieve
extraction of charged lipids from the outer lipid coating or from
the outer leaflet of the outermost lipid coating of the lipid
particles. The nature of the lipid-DNA particle after incubation
with an incubation medium comprising a suspension of neutral
liposomes is shown as particle 100 in FIG. 2B. Incubation of the
particle resulted in removal of cationic lipids from the outer
lipid leaflet 102, as denoted in the schematic by fewer lipids with
a solid head group relative to particle 80 prior to incubation. The
difference in composition between the outer lipid leaflet and the
inner lipid structures lends an asymmetric lipid composition to the
particle. The asymmetric particle 100 has a reduced surface charge
relative to particle 80 prior to incubation. In one embodiment of
the invention, the incubation is sufficient to remove the charged
lipids from the outer lipid leaflet and from the inner lipid
structures abutting the outer lipid leaflet. In another embodiment,
the incubation is performed in such a way, e.g. by varying the
incubation time, temperature, and/or medium, to remove charged
lipids primarily from the outer lipid leaflet. This latter
embodiment is illustrated in the particle 110 in FIG. 2B, and will
be discussed more fully below. In brief, in this latter embodiment,
charged lipids are present in the inner lipid structure, such as
lipid leaflet 104 of particle 100, and available for translocation
or "flip-flop" to the outer lipid leaflet. The translocation is
accomplished by incubating the particles at a temperature
sufficient to permit movement of the lipids, typically at a
temperature above the lipid composition's phase transition
temperature. The higher concentration of charged lipids in the
inner lipid layers permits translocation of the charged lipids to
the region of lower concentration in the outer lipid leaflet. The
translocation results in a regeneration of surface charge on the
lipid-DNA particle, as illustrated in particle 110 by the increased
presence of cationic lipids (represented by the solid polar head
groups) relative to that in particle 100.
[0042] It is also possible to generate asymmetric lipid particles
by first preparing the lipid vesicles having an asymmetric outer
lipid coating and then complexing the asymmetric vesicles with a
charged drug. In this embodiment, lipid particles comprised of a
charged lipid are incubated under conditions suitable for removal
of a majority of charged lipids from the outer lipid coating or
leaflet, thus generating asymmetric vesicles. After formation of
the asymmetric lipid coating, the asymmetric lipid vesicles are
subsequently complexed with a drug to form asymmetric lipid-drug
particles.
[0043] 1. Preparation of Lipid Vesicles
[0044] Lipid vesicles, typically unilamellar or multilamellar
liposomes, are prepared from a lipid composition that includes a
charged lipid, preferably a cationic lipid. The cationic lipid can
be the sole vesicle-forming lipid in the composition, or can be one
of two or several lipids, vesicle-forming or non-vesicle-forming,
in the composition. Exemplary unilamellar vesicles prepared in
support of the invention were prepared from a lipid composition
comprised of a cationic vesicle-forming lipid, neutral lipids, and
a vesicle-forming lipid derivatized with a hydrophilic polymer.
[0045] A cationic vesicle-forming lipid is one having a polar head
group with a net positive charge, at the operational pH, e.g., pH
4-9. Exemplary cationic lipids include
1,2-diolelyloxy-3-(trimethylamino) propane (DOTAP);
N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium
bromide (DMRIE);
N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium
bromide (DORIE);
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA); 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP);
dioleoylphosphatidylcholine (DOPC);
3.beta.[N--(N',N'-dimethylaminoethane) carbamoly]cholesterol
(DC-Chol); dimethyldioctadecylammonium (DDAB), cationic
surfactants, sterol amines, and others. It is also possible to
render a neutral or negatively charged lipid cationic by
derivatization with a cationic moiety. For example, a phospholipid,
such as phosphatidylethanolamine, can be derivatized at its polar
head group with a positive moiety, e.g., lysine, as illustrated,
for example, for the lipid DOPE derivatized with L-lysine
(LYS-DOPE) (Guo, L. et al., Journal of Liposome Research 3(1):51-70
(1993)). Also included in this class of cationic lipids are the
glycolipids, such as cerebrosides and gangliosides having a
cationic polar head-group. Another cationic vesicle-forming lipid
which may be employed is cholesterol amine and related cationic
sterols.
[0046] It will be appreciated that the charged lipid included in
formation of the lipid vesicles can be an anionic lipid, such as
dimyristoyl phosphatidylglycerol (DMPG);
dioleoylphosphatidylglycerol (DOPG);
dioleoylphosphatidylethanolamine (DOPE);
dioleoylphosphatidylcholine (DOPC); and others.
[0047] The lipid composition for preparation of the lipid vesicles,
in addition to a charged lipid species, may include other lipids.
Typically, the composition will include a vesicle-forming lipid,
which intends a lipid that can form spontaneously into bilayer
vesicles in an aqueous medium, as exemplified by the phospholipids.
The lipid composition can also include lipids that are stably
incorporated into lipid bilayers, with its hydrophobic moiety in
contact with the interior, hydrophobic region of the bilayer
membrane, and its head group moiety oriented toward the exterior,
polar surface of the bilayer lipid membrane. Vesicle-forming lipids
are preferably ones having two hydrocarbon chains, typically acyl
chains, and a head group, either polar or nonpolar. There are a
variety of synthetic vesicle-forming lipids and naturally-occurring
vesicle-forming lipids, including the phospholipids, such as
phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid,
phosphatidylinositol, and sphingomyelin, where the two hydrocarbon
chains are typically between about 14-22 carbon atoms in length,
and have varying degrees of unsaturation. Phospholipids with acyl
chains having varying degrees of saturation can be obtained
commercially or prepared according to published methods.
[0048] In one embodiment, the vesicle-forming lipid is selected to
achieve a specified degree of fluidity or rigidity to control the
conditions effective for insertion of a targeting lipid-ligand
conjugate and/or to permit translocation of charged lipids from the
inner lipid structure to the outer lipid leaflet upon in vivo
administration of the lipid particles, as will be described. Lipid
particles having a more rigid lipid coating are achieved by
incorporation of a relatively rigid lipid, e.g., a lipid having a
relatively high phase transition temperature, e.g., up to
60.degree. C. Rigid, i.e., saturated, lipids contribute to greater
membrane rigidity in the lipid coating. Other lipid components,
such as cholesterol, are also known to contribute to membrane
rigidity in lipid structures. Exemplary rigid lipids include
distearyl phosphatidylcholine (DSPC), which has a phase transition
temperature of 62.degree. C., and hydrogenated soy
phosphatidylcholine (HSPC), which has a phase transition
temperature of 58.degree. C.
[0049] A more fluid bilayer is achieved by incorporation of a
relatively fluid lipid, typically one having a lipid phase with a
relatively low liquid to liquid-crystalline phase transition
temperature, e.g., at or below body temperature of about
37-38.degree. C. Examples of lipids having a phase transition
temperature below 38.degree. C. are egg phosphatidylcholine (-15 to
-7.degree. C.), dimyristoylphosphatidylcholine (23.degree. C.),
1-myristoyl-2-palmitoylphosphatidylcholine (27.degree. C.),
1-palmitoyl-2-myristoylphosphatidylcholine (35.degree. C.),
dimyristoylphosphatidylglycerol (23.degree. C.), brain
sphingomyelin (32.degree. C.) (Szoka, F. et al., Ann. Rev. Biophys.
Bioeng., 9:467 (1980)). Phase transition temperatures of many
lipids are tabulated in a variety of sources, such as Szoka &
Papahadjopoulos, Ann. Rev. Biophys. Bioeng., 9:467-508 (1980),
Avanti Polar Lipids catalogue, and Lipid Thermotropic Phase
Transition Database (LIPIDAT, NIST Standard Reference Database
34).
[0050] As mentioned above with respect to FIG. 1D, the lipid
particles can optionally include a surface coating of hydrophilic
polymer chains and/or lipid-anchored targeting conjugates. Either
of these features can be incorporated into the lipid particles by
including a polymer-derivatized lipid and/or a ligand-derivatized
lipid in the lipid composition used for formation of the lipid
vesicles in the initial step of the method. It is also possible to
incorporate these features into the lipid particles in the third
step of the process, when the lipid particles are incubated in a
medium. In this case, the polymer-derivatized lipid and/or a
ligand-derivatized lipid is included in the incubation medium and
becomes inserted into the outer lipid coating of the lipid
particles during incubation. This so-called "insertion" method has
been described in U.S. Pat. Nos. 5,891,468, 6,056,973, and
6,210,707.
[0051] Lipids derivatized with a hydrophilic polymer, and liposomes
containing polymer-derivatized lipids have been described (U.S.
Pat. No. 5,013,556; U.S. Pat. No. 5,395,619). Polymer-derivatized
lipids incorporated into a lipid coating forms a surface coating of
hydrophilic polymer chains around the lipid vesicle. The surface
coating of hydrophilic polymer chains is effective to increase the
in vivo blood circulation lifetime of the lipid particles when
compared to lipid particles lacking such a coating. Vesicle-forming
lipids suitable for derivatization with a hydrophilic polymer
include any of those lipids listed above, and, in particular
phospholipids, such as distearoyl phosphatidylethanolamine
(DSPE).
[0052] Hydrophilic polymers suitable for derivatization with a
vesicle-forming lipid include polyvinylpyrrolidone,
polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,
polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide,
polymethacrylamide, polydimethylacrylamide,
polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,
hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol,
polyaspartamide and hydrophilic peptide sequences. The polymers may
be employed as homopolymers or as block or random copolymers.
[0053] A preferred hydrophilic polymer chain is polyethyleneglycol
(PEG), preferably as a PEG chain having a molecular weight between
500-10,000 Daltons, more preferably between 1,000-5,000 Daltons.
Methoxy or ethoxy-capped analogues of PEG (e.g., mPEG) are also
preferred hydrophilic polymers, commercially available in a variety
of polymer sizes, e.g., 120-20,000 Daltons.
[0054] Preparation of vesicle-forming lipids derivatized with
hydrophilic polymers has been described, for example in U.S. Pat.
No. 5,395,619. Preparation of liposomes including such derivatized
lipids has also been described, where typically, between 1-20 mole
percent of such a derivatized lipid is included in the liposome
formulation.
[0055] Lipids derivatized with targeting ligands have also been
described (U.S. Pat. Nos. 5,891,468, 6,056,973, and 6,210,707).
Targeting ligands are typically moieties that are part of a
receptor-ligand binding pair, where the ligand of the pair is
attached to the lipid particles to enable the particles to
specifically bind to a particular target bearing its receptor pair.
Exemplary ligands are set forth in U.S. Pat. No. 5,891,468, and are
incorporated by reference herein. Particularly preferred ligands
are those that upon binding to a cell receptor are internalized by
the cell. Such ligands permit intracellular delivery of the lipid
particles' contents.
[0056] 2. Formation of Lipid Particles via Complexation of Lipid
Vesicles with a Therapeutic Agent
[0057] With continuing reference to FIG. 2, after formation of
charged lipid vesicles, the vesicles are mixed with a therapeutic
agent to form lipid particles. As used herein, "lipid vesicles"
refers to lipid structures, which may be small or large unilamellar
or multilamellar liposomes or may be lipid structures having less
defined lipid layers. "Lipid particles" refers to the lipid
vesicles complexed with a therapeutic agent, and more particularly
with a charged therapeutic agent.
[0058] As noted above, the lipid vesicles include a charged lipid,
to impart an overall charge to the vesicle. The overall charge can
be negative, by inclusion of anionic lipids, or positive, by
inclusion of cationic lipids. The therapeutic agent mixed with the
charged lipid vesicles is also charged, and more specifically,
carries a charge opposite to the charge of the lipid vesicles.
Cationic lipid vesicles mixed with a negatively charged therapeutic
agent complex to form lipid particles. Similarly, anionic lipid
vesicles mixed with a positively charged therapeutic agent complex
to form lipid particles.
[0059] Positively and negatively charged therapeutic agents are
known in the art. A preferred negatively charged therapeutic agent
is a nucleic acid, either DNA or RNA, single strand or double
strand. In one embodiment, the nucleic acid is an antisense DNA
oligonucleotide composed of sequences complementary to its target,
usually a messenger RNA (mRNA) or a mRNA precursor. The mRNA
contains genetic information in the functional, or sense,
orientation and binding of the antisense oligonucleotide
inactivates the intended mRNA and prevents its translation into
protein. Such antisense molecules are determined based on
biochemical experiments showing that proteins are translated from
specific RNAs and that once the sequence of the RNA is known, an
antisense molecule that will bind to it through complementary
Watson-Crick base pairs can be designed. Such antisense molecules
typically contain between 10-30 base pairs, more preferably between
10-25, and most preferably between 15-20. The antisense
oligonucleotide can be modified for improved resistance to nuclease
hydrolysis, as phosphorothioate, methylphosphonate, phosphodiester
and p-ethoxy oligonucleotides (WO 97/07784).
[0060] Nucleic acids are useful as therapeutic agents for a variety
of therapies, including, but not limited to, treatment of viral,
malignant and inflammatory diseases and conditions, such as, cystic
fibrosis, adenosine deaminase deficiency and AIDS. Treatment of
cancers by administration of tumor suppressor genes, such as APC,
DPC4, NF-1, NF-2, MTS1, RB, p53, WT1, BRCA1, BRCA2, VHL, or
administration of oncogenes, such as PDGF, erb-B, erb-B2, RET, ras
(including Ki-ras, N-ras), c-myc, N-myc, L-myc, Bcl-1, Bcl-2 and
MDM2, are contemplated. Administration of the following nucleic
acids for treatment of the indicated conditions are also
contemplated: HLA-B7, tumors, colorectal carcinoma, melanoma; IL-2,
cancers, especially breast cancer, lung cancer, and tumors; IL-4,
cancer; TNF, cancer; IGF-1 antisense, brain tumors; IFN,
neuroblastoma; GM-CSF, renal cell carcinoma; MDR-1, cancer,
especially advanced cancer, breast and ovarian cancers; Factor
VIII, hemophilia, and HSV thymidine kinase, brain tumors, head and
neck tumors, mesothelioma, and ovarian cancer.
[0061] In addition to nucleic acids, charged organic drug molecules
are also suitable for complexing with the lipid vesicles. A variety
of charged drugs are known in the art and readily recognized by
those of skill.
[0062] 3. Incubation of Lipid Particles to Create Asymmetric Outer
Lipid Coating
[0063] With continuing reference to FIG. 2, after complexing the
lipid vesicles with the therapeutic agent, the lipid particles are
incubated under conditions effective to extract at least a portion
of the charged lipids from the outer lipid coating layer or from
the outer lipid leaflet of the outer lipid coating. As discussed
above in FIG. 1, the lipid particles include an outer lipid coating
comprised of an inner lipid structure and an outer lipid surface.
The outer lipid surface or leaflet is in contact with the external,
incubating medium. The lipid particles are incubated to achieve
removal of substantially all of the charged lipids from the outer
leaflet or from the outermost lipid coating, thus rendering the
outer lipid coating asymmetric. The extent of removal of the
charged lipids is determined and controlled by such factors as
incubation time, temperature, and medium, as will be further
described.
[0064] Removal of the charged lipids from the outer lipid coating
or leaflet is achieved by placing the lipid particles in a medium
into which the charged lipids partition. The conditions to effect
partitioning of the lipids from the particles into the medium are
variable, and include selection of the incubation medium,
temperature of the incubation medium, and time of incubation. In
studies performed in support of the invention, an incubation medium
comprised of an aqueous suspension of neutral lipid vesicles was
effective to cause partitioning of cationic lipids from the outer
lipid coating or leaflet of the particles' lipid coating. An
incubation medium containing neutral lipid vesicles serves as a
sink for the cationic lipids, causing movement of the cationic
lipids from high concentration in the lipid particle outer coating
to low concentration in the incubation medium. A preferred
incubation medium contains the same neutral lipid present in the
lipid particles, so that no substantial movement of neutral lipid
from the particles to the incubation medium occurs. Other exemplary
incubation media are those that include a negatively charged lipid,
a surfactant, polymer particles, or other materials capable of
drawing out a charged lipid from the lipid particles.
[0065] In another embodiment, after incubation of the lipid
particles to reduce the cationic surface charge, the particles are
subsequently incubated in a second medium that includes a
negatively charged lipid species to introduce a negative charge to
the outer lipid leaflet of the lipid particles.
[0066] As noted above, the lipid particles can optionally include a
surface coating of hydrophilic polymer chains and/or lipid-anchored
targeting conjugates. Polymer-derivatized lipids or
ligand-derivatized lipids can be incorporated into the lipid
particles by including one or both of these conjugates in the
incubation medium. The conjugates insert into the outer lipid
coating of the lipid particles during incubation. Insertion of a
lipid-polymer conjugate and/or a lipid-targeting ligand conjugate
during incubation of the lipid particles can be tailored according
to the composition of the lipid bilayer, the targeting ligand, and
other factors. For example, a rapid rate of insertion can be
achieved by a higher incubation temperature, but must be balanced
against the temperature to which the ligand can be safely heated
without affecting its activity. The phase transition temperature of
the lipids in the lipid composition will also dictate the
temperature suitable for insertion. It will also be appreciated
that insertion can be varied by the presence of solvents, such as
amphipathic solvents including polyethyleneglycol and ethanol, or
detergents.
[0067] B. Characterization of Lipid Particles
[0068] Lipid particles were prepared as described in Example 1.
Briefly, cationic small unilamellar vesicles (SUVs) were prepared
from a lipid composition of DMTAP, DOPE, cholesterol, and
mPEG-DSPE. The cationic lipid vesicles were complexed with a DNA
plasmid bearing a luciferase reporter gene to form lipid particles.
Complexation of the cationic SUVs and the nucleic acid was done at
a temperature of about 0.degree. C. The lipid particles were
separated from uncomplexed cationic SUVs and/or nucleic acid. Then,
the lipid particles were incubated in an incubation medium
comprised of neutral SUVs (POPC, cholesterol, and mPEG-DSPE) at a
temperature of 4.degree. C. for 24 hours. After incubation, the
lipid particles, now with an asymmetric outer lipid bilayer, were
isolated from the other lipid components in the incubation medium
by sucrose density gradient ultracentrifugation for analysis of
charge by zeta potential.
[0069] Zeta potential values provide a measure of the apparent
charge on the outer surface of the particles. More specifically,
the zeta potential is a measure of the potential that arises across
the interface between a liquid boundary layer in contact with a
solid and the movable diffuse layer in the body of the liquid,
e.g., the slipping plane. Zeta potential values were measured as
set forth in the methods section below, using a commercially
available apparatus. FIG. 3 shows the relationship between zeta
potential, in mV, for cationic lipid vesicles comprised of DOTAP (x
mol %), POPC (55-x mol %), cholesterol (40 mol %), and polyethylene
glycol derivatized distearoyl (PEG-DS, 5 mol %), where the amount
of DOTAP is indicated along the x-axis of the graph in mol %. The
lipid vesicles were prepared at the compositions indicated and
extruded to a size of about 100 nm. Zeta potential was measured in
5 mM NaCl at 25.degree. C. The zeta potential increases as a
function of concentration of cationic lipid, DOTAP, with a rapid
increase in zeta potential observed as the concentration increases
from 0-10 mole percent, and a slower increase for compositions
having greater than 10 mole percent DOTAP.
[0070] In another study, lipid particles were prepared according to
Example 1. The lipid composition consisted of DMTAP (50 mol %),
DOPE (24 mol %), cholesterol (24 mol %), and PEG-DS (2 mol %).
After complexing the lipid vesicles with DNA, but prior to
incubation for generation of the asymmetric lipid coating, a sample
of the lipid particles was reserved for zeta potential analysis as
a comparative control. The remaining particles were incubated in a
medium containing neutral lipid vesicles (Example 1) for various
times to generate an asymmetric lipid coating. The asymmetric lipid
particles were separated from the other lipid vesicles in the
incubation medium using sucrose density gradient centrifugation and
the zeta potential was measured. The results are shown in Table 1A,
along with the size of the particles, determined by dynamic light
scattering. TABLE-US-00001 TABLE 1A Zeta Potential of Lipid
Particles and Asymmetric Lipid Particles Treated Under Various
Conditions Particle Zeta Size at Potential Lipid Particle
90.degree. (nm) (mV) lipid particle with no asymmetric lipid 272
17.27 coat, control lipid particle with asymmetric lipid 311 8.30
coating formed by incubation in medium with neutral vesicles for 24
hr, 25.degree. C. lipid particle with asymmetric lipid 295 8.63
coating formed by incubation in medium with neutral vesicles for
3.5 hr, 37.degree. C.
[0071] The zeta potential of the lipid particles with no asymmetric
lipid coating was 17.27 mV, indicating a positive charge on the
external surface of the particles. Incubation of the particles in
an incubation medium containing neutral lipid vesicles for 24 hours
at 25.degree. C. and for 3.5 hours at 37.degree. C. was effective
to reduce the zeta potential to 8.30 mV and 8.63 mV, respectively,
indicating the surface charge has been reduced significantly.
[0072] A similar study was conducted using lipid particles
comprised of DMTAP (50 mol %), POPC (24 mol %), cholesterol (24 mol
%), and PEG-DS (2 mol %). Zeta potential measurements were made on
a sample of the lipid particles after complexing the lipid vesicles
with DNA, but prior to incubation for generation of the asymmetric
lipid coating, as a control. Particles were incubated in an
incubation medium at different times and temperatures to generate
an asymmetric lipid coating. The zeta potential measurements and
size of the particles, determined by dynamic light scattering, are
shown in Table 1B. TABLE-US-00002 TABLE 1B Zeta Potential of Lipid
Particles and Asymmetric Lipid Particles Treated Under Various
Conditions Particle Zeta Size at Potential Lipid Particle
90.degree. (nm) (mV) lipid particle with no asymmetric lipid 299
23.07 coat, control lipid particle with asymmetric lipid 349 4.50
coating formed by incubation in medium with neutral vesicles for 24
hr, 25.degree. C. lipid particle with asymmetric lipid 294 7.23
coating formed by incubation in medium with neutral vesicles for
3.5 hr, 37.degree. C.
[0073] Incubation of the lipid particles in a medium containing
neutral lipid vesicles was effective to extract cationic lipids
from the outer lipid leaflet, as evidenced by the decreased zeta
potential in the asymmetric lipid particles relative to the control
particles.
[0074] In summary, lipid particles having a charged lipid in the
lipid coating composition were incubated in a medium to extract the
charged lipid from the outer coating, as evidenced by the zeta
potential measurements of the asymmetric lipid particles. The
presence of the charged lipid during particle formation is
advantageous in that charge-charge interaction between the lipid
and the charged therapeutic agent permits efficient formation of
the particles. Removal of the charged lipid from the outer lipid
coating is advantageous in that upon in vivo delivery a reduced or
absent surface charge permits a longer blood circulation time for a
more widespread biodistribution.
[0075] To determine whether the lipid particles described above
with respect to Tables 1A, 1B, and in Example 1 would remain
uncharged after in vivo administration, the asymmetric lipid
particles were placed in a 37.degree. C. temperature for 15 hours,
to simulate conditions after in vivo administration. The zeta
potential of the particles was measured after the 15 hour period,
and the results are shown in Tables 2A, 2B. Also, to determine the
extent that the lipid coating surrounded and protected the
entrapped DNA, a dye (PicoGreen.RTM. dsDNA quantitation reagent)
that emits fluorescence when in contact with DNA was added to an
aliquot of each preparation. The percent of DNA protection was
determined by comparing the fluorescent emission of the lipid
particles to that of naked DNA treated with the dye. The percent of
DNA protection is also shown in Tables 2A, 2B. TABLE-US-00003 TABLE
2A Particle Size, Percent of DNA Protected, and Zeta Potential of
Asymmetric Lipid Particles Treated Under Various Conditions
Particle DNA Zeta Size at Protection Potential Lipid Particle.sup.1
90.degree. (nm) (%) (mV) lipid particle with no asymmetric 272 93.8
17.27 lipid coat, control lipid particle with no asymmetric 319 --
25.93 lipid coat, control, after in vivo simulation (15 hr, at
37.degree. C.) lipid particle with asymmetric 311 82.4 8.30 lipid
coating formed by incubation in medium with neutral vesicles for 24
hr, 25.degree. C. lipid particle with asymmetric 314 -- 9.63 lipid
coating formed by incubation in medium with neutral vesicles for 24
hr, 25.degree. C., after in vivo simulation (15 hr, at 37.degree.
C.) lipid particle with asymmetric 295 81.0 8.63 lipid coating
formed by incubation in medium with neutral vesicles for 3.5 hr,
37.degree. C. lipid particle with asymmetric 291 -- 9.73 lipid
coating formed by incubation in medium with neutral vesicles for
3.5 hr, 37.degree. C., after in vivo simulation (15 hr, at
37.degree. C.) .sup.1Lipid particles prepared from a lipid
composition of DMTAP/DOPC/cholesterol/mPEG-DS (50/24/24/2).
[0076] TABLE-US-00004 TABLE 2B Particle Size, Percent of DNA
Protected, and Zeta Potential of Asymmetric Lipid Particles Treated
Under Various Conditions Particle DNA Zeta Size at Protection
Potential Lipid Particle.sup.1 90.degree. (nm) (%) (mV) lipid
particle with no asymmetric 299 97.2 23.07 lipid coat, control
lipid particle with no asymmetric 432 -- 26.37 lipid coat, control,
after in vivo simulation (15 hr, at 37.degree. C.) lipid particle
with asymmetric 349 78.4 4.50 lipid coating formed by incubation in
medium with neutral vesicles for 24 hr, 25.degree. C. lipid
particle with asymmetric 329 -- 4.40 lipid coating formed by
incubation in medium with neutral vesicles for 24 hr, 25.degree.
C., after in vivo simulation (15 hr, at 37.degree. C.) lipid
particle with asymmetric 294 78.7 7.23 lipid coating formed by
incubation in medium with neutral vesicles for 3.5 hr, 37.degree.
C. lipid particle with asymmetric 307 -- 7.40 lipid coating formed
by incubation in medium with neutral vesicles for 3.5 hr,
37.degree. C., after in vivo simulation (15 hr, at 37.degree. C.)
.sup.1Lipid particles prepared from a lipid composition of
DMTAP/POPC/cholesterol/mPEG-DS (50/24/24/2).
[0077] The zeta potential of lipid particles having cationic lipids
in the outer lipid leaflet (control particles) increased during the
in vivo simulation conditions, indicating the increased presence of
charge on the outer particle surfaces. The zeta potential of
asymmetric lipid particles had no significant change after exposure
to the in vivo simulation conditions of 15 hours at 37.degree. C.
For example, asymmetric lipid particles comprised of DMTAP, DOPE,
cholesterol, and mPEG-DS (Table 2A) had a zeta potential of 8.30 mV
after formation. That the zeta potential of the particles changed
very little upon incubation at 37.degree. C. suggests that the
initial incubation at 25.degree. C. for 24 hours or at 37.degree.
C. for 3.5 hours was sufficient to remove the cationic lipids from
the outer lipid coating.
[0078] The ability of the asymmetric lipid particles to transfect
cells in vitro was evaluated. The lipid particle compositions
described above were contacted with cells in vitro according to the
procedure described in Example 2. Luciferase expression of the
cells was determined as an indication of transfection. Tables 3A
and 3B show the luciferase expression of cells transfected with
asymmetric particles prepared as described in Example 1.
TABLE-US-00005 TABLE 3A Luciferase Expression after in vitro
transfection of a luciferase-encoding plasmid entrapped in
Asymmetric Lipid Particles Treated Under Various Conditions
Particle Zeta Luciferase Size at Potential Expression Lipid
Particle.sup.1 90.degree. (nm) (mV) (pg/mg) lipid particle with no
asymmetric 272 17.27 98,778 lipid coat, control lipid particle with
no asymmetric 319 25.93 755,671 lipid coat, control, after in vivo
simulation (15 hr, at 37.degree. C.) lipid particle with asymmetric
311 8.30 3,274 lipid coating formed by incubation in medium with
neutral vesicles for 24 hr, 25.degree. C. lipid particle with
asymmetric 314 9.63 1,319 lipid coating formed by incubation in
medium with neutral vesicles for 24 hr, 25.degree. C., after in
vivo simulation (15 hr, at 37.degree. C.) lipid particle with
asymmetric 295 8.63 981 lipid coating formed by incubation in
medium with neutral vesicles for 3.5 hr, 37.degree. C. lipid
particle with asymmetric 291 9.73 1,757 lipid coating formed by
incubation in medium with neutral vesicles for 3.5 hr, 37.degree.
C., after in vivo simulation (15 hr, at 37.degree. C.) .sup.1Lipid
particles prepared from a lipid composition of
DMTAP/DOPC/cholesterol/mPEG-DSPE (50/24/24/2).
[0079] TABLE-US-00006 TABLE 3B Luciferase Expression after in vitro
Transfection of a Luciferase-Encodinq Plasmid Entrapped in
Asymmetric Lipid Particles Treated under Various Conditions
Particle Zeta Luciferase Size at Potential Expression Lipid
Particle.sup.1 90.degree. (nm) (mV) (pg/mg) lipid particle with no
asymmetric 299 23.07 146,957 lipid coat, control lipid particle
with no asymmetric 432 26.37 206,723 lipid coat, control, after in
vivo simulation (15 hr, at 37.degree. C.) lipid particle with
asymmetric 349 4.50 739 lipid coating formed by incubation in
medium with neutral vesicles for 24 hr, 25.degree. C. lipid
particle with asymmetric 329 4.40 274 lipid coating formed by
incubation in medium with neutral vesicles for 24 hr, 25.degree.
C., after in vivo simulation (15 hr, at 37.degree. C.) lipid
particle with asymmetric 294 7.23 120 lipid coating formed by
incubation in medium with neutral vesicles for 3.5 hr, 37.degree.
C. lipid particle with asymmetric 307 7.40 147 lipid coating formed
by incubation in medium with neutral vesicles for 3.5 hr,
37.degree. C., after in vivo simulation (15 hr, at 37.degree. C.)
.sup.1Lipid particles prepared from a lipid composition of
DMTAP/DOPC/cholesterol/mPEG-DSPE (50/24/24/2).
[0080] Tables 3A and 3B show that the asymmetric lipid particles
have a reduced ability to transfect relative to the control
particles that bear a positive surface charge. The lower
transfection rate provides further evidence of the reduced surface
charge on the asymmetric lipid particles.
[0081] In summary, the data in Table 1A-1B, 2A-2B, and 3A-3B
illustrate a first aspect of the invention where a lipid particle
composition is prepared from a charged lipid, and the particles are
incubated to reduce the surface charge, relative to the surface
charge prior to incubation. The particles are formed under
conditions where a substantial portion of the charged lipids are
removed from the outer lipid coating. The particles have a reduced
charge relative to particles of the same lipid composition but
untreated for removal of all or a portion of charged lipids from
the outer lipid coating.
[0082] In another aspect, the invention provides an asymmetric
lipid particle that has low or minimal surface charge after
formation, but is able to regain or generate an external surface
charge after exposure to in vivo conditions. This aspect was
discussed briefly above with respect to particle 110 in FIG. 2B.
After distribution of lipid particles in vivo, the presence of a
surface charge can be beneficial. For example, after distribution
and entry into a tumor, the presence of a surface charge to cause
binding of the lipid particles with cell membranes would be
desirable. Lipid particles that have an asymmetric lipid coating,
where the outer lipid leaflet of the coating is uncharged and the
inner lipid structure is charged after asymmetric lipid particle
formation, prepared as described above, are capable of
translocation of the cationic lipids after particle formation. Due
to the concentration gradient of charged lipids in the lipid
particle, where a higher concentration of charged lipids is present
in the interior of the particle than on the external surface of the
particle, a gradual transfer of the charged lipid occurs at body
temperature. The gradual transfer, or translocation, of charged
lipid from the inner lipid structures to the outer lipid leaflet
was demonstrated in various studies, now to be described.
[0083] In this aspect of the invention, a lipid particle
composition is provided where the lipid particles are prepared from
a charged lipid, but the particles have no appreciable surface
charge at a first temperature, typically a temperature lower than
the phase transition temperature of the lipid coating. Yet, after
exposure to a temperature higher than the phase transition of the
lipid coating, the particles have a measurable surface charge.
Translocation of the charged lipids from the inner lipid structures
to the outer lipid leaflet was discussed above with respect to FIG.
2B and is illustrated by particles 100, 110 in FIG. 2B. With
respect to the particles prepared for the study in Tables 2A, 2B,
the lipid composition was comprised of 50 mole percent DMTAP which
has a gel-liquid crystalline phase transition (Tc) of between about
20-24.degree. C. (Zelphati et al., Proc. Natl. Acad. Sci. USA,
93:11493 (1996)). DMTAP is thus characterized as a fluid lipid, and
the addition of cholesterol makes the lipid composition more rigid.
DOPE at temperature above about 11.degree. C. is in a hexagonal
phase. Incubation of the particles at 37.degree. C. is expected to
bring the DMTAP/DOPE/cholesterol/mPEG-DS composition above its
phase transition, where the lipids are fluid. Translocation of
lipids from one lipid leaflet to another readily occurs when the
lipids are in this fluid state above their phase transition. Thus,
the asymmetric particles which have no appreciable charge after
formation become charged when the lipids in the lipid coating are
brought to a temperature above their phase transition, as
evidenced, for example, by zeta potential measurements.
[0084] Translocation of cationic lipids from the inner lipid
leaflet to the outer lipid leaflet is illustrated in FIGS. 4A-4B.
Lipid particles were prepared as described in Example 3 to have a
lipid coating of DMTAP/DOPE/cholesterol/mPEG-DS (50:24:24:2), and
were rendered asymmetric by incubation for 24 hours at 04.degree.
C. in a medium comprised of neutral lipid vesicles having a lipid
composition of DOPE/cholesterol/mPEG-DS. The asymmetric lipid
particles were then held at 37.degree. C. for up to 90 hours, and
at selected times, a sample was taken for zeta potential
measurements. The medium in which the asymmetric lipid particles
were incubated at 37.degree. C. was water alone or was a suspension
of neutral lipid vesicles formed of POPC/cholesterol/mPEG-DS
(58:40:2). The zeta potential measurements as a function of
incubation time, in hours, are shown in FIG. 4A. The asymmetric
lipid particles incubated in the medium containing neutral lipid
vesicles (triangles) had an initial zeta potential of about 11 mV.
After incubation at 20 hours at 37.degree. C., the zeta potential
increased to about 16 mV. By 42 hours of incubation, the zeta
potential had increased to 18 mV, with no further increase
observed. Asymmetric lipid particles incubated in buffer alone
(diamonds) also showed an increase in zeta potential over the
incubation period, indicating translocation or "flip-flop" of
cationic lipids from the inner to outer lipid leaflets.
[0085] FIG. 4B is a similar graph for asymmetric lipid particles of
a slightly different lipid composition;
DMTAP/DOPE/cholesterol/mPEG-DS (50:25.5:22.5:2). Here, asymmetric
lipid particles incubated in the presence of neutral lipid vesicles
(squares) increased in zeta potential over the 75 hour incubation
period. A similar result was observed for the asymmetric lipid
particles incubated in buffer alone (diamonds).
[0086] The stability of the asymmetric lipid particles was analyzed
in another study. Lipid particles prepared as described in Example
1 were prepared and stored at 4.degree. C. for two months. After
the two month storage, the particles were held in a medium at
37.degree. C., with and without neutral lipid vesicles, for about
100 hours. The zeta potential of the asymmetric lipid particles was
evaluated over the 100 hour incubation time to monitor
translocation of cationic lipid from the inner to outer lipid
leaflet. The results are shown in FIG. 5.
[0087] The zeta potential of asymmetric lipid particles increased
over the incubation time, when incubated in buffer alone (diamonds)
or in buffer containing neutral lipid vesicles (squares). The
increase in zeta potential is indicative of movement of cationic
lipids from the inner leaflet to the outer leaflet, showing that
the asymmetric lipid coating was stable during the 2 month storage
period.
[0088] An in vitro transfection study was conducted an asymmetric
lipid particles that had been stored at 4.degree. C. for two
months. An asymmetric lipid particle composition comprised of
DMTAP/DOPE/cholesterol/mPEG-DS (50:24:24:2) was prepared as
described in Example 1. The asymmetric lipid particles were then
held at 4.degree. C. for two months. A control composition was
prepared comprised of the same lipids, but which was not subjected
to the incubation set to generate an asymmetric lipid bilayer. The
control composition was also stored at 4.degree. C. for two months.
After storage, the two formulations were incubated at 37.degree. C.
Samples of the formulations after 37.degree. C. incubation of 0
hours, 48 hours, and 60 hours were contacted with cells in vitro
and luciferase expression measured. The results are shown in FIG.
6.
[0089] In FIG. 6, formulation numbers 1, 2, and 3 correspond to the
control lipid particles that lack an asymmetric lipid coating.
Formulation numbers 4, 5, and 6 correspond to asymmetric lipid
particles. Formulations 1 and 4 show the luciferase expression
prior to incubation at 37.degree. C. (0 hours incubation at
37.degree. C.). The asymmetric lipid particle has a lower
luciferase expression, thus a lower transfection ability, due to
the absence of positive charge on the outer particle surface.
Formulations 2 and 5 correspond to the control formulation and the
asymmetric lipid particle formulation after incubation at
37.degree. C. for 48 hours. The luciferase expression of
Formulation 5 has increased relative to that of Formulation 4 due
to translocation of cationic lipids from the inner leaflet to the
outer leaflet during the 48 hour incubation period, which simulates
in vivo conditions. Formulations 3 and 6 correspond to the control
formulation and the asymmetric lipid particle formulation after
incubation at 37.degree. C. for 60 hours. The luciferase expression
of Formulation 6 has increased relative to that of Formulations 4
and 5 due to further translocation of cationic lipids from the
inner leaflet to the outer leaflet during the 60 hour incubation
period. The luciferase expression of the control formulations 2 and
3 decreased as a result of incubation at 37.degree. C.
[0090] The data presented in FIG. 6 shows that the asymmetric lipid
particle composition has a low rate of transfection initially, due
to the absence of charge on the particle surface. Exposure of the
particles to a temperature close to, at, or above the phase
transition temperature of the lipid composition allows for
translocation, or "flip-flop" of charged lipids from the inner
lipid leaflet to the outer lipid leaflet, generating a surface
charge on the asymmetric lipid particles. Presence of the charge
improves transfection since the charge enhances binding between the
particles and the cells.
EXAMPLES
[0091] The following examples further illustrate the invention
described herein and are in no way intended to limit the scope of
the invention.
Methods
[0092] Measurement of Zeta potential: The zeta potential values of
cationic liposomes and lipid-DNA particles were measured by
Zetasizer 2000 (Malvern Ins.). Specifically, 50 .mu.L of liposomes
was added to 5 mL aqueous solution containing 5 mM NaCl (made from
30 fold dilution of USP saline with Milli-Q water) and injected
into the sample chamber according to the procedure give by the
instrument vendor. Three measurements were made for each sample at
25.degree. C.
[0093] Dynamic Light Scattering: Lipid particle sizes were
determined were obtained by dynamic light scattering (DLS) using a
Coulter N4MD instrument, operated according to the manufacturer's
instructions. The results were expressed as the mean diameter in nm
and standard deviation of a Gaussian distribution of particles by
relative volume.
Example 1
Preparation of Asymmetric Lipid Particles
[0094] Cationic liposomes (small unilamellar vesicles) were
prepared from a lipid composition of DMTAP/DOPE/CHOL/PEG-DS
(50/24/24/2 mol/mol). Individual lipid stocks were made in
chloroform/methanol (90:10 v/v) at 10 mg/mL for DMTAP (Avanti Polar
Lipids, 890860, 20 mg/mL for DOPE (Avanti Polar Lipids, 850725), 20
mg/mL for cholesterol, and 10 mg/mL for mPEG-DS
(methoxy-polyethyleneglycol-distearoyl, mPEG molecular weight 2000
Daltons, Shearwater Polymers Inc). Aliquots of solvent solutions
containing appropriate amount of lipids for a final lipid
suspension of 2 mL at 20 mM lipid concentration were taken using
positive displacement pipettes and mixed in 10 mL round bottom
flasks. The solvent was slowly removed by rotary evaporation at
about 45.degree. C. to form a thin film around the flask. The
residual solvent was removed by vacuum overnight. The lipid was
then hydrated by adding 2 mL of deionized water at 50.degree. C.
for 0.5-1 hour with stirring. The lipid suspension was then subject
to extrusion through a Lipex.RTM. extruder (10 mL volume, Northern
Lipids, Inc.) with double polycarbonate filters (0.8 .mu.m over 0.1
.mu.m) for 10 passes at 50.degree. C. The final liposome diameter
was 117.+-.30 nm as measured by Coulter submicron particle sizer
(model N4MD). After the extrusion, 2 mL deionized water was added
into the extruder and was pushed through the filter to rinse off
the remaining liposomes. This rinse was mixed with the liposomes to
a final volume of 4 mL. The final lipid concentration was
approximately 10 mM.
[0095] A. Preparation of Neutral Liposomes for Incubation
[0096] Neutral liposomes with a composition of POPC/DOPE/CHOUPEG-DS
(58/40/2 mol/mol) were prepared by a procedure similar to that
given in 1. above. The solvent stock solution was made at 40 mg/mL
POPC (Avanti Polar Lipids, 850457). The final lipid concentration
was 47.1 mM and the particle mean diameter was 108.+-.40 nm.
[0097] B. Preparation of Lipid-Plasmid DNA Complex
[0098] A DNA plasmid (pCC-luciferase) at 1 mg/mL concentration in
water was slowly injected into the cationic liposome suspension (10
mM of neutral lipid/DOPE/cholesterol/mPEG-DS, 50/24/24/2 mol/mol,
as prepared in Example 1, where the neutral lipid was either DOPE
or POPC) set in an ice-water container. The DNA injection was
performed using an infusion pump set at a rate of 10 .mu.L/min with
constant stirring. The final volume of the complexes was 1.6 mL
with DNA concentration of 0.5 mg/mL and lipid concentration of 5
mM, i.e the DNA/lipid ratio of 1 .mu.g per 20 nmoles lipid.
[0099] C. Preparation of Asymmetric Lipid-DNA Particles
[0100] The asymmetric lipid-DNA complexes were prepared by mixing
lipid-DNA complex particles with 10 fold excess neutral liposomes
("sink liposomes") prepared as described in A. above. Specifically,
1.28 mL of the lipid-DNA complexes prepared as in B. above
containing 6.4 .mu.moles lipids was mixed slowly with 2 mL neutral
incubation liposomes (POPC/CHOL/mPEG-DS, 58/40/2 mol/mol)
containing 64 .mu.moles lipids. The mixtures were set in an
ice-water bath before incubation under various conditions as shown
in FIGS. 4-6.
[0101] After the completion of the incubation, the sink liposomes
were then separated by a sucrose density gradient centrifugation
method. Specifically, a step-gradient of sucrose was loaded in
clear ultracentrifugation tubes (4.0 mL 11.times.60 mm, Beckman cat
344062 for SW 60 Ti rotor). The gradients were 25, 20, 15, 10, and
5 wt % sucrose (bottom to top). The amount of samples loaded was
typically 0.8-1 mL. The centrifugation was typically done at 40,000
rpm for 3 hour at 20.degree. C. The lipid-DNA complexes were
typically located in a weak band at the 10-15% interface. The sink
liposomes were usually retained above the 10% sucrose region. The
DNA complex band was then carefully removed using a pipette. For
the preparation of a large volume of the asymmetric lipid-DNA
particles, 40 mL centrifugation tubes were used with rotor SW28
(Beckman). The volume of sample loaded onto the tube was 4 mL. The
centrifugation took typically 15-20 hour at 40,000 rpm at
20.degree. C.
[0102] The final DNA concentration in the asymmetric lipid-DNA
particles was determined by a fluorescent assay using
PicoGreen.RTM. dsDNA quantitation reagent (Molecular Probes,
P-7581). The standard curve was generated from a series of plasmid
DNA solutions up to 2000 ng/mL DNA (pCC-Luc) in 10 mM Tris HC1 and
1 mM EDTA at pH 7.0. The linear range was found up to 1000
ng/mL.
Example 2
In Vitro Transfection
[0103] Asymmetric lipid-DNA particles and various controls prepared
as described in Example 1 were compared in vitro. BHK cells were
seeded in 6-well plates at 1.13.times.104 cells/well and incubated
at 37.degree. C., 5% CO2, for 48 hours with complete MEM media.
Before the transfection, the cells were rinsed twice with 1.0 mL
serum-free MEM media. An aliquot of the transfection sample was
mixed with serum-free MEM media to achieve a desired concentration
of plasmid DNA (typically 60-200 .mu.g/mL pCC-Luc). For
transfection, 1 mL was than overlayed onto the rinsed cells
followed by incubation at 37.degree. C. for 5 hours. After
incubation, the sample-containing media was aspirated and replaced
with 1.0 mL of complete MEM media and the incubation was continued
under the same condition for an additional 16.5 hours.
[0104] The luciferease activity was assayed using Promega
Luciferase Assay System (cat# E1500). The cells were rinsed twice
with phosphate buffered saline (PBS) and then 250 .mu.L of Cell
Culture Lysis 1.times. Reagentlysis was added. The lysed cells were
transferred to microcentrifuge tubes after two 8 minute incubations
(with swirling the plates) at room temperature. Then the tubes were
spun 14 K for 10 minutes. Luciferase activity was assayed
immediately using 20 .mu.L of the sample by a luminometer (100
.mu.L of luciferin and ATP containing assay buffer, 10 second
measurement). The relative light unit was normalized by the amount
of protein in the extracts.
[0105] The protein content was assayed using the BioRad protein
reagent. Ten microliters of lysed cells was transferred onto 96
well flat bottom plate and added with 200 .mu.L of reagent.
Absorbance at 595 nm was measured using a Molecular Devices plate
reader.
Example 3
Preparation of Asymmetric Lipid Particles
[0106] Lipid particles were formed as described in Example 1,
except that the temperature of the incubation solution comprised of
neutral SUVs (see step 4 C in Example 1) was maintained between
0-4.degree. C. with an ice bath.
[0107] Although the invention has been described with respect to
particular embodiments, it will be apparent to those skilled in the
art that various changes and modifications can be made without
departing from the invention.
* * * * *