U.S. patent application number 10/445732 was filed with the patent office on 2004-12-16 for targeted polymerized liposome diagnostic and treatment agents.
This patent application is currently assigned to Targesome, Inc.. Invention is credited to Bednarski, Mark David, Kuniyoshi, Jeremy Kenji, Li, Henry Y., Li, King Chuen, Sipkins, Dorothy Anna, Song, Curtis Kang Hoon, Storrs, Richard Wood, Tropper, Francois Daniel.
Application Number | 20040253184 10/445732 |
Document ID | / |
Family ID | 26963903 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253184 |
Kind Code |
A1 |
Li, King Chuen ; et
al. |
December 16, 2004 |
Targeted polymerized liposome diagnostic and treatment agents
Abstract
Polymerized liposome particles which are linked to a targeting
agent and may also be linked to a contrast enhancement agent and/or
linked to or encapsulating a treatment agent. The targeting imaging
enhancement polymerized liposome particles interact with biological
targets holding the image enhancement agent to specific sites
providing in vitro and in vivo study by magnetic resonance,
radioactive, x-ray or optical imaging of the expression of
molecules in cells and tissues during disease and pathology.
Targeting polymerized liposomes may be linked to or encapsulate a
treatment agent, such as, proteins, drugs or hormones for directed
delivery to specific biological sites for treatment.
Inventors: |
Li, King Chuen; (Bethesda,
MD) ; Bednarski, Mark David; (Los Altos, CA) ;
Storrs, Richard Wood; (San Diego, CA) ; Li, Henry
Y.; (Visalia, CA) ; Tropper, Francois Daniel;
(Toronto, CA) ; Song, Curtis Kang Hoon;
(Sunnyvale, CA) ; Sipkins, Dorothy Anna; (Palo
Alto, CA) ; Kuniyoshi, Jeremy Kenji; (Cupertino,
CA) |
Correspondence
Address: |
SWANSON & BRATSCHUN L.L.C.
1745 SHEA CENTER DRIVE
SUITE 330
HIGHLANDS RANCH
CO
80129
US
|
Assignee: |
Targesome, Inc.
|
Family ID: |
26963903 |
Appl. No.: |
10/445732 |
Filed: |
May 27, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10445732 |
May 27, 2003 |
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10083422 |
Feb 26, 2002 |
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6569451 |
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10083422 |
Feb 26, 2002 |
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09650276 |
Aug 29, 2000 |
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6350466 |
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09650276 |
Aug 29, 2000 |
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08629056 |
Apr 8, 1996 |
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6132764 |
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08629056 |
Apr 8, 1996 |
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08286555 |
Aug 5, 1994 |
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5512294 |
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Current U.S.
Class: |
424/9.363 ;
424/450 |
Current CPC
Class: |
Y10S 977/927 20130101;
Y10S 977/928 20130101; A61K 49/1812 20130101; A61K 49/22 20130101;
Y10S 977/93 20130101; G01N 33/586 20130101; Y10S 977/795 20130101;
Y10S 977/907 20130101; Y10S 977/775 20130101; Y10S 977/808
20130101; Y10S 977/918 20130101; Y10S 424/812 20130101; A61K
51/1237 20130101; Y10S 977/897 20130101; Y10S 436/829 20130101 |
Class at
Publication: |
424/009.363 ;
424/450 |
International
Class: |
A61K 049/00; A61K
009/127 |
Claims
What is claimed is:
1. A functional lipid having an active hydrophilic head group
linked by a bifunctional linker portion to a liposome forming lipid
with a hydrophobic tail group having a polymerizable functional
group.
2. A functional lipid according to claim 1 wherein said hydrophilic
head group comprises a lanthanide-diethylenetriamine pentaacetic
acid chelate and said variable length linker group is selected from
the group consisting of bifunctional aliphatic compound and
bifunctional aromatic compound.
3. A functional lipid according to claim 1 wherein said hydrophilic
head group comprises a lanthanide-diethylenetriamine pentaacetic
acid chelate and said linker group is selected from the group
consisting of polyethylene glycol, propylene glycol and
polyglycine.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/083,422, filed Feb. 26, 2003, entitled "Targeted Polymerized
Liposome Diagnostic and Treatment Agents," now U.S. Pat. No.
6,569,451 which is a continuation of U.S. application Ser. No.
09/650,276, filed Aug. 29, 2000, entitled "Targeted Polymerized
Liposome Diagnostic and Treatment Agents," now U.S. Pat. No.
6,350,466 which is a continuation of U.S. application Ser. No.
08/629,056, filed Apr. 8, 1996, entitled "Targeted Polymerized
Liposome Diagnostic and Treatment Agents," now U.S. Pat. No.
6,132,764, which is a continuation-in-part of U.S. application Ser.
No. 08/286,555, filed Aug. 5, 1994, entitled "Targeted Polymerized
Liposome Contrast Agents," now U.S. Pat. No. 5,512,294. The
disclosure of each of these applications, and of all other patents,
patent applications and publications referred to herein, is
incorporated by reference herein, in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to polymerized liposomes which are
linked to a targeting agent and may also be linked to at least one
of an image contrast enhancement agent and a therapeutic or
treatment agent to provide targeted polymerized liposome diagnostic
agents and targeted polymerized liposome therapeutic agents,
respectively. In one embodiment, this invention relates to liposome
s which may be linked to contrast ions for magnetic resonance
imaging and radioisotope imaging or optical imaging by using
chromophores attached to the liposome or chromophores inherent in
the particle in which the polymerization adds stability in vivo.
The paramagnetic or radioactive polymerized liposomes may also be
linked to antibodies and ligands for specific interaction with
biological targets holding the contrast agent to specific
biological sites, providing in vitro and in vivo study of the
expression of molecules in or on the surface of cells and tissues
during disease and pathology. In another embodiment, targeted
polymerized liposomes may be linked to or encapsulate a therapeutic
agent, such as, for example, proteins, hormones and drugs, for
directed delivery of a treatment agent to specific biological
locations for localized treatment.
[0004] 2. Description of Related Art
[0005] Liposomes have been used as carriers for administration of
drugs and paramagnetic contrast agents. U.S. Pat. Nos. 5,077,057
and 5,277,914 teach preparation of liposome or lipidic particle
suspensions having particles of a defined size, particularly lipids
soluble in an aprotic solvent, for delivery of drugs having poor
aqueous solubility. U.S. Pat. No. 4,544,545 teaches phospholipid
liposomes having an outer layer including a modified cholesterol
derivative to render the liposome more specific for a preselected
organ. U.S. Pat. No. 5,213,804 teaches liposome compositions
containing an entrapped agent, such as a drug, which are composed
of vesicle-forming lipids and 1 to 20 mole percent of a
vesicle-forming lipid derivitized with hydrophilic biocompatible
polymer and sized to control its biodistribution and recirculatory
half life. U.S. Pat. No. 5,246,707 teaches phospholipid coated
microcrystalline particles of bio-active material to control the
rate of release of entrapped water soluble biomolecules, such as
proteins and polypeptides. U.S. Pat. No. 5,158,760 teaches liposome
encapsulated radio-active labeled proteins, such as hemoglobin.
[0006] The use of magnetic resonance imaging contrast enhancement
agents or radioactive isotopes in the body is practiced by a
variety of methods. U.S. Pat. No. 5,135,737 teaches magnetic
resonance imaging enhancement agents of paramagnetic metal ion
chelates attached to polymers such as polyamine based molecules
with antibodies attached for concentration at desired sites in the
body. U.S. Pat. Nos. 4,938,947 and 5,017,359 teach an aerosol
composition containing soluble fragments of bacterial wall or cell
peptidoglycan which may be labeled with a paramagnetic element and
encapsulated in liposomes which may be administered as an aerosol.
U.S. Pat. No. 5,078,986 teaches magnetic resonance imaging agents
of a chelate of a paramagnetic element carried by or within the
external surface of a liposome and released at a desired organ or
tissue site. PCT Publication Number WO 92/21017 teaches specific
liposomes complexed with paramagnetic ions to prolong their blood
pool half life and control magnetic resonance relaxivity. Liposomes
as MR contrast agents has been reviewed by Unger, E. C., Shen, D.
K., and Fritz, T. A., Status of Liposomes as MR Contrast Agents,
JMRI, 3, 195-198, (1993).
[0007] The need for recirculation of paramagnetic contrast agents
in the body, that is avoidance of rapid endocytosis by the
reticuloendothelial system and avoidance of rapid filtration by the
kidney, to provide sufficient concentration at a targeted site to
afford necessary contrast has been recognized. The use of small
molecules, such as gadolinium diethylenetriaminepentaacetic acid,
is restricted due to rapid renal excretion while most liposomes,
having diameters >800 nm, are quickly cleared by the
reticuloendothelial system. Attempts to solve these problems have
involved use of macromolecular materials, such as gadolinium
diethylenetriaminepentaacetic acid derived polysaccharides,
polypeptides, and proteins. These agents have not achieved the
versatility in chemical modification to provide for both long
recirculation times and active targeting.
[0008] Prior attempts to construct bifunctional, ligand-bearing
magnetic resonance contrast agents have not been satisfactory due
to insufficient sensitivity, poor target specificity and lack of
characterization. Gore, J. C. and Smith, F. W., Special Issue:
Contrast Agents, Magn. Reson. Img., 3, 1-97, (1985); Hasso, A. N.
and Stark, D. D., Special Issue: Contrast Agents, JMRI, 3, 137-310,
(1993); and Wehrli, F. W., SMRM Workshop: Contrast Enhanced
Magnetic Resonance, Magn. Reson. Med., 22, 177-378, (1991).
[0009] Receptor-directed contrast agents for MRI have been
attempted using iron oxide particles, but the chemistry and
characterization of the particle has been poorly defined and thus
it has been difficult to achieve control over non-specific
adhesion, blood pool half life and the versatility for both T1 and
T2* imaging modes. In addition, no radioisotope imaging is possible
using these iron-based agents which further limits their
usefulness. Reimer, P., Weissleder, R., Brady, T. J., Baldwin, B.
H., Tennant, B. C., and Wittenberg, J., Experimental Hepatocellular
Carcinoma: MR Receptor Imaging, Radiology, 180, 641-645 (1991),
Reimer, P., Weisslender, R., Lee, A. S., and Brady, T. J., Receptor
Imaging: Application to MR Imaging of Liver Cancer, Radiology, 177,
729-734 (1990), Reimer, P., Weissleder, R., Wittenberg, J., and
Brady, T. J., Receptor-birected Contrast Agents for MR Imaging:
Preclinical Evaluation With Affinity Assays, Radiology, 182,
565-569 (1992), and Weissleder, R., Reimer, P., Lee, A. S.,
Wittenberg, J. and Brady, T. J., MR Receptor Imaging: Ultrasmall
Iron Oxide Particles Targeted to Asialoglycoprotein Receptors, AJR,
155, 1161-67, (1990).
[0010] Antibody MR imaging has been described by Unger, E. C.,
Totty, W. G., Neufeld, D. M., Otsuka, F. L., Murphy, W. A., Welch,
M. S., Connett, J. M., and Philpott, G. W., Magnetic Resonance
Imaging Using Gadolinium labeled Monoclonal Antibody, Invest.
Radiol., 20, 693-700. (1985), and Weissleder, R., Lee, A. S.,
Fischman, A. J., Reimer, P., Shen, T., Wilkinson, R., Callahan, R.
J., and Brady, T. J., Polyclonal Human Immunoglobulin G Labeled
with polymeric Iron oxide: Antibody MR Imaging, Radiology, 181,
245-249, (1991). In the former case, one is limited by the amount
of contrast enhancement that can be achieved by direct attachment
of chelator to an antibody. In the latter case, the iron oxide
particle is not amenable to control over surface functionality
needed to reduce non specific adhesion and the particle is not well
characterized or well tolerated in vivo.
[0011] The economic driven requirement for improved in vitro
diagnostic techniques for medicine is also well recognized. Hannon,
Robert E., Future Practices in Diagnostic Medicine, Arch Pathol Lab
Med; Vol 119, pg 890-893 (October 1995) A common technique
presently used in diagnostic medicine for detection of the presence
of specific antigens in solution is addition of latex beads coated
with antibodies to the solution and detection of micro-agglutinated
products, as described in Microparticle Immunoassay Techniches, 2nd
Ed., Seradyn, Inc., Particle Technology Division, P.O. Box 1210,
Indianapolis, Ind. 46206 (1993) and U.S. Pat. Nos. 4,801,504 and
5,053,443. However, detection of micro-agglutinated products,
approximately 1 .mu.m in size, is very difficult.
[0012] Currently used in vitro enzyme linked immunoassays (ELISA)
have a sensitivity in the order of 1 picomolar concentration (0.5
.mu.g/10 mL). Other in vitro assay technologies, including
radioactive immunoassay systems, have sensitivities 2 to 3 orders
of magnitude more sensitive than ELISA assays. While polymerase
chain reaction (PCR) based technologies have the technology is
limited to detection of nucleic acids.
[0013] The expression of glycoproteins on a cell surface is
currently detected using assays requiring multiple steps and
frequently resulting in low sensitivity. For example, for assays of
protein expression on activated endothelial cells, a first step
involves the use of an antibody against the cell surface protein
followed by multiple steps to amplify the ability for detection of
the resulting complexes using flourescent techniques, such as, for
example, fluorescent antivated cell flow cytometry, fluorescent
antivated cell sorting, and fluorescent microscopy.
[0014] It has been recognized that unique proteins called cell
adhesion molecules (CAMs) are expressed by endothelial cells during
a variety of physiological and disease processes. Reisfeld, R. A.,
Monoclonal Antibodies in Cancer Immunotherapy, Laboratory
Immunology II, Vol. 12, No. 2, pgs. 201-216, (June 1992) and
Archelos, J. J., Jung, S., Maurer, M., Schmied, M., Lassmann, H.,
Tamatani, T., Miyasaka, M., Toyka, K. V. and Hartung, H. P.,
Inhibition of Experimental Autoimmune Encephalomylitis by the
Anitbody to the Intercelluler Adhesion Molecule ICAM-1, Ann. of
Neurology, Vol. 34, No. 2, pgs. 145-154 (1993) Multiple endothelial
ligands and receptors, including CAMs, are known to be upregulated
during various pathologies, such as inflammation and neoplasia.
Currently, the evaluation of the pathophysiology of the cell
adhesion molecules is generally limited to in vitro assays.
SUMMARY OF THE INVENTION
[0015] This invention, in one embodiment, relates to nanoscale
polymerized liposome particles based upon lipids having a
polymerizable functional group and a metal chelator to attach an
imaging enhancement agent, such as paramagnetic or radioactive
ions, which assemble to form imaging enhancement polymerized
liposomes. In preferred embodiments, the imaging enhancement
polymerized liposomes are derivatized with antibodies and/or
ligands for in vivo binding to cell surface receptors of targeted
cells. In particular, these receptors can be located on the
endothelium which eliminates the need for distribution of the
active agent out of the blood pool. Paramagnetic polymerized
liposomes according to this invention have been found to be well
tolerated by rabbits, mice and rats, even on repeated
administration, and effectively recirculate in the bloodstream,
avoiding rapid endocytosis by the reticuloendothelial system. These
materials provide good magnetic resonance imaging signal
enhancement of targeted cells, liver and kidney, for long periods
of time, of 90 minutes and more.
[0016] The polymerized liposomes of this invention are stable in
vivo and provide for effective control of particle size, surface
functionality, active ion density and water accessibility to
maximize their effective relaxivity for T1 and T2* magnetic
resonance imaging enhancement of specific biological systems. For
example, the polymerized liposomes of this invention may have a
plurality of metal ions for high relaxivity per particle providing
highly effective magnetic resonance imaging enhancement and may
also have attached antibodies or ligands specific for cellular
receptors, resulting in a sensitive probe for areas of vascular
tissue expressing these cell surface molecules. Receptors of
protein adhesins on the endothelium surface are of particular
interest in this targeting scheme because they are expressed
extensively during pathological processes of inflammation for the
recruitment of leukocytes or in the process of angiogenesis for
vascularization of diseased tissue, such as tumors. Targeted
polymerized liposomes provide for in vivo magnetic resonance
imaging histology that enables early evaluation of changes in the
endothelium in disease processes due to the attachment of a high
concentration of paramagnetic or superparamagnetic ions to specific
receptors on specifically targeted tissue or endothelium of
concern.
[0017] This invention provides various methods for in vitro assays.
For example, antibody-conjugated polymerized liposomes, according
to this invention, provide an ultra-sensitive diagnostic assay for
specific antigens in solution. Polymerized liposomes of this
invention having a chelator head group chelated to
spectroscopically distinct ions provide high sensitivity for enzyme
linked immunoassays. Polymerized liposomes of this invention having
a fluorophore head group provide a method for detection of
glycoproteins on cell surfaces.
[0018] In one embodiment of this invention, a targeting polymerized
liposome particle comprises: an assembly of a plurality of liposome
forming lipids each having an active hydrophilic head group linked
by a bifunctional linker portion to the liposome forming lipid, and
a hydrophobic tail group having a polymerizable functional group
polymerized with a polymerizable functional group of an adjacent
hydrophobic tail group of one of the plurality of liposome forming
lipids, at least a portion of the hydrophilic head groups having an
attached targeting active agent for attachment to a specific
biological molecule. In another embodiment, the targeting
polymerized liposome particle has a second portion of the
hydrophilic head groups with functional surface groups attached to
an image contrast enhancement agent to form a targeting image
enhancing polymerized liposome particle. In yet another embodiment,
a portion of the hydrophilic head groups have functional surface
groups attached to or encapsulating a treatment agent for
interaction with a biological site at or near the specific
biological molecule to which the particle attaches, forming a
targeting delivery polymerized liposome particle or a targeting
image enhancing delivery polymerized liposome particle.
[0019] This invention provides a method of assaying abnormal
pathology in vitro comprising, introducing a plurality of targeting
polymerized liposome particles targeted to a molecule involved in
the abnormal pathology into a fluid contacting the abnormal
pathology, the targeting polymerized liposome particles attaching
to a molecule involved in the abnormal pathology, and detecting in
vitro the targeting polymerized liposome particles attached to
molecules involved in the abnormal pathology.
[0020] This invention also provides a method of diagnosing abnormal
pathology in vivo comprising, introducing a plurality of targeting
image enhancing polymerized particles targeted to a molecule
involved in the abnormal pathology into a bodily fluid contacting
the abnormal pathology, the targeting image enhancing polymerized
particles attaching to a molecule involved in the abnormal
pathology, and imaging in vivo the targeting image enhancing
polymerized particles attached to molecules involved in the
abnormal pathology.
[0021] This invention further provides a method of therapeutic
treatment comprising, introducing into a bodily fluid contacting an
area of desired treatment a plurality of targeting delivery
polymerized liposome particles targeted to a molecule at or near
the site of desired treatment and having a desired therapeutic
agent attached or encapsulated, the targeting delivery polymerized
liposome particles attaching to molecules at or near the site of
desired treatment rendering the therapeutic agent available at the
site of desired treatment.
[0022] Further details of preparation and use of the targeting
paramagnetic polymerized liposomes of this invention in magnetic
resonance imaging is described in: Storrs, R. W., Tropper, F. D.,
Li, H. Y., Song, C. K., Kuniyoshi, J. K., Slpkins, D. A., Li, K. C.
P. and Bednarski, M. D., Paramagnetic Polymerized Liposomes:
Synthesis, Characterization, and Applications for Magnetic
Resonance Imaging, J. Am. Chem. Soc., Vol. 117, No. 28, pgs.
7301-7306, (Aug. 1995) and Storrs, R. W., Tropper, F. D., Li, H.
Y., Song, C. K., Sipkins, D. A., Kuniyoshi, J. K., Bednarski, M.
D., Strauss, H. W. and Li, K. C. P., Paramagnetic Polymerized
Liposomes as New Recirculating MR Contrast Agents, JMRI, Vol. 5,
No. 6, pgs.719-724, (November/December 1995), which publications
are incorporated herein by reference in their entireties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The invention will be described in detail with reference to
the drawings wherein:
[0024] FIG. 1 schematically shows the action of targeted
paramagnetic polymerized liposomes according to this invention;
[0025] FIGS. 2 and 3 schematically show polymerizable lipid
molecules according to one embodiment of this invention;
[0026] FIG. 4 shows the synthesis of a metal chelated lipid
according to one embodiment of this invention;
[0027] FIGS. 5. and 6 show formation of paramagnetic polymerized
liposomes from the metal chelated lipid shown in FIG. 4 with filler
lipids DAPC, DAPE or PDA according to one embodiment of this
invention;
[0028] FIG. 7 is a transmission electron micrograph of polymerized
liposome particles;
[0029] FIG. 8 is an atomic force micrograph in color of polymerized
liposome particles;
[0030] FIG. 9 shows the synthesis of biotinylated paramagnetic
chelated lipids according to one embodiment of this invention;
[0031] FIGS. 10 and 11 show formation of biotinylated paramagnetic
polymerized liposomes using PDA and DAPC or DAPE;
[0032] FIG. 12 shows formation of paramagnetic polymerized
liposomes having positively charged functional groups;
[0033] FIG. 13 shows formation of paramagnetic polymerized
liposomes having negatively charged functional groups;
[0034] FIG. 14 shows formation of paramagnetic polymerized
liposomes having zwitter ionic functional groups;
[0035] FIG. 15 shows formation of paramagnetic polymerized
liposomes having lactose targeting groups;
[0036] FIG. 16 schematically shows formation of paramagnetic
polymerized liposomes having antibodies attached;
[0037] FIGS. 17 and 18 show formation of liposomes that can be used
for direct attachment of oxidized antibodies by an amine via
reductive amination and hydrazone formation via alkyl
hydrazine;
[0038] FIGS. 19-21 are magnetic resonance images of rat livers as
described in Example III;
[0039] FIGS. 22-24 are magnetic resonance images of rat kidneys as
described in Example III;
[0040] FIG. 25 is a graph showing average enhancement of magnetic
resonance image intensity in rat kidneys versus time;
[0041] FIG. 26 is a graph showing average enhancement of magnetic
resonance image intensity in rat livers versus time;
[0042] FIG. 27 is a graph showing the ratio of enhancement shown in
FIGS. 25-26 of kidneys to liver, relative to precontrast
enhancement versus time;
[0043] FIG. 28 is a schematic showing of an antibody-conjugated
paramagnetic polymerized liposome as prepared in Example VIII;.
[0044] FIG. 29 is a photograph in color of gel electrophoresis
using anti-avidin alkaline phosphatase as described in Example
IX;
[0045] FIG. 30 is a photograph in color of gel electrophoresis
using anti-IgG alkaline phosphatase as described in Example IX;
[0046] FIG. 31 is a fluorescence micrograph in color showing cell
binding of fluorescent antibody-conjugated paramagnetic polymerized
liposomes as described in Example X;
[0047] FIG. 32 shows schematically the cell binding shown in FIG.
31;
[0048] FIG. 33 is a fluorescence micrograph in color of mouse
cerebellum showing anti-ICAM-1 antibody-conjugated polymerized
liposomes bound to capillaries as described in Example XI;
[0049] FIG. 34 is a magnetic resonance image of a brain slice of an
experimental autoimmune encephalitis mouse without injection of
polymerized liposomes as described in Example XII;
[0050] FIG. 35 is a magnetic resonance image of a brain slice of an
experimental autoimmune encephalitis mouse injected with
anti-ICAM-1 antibody-conjugated paramagnetic polymerized liposomes
as described in Example XII;
[0051] FIG. 36 is a magnetic resonance image of a brain slice of a
healthy mouse injected with anti-ICAM-1 antibody-conjugated
paramagnetic polymerized liposomes as described in Example XII;
and
[0052] FIG. 37 is a bar chart showing magnetic resonance image
intensity measurements as described in Example XII.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0053] The polymerized liposomes of this invention are
self-assembled aggregates of lipid molecules which offer great
versatility in particle size and surface chemistry. The size of the
polymerized liposomes can be controlled by extrusion. The
polymerized liposomes can be a mixture of lipids which provide
different functional groups on the hydrophilic exposed surface. For
example, some hydrophilic head groups can have functional surface
groups, for example, biotin, amines, cyano, carboxylic acids,
isothiocyanates, thiols, disulfides, .alpha.-halocarbonyl
compounds, .alpha.,.beta.-unsaturated carbonyl compounds and alkyl
hydrazines for attachment of targeting active agents, such as
antibodies, ligands, proteins, peptides, carbohydrates, vitamins,
drugs, and combinations of these materials to form antigenic
determinants for specific targeting and attachment to desired cell
surface molecules. Other such head groups may have an attached or
encapsulated treatment agent, such as, for example, antibodies,
hormones and drugs for interaction with a biological site at or
near the specific biological molecule to which the polymerized
liposome particle attaches. Other hydrophilic head groups can have
a functional surface group of diethylenetriamine pentaacetic acid,
ethylenedinitrile tetraacetic acid, tetraazlocyclododecane
1,4,7,10-tetraacetic acid, porphoryin chelate and
cyclohexane-1,2,-diamino-N,N'-diacetate for attachment to an image
contrast enhancement agent, such as, specifically, a
lanthanide-diethylenetriamine pentaacetic acid chelate for coupling
a metal which provides for the paramagnetism and magnetic resonance
contrast properties or for chelation of radioactive isotopes or
other imaging materials.
[0054] These lipids can be combined in varied proportions to
produce image enhancing paramagnetic or radioactive polymerized
liposomes with a broad spectrum of chemical and biological
properties. The magnetic resonance imaging R1 and R2* relaxivities
can be controlled by the nature of the metal chelate and the
distance of the metal from the surface of the particle. The
hydrophobic tail groups of the lipids are derivatized with
polymerizable groups, such as diacetylene groups, which
irreversibly cross-link, or polymerize, when exposed to ultaviolet
light or other radical, anionic or cationic, initiating species,
while maintaining the distribution of functional groups at the
surface of the liposome. The resulting polymerized liposome
particle is stabilized against fusion with cell membranes or other
liposomes and stabilized towards enzymatic degradation. In this
manner, many thousands of active lanthanide ions or radioisotopes
may be attached to one particle that may also bear several to
hundreds of ligands for in vivo adherence to targeted surfaces. For
T1 contrast agents the polymerized liposomes suitably have about 7
to about 30 percent metal chelating lipids, while for T2* contrast
agents the polymerized liposomes have about 50 to about 99 percent
metal chelating lipids. The large number of lanthanide ions renders
the paramagnetic polymerized liposomes of this invention very
sensitive magnetic resonance contrast agents with high R1 and R2*
molar relaxivities and high ion concentration while the multiple
ligand binding sites improves in vivo binding affinity and
specificity. This improved binding can also be utilized
therapeutically to block cell adhesion to endothelial receptors in
vivo. Blocking these receptors can be useful to control
pathological processes, such as inflamation and control of
metastatic cancer. For example, multi-valent sialyl Lewis X
derivatized liposomes can be used to block neutrophil binding and
antibodies against VCAM-1 on polymerized liposomes can be used to
block lymphocyte binding, for example T-cells.
[0055] FIG. 1 schematically shows the action of the polymerized
liposome contrast agents of this invention. Polymerized liposome
contrast agent core 50 has attached to its exterior surface
contrast ions 51 for imaging enhancement, such as Gd.sup.3+ for T1
MRI agents, Dy.sup.3+ for T2* MRI agents and Tc or In ions for
radioisotope imaging, and targeting groups 52, such as antibodies
and ligands, tailored for attachment to cell surface molecules 53,
such as receptors, ligands and antigenic determinants.
[0056] Suitable image enhancing polymerized liposomes for use in
this invention are those in which a contrast agent, paramagnetic
ion or radioisotope, is provided at the surface of the particle.
Preferably, the particle also contains groups to control
nonspecific adhesion and reticuloendothelial system uptake with an
agent to target the particle to areas of pathology related to
changes in the endothelium, providing identification of changes in
the endothelium during disease and to sequester the liposome in
these areas without the need for the particle to leave the
circulatory system. The polymerized liposomes of this invention
provide: controlled surface functionality and particle rigidity to
prolong blood pool half life and retain the particle in the
circulatory system, as desired; a targeting group, such as a ligand
or antibody, to direct the particle to the desired region of
interest; and a contrast enhancement material, such as a
paramagnetic ion for MRI, a radioisotope, such as Tc or In, for
radioisotope imaging, or a heavy metal, such as lead or barium, for
standard x-ray analysis or a chromophore for optical imaging, to
detect the presence of the particles in vivo.
[0057] The component lipids of the polymerized liposomes of this
invention may be purified and characterized individually using
standard, known techniques and then combined in controlled fashion
to produce the final particle. The polymerized liposomes of this
invention can be constructed to mimic native cell membranes or
present functionality, such as ethylene glycol derivatives, that
can reduce their potential immunogenicity. Additionally, the
polymerized liposomes of this invention have a well defined bilayer
structure that can be characterized by known physical techniques
such as transmission electron microscopy and atomic force
microscopy.
[0058] FIGS. 2 and 3 schematically show a polymerizable lipid
molecule for use in this invention. The amphiphilic lipid molecule
has a polar head group 60 and a hydrophobic tail group 61. The tail
portion of the lipid has a polymerizable functional group 62, such
as diacetylene, olefins, acetylenes, nitriles, alkyl styrenes,
esters, thiols, amides and .alpha.,.beta. unsaturated carbonyl
compounds forming liposomes that will polymerize upon irradiation
an electromagnetic source, such as, with UV light or by chemical or
thermal means. FIG. 2 shows polymerizable functional groups which
may be located at specific positions A, B and C on tail group 61.
As shown in FIG. 3, the head group and tail group are joined by
variable length linker portion 63. The length of the linker
portion, indicated by m, controls the distance of the active agent
from the surface of the particle to make it more available for its
active function. The linker portion may be a bifunctional aliphatic
compounds which can include heteroatoms or bifunctional aromatic
compounds. Preferred linker portions are compounds such as, for
example, variable length polyethylene glycol, polypropylene glycol,
polyglycine, bifunctional aliphatic compounds, for example amino
caproic acid, or bifunctional aromatic compounds. The head group
has a functional surface group 64, such as diethylenetriamine
pentaacetic acid (DPTA), ethylenedinitrile tetraacetic acid (EDTA),
tetraazocyclododecane 1,4,7,10-tetraacetic acid (DOTA),
cyclohexane-1,2- diamino-N,N'-diacetate (CHTA) for chelating a
paramagnetic or radioactive intensifying agent for contrast
enhancement, or biotin, amines, carboxylic acids and alkyl
hydrazines for coupling biologically active targeting agents, such
as ligands, antibodies, peptides or carbohydrates for specific cell
surface receptors or antigenic determinants.
[0059] Generally, the lipids suitable for use in this invention
have: an active head group, for at least one of targeting, image
contrast enhancement and/or for treatment, a linker portion for
accessibility of the active head group; a hydrophobic tail for
self-assembly into liposomes; and a polymerizable group to
stabilize the liposomes.
[0060] A unique lipid is synthesized containing pentacosadiynoic
acid conjugated to diethylenetriamine pentaacetic acid via a
variable length polyethylene glycol linker as shown in FIG. 4.
These amphipathic molecules have metal chelates as head groups
connected to a lipid tail which contains a polymerizable
diacetylene moiety. The linker length can be controlled by the
choice of commercially available variable length polyethylene
glycol derivatives.
[0061] Specifically, we have synthesized compounds, such as shown
in FIG. 4, by reacting the NHS ester of the lipid pentacosadiynoic
acid (PDA) with triethyleneglycol-diamine and
tetraethyleneglycol-diamine linkers to form the corresponding
PEG.sub.m-PDA amides, m=1 or 2, then reacting the PEG.sub.m-PDA
amide with diethylenetriamine pentaacetic acid dianhydride (DTPAA)
to form diethylenetriamine pentaacetic acid-bis(tri or
tetraethylene glycol-pentacosadiynoic acid) diamide
(DTPA-bis-(PEG.sub.m-PDA), m=1 or 2 diamide). The diamide is then
treated with a metal ion source M, such as gadolinium trichloride,
dysprosium trichloride or a technicium or indium derivative to form
the amphiphilic metal chelate as shown in FIG. 4 with a
polyethylene linker (m=1 and m=2). The diamide-lanthanide chelate,
shown in FIG. 4 and as a reactant in FIG. 5, is mixed with a matrix
lipid of diacetylenic choline (DAPC, R.dbd.CH.sub.3) or
diacetylinic ethanolamine (R.dbd.H), shown in FIG. 5,
pentacosadiynoic acid (PDA) or derivatives of PDA in an amount to
result in the desired surface density of contrast agent on the
polymerized liposomes. The matrix lipid forms polymerizable
liposomes under a variety of conditions and closely mimics the
topology of in vivo cell membranes.
[0062] To form the paramagnetic polymerized liposome shown as the
product in FIGS. 5 and 6, the metal chelated diamide shown in FIG.
4 is doped into the DAPC, as shown in FIG. 5, or PDA, as shown in
FIG. 6, matrix in organic solvent. The organic solvent is
evaporated and the dried lipid film is hydrated to a known lipid
density, such as 15 mM total lipid, with the desired buffer or
water. The resulting suspension is sonicated at temperatures above
the gel-liquid crystal phase transition for DAPC or PDA,
T.sub.m.apprxeq.40.degree. C., with a probe-tip sonicator. A nearly
clear, colorless solution of emulsified vesicles, or liposomes, is
produced. We have determined by transmission electron microscopy
and atomic force microscopy that these liposomes are on average 30
to 200 nm in diameter. Their size can be reduced by extrusion at
temperatures greater than T.sub.m through polycarbonate filters
with well defined porosity. The liposomes are polymerized by
cooling the solution to 4.degree. C. on a bed of ice and
irradiating at 254 nm with a UV lamp. Alternatively, the liposomes
can be irradiated at room temperature and then cooled while
continuing UV irradiation. The resulting paramagnetic polymerized
liposomes, diagramatically shown as the products in FIGS. 5 and 6,
are orange in color when using DAPC with two visible absorption
bands centered at 490 nm and 510 nm arising from the conjugated
ene-yne diacetylene polymer and generally blue in color when using
PDA with absorption bands around. 540 nm and 630 nm. These
liposomes can undergo a blue to red transition when molecules bind
to their surface after heating or resonication or after standing at
room temperature for extended times or being treated with organic
solvents. This transition may be useful for developing a detection
system for these conditions.
[0063] We have constructed paramagnetic polymerized liposomes using
the above techniques with 2.5 to 100 mol % of the gadolinium
chelated diamide shown in FIG. 4, with the polyethylene glycol
linker m=1 and m=2, and sizes ranging from 30 nm to 200 nm in
diameter. We have determined the maximum T1 relaxivity, showing the
best contrast, is obtained with 15% gadolinium chelated diamide and
85% DAPC with m=2 and having 200 nm particle size. High relaxivity
is also observed with 30% gadolinium chelated diamide and 70% PDA
with m=1 and having a variable particle size of about 10 to about
200 nm. Results of a variey of these measurements using DAPC matrix
lipid are shown in Tables 1 and 2.
1TABLE 1 Gadolinium-diamide (%) Size (nm) R1 (s.sup.-1mM.sup.-1) R2
(s.sup.-1mM.sup.-1) 5.7 (m = 1) 200 5.7 Not Det. 5.7 (m = 2) 200
8.3 Not Det. 10 (m = 2) 200 9.2 Not Det. 15 (m = 2) 200 14.6 Not
Det. 20 (m = 2) 200 8.9 Not Det. 30 (m = 2) 200 7.7 Not Det. 10 (m
= 2) 100 10.9 16.0 10 (m = 2) 80 9.6 Not Det. 10 (m = 2) 50 8.6
18.3 10 (m = 2) 30 7.8 19.2 Gd (DTPA) Magnevist, 4.4 1.9 Lab.,
Wayne N.J. Berle
[0064] To demonstrate the dependence on linker length, it is seen
from Table 1 that when m=2 (R1=8.3) the metal ion appears to be
suspended off the surface of the polymerized liposome allowing
greater aqueous accessibility and hence greater relaxation than
when m=1 (R1=5.7).
[0065] Similar measurements were made using PDA liposomes as the
matrix lipid and the gadolinium chelated diamide (m=1). The results
are shown in Table 2.
2 TABLE 2 Gadolinium-diamide (%) R1 (s.sup.-1mM.sup.-1) 10 8.86 30
8.67 50 4.34 50* 4.19 100 3.4 *1% biotin-DAPE
[0066] It is seen from Table 2 that liposome formulations of 10%
and 30% metal chelator diamide and 90% and 70% PDA, respectively,
exhibited the highest relaxivity of over 8 mM.sup.-1sec.sup.-1,
while formulations of 50, and 100% metal chelator diamide had lower
relaxivities. It is desired that the paramagnetic polymerized
liposomes of this invention have a long half life in the
recirculating blood pool to find their desired targeted receptors
in vivo. To aid in retention in the blood pool, the overall size of
the paramagnetic polymerized liposomes can be controlled by
extrusion to reduce elimination from the blood pool by the
reticuloenthelial system. Additionally, the surface chemistry of
the polymerized liposomes can be modified to evade the hepatica and
immune systems, for example, liposomes derivatized with
polyethylene glycol decrease the rate of elimination by the
reticuloendothelial system. Particle rigidity can also be
controlled by the polymerization time and method which modifies
recirculation time.
[0067] In a similar manner as described above with respect to FIGS.
5 and 6, dysprosium chelated lipids may be used to construct T2*
untargeted or targeted paramagnetic polymerized liposomes according
to this invention. Dysprosium is a desirable metal for T2* contrast
since its magnetic susceptibility is the largest of any element and
it is easily incorporated into a diethylenetriamine pentaacetic
acid chelate. It may not be desired to use a matrix lipid to
separate the paramagnetic metal centers, as found desirable for T1
paramagnetic polymerized liposomes. The chelator lipid described in
FIG. 4 can be treated with dysprosium trichloride in sodium
bicarbonate to produce the Dy-diacetylene lipid having M=Dy.sup.+3
in FIG. 4. Single component paramagnetic polymerized liposomes can
be constructed from these compounds by sonication, extrusion and
polymerization in the manner described above, as shown in FIG. 6,
with x=0. Alternatively, the dysprosium lipid reactant can be doped
into DAPC, DAPE or PDA lipids at varying percentages.
[0068] For transmission electron microscopy (TEM), a polymerized
liposome dispersion was deposited by freeze-drying onto the sample
grid of the microscope and stained with osmium tetraoxide for 15
minutes. The micrograph shown as FIG. 7 was taken at a
magnification of 21000 times and shows the polymerized liposome
particles as ellipsoids having diameters of about 50 to 200 nm.
[0069] For Atomic Force Microscopy (AFM), samples were prepared by
covering freshly cleaved mica with a solution of paramagnetic
polymerized liposomes, 15 mM total lipid, for 1 to 2 minutes. The
solution was recovered by pipet and the mica surface rinsed with a
stream of distilled water. AFM images were obtained on an Explorer
Life Sciences model 200 (Topometrix, Santa Clara, Calif.). The AFM
was operated in the contact mode using the minimum force necessary
to prevent hopping of the cantilever tip. The raw images were
flattened either line-by-line or through a user-defined baseline
plane, as appropriate, using software supplied by Topometrix. The
paramagnetic polymerized liposomes, as shown in the AFM micrograph
of FIG. 8, were readily observed as flattened ellipsoids with
in-plane dimension similar to the 50 to 200 nm in diameter obtained
by transmission electron microscopy (TEM). With non-extruded
paramagnetic polymerized liposomes, smaller particles were more
abundant than observed by TEM due to the higher resolution of AFM
relative to TEM. The AFM provides more accurate sampling of
particle sizes than TEM due to its higher resolution. Confidence in
the uniformity of sampling particle sizes using AFM is enhanced
since forward and reverse scanned images appeared identical within
the resolution of the technique. We have found that AFM provides, a
simple and reliable method to assay particle sizes of the
paramagnetic polymerized liposomes of this invention.
[0070] We have found that the metal chelate lipid, such as DTPA, is
necessary to obtain images when mounting the sample on cleaved
mica. Polymerized lipids lacking the metal chelate lipid did not
produce AFM images using the above-described method. It is believed
that the metal chelate lipid serves as a unique functionality for
attachment of these materials to the mica, probably by chelating
DTPA to metals on the cleaved mica. The metal chelate lipid
molecule may be used to provide a unique functionality for
attachment of other biomolecules to the surface of mica for AFM
imaging.
[0071] Targeted paramagnetic polymerized liposomes were produced
from biotinylated or negatively charged liposomes to which
biotinylated antibodies are attached through avidin, which has a
high affinity for biotin and a high positive charge. In addition to
biotin-avidin crosslinking, antibody-avidin conjugates can be
attached to the polymerized liposome via charge-charge interactions
similar to ion exchange. Commercially available diacetylene
glycerophosphoethanolamine (DAPE) lipid is converted to its
biotinylated analog by acylation of the amine terminated lipid with
commercially available biotinylating agents, such as
biotinamidocaproate N-hydroxysuccinimide ester or paranitrophenol
esters, as shown in FIG. 9. The biotinylated paramagnetic
polymerized liposomes are produced by incorporating the
biotinylated lipid in an matrix of lipids of either PDA, DAPE or
DAPC as shown in FIGS. 10 and 11, respectively. Negatively charge
polymerized liposomes may be constructed by using pentacosadiynoic
acid as a matrix lipid.
[0072] This invention includes a broad based group of agents having
varied functionality which includes liposomes containing positively
charged groups, such as amines as shown in FIG. 12, negatively
charged groups, such as carboxylates as shown in FIG. 13, and
neutral groups, such as zwitterions as shown in FIG. 14. These
groups are important to control biodistribution blood pool
half-life and non-specific adhesion of the particles.
[0073] Targeting groups of polymerized liposomes according to this
invention may be ligands, such as carbohydrates, proteins, such as
antibodies, peptides, antigenic determinants, or other receptor
targeting groups. These head groups can be used to control the
biodistribution, non-specific adhesion, and blood pool half life of
the polymerized liposomes. For example, .beta.-D-lactose has been
attached on the surface, as shown in FIG. 15, to target the
aloglysoprotein (ASG) found in liver cells which are in contact
with the circulating blood pool. Targeting glycolipids are formed
by converting the commercially available lipid (DAGPE) or the
PEG-PDA amine shown in FIG. 4 into its isocyanate followed by
treatment with triethylene glycol diamine spacer to produce the
amine terminated thiocarbamate lipid which by treatment with the
para-isothiocyanophenyl glycoside of the carbohydrate ligand
produces the desired targeting glycolipids. This synthesis provides
a water soluble flexible linker molecule spaced between the lipid
that will form the internal structure or core of the liposome and
the ligand that binds to cell surface receptors, allowing the
ligand to be readily accessible to the protein receptors on the
cell surfaces. The carbohydrate ligands can be derived from
reducing sugars or glycosides, such as para-nitrophenyl glycosides,
a wide range of which are commercially available or easily
constructed using chemical or enzymatic methods. Paramagnetic
polymerized liposomes coated with carbohydrate ligands can be
produced by mixing appropriate amounts of individual lipids
followed by sonication, extrusion and polymerization and filtration
as described above and shown in FIG. 15. Suitable carbohydrate
derivatized paramagnetic polymerized liposomes have about 1 to
about 30 mole percent of the targeting glycolipid and filler lipid,
such as PDA, DAPC or DAPC, with the balance being metal chelated
lipid. Other lipids may be included in the polymerized liposomes to
assure liposome formation and provide high contrast and
recirculation.
[0074] Antibodies may be attached to the particle by the
biotin-avidin biotinylated antibody sandwich, as shown in FIG. 16,
to allow a variety of commercially available biotintylated
antibodies to be used on the polymerized liposome particles of this
invention.
[0075] Biotinylated paramagnetic polymerized liposomes with a
biotinylated anti-VCAM-1 antibody attached via a biotin avidin
sandwich were produced in the manner described above. This targeted
paramagnetic polymerized liposome binds to VCAM-1, a leukocyte
adhesion receptor on the endothelial surface which is upregulated
during inflammation. In vitro histology demonstrated specific
interactions between the polymerized liposomes and the inflammed
brainstem tissue from a mouse with allergic autoimmune
encephalitis. The formation of such biotinylated antibody coated
polymerized liposomes and their attachment to in vivo cell
receptors is schematically shown in FIG. 16. As shown in FIG. 16,
the biotinylated antibody 70 having functional group 74 is attached
to the biotinylated lipid surface 71 through bridge 72 of avidin or
streptavidin to form antibody coated polymerized liposomes 73. The
functional group 74 of antibody 70 is attached in vivo to an
endothelium receptor 75, thereby attaching the, paramagnetic
polymerized liposome to the endothelium for external detection.
[0076] Antibodies may also be attached by "direct" methods. In
particular, wherein the liposome contains a group, such as an amine
or hydrazine derivatives, that reacts with aldehydes on oxidized
antibodies and olgosaccharides. We have constructed liposomes
containing amine, FIG. 17, and hydrazine, FIG. 18, head groups for
this purpose. Antibodies can also be attached by charge-charge
interaction such as ion exchange. In this case, the antibody is
bound to a positively charged protein, such as, for example, avidin
and this complex ion exchanged onto negatively charged polymerized
liposomes.
[0077] Although the embodiments of this invention using image
intensifiers have been described and specifically exemplified
primarily with respect to polymerized liposomes having an attached
metal for magnetic resonance imaging, it should be clear to one
skilled in the art that other detection materials may be attached
in a similar manner, such as a radioisotope for radioisotope
imaging, a heavy metal for x-ray imaging, or a chromophore for
optical imaging and are meant to be included in this invention. We
have attached Indium to paramagnetic polymerized liposomes, as
described in Storrs, Richard W., et al, JMRI, (1995) incorporated
herein by reference, supra. Likewise, in other embodiments, any
suitable functional group may be attached to liposomes incorporated
into the polymerized liposomes of this invention to provide
attachment to specified targets, in vitro and in vivo, to obtain
concentration of the image contrast agent at the specified target
site.
[0078] We have found that use of antibody-conjugated polymerized
liposomes, according to this invention, provides an in vitro
ultra-sensitive diagnostic assay of the presence of specific
antigens in solution. Monoclonal antibodies are conjugated to the
surface of polymerized liposomes by combintion of avidin with
biotinylated antibody followed by addition of polymerized liposomes
to form antibody-conjugated polymerized liposomes. The size
distribution of these particles is then determined by photon
correlation spectroscopy (PCS), using for example, a Coulter N4+
submicron particle analyzer. The sample containing an antigen of
interest is added to the antibody-conjugated polymerized liposomes
and allowed to incubate in solution. The multivalent
antibody-conjugated polymerized liposomes recognize the antigen and
microscopic agglutination occurs and is detected by a change in
size distribution as detected by photon correlation
spectroscopy.
[0079] Polymerized liposomes of this invention having a chelator
head group chelated to spectroscopically distinct ions, such as
Europium, provide high sensitivity methods for ELISA based in vitro
assays which do not require radiochemistry. Europium labelled
polymerized liposomes of this invention, in an ELISA based system,
can be detected at concentrations of 10.sup.31 21 molar using
time-resolved flourscence spectroscopy.
[0080] The expression of glycoproteins on cell surfaces may be
detected according to this invention by use of polymerized
liposomes having a head group bearing a fluorophore. Polymerized
liposomes may be constructed according to this invention having a
negative charge so that they adhere to cell surfaces expressing
positively charged proteins. Pentacosadiynoic acid or other
carboxylic acid terminated lipids can be used for this purpose. In
similar manner, polymerized liposomes may be constructed having a
positive charge. In another embodiment, a lipid containing a
fluorophore head group, such as Texas Red, was constructed and
incorporporated into polymerized liposomes. Fluorophore head group
polymerized liposomes may be constructed by incorporating an amine
containing lipid, such as pentacosadiynoic
acid-(PEG).sub.4-NH.sub.2, into the liposome and having a
commercially available fluorophore attached to the liposome via the
amine.
[0081] Antibody-conjugated paramagnetic polymerized liposomes of
this invention achieve in vitro and in vivo targeting of specific
molecules associated with specific body tissues and specific
molecules associated with specific bodily functions and pathologies
to provide sufficient signal enhancement for detection by magnetic
resonance imaging. Such in vivo imaging of various disease or
developmentally associated molecules permits following the
relationship of these molecules to disease progression, their time
course of progression, and their response to pharmacologic
interventions. Characterization of these responses in individual
animals simplifies assessment of the interventions, since
expression and regression of the target can be confirmed as it
relates to disease outcomes. As a diagnostic tool, this technique
detects disease at early stages, thereby enabling more effective
treatment. The paramagnetic polymerized liposomes of this invention
are suitable for combination of imaging and delivery of drugs for
therapeutic treatments. Various agents can be encapsulated or
attached to the surface of polymerized Iiposomes for delivery to
specific sites in vivo. By using target-specific drug/paramagnetic
polymerized liposomes of this invention, the drug delivery can be
simultaneously visualized by magnetic resonance imaging.
[0082] The targeted paramagnetic polymerized liposomes of this
invention provide non-invasive in vivo investigation by providing
magnetic resonance imaging of tissues to visualize endothelial
antigens which characterize disease at the molecular level in
nearly real time. Endothelial cell adhesion molecules serve as a
suitable target for magnetic resonance imaging, since the early and
specific upregulation of vascular cell adhesion molecules occurs in
a variety of diseases,. Targeted paramagnetic polymerized liposomes
which recirculate in the vasculature may include endothelial
antigens which interact with the cell adhesion molecules to retain
a number of the the targeted paramagnetic polymerized liposomes at
the desired location. The high concentration of magnetic resonance
image enhancement agents in the polymerized liposomes of this
invention render possible in vivo non-invasive magnetic resonance
imaging of pathologic changes. To our knowledge, no magnetic
resonance image contrast enhancement agent specific for changes in
endothelial receptor expression have been described. The
polymerized liposomes of this invention are particularly well
suited since they maintain their integrity in vivo, recirculate in
the blood pool, are rigid and do not easily fuse with cell
membranes, and serve as a scaffold for attachment of both the
antibodies and the paramagnetic contrast ion for magnetic resonance
imaging. The size distribution, particle rigidity and surface
characteristics of the polymerized liposomes can be tailored to
avoid rapid clearance by the reticuloendothelial system and the
surface can be modified with ethylene glycol to further increase
intravascular recirculation times. We have found that the
paramagnetic polymerized liposomes have blood pool half-lives of
about 20 hours in rats.
[0083] In one embodiment, the site-specific paramagnetic
polymerized liposome having attached monoclonal antibodies for
specific receptor targeting may be used to visualize intercellular
adhesion molecule-1, ICAM-1, upregulation in murine experimental
autoimmune encephalitis, an animal model for multiple sclerosis.
Such an agent can also enable imaging of endothelial antigen
expression and regulation in vivo, permitting early detection and
treatment of diseases like multipe sclerosis in humans.
[0084] The following specific examples are set forth in detail to
illustrate the invention and should not be considered to limit the
invention in any way.
EXAMPLE I
[0085] Paramagnetic polymerizable lipids having Gd.sup.+3 and PDA
headgroups were synthesized by first preparing the succinimidyl
ester by stirring pentacosadiynoic acid (PDA, Lancaster; 10.0 g,
26.7 mmol), N-hydroxysuccinimide (NHS, Aldrich; 5.90 g, 43.4 mmol)
and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC, Aldrich; 6.01 g, 31.3 mmol) in 660 ml CH.sub.2Cl.sub.2 at
room temperature and shielded from light. The reaction was followed
by thin layer chromatography (CHCl.sub.3/MeOH; 8/1) and deemed
complete after approximately 5 hours. The solution was washed with
water, 1% HCl, saturated sodium bicarbonate and brine. The organic
phase was then dried with MgSO.sub.4, filtered, and concentrated
under reduced pressure to yield the N-succinimidyl
10,12-pentacosadiynoic acid ester as a slightly yellow solid (10.84
g; 23.0 mmol; 86%).
[0086] The succinimidyl ester was dissolved in CH.sub.2Cl.sub.2
(250 ml) and then slowly added, in dropwise fashion, to a stirred
solution of 1,11-diamino-3,6,9-trioxyundecane (9.13 g, 61.6 mmol;
Texaco) in CH.sub.2Cl.sub.2 (110 ml) over a 16 hour period at room
temperature and shielded from light. The resulting solution was
concentrated to a thick slurry and chromatographed on silica gel
using a gradient of CHCl.sub.3/MeOH (1/0 to 8/1). The homogeneous
fractions were pooled and evaporated under reduced pressure to
result in the desired lipid,
(1'-N-,11'-amino-3',6'-dioxyundecanoyl)-10,12-pentcosadiynamide, as
a white solid (4.40 g; 38.1%). This product must be handled with
care as it spontaneously polymerizes in the solid state when it is
pure. It is more stable in solution at 4.degree. C., but should be
used as soon as possible after preparation.
[0087] The above-prepared aminoamide (4.40 g; 8.78 mmol) and DTPAA
(1.56 g; 4.37 mmol) were stirred in pyridine (25 ml) overnight,
shielded from the light. The solvent was evaporated and the residue
coevaporated with methanol to dryness twice to result in an oil
free from pyridine.. The residue was dissolved in acetone and the
product allowed to precipitate from solution after overnight
storage at 4.degree. C. Filtration resulted in the desired chelator
lipid, bis-N-[2-ethyl-N-'carboxymethyl,N'-carboxy-
methyl(1'-N-'",11'-N""-3',6'-dioxyundecanoyl)amide-1",12"-pentacosadiynami-
de]-glycine, as a white amorphous powder (3.30 g; 55%). Further
purification can be achieved by crystallization from methanol
[0088] (40 mg/ml; m.p. 128.5-129.5.degree. C. (decomp.)).
[0089] The chelator lipid, as prepared above, was heated with
GdCl.sub.3,6H.sub.2O or DyCl.sub.3.6H.sub.2O (0.95-0.98 equiv.) in
methanol. The solvent was evaporated and the residue coevaporated
with methanol to remove all traces of generated HCl. The resulting
lanthanide chelate lipids,
bis-N-[2-ethyl-N-'carboxymethyl,N'-carboxymethyl(1'-N-'",-
11'-N""-3',6'-dioxyundecanoyl)amide-1",12"-pentacosadiynamide]-glycine-lan-
thanide,gadolinium or dysprosium, complexes, were then stored as
methanolic solutions at 4.degree. C., shielded from light. The
identity of the synthesized chelates was confirmed by FAB-MS.
[0090] Paramagnetic polymerized lipids were formed by mixing a 1:9
molar ratio of the above prepared paramagnetic polymerizible lipids
with di-tricosadiynoyl phosphatidyl choline (Avanti Polar Lipids,
Birmingham, Ala.) in an organic solvent methyl alcohol and
chloroform (1/3) and evaporating- the solvent and rehydrating with
distilled water to 30 mM diacetylene (15 mM total lipid). Following
sonication with a 450 W probe-tip sonicator (Virsonic 475, Virtis
Corp., Gardiner, N.Y.) set at a power setting of 21/2 units for 30
to 60 minutes without temperature control, the suspension of lipid
aggregates was extruded ten times through two polycarbonate filters
with pores of 0.1 .mu.m diameter (Poretics, Livermore, Calif.) at
56.degree. C. using a thermobarrel extruder (Lipex Biomembranes,
Vancover, BC). This solution was spread thinly on a petri dish in a
wet ice slush and irradiated with a UV lamp, 2200 .mu.Watt/cm.sup.2
held 1 cm over the solution while stirring. The solution turned
orange using DAPC over the course of a one hour irradiation, due to
the absorption of visible light by the conjugated ene-yne system of
the polymer. The paramagnetic polymerized liposomes passed easily
through a 0.2 .mu.m sterilizing filter and were stored in solution
until use. The paramagnetic polymerized lipid suspensions prepared
in this manner have been found to be stable for many weeks at
4.degree. C.
[0091] The size and shape of the paramagnetic polymerized liposomes
have been ascertained by transmission electron microscopy and by
atomic force microscopy, as shown in FIGS. 7 and 8. They appear as
prolate ellipsoids with minor axes on the order of the membrane
pore and major axes about 50 percent greater.
EXAMPLE II
[0092] The procedures of Example I were followed except that
instead of using DAPC, pentacosydiynoic acid (PDA) was used as the
filler lipid. The solution turned blue over the course of one hour
irradiation. The resulting polymerized liposomes had the same
general properties as reported in Example I.
EXAMPLE III
[0093] Two month old Lewis rats were anaesthetized either with
40-75 mg/Kg dose of sodium pentobarbitol i.p. or by 1.5% isoflurane
by inhalation. Paramagnetic polymerized liposome, as prepared in
Example I, was administered i.v. over. 60-90 seconds through a 24 G
catheter in a laterial tail vein at a dose of 0.015 mmol
Gd.sup.+3/kg body weight.
[0094] Axial magnetic resonance images of the abdomen were obtained
prior to paramagnetic polymerized liposome administration and
periodically for up to 2 hours post administration. All magnetic
resonance images were obtained using an OMEGA-CSI imager (GE,
Milwaukee, Wis.) at field strengths of 2.0 or 4.7 Telsa using the
standard spin-echo acquisition sequence. T1 weighted images were
obtained using a repetition time (TR) of 400 ms, echo time (TE) of
18 ms, and 2 excitations (NEX) per 128 phase encoding steps,
completing a 256.times.128 data matrix in under 2 minutes. Slice
thickness (ST) was 2 mm and the interslice gap was 2 mm. Four axial
slices were acquired in multislice mode, with the slice position
chosen so that the liver appeared in the first two slices and the
kidneys appeared in the fourth, most inferior slice. These images
were often supplemented by a second set of images interleaving the
first set. Two phantoms, test tubes containing 10 mM Ni(NO.sub.3)
or 1 mM GdCl.sub.3, were placed longitudinally beneath the rats and
were imaged concurrently to monitor instrumental variations. The
image intensity of the phantoms varied less than 5% in all of the
experiments.
[0095] Data analysis was performed using the program XCINEMA (Lucas
MRS Center, Stanford University, Stanford, Calif.) Region of
interests (ROI) were drawn conservatively within each organ, and
the intensity of the same region at each time point was measured.
The intensity data post contrast was normalized to the intensity of
the ROI prior to contrast administration and the normalized data
for each time point averaged across six experiments on four rats
each.
[0096] The injected paramagnetic polymerized liposomes were well
tolerated by the rats with no significant adverse effects observed.
The rats continued to gain weight in the days succeeding
administration and exhibited normal behavior and activity.
Hematuria was observed only in the first urination following
recovery from the anesthesia, likely as a result of osmotic shock
since the injection preparation contained no added salts. Repeated
administration of the paramagnetic polymerized liposome preparation
to the same rat did not affect tolerance or contrast
enhancement.
[0097] Representative magnetic resonance images of the rat liver
and kidneys are shown in FIGS. 19-21 and 22-24, respectively: prior
to administration shown in FIGS. 19 and 22; 5 minutes after
administration shown in FIGS. 20 and 23; and 60 minutes after
admistration shown in FIGS. 21 and 24. The increase in T1-weighted
signal intensity is readily apparent in both the liver and kidneys
and has been found to persist throughout a 90 minute period.
[0098] The average enhancement of magnetic resonance intensity for
ROIs within the kidney and liver for all six experiments are shown
in FIGS. 25 and 26, respectively. These data were not corrected for
the intensity variation of the phantoms. The kidneys enhanced an
average of 34% over 90 minutes, reaching a maximum of 45%
enhancement at about 30 minutes. The liver enhanced an average of
about 20% over 90 minutes, reaching a maximum of about 23% at about
5 to 40 minutes. FIG. 27 shows the ratio of enhancement, relative
to precontrast enhancement, of the kidneys to liver, showing the
time course of enhancement of these two organs to be similar,
indicating that the enhancement agent was not selectively
eliminated by either of these organs during the 90 minute
experimental time period. This indicates recirculation of the
enhancement agent in the blood pool of the rats.
[0099] This Example illustrates that the enhancement seen in the
liver and kidneys, both highly vascularized organs, is easily
visible even at a dose of 0.015 mmol Gd.sup.+3/kg, one tenth the
normal clinical dose of Gd-diethylenetriamine pentaacetic acid for
magnetic resonance imaging. The high magnetic resonance sensitivity
of the paramagnetic polymerized lipid preparation results from: (1)
The particulate nature of the polymerized lipid slows the
correlation time for reorientation of the Gd.sup.+3 ion, which
concentrates the power of the relaxation-effecting magnetic
fluctuations in the regime of the water proton Larmor frequency and
results in a higher molar relaxivity per Gd.sup.3 ion of 11.2
mM.sup.-1s.sup.-1, as compared with Gd-diethylenetriamine
pentaacetic acid of 4.2 mM.sup.-1s.sup.-1; and (2) The paramagnetic
polymerizied lipid particles are confined to the blood pool and do
not leak into the interstitial spaces, as does
Gd-diethylenetriamanine pentaacetic acid. The reduced volume of
distribution leads to a relatively increased blood pool
concentration of gadolinium for the paramagnetic polymerized
liposomes, as compared to a similar body weight dosage of
Gd-diethylenetriamine pentaacetic acid.
[0100] Extended recirculation of the paramagnetic polymerized
liposomes and their lack or absence of retention by the kidneys and
liver is evident from the prolonged magnetic resonance intensity
enhancement and the constant ratio of enhancement for these organs,
as compared to Gd-diethylenetriamine pentaacetic acid, which is
eliminated from the blood pool within a few minutes. The prolonged
recirculation of the paramagnetic polymerized liposomes reults from
reduction in phagocytosis by macrophages of the reticuloendothelial
system by selection and control of the particle size, particle
rigidity and, perhaps, by use of polyethylene linkers for
attachment of the Gd.sup.+3 ion. Evasion of the reticuloendothelial
system is probably complemented by evasion of the immune system by
use of surface groups, such as, for example, choline, which is the
major component of mammalian cells, as the matrix for presentation
of the paramagnetic and ligand-bearing paramagnetic polymerized
liposomes of this invention.
EXAMPLE IV
[0101] Antibodies towards the specific immunoglobin, anti-goat
.gamma.-IgG, were conjugated to polymerized liposomes to form
antibody-conjugated polymerized liposomes for use in in vitro
diagnostic applications.
[0102] Lipid components of: 60% pentacosadiynoic acid filler lipid,
29.5% chelator lipid, 10% amine terminated lipid and 0.5%
biotinylated lipid were combined in the indicated amounts and the
solvents evaporated. Water was added to yield a solution that was
30 mM in acyl chains. The lipid/water mixture was then sonicated
for at least one hour. During sonication, the pH of the solution
was maintained between 7 and 8 with NaOH and the temperature was
maintained above the gel-liquid crystal phase transition point by
the heat generated by sonication. The liposomes were transferred to
a petri dish resting on a bed of wet ice and irradiated at 254 nm
for at least one hour to polymerize. The polymerized liposomes were
collected after passage through a 0.2.mu. filter. To form the
antibody conjugated polymerized liposomes, 2.3 .mu.g avidin was
combined with 14.9 .mu.g biotinylated antibody in phosphate
buffered saline in about 1:3 molar ratio and incubated at room
temperature for 15 minutes. This solution was combined with 150
.mu.L of the above formed polymerized liposomes and incubated at
4.degree. C. overnight to form the antibody-conjugated polymerized
liposomes. The total number of antibody-conjugated polymerized
liposomes in a 40 .mu.l aliquot was found to be about
1.4.times.10.sup.11 as determined by light scattering and
theoretical calculations based on the size of the particles and
protein and amount of lipid used in the preparation. The
antibody-conjugated polymerized liposomes were analyzed by photon
correlation spectroscopy using a Coulter N4+ submicron particle
analyzer and shown to have a mean diameter of 262 nm. Then 9.6
.mu.g of agglutinating antibody, goat IgG, was added to a 40 .mu.l
aliquot of anti-goat .gamma.-IgG-conjugated polymerized liposomes,
as prepared above, and incubated for about 1 hour. After this
incubation, 53% of the antibody-conjugated polymerized liposomes
had agglutinated as demonstrated by the appearance of a new group
of particles with a mean diameter of 1145 nm, as determined by
photon correlation spectroscopy. The antibody-conjugated
polymerized liposomes thereby provide a simple and very sensitive
in vitro assay for the presence of specific antigens in
solution.
EXAMPLE V
[0103] Lipids containing a DTPA chelator head group were
constructed as described in Storrs, et al, JACS, (1995)
incorporated herein supra, paragraph spanning pages 7305-7506, for
compound 4 and 1b and chelated to Eu.sup.+3 ions and formed into
polymerized liposomes at a level of 1%. A wide variety of suitable
chelating agents for spectroscopically distinct ions are known to
the art as, for example, as described in U.S. Pat. Nos. 4,259,313;
4,859,777; 4,801,504; 4,784,912; and 4,801,722. The Europium
labelled polymerized liposomes were serially diluted with buffer
and detected using time-resolved flourescence spectroscopy,
detecting Eu.sup.+3 labeled polymerized liposomes down to
concentrations of 10.sup.-21 molar in an ELISA based system.
EXAMPLE VI
[0104] Polymerized liposomes based upon pentacosadiynoic acid were
constructed having a negative charge. No exogenous fluorescent
probes were used and only the intrinsic fluorescence of the
polymerized liposomes, emission at 530-680 nm, was relied upon for
detection. The polymerized liposomes were incubated with
endothelial cells expressing P-Selectin, a protein that binds
charged entities, and then analyzed using flow cytometry. Flow
cytometry detected the polymerized liposomes adhered to the
endothelial cells.
EXAMPLE VII
[0105] A lipid containing a fluorophore head group, such as, for
example, Texas Red, was constructed. Suitable lipids are, for
example, PDA(PEG).sub.3-NH.sub.2/carboxylic acids and hydraziene
derivatives and suitable fluorophore head groups are, for example,
Texas Red and FITC. This material was incorporated into polymerized
liposomes at a level of 0.5%. 200 .mu.g Texas Red sulfonyl chloride
in acetonitrile was added to 600 .mu.l polymerized liposomes, 30 mM
in acyl chain, on 0.01M sodium bicarbonate buffer, pH9, and reacted
at room temperature for 2 hours. The labeled polymerized liposomes
were then purified by gel filtration (Sephadex G-25, Sigma, St.
Louis, Mo.) using PBS as eluent. An anti-ICAM-1 antibody was then
attached to the Texas Red labelled polymerized liposomes in the
same manner as described in Example IV and then incubated with
activated endothelial cells expressing ICAM-1 and analyzed using
fluorescent microscopy. Using this approach, 10.sup.5 to 10.sup.6
Texas Red molecules can be linked to each antibody resulting in
dramatic increase in sensitivity of the assay. The antibody
conjugated polymerized liposomes can be easily seen bound to the
activated endothelium, thus simplifying the methodology for
assaying cell surface glycoproteins.
EXAMPLE VIII
[0106] To conjugate monoclonal antibodies to paramagnetic
polymerized liposomes, we constructed paramagnetic polymerized
liposomes containing biotinylated lipids. Avidin, a biotin binding
protein, was then used to bridge biotinylated antibodies to biotin
on the particle surface. Alternatively, anionic polymerized
liposome particles may be constructed and antibodies conjugated to
cationic proteins, such as avidin, are then exchanged onto the
particles.
[0107] Lipid components of: 60% pentacosadiynoic acid filler lipid,
29.5% Gd.sup.+3 chelator lipid, 10% amine terminated lipid and 0.5%
biotinylated lipid were combined in the indicated amounts and the
solvents evaporated. Water was added to yield a solution 30 mM in
acyl chains. The lipid/water mixture was then sonicated for at
least one hour. During sonication, the pH of the solution was
maintained between 7 and 8 with NaOH and the temperature was
maintained above the gel-liquid crystal phase transition point by
the heat generated by sonication. The liposomes were transferred to
a petri dish resting on a bed of wet ice and UV irradiated at 254
nm for at least one hour to polymerize. The paramagnetic
polymerized liposomes were collected after passage through a
0.2.mu. filter. The resulting paramagnetic polymerized liposomes
were dark blue and exhibited absorption bands at 544 nm, 588 nm and
638 nm (.lambda..sub.max). Gentle heating turned the paramagnetic
polymerized liposomes red having absorption maxima at 498 nm and
538 nm. All paramagnetic polymerized liposomes used in this study
were converted to the red form.
[0108] To form antibody conjugated paramagnetic polymerized
liposomes, 2.3 .mu.g avidin was combined with 14.9 .mu.g
biotinylated antibody in phosphate buffered saline in about 1:3
molar ratio and incubated at room temperature for 15 minutes. This
solution was combined with 150 .mu.L of the above formed
paramagnetic polymerized liposomes, 5.6 mM in acyl chains, and
incubated at 4.degree. C. overnight to form the anti-cell adhesion
molecule antibody-avidin conjugation to the biotinylated
polymerized liposomes. FIG. 28 schematically shows the
antibody-conjugated paramagnetic polymerized liposome (ACPL) formed
as described above.
EXAMPLE IX
[0109] Attachment of the monoclonal antibodies to the biotinylated
paramagnetic polymerized liposomes, as prepared in Example VIII,
was confirmed using gel electrophoresis and immunodetection
techniques.
[0110] For gel electrophoresis, samples were run on 0.65% agarose
gels under non-denaturing conditions, running buffer 25 mM Tris,
190 mM glycine, pH 7.5. Gels were fixed in a solution of 45%
methanol and 10% acetic acid for 15 minutes, rinsed overnight in
water, incubated in 1% rabbit normal serum for 2 hours at room
temperature, and incubated overnight at 4.degree. C. with a 1:1000
dilution in PBS of alkaline phosphatase-conjugated antibodies
against avidin (Sigma) or .gamma.-immunoglobulin (Victor
Laboratories, Burlingame, Calif.). After rinsing in several changes
of PBS, gels were incubated at room temperature in the enzyme
substrate, 5-bromo 4-chloro 3-indolyl phosphate 0.16 mg/ml and
nitro blue tetrazolium 0.32 mg/ml (Sigma) in 0.1M NaCl, 0.1 M Tris,
50 mM MgCl.sub.2, pH 9.5, until the gel was adequately developed.
The reaction was stopped by rinsing in 1 mM EDTA. The paramagnetic
polymerized liposomes contain a chromophore and were therefore
visible without staining.
[0111] Gel electrophoresis, using anti-avidin alkaline phosphatase,
in FIG. 29, showed in Lane 1 intense staining of 0.5 .mu.g avidin,
which, apparently at its isoelectric point, moved slowly from the
loading well. Lane 2 showed a 5 .mu.L sample of paramagnetic
polymerized liposomes moved as a discrete band toward the positive
pole. A solution of approximately 1:3 molar ratio of avidin, 4
.mu.g, and unbiotinylated anti-CAM antibody, 26.25 .mu.g, was
incubated in a total volume of 60.5 .mu.L PBS at 4.degree. C. for
48 hours. A 3.2 .mu.L aliquot of this solution was added to 16
.mu.L of paramagnetic polymerized liposomes and incubated for
approximately 1 week at 4.degree. C. A 5 .mu.L sample of
paramagnetic polymerized liposomes pre-incubated with avidin and
unbiotinylated anti-CAM antibody, as prepared above, showed, in
Lane 3, avidin co-migrated with the liposome band, indicating the
avidin was bound to the surface of the paramagnetic polymerized
liposomes. No free avidin was detected near the well.
Antibody-conjugated paramagnetic polymerized liposomes were
prepared in the manner described above, except that biotinylated
anti-CAM antibody was used, allowing conjugation of the antibody to
the avidin-paramagnetic polymerized liposome complex to form
antibody-conjugated paramagnetic polymerized liposomes. A 5 .mu.L
sample of the biotinylated anti-CAM antibody-conjugated polymerized
liposomes showed, in Lane 4, no. free avidin detected indicating
that the avidin was bound to the paramagnetic polymerized
liposomes. However, no avidin band appeared with the liposomes,
suggesting that antibody conjugation to the particle surface
sterically hindered binding of the anti-avidin alkaline phosphatase
immunodetection antibody to the complex.
[0112] For immunodetection by anti-IgG alkaline phosphatase to
assess antibody binding to the paramagnetic polymerized liposomes,
paramagnetic polymerized liposome preparations and antibody/avidin
incubations were performed as described above for the anti-avidin
alkaline phosphatase immunodetection. FIG. 30 shows a 2.5 .mu.g
aliquot of biotinylated anti-CAM antibody moved as a distinct band
in Lane 1 toward the negative pole. A 5 .mu.L sample of
paramagnetic polymerized liposome, as above, showed in Lane 2,
movement toward the positive pole, being visible due to its
intrinsic chromophore. A 5 .mu.L sample of paramagnetic polymerized
liposomes pre-incubated with avidin and unbiotinylated antibody,
2.2 .mu.g total antibody, exhibited a free antibody band, in Lane
3, indicating that unbiotinylated antibody did not bind with the
avidin-paramagnetic polymerized liposome complex. A 5 .mu.L sample
of paramagnetic polymerized liposomes pre-incubated with avidin and
biotinylated antibody, 2.2 .mu.g total antibody, in Lane 4,
exhibited no detection of a free antibody band, demonstrating
conjugation of the biotinylated antibody to the avidin-paramagnetic
polymerized liposomes forming antibody-conjugated paramagnetic
polymerized liposomes.
[0113] This Example shows that the antibody-conjugated paramagnetic
polymerized liposome is functional in a competitive ELISA assay.
Anti-ICAM-1 antibody-conjugated paramagnetic polymerized liposomes
incubated on ELISA plates coated with soluble ICAM-1 demonstrated
inhibition of free monoclonal anti-ICAM-1 antibody binding.
EXAMPLE X
[0114] Cell-binding assays using fluorescently-tagged
antibody-conjugated paramagnetic polymerized liposomes were
conducted to show that the anti-ICAM-1 antibody-conjugated
paramagnetic polymerized liposomes could recognize antigens in
vitro. Paramagnetic polymerized liposomes, as prepared in Example
VIII, were coupled to Texas Red flurophore (Pierce, Rockford,
Ill.). 200 .mu.g Texas Red sulfonyl chloride in acetonitrile was
added to 600 .mu.l paramagnetic polymerized liposomes, 30 mM in
acyl chain, in 0.1M sodium bicarbonate buffer, pH 9, and reacted at
room temperature for 2 hours. The labeled paramagnetic polymerized
liposomes were then purified by gel filtration (Sephadex G-25,
Sigma, St. Louis, Mo.) using PBS as eluent. Fluorescent
paramagnetic polymerized liposomes were then conjugated to
anti-ICAM-1 antibodies as described in the prior example.
[0115] Endothelial cells, bEnd 3, were plated onto 100 mm plastic
petri dishes and grown until confluent. Cells were stimulated with
1 .mu.g/ml bacterial lipopolysaccharide about 24-48 hours prior to
use to elicit expression of ICAM-1. Unstimulated cells
constitutively expressing only low levels of adhesion molecules
were used as controls. Media was aspirated from cells and the
plates were rinsed with Hank's balanced salt solution for 30
minutes, washed three times with PBS and then divided in 1 cm.sup.2
wells. The wells were pre-incubated with 0.5% bovine serum albumin
in PBS for approximately 3 hours at room temperature following
which aliquots of 50 .mu.L each of 1:100 and 1:1000 dilutions of
antibody-conjugated paramagnetic polymerized liposomes were added
to cover the wells. Antibody-conjugated paramagnetic polymerized
liposomes were incubated with the cells for 2 hours at room
temperature and then washed two times for five minutes with 0.5%
BSA-PBS and four times for five minutes with PBS. Using
fluorescence microscopy, fluorescently tagged anti-ICAM-1
antibody-conjugated paramagnetic polymerized liposomes were seen
bound to the cultured endothelial cells stimulated with bacterial
lipopolysaccharide to elicit ICAM-1 expression, outlining the
morphology of individual cell membranes, as shown in FIG. 31. This
binding is shown schematically in FIG. 32. No binding of
fluorescent antibody-conjugated paramagnetic polymerized liposomes
to stimulated cells was observed when a non-specific
anti-immunoglobulin antybody was substituted for anti-ICAM-1.
Similarly, unstimulated cells that express only low levels of
ICAM-1 did not bind anti-ICAM-1 fluorescent antibody-conjugated
paramagnetic polymerized liposomes.
EXAMPLE XI
[0116] To show that antibody-conjugated paramagnetic polymerized
liposomes could both successfully target endothelial CAMs in vivo
and also provide substantial magnetic resonance image contrast
enhancement, a well documented model of cerebral inflammation in
mice was examined.
[0117] Experimental autoimmune encephalitis is an ascending
encephalomyelitis characterized by an intense perivascular
lympho-/monocytic inflammatory process in the central nervous
system white matter, primarily the cerebellum, brain stem and
spinal cord. This system is of clinical interest as an animal model
for multiple sclerosis and the nature of the receptors involved in
inflammatory cell trafficking in experimental
autoimmune-encephalitis have been well investigated. ICAM-1
expression on the experimental autoimmune encephalitis mouse brain
microvasculature has been shown to be upregulated at the onset of
clinical disease. The ICAM-1 receptor mediates the attachment of
leukocytes to inflamed endothelium and is present on both activated
leukocytes and stimulated endothelium of capillaries and venules
throughout the central nervous system. Its expression is not
limited to vessels involved by inflammatory infiltrates. Histologic
studies have previously shown that the blood-brain barrier
maintains integrity during the onset of disease and for 48 hours
after paralysis is apparent. Prior magnetic resonance and
fluorescence microscopy studies of liposome transit across the
blood-brain barrier in acute experimental autoimmune encephalitis
guinea pigs have shown that liposomes were unable to penetrate
compromised blood-brain barrier and enter brain parenchyma.
Therefore, we targeted the ICAM-1 receptor in the early phase of
its upregulation in experimental autoimmune encephalitis, when
expression of ICAM-1 is increased ten-fold.
[0118] Fluorescently labeled anti-ICAM-1 antibody-conjugated
paramagnetic polymerized liposomes were shown in vivo to bind to
cerebellar vasculature of mice with grade 2 experimental autoimmune
encephalitis by showing location of the particle as seen by high
resolution magnetic resonance could be confirmed with fluorescence
microscopy.
[0119] Experimental autoimmune encephalitis was induced in SJL/J
mice according to a proteolipid protein immunization protocol. When
clinical signs of grade 2 disease were apparent, tail paralysis and
limb weakness, the fluorscent anti-ICAM-1 antibody-conjugated
paramanetic polymerized liposomes, as prepared in the prior
example, were injected via a tail vein, 10 .mu.L/g representing 1.2
mg/kg Gd.sup.+3 and 890 kg antibody/kg, and allowed to recirculate
for 24 hours. Mice were then sacrificed and perfused with PBS. The
brains were removed and cut in half sagitally, one half frozen for
direct fluorscence microscope analysis of 10 .mu.m thin sections
and the other half fixed in 4% paraformaldehyde in PBS, pH7.4, and
used for high resolution magnetic resonance imaging.
[0120] In three separate tests, a total of seven diseased mice were
injected with fluorescent anti-ICAM-1 antibody-conjugated
paramagnetic polymerized liposomes and all were shown to be
positive for the antibody conjugated-polymerized liposome binding
to central nervous system vasculature by fluorescence microscopic
analysis of cerebellum, brainstem and spinal cord. FIG. 33 is a
typical fluorscence micrograph of mouse cerebellum counterstained
with haematoxylin showing multiple vessels surrounded by an
inflammatory infiltrate. Anti-ICAM-l antibody-conjugated
paramagnetic polymerized liposomes, indicated by arrows, are seen
by fluorescence to be bound to small capillaries (Sv), but not
bound to large central arteriole (LV) which is seen to be negative
for fluorescence. This is consistent with expression of ICAM-1
which is upregulated on endothelium of venules and capillaries, but
not expressed on arterioles or larger vessels. We also noted
fluorescent anti-ICAM-1 polymerized liposomes bound to microvessels
that are not associated with inflammatory infiltrates, which is
consistent with histological findings of ICAM-1 expression on both
infiltrated and non-infiltrated vessels.
[0121] Six controls: three healthy animals injected with
anti-ICAM-1 antibody-conjugated paramagnetic polymerized liposomes;
two diseased animals administered anti-trinitrophenol
antibody-conjugated paramagnetic polymerized liposomes, and one
diseased animal administered anti-V.beta.11 T-cell receptor
antibody-conjugated paramagnetic polymerized liposomes, targeted to
an antigen not expressed in the SJL/J mouse, were all found by
fluorescence microscopy to show no polymerized liposome
binding.
EXAMPLE XII
[0122] High resolution magnetic resonance images were made of the
complimentary half of two mouse brains from mice having grade 2
experimental autoimmune encephalitis used in the previous example
containing anti-ICAM-1 antibody-conjugated paramagnetic polymerized
liposomes. High resolution T1 and T2-weighted images of the intact
half brains were obtained by using a 9.4 T MR scanner (General
Electric) using 3 DFT spin echo pulse sequences. Parameters for
T1-weighted images were TR 200 ms, TE 4 ms, 1 NEX, matrix
256.times.256.times.256, and a field of view of 1 cm, resulting in
a voxel size of approximately 40 .mu.m in each dimension.
T1-weighted acquisitions times were approximately 7 hours per scan.
T2-weighted parameters were TR 100 ms, TE 20 ms, 8 NEX, matrix
256.times.256.times.256. T2-weighted scan times were approximately
12 hours. FIG. 34 shows a T2-weighted scan of an experimental
autoimmune encephalitis mouse, without injection of polymerized
liposomes, cerebrum (coronal) and cerebellum (axial) to define
normal anatomy. FIG. 35 shows a representative slice from a
T1-weighted scan of an autoimmune encephalitis mouse injected with
anti-ICAM-1 antibody-conjugated paramagetic polymerized liposomes.
Diffuse perivascular enhancement is seen throughout the brain, in
the cerebellum and cerebrum, lending particularly significant
contrast between the meagerly vascularized cerebellar white (W) and
the highly vascular grey (g) matter. FIG. 36 shows a representative
slice from a T1-weighted scan of a healthy mouse similarly injected
with anti-ICAM-1 antibody-conjugated paramagnetic polymerized
liposomes showed no enhancement.
[0123] Signal intensity measurements were made using the image
analysis program Voxel View/Ultra 2.2 (Vital Images, Inc.,
Fairfield, Iowa). For each mouse brain, three slices were chosen
for analysis. For each slice, the signal intensity of cerebral
gray, cerebellar gray, and cerebellar white matter was determined
by manually drawing at least five large region-of-interest paths
within each of these tissues. Signal intensity measurements from
the three slices were averaged to give a mean signal intensity
value for each tissue type, means weighted according to standard
deviation of individual signal intensity values. The differences in
tissue signal intensities between mouse brains were assesed using
the two-tailed Stundent's t-test. The statistical significance
level was set at P<0.05. The results are shown in FIG. 37.
Compared to the controls, the magnetic resonance scans of the
experimental autoimmune encephalytis infected mice injected with
anti-ICAM-1 antibody-conjugated paramagnetic polymerized liposomes
showed substantial increases in magnetic resonance signal intensity
of about 32% in the cerebellar, 28% in the cerebral cortex and, to
a lesser extent, about 18% in the cerebellar white matter. As a
result of the enhanced gray matter signal, contrast between gray
and white matter was improved. This was particularly pronounced in
the cerebellum which was actively affected-by experimental
autoimmune encephalytis.
[0124] The above examples have demonstrated that
antibody-conjugated paramagnetic polymerized liposomes can be
delivered to cell adhesion molecules upregulated in disease. This
provides a new target-specific magnetic resonance contrast
enhancement agent for providing in vivo imaging studies of specific
targeted physiological activies, such as, for example, endothelial
antigens involved in numerous pathologies.
[0125] While in the foregoing specification this invention has been
described in relation to certain preferred embodiments thereof, and
many details have been set forth for purpose of illustration it
will be apparent to those skilled in the art that the invention is
susceptible to additional embodiments and that certain of the
details described herein can be varied considerably without
departing from the basic principles of the invention.
* * * * *