U.S. patent application number 11/796064 was filed with the patent office on 2007-08-30 for blood clot-targeted nanoparticles.
This patent application is currently assigned to Barnes-Jewish Hospital. Invention is credited to Gregory Lanza, Samuel A. Wickline.
Application Number | 20070202040 11/796064 |
Document ID | / |
Family ID | 31946292 |
Filed Date | 2007-08-30 |
United States Patent
Application |
20070202040 |
Kind Code |
A1 |
Lanza; Gregory ; et
al. |
August 30, 2007 |
Blood clot-targeted nanoparticles
Abstract
Emulsions comprising nanoparticles formed from high boiling
perfluorochemical substances, said particles coated with a
lipid/surfactant coating are made target-specific by directly
coupling said nanoparticles to a targeting ligand. The
nanoparticles may further include biologically active agents,
radionuclides, and/or other imaging agents.
Inventors: |
Lanza; Gregory; (St. Louis,
MO) ; Wickline; Samuel A.; (St. Louis, MO) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE
SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Assignee: |
Barnes-Jewish Hospital
St. Louis
MO
63110
|
Family ID: |
31946292 |
Appl. No.: |
11/796064 |
Filed: |
April 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10225024 |
Aug 20, 2002 |
7220401 |
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11796064 |
Apr 25, 2007 |
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09404963 |
Sep 24, 1999 |
6548046 |
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10225024 |
Aug 20, 2002 |
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Current U.S.
Class: |
424/1.33 ;
424/1.29 |
Current CPC
Class: |
A61K 49/0471 20130101;
A61K 49/227 20130101; A61K 47/6843 20170801; A61K 49/1812 20130101;
A61K 49/1806 20130101; A61K 51/1227 20130101; A61K 47/6907
20170801; B82Y 5/00 20130101 |
Class at
Publication: |
424/001.33 ;
424/001.29 |
International
Class: |
A61K 51/12 20060101
A61K051/12; A61K 103/10 20060101 A61K103/10 |
Claims
1. A composition for performing an imaging and/or delivery function
with respect to a blood clot in a subject, said composition
comprising an emulsion of nanoparticles, wherein said nanoparticles
comprise a core consisting of liquid perfluorocarbon which core is
coated with lipid/surfactant and which nanoparticles are coupled
directly to at least one targeting ligand that is specific for at
least one component characterizing blood clots, and wherein the
perfluorocarbon is selected from the group consisting of
perfluorodecalin, perfluorooctane, perfluorodichlorooctane,
perfluoro-n-octyl bromide, perfluoroheptane, perfluorodecane,
perfluorotripropylamine, perfluortributylamine, and mixtures
thereof, and wherein said nanoparticles further include at least
one chelator for a paramagnetic metal ion as a magnetic resonance
imaging (MRI) contrast agent, and/or wherein said nanoparticles
further include at least one biologically active agent, and/or
wherein said nanoparticles further contain at least one
radionuclide.
2. The composition of claim 1, wherein said chelating agent is
diethylenetriaminepentaacetic acid and the paramagnetic ion is
gadolinium ion.
3. The composition of claim 1, wherein said biologically active
agent is a hormone or pharmaceutical.
4. The composition of claim 1, wherein said radionuclide is
.sup.99Tc.
5. The composition of claim 1, wherein said ligand binds
specifically to fibrin.
6. The composition of claim 1, wherein said targeting ligand is an
antibody, a fragment of an antibody, an aptamer, a hormone,
peptidomimetic or a receptor ligand.
7. The composition of claim 6, wherein the targeting ligand is an
antibody or fragment of an antibody.
8. The composition of claim 7, wherein said antibody or fragment is
humanized.
9. A method to obtain a magnetic resonance image of a blood clot in
a subject, which method comprises administering to said subject the
composition of claim 1; allowing the targeting ligand to couple to
the blood clot; and obtaining an image of said blood clot, while
said nanoparticles consist of components in the liquid state.
10. The method of claim 9, which further includes verifying the
location of said nanoparticles by detecting a .sup.19 F magnetic
resonance signal.
11. A method to deliver a biologically active agent to a blood clot
in a subject, which method comprises administering to a subject in
need of the presence of such biologically active agent at a blood
clot the composition of claim 7; and permitting the nanoparticles
of said composition to locate in said blood clot.
12. A method to deliver a radionuclide to a blood clot in a
subject, which method comprises administering to a subject the
composition of claim 1; and permitting the nanoparticles of said
composition to locate in said blood clot, whereby said radionuclide
is delivered to said blood clot.
13. The method of claim 12, which further comprises imaging the
blood clot by emissions from said radionuclide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of U.S. Ser. No. 10/225,024 filed 20
Aug. 2002 and now allowed, which is a continuation-in-part of U.S.
Ser. No. 09/404,963 filed 24 Sep. 1999, now U.S. Pat. No.
6,548,046. The contents of these applications are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The invention is directed to nanoparticles which home to
specific blood clots and which carry to these targets substances
useful in diagnosis or treatment. More specifically, the invention
concerns nanoparticles to which ligands specific for thromboses are
directly bound and which further contain imaging agents and/or
bioactive materials.
BACKGROUND ART
[0003] U.S. Pat. Nos. 5,690,907, 5,780,010 and 5,958,371, the
disclosures of which are incorporated herein by reference, describe
biotinylated lipid-encapsulated perfluorocarbon nanoparticles which
are useful for the delivery of radionuclides, and magnetic
resonance imaging agents to specific locations through a
biotin-avidin system. Bioactive agents may also be included. In
this approach, the target location is coupled to a target-specific
ligand which is also coupled to biotin. Avidin is then employed to
bridge the now biotinylated target with biotin derivatized
nanoparticles contained in an emulsion. Included among the targets
are blood clots; however, these blood clots are first labeled with
antifibrin antibodies to which biotin is then bound. No direct
targeting of blood clots with ligands specific for such clots is
disclosed.
[0004] This is in contrast to the compositions of the present
invention wherein a ligand specific for thromboses is directly
coupled, initially, to the nanoparticles in the emulsion. Thus, the
emulsion, when administered, is target-specific by virtue of
bearing the target-specific ligand at its surface.
[0005] Fluorochemical emulsions with specification binding moieties
have been described in U.S. Pat. No. 5,401,634 for use as labels in
in vitro analytical procedures. However, in vivo uses, for example,
for acoustic imaging, drug delivery or delivery of imaging agents
or nuclides is not contemplated. In addition, consistent with the
failure to envision in vivo use, no modification of these particles
for binding to thromboses is mentioned.
[0006] Others have described drug delivery using particulate
supports which differ from the nanoparticles of the present
invention. For example, PCT publication WO 95/03829 describes oil
emulsions where the drug is dispersed or solubilized inside an oil
droplet and the oil droplet is targeted to a specific location by
means of a ligand. U.S. Pat. No. 5,542,935 describes site-specific
drug delivery using gas-filled perfluorocarbon microspheres. The
drug delivery is accomplished by permitting the microspheres to
home to the target and then effecting their rupture. Low boiling
perfluoro compounds are used to form the particles so that the gas
bubbles can form.
[0007] In contrast to the compositions described above, the
compositions of the invention are ligand-bearing liquid emulsions
based on high boiling perfluorocarbon liquids. The compositions of
the invention provide facile means to deliver materials contained
in their surface to blood clots.
[0008] An article reporting the work of the present inventors,
Flacke, S., et al., Circulation (2001) 104:1280-1285 appeared in
September of 2001 and described molecular imaging of thrombus using
nanoparticles formulated with GD-DTPA-BOA. The particles were
covalently coupled to antifibrin monoclonal antibody and used to
obtain magnetic resonance images of blood clots.
[0009] The present invention expands the concept set forth in this
article and provides nanoparticles which target blood clots
specifically and which may provide, in addition to magnetic
resonance imaging agents, means for acoustic imaging, therapeutic
agents, and radionuclides.
DISCLOSURE OF THE INVENTION
[0010] The invention provides compositions which are liquid
emulsions. The liquid emulsions contain nanoparticles comprised of
liquid, relatively high boiling perfluorocarbons surrounded by a
coating which is composed of a lipid and/or surfactant. The
surrounding coating is able to couple directly to a moiety that
targets blood clots or can entrap an intermediate component which
is covalently coupled to the said moiety, optionally through a
linker. Alternatively, the coating may be cationic so that
negatively charged blood clot targeting agents such as nucleic
acids, in general or aptamers, in particular, can be adsorbed to
the surface.
[0011] In addition to the targeting agent or ligand, the
nanoparticles may contain at their surface a radionuclide, a
contrast agent for magnetic resonance imaging (MRI) and/or a
biologically active compound. The nanoparticles themselves can
serve as contrast agents for ultrasound imaging.
[0012] As the emulsions of the invention are intended to target
blood clots or thromboses, especially in vivo, components of these
clots are used as suitable targets. Among these markers or targets
are fibrin, tissue factor, gpIIb/IIIa, tissue factor/VIIA complex,
activated clotting factor Xa, activated clotting factor IXa, and
the fibrin condensation product, d-dimer. Tissue factor is present
but not preferred as it is relatively nonspecific.
[0013] Thus, in one aspect, the invention is directed to a
composition comprising an emulsion of liquid, high boiling
perfluorocarbon-based nanoparticles, said nanoparticles further
comprising a coating of a lipid/surfactant in which is embedded, or
to which is directly covalently bound at least one ligand that
targets blood clots, and optionally at least one biologically
active compound, at least one radionuclide, and/or at least one
imaging agent.
[0014] In other aspects, the invention is directed to methods to
administer drugs to, and to obtain images of blood clots,
especially in vivo, using the compositions of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A and 1B show acoustic images obtained with
fibrin-specific and non-fibrin-specific paramagnetic nanoparticles
respectively. FIG. 1C shows similar images but with fat
suppression.
MODES FOR CARRYING OUT THE INVENTION
[0016] The carrier system that is the basis for the present
invention is a nanoparticulate system containing a high boiling
perfluorocarbon as a core and an outer coating that is a
lipid/surfactant mixture which provides a vehicle for binding a
multiplicity of copies of one or more desired components to the
nanoparticle. The construction of the basic particles and the
formation of emulsions containing them, regardless of the
components bound to the outer surface is described in the
above-cited patents to the present applicants, U.S. Pat. Nos.
5,690,907; 5,780,010; and patents issued on daughter applications
U.S. Pat. Nos. 5,989,520 and 5,958,371 and incorporated herein by
reference.
[0017] The high boiling fluorochemical liquid is such that the
boiling point is higher than that of body temperature--i.e.,
37.degree. C. Thus, fluorochemical liquids which have boiling
points at least 30.degree. C. are preferred, more preferably
37.degree. C., more preferably above 50.degree. C., and most
preferably above about 90.degree. C. The "fluorochemical liquids"
useful in the invention include straight and branched chain and
cyclic perfluorocarbons including perfluorinated compounds which
have other functional groups. Perfluorinated compounds are
preferred. Particularly preferred are compounds which will remain
in the liquid state when they serve their function in the subject;
for example, when used to obtain an acoustic image.
[0018] Useful perfluorocarbon emulsions are disclosed in U.S. Pat.
Nos. 4,927,623, 5,077,036, 5,114,703, 5,171,755, 5,304,325,
5,350,571, 5,393,524, and 5,403,575 and include those in which the
perfluorocarbon compound is perfluorodecalin, perfluorooctane,
perfluorodichlorooctane, perfluoro-n-octyl bromide,
perfluoroheptane, perfluorodecane, perfluorocyclohexane,
perfluoromorpholine, perfluorotripropylamine,
perfluortributylamine, perfluorodimethylcyclohexane,
perfluorotrimethylcyclohexane, perfluorodicyclohexyl ether,
perfluoro-n-butyltetrahydrofuran, and compounds that are
structurally similar to these compounds and are partially or fully
halogenated (including at least some fluorine substituents) or
partially or fully perfluorinated including perfluoroalkylated
ether, polyether or crown ether.
[0019] The coating which comprises lipid/surfactant to form an
outer coating on the nanoparticles which will contain the coupled
ligand or entrap reagents for binding desired components to the
surface include natural or synthetic phospholipids, fatty acids,
cholesterols, lysolipids, sphingomyelins, and the like, including
lipid conjugated polyethylene glycol. Various commercial anionic,
cationic, and nonionic surfactants can also be employed, including
Tweens, Spans, Tritons, and the like. Some surfactants are
themselves fluorinated, such as perfluorinated alkanoic acids such
as perfluorohexanoic and perfluorooctanoic acids, perfluorinated
alkyl sulfonamide, alkylene quaternary ammonium salts and the like.
In addition, perfluorinated alcohol phosphate esters can be
employed. Cationic lipids included in the outer layer may be
advantageous in entrapping ligands such as nucleic acids, in
particular aptamers. Typical cationic lipids may include DOTMA,
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride;
DOTAP, 1,2-dioleoyloxy-3-(trimethylammonio)propane; DOTB,
1,2-dioleoyl-3-(4'-trimethyl-ammonio)butanoyl-sn-glycerol,
1,2-diacyl-3-trimethylammonium-propane;
1,2-diacyl-3-dimethylammonium-propane;
1,2-diacyl-sn-glycerol-3-ethyl phosphocholine; and
3.beta.-[N',N'-dimethylaminoethane)-carbamol]cholesterol-HCl.
[0020] The lipid/surfactant coated nanoparticles are typically
formed by microfluidizing a mixture of the fluorocarbon lipid which
forms the core and the lipid/surfactant mixture which forms the
outer layer in suspension in aqueous medium to form an emulsion.
Sonication or other techniques may be required to obtain a
suspension of the lipid/surfactant in the aqueous medium.
Typically, at least one of the components of the lipid/surfactant
outer layer comprises a linker or functional group which is useful
to bind the targeting ligand or the targeting ligand may already be
coupled to the component at the time the emulsion is prepared. The
components of the outer layer may also be coupled to imaging agents
or radionuclides. The components may also include biologically
active materials.
[0021] For coupling by covalently binding the targeting ligand or
other organic moiety (such as a chelating agent for a paramagnetic
metal) to the components of the outer layer, various types of bonds
and linking agents may be employed. Typical methods for forming
such coupling include formation of amides with the use of
carbodiimides, or formation of sulfide linkages through the use of
unsaturated components such as maleimide. Other coupling agents
include, for example, glutaraldehyde, propanedial or butanedial,
2-iminothiolane hydrochloride, bifunctional N-hydroxysuccinimide
esters such as disuccinimidyl suberate, disuccinimidyl tartrate,
bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, heterobifunctional
reagents such as N-(5-azido-2-nitrobenzoyloxy)succinimide,
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and
succinimidyl 4-(p-maleimidophenyl)butyrate, homobifunctional
reagents such as 1,5-difluoro-2,4-dinitrobenzene,
4,4'-difluoro-3,3'-dinitrodiphenylsulfone,
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene,
p-phenylenediisothiocyanate, carbonylbis(L-methionine p-nitrophenyl
ester), 4,4'-dithiobisphenylazide, erythritolbiscarbonate and
bifunctional imidoesters such as dimethyl adipimidate
hydrochloride, dimethyl suberimidate, dimethyl
3,3'-dithiobispropionimidate hydrochloride and the like. A
multiplicity of ways to couple, covalently, a desired ligand to one
or more components of the outer layer is well known in the art. The
ligand itself may be included in the surfactant layer if its
properties are suitable. For example, if the ligand contains a
highly lipophilic portion, it may itself be embedded in the
lipid/surfactant coating. Further, if the ligand is capable of
direct adsorption to the coating, this too will effect its
coupling. For example, nucleic acids, because of their negative
charge, adsorb directly to cationic surfactants.
[0022] By "direct binding" of the ligand to the nanoparticle is
meant that the ligand specific for a component characteristic of
blood clots is associated with the nanoparticle itself, as opposed
to indirect binding effected through biotin/avidin. In the
biotin/avidin mediated targeting methods of the art, the
clot-specific ligand is coupled not to the emulsion, but rather
coupled, in biotinylated form to the targeted tissue. A component
"characteristic of" blood clots does not include tissue factor.
[0023] The targeting ligands cover a range of suitable moieties
which bind to components of blood clots. In general, a component
may itself be used to generate a ligand by using the component to
raise antibodies or to select aptamers that are specific binding
partners for the component. Alternatively, a suitable ligand may be
known in the art. More generically, however, antibodies can be
raised to desired components by conventional techniques and can be
provided, preferably, as monoclonal antibodies or fragments
thereof, or as single chain antibodies produced recombinantly. If
the subject to be administered the compositions of the invention is
human, it may be desirable to humanize antibody-type ligands using
techniques generally known in the art. Further, suitable proteins
which bind to targets can be discovered through phage-display
techniques or through the preparation of peptide libraries using
other appropriate methods. Selective aptamers which are able
selectively to bind desired targets may also be prepared using
known techniques such as SELEX.TM.. (Aptamers are oligonucleotides
which are selected from random pools for their ability to bind
selected targets.)
[0024] In addition to the foregoing, peptidomimetics, which are
small organic molecules intended to mimic peptides of known
affinities can also be used as targeting agents. Particularly
preferred are targeting agents that bind to fibrin, as fibrin is a
particularly characteristic element included in blood clots.
Antifibrin antibodies are particularly preferred, including
fragments thereof, such as the F.sub.ab, F.sub.(ab')2 fragments,
single chain antibodies (F.sub.v) and the like. In one preferred
embodiment, when the emulsion includes an MRI imaging agent, such
as a chelated transition metal, the targeting agent targets
components of the blood clot other than fibrin, such as gpIIb/IIIa,
clotting factors Xa and IXa and the like.
[0025] In addition to the ligand designed to bind the emulsion to
blood clots, additional components of the emulsion can be bound to
the nanoparticles in ways similar to those which are used to bind
the ligands.
[0026] Other components which may be coupled to the nanoparticles
through entrapment in the coating layer include radionuclides.
These radionuclides include, for example, .sup.99Tc. The
radioactive ions can be provided to the preformed emulsion in a
variety of ways. For example, .sup.99Tc-pertechnate may be mixed
with an excess of stannous chloride and incorporated into the
preformed emulsion of nanoparticles, followed by removal of unbound
.sup.99Tc-pertechnate by repeated centrifugation and washing.
Stannous oxinate can be substituted for stannous chloride. In
addition, commercially available kits, such as the HM-PAO
(exametazine) kit marketed as Ceretek.RTM. by Nikomed Amersham can
be used. Means to attach various radioligands to the nanoparticles
of the invention are understood in the art.
[0027] In addition to incorporation of radionuclides, chelating
agents containing paramagnetic metals for use in magnetic resonance
imaging can also be employed. Typically, a chelating agent
containing a paramagnetic metal is associated with the
lipids/surfactants of the coating on the nanoparticles and
incorporated into the initial mixture which is sonicated. The
chelating agent can be coupled directly to one or more of
components of the coating layer. Suitable chelating agents include
a variety of multi-dentate compounds including EDTA, DPTA, and the
like. These chelating agents can be coupled directly to functional
groups contained in, for example, phosphatidyl ethanolamine,
bis-oleate, and the like.
[0028] The paramagnetic metals useful in the MRI contrast agents of
the invention include rare earth metals, typically, lanthanum,
ytterbium, gadolinium, europium, and the like. Iron ions may also
be used.
[0029] Also included in the surface of the nanoparticle, in some
embodiments of the invention, are biologically active agents. These
biologically active agents can be of a wide variety, including
proteins, nucleic acids, pharmaceuticals, and the like. Thus,
included among suitable pharmaceuticals are antineoplastic agents,
hormones, analgesics, anesthetics, neuromuscular blockers,
antimicrobials or antiparasitic agents, antiviral agents,
interferons, antidiabetics, antihistamines, antitussives,
anticoagulants, and the like.
[0030] In a typical procedure for preparing the emulsions of the
invention, the fluorochemical liquid and the components of the
lipid/surfactant coating are fluidized in aqueous medium to form an
aqueous emulsion. The functional components of the surface layer
may be included in the original emulsion, or may later be
covalently coupled to the surface layer subsequent to the formation
of the nanoparticle emulsion. In one particular instance, for
example, where a nucleic acid targeting agent or drug is to be
included, the coating may employ a cationic surfactant and the
nucleic acid adsorbed to the surface after the particle is
formed.
[0031] When appropriately prepared, the particles contain a
multiplicity of functional reagents at their outer surface, the
nanoparticles typically contain thousands of molecules of the
biologically active agent, targeting ligand, radionuclide and/or
MRI contrast agent. Desirably, the number of copies of a component
to be coupled to the nanoparticle is in excess of 1,000 copies per
particle, more preferably 5,000 copies per particle, still more
preferably 10,000, and still more preferably 50,000 copies per
particle.
[0032] In general, the targeted particles, directly coupled to a
target-specific ligand, are useful themselves as ultrasound
contrast agents. However, the inclusion of other components in
multiple copies renders them useful in other respects. For
instance, the inclusion of a chelating agent containing a
paramagnetic ion makes the emulsion useful as a magnetic resonance
imaging contrast agent. Because the particles comprise large
amounts of fluorine, the addition of a paramagnetic ion is not
necessary to make these particles useful for MRI. The inclusion of
biologically active materials makes them useful as drug delivery
systems. The inclusion of radionuclides makes them useful either as
therapeutics for radiation treatment or as diagnostics for imaging
or both. A multiplicity of such activities may be included; thus,
images can be obtained of targeted tissues at the same time active
substances are delivered to them. Finally, because the particles
have a fluorocarbon core, .sup.19 F magnetic resonance imaging can
be used to track the location of the particles concomitantly with
their additional functions described above.
[0033] The emulsions can be prepared in a range of methods
depending on the nature of the components to be included in the
coating. In a typical procedure, used for illustrative purposes
only, the following procedure is set forth: Perfluorooctylbromide
(40% w/v, PFOB, 3M), and a surfactant co-mixture (2.0%, w/v) and
glycerin (1.7%, w/v) is prepared where the surfactant co-mixture
includes 64 mole % lecithin (Pharmacia Inc), 35 mole % cholesterol
(Sigma Chemical Co.) and 1 mole %
dipalmitoyl-L-alpha-phosphatidyl-ethanolamine, Pierce Inc.)
dissolved in chloroform. A drug is suspended in methanol (.about.25
.mu.g/20 .mu.l) and added in titrated amounts between 0.01 and 5.0
mole % of the 2% surfactant layer, preferably between 0.2 and 2.0
mole %. The chloroform-lipid mixture is evaporated under reduced
pressure, dried in a 50.degree. C. vacuum oven overnight and
dispersed into water by sonication. The suspension is transferred
into a blender cup (Dynamics Corporation of America) with
perfluorooctylbromide in distilled or deionized water and
emulsified for 30 to 60 seconds. The emulsified mixture is
transferred to a Microfluidics emulsifier (Microfluidics Co.) and
continuously processed at 20,000 PSI for three minutes. The
completed emulsion is vialed, blanketed with nitrogen and sealed
with stopper crimp seal until use. A control emulsion can be
prepared identically excluding the drug from the surfactant
commixture. Particle sizes are determined in triplicate at
37.degree. C. with a laser light scattering submicron particle size
analyzer (Malvern Zetasizer 4, Malvern Instruments Ltd.,
Southborough, Mass.), which indicate tight and highly reproducible
size distribution with average diameters less than 400 nm.
Unincorporated drug can be removed by dialysis or ultrafiltration
techniques. To provide the targeting ligand, an F.sub.(ab) fragment
is coupled covalently to the phosphatidyl ethanolamine through a
bifunctional linker in the procedure described above.
[0034] The following examples are intended to illustrate but not to
limit the invention.
EXAMPLE 1
Preparation of Nanoparticles
[0035] Nanoparticles are prepared that comprise
perfluorooctylbromide (40% w/v, PFOB), a surfactant co-mixture
(2.0%, w/v) and glycerin (1.7%, w/v) and optionally an "oil" (2 to
10% w/v, substituted for the PFOB).
[0036] For various applications, the surfactant co-mixture includes
therapeutic agents, dipalmitoylphosphatidyl choline, cholesterol,
phosphoethanolamine-N-4 PEG(2000)-(p-maleimidophenyl)butyramide
(MPB-PEG-PE) or phosphoethanolamine-(p-maleimidophenyl)butyramide,
phosphatidylethanolamine, and sphingomyelin in varying molar
ratios, which are dissolved in chloroform/methanol, evaporated
under reduced pressure, dried in a 50.degree. C. vacuum oven
overnight and dispersed into water. For paramagnetic formulations,
the surfactant co-mixture includes varying amounts of gadolinium
lipophilic chelates such as gadolinium
diethylene-triamine-pentaacetic acid-bis-oleate (Gd-DTPA-BOA) or
gadolinium diethylene-triamine-pentaacetic
acid-phosphatidylethanolamine (Gd-DTPA-PE) at overall
concentrations of 0, to 50 mole %.
[0037] Oil (i.e., vegetable oil, vitamin E or other biocompatible
"oil") may be added alone or may incorporate therapeutic agents.
Lipophilic and hydrophobic therapeutic agents may be dissolved into
the oil component up to supersaturating concentrations to increase
total drug payload.
[0038] The above suspension is combined with PFOB and distilled,
deionized water, blended and then emulsified at 10,000-20,000 PSI
for three minutes.
[0039] The thiolated ligands are coupled to the maleimide
derivatized phospholipid (or lipophilic substitute) in 50 mM
phosphate, 10 mM EDTA buffer at pH 6.65 overnight under an
non-oxidative atmosphere (i.e., nitrogen, argon). Small peptides
and non-peptide molecules are coupled to the lipid moiety prior to
emulsification.
[0040] Antibodies are reacted with N-succinimidyl
S-acetylthioacetate (SATA) for 30 min, dialyzed overnight,
deprotected with hydroxylamine, dialyzed in oxygen free buffers,
then coupled to the nanoparticles at room temperature.
Alternatively, antibodies are enzymatically digested with papain or
pepsin to yield F.sub.(ab) fragments isolated by routine affinity
chromatography.
[0041] Particle sizes are determined in triplicate at ambient
temperature with a laser light scattering submicron particle size
analyzer (Malvern Zetasizer 4, Malvern Instruments Ltd,
Southborough, Mass.), which typically indicates a highly
reproducible size distribution with average diameters around 250
nm.
EXAMPLE 2
Targeting Canine In Situ Fibrin In Vivo
[0042] A perfluorocarbon nanoparticle contrast agent incorporates
1,2-dipalmitoyl-sn
glycero-3-phosphoethanolamine-N-4-(p-maleimidophenyl)butyramide
(MPB-PE; Avanti Polar Lipids, Alabaster, Ala.) into the outer lipid
monolayer of the emulsion to accommodate subsequent ligand
conjugation. Gd-DTPA-phosphatidylethanolamine (Gd-DTPA-PE) was
added to the surfactant mixture at 0 or 20 mole % as described
above.
[0043] Anti-fibrin monoclonal antibody (NIB 1H10, NIB 5F3) is
produced and purified by conventional methods. A fibrin-targeted
nanoparticle contrast agent is created by the covalent bonding of
anti-fibrin F.sub.(ab)' fragments to the outer lipid membrane
surface. Anti-fibrin F.sub.(ab)' fragments are generated (Pierce,
Rockford, Ill.) and combined with the MPB-PE derivatized emulsion
(1-2 mg F.sub.(ab)'/ml of 40% perfluorocarbon emulsion) at pH 6.7
under nitrogen overnight. The conjugated nanoparticles are
dialyzed, vialed and stored at 4.degree. C. A nonspecific control
emulsion is prepared using irrelevant IgG F.sub.(ab)'
fragments.
[0044] The detection of clots in a flowing intravascular
environment is evaluated in canines. Thrombi are formed within the
open circulation, targeted with system in situ within isolated
vascular segments, then exposed to the systemic circulation for
magnetic resonance imaging. Animal protocols are approved by the
Animal Studies Committee at Washington University.
[0045] Two dogs (.about.20 kg) were pretreated with tranexamic acid
(0.25 g/hr) to inhibit endogenous thrombolysis. Each animal was
anesthetized (sodium pentothal/isofluorane), prepped for surgery
and the external jugular veins exposed. Nylon monofilament (4-0)
with 10, 0.5 cm strands of thrombin-soaked cotton fibers were
positioned by ultrasound (Acuson Sequoia, Mountainview, Calif.).
Following clot formation, thrombi were entrapped between snare
closures and one ml of fibrin-targeted gadolinium or control
nanoparticles was infused into the isolated segment. After contrast
incubation (1 hr), the thrombi were reintroduced to the general
circulation and imaged. At the conclusion of the acute procedure,
animals were euthanized and the vessels retrieved for routine
immunohistopathology of fibrin within the thrombus.
[0046] Canine thrombi created within the external jugular vein were
imaged with a 3-D, fat-suppressed, T1-weighted fast gradient echo
(TE/TR/a: 8.1/24135f, FOV 180 mm, matrix 205.times.256). Flow
within vessels and thrombi (as a flow deficit) were imaged with a
3-D phase contrast, T1-weighted fast gradient echo angiogram
(TE/TR/a: 5.3/15115f, FOV 200 mm, matrix 192.times.256).
[0047] The magnitude of contrast-enhancement expected in vivo with
open circulation conditions was evaluated in dogs. Control or 20
mole % (Gd-DTPA-PE) anti-fibrin nanoparticles were administered to
thrombus created within the external jugular vein. Thrombus was
imaged with a 3-D T1-weighted, fat suppression, fast gradient echo
sequence and detectability of targeted clot was markedly enhanced
by the fibrin-specific paramagnetic nanoparticles relative to
control thrombus (FIGS. 1A and 1B). Phase contrast angiography
revealed the clots as flow deficits in both external jugular veins.
Corresponding gradient echo images revealed a selective enhancement
of the treated clot yielding a signal intensity (1780.+-.327)
higher than the bright fat signal (1360.+-.140), whereas, the
control clot had a signal intensity (815.+-.41) similar to that of
the adjacent muscle (768.+-.47). On T1-weighted gradient recalled
echo images with fat suppression, the targeted clot showed the
brightest image signal (FIG. 1C). The CNR between the targeted clot
and blood using nanoparticles with 20 mole % Gd-DTPA measured with
this sequence was approximately 118.+-.21. The CNR between the
targeted clot and the control clot was 131.+-.37. Fibrin
immunostaining of the excised vessel and clot confirmed the
abundance and localization of fibrin corresponding to the contrast
enhancement in vivo.
EXAMPLE 3
Targeting Canine Circulating Fibrin
[0048] The perfluorocarbon nanoparticle contrast agent used in vivo
(circulating) was produced by incorporating 1,2-dipalmitoyl-sn
glycero-3-phosphoethanolamine-N-4-(p-maleimidophenyl)butyramide
(MPB-PE; Avanti Polar Lipids, Alabaster, Ala.) into the outer lipid
monolayer of the emulsion to accommodate subsequent ligand
conjugation 20. Gd-DTPA-phosphatidylethanolamine (Gd-DTPA-PE) was
added to the surfactant mixture at 20 mole % as described
above.
[0049] Anti-fibrin monoclonal antibody (NIB 1H10, NIB 5F3) was
produced and purified. A fibrin-targeted nanoparticle contrast
agent was created by the covalent bonding of anti-fibrin
F.sub.(ab)' fragments to the outer lipid membrane surface.
Anti-fibrin F.sub.(ab)' fragments were generated (Pierce, Rockford,
Ill.) and combined with the MPB-PEG-PE derivatized emulsion (1-2 mg
F.sub.(ab)'/ml of 40% perfluorocarbon emulsion) at pH 6.7 under
nitrogen overnight. The conjugated nanoparticles were dialyzed,
vialed and stored at 4.degree. C.
[0050] Two dogs (.about.20 kg) were pretreated with tranexamic acid
(0.25 g/hr) to inhibit endogenous thrombolysis. Each animal was
anesthetized (sodium pentothal/isofluorane), prepped for surgery
and the external jugular veins exposed. Nylon monofilament (4-0)
with 10, 0.5 cm strands of thrombin-soaked cotton fibers were
positioned by ultrasound (Acuson Sequoia, Mountainview, Calif.).
Following clot formation, thrombi were entrapped between snare
closures and one ml of fibrin-targeted gadolinium or control
nanoparticles was infused into the isolated segment. After contrast
incubation (1 hr), the thrombi were reintroduced to the general
circulation and imaged. At the conclusion of the acute procedure,
animals were euthanized and the vessels retrieved for routine
immunohistopathology of fibrin within the thrombus. Canine thrombi
within the external jugular vein were imaged with a 3-D,
fat-suppressed, T1-weighted fast gradient echo (TE/TR/a:
8.1/24135f, FOV 180 mm, matrix 205.times.256). Fibrin-targeted
paramagnetic nanoparticles were injected intravenously through
peripheral access. After 30 minutes, T1-weighted contrast of the
clot was noted. Contrast single level continued to increase up to
60 minutes.
EXAMPLE 4
Coupling Antibody to Fibrin to Perfluorocarbon Emulsion
Particle
[0051] Preparation of Emulsion: The perfluorocarbon nanoparticle
contrast agent is, produced by incorporating
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-4-(p-maleimidophenyl)b-
utyramide (MPB-PE) into the outer lipid monolayer of the emulsion.
The emulsion is comprised of perfluorodichlorooctane, safflower
oil, a surfactant co-mixture and glycerin. The surfactant
co-mixture includes lecithin, cholesterol and MPB-PE which is
dissolved in chloroform. The chloroform-lipid mixture is evaporated
under reduced pressure, dried in a 50.degree. C. vacuum oven
overnight and dispersed into water by sonication. The suspension is
transferred into a blender cup with perfluorodichlorooctane,
safflower oil and distilled, deionized water and emulsified for 30
to 60 seconds. The pre-emulsified mixture is transferred to a
micro-emulsifier and continuously processed at 10,000 PSI for three
minutes. The completed emulsion is vialed, blanketed with nitrogen
and sealed with stopper crimp seal until use. A negative control
emulsion is prepared identically, except a non-derivatized
phosphatidylethanolamine is substituted into the surfactant
co-mixture. Particle sizes are determined in triplicate at
30.degree. C. with a laser light scatter submicron particle size
analyzer.
[0052] Conjugation of fibrin F.sub.(ab)' With MPB-PE Derivatized
Emulsion: F.sub.(ab)' fractions are pooled and combined with the
MPB-PE derivatized emulsion (0.01 to 5.0 mg F.sub.(ab)'/ml of
emulsion, preferably 1 to 2 mg F.sub.(ab)'/ml of emulsion). The
mixture is adjusted to pH 6.7, sealed under nitrogen and allowed to
react overnight at ambient temperatures with gentle, continuous
mixing. The mixture may be subsequently dialyzed with a 300,000
MWCO Spectra/Por DispoDialyzer (Laguna Hills, Calif.) against 10 mM
phosphate buffer (pH 7.2) to remove unconjugated F.sub.(ab)'
fragments. The final emulsion is vialed-under nitrogen and stored
at 4.degree. C. until use.
EXAMPLE 5
In Vitro Targeting of Fibrin-Rich Plasma Thrombi Using A
Fibrin-Targeted, Acoustic Contrast System
[0053] Whole blood was obtained fresh and anti-coagulated (9:1,
v/v) with sterile sodium citrate. In a series of trials, plasma
clots (9) were produced by combining plasma and 100 mM calcium
chloride (3:1, v/v) with 5 units of thrombin (Sigma Chemical
Company, St. Louis, Mo.) in a plastic tube overlying nitrocellulose
membranes. The plasma was allowed to coagulate slowly at room
temperature.
[0054] Plasma clots were incubated with anti-fibrin (F.sub.ab)
conjugated or non-conjugated control emulsion contrast using
antifibrin monoclonal antibodies (NIB-5F3 or NIB-1H10) (Tymkewycz,
et al. (1992); Tymkewycz, et al. (1993)). Half of the clots (5)
were incubated individually with 150 .mu.g biotinylated antifibrin
monoclonal antibody in 10 ml PBS with 1% bovine serum albumin,
(crystallized, Sigma Chemical Company, St. Louis, Mo.) for two
hours; the remaining clots (4) were maintained in PBS with 1%
bovine serum albumin. Bovine serum albumin was added during
antibody incubations to minimize nonspecific protein binding to the
polystyrene petri dish walls. The anti-fibrin targeted emulsion was
incubated with clots (0.2 ml) for 30 minutes. Control clots were
treated similarly with a nontargeted control perfluorocarbon
emulsion (0.2 ml) for 30 minutes. The plasma clots on
nitrocellulose were insonified using an acoustic microscope to
assess the change in ultrasonic backscattered power attributable to
the control and targeted emulsions.
[0055] The microscope consisted of a 50 MHz broadband, focused,
piezoelectric delay-line transduce (1/4 inch diameter, 1/2 inch
focal length, Model V390, Panametrics Co., Waltham, Mass.) operated
in the pulse-echo mode. A Tektronix DSA 601 digitizing oscilloscope
(Beaverton, Oreg.) was used to digitize backscattered
radiofrequency data at 500 mega-samples per second with 8-bit
resolution. Radiofrequency data collected from each site was
averaged 32 times. Averaged radiofrequency data were acquired from
approximately 400 independent sites with 50 micron lateral step
resolution. The radiofrequency data are stored in a low resolution
raster scan format and analyzed with custom software. Segments of
the radiofrequency lines, 500 nsec in duration and encompassing
surface reflection are gated for analysis. The gated data are
multiplied by a Hamming window and their power spectra determined
by fast-Fourier transformation.
[0056] The power spectra from each specimen was referenced to the
power spectrum backscattered from a near-perfect steel plate
reflector to compute the apparent frequency-dependent backscatter
transfer function. The backscatter transfer function for the
acoustic reflectivity of the smooth cells, B(f), was expressed in
decibels relative to the power reflected from the steel plate:
B(f).sup.2=10 log
[V.sub.(f).sup.2.sub.tissue]/[V.sub.(f).sup.2.sub.steel plate]
where V.sub.(f).sup.2.sub.tissue is the power at selected frequency
of the gated rf backscattered from the cells and
V.sub.(f).sup.2.sub.steel plate is the power at the same frequency
of the gated rf backscattered from the steel plate. Integrated
backscatter (IB) was computed from the average of the
frequency-dependent backscatter transfer function over the useful
bandwidth of the transducer.
* * * * *