Note: Descriptions are shown in the official language in which they were submitted.
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Articles Comprising Magnetic Material and Bioactive Agents
FIELD OF THE INVENTION
The present invention is directed to the field of delivery of bioactive agents
via
nanoarticles comprising magnetic material, and the use thereof in separation
techniques,
magnetic resonance imaging, and therapeutic treatments.
STATEMENT OF RIGHTS TO INVENTIONS MADE
UNDER FEDERALLY SPONSORED RESEARCH
This invention was made with government support under a grant supported by the
National Institutes of Health. The government has certain rights in the
invention.
BACKGROUND OF THE INVENTION
Chemotherapeutics are widely used in the treatment of cancer. While somewhat
efficacious, the toxicity of the chemotherapeutics is severe and harmful,
remission is often
incomplete, and the eventual regrowth and spread of cancerous tissue is the
norm. Efforts at
localizing chemotherapeutics to the cancerous tissues, for instance through
the attachment
of chemotherapeutics to monoclonal antibodies which bind to receptors over-
expressed on
cancer cells, has thus far been a modest success.
To reduce side effects, drug molecules have been directly conjugated to
monoclonal
antibodies (mAb). These constructs are known as immunoconjugates. Despite
improvement
2o in biodistribution, immunoconjugates still struggle to deliver an effective
dose to tumors and
not to healthy tissue.
Hyperthermia, in which the temperature of cancerous tissue is raised, has
demonstrated some efficacy in treating multiple types of cancer. One way that
the
temperature can be raised is through first localizing magnetic articles within
a tumor, and
subsequently heating the magnetic articles by subjecting them to an
alternating magnetic
field. Magnetic articles used for this type of therapeutic regimen may be made
using several
methods; one such method is described by Tan, et.al., in PCT application WO
01/88540 A1.
It has been reported that certain magnetic particles can reach temperatures in
excess of 150
°C. However, to effectively heat the tumor mass, small particles
require a high level of
so accumulation that is very difFicult to achieve.
Cancer cells are under intrinsic oxidative stress and as such are vulnerable
to free
radical-induced apoptosis. The use of free radicals / free radical-producing
agents as a
therapeutic for the treatment of cancer has been investigated. For instance,
hydrogen
peroxide (H202) and superoxide anion ( O~(-) ) are known to be involved in the
cytotoxic
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action of a number of drugs (Akiyama and Natori, CancerSci., 2003, 94: 400-
404; Pelicano
et al., J Biol Chem., 2003, 278: 37832-9.) A further example is calicheamicin,
an enediyne
antibiotic capable of binding DNA which, following activation, results in
cleavage of the
double strand of the oligopyrimide-oligopurine sections of DNA. Recently, an
immunoconjugate of this drug, gemtuzumab ozogamicin, has been approved by the
FDA for
the treatment of acute myelogenous leukemia. Thus, drug formulations that
produce free
radicals have demonstrated clear therapeutic benefit. If properly targeted to
the tumor, the
potential side effects of such formulations may be minimized.
Thus, although all the above cancer treatment methods are somewhat
efficacious,
io improved cancer treatments are urgently needed.
SUMMARY OF THE INVENTION
This invention is directed to discrete particles, preferably nanoparticles,
comprising
magnetic, preferably superparamagnetic, material and a polymeric scaffold
outer layer.
Preferably, the superparamagnetic material is selected from the iron oxides,
such as
1s magnetite, maghemite, and greigite. The magnetoarticles may further
comprise bioactive
agents (such as, for example, chemotherapeutics, cytotoxics, free radical-
generating agents,
other toxic agents and other therapeutic agents). The particles of the
invention will be
referred to herein as magnetic therapeutic nanoparticles, or MTNPs.
More particularly, the invention, in a first embodiment, is directed to MTNPs
2o comprising a superparamagnetic material core encapsulated by a crosslinked
polymeric
scaffold and having least some of the bioactive agents bound to the polymer
matrix.
In a second embodiment, the MTNPs comprise a superparamagnetic material core
encapsulated by a polymer material, preferably selected from hydrophilic
polycarboxylates.
The superparamagnetic material is bound to the polymer matrix by coordination
bonds with
2s carboxylate moieties on the polymeric material. The polymer matrix further
incorporates
bioactive agent molecules, preferably chemotherapeutic molecules, within the
matrix.
Preferred chemotherapeutic molecules for this embodiment are comprised of
platinum
atoms, at least some of the platinum atoms forming coordination bonds with
carboxylate
moieties on the polymer material.
3o In a third embodiment of the invention, the MTNPs comprise a
superparamagnetic
core and a polymeric scaffold where the polymer scaffold itself is comprised
of free radical-
generating agents, the bonds of which break apart upon heating to release free
radicals. In
this embodiment, other bioactive agents may or may not also be attached to the
MTNP.
The various MTNPs of the invention may further comprise recognition el ements
(REs)
3s to facilitate targeting and/or delivery by binding to certain biomolecules
found in pathogenic
2
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tissue, such as certain cellular receptors that are overexpressed on the
surface of some
cancer cells. The articles may also optionally comprise polyethylene glycol
(PEG)-based
molecules. The PEG chains may be used to extend the circulation time of the
article in vivo;
they may also serve as linkers or tethers, with one end attached to the
article scaffold and
the other end functionalized with a recognition element or another
nanoarticle.
At least four mechanisms may localize the magnetoarticles of the invention in
a
particular environment, such as pathogenic tissue. First, the article size is
such that
preferential tumor accumulation can occur through the well-documented enhanced
permeability and retention (EPR) effect. Second, binding elements on the
surface of the
io nanoarticles can bind to tumor-associated antigens. Several different
recognition elements
(REs) may be utilized, including specific small molecule or peptide ligands,
antibodies or
antibody fragments (e.g., Fab and scFv fragments) and other proteins,
including natural
proteins and engineered proteins, as well as carbohydrates. Third, application
of a localized
magnetic field to a specific tissue may cause the retention of magnetic
nanoarticles in this
15 tissue. Fourth, application of an alternating magnetic field (AMF) of an
appropriate strength
and frequency may be directed to the pathogenic tissue and will heat the
articles in the area
of application, and less so systemically where the field is not applied, the
heating causing the
release of a bioactive agent included with the MTNP.
The MTNPs of the present invention may be used in the treatment of diseases
such
2o as cancer, inflammatory diseases, and infectious diseases. Several types of
pathogenic
tissues may be treated with the articles disclosed in the present invention
containing
appropriate bioactive agents. For instance, antibiotics may be incorporated
for the treatment
of fungal and bacterial infections, anti-inflammatory agents may be
incorporated for the
treatment of inflammatory diseases such as rheumatoid arthritis, and
chemotherapeutic
2s agents may be incorporated for the treatment of cancer.
In the case of chemotherapeutics, elevated temperature is known to increase
the
toxicity and anti-cancer potency of chemotherapeutics, so the localized
heating generated by
the magnetic material is expected to increase the potency of the released
chernotherapeutic.
The heating itself can result in the death of a portion of cancer cells in a
tumor. Additionally,
3o the heating of the tumor tissue is expected to aid in the permeation of the
chernotherapeutic
into the tumor, resulting in the killing of more cancer cells. Without an
applied magnetic field
AMF of sufficient strength and appropriate frequency range wherein the
articles are heated,
the articles of the invention will release the chemotherapeutic toxins to a
lesser extent
compared to articles subject to an alternating magnetic field. The toxicity of
the
35 chemotherapeutic both systemically and in certain organs, such as in the
heart, liver, kidney,
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and lung, can be substantially reduced by applying the alternating magnetic
field locally to
pathogenic tissue.
Advantageously, the location of the articles of the instant invention within
the
mammalian body may be determined using magnetic resonance imaging (MRI) of the
superparamagnetic cores. In addition to the utility of enabling release of
bioactive agents
under the application of heat, preferably created by an AMF, and the utility
of enhancing MRI
images, the incorporation of iron oxides into the articles of the instant
invention can be used
advantageously in several additional ways. For instance, the superparamagnetic
core allows
for expedient purification of therapeutic agent-containing MTNPs from
reactants after vari ous
to reaction and fabrication steps. For instance, therapeutic-loaded MTNPs may
be separated
from unattached therapeutics by using a separation scheme wherein after the
therapeutic
incorporation procedure is completed, MTNPs are retained in a reaction vessel
by a
permanent magnetic field, while the solution (with therapeutic agents that are
unassociated
with the articles) is decanted off, siphoned off, or otherwise removed.
15 Thus, the invention is further directed to methods of using these MTNPs, as
well as to
methods of synthesizing the articles. The invention is also directed to a
composition for
treatment of pathogenic tissue comprising a plurality of MTNPs, each MTNP
having a
superparamagnetic material core and a polymeric outer layer and optionally, a
bioactive
agent and/or recognition elements, and to a therapeutic drug delivery system
comprising
2o such a MTNP-containing composition.
DETAILED DESCRIPTION OF THE INVENTION
The terms "a" and "an" mean "one or more" when used herein.
By "soluble" is meant, herein and in the appended claims, having a solubility
in water
of greater than 1 mg/mL, preferably greater than 10 mg/mL, and more preferably
greater
2s than 50 mg/mL.
Disclosed herein are MTNPs comprising magnetic material, a polymeric outer
layer or
scaffold encapsulating the magnetic material, and bioactive agents.
Preferred magnetic cores are superparamagnetic, which are magnetic only when a
magnetic field is applied. Preferred superparamagnetic materials are iron
oxides, such as
3o magnetite, maghemite, and greigite.
The MTNP of the invention may be further comprised of targeting agents or
recognition elements that bind to certain biomolecules found in pathogenic
tissue, for
instance cancerous tissue, such as certain cellular receptors that are
overexpressed on the
surface of some cancer cells, including growth factor receptors, or in the
tumor vasculature,
35 such as integrins or growth factor receptors. Targeting agents that can be
used include, but
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are not limited to, small molecules; vitamins, such as folate; peptides, such
as those that
target receptors; proteins, such as transferrin; and monoclonal antibodies and
monoclonal
antibody fragments. Unless otherwise specifically indicated, the terms
"targeting agent"
"recognition element" are used interchangeably herein.
While the MTNPs of the invention may be larger in size, they are preferably
from
about 5 nm to about 1000 nm, more preferably from about 10 nm to about 500 nm,
in
diameter. Because of their size and structure, the nanoarticles may circulate
in the blood
stream without being eliminated by the kidney or taken up by the reticulo-
endothelial system
system, and may localize in pathological tissue via passage through the
pathological tissue's
io leaky vasculature; the incorporation of targeting agents can further
increase article
accumulation in tissue to be treated, as described below. Larger MTNPs may be
better
retained in a desired location in the body, for instance in pathological
tissue, through the
application of a high strength non-oscillating magnetic field.
Magnetorticles of the present invention can be made through the incorporation
of
1s bioactive agents, such as chemotherapeutic molecules or free radical-
generating agents,
with certain iron oxide articles. Iron oxides may be incorporated into the
articles in several
ways. Magnetic articles useful in the present invention include dextran-
magnetite
nanoparticles. These particles may be fabricated or may be purchased, for
instance from
companies such as Micromod Partikeltechnologie (Rostock, Germany).
Magnetosomes from
2o bacteria may also be used in the invention. In most magnetotactic bacteria,
magnetosomes
comprise membrane-bound crystals of magnetite, Fe304. Predominantly, members
of the
bacterial species Magnetospirillum form these highly ordered magnetite
crystals to allow their
orientation within the earth's magnetic field. The bacterial magnetosomes are
characterized
by narrow size distributions and uniform crystal habits, which are unknown
from magnetite
25 particles produced abiotically. Magnetosomes range in size from 35-100 nm.
Magnetic Colloid Formation: Magnetic cores, such as those comprised of
magnetite or
maghemite, can be formed using reverse microemulsions. For instance, Fe(II) is
prepared
by dissolving FeS047H20 in water and an Fe(III) solution is prepared by
dissolving
so FeC136HZ0. These aqueous phases can then be combined in a reverse
microemulsion.
Addition of a base results in the formation of a magnetite colloid. This
colloid can then be
incorporated into a polymeric matrix as described below. Prior to
incorporation into the
polymer matrix, the magnetic colloid can be encapsulated in silica, and may be
further
functionalized via alkoxysilane derivatives to provide desired surface
functionalities, such as
3s acids and amines that can provide greater water solubility, covalent
ligation to chemo-loaded
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articles, and/or facilitate hydrogel incorporation. Silica encapsulation is
discussed by
Grasset, et.al. in European Cells and Materials, Vol. 3, Suppl. 2, 2002, pp.
110-113.
The iron oxide colloids may also be formed following the procedure published
by
Hyeon, et al. (J Am Chem Soc (2001) 123 12798-12801). Compared to other
methods, this
method allows careful control of the iron oxide surface chemistry. This method
results in
oleic acid coupled to the surface of the iron oxide with a carboxylate moiety
forming
coordination bonds with iron atoms on the surface of the iron oxide core. The
hydrocarbon
chain of the oleic acid is then exchanged for a water-soluble coating, for
instance with
hydrophilic polycarboxylates, to provide for aqueous solubility.
Article Scaffold Fabrication in Reverse Microemulsions: Components of the
present
invention can be fabricated using reverse microemulsions. In one embodiment,
magnetic
colloids are formed in the dispersed aqueous phase of a reverse microemulsion.
Next,
polymer scaffolds are formed around the magnetic core through the addition of
hydrophilic
building blocks to the reverse microemulsion containing the magnetic colloids.
The
hydrophilic building blocks are then polymerized, forming a magnetic colloid-
containing
hydrogel nanoarticle. The organic solvent and non-reactive surfactants are
removed after
polymerization to yield crosslinked, water-soluble, iron oxide-cored
nanoscopic articles.
Bioactive agents, if included in the final nanoarticle, may be incorporated
either during or
2o after hydrogel formation.
As used herein, the terms "nanoarticle scaffold", "hydrogel scaffold" and
"scaffold" are
used interchangeably and refer to the portion of the nanoarticle (the
polymeric matrix
structure) that incorporates the magnetic material.
Reverse microemulsions for magnetic colloid and scaffold fabrication are
formed by
2s combining aqueous buffer or water, building blocks, organic solvent,
surfactants and initiators
in the appropriate ratios to yield a stable phase of surfactant-stabilized
aqueous nanodroplets
dispersed in a continuous oil phase. Stable reverse microemulsion formulations
can be
found using known methods by those skilled in the art. They are discussed, for
example, in
Microemulsion Systems, edited by H. L. Rosano and M. Clausse, New York, N.Y.,
M.
3o Dekker, 1987; and in Handbook of Microemulsion Science and Technology,
edited by P
Kumar and K.L. Mittel, New York, N.Y., M. Dekker, 1999. In this invention, an
aqueous
phase with solubilized hydrophilic building blocks is added to an organic
solvent containing
one or more solubilized surfactants to form a reverse microemulsion.
The dispersed aqueous phase includes hydrophilic building blocks solubilized
at
35 about 1 to about 65 wt%, preferably about 5 to about 25 wt%, most
preferably 10 to 20 wt%.
While not wishing to be bound by theory, the use of high water-content
hydrogel scaffolds
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also may reduce immunogenicity in end uses, because there is less foreign
surface for
immune system components to recognize. The high water content also provides
compliancy
through a more flexible scaffold. Thus, when attaching to cell surface
receptors, the articles
are able to conform to the cell surface, allowing more surface receptors to be
bound. Binding
more receptors may allow the article to better function to localize in desired
tissues and bind
to desired cells. Additionally, while not wishing to be bound by theory, it is
believed that
article cell surface coverage can inhibit other cell signaling pathways.
Polymerization of the building blocks in the nanodroplets of the dispersed
aqueous
phase of the reverse microemulsion follows procedures known to those skilled
in the art (see,
io for example, Odian G.G., Principles ofPolymerization, 3rd Ed., Wiley, New
York, 1991; L.H.
Sperling; Introduction to Physical Polymer Science, Chapter 1, pp. 1-21, John
Wiley and
Sons, New York, 1986; and R.B. Seymour and C.E. Carraher, Polymer Chemistry,
Chapters
7-11, pp. 193-356, Dekker, New York, 1981). Polymerization has been performed
in the
dispersed phase of microemulsions and reverse microemulsions (for a review,
see Antonietti,
1s M.; and Basten, R., Macromol. Chem. Phys. 1995, 196, 441; for a study of
the polymerization
of a hydrophilic monomer in the dispersed aqueous phase of a reverse
microemulsion, see
Holtzscherer, C.; and Candau, F., Colloids and Surfaces, 1988, 29, 411). Such
polymerization may yield articles in the 5 nm to 50 nm size range.
The size of the nanodroplets of the dispersed aqueous phase is determined by
the
2o relative amounts of water, surfactant and oil phases employed. Surfactants
are utilized to
stabilize the reverse microemulsion. These surfactants do not include
crosslinkable moieties;
they are not building blocks. Surfactants that may be used include
commercially available
surfactants such as Aerosol OT (AOT), polyethyleneoxy(n)nonylphenol (IgepaITM,
Rhodia Inc.
Surfactants and Specialties, Cranbrook, NJ), sorbitan esters including
sorbitan monooleate
2s (Span~ 80), sorbitan monolaurate (Span~ 20), sorbitan monopalmitate (Span~
40), sorbitan
monostearate (Span~ 60), sorbitan trioleate (Spank 85), and sorbitan
tristearate (Span~
65), which are available, for example, from Sigma (St Louis, MO). Sorbitan
sesquioleate
(Span~ 83) is available from Aldrich Chemical Co., Inc. (Milwaukee, WI). Other
surfactants
that may be used include polyoxyethylenesorbitan (Tween~) compounds, including
3o polyoxyethylenesorbitan monolaurate (Tween~ 20 and Tween~ 21 ),
polyoxyethylenesorbitan monooleate (Tween~ 80 and Tween~ 80R),
polyoxyethylenesorbitan monopalmitate (Tween~ 40), polyoxyethylenesorbitan
monostearate (Tween~ 60 and Tween~ 61 ), polyoxyethylenesorbitan trioleate
(Tween~ 85),
and polyoxyethylenesorbitan tristearate (Tween~ 65), which are available, for
example, from
3s Sigma (St Louis, MO). Other exemplary commercially available surfactants
include
polyethyleneoxy(40)-sorbitol hexaoleate ester (Atlas G-1086, ICI Specialties,
Wilmington
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DE), hexadecyltrimethylammonium bromide (CTAB, Aldrich), and linear
alkylbenzene
sulfonates (LAS, Ashland Chemical Co., Columbus, OH).
Other exemplary surfactants include fatty acid soaps, alkyl phosphates and
dialkylphosphates, alkyl sulfates, alkyl sulfonates, primary amine salts,
secondary amine
salts, tertiary amine salts, quaternary amine salts, n-alkyl xanthates, n-
alkyl ethoxylated
sulfates, dialkyl sulfosuccinate salts, n-alkyl dimethyl betaines, n-alkyl
phenyl
polyoxyethylene ethers, n-alkyl polyoxyethylene ethers, sorbitan esters,
polyethyleneoxy
sorbitan esters, sorbitol esters and polyethyleneoxy sorbitol esters.
Other surfactants include lipids, such as phospholipids, glycolipids,
cholesterol and
1o cholesterol derivatives. Exemplary lipids include fatty acids or molecules
comprising fatty
acids, wherein the fatty acids include, for example, palmitate, oleate,
laurate, myristate,
stearate, arachidate, behenate, lignocerate, palmitoleate, linoleate,
linolenate, and
arachidonate, and salts thereof such as sodium salts. The fatty acids may be
modified, for
example, by conversion of the acid functionality to a sulfonate by a coupling
reaction to a
1s small molecule containing that moiety, or by other functional group
conversions known to
those skilled in the art.
Additionally, polyvinyl alcohol (PVA), polyvinylpirolidone (PVP), starch and
their
derivatives may find use as surfactants in the present invention.
Cationic lipids may be used as cosurFactants, such as cetyl trimethylammonium
2o bromide/chloride (CTAB/CTAC), dioctadecyl dimethyl ammonium
bromide/chloride
(DODAB/DODAC), 1,2-diacyl-3-trimethylammonium propane (DOTAP), 1,2-diacyl-3-
dimethyl
ammonium propane (DODAP), [2,3-bis(oleoyl)propyl] trimethyl ammonium chloride
(DOTMA), and [N-(N'-dimethylaminoethane)-carbamoyl]cholesterol, dioleoyl) (DC-
Chol).
Alcohols may also be used as cosurfactants, such as propanol, butanol,
pentanol, hexanol,
2s heptanol and octanol. Other alcohols with longer carbon chains may also be
used.
Polymer Encapsulant Formation in Reverse Microemulsions: In one embodiment of
the
invention, a crosslinked polymeric scaffold that incorporates magnetic
material is formed by
crosslinking builiding blocks around iron oxide colloids in the dispersed
aqueous phase of a
3o reverse microemulsion. Hydrophilic building blocks with polymerizable
groups are employed
to form stable nanoarticle scaffolds. In this embodiment, preferred building
blocks are
comprised of carbohydrate or derivatized carbohydrate. For example, the
carbohydrate
region may be derived from simple sugars, such as N-acetylglucosamine, N-
acetylgalctosamine, N-acetylneuraminic acid, neuraminic acid, galacturonic
acid, glucuronic
3s acid, ioduronic acid, glucose, ribose, arabinose, xylose, lyxose, allose,
altrose, apiose,
mannose, gulose, idose, galactose, fucose, fructose, fructofuranose, rhamnose,
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arabinofuranose, and talose; a disaccharide, such as maltose, sucrose,
lactose, or trehalose;
a trisaccharide; a polysaccharide, such as cellulose, starch, glycogen,
alginates, inulin,
pullulan, dextran, dextran sulfate, chitosan, glycosaminoglycans, heparin,
heparin sulfate,
hyaluronates, tragacanth gums, xanthan, other carboxylic acid-containing
carbohydrates,
uronic acid-containing carbohydrates, lactulose, arabinogalactan, and their
derivatives, and
mixtures of any of these; or modified polysaccharides. Other representative
carbohydrates
include sorbitan, sorbitol, chitosan and glucosamine.
The carboxyl, amine and hydroxyl groups of the carbohydrates can be modified,
or
replaced, to include crosslinking groups, other functionalities, or
combinations thereof.
1o Carbohydrate-based building blocks may be prepared from the carbohydrate
precursor (e.g., sucrose, inulin, dextran, pullulan, etc.) by coupling
technologies known in the
art of bioorganic chemistry (see, for example, G Hermanson, Bioconjugation
Techniques,
Academic Press, San Diego, 1996, pp 27-40,155, 183-185, 615-617; and S.
Hanesian,
Preparative Carbohydrate Chemistry, Marcel Dekker, New York, 1997). For
example, a
1s crosslinkable group may be attached to a carbohydrate via the dropwise
addition of acryloyl
chloride to an amine-functionalized sugar. Amine-functionalized sugars can be
prepared by
the reaction of ethylene diamine (or other amines) with 1,1'-
carbonyldiimidazole-activated
sugars. Ester-linked reactive groups can be synthesized through the reaction
of acrylic or
methacrylic anhydrides with the hydroxyl group of a carbohydrate such as
inulin in pyridine.
2o Aldehyde- and ketone-functionalized carbohydrates can be obtained by
selective reduction of
the sugar backbone or addition of a carbonyl-containing moiety. Other
reactions that
introduce an amine on the carbohydrate may also be used, many of which are
outlined in
Bioconjugation Techniques (supra).
Carbohydrate-based building blocks may also be prepared by the partial (or
2s complete) functionalization of the carbohydrate with moieties that are
known to polymerize
under free radical conditions. For example, methacrylic esters may be placed
on a
carbohydrate at varying substitution levels by the reaction of the
carbohydrate with
methacrylic anhydride or glycidyl methacrylate (Vervoort, L.; Van den Mooter,
G.; Augustijins,
P.; Kinget, R. International Journal of Pharmaceutics, 1998, 172, 127-135).
3o Carbohydrate-based building blocks may also be prepared by chemoenzymatic
methods (Martin, B. D. et. al., Macromolecules, 1992, 25, 7081), for example
in which
Pseudomonas cepacia catalyzes the transesterification of monosaccharides with
vinyl
acrylate in pyridine or by the direct addition of an acrylate (Piletsky, S.,
Andersson, H.,
Nicholls, Macromolecules, 1999, 32, 633-636). Other functional groups may be
present, as
3s numerous derivatized carbohydrates are known to those familiar with the art
of carbohydrate
chemistry.
9
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Carbohydrates may be derivatized with allyl functionalities, including
acrylates,
methacrylates, acrylamides and methacrylamides to produce compounds such as
inulin
multi-methacrylate (IMMA). In a presently preferred embodiment, inulin with an
average
degree of polymerization (DOP) of about 10 to about 20 is used. The extent to
which inulin is
functionalized with methacrylate moieties, that is, the number of hydroxyl
moieties on inulin
that are converted to methacrylic esters to produce IMMA, is a statistical
process governed
by the concentrations and weight ratios of inulin and methacrylic anhydride
starting material.
The extent of functionalization may range from one methacrylate for every 1 to
100
monosaccharide repeat units, more preferably one methacrylate for every 3 to
20
1o monosaccharide repeat units. The number of monosaccharide repeat units in
the IMMA may
be from about 1 to about 100 or more, and is preferably from about 5 to about
50. The ester
linkage to inulin may advantageously function as a site of degradation in
vivo, allowing the
article to degrade and be cleared from the body. Dextran multimethacrylamide
and pullulan
multimethacrylamide are additional preferred building blocks that may be
prepared using
15 methods similar to those for preparing IMMA. MTNP scaffolds may also be
formed using
inulin multibenzaldehyde or oxidized dextran, each of which may be synthesized
by methods
known in the art.
The carbohydrate structures are chosen in part for their hydrophilicity. Nano-
articles
that incorporate magnetic cores must possess highly hydrophilic scaffolds in
order that high
2o water solubility is maintained. MTNPs of the invention in one embodiment
have a high water
content for high water solubility. "High water content", as used herein, means
an article
comprised of about 65 to about 98 wt% water, more preferably about 75 to about
98 wt%
water, and most preferably about 80 to 97 wt% water. Thus, the amount of
breakdown
products is less than articles with a higher polymer concentration. The high
water content
25 scaffolds also can reduce immunogenicity, because there are fewer surfaces
for immune
system components to interact with.
Besides carbohydrate-based building blocks, other examples of acrylate- or
acrylamide-derivatized polymeric building blocks include polyethylene glycol-
based
molecules, such as polyethyleneglycol multiacrylates of molecular weights
ranging from 200
3o to 10,000 daltons.
In one embodiment of the invention, to facilitate metabolism of the MTNP
scaffold,
degradable linkages are included within the crosslinked scaffold. Degradable
linkages can
be included through the use of polylactide, polyglycolide, poly(lactide-co-
glycolide),
polyphosphazine, polyposphate, polycarbonate, polyamino acid, polyanhydride,
and
3s polyorthoester- based building blocks, among others. Additionally,
degradable linkages
may be used to attach polymerizable moieties to carbohydrates. For instance,
inulin multi-
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methacrylate (IMMA) contains ester moieties that connect the inulin
carbohydrate backbone
to the alkyl chain that is formed upon free radical polymerization used to
generate the
scaffold of the present invention. Additionally, small molecule crosslinking
agents containing
similar hydrolyzable moieties as the polymers such as carbonates, esters,
urethanes,
orthoesters, amides, imides, imidoxy, hydrazides, thiocarbazides, and
phosphates may be
used as building blocks. To function as degradable components in the hydrogel
scaffold,
these building blocks must be functionalized with two or more polymerizable
moieties. For
example, polyglycolide diacrylate, polyorthoester diacrylate and acrylate-
substituted
polyphosphazine, acrylate-substituted polyamino acid, or acrylate-substituted
polyphosphate
1o polymers can be used as degradable building blocks.
Methacrylate or acrylamide moieties can be employed instead of acrylate
moieties in
the above examples. Similarly, small molecules containing a hydrolyzable
segment and two
or more acrylates, methacrylates, or acrylamides may be used. Such degradable
polymers
and small molecule building blocks may be functionalized with acrylate,
methacrylate,
15 acrylamide or similar moieties by methods known in the art.
Other agents can also be incorporated into the polymer matrix. These agents or
"functional building blocks" have reactive groups, and such functional
building blocks include,
but are not limited to, N,N'-cystinebisacrylamide (CiBA), sodium acrylate
(NaA), N-(3-
aminopropyl)methacrylamide hydrochloride (APMA), N[ethylamino]-3-amino-
2o propylmethacrylamide hydrochloride, polyethylene imine (PEI), polylysine,
polyamido-
acrylamide derivatives, and protamine sulfate. The composition of the
nanoarticles can be
manipulated using functional building blocks to produce articles with a
desirable
characteristic, such as charge (positive, negative or neutral) or degree of
crosslinking.
Additionally, functional building blocks may be chosen to achieve a desired
content of certain
25 functionalities in the article scaffold. Such functionalities can improve
solubility and may also
be used as points of attachment for REs or PEG chains. For instance, APMA may
be used
to introduce amines, sodium acrylate may be used to introduce carboxylates,
and diacetone
acrylamide (DAA) may be used to incorporate ketones. The disulfide linkage of
the CiBA
monomer, which has the following formula I, provides, after reduction, free
thiols for linker
30 attachment.
CO~ Na+ O
N /S~
~S ~ \N
H
O COZ Na+
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WO 2005/070471 PCT/US2005/001755
The MTNP scaffolds and the scaffold breakdown products of this invention are
designed to be non-toxic and eliminated from the body. They may have
degradable,
preferably carbohydrate-based, polyamino acid-based, polyester-based, or PEG-
based
cores, with the rate of degradation controlled by the identity of the sugar,
crosslink density,
and other features. Thus, the articles can be metabolized in the body,
preventing
undesirable accumulation in the body.
Polymer Encapsulant Formation in Aqueous Solutions. In one preferred
embodiment,
to polycarboxylate chains of a polymer scaffold are attached via coordination
bonds of a
fraction of the carboxylate moieties to iron atoms in the iron oxide colloid
core. This
attachment can be accomplished in an aqueous solution.
Incorporation of Bioactive Agent Molecules into Articles: The terms "bioactive
agent"
is and "therapeutic agent" are used interchangeably herein and include, but
are not limited to,
chemotherapeutics, cytotoxics, free radical-generating agents, other drugs and
toxic agents,
and other therapeutic agents. A MTNP may comprise from one to up to 100 or
more
bioactive agents. A "plurality of bioactive agents" in a MTNP may all be the
same bioactive
agent or they may be two or more different bioactive agents; that is, the MTNP
may comprise
2o a plurality of one or more types of bioactive agents.
Drugs that may find use in the present invention include those that act on the
peripheral nerves, adrenergic receptors, cholinergic receptors, nervous
system, skeletal
muscles, cardiovascular system, smooth muscles, blood circulatory system,
synaptic sites,
neuro-effector functional sites, endocrine system, hormone systems,
immunological system,
2s reproductive system, skeletal system, autocoid systems, alimentary and
excretory systems,
histamine systems, respiratory system, reticuloendothelial system, skeletal
system, skeletal
muscles, smooth muscles, immunological system, reproductive system, cancerous
tissues,
and the like. The active drug that can be delivered for acting on these
recipients includes, but
is not limited to, anticonvulsants, analgesics, anti-parkinsons, anti-
inflammatories, calcium
3o antagonists, anesthetics, antimicrobials, antimalarials, antiparasitics,
antihypertensives,
antihistamines, antipyretics, alpha-adrenergic agonists, alpha-blockers,
biocides,
bactericides, bronchial dilators, beta-adrenergic blocking drugs,
contraceptives,
chemotherpeutics, cardiovascular drugs, calcium channel inhibitors,
depressants,
diagnostics, diuretics, electrolytes, enzymes, hypnotics, hormones,
hypoglycemics,
3s hyperglycemics, muscle contractants, muscle relaxants, neoplastics,
glycoproteins,
nucleoproteins, lipoproteins, ophthalmics, psychic energizers, sedatives,
steroids,
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WO 2005/070471 PCT/US2005/001755
sympathomimetics, parasympathomimetics, tranquilizers, urinary tract drugs,
vaccines,
vaginal drugs, vitamins, nonsteroidal anti-inflammatory drugs, angiotensin
converting
enzymes, polynucleotides, polypeptides, polysaccharides, and the like.
In a presently preferred embodiment, drugs that may be advantageously employed
in
the present invention include, but are not limited to, chemotherapeutics such
as cisplatin,
oxaliplatin, doxorubicin, paclitaxel, gemcitibine, vincristine, chlorambucil,
topotecan,
methotrexate, bortezomib, and any other FDA-approved chemotherapeutic, as well
as
molecules that may act as chemotherapeutics but that are not yet
commercialized, and
derivatives and analogues of all of the above chemotherapeutics.
For example, the chemotherapeutic doxorubicin may be attached to the scaffold
through a EDC coupling reaction between the amine moiety on doxorubicin and a
carboxylic
acid moiety included in the hydrogel scaffold, for example by using sodium
acrylate (NaA),
malonate acrylamide (MalAc) or N,N'-cystinebisacrylamide (CiBA; synthesis
described in
PCT Publn. WO 03/101425) as a building block. In another embodiment,
doxorubicin may
1s be attached via an imine bond by reacting doxorubicin's amine moiety with
an aldehyde
moiety of the hydrogel scaffold. An aldehyde may be created by first using a
carbohydrate-
based building block to form the article, and then oxidizing the carbohydrate
after the article
is formed. In another embodiment, doxorubicin may be attached to the article
matrix through
its ketone moiety.
2o Carbohydrazide or other dihydrazide or di-amino-oxy functionalized
structures may be
used to link doxorubicin to a scaffold that contains an aldehyde or ketone
through the
formation of a hydrazone bond. An aldehyde or ketone may be incorporated into
the scaffold
through the use of a ketone-containing acrylate building block such as
diacetone acrylamide
(DAA). A hydrazone bond may favorably release the therapeutic compound under
the mildly
2s acidic physiological conditions encountered upon article endocytosis and
entrance into
lysosomes.
In another embodiment, nanoarticle scaffolds comprised of amino groups, for
example through the inclusion of N-(3-aminopropyl)methacrylamide (APMA) or
methacrylate-
functionalized short peptide (prepared according to US patent 5,037,883)
building blocks,
3o may be used to covalently attach cyclosporins that contain carboxylate
linkages. Cyclosporin
drugs may find applications for pathologies that benefit from
immunosuppression, such as
inflammatory diseases, and for organ transplantation.
In another embodiment, nanoarticle scaffolds comprised of aldehyde or ketone
groups (for example incorporated through the inclusion of DAA, levulinic
acrylamide or
3s oxidized carboxylates such as inulin or dextran building blocks) may be
used to covalently
attach drugs or drug derivatives that contain a moiety, for instance
calicheamicin, through the
13
CA 02553647 2006-07-18
WO 2005/070471 PCT/US2005/001755
use of a hydrazone coupling scheme. This coupling scheme results in a
hydrazone bond
that is hydrolytically labile, especially at low pH found in lysosomes
(Bernstein L, et al.,
Bioconjugate Chem., 2002, 13, 40-46).
In another embodiment, nanoarticle scaffolds comprised of acid or anhydride
groups,
for example incorporated through the inclusion of sodium acrylate or anhydride
building
blocks, may be used to covalently attach dexamethasone, through the use of an
amide
coupling scheme.
Nanoarticle scafFolds comprised of carboxylate groups (for example,
incorporated
through the inclusion of NaA, CiBA or MAIAc building blocks) may be used to
covalently
1o attach drugs or drug derivatives that contain an amine moiety, for instance
peptide-modified
camptothecin (Frigerio E., et al., J. Controlled Release, 2000, 65, 105-119)
through an EDC-
NHS coupling scheme. Carboxylate moieties can also be employed to form
coordination
bonds with platinum atoms found in bioactive platinum molecules.
In another embodiment, when the nanoarticle scaffolds are comprised of
aldehyde or
15 ketone groups (which may be incorporated through the use of DAA, levulinic
acrylamide or
oxidized carboxylates such as inulin or dextran), a drug or drug derivative
possessing an
amine, such as gemcitabine, may be incorporated through the use of a "Schiff
base" coupling
scheme. The imide bond formed from the attachment of gemcitabine to DAA can be
cleaved
in acidic media. During internalization, the drug is taken up by the cell,
where it is exposed to
2o the acidic environment of the lysosome, thereby releasing gemcitabine in
its unmodified
form.
In another embodiment, nanoarticle scaffolds comprised of carboxyl groups (for
example, incorporated through the inclusion of CiBA, MalAc or NaA building
blocks) may be
used to covalently attach drugs or drug derivatives that contain a moiety, for
instance
25 salicylic acid, through the use of an EDGNHS coupling scheme. For instance,
the hydroxyl
group of salicylic acid will react with the carboxyl group of the CiBA, MalAc
or NaA to form an
ester link. Hydrolysis or the enzyme esterase will cleave the ester bond
between salicylic
acid and the carboxylic acid groups of CiBA or NaA, releasing salicylic acid
in an unmodified
form.
3o In another embodiment, a drug structure may be modified to facilitate
attachment to a
nanoarticle scaffold. For instance, the 2'-hydroxyl group of paclitaxel can be
reacted with
multiple linkers that enable the coupling to nanoarticle scaffolds. For
example, the acid
moiety of a resin-immobilized glycine linker can be attached to paclitaxel
using a
carbodiimide; the resulting compound can be cleaved at the site of the amine
using 1 % TFA,
3s producing a free amine which can be conjugated with nanoarticles possessing
carboxylates
using an EDC coupling scheme.
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WO 2005/070471 PCT/US2005/001755
In another embodiment, paclitaxel-2'-succinate (Deutsch H., et al., J Med.
Chem.,
1989, 32, 788-792) conjugation to the nanoarticle is possible using a
carbodiimide-mediated
amide coupling. This coupling occurs between the paclitaxel-2'-succinate group
and an
amine group of the APMA component of the nanoarticle to form a labile ester.
In another embodiment, the nanoarticle can be directly coupled to paclitaxel
by
reacting the acid-functionalized (e.g., NaA) nanoarticle to the 2'-hydroxyl
group of paclitaxel.
This chemical pathway has been previously described using a poly(L-glutamic
acid)-
paclitaxel conjugate (Li H., et al., Cancer Res., 1998, 58, 2404-2409).
In another embodiment, nanoarticle scafFolds containing carboxylic acids, for
example
io incorporated through the inclusion of NaA building blocks, may be used to
covalently attach
drugs or drug derivatives that contain a moiety, for instance 5-flourouracil
(5FU) (or
derivatives) through the use of an amide-forming coupling reaction between an
amine-
functionalized 5FU derivative and the carboxylic acids located on the
nanoarticle. The
synthesis of 1-alkylcarbonyloxymethyl derivatives of 5FU has been previously
described and
15 those materials have been demonstrated to release 5FU in an unmodified form
(Taylor H.E.;
Sloan K.B., Journal ofPharmaceutical Sciences, 1998, 87, 15). The application
of this
synthetic route will yield the necessary amine-functionalized 5FU, whilst
realizing a similar
release profile.
Nanoarticle scaffolds comprised of carboxylate groups (for example
incorporated
2o through the inclusion of CiBA, MalAc and NaA building blocks) may be
further used to
covalently attach drugs or drug derivatives that contain a carboxylate moiety,
for instance
methotrexate, by first coupling the drug or drug derivative to boc-protected
ethanolamine to
form an ester, and then coupling to the nanoarticle through an EDC coupling
scheme after
deprotecting the modified drug. This ester conjugate is known to hydrolyze at
low pH,
25 releasing the drug in its original form (Wilson J.M., et al., Biochem
Biophys. Res. Commun.,
1992, 184, 300-305; Ohkuma S., Poole B., Proc. Natl. Acad. Sci. USA, 1978, 75,
3327-
3331). Such conditions of low pH are found in cellular lysosomes. These
nanoarticles may
find use in the treatment of multiple pathologies, including cancer and
inflammatory
conditions such as rheumatoid arthritis and inflammatory bowel disease.
3o In another embodiment, platinum is complexed to the nanoarticle polymer
matrix via
O,N-ligation, which is expected to yield a more stable compound. This can be
accomplished
preferentially for nanoarticles obtained by free-radical polymerization, and
containing a
combination of acid functions (such as from NaA) and amines (such as from
APMA) or amide
moieties (such as acrylamide), or building blocks carrying both types of
functions such as
35 CIBA, MalAc or methacryloylate-functionalized short peptides made according
to US
CA 02553647 2006-07-18
WO 2005/070471 PCT/US2005/001755
5,037,883. Such moieties provide attachment points to generate a O,N-cis
platinum
nanoarticle conjugate, and also allow the possibility of targeting the
nanoarticle.
In all embodiments, the bonds attaching the bioactive agents to the MTNP
scaffold
are heat-labile and are substantially disrupted at moderate temperatures, such
that the
bioacfiive agenfis are released from the articles upon application of moderate
heat.
Platinum Incorporation into Polycarboxyate Scaffold. In a preferred embodiment
an iron
oxide colloid is encapsulated by a polymer-platinum complex. The polymer is
preferably a
polycarboxylate comprised of multiple carboxylate moieties. As described
previously herein,
to the carboxylates can bind to surface iron atoms. In addition, carboxylates
that are not bound
to surface iron atoms can be used to form coordination bonds to platinum
molecules.
As referred to herein, "platinum", typically used in the context of a platinum
complex
or compounds, refers to a platinum metal atom bound to one or more ligands.
The platinum
atom may carry a formal charge, such as in the case of platinum salts such as
K~PtCl4,
1s potassium tetrachloroplatinate, in which the platinum carries a formal
charge of (-2), or may
carry no formal charge, as in cisplatin, PtCl2(NH3)2. The platinum metal atom
may exist in
various oxidation states, such as Pt(0), Pt(II), or Pt(IV). The platinum
species can be in any
coordination state, but is typically four-coordinate for Pt(II) complexes and
six-coordinate for
Pt(IV) complexes.
2o Presently preferred platinum chemotherapeutic agents are in the II"d or
IVt" oxidation
state. In one preferred embodiment, platinum (II) compounds are incorporated
into the article
through carboxylate coordination bonds with the polymer scaffold and can,
preferably, act as
a crosslinking agent. Preferred platinum (II) compounds are of the general
formula cis-
[PtX2(NHR~R~)(NHR3R4)] where X is an anion, such as chloride or nitrate ion,
where one or
2s both of the anions coordinated with a particular platinum atom are
displaced by carboxylate
ligands in the process of incorporation into the nanoarticles of the
invention. Each of R~, R2,
R3, and R4 is independently selected from the group consisting of hydrogen,
lower alkyl
unsubstituted or substituted with a halo group or an alkyl group, lower
alkenyl unsubstituted
or substituted with a halo group or an alkyl group, lower cycloalkyl
unsubstituted or
3o substituted with a halo group or an alkyl group, and lower cycloalkenyl
unsubstituted or
substituted with a halo group or an alkyl group, or R~ and R~ together form an
alkyl or an
alkenyl bridge, or R3 and R4 together form an alkyl or alkenyl bridge.
In another embodiment of the invention, multinucleate platinum agents are
incorporated into the nanoarticles. Such agents may form more stable networks
by forming
3s three or more coordination bonds with polyanion components.
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WO 2005/070471 PCT/US2005/001755
Preferred scaffold polymers for platinum incorporation are comprised of
multiple
carboxylate moieties ("polycarboxylates"). The carboxylate moieties of this
polymer can
serve multiple purposes, first to form coordination bonds with platinum and
with the magnetic
colloids, as well as play an active role in favoring the nanoarticle
localization and/or
preferential uptake by cancer cells, or have intrinsic antiproliferation,
antiangiogenic or
general anticancer properties.
Polycarboxylate materials that may be used in the instant invention include
carbohydrates with each chain comprised of multiple carboxylate moieties.
Polycarboxylate
materials that may be used in the instant invention include acrylic polymers
with carboxylate
io side chains, such as polyacrylate and polymethacrylate. Polyethylene glycol
can be attached
to polycarboxylates to allow extended circulation, reduced binding by
opsonization-promoting
biomolecules and reduced reticulo-endothelial system uptake in vivo, as is
well-described in
the literature, and also serve as a tether for attaching targeting ligands or
bioactive agent-
containing nanoarticles to the MTNPs. Preferred copolymers for use in the
instant invention
1s are polyethylene glycol (PEG) - polycarboxylates, more preferably
polyethylene glycol -
poly(meth)acrylates, and most preferably polyethylene glycol -
polymethacrylate (sodium
salt) or polyethylene glycol - polyacrylate (sodium salt). For the PEG chain,
a preferred
molecular weight is in the range of 2000 to 10,000 Daltons. For the acrylic
polymer, a
preferred molecular weight is in the range of 2000 to 6000 Daltons.
2o Preferred carbohydrate polycarboxylates are inulin derivatives, polysialic
acid,
hyaluronic acid, and colominic acid. Pectins and alginates, both multi-acid
carbohydrates
that have been used for various surgicalideviceldelivery purposes, are avoided
due to their
high viscosity and lack of information on their use in circulating materials.
Additional
carbohydrates, particularly those with targeting or anti-cancer properties
unto themselves,
2s such as schizophyllan, also may be employed.
Inulin, consisting mainly of linear (3-1,2 linked polyfructose with a
glucopyranose unit
at the reducing end, has been used extensively as an i.v. injection to assess
kidney function.
It can be readily modified with carboxylate groups through the use of cyclic
anhydrides such
as succinic anhydride and aconitic anhydride. Presently preferred
functionalization levels
3o include one acid group per saccharide repeat. Inulin with 10 repeats (that
is, Degree of
Polymerization (DP) = 10) to DP = 70 or higher may be used.
Polysialic acid (a-2,8 linkage) is very water soluble, behaving similarly to
inulin.
Polysialic acid is commercially available at a MW of 10,000 Da, which is a
good size for
constructing the nanoarticles of the instant invention. Advantages suggesting
use are its
35 natural occurrence in humans and its use as "nature's stealth" by bacteria.
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WO 2005/070471 PCT/US2005/001755
Hyaluronic acid, a [3-1,4 linked D-glucuronic acid and ~i-1,3 linked N-acetyl-
D-
glucosamine polysaccharide, is commonly used for ophthalmic surgery and as an
injectable
into joints to ease osteoarthritis. Several types of cancer strains are
characterized by an
altered metabolism of hyaluronic acid, which can be used for advantageous
purposes. In
particular, some cancer cells overexpress the receptor CD-44 and some of its
more active
variants, which bind and in some cases rapidly internalize hyaluronic acid.
Tumors in their
invasive or metastasizing stages present increased expression of factors and
receptors
modifying their adhesion and motility abilities. One of such adhesion factors
overexpressed
in some cancerous phases is RHAMM, which also binds hyaluronic acid. Thus, the
inclusion
of hyaluronic acid in the MTNP scaffold may help localize the articles in
these cancers.
Furthermore, hyaluronan oligosaccharides have been shown to induce apoptosis
and reduce
tumor growth in vivo. Although not wishing to be bound by theory, we expect
the degradation
process of the nanoarticle to release such hyaluronan oligomers, which will
compete with
endogenous polymeric hyaluronans, leading to proapoptotic events. Finally,
hyaluronans,
is especially shorter oligomers, have the potential to reverse mufti-drug
resistance, probably via
interterence with cell survival signaling pathways.
Additonal polycarboxylate polymers that may be used in the present invention
include
polyaminoacids where the amino acid chains are comprised of aspartic or
glycolic acid
residues.
2o One or more PEG chains can be attached to all of the above polycarboxylate
polymers to form PEG-polycarboxylate copolymers. For the PEG chains, preferred
molecular weights are in the range of 2000 to 10,000 Daltons. For the
polycarboxylates,
preferred molecular weight is in the range of 1000 to 25,000 Daltons,
preferably 2500 to
10,000 Daltons.
Radical-generating iron-oxide containing MTNPs: Polymer-coated magnetic
nanoarticles
that degrade to form radicals can be synthesized by two synthetic strategies.
In the first
strategy, radical-generating functionalities are attached to the polymeric
matrix surrounding a
MTNP (Reaction Scheme 1).
Reaction Scheme 1. Two synthetic routes which could be used to attach a
radical generating species to a hydrogel coated nanoparticle.
carbodiimide p
p coupling ~N,. ~OH
1. ~ 'E' HO~N~ OH > ~ N IOI
O
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WO 2005/070471 PCT/US2005/001755
O O
2. HO~N.N~OH -~- 2 ~N~ L~ >/ O~N~~O
O
N
OOH ~ N\ ~ N N hydrolysis O~O~N.N~OH
+ N O~ N ~- ~
O 0
With the variety of temperature sensitivities that radical sources can display
(see,
s e.g., Odian, G. Principles of Polymerization 2~d Edition, Wiley-
Interscience, 1981, 196), a
radical-producing structure that would be satisfactory for the temperature
profile of the target
product would be selected. Radical-generating functionalities, such as azo or
peroxides, can
be attached to the hydrogel through a variety of chemical bonds such as amides
or
carbonates. The exact strategy used would depend on the properties of the
radical initiator
1o being attached. For example, the use of peroxide radical sources would
preclude the use of
amine-containing hydrogels, because of the increased instability of those
compounds in the
presence of an amine. However, azo-containing compounds would be generally
useful in
either proposed attachment method. Two commercially available radical-
generating azo
compounds have been identified:
1s
HO~~N N~ ~OH ~N N-~-
N~ ~ ~ ~N H02C~ ~CN CN C02H
2o In the second strategy, the MTNP's polymeric scaffold itself degrades to
form radical
species. In this strategy, the unique reactivity of alkoxyamine compounds to
generate radical
species is used (for a general discussion of alkoxy amines, their thermal
behavior, stability,
and use in polymer chemistry, see Lizotte, J., "Synthesis and Characterization
of Tailored
Macromolecules via Stable Free Radical Polymerization Methodologies",
Dissertation
2s Virginia Polytechnic Institute and State University, 2003, Chapter 2)
(Reaction Scheme 2).
Reaction Scheme 2. On heating, alkoxyamine compounds degrade to radical
species. In this nonspecific example, the stable nitroxyl compound TEMPO is
released.
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WO 2005/070471 PCT/US2005/001755
N~ heat ,
> ~ +
O O"OR Zi
~ O
O"OR
Magnetic colloids can be coated with an alkoxyamine-containing hydrogel by a
s strategy similar to that described above, using reverse microemulsions. In
this procedure, a
2,2,6,6-tetramethyl-1-piperdinyloxy (TEMPO)-derivatized polysaccharide,
radical initiator,
acrylate-functionalized polyethylene glycol), and acrylic acid monomer are
dissolved in the
aqueous phase of the magnetic colloid-containing reverse microemulsion. Unlike
typical
polymerizations, the radical initiator would have a stoichiometry close to
that of the total
to acrylate concentration of the monomers. On photolysis, the radical
initiators initiate
polymerization of the monomer species. However, the high concentration of the
TEMPO
nitroxyl (equal to that of the total acrylate concentration) would quickly
react with the
monomer-centered radical, making the stable alkoxy amine (Reaction Schemes 3
and 4).
is
Reaction Scheme 3. A representative reaction in which TEMPO is used to make
a stable alkoxyamine (Hawker, C. et al., Macromolecules, 1996, 29, 5245).
The alkoxyamine product, on heating, generates radical species, which will
initiate a polymerization reaction.
-I-CH2 O-N
~N=~ + - + ~ > CN ~
CN CN ~ O I
Reaction Scheme 4. The reaction of a mixture containing a radical initiator,
TEMPO-derivatized carbohydrate, acrylic acid, and PEG diacrylate. This
2s reaction, if done in a reverse microemulsion containing a magnetic colloid,
will
make a hydrogel-coated nanoarticle.
CA 02553647 2006-07-18
WO 2005/070471 PCT/US2005/001755
~N ~ +
CN CN
.o-
~O
~' ~O ~ I
\\ ,O + / PEG
O~ PEG~O~
OH
O cross-linked hydrogel
The alkoxyamine-containing MTNPs, when heated, will degrade, releasing
radicals.
s
Incorporation of Recognition Elements: MTNPs may be functionalized with
recognition
elements ("REs"). The REs can target a multitude of disease-associated
biomolecules.
Tumor-associated targets include folate receptors, transferrin receptors,
erbB1, erbB2,
erbB3, erbB4, CMET, CEA, EphA2, carcinoembryonic (CEA) antigen, mucin
antigens,
to including Muc-1, cellular adhesion, of the cluster differentiation (CD)
antigen family.
Vascular targets associated with multiple pathologies, including cancer,
include VEGFR-1,
VEGFR-2, and integrins, including integrin av~33, and integrin av(31.
Additional targets are
extracellular proteins such as matrix metalloproteinases (MMPs), the collagen
family, and
fibrin.
1s The REs can be linked either directly or through a linker molecule to the
nanoarticle.
In a linker configuration, part or all of the REs are "displayed" at the end
terminus of the
tether. Therefore, in one application of the invention, the articles consist
of REs displayed on
a polymer scaffold. In another embodiment of the invention, the articles
consist of an RE,
such as a high affinity peptide, linked to the surface of the article core
scaffold via a linker
2o molecule, the linker comprising, in a preferred embodiment, polyethylene
glycol (PEG). The
PEG linker can be linear with reactive functionalities at both of the chain
terminals; the PEG
linker can also be multi-armed, for instance possessing three, four, five,
six, eight arms or
more, with two or more of the arms possessing reactive functionalities that
can be used to
attach the PEG to the nanoarticle scaffold and the RE to the PEG.
2s For each of these embodiments, it is possible to functionalize the articles
with several
coupling strategies, varying both the order of addition of the different
components and the
reactive chemical moieties used for the coupling.
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The components may be attached to one another in the following sequences. The
polymer scaffold is first reacted with a di-functional PEG-containing tether,
followed by
functionalization of the free terminus of a portion of the PEG chain with a
RE. Alternatively,
the RE is coupled first to the PEG-containing tether, followed by the
attachment of the other
PEG terminus to the scaffold.
Several combinations of reactive moieties can be chosen to attach the RE to
the
tether and to attach the tether to the nanoarticle scaffold. In using a series
of orthogonal
reaction sets, varying some of the scaffold building blocks and/or tethering
arms, it is also
possible to attach REs with different molecular structures that bind to
different receptors,
onto the same article scaffold in well-controlled proportions. Reactions using
orthogonal
reactive pairs can be done simultaneously or sequentially.
It is preferable to functionalize the articles in an aqueous system. The
surfactants and
the oil phase, residual from the synthesis of the polymer scaffold, can be
removed through
the use (singularly or in combination) of solvent washing, for instance using
ethanol to
solubilize the surfactant and oil while precipitating the articles; surfactant-
adsorbing beads;
dialysis; or the use of aqueous systems such as 4M urea. Methods for
surfactant removal are
known in the art.
The RE must contain a functionality that allows its attachment to the article.
Preferentially, although not necessarily, this functionality is one member of
a pair of
2o chemoselective reagents selected to aid the coupling reaction (Lemieux, G.,
Bertozzi, C.,
Trends in Biotechnology, 1998, 16, 506-513). For example, when the article
surface (and/or
linkers grafted to its surface) displays a halo acetal, a peptide RE may be
attached through a
sulfhydryl moiety. A sulfhydryl moiety in the RE structure can be accomplished
through
inclusion of a cysteine residue.
Coupling is also possible between a primary amine on the article or the linker
terminus and a carboxylic acid on the RE. A carboxylate in the peptide
structure can be
found either on its terminal amino acid, for linear peptides, or through the
inclusion of
aspartic or glutamic acid residues. The opposite configuration, where the
carboxylic acid is
on the article and a primary amine belongs to the peptide, is also easily
accessible. Many
3o polymerizable building blocks contain acidic moieties, which are accessible
at the surface of
the beads after their polymerization. As for poly(amino acid)-based REs, a
primary amine
function can be found either at its N-terminus (if it is linear) and/or via
introduction of a lysine
residue.
Another example of reactive chemical pairs consists of the coupling of a
sulfhydryl
3s with a halo acetal or maleimide moiety. The maleimide function can be
easily introduced,
either on a peptide, a linker, or the surface of the articles, by reacting
other common
22
CA 02553647 2006-07-18
WO 2005/070471 PCT/US2005/001755
functionalities (such as carboxylic acids, amines, thiols or alcohols) with
linkers through
methods known to one of skill in the art, such as described for example by G.
T. Hermanson
in Bioconjugate Technigues, Academic Press Ed., 1996. In a preferred
embodiment, the
inclusion of CiBA or other disulfide-containing building blocks, in the
scaffold facilitates the
attachment of REs through thiol reactive moieties. After scaffold formation,
reduction of the
disulfide linkage in CiBA produces free thiols. Linker molecules containing
groups that are
reactive with thiol, such as bromoacetamide or maleimide, are added to the
reduced
therapeutic agent-containing article to attach the linker to the article
scaffold. REs are then
added, which react with the free terminus of the linker molecules to give RE-
functionalized
1o articles. Alternatively, the RE may be attached to one end of the linker
molecule prior to
attachment of the linker molecule to the reduced article.
Peptides can also be coupled to the article and/or the tether with a reaction
between
an amino-oxy function and an aldehyde or ketone moiety. The amino-oxy moiety
(either on
the articles or in the peptide) can be introduced, starting from other common
functionalities
1s (such as amines for example), by a series of transformations known to those
skilled in the
art. In the same way, aldehyde- or ketone-containing articles and aldehyde-
containing
peptides are readily synthesized by known methods.
The resulting RE-functionalized, bioactive agent-containing articles may be
used
immediately, may be stored as a liquid solution, or may be lyophilized for
long-term storage.
2o The REs may be any small or large molecular structure that provides the
desired
binding interactions) with the cell surface receptors of the targeted
molecule. The number of
recognition element moieties per article can range from 2 to about 1000,
preferably from 2 to
500, and most preferably from 2 to 100. The articles may optionally further be
comprised of
more than one type of RE. As used herein, a RE "type" is defined as a specific
molecular
25 structure.
In one embodiment REs are comprised of peptides. Peptides used as REs
according
to this invention will generally possess dissociation constants between 10-4
and 10-9 M or
lower. Such REs may be comprised of known peptide ligands. For instance,
Phoenix
Peptides' peptide ligand-receptor library
30 (htt~://www.phoenixpeptide.com/Peptidelibrarylist.htm) contains thousands
of known peptide
ligands to receptors of potential therapeutic value. The peptides may be
natural peptides
such as, for example, lactams, dalargin and other enkaphalins, endorphins,
angiotensin II,
gonadotropin releasing hormone, melanocyte-stimulating hormone, thrombin
receptor
fragment, myelin, and antigenic peptides. Peptide building blocks useful in
this invention
3s may be discovered via high throughput screening of peptide libraries (e.g.
phage display
libraries or libraries of linear sequences displayed on beads) to a protein of
interest. Such
23
CA 02553647 2006-07-18
WO 2005/070471 PCT/US2005/001755
screening methods are known in the art (for example, see C.F. Barbas, D. R.
Burton, J. K.
Scott, G. J. Silverman, Phage Display, 2001, Cold Spring Harbor Laboratory
Press, Cold
Spring Harbor, NY). The high affinity peptides may be comprised of naturally-
occurring
amino acids, modified amino acids or completely synthetic amino acids. The
length of the
recognition portion of the peptide can vary from about 3 to about 100 amino
acids.
Preferably, the recognition portion of the peptide ranges from about 3 to
about 15 amino
acids, and more preferably from 3 to 10 amino acids. Shorter sequences are
preferred
because peptides of less than 15 amino acids may be less immunogenic compared
to longer
peptide sequences. Small peptides have the additional advantage that their
libraries can be
1o rapidly screened. Also, they may be more easily synthesized using solid-
state techniques.
Particular peptides of interest are comprised of the amino acid sequence
YCPIWKFPDEECY, or other sequences found in Greene, et.al., J. Biol. Chem.,
2002,
277(31), 28330-28339, that bind to erbB1; peptides comprised of the amino acid
sequence
CdFCDGFdYACYMDV, where dF and dY representing the D isomer of the amino acid
15 residues or other sequences delineated in Murali, J. Med. Chem., 2001, 44,
2565 - 2574, as
REs; peptides disclosed in PCT WO 01/74849 that bind to CEA; and peptides
comprised of
the amino acid sequence ATWLPPR, as described in Demangel, et.al., EMBO J.,
2000,
19(7), 1525-1533.
REs may be comprised of a variety of other molecular structures, including
vitamins
2o such as folate, growth factors such as EGF, proteins such as transferrin,
antibodies, antibody
fragments, lectins, nucleic acids, and other receptor ligands. Humanized or
fully human
antibodies, and humanized or fully human antibody fragments are preferred for
use in the
present invention.
Additionally, it will be possible to design other non-protein compounds to be
2s employed as the binding moiety, using techniques known to those working in
the area of
drug design. Such methods include, but are not limited to, self consistent
field (SCF)
analysis, configuration interaction (CI) analysis, and normal mode dynamics
computer
programs, all of which are well described in the scientific literature. See,
Rein et al.,
Computer-Assisted Modeling of Receptor-Ligand Interactions, Alan Liss, New
York (1989).
3o Preparation of non-protein compounds and moieties will depend on their
structure and other
characteristics and may normally be achieved by standard chemical synthesis
techniques.
See, for example, Methods in Carbohydrate Chemistry, Vols. I-VII; Analysis and
Preparation
of Sugars, Whistler et al., Eds., Academic Press, Inc., Orlando (1962), the
disclosures of
which are incorporated herein by reference.
35 The use of multiple RE molecules of the same molecular structure or of
different
molecular structure to make up the article can increase the avidity of the
article. As used in
24
CA 02553647 2006-07-18
WO 2005/070471 PCT/US2005/001755
the present invention, "high affinity" means a binding of a single RE to a
single target
molecule with a binding constant stronger than 10-4 M, while "avidity" means
the binding of
two or more such RE units to two or more target molecules on a cell or
molecular complex.
Use of two different REs to two different target molecules on the surface of a
cell may have
an advantage to select diseased tissue over normal tissue.
Pharmaceutical Compositions. For the herein-described uses, the MTNPs of the
invention are
provided as pharmaceutical preparations. The articles can be administered by
injection
(subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal,
intracerebral, or
io parenteral), with intraveneous injection being a preferred route. The
articles may also be
suitable for nasal, pulmonary, vaginal, ocular delivery and oral
administration. A
pharmaceutical preparation of a MTNP may be administered alone or in
combination with
pharmaceutically acceptable carriers, in either single or multiple doses.
Suitable
pharmaceutical carriers include inert solid diluents or fillers, sterile
aqueous solution and
15 various organic solvents. The pharmaceutical compositions formed by
combining a
nanoarticle of the present invention and the pharmaceutically acceptable
carriers are then
easily administered in a variety of dosage forms such as injectable solutions.
For parenteral administration, solutions of the nanoarticle in aqueous
propylene glycol
or in sterile aqueous solution may be employed. Such aqueous solutions should
be suitably
2o buffered if necessary and the liquid diluent first rendered isotonic using,
for example, saline
or glucose. These particular aqueous solutions are especially suitable for
intravenous,
intramuscular, subcutaneous and intraperitoneal administration. In this
connection, sterile
aqueous media which can be employed will be known to those of skill in the art
in light of the
present disclosure.
2s The pharmaceutical forms suitable for injectable use include sterile
aqueous solutions
or dispersions and sterile powders for the extemporaneous preparation of
sterile injectable
solutions or dispersions. In all cases, the form must be sterile and must be
fluid to the extent
that easy use with a syringe exists. It must be stable under the conditions of
manufacture
and storage and must be preserved against the contaminating action of
microorganisms,
3o such as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid
polyethylene glycol, and the like), and suitable mixtures thereof. The
prevention of the action
of microorganisms can be brought about by various antibacterial and antifungal
agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the
like. In many
3s cases, it will be preferable to include isotonic agents, for example,
sugars such as mannitol
or dextrose or sodium chloride.
CA 02553647 2006-07-18
WO 2005/070471 PCT/US2005/001755
Sterile injectable solutions are prepared by incorporating the active
compounds in the
required amount in the appropriate solvent with various of the other
ingredients enumerated
above, as required, followed by filtered sterilization. Generally, dispersions
are prepared by
incorporating the various sterilized active ingredients into a sterile vehicle
which contains the
basic dispersion medium and the required other ingredients from those
enumerated above.
In the case of sterile powders for the preparation of sterile injectable
solutions, the preferred
methods of preparation are vacuum-drying and freeze-drying techniques which
yield a
powder of the active ingredient plus any additional desired ingredient from a
previously
sterile-filtered solution thereof.
1o As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic
agents and the. like.
The use of such media and agents for pharmaceutically active substances is
well known in
the art. Except insofar as any conventional media or agent is incompatible
with the
nanoarticle components, its use in the therapeutic compositions is
contemplated.
15 Supplementary active ingredients can also be incorporated into the
compositions.
Reagents and starting materials in some embodiments can be obtained
commercially
from chemical distributors such as Sigma-Aldrich (St Louise, MO and Milwaukee,
WI), Kodak
(Rochester, N~, Fisher (Pittsburgh, PA), Pierce Chemical Company (Rockford,
IL),
Carbomer Inc. (Vllestborough, MA), Radcure (Smyrna, GA), and Polysciences
(Niles, IL).
2o PEG compounds may be purchased through companies such as NOF America
Corporation
(White Plains, Nl~, and Nektar (Birmingham, AL). Peptides to be used as REs
can be
purchased from many sources, one being Bachem (King of Prussia, PA). Proteins
may be
obtained from sources such as Calbiochem (San Diego, CA).
2s Uses of MTNPs. The MTNPs of the present invention may be used for targeted
bioactive
agent delivery to either an in vivo or an in vitro environment. A plurality of
bioactive agent-
containing MTNPs are administered to the environment to be treated, and heat
sufficient for
release of bioactive agent from the MTNPs is applied to the environment. The
bioactive
agents are incorporated into the MTNP through heat-labile covalent or
coordination linkages
3o that are substantially disrupted at moderate temperatures (that is,
temperatures above body
temperature but lower than several hundred °C), causing the bioactive
agents to be released
from the article. Preferably, the MTNP is capable of releasing at least about
50% and
preferably substantially all of its bioactive agent payload upon the
application of heat, which
in the locality of the MTNP is greater than 42 °C for longer than 10
minutes, and where the
35 heat is preferably generated from within the iron oxide core through the
application of an
AMF.
26
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WO 2005/070471 PCT/US2005/001755
Under the influence of a source of heat, the temperature of the MTNP is
raised,
resulting in an acceleration of release of bioactive agent molecules from the
article and/or,
where the nanoarticle includes free radical-generating components, the
generation of free
radicals. In a preferred embodiment, the source of heat is an applied magnetic
field, most
s preferably an alternating magnetic field, the field being generated external
to the environment
in which the MTNPs have been administered. Additionally, as elevated
temperature is
known to increase the toxicity and anti-cancer potency of chemotherapeutics,
the localized
heating generated by the magnetic material can increase the potency of the
released
chemotherapeutic. This heating is expected to result in the death of at least
a portion of
1o cancer cells in a tumor. Additionally, the heating of the surrounding
tissue is expected to aid
in the diffusion of the chemotherapeutic or free radicals into the tumor,
resulting in more
complete cancer cell destruction. Without the application of the applied
magnetic field, the
articles of the invention will release the bioactive agents to a lesser degree
and in a less
controllable manner. Thus, with AMF application, the toxicity of the bioactive
agent both
is systemically and in certain organs, such as the kidney, is reduced.
Utilizing the MTNPs of the present invention, it is not necessary to rely on
heating as
the sole and primary mechanism for tumor destruction. The superparamagnetic
material
needs to generate only enough heat to release the bioactive molecules, which
are localized
to the radius of the article itself. It is not required, although it will be
of additional benefit, to
2o heat up the surrounding tumor tissue.
The superparamagnetic particles used in the present invention are of a size
smaller
than a magnetic domain (1-100 nm). These subdomain superparamagnetic particles
produce substantially more heat, especially at low amplitudes of alternating
magnetic field
(AMF). When suitable, physiologically acceptable frequencies and field
strength
2s combinations are used for the AMF, no interaction is observed between the
human body and
the field; hence tolerable low power absorption is obtained. The frequency of
magnetic field
used to heat the superparamagnetic material-containing MTNPs should be greater
than that
sufficient to cause any appreciable neuromuscular response, and less than that
capable of
causing any detrimental eddy current heating or dielectric heating of healthy
tissue.
3o Frequencies of around 50-200 kHz and a magnetic field strenght of around 50-
100 kA/m is
well-suited for human application (Jordan et al. (1999) Journal of Magnetism
and Magnetic
Materials 201, 413-419); mice can tolerate substantially higher frequencies
(greater than 1
MHz). Instrumentation is available that would allow the application of
hyperthermia treatment
in both animals and humans. Commercial source include Comdel, Inc.
(Gloucester, MA),
3s Bell Electrons NW, Inc., and ICandel Electronics. For frequencies suitable
for both mice and
humans, a frequency range of 20-450 kHz and output wattage of 500 W is
adequate.
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WO 2005/070471 PCT/US2005/001755
A key to efficient heating of an environment, such as a tumor, is to produce
soluble
particles that selectively locate to the tumor. At least three targeting
mechanisms localize the
MTNPs of the invention at the tumor site. First, the article size will be
designed such that
preferential tumor accumulation occurs through the well-documented enhanced
permeability
and retention (EPR) effect. Second, targeting (i.e., recognition) elements on
the surFace of
the nanoarticles will help localize the articles to the tumor site by binding
tumor-associated
antigens. Several different targeting agents or REs may be utilized, including
specific small
molecule or peptide ligands, as well as antibodies or antibody fragments
(e.g., scFv). Finally,
application of a localized magnetic field (which may be a substantially
constant magnetic field
io or an alternating magnetic field) at the tumor site can be used to cause
the articles to
accumulate at the magnetized tumor site. This in turn can result in localized
release of the
bioactive agent. The magnetic field will also lead to heating within the
cancer tissue, killing
some tumor cells by this mechanism and, perhaps more importantly, increasing
the
vulnerability of the tumor cells to the released cytotoxic payload.
1s In a further aspect of the present invention, a MTNP as described herein
can be
utilized in an imaging method comprising administering to a subject (which can
be a human
or an animal) an amount of MTNPs, said amount being effective as a MR contrast
or image-
brightening agent, and imaging the subject using a magnetic resonance device.
In addition to the utility of enabling release of therapeutic agents under the
application
20 of an AMF and the utility of enhancing MRI images, the incorporation of
iron oxides into the
articles of the instant invention can be used advantageously in several
additional ways. For
instance, the superparamagnetic core allows for expedient purification of
bioactive agent-
containing MTNPs from reactants after various reaction and fabrication steps.
For instance,
agent-loaded particles may be separated from unattached bioactive agents by
using a
25 separation scheme wherein after the agent incorporation procedure is
completed, the
MTNPs are retained in a reaction vessel by a permanent magnetic field, while
the solution
that is unassociated with the articles is decanted off, siphoned off, or
otherwise removed.
Advantageously, the location of the MTNPs of the instant invention within the
mammalian
body may be determined using magnetic resonance imaging (MRI) of the
superparamagnetic
30 cores.
The following non-limiting examples are provided to further describe how the
invention may be practiced.
2s
CA 02553647 2006-07-18
WO 2005/070471 PCT/US2005/001755
EXAMPLES
Example 1. Formation of maghemite nanoparticles
Iron pentacarbonyl (0.74 mL) was added to a mixture of oleic acid (4.9 g) in
octyl
ether (28 mL) under a slow flow of NZ gas at 100 °C. After 10 min, the
solution was heated to
290 °C over one hour and held at this temperature until it turned
black. After a further 30
min, heating was discontinued and the mixture was allowed to return to room
temperature.
Trimethylamine oxide (1.26g) was added to the mixture and heated to 130
°C (over approx.
20min), and held at this temperature for two hours. After heating to 290
°C over the next
1o hour, and maintaining at this temperature for one hour, heating was
discontinued. The
product was recovered by precipitation with wash alcohol and centrifugation.
Precipitation
was repeated twice by dissolving the product in a minimum of hexanes (to which
a few drops
of oleic acid had been added), filtration through a 0.2 ~,m membrane filter,
followed by
addition of alcohol and subsequent centrifugation. Very fine suspensions of
particles can be
15 more easily isolated by placing the hexanes/alcohol solution in a beaker on
a strong
magnetic source, such as a NdFeB magnet (typically 8-12 KGauss), followed by
decanting
and/or pipetting the bulk solution for the solid precipitate. Transmission
electron microscopy
demonstrates the nanoparticles are of a narrow size distribution arround
~10nm.
2o Examule 2. Exchange of oleic acid capping groups for tetraalkylammonium
hydroxide
Oleic acid-stabilized iron oxide particles, from Example 1, were stirred in
20% aq.
tetraethylammonium hydroxide (~10 mg particles/mL base solution) until the
black solid
dissolved. Mild heating (~50 °C) was applied as necessary to speed
dissolution. The
25 particles can be concentrated by spin filtration (such as with a Millipore
Amicon-4 centrifugal
filtration device), and washed with DI water to remove excess tetra-
alkylammonium salts.
The particles can alternately be concentrated and washed by magnetic
separation methods.
Thus, inert-polymer coated metallic beads (such as parylene coated steel
beads, PTFE
coated iron beads, etc) are added to the cooled solution and the vessel is
transferred to a
3o strong magnetic source such as a NdFeB magnet (typically 8-12 KGauss). A
pair of
magnets separated by a distance corresponding to the width of the container
holding the
nanoparticles solution is optimal. After the solution becomes transparent
(with the
nanoparticles coated onto the included beads), the bulk solution is removed.
Removal of the
container from the magnetic source releases the particles into solution which
can then be
3s diluted and the process of magnetic concentration and purification repeated
as desired.
29
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WO 2005/070471 PCT/US2005/001755
Example 3. Exchange of oleic acid capping groups for tetraalkylammonium
hydroxide
Oleic acid-stabilized iron oxide particles, from Example 1, were stirred in
water (~5
mg/mL) at 50 °C. A tetra-alkylammonium hydroxide solution (e.g. 20% aq
tetraethylammonium hydroxide) was added drop-wise until the black solid
started to dissolve
to give a dark brown solution. Further hydroxide addition was made if the
particles had not
completely dissolved after 10 min. The final solution was approximately pH 12.
The solution
was cooled to room temperature and filtered (0.2 pm membrane). The particles
were
isolated and purified by the techniques described in Example 2.
Example 4. Hyaluronic Acid reaction with coated iron oxide particles
To a 1 % wtlvol solution of tetraalkylammonium hydroxide coated iron oxide
particles
from Example 2 or 3, hyaluronic acid (10,000 MW, 10 wt. equiv.) was added and
the mixture
agitated. A dark-brown precipitate formed rapidly. This material could be
dissolved by
sonicating for several minutes, however, upon standing, a suspension reformed.
Example 5. PEG-Hyaluronic Acid reaction with coated iron oxide particles
To a 1 % wt/vol solution of tetraalkylammonium hydroxide coated iron oxide
particles
from Example 2 or 3, PEG-Hyaluronic acid (PEG=5000 MW, HA=10,000 MW, 1 wt.
equiv.)
2o was added and the mixture agitated. A dark-brown precipitate formed
rapidly. This material
could not be re-dissolved in aqueous solution.
Example 6. Polyethylene oxide (PEO)-polymethylmethacrylate (PMMA) block
copolymer-coated iron oxide particles
2s To a 1 % wt/vol solution of tetraalkylammonium hydroxide-coated iron oxide
particles
from Example 2 or 3, PEO(7.8K)-b -PMAA(2K) (1 wt. equiv.) was added and the
mixture
agitated for several days. The mixture was filtered (0.2 ~m membrane) and
purified by spin
filtration or magnetically concentrated and purified as in the above example
the resulting
MTNPs.
Example 7. Incorporation of platin into polyethylene oxide-
polymethylmethacrylate
block copolymer-coated iron oxide MTNPs
To a solution of purified PEO(7.8K)-b-PMAA(2K)-coated iron oxide nanoparticles
from
Example 6, at approx. 10 mg/mL, was added 2.5 mg/mL of cis-
diamminoplatinum(II) nitrate
(from a stock solution of 1-10% wt/vol). The reaction was agitated at room
temperature for 1
day, after which the platinated PEO(7.8K)-b-PMAA(2K)-coated iron oxide MTNPs
were
CA 02553647 2006-07-18
WO 2005/070471 PCT/US2005/001755
separated and purified by the magnetic techniques described above. Analysis by
ICP
indicated 4-5% wt incorporation of Pt.
Example 8. Direct synthesis of platinated polyethylene oxide-polymethyl-
s methacrylate block copolymer-coated iron oxide MTNPs
To a solution of the tetraalkylammonium hydroxide-coated iron oxide particles
from
Example 2 or 3 (10 mgimL) was added PEO(7.8K)-b -PMAA(2K) (1 wt. equiv.), and
the
mixture was agitated at 40 °C overnight, followed by addition of cis-
diamminoplatinum(II)
nitrate (2.5 mg per 10mg of iron oxide particles from a stock solution of 1-
10% wtivol platin).
to After 24 hr, the platinated PEO(7.8K)-b-PMAA(2K)-coated iron oxide MTNPs
were separated
and purified by the magnetic techniques described above. Analysis by ICP
indicated 4-6%
wt incorporation of Pt
Examale 9. Preparation of Silica-Coated Magnetite Colloids in Microemulsion
1s A stock solution of 1 M Fe(II) was prepared by dissolving 0.278g FeS047H~0
in 1 mL
of nitrogen-purged deionized water. Similarly, a stock solution of 1.5M
Fe(III) was prepared
by dissolving 0.4O55g FeC136H2O in 1 mL of nitrogen-purged deionized water. An
oil phase
solution containing cyclohexane, Igepal CO-210 and Igepal CO-720 surfactants
was
prepared with a weight percent of 83.35%, 7.3% and 9.35%, respectively. In a
glass
2o container, 25p,L of the 1 M Fe(II) solution and 25~.L of the 1.5M Fe(III)
solution were added to
a 5mL solution of the oil phase under a nitrogen atmosphere, and the mixture
was
magnetically stirred for 1 hr to form a microemulsion. In another container,
100p.L of NH40H
(28-30 wt%) was added to a 5mL solution of the oil phase, and the mixture was
magnetically
stirred for 1 hr to form a NH40H microemulsion. In the absence of a magnetic
field, the
2s NH40H microemulsion was added dropwise to the Fe microemulsion with
vigorous
mechanical stirring for 1 hr to form magnetite nanoparticles.
50p,L of tetraethylorthosilicate (TEOS) was then added to the magnetite
nanoarticles
solution and mecflanically stirred for additional 24 hrs. Acetone was added to
the colloidal
microemulsion to precipitate the silica-coated nanoarticles. The nanoarticles
were washed
3o with acetone and ethanol several times, then dissolved in water and
lyophilized to obtain
magnetite nanoparticles in powder form.
Examale 10. Preparation of Inulin Magnetite MTNPs in Microemulsion:
Coating Crosslinked by Free Radical Polymerization
3s A stock solution of 1 M Fe(II) was prepared by dissolving 0.278g FeS047H~0
in 1 mL
of nitrogen-purged deionized water. Similarly, a stock solution of 1.5M
Fe(III) was prepared
31
CA 02553647 2006-07-18
WO 2005/070471 PCT/US2005/001755
by dissolving 0.4055g FeC136H20 in 1 mL of nitrogen-purged deionized water. An
oil phase
solution containing cyclohexane, Igepal CO-210 and Igepal CO-720 surfactants
was
prepared with a weight percent of 83.35%, 7.3% and 9.35%, respectively. In a
glass
container, 25p.L of the 1 M Fe(II) solution and 25p,L of the 1.5M Fe(III)
solution were added to
s a 5mL solution of the oil phase under a nitrogen atmosphere, and the mixture
was
magnetically stirred for 30 minutes to form a Fe microemulsion. In another
container, 100p.L
of NH40H (28-30 wt%) was added to a 5mL solution of the oil phase, and the
mixture was
magnetically stirred for 30 minutes to form NH40H microemulsion. In the
absence of a
magnetic field, the NH40H microemulsion was added dropwise to the Fe
microemulsion with
to vigorous mechanical stirred for 1 hr to form magnetite nanoarticles.
166pL of monomers solution comprised of 25% inulin multi-methacrylate (IMMA),
2%
cystine bisacrylamide (CiBA) and 1 % sodium acrylate (NaA) in 10mM sodium
phosphate
buffer at pH7.2 was added to the magnetite nanoarticles solution, followed by
3p,L of sodium
persulfate (50mg/mL water) and 3p,L of TEMED (5% solution). The solution was
degassed
1s using a water pump aspirator and mechanically stirred for 2 hrs to form a
crosslinked
scaffolding comprised of derivitized inulin. Ethanol was added to the
microemulsion to
precipitate the nanoarticles. The articles were then dissolved in water and
purified using ion
exchange BioBeads SM-2 for 2 hrs. The articles were filtered and lyophilized
to obtain
magnetite nanoarticles in powder form.
Example 11. Incorporation of Doxorubicin and Recognition Elements
Reduction of CiBA-Containing Nanoarticles: Dissolve 1.0 g nanoarticles as
prepared
in Example 10 in 6.16 mL PBS at 65 mg/mL. Add 268 mg of DTT (FW 154; 24-fold
excess
per mole of CiBA) into the nanoarticle solution. Agitate the reaction for 2
hours at room
2s temperature. Pass the nanoarticle solution through three FPLC desalting
columns to remove
excess DTT, using PBS as buffer. Collect the nanoarticle fractions and
concentrate to a total
of 10 mL of buffer (50 mg/mL of nanoarticles) using Amicon Ultra-15, MWCO 50k
centrifugal
filters (2.5 mL per filter) spun at 4000 rpm.
Linker Attachment: Add 233.6 mg of PEG4ooDBA (FW 641.86; 2.5-fold excess per
3o mole of thiol) to the nanoarticle solution. Add 81.6 mg N-(s-
maleimidocaproic acid) hydrazide
(EMCH) (FW 225.24; 2.5-fold excess per mole of thiol) 5 minutes after
PEG4ooDBA addition
to the nanoarticle solution. Agitate the reaction for 2 hrs after this step.
Remove unreacted
linkers by centrifuging in Amicon Ultra-15, MWCO 5010 centrifugal filters spun
at 4000 rpm,
until about 250 p,L remain. Reconstitute the nanoarticle retentate in 2.5 mL
of 0.1 M PBS, pH
3s 7.2 and repeat the centrifugation. Re-dissolve the retentate in each tube
in 2.5 mL of 0.1 M
PBS, pH 7.2.
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Ligand Attachment: Add 42.08 mg of the peptide RGDdFC (FW 578; 0.5 equivalent
of
thiol) to the PEG4ooDBA-nanoarticles (any peptide or ligand with a free thiol,
readily
incorporated via a cysteine residue, can be attached to the nanoarticle
through reaction with
a PEG multibromoacetate linker). Take a 10 pL aliquot at t = 0 and t = 1 hr
after peptide
addition for HPLC analysis. Agitate the reaction for 1 hr. Cap unreacted
bromoacetamide
with 17.6 mg of cysteine (FW 121.16; 1 equivalent of thiol) to each solution.
Agitate the
reaction for 10 minutes. Remove unreacted ligands by centrifuging each
nanoarticle solution
in Amicon Ultra-15, MWCO 50iC centrifugal filters spun at 4000 rpm, until
about 250 wL
remain. Reconstitute each nanoarticle retentate in 2 mL of 0.1 M sodium
phosphate buffer,
1o pH 5, and repeat the centrifugation. Re-dissolve the retentate in 2.5 mL of
0.1 M sodium
phosphate buffer, pH 5.
Doxorubicin attachment: Add 41.6 mg of doxorubicin (FW 579.99; 1 theoretical
equivalent per 2 moles of thiol) to the above nanoarticle solution (at 50 mg
NP/mL (5 mL
buffer)), first pre-dissolving doxorubicin in 8 mL of de-ionized water at 5.2
mg/mL. React at
1s 37°C for 20 h. Remove unreacted doxorubicin using Amicon Ultra-15,
MWCO 501<
centrifugal filters spun at 4000 rpm, until about 250 p.L remain. Reconstitute
the nanoarticle
retentate in 2.5 mL de-ionized water and repeat the centrifugation. Re-
dissolve the retentate
in 50 mL de-ionized water (5 mg/mL for lyophilization). Lyophilize the
doxorubicin-containing
MTNP solution overnight.
Example 12:
Add 5 fold excess 5/8 arm-PEGZOOOBA linker to reduced CiBA-containing
nanoarticles
(prepared as in Example 11 ) in 0.1 M PBS, 1.2 g. Allow to react 2 hours at
room temperature
with agitation. Remove unreacted linker on FPLC with three 26/10 desalting
columns in-line,
2s equilibrated and run with 0.1 M PBS pH 7.2.
Example 13: Preparation of Carbohydrate-Coated Magnetite MTNPs:
Coating Crosslinked by Michael-type addition reaction
A stock solution of 1 M Fe(II) was prepared by dissolving 0.278g FeSO47H20 in
1 mL
of nitrogen-purged deionized water. Similarly, a stock solution of 1.5M
Fe(III) was prepared
by dissolving 0.4055g FeC136H20 in 1 mL of nitrogen-purged deionized water. An
oil phase
solution containing cyclohexane, Igepal CO-210 and Igepal CO-720 surfactants
was
prepared with a weight percent of 83.35%, 7.3% and 9.35%, respectively. In a
glass
container, 25pL of the 1 M Fe(II) solution and 25p,L of the 1.5M Fe(III)
solution were added to
3s a 5mL solution of the oil phase under a nitrogen atmosphere, and the
mixture was
magnetically stirred for 1 hr to form an Fe microemulsion. In another
container, 100pL of
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NH40H (28-30 wt%) was added to a 5mL solution of the oil phase, and the
mixture was
magnetically stirred for 1 hr to form a NH40H microemulsion. In the absence of
a magnetic
field, the NH40H microemulsion was added dropwise to the Fe microemulsion with
vigorous
mechanical stirred for 1 hr to form magnetite nanoparticles.
150uL of 30%IMMA in 10mM sodium phosphate buffer at pH7.2 was added to the
magnetite nanoarticles solution, followed by 5.7p.L of PEG3aoodithiol. The
solution was
mechanically stirred for 24 hrs. Ethanol was added to the microemulsion to
precipitate the
nanoparticles. The articles were then dissolved in water and purify using ion
exchange
BioBeads SM-2 for 2 hrs. The articles were filtered and lyophilized to obtain
iron oxide-
1o containing nanoarticles in powder form.
Example 14: '
Combine 1.0 mL of a 2.0 M FeS04 in 2 M HCI with 4.0 mL of a 1.OM FeCl3 in 2 M
HCI
and stir with a magnetic stir bar. Add 50 mL of a 0.7 M NH3 solution dropwise
to the stirring
is solution. Allow the magnetite to settle and decant some of the liquid
before centrifuging for 1
minute at 1000 rpm. Add 10 mL of 20% tetraethylammonium hydroxide to the
precipitate
and resuspend the magnetite. Use an aspirator vacuum to remove excess ammonia
from
the solution. Pour off some of the liquid and pour the magnetite-covered stir
bar into a weigh
boat. Using a strong magnet under the weigh boat to attract the magnetite,
remove the stir
2o bar and any excess liquid. Let the magnetite dry over the weekend in the
hood. A ferrofluid
solution of the magnetite was prepared containing 100mg/mL of deionized water.
Aqueous monomers solution was prepared containing 30 wt% IMMA, 4wt% CiBA and
2 wt% NaA in deionized water. The oil phase was prepared containing
cyclohexane, Igepal
CO-720 and Igepal CO-210 with a ratio of 83.35, 9.35 and 7.3 wt%,
respectively. Thermal ,
2s initiator, sodium persulfate (NaPS), solution was prepared containing
250mg/mL of deionized
water. N,N,N',N'-Tetramethylethylenediamine (TEMED) was used as purchased.
1mL of aqueous ferrofluid was mixed with 1mL of aqueous monomers. The
combined solution was then added dropwise to the oil phase with mechanical
stirring. 50p,L
of NaPS 250mg/mL solution and 12.5wL of TEMED were added to the resulting
3o microemulsion while stirring. The microemulsion was then transferred to a
100-mL Schlenk
tube and degassed in an ice bath using a water pump aspirator. The degassed
solution was
placed on the shaker for 18 hrs. Ethanol was then added to the microemulsion
to precipitate
the nanoparticles. The particles were dissolved in water and purify using ion
exchange
(BioBeads SM-2) for 2 hrs. The particles were filtered and lyophilized to
obtain polymer-
35 coated magnetite nanoparticles.
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Example 15:
A 0.27 M bis(2-ethylhexyl)sulfosuccinate sodium salt (Aerosol OT or AOT)
solution
was prepared by dissolving 12g AOT in 10mL isooctane. An aliquot of ultra-pure
water was
purged for one hour with NZ gas. A stock solution of 1 M Fe(II) was prepared
by dissolving
s 0.278g FeS04.7Hz0 in 1 mL of the nitrogen purged water. Similarly, a stock
solution of 1.5 M
Fe(III) was prepared by dissolving 0.4055g FeCl3.6 H20 in 1 mL of the nitrogen
purged water.
In a glass container, 25uL of the 1 M Fe(II) solution and 25uL of the 1.5 M
Fe(III) solution
were added to a 5mL aliquot of the AOT solution under a nitrogen atmosphere,
and the
resulting FeIAOT mixture was magnetically stirred for 1 hr to form a Fe/AOT
solution. In
1o another container, 100f.~,L NH40H (28-30 wt %) was added to another 5mL
aliquot of the AOT
solution, and the resulting NH40H/AOT mixture was magnetically stirred for 1
hr to form a
NH40H /AOT solution. In absence of magnetic field, the NH40H/AOT solution was
added
dropwise to the Fe/AOT solution with vigorous mechanical stirring for 1 hr.
50p.L of
tetraethylorthosilicate (TEOS) was then added to the resulting brown solution
and
1s mechanical stirring was continued for an additional 24 hrs to give a
magnetite microemulsion.
Aqueous monomers solution was prepared containing 25 wt% IMMA, 2 wt% CiBA
and 1 wt% NaA in 10mM sodium phosphate pH 7.2 buffer. The oil phase was
prepared
containing cyclohexane, Igepal CO-720 and Igepal CO-210 with a ratio of 83.35,
9.35 and
7.3 wt%, respectively. Sodium persulfate (NaPS) thermal initiator solution was
prepared
o containing 250mg/mL of deionized water. N,N,N',N'-tetramethylethylenediamine
(TEMED)
was used as purchased.
In a glass container, 0.5mL of aqueous monomer solution was added dropwise to
10mL of oil phase while stirring. 12.5pL of NaPS 250mg/mL solution and 5pL of
TEMED
were added to the microemulsion while stirring. With the magnetic stirrer
removed, the
2s monomer microemulsion was mixed with the magnetite microemulsion. The
combined
microemulsion was transferred to a 100-mL Schlenk tube and degassed in an ice
bath using
a water pump aspirator. The degassed solution was placed on the shaker for 18
hrs.
Acetone was used to precipitate the nanoparticles. The nanoparticles were
washed with
acetone and ethanol several times with each solvent, then dissolved in water
and lyophilized
3o to obtain polymer-coated magnetite nanoparticles.
Example 16: Attachment of 4,4'-azobis(4-cyanovaleric acid) (ACVA) to amine-
containing MTNPs
One gram of magnetic cored nanoarticles having a polymer scaffold composition
of
3s 25/2/1 IMMA/CiBA/APMA is dispersed in 50 mL of 0.2 M pH=7.5 HEPES buffer. N-
hydroxysuccinimide (NHS, 0.109 g) and ACVA (0.133 g) are dissolved in a second
50 mL
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quantity of the same buffer. To the solution containing the NHS and ACVA is
added 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC, 0.182 g). After
stirring the
NHS/ACVA/EDC solution at room temperature for 10 minutes with a mechanical
stirrer, the
magnetic nanoarticles are added over the course of 1 minute. One hour later,
with the
s aqueous mixture being continuously agitated by a mechanical stirrer over
that interval, a
second quantity of EDC (0.182 g) is added. One hour later, a third quantity of
EDC (0.182 g)
is added. After one more hour, the particles are isolated by centrifugation or
magnetic
separation. Excess reagents and side products are removed from the particles
by three re-
suspension/isolation cycles, and the nanoarticle products are isolated by
lyophilization.
Example 17: TEMPO-functionalized inulin
The product (1.0 g) of the reaction of 4.-hydroxy-2,2,6,6-tetramethyl-
piperdinyloxy,
free radical (4-hydroxy-TEMPO) with 1,1'-carbonyldiimidazole (CDI) is added to
an
anhydrous DMSO (25 mL) solution containing inulin (DP=20, 1.22 g). The
resulting mixture
1s is stirred under a nitrogen atmosphere for two days, and is then poured
into toluene (400 mL)
with rapid stirring. The precipitated product is dried under vacuum, dissolved
in de-ionized
water, and dialyzed in a 500 MW cutoff dialysis membrane against de-ionized
water. The
product is isolated after lyophilization.
2o Examale 18: Preparation of magnetic colloids
Combine 1.0 mL of a 2.0 M FeS04 in 2 M HCI with 4.0 mL of a 1.OM FeCl3 in 2 M
HCI
and stir with a magnetic stir bar. Add 50 mL of a 0.7 M NH3 solution dropwise
to the stirring
solution. Allow the magnetite to settle and decant some of the liquid before
centrifuging for 1
minute at 1000 rpm. Add 10 mL of 20% tetraethylammonium hydroxide to the
precipitate and
2s re-suspend the magnetite. Use an aspirator vacuum to remove excess ammonia
from the
solution. Pour off some of the liquid and pour the magnetite-covered stir bar
into a weigh
boat. Using a strong magnet under the weigh boat to attract the magnetite,
remove the stir
bar and any excess liquid. Let the magnetite dry for two days in a fume hood.
3o Example 19: Synthesis of radical generating polymer-coated MTNPs
An oil phase is prepared containing cyclohexane, Igepal CO-720 and Igepal CO-
210
with a ratio of 83.35, 9.35 and 7.3 wt%, respectively. An aqueous phase is
prepared by
adding TEMPO-functionalized inulin (0.491 g) from Example 17, polyethylene
glycol)
diacrylate (formula weight 575, 0.100 g), sodium acrylate (0.022 g), and ACVA
(0.162 g) to
3s 2.4 mL deionized water. Two mL of the resulting solution is added to 100 mg
of the dry
magnetite colloids from Example 18. After thorough mixing, the aqueous phase
is then
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added dropwise to the oil phase with mechanical stirring. The microemulsion is
transferred to
a 100-mL Schlenk tube and degassed in an ice bath using a water pump
aspirator. The
degassed solution is placed on a shaker for 2 hrs while being exposed to UV
light. Ethanol is
added to the microemulsion to precipitate the nanoparticles. The particles are
dissolved in
water and purified using ion exchange (BioBeads SM-2) for 2 hrs. The
nanoarticles are
filtered and lyophilized to obtain radical generating MTNPs.
Example 20. Pt release from platinated polyethylene oxide -
polymethylmethacrylate
block copolymer coated maghemite MTNPs.
to A solution (~10mg/mL) of MTNPs such as prepared in Example 8 were heated to
80
°C for 30 min. The low molecular weight species were separated from the
articles with a
centrifugal membrane filtration device with a nominal molecular weight cut-off
of 5000.
Platinum release was monitored by an increase in absorbance at 300 nm and by
%Pt
determined by ICP. Compared to a sample stored at room temperature, heating at
80 °C
15 increases Pt release at least ~2-5-fold over this 30 min period.
Example 21. Effect on in vitro toxicity by heat treatment of platin-containing
polyethylene oxide-polymethylmethacrylate block copolymer-coated
iron oxide MTNP.
2o A platin-containing MTNP (prepared as in Example 7) was heated at 80
°C for 30 min
and cooled to room temperature, prior to incubating with an A-498 renal cell
cancer line.
Compared to a control of an identical, but non-heated MTNP, the heated sample
demonstrated thirty times the potency in cell proliferation inhibition as
measured a
sulforhodamine B assay.
37
