U.S. patent application number 10/335790 was filed with the patent office on 2003-07-03 for amine functionalized superparamagnetic nanoparticles for the synthesis of bioconjugates and uses therefor.
Invention is credited to Gaw, Debra A., Josephson, Lee.
Application Number | 20030124194 10/335790 |
Document ID | / |
Family ID | 23354146 |
Filed Date | 2003-07-03 |
United States Patent
Application |
20030124194 |
Kind Code |
A1 |
Gaw, Debra A. ; et
al. |
July 3, 2003 |
Amine functionalized superparamagnetic nanoparticles for the
synthesis of bioconjugates and uses therefor
Abstract
Amine functionalized magnetic nanoparticle compositions and
processes for synthesizing the same are described. The process
consists of obtaining a carboxylated polymer in substantially pure
form, which is used to prepare a substantially size homogeneous,
polymer coated carboxyl, functionalized magnetic nanoparticle. The
carboxyl groups are converted to reactive primary amino groups by
the use of a water-soluble carbodiimide followed by reaction of a
large excess of a diamine. The amine-terminated nanoparticles are
then reacted with bifunctional crosslinking agents and with various
biomolecules to make nanoparticles for in vitro assays, cell
sorting applications and target specific MR contrast agents.
Inventors: |
Gaw, Debra A.; (Reading,
MA) ; Josephson, Lee; (Reading, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Family ID: |
23354146 |
Appl. No.: |
10/335790 |
Filed: |
January 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60345233 |
Jan 2, 2002 |
|
|
|
Current U.S.
Class: |
424/491 ;
427/2.13 |
Current CPC
Class: |
A61K 9/5026 20130101;
A61K 9/5094 20130101; A61K 49/1854 20130101; B82Y 5/00
20130101 |
Class at
Publication: |
424/491 ;
427/2.13 |
International
Class: |
A61K 009/14; A61K
009/16; A61K 009/50; B05D 003/00 |
Claims
We claim:
1. A nanoparticle comprising: (i) magnetic core having one or more
magnetic metal oxide crystals; and (ii) a noncrosslinked polymer
coating associated with the core, wherein the polymer coating has a
plurality of carboxyl groups and plurality of reactive primary
amino groups, wherein a portion of the carboxyl groups are
associated with the crystals.
2. The nanoparticle of claim 1 wherein reactive primary amino
groups are associated with polymer through a peptidyl linkage of
the formula:--O--(CH.sub.2).sub.m--CONH--[X]wherein X is
--(CH.sub.2).sub.n NH.sub.2, --(CH.sub.2).sub.oCH NHCOO--,
--(CH.sub.2).sub.3NH (CH.sub.2).sub.4NH.sub.2 or
--(CH.sub.2).sub.3NH(CH.sub.2).sub.4NH(CH.sub- .2).sub.3
NH.sub.2--wherein m=1,2, or 3; n=2,3, 6, and o=3 or 4.
3. The nanoparticle of claim 1, wherein the magnetic core comprises
one or more superparamagnetic iron oxide crystals.
4. The nanoparticle of claim 3 wherein the superparamagnetic core
has a diameter between about 1 nm and about 25 nm.
5. The nanoparticle of claim 4 where in the superparamagnetic core
has a diameter between about 3 nm and about 10 nm.
6. The nanoparticle of claim 5 wherein the core has a diameter
about 5 nm.
7. The nanoparticle of claim 1 wherein the nanoparticle has
diameter between about 15 nm and 100 nm.
8. The nanoparticle of claim 7 wherein the nanoparticle has a
diameter between about 20 nm and about 100 nm.
9. The nanoparticle of claim 1 wherein the coating is selected from
the group of polymers consisting of natural polymers, synthetic
polymers and derivatives thereof.
10. The nanoparticle of claim 1 wherein the polymer is
carboxymethyldextran.
11. The nanoparticle of claim 1 wherein the nanoparticle is
conjugated with a biomolecule.
12. The nanoparticle of claim 11 wherein the biomolecule has at
least one sulfhydryl group wherein the sulfhydryl group reacts with
an amino group on the coating.
13. A nanoparticle-bioconjugate molecule comprising: (i) a
nanoparticle, the nanoparticle comprising (a) a magnetic core
having one or more magnetic metal oxide crystals; (b) a
noncrosslinked polymer coating associated with the core, wherein
the polymer coating has a plurality of carboxyl groups and
plurality of reactive primary amino groups, wherein a portion of
the carboxyl groups are associated with the crystals; (ii) a
biomolecule linked to the nanoparticle, wherein the biomolecule has
at least one sufhydryl group linked to the amino group.
14. The a nanoparticle-bioconjugate of claim 13 wherein the amino
groups are associated with polymer through a peptidyl linkage of
the formula:--O--(CH.sub.2).sub.m--CONH--[X]wherein X is
--(CH.sub.2).sub.n NH.sub.2, --(CH.sub.2).sub.oCH NHCOO--,
--(CH.sub.2).sub.3NH (CH.sub.2).sub.4NH.sub.2 or
--(CH.sub.2).sub.3NH(CH.sub.2).sub.4NH(CH.sub- .2).sub.3 NH.sub.2
wherein m=1, 2, or 3; n=2, 3, 6, and o=3 or 4.
15. A process for synthesizing an amine functionalized magnetic
metal oxide nanoparticle comprising: i. obtaining a polymer having
a plurality of carboxyl groups attached thereto, ii. contacting the
polymer with magnetic metal oxide to produce a coated magnetic
metal oxide wherein a portion of the carboxyl groups are associated
with the metal oxide; iii. reacting the coated magnetic metal oxide
of step (ii) with a diamine.
16. The process of claim 15 wherein the diamine is ethylene
diamine.
17. The process of claim 15 wherein the diamine is hexane
diamine.
18. The process of claim 15 wherein step (ii) further comprises the
steps of (a) providing a solution of soluble iron salts; (b)
converting the iron salts into iron oxide crystals; and (c)
removing unassociated polymer.
19. The process of claim 18 wherein the step of converting the iron
salts into iron oxides further includes the steps of: (d) heating
the iron salts to a temperature between about 4.degree. C. and
about 20.degree. C.; (e) adding an amount of base to raise the pH
to about 8 or higher to form iron oxides.
20. The process of claim 19 further including the step of heating
the iron oxides to a temperature about 60.degree. C. or more for at
least 30 minutes.
21. A pharmaceutical composition comprising: (A) A
nanoparticle-bioconjuga- te molecule comprising: (i) a
nanoparticle, the nanoparticle comprising (a) a magnetic core
having one or more magnetic metal oxide crystals; (b) a
noncrosslinked polymer coating associated with the core, wherein
the polymer coating has a plurality of carboxyl groups and
plurality of reactive primary amino groups, wherein a portion of
the carboxyl groups are associated with the crystals; (ii) a
biomolecule linked to the nanoparticle, wherein the biomolecule has
at least one sufhydryl group linked to at least one amino group;
and (B) a pharmaceutically acceptable carrier.
Description
RELATED APPLICATIONS
[0001] This Application claims priority under 35 USC 119(e) to
provisional patent application U.S. No. 60/345,233 filed Jan. 2,
2002.
TECHNICAL FIELD
[0002] The present invention relates to novel superparamagnetic
nanoparticle compositions, synthesis and uses thereof.
BACKGROUND
[0003] All patents, patent applications and references cited in
this specification are incorporated herein by reference.
[0004] Carboxyl bearing polymers have found a wide usage in food,
drugs, and industrial applications. A few well known examples
include the carboxymethyl (`CM`) polysaccharides CM-cellulose,
CM-dextran, and CM-arabinogalactan (U.S. Pat. No. 5,981,507). The
CM polysaccharides are produced from a reaction of the
polysaccharide with haloacetic acids in base.
[0005] Carboxyl bearing polymers have been used in the synthesis of
magnetic nanoparticles. Carboxylated dextrans of two major types
have been used in the synthesis of superparamagnetic iron oxide
nanoparticles. Carboxydextrans have a single terminal carboxyl
group on each dextran molecule obtained by treatment with base
(Hasegawa U.S. Pat. No. 4,101,435; Hasegawa U.S. Pat. No.
5,424,419, column 2, line 17). Carboxymethylated dextrans have
numerous carboxymethyl groups attached per mole of dextran by
reaction of alkyl halogenated acids in base (Maruno U.S. Pat. No.
5,204,457; Groman WO 00/61191).
[0006] Magnetic nanoparaticles used for the attachment of
biomolecules have been described by Molday (U.S. Pat. No.
4,452,773). A dextran coated magnetic nanoparticle is formed and
then treated with periodate to produce aldehyde groups. The
aldehydes react with amino groups on a biological molecule, to form
a Schiff base. The Schiff base maybe stabilized by treatment of
with a reducing agent like sodium borohydride. After treatment with
a reducing agent a methylene amino linker connects the biomolecule
to the nanoparticle. As shown in FIG. 2B, there are no peptidyl
bonds in such linkages. A drawback of this method is the difficulty
controlling the number and position of amino groups on the
biomolecule that are available to react with the reactive aldehyde
groups on the nanoparticle.
[0007] Other methods of attaching biomolecules to nanoparticles
also use the reactivity of the aldehyde group. Rembaum and
coworkers have utilized this approach, synthesizing nanoparticles
with glutaraldehyde (U.S. Pat. Nos. 4,438,239; 4,369,226).
[0008] The development of amine functionalized crosslinked iron
oxide nanoparticle ("amino-CLIO", FIG. 2) by one of the inventors
has proven to be an excellent method of synthesizing magnetic
particle-biomolecule conjugates. Amino-CLIO is prepared by first
synthesizing a dextran coated magnetic nanoparticle, followed by
crosslinking the dextran with epichlorohydrin. Finally the amine
groups are incorporated by reacting the dextran with ammonia (see
Josephson et al, (1999) Bioconjug Chem 10, 186-91; Josephson et. al
(2001) Angwandte Chemie 40, 3204-3206;).
[0009] Amino-CLIO is an excellent label for the attachment of
biomolecules, and for the synthesis of magnetic
nanoparticle-biomolecule conjugates, for two reasons. First it
provides an amine group for reaction with many bifunctional
conjugation reagents that consist of N-hydroxysuccinimide esters
that react first with an amine group and have a second group that
reacts with sulfhydryl groups on a biomolecule. Examples of these
bifunctional conjugating reagents are SPDP, SIA, SMCC and MBS.
These reagents are available commercially (Pierce Chemical or
Molecular Biosciences). Examples of biomolecules that have been
attached to amino-CLIO include peptides (Josephson et al, (1999)
Bioconjug Chem 10, 186-91), oligonucleotides (Josephson et. al
(2001) Angwandte Chemie 40, 3204-3206;) and proteins (Hogemann et
al. (2000). Bioconjug Chem 11, 941-6). Second, amino-CLIO is highly
stable due to the fact that the crosslinking forms a shell of
dextran around a core of iron oxide. This allows storage of either
amino-CLIO or bioconjugates based on amino-CLIO under a wide range
of conditions (temperature, pH, ionic strength). By covalently
joining polymeric molecules of the coating, crosslinking is
associated with a pronounced increase in the molecular weight of
the polymeric coating.
[0010] This amino-CLIO based chemistry has one major drawback,
however, which arises precisely because of the extraordinary
stability achieved by using a crosslinked-stablilized dextran on
the nanoparticle surface. For human parenteral applications, such
as for a magnetic label for targeted MR contrast agents, the
degradation or elimination of the agent, including the coating, is
required. However, when the iron oxide of an amino-CLIO based MR
contrast is dissolved or biodegraded, the crosslinked dextran
remains as a non-degradable sphere of polysaccharide. Similarly,
non-degradability occurs with micron-sized magnetic microspheres
where iron oxide is entrapped in a non-biodegradable polymeric
shell (see U.S. Pat. Nos. 4,654,267; 5,512,439).
[0011] CM-polymers can also function as starting materials for the
synthesis of drugs conjugates or for the attachment of various
biological molecules. As drug conjugates, CM-arabinogalactan,
CM-dextran and polyvinyl alcohol were used as carriers for
nucleotide analogues (U.S. Pat. No. 5,981,507). The carboxyl groups
were converted to primary amino groups by reaction with diamines.
Biological molecules like araAMP were then attached to the primary
amine of the aminated arabinogalactan, see Josephson, et al. (1996)
Antivir Ther 1, 147-56 and U.S. Pat. No. 5,478,576.
[0012] In these examples, the CM-polymers, such as
CM-arabinogalactan, exist as macromolecules in solution, which
allows conditions to be employed that insure the nearly
quantitative conversion of carboxyl groups to amino groups. The
absence of protected carboxyl groups allows essentially all
carboxyl groups to be chemically reactive.
[0013] There is a need for a improved magnetic nanoparticles to
which biomolecules can be attached for use in cell sorting
applications, in vitro assays, and which can be used as an
intravenously administerable, MR contrast agents. The ideal
nanoparticle must have a surface chemistry amenable to the
efficient attachment of biomolecules with retention of their
biological activity. It must be highly stable in vitro, both before
and after the attachment of biomolecules. Yet it must be labile or
degradable in vivo, with the utilization or elimination of all of
its components.
SUMMARY
[0014] The present invention relates to aminofunctionalzied
nanoparticles. In one embodiment, the nanoparticles have a magnetic
core having one or more magnetic metal oxide crystals, and a
noncrosslinked polymer coating associated with the core. The
polymer coating has a plurality of carboxyl groups and plurality of
reactive primary amino groups. A portion of the carboxyl groups are
associated with the crystals. In one embodiment, a portion of the
amino groups are associated with polymer through a peptidyl linkage
of the formula:
--O--(CH.sub.2).sub.m--CONH--[X]
[0015] wherein X is --(CH.sub.2).sub.nNH.sub.2,
--(CH.sub.2).sub.oCH NHCOO--,
--(CH.sub.2).sub.3NH(CH.sub.2).sub.4NH.sub.2 or
--(CH.sub.2).sub.3NH(CH.sub.2).sub.4NH(CH.sub.2).sub.3
NH.sub.2--
[0016] wherein m=1, 2, or 3; n=2, 3, 6, and o=3 or 4.
[0017] In one embodiment, the magnetic core has one or more
superparamagnetic iron oxide crystals. The superparamagnetic core
has a diameter between about 1 nm and about 25 nm, preferably
between about 3 nm and about 10 nm, and more preferably about 5
nm.
[0018] The nanoparticle (core and polymer) has diameter between
about 15 nm and 100 nm, preferably between about 20 nm and about
100 nm.
[0019] The polymer coating may be made from natural polymers, or
synthetic polymers, or derivatives or each. Nonlimiting examples
include polyvinyl alcohol and carboxymethyldextran In one aspect of
the invention, the nanoparticle can be conjugated to a
biomolecule.
[0020] In another aspect of the invention, there is provided a
nanoparticle biomolecule conjugate. The nanoparticle portion
includes a magnetic core having one or more magnetic metal oxide
crystals, a noncrosslinked polymer coating associated with the
core. As in the previous emboidiment, the the polymer coating has a
plurality of carboxyl groups and plurality of reactive primary
amino groups. A portion of the carboxyl groups are associated with
the crystals, In one embodiment, the amino groups are associated
with polymer through a peptidyl linkage of the formula:
--O--(CH.sub.2).sub.m--CONH--[X]
[0021] wherein X is --(CH.sub.2).sub.nNH.sub.2,
--(CH.sub.2).sub.oCH NHCOO--,
--(CH.sub.2).sub.3NH(CH.sub.2).sub.4NH.sub.2 or
--(CH.sub.2).sub.3NH(CH.sub.2).sub.4NH(CH.sub.2).sub.3NH.sub.2--
[0022] wherein m=1, 2, or 3; n=2, 3, 6, and o=3 or 4; and
[0023] whereby a a biomolecule is linked to the nanoparticle
through the amino group.
[0024] In another aspect of the invention, there is provided a
process for synthesizing an amine functionalized magnetic metal
oxide nanoparticle via the steps of (i) obtaining a polymer having
a plurality of carboxyl groups attached thereto, (ii) contacting
the polymer with magnetic metal oxide to produce a coated magnetic
metal oxide wherein a portion of said carboxyl groups are
associated with the metal oxide; and (iii) reacting the coated
magnetic metal oxide of step (ii) with a diamine. In one
embodiement the diamine is ethylene diamine; in another embodiment
the diamine is hexane diamine.
[0025] In another aspect of the invention, the contacting step in
the synthesis further includes the steps of (a) providing a
solution of soluble iron salts; (b) converting the iron salts into
iron oxide crystals; and (c) removing unassociated polymer. In yet
another aspect of the invention, the converting process includes
the further steps of (a) heating the iron salts to a temperature
between about 4.degree. C. and about 20.degree. C.; and adding an
amount of base to raise the pH to about 8 or higher to form iron
oxides. In another aspect, the process includes the step of heating
the iron oxides to a temperature about 60.degree. C. or more for at
least 30 minutes.
[0026] Another aspect of the invention provides a pharmaceutical
composition of the nanoparticle biomolecule conjugates and a
pharmaceutically acceptable carrier. This composition is
particularly useful in targeted MR imaging applications.
BRIEF DESCRIPTION OF THE FIGURES
[0027] In all figures, the large sphere represents the core
magnetic material.
[0028] FIG. 1 shows a reaction scheme for synthesis of amino
functionalized magnetic nanoparticles. First, a polymer 1 having
multiple carboxyl groups is obtained. Next, the polymer is used to
synthesize a coated nanoparticle. The core magnetic material is
shown as a dark sphere 2. Carboxyl groups face (i) both the
magnetic core which are blocked (arrow 3b) and (ii) the solvent
(arrow 3a) and are available for further reaction. Third, the free
carboxyl groups are reacted, here with carbodiimide, to produce the
amine functionalized nanoparticle 4.
[0029] FIGS. 2A-C are comparisons of the prior art nanoparticles
(FIGS. 2A and 2B) with the nanoparticles of the invention (2C).
[0030] In FIG. 2A, the prior art nanoparticles use crosslinked
dextran and ammonia. This produces a functionalized particle in
which every nitrogen is a primary amine. The polymer is associated
with the magnetic core (large sphere) via hydroxyl groups.
[0031] In FIG. 2B, the prior art nanoparticles use non-crosslinked
dextran which has been reacted with periodate to form aldehydes,
followed with EDA and a reducing agent. There are two nitrogen
atoms for every primary amine: a terminal amino group (1) linked to
the polymer through methyl amine groups (the dashed box, showing
second amine 2). There are no peptidyl linkages. The polymer is
associated with the magnetic core (large sphere) via hydroxyl
groups.
[0032] FIG. 2C shows the nanoparticles of the present invention.
The polymer is noncrosslinked, and the amino group is linked to the
polymer via a peptide bond. There are two nitrogen atoms for each
primary amine: terminal amine 1 and at peptidyl linkage 2. The
noncrosslinked polymer is associated with the magnetic core (large
sphere) via carboxyl groups.
DETAILED DESCRIPTION
[0033] The present invention meets the aforementioned requirements
by providing novel compositions of amino functionalized
nanoparticles, and their methods of synthesis. These novel amino
functionalized nanoparticles can serve further as magnetic
nanoparticles for the development biomolecule-magnetic nanoparticle
conjugates useful in a variety of in vitro, and in vivo
applications.
[0034] Definitions
[0035] A nanoparticle as described and claimed herein is a material
with a "core" of magnetic material associated with a polyfunctional
noncrosslinked polymer. The polymer coating displays a plurality of
carboxyl groups and reactive primary amino groups; a portion of the
carboxyl groups are associated with the magnetic core; the reactive
primary amino groups are available for subsequent covalent
reactions, e.g., for the attachment of biomolecules. The
nanoparticles have an overall size less than about 100 nm, before
conjugation to biomolecules. The overall size of the nanoparticles
is about 15 to 100 nm, preferably about 20 to 100 nm, more
preferably about 40 to 60 nm; about 50 nm is the most preferred.
The polymeric coating can be about 5 to 20 nm thick or more. Size
can be determined by laser light scattering by atomic force
microscopy or other suitable techniques.
[0036] The nanoparticle core can be monodisperse (a single crystal
of a magnetic material, e.g., metal oxide, such as
superparamagnetic iron oxide, per particle) or polydisperse (a
plurality of crystals, e.g., 2, 3, or 4, per particle). The metal
oxides are crystals of about 1-25 nm, preferably about 3-10 nm, and
most preferably about 5 nm in diameter. The magnetic metal oxide
can also comprise cobalt, magnesium, zinc, or mixtures of these
metals with iron. The term "magnetic" as used in this specification
and the accompanying claims means materials of high positive
magnetic susceptibility.
[0037] In a preferred embodiment, a superparamagnetic form of iron
oxide is used. Superparamagnetic iron oxide is one of the highly
magnetic forms (magnetite, non-stoichiometric magnetite,
gamma-ferric oxide) that has a magnetic moment of greater than
about 30 EMU/gm Fe at 0.5 Tesla and about 300 K. When magnetic
moment is measured over a range of field strengths, it shows
magnetic saturation at high fields and lacks magnetic remanence
when the field is removed.
[0038] The "polymer coating" is a natural or synthetic polymer
associated with the magnetic core that functions to keep the metal
oxides dispersed from each other. In one embodiment the polymer
"coating" is not a continuous film around the magnetic metal oxide,
but is a "mesh" or "cloud" of extended polymer chains attached to
and surrounding the metal oxide. The polymer coating may be a
natural polymer, a synthetic polymer. The polymer maybe linear, or
moderately or highly branched. In one embodiment, the polymer
coating can be carboxy dendrimers, commercially available from
Sigma-Aldrich, which are highly branched polycarboxyl polymers.
Other embodiments of the polymer coating are described in detail
below.
[0039] A natural polymer is obtained when a pure polymer, such as a
polysaccharide, is synthesized by a microorganism, plant or animal
and extracted in substantially pure form. Non limiting examples of
natural polymers include polysaccharides, such as dextran. A
synthetic polymer is obtained from nonbiological syntheses, by
using standard polymer chemistry techniques known to those in the
art to react monomers into polymers. The polymers may be
homopolymers, i.e, synthesized from a single type of monomer, or
co-polymers, i.e., synthesized from two or more types of monomers.
Non-limiting examples of synthetic polymers include
polymethylmethacrylate polymers and polyvinyl alcohol polymers.
[0040] A crosslinked polymer is one in which functional groups on a
polymer chain and/or branches has reacted with functional groups on
another polymer to form polymer networks. Crosslinked polymers are
characterized herein as being heat stable and resistant to
breakdown in biological systems. A crosslinked polymer has a
molecular weight significantly higher than the original starting
polymer.
[0041] A non-crosslinked polymer is described and claimed herein as
a polymer in which few or no individual polymer chains have reacted
with the functional groups of another polymer chain to form the
interconnected polymer networks. A non-crosslinked polymer that has
been functionalized is reasonably size homogeneous as compared to
the starting polymer, i.e., the polymer before and after
incorporation of amine and carboxyl functional groups have similar
molecular weights and molecular weight distributions. A small
increase in molecular weight of the polymer is seen generally as a
result of the incorporation of functional groups onto the polymer
and the few crosslinks that may occur on a statistical basis.
[0042] A polycarboxyl (or carboxyl) polymer is a polymer with more
than one carboxyl group per polymer.
[0043] A polyfunctionalpolymer is one with different functional
groups, such as amino and carboxyl groups attached to a
polymer.
[0044] Description
[0045] We have surprisingly discovered that amino functionalized
nanparticles can be synthesized using non crosslinked, carboxylated
polymers, and that these polymers permit the addition of reactive
primary amine groups to the polymer. These reactive primary amines
are attached to the polymer via peptidyl linkages.
[0046] When the noncrosslinked carboxylated polymers are used in
the synthesis of the nanoparticles, the resulting polymer coated
nanoparticle has two classes of carboxyl groups with very different
chemical reactivities. Some carboxyl groups are shielded from
further chemical reaction by forming a strong bond between the
polymer and the surface of the iron oxide. Some surface carboxyl
groups, facing the bulk solvent, can be activated with carbodiimide
and converted to reactive primary amino groups (FIG. 1). These
reactive primary amines can be reacted with bifunctional
conjugating agents and then with biomolecules to form targeted MR
contrast agents, or probes for used in biosensors.
[0047] As shown in FIG. 1, the process for synthesizing magnetic
nanoparticies involves three general steps: (i) obtaining a
polycarboxylated polymer; (ii) synthesizing a polycarboxylated
coated magnetic nanoparticle; and (iii) converting the surface
carboxyl groups to reactive primary amine groups while the
remaining carboxyl groups bind the polymer to the nanoparticle
(FIG. 1).
[0048] As a result of this procedure, the magnetic nanoparticles of
the invention have polyfunctional polymeric coating, i.e. one that
contains both amino and carboxyl groups. Since the carboxylated
polymer used to synthesize the magnetic nanoparticle in step (ii)
possesses only a single class of carboxyl groups, it is highly
surprising that the resulting polymer coated magnetic nanoparticle
consists of two distinct types of carboxyl groups. One type is
bound to the surface of the iron oxide (blocked from further
chemical reaction). A second type is exposed to the bulk solvent
and available for conversion to reactive primary amino groups, see
FIG. 1. Moreover, the chemistry used to incorporate the functional
groups results in a polymer coating that is noncrosslinked (i.e,
uncrosslinked or minimnally crosslinked).
[0049] The presence of both reactive primary amino and carboxyl
groups on the polymeric coating of the magnetic nanoparticles is
one of the distinctive features of the invention. Polymers
containing both reactive primary amino groups and carboxyl groups
are difficult to develop because of the propensity for
self-reaction. In solution at neutral pH, the positively charged
carboxyl group and negatively charged amino group form non-covalent
electrostatic bonds. When covalent modification of amino and
carboxyl group containing polymers is attempted, activation of the
carboxyl groups with a carbodiimide results in the formation of
peptidyl bonds by reaction amino groups on the same molecule. This
results in intrachain crosslinking when the reactive amino groups
are on the same polymer, or interchain crosslinking when the amino
group is on a second polymer molecule. Such interchain
cross-linking can be induced in proteins, which present a mixture
of amino and carboxyl groups on the surface, by activating agents
like carbodiimide and is used to make aggregates of soluble
proteins. For this reason the synthesis of polymers such as
peptides containing amino and carboxyl groups requires use of
protecting groups followed by deprotection. The synthesis used here
eliminates the protection-deprotection reaction steps.
[0050] Another feature of the amine functionalized nanoparticles of
the invention is that they can be readily degraded to yield their
metal salts and polymer coating. In one embodiment, the degradation
yields iron salts and the polyfunctional polymeric coating. In
vivo, this results in the utilization of iron oxide, by
incorporation of iron into red blood cells, and by the excretion
and/or degradation of the polycarboxylated polymer. In vitro, the
conditions of biodegradation can be simulated by exposing
nanoparticles to mildly acidic pH (3-6) in the presence of a metal
chelator, e.g. citrate or EDTA. This yields ferric ion chelates and
soluble polyfunctional polymers. The molecular weight of the
polyfunctional polymers, bearing amino and carboxyl groups, will be
slightly larger than the polycarboxylated polymers used to
synthesize the nanoparticles due the addition of reactive primary
amino groups.
[0051] The amine functionalized nanoparticles of the invention are
synthesized by activation of free carboxyl groups of the
nanoparticle with a water soluble carbodiimide, followed by
reaction with a large excess of a diamine. The nature of the
diamine provides a linker arm of varying lengths and chemistries
for the attachment of biomolecules. Nonlimiting examples of
diamines include ethylenediamine (EDA), propyldiamine, spermidine,
spermine, hexanediamine, and diamine amino acids, such as lysine or
omithine.
[0052] Unlike the synthesis for other amine functionalized
particles, ammonia is not used to make the amine functionalized
nanoparticles of the invention. For example, in the synthesis of
amino-CLIO, dextran coated magnetic nanoparticles are reacted with
epichlorohydrin, followed by reaction with ammonia. This reaction
produces a dextran crosslinked, amine functionalized nanoparticle
bearing primary amino groups
(H.sub.2N--CH.sub.2--CHOH--CH.sub.2--O-Polymer), as shown in FIG.
2A. If the carbodiimide activated carboxylated nanoparticles of
this invention were reacted with ammonia, an amide would be
obtained (H.sub.2N--CO--CH.sub.2--O-Polymer). The nitrogen atoms of
amides are far less reactive than primary amino groups and not
satisfactory for reaction with the bifunctional conjugating
reagents used to attach biomolecules.
[0053] The reaction with diamine is performed under conditions that
prevent crosslinking between nanoparticles. This is accomplished by
using a large excess of diamine. In general the moles of diamine
used will exceed the number of carboxyl groups present by a factor
of at least 10. Diamines are cheap and can be used in very large
excess. Unreacted diamine (MW<2 kDa) can be separated from amino
functionalized nanoparticle (MW>500 kDa) by ultrafiltration.
Alternatives to ultrafiltration for the removal of unreacted
diamine include gel permeation chromatography, dialysis, and
precipitation and resolubilization of the nanoparticle.
[0054] When the carbodiimide activated carboxylated nanoparticles
of the invention are reacted with a large excess of diamine, one of
the nitrogen atoms reacts with the carboxyl group to provide a
peptide bond, while a second exists as a primary amine suitable for
further chemistry. Hence the amino functionalized nanoparticles of
the invention have a characteristic general structure that includes
a peptidyl bond and a primary amino group, see FIGS. 1 and 2C. This
characteristic structure is not found with amino functionalized
amino-CLIO nanoparticle. Similarly, a peptide bond is not obtained
when dextran coated magnetic iron oxides are activated by treatment
with periodate, followed by reaction with a primary amine and
treatment with a reducing agent. In this case a methyl amine
linkage is obtained (FIG. 2B).
[0055] The presence of primary amino groups on the magnetic
nanoparticles can be readily ascertained by reaction with amine
specific reagents such as TNBS or ninhydrin or SPDP with the in
tact magnetic nanoparticle. Since the carboxyl groups are protected
by the metal oxide, they can be most easily analyzed after
digestion of the metal oxide core and isolation of the polymeric
coating. Digestion of metal oxide core can be accomplished by
treatment with acid and chelator. Typically a pH below 5, or
between 2 and 5, is sufficient. Chelators like citrate or EDTA
enhance the solubility of iron and are added an amount sufficient
to bind all metal ions. After digestion, the metal is removed by
passage over a cation exchange column or metal removing chelating
column such as Chelex. The polymer is then analyzed by IR and shows
characteristic peaks from carboxyl groups. Polymers with carboxyl
groups have characteristic absorption frequencies from the carbonyl
group (C.dbd.O) of the carboxyl (1780 to 1710 cm-1, strong) and the
hydroxyl group (3000 to 2500 cm-1, broad, variable).
[0056] The polymer obtained after digestion and removal of iron can
also be characterized by size and compared with the polymer used in
the nanoparticle synthesis. The polymer will be slightly larger
than the starting polymer due to addition of amino groups.
[0057] A second property of the amine functionalized polymers of
the invention is presence of at least two nitrogen atoms for each
primary amine due to characteristic general structure
(H.sub.2N--X--NH--CO--), see also FIG. 2C. X can be any structure
connecting the two primary amines of the diamine. Non limiting
examples include hexamine diamine, ethylene diamine, spermidine or
spermine, and amino acids like omithine or lysine, which are of
interest because of their negatively charged carboxyl group. The
total number of nitrogen groups attached to the purified polymer
can be obtained by submitting the purified polymer to elemental
analysis of nitrogen, i.e., determination of the content of all
nitrogen atoms. The number of reactive primary amino groups can be
determined by the TNBS method. A property of the amine
functionalized polymers of the invention is the amount of total
nitrogen will exceed the amount of nitrogen present as a primary
amine. For example, when ethylene diamine (EDA) has been used, the
total nitrogen content will be twice the nitrogen content obtained
with methods determining the amount of primary amine.
[0058] Properties and sources of polycarboxylated polymers for the
synthesis of coated nanoparticle. The polycarboxylated polymers can
be obtained by a variety of routes and have a variety of
compositions. They may be man made or naturally occurring and may
be highly branched or linear.
[0059] The polycarboxylated polymers have a molecular weight
between about 5 and 200 kDa, more preferably between 5 and 50 kDa.
Smaller polymers lack a sufficient number of carboxyl groups to
both strongly bind to the iron oxide and have the numerous free
carboxyl groups available for conversion to amino groups (FIG. 1).
The polymers must contain more than about five moles of carboxyl
group per mole of polymer. The number of carboxyl groups can be
determined by titration. The polycarboxylated polymers should have
a high water solubility over a wide range of pH's to be employed in
the synthesis of water soluble polymer coated magnetic
nanoparticles.
[0060] To be used in the synthesis of polymer coated magnetic
nanoparticles, unreacted polymer must be separated from polymer
coated nanoparticle. To do this easily and efficiently, it is
preferred that the polymer maintain a homogenous size distribution,
which can be determined by the usual methods of polymer chemistry
including light scattering, gel permeation chromatography. For
example, unreacted carboxymethylated dextran (MW=20 kDa) can be
readily separated from the coated nanoparticle (MW>500 kDa) by
ultrafiltration using a membrane with a cutoff of 100 kDa (Groman
WO 00/61191). Polymers larger than about 200 kDa can used but they
are more difficult and more expensive to separate from the polymer
coated nanoparticles as the larger molecular weight polymers and
the polymer coated nanoparticles pass through the same
membrane.
[0061] A preferred method of synthesizing polycarboxylated polymers
is the reaction of a water soluble polymer containing multiple
amino or hydroxyl groups with an alkyl halogenated acid in aqueous
strong base. This method has several advantages. First, the size
and size distribution of the polymer obtained will be determined
the size of the starting polymer. Hence by selecting a size
homogeneous polymer, the size homogenity of the carboxylated
polymer is achieved. The size of the polycarboxylated polymer will
be slightly larger than the starting polymer due to the addition of
carboxyl groups. For details see examples 1, 4 and 6, of Josephson
et al WO97/21452. Second, polymers with varying number of carboxyl
groups on them can be synthesized by varying the amount of
halogenated acid, to determine the optimum level of carboxylation
required for the synthesis of polymer coated magnetic nanoparticle,
see Groman WO 00/61191. An alternative method is the direct
synthesis of polycarboxylic functional polymers, such as
polymethacrylic acid based polymers.
[0062] Naturally occurring hydroxylated polymers that can be used
in the synthesis of polycarboxylated polymers include
polysaccharides like dextran, starch or cellulose. Polyvinyl
alcohol is a synthetic hydroxylated polymer that can replace
naturally occurring polysaccharides. These hydroxyl group-bearing
polymers are reacted with halogenated acids like bromoacetic acid,
chloroacetic acid, bromohexanoic acid and chlorohexanoic acid in
the present of strong base, typically 1-8 M NaOH. The
polycarboxylated polymer is then purified by ultrafiltration or by
precipitation. Alternatively anhydrides like succinic anhydride can
be used for carboxylation polyhydroxylated polymers, but these
result in ester linkages which can undergo slow hydrolysis.
[0063] Reaction of positively charged polymers like polylysine or
poly vinyl amine with an anhydride (succininc anhydride, maleic
anhydride, DTPA anhydride) provide another method of synthesizing
carboxylated polymers.
[0064] Hydrolysis of an anhydride containing polymer, such as
polytheyelene-alt-maleic anhydride (Sigma) is another method that
can yield a suitable carboxylated polymer.
[0065] Carboxyl group bearing polyamino acids can also be employed
as polycarboxylated polymers., e.g. polyaspartate or polyglutamate.
Carboxylated dendrimers are available commercially and are highly
branched synthetic polymers and can be used in the synthesis of
nanoparticles.
[0066] Synthesis of carboxyl terminated, polymer coated
nanoparticles.
[0067] Carboxy terminated nanoparticles can be synthesized by
mixing the carboxyl terminated polymer with ferrous and ferric
salts. Metals other than iron can be used in the synthesis of
magnetic metal oxides. For example, zinc, manganese or cobalt can
partially or completely replace the ferrous ion during the
synthesis of magnetic metal oxides.
[0068] The mixture is enclosed in a jacked reactor for temperature
control and stirred. It is covered to control access of oxygen. The
mixture is then brought to controlled temperature between 4 and 20
C., and a base, such as ammonia, is added. Base is added in a
highly controlled fashion, either by pumping or by drop wise
addition. Sufficient base is added to bring the pH to higher than
pH 8, which causes the formation of iron oxides. The resulting gel
or colloid maybe heated, to induce formation of the highly magnetic
iron oxide. The temperature of the mixture is heated to above 60 C.
for more than 30 minutes. Finally, the colloid is allowed to cool
and unreacted polymer removed from the polymer coated nanoparticle.
The preferred technique for removal is ultrafiltration, using a
membrane that has a cutoff that permits the carboxylated polymer to
pass through, while the larger coated nanoparticle is retained.
Alternatives to ultrafiltration are gel filtration and magnetic
separation. Citrate maybe added as stabilizer but it must be
removed by ultrafiltration before use of carbodiimide because of
its carboxyl groups. Details methods for the synthesis of
polycarboxylated polymer coated nanoparticles can be found in
Groman and Manro
[0069] Synthesis of amine terminated, polymer coated
nanoparticles.
[0070] The carboxyl groups of the carboxyl terminated nanoparticle
are then activated by the use of a water soluble carbodiimide.
Typically this is done in a non-amine containing buffer between pH
4.5 and 7. The activation with 0.1 M TEMED, pH 4.8 has been found
to satisfactory. Activation is typically done at 20-40 C. EDA) is
added in vast excess to prevent the formation of crosslinks between
the carboxyl groups. Excess diamine can be separated from the
aminated, polymer coated nanoparticle using ultrafiltration. The
use of carbodiimide results in the formation of a peptide bond
between the diamine linker and polymeric nanoparticle coating. The
number of primary amines on the particle can be monitored by
reaction with trinitrobenze. A variety of diamines can be used such
as hexamine diamine, ethylene diamine, spermidine or spermine.
Amino acids like omithine or lysine are diamines of interest
because of their negatively charged carboxyl group.
[0071] The aminofunctionalized nanoparticles can be used as
attachment substrates to form a variety of nanoparticle conjugates
for in vivo or in vitro applications. Example applications include
cell sorting, in vitro assays and in vivo applications such as
magnetic resonance imaging. A non limiting exemplary conjugate is a
biomolecule nanoparticle conjugate.
[0072] The amino terminated nanoparticle is then reacted with a
bifunctional conjugation reagent designed to react with amino
groups. Preferred conjugation reagents are NHS esters, which react
with the amine group of the nanoparticle, and which have second
moiety that can react with the sulfhydryl group of the biomolecule.
Such crosslinking agents include, for example, SPDP, long
chain-SPDP, SIA, MBS, SMCC, and others that are well known in the
art and are available from Piece Chemical Company. Detailed
procedures for their are available from the Piece Chemical web
site, see http://www.piercenet.com/ and the attached pdf files
downloaded from that site.
[0073] The activated biomolecule, preferrably with a single
sulfhydryl group distal from the site of bioactivity, is allowed to
react with the activated nanoparticle. Separation of unreacted
biomolecule from the biomolecule-nanoparticle conjugates can be
accomplished by gel filtration, ultrafiltration, dialysis or
magnetic separation methods. Examples of thiolated biomolecules
that have been attached to SPDP activated crosslinked magnetic
nanoparpticles include transferrin, (Hogemann, (2000) Bioconjug
Chem 11, 941-6), tat peptides (Josephson, (1999) Bioconjug Chem 10,
186-91; Zhao (2002) Bioconjug Chem 13, 840-4), oligonucleotides
(Josephson (2001) Agnew Chem Int Ed 40, 3204-3206; Perez, (2002) J
Am Chem Soc 124, 2856-7), antibodies (Kang,. (2002) Bioconjug Chem
13, 122-7) and proteins (Perez, Nature Biotechnol 20, 816-20). For
peptides (1-2 kDa), 5-25 peptides can be attached per 2000 Fe
atoms. For proteins, such as transferrin or antibodies (50-200 kDa)
1-4 biomolecules can be attached per 2000 Fe atoms.
[0074] Uses of the biomolecule-nanoparticle conjugates
[0075] The magnetic nanoparticles of the invention have various
uses with in vitro ligand binding assays. The nanoparticles can be
used in magnetic detection based assays (see Simmonds U.S. Pat.
Nos. 6,046,585 and 6,275,031, Rohr U.S. Pat. No. 5,445,970;
Ebersole, U.S. Pat. No. 4,219,335, Chemla, et. al. (2000)
Ultrasensitive magnetic biosensor for homogeneous immunoassay. Proc
Natl Acad Sci USA 97, 14268-72.). They can also be used in magnetic
resonance based ligand binding assays such as Josephson U.S. Pat.
No. 5,164,297 and Perez et al Nature Biotechnol. 2002
August;20(8):816-20.
[0076] The magnetic nanoparticles of the invention of the invention
are also suitable for cell sorting applications. Magnetic
nanoparticles were described by Molday U.S. Pat. No. 4,452,773 and
commercially available (Miltenyi Biotech, Auburn Calif., and
Molecular Probes, Eugene Oreg.).
[0077] Finally the magnetic nanoparticles of the invention can be
used as for targeted MR imaging applications.
[0078] For in vivo uses, the biomolecule-nanoparticle conjugates
are formulated and sterilized according to the published methods
for sterilizing parenterally administered MRI contrast agents. For
parenteral applications, sterilization can be achieved by filtering
the colloid through a 220 nm filter (filter sterilization) or by
heat sterilization (terminal sterilization). Depending on the
method of sterilization various excipients, such as
monosaccharides, polysaccharides, salts, can be added to stabilize
the colloid during heat stress or storage. Excipients can also
serve to bring the ionic strength and pH of the preparation into
the physiological range. See Josephson U.S. Pat. No. 5,160,726,
Groman U.S. Pat. No. 5,248,492.
EXAMPLES
Example 1
[0079] Synthesis of a carboxymethylated polyhydroxylated
polymer
[0080] Carboxymethylated polymers are prepared by reaction of a
halo acetic acid with a polymer in strong base, usually NaOH. The
polymer should be of sufficient molecular weight to allow
separation from unreacted haloacetic acid from the
carboxymethylated polymer. The polymer is preferably between 5 kDa
and 100 kDa. The separation can be accomplished by dialysis,
ultrafiltration or precipitation. The polymer is then dried by
lyophilization, vacuum drying or spray drying. The polymer should
be of sufficient molecular weight to allow separation of dextran
from dextran coated iron oxide. For example, when the nanoparticles
have molecular weights of greater than 500 kDa, and the polymer is
preferably less than 100 kDa, this separation can be accomplished
by ultrafiltration.
[0081] (Poly)vinyl alcohol (100 g, MW=15 kDa) was dissolved in 1000
ml of hot water. After reaching room temperature, 600 ml of 8M NaOH
solution was added to it with stirring and again equilibrates to
room temperature. 100 g of bromo-acetic acid was then added and
mixture stirred for two hours. The polymer was neutralized with 6M
HCL It was then dried under vacuum overnight at room temperature.
It is denoted CM-PVA.
Example 2
[0082] Synthesis of carboxymethylated polymer coated
nanoparticle
[0083] One hundred milliliters of a solution of 12 mmoles of ferric
chloride (hexahydrate) and 6 grams of of CM-PVA. was prepared. The
solution was filtered and cooled to 2-4 C. To the mixture was added
6 mmoles of ferrous chloride (tetrahydrate) dissolved in 5 mL of
water. While being stirred rapidly, 4.5 mLs of 28-30% ammonium
hydroxide (2-4 C.) was added dropwise. The mixture was then heated
to between 70 and 90 C. and maintained at the higher temperature
for 2 hours. Unreacted CM-PVA was removed by ultrafiltration using
a 100 kDa cutoff membrane. The colloid had a size of 54 nm by light
scattering and an R2 of 60 mM-1 sec-1. The procedure was repeated
using 3 g CM-PVA to give a colloid with 65 nm and an R2 of 160 mM-1
sec-1.
Example 3
[0084] Conversion of carboxyl groups on the magnetic nanoparticle
to amino groups
[0085] To 10 mLs of 10 mg Fe/mL of the carboxylated polymer coated
nanoparticle in 0.1 M TEMED buffer, pH 4.8, was added 0.2 g of
1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride at room
temperature. After 15 mintues, 0.5 mL 1,2 ethylene diamine was
added. After 24 hours the mixture was put in dialysis bag and
dialyzed until the dialysate was free of amine by the TNBS
assay.
Example 4
[0086] Reaction of amino-functionalized magnetic nanoparticles with
a biomolecule
[0087] Nanoparticles were reacted with N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP). To 1 mL of amino
functionalized magnetic nanoparticle (10 mg Fe) was added 1 mL of
0.1 M phosphate buffer, pH 7.4, and 2 mL of 25 mM SPDP in DMSO (50
umoles SPDP). The mixture was allowed to stand for 60 min at room
temperature. Low molecular impurities were removed by PD-10 columns
(Sigma Chemical, St. Louis, Mo.) equilibrated with 0.01 MTris and
0.02 Mcitrate, pH 7.4 buffer. The number of amine groups can be
obtained for the amount of 2PT released assayed by addition of
dithiothreitol. (Zhao, (2002) Bioconjug Chem 13, 840-4).
[0088] It will be apparent to those skilled in the art that other
changes and modifications may be made in the above-described
compounds, compositions, and methods for making and using the same,
without departing from the scope of the invention herein, and it is
intended that all matter contained in the above description shall
be interpreted in an illustrative and not in a limiting sense.
* * * * *
References