Polymer coated magnetic particles

The invention relates to a polymer comprising a segment of Formula (I): wherein, R is either absent or a linking group, n is an integer greater than 0; and m is an integer from 1 to 6.

RELATED APPLICATIONS

This application is a U.S. national stage application under §371 of International Application No. PCT/SG2010/000415, filed on Oct. 28, 2010, which claims benefit of, and priority from, Singapore patent application No. 200907163-0, filed on Oct. 28, 2009, each of which is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to polymer-coated magnetic particles.

BACKGROUND

Magnetic particles (MPs) have received a great deal of interest because of their potential use in various biomedical applications requiring magnetism, such as magnetic resonance imaging. Recent advances in synthesis have enabled the size and shape control of MPs for applications in catalysis and bioimaging. On the other hand, quantum dots (QDs) are emerging as potential biomarkers and have been revolutionising the field of bioimaging in recent years.

Most MPs and QDs are synthesised in organic solvents using hydrophobic surfactants. The resulting nanoparticles are only dispersible in organic solvents i.e. they are hydrophobic. In order to use these particles for biomedical applications, they have to be dispersible in aqueous media and buffer media i.e. they need to be hydrophilic. Different coating strategies exist in the literature to impart the QDs and MPs with hydrophilicity and colloidal stability. Apart from small molecule coatings (e.g. thiols and carboxylic acids), silica and polymer coatings dominate the surface functionalisation methods.

Magnetic resonance imaging (MRI) is regarded as a powerful tool in medicine because of its spatial resolution and its capability to enhance contrast differences between healthy and pathological tissues. However, its low signal sensitivity is a major problem. The combination of MRI and fluorescence imaging has the potential to enhance the sensitivity and resolution, resulting in better disease diagnosis. Multifunctonal nanoparticles are, therefore, emerging as an interesting class of materials. Multifunctional nanoparticles with multiple capabilities (imaging, targeting and delivery) have potential in bio-labelling, MRI and drug delivery applications.

Accordingly there is a need for new hydrophilic nanomaterials for use as contrast agents in MRI and for labelling cells. Furthermore, there is a need for polymers that are hydrophilic, biodegradable, non-toxic and biocompatible, that can be used to impart these properties to nanomaterials. It would be a further advantage if such polymers could be prepared by environmentally friendly and economically inexpensive routes.

SUMMARY

In a first aspect of the invention there is provided a polymer comprising a segment of Formula (I):

wherein,
R is either absent or is a linking group,
n is an integer greater than 0; and
m is an integer from 1 to 6, optionally from 1 to 4.

According to a second aspect there is provided a polymer comprising a segment of Formula (III):

wherein,
R is either absent or a linking group,
R′ may be R—NH2or it may be different, optionally R may individually be selected from hydrogen, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, heterocyclene, ether, thioether and amine, each of which may be optionally substituted,
n is an integer greater than 0; and
m is an integer from 1 to 6, optionally from 1 to 4.

The following options may be used in conjunction with either the first or the second aspect, either individually or in any suitable combination.

R may comprise one or more of alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, heterocyclene, ether, thioether and amine, each of which may, independently, be optionally substituted. The polymer may comprise a segment of Formula (I) wherein R is 1-(ethylenethio)-1,3-propanediyl.

m may be 4.

The polymer may consist only of a segment of Formula (I) and terminal groups. In this case, n may be at least 6.

The polymer may be hydroxyl terminated. It may have hydroxyl groups at both ends.

n may be between about 35 and about 40.

The molecular weight of the polymer may be between about 15000 and about 15500 Da.

The polymer may be on the surface of a material. The material may be rendered hydrophilic by said polymer. The material may be rendered biocompatible by said polymer.

The material may be on the surface of a material that is a nanomaterial. The nanomaterial may comprise at least one nanoparticle. The at least one nanoparticle may be a γ-Fe2O3nanoparticle. It may be a magnetic nanoparticle. It may be a magnetic γ-Fe2O3nanoparticle. The nanomaterial may have a saturation magnetisation of greater than about 20 emu/g. The nanomaterial may have a saturation magnetisation of between about 25 and about 35 emu/g. The nanomaterial may be used as a positive T1contrast agent in magnetic resonance imaging. The nanomaterial may comprise one or more quantum dots. The one or more quantum dots may be CdSe/ZnS core-shell particles (i.e. they may have a core of CdSe and a shell of ZnS at least partially, optionally completely, surrounding the core). The nanomaterial containing quantum dots may be used for labelling a biological molecule. The biological molecule may be a cancer cell. The cancer cell may be a Hep G2 human liver cancer cell. The diameter of the nanoparticles of the nanomaterial may be between about 1 and about 20 nm. The diameter of the nanoparticles may be between about 5 and about 15 nm.

The polymer may be used as a taste masking agent.

The polymer may be prepared by a process comprising the steps of:

a) providing a dicarboxylic acid monomer having a pendant hydroxyl group and a polyhydric alcohol monomer of Formula (II):

wherein m is an integer from 1 to 6;

b) combining said dicarboxylic acid monomer and said polyhydric alcohol monomer at a temperature above the melting point of said dicarboxylic acid monomer and said polyhydric alcohol monomer to form a homogeneous solution;

c) adding a lipase to said homogeneous solution so as to catalyse polycondensation of said dicarboxylic acid monomer and said polyhydric alcohol monomer to form a hydroxyl-functionalised polymer;

d) reacting the pendant hydroxyl group of the dicarboxylic acid monomer residue of the hydroxyl-functionalised polymer to form an ally-ether-substituted polymer; and

e) reacting the allyl group of the allyl-ether-substituted polymer with an amino-functionalised compound to form an amino-functionalised polymer.

The polymer may be prepared by a process comprising the steps of:

a) providing a malic acid monomer and polyhydric alcohol monomer of Formula (II):

wherein m is an integer from 1 to 6.

b) combining the malic acid monomer and the polyhydric alcohol monomer, optionally at a temperature above the melting points of both the malic acid monomer and the polyhydric alcohol monomer, to form a homogeneous solution;

c) adding a lipase to the homogeneous solution so as to catalyse polycondensation of the malic acid monomer and the polyhydric alcohol monomer to form a hydroxyl-functionalised polymer;

d) reacting the pendant hydroxyl group of the malic acid monomer residue of the hydroxyl-functionalised polymer to form an allyl-ether-substituted polymer, e.g. by reacting said hydroxyl group with an allyl compound comprising a leaving group; and

e) reacting the allyl group of the allyl-ether-substituted polymer with an amino-functionalised compound to form an amino-functionalised polymer.

The polyhydric alcohol monomer may be a sorbitol monomer. The sorbitol monomer may be D-sorbitol. The malic acid monomer may be L-malic acid. The lipase may beCandida antarcticalipase B.

The step of reacting the pendant hydroxyl group may comprise forming an allyl ether of the hydroxyl group and reacting the terminal olefin groups of the resulting polymer with a thiol functional amine in the presence of a free radical source so as to form the aminofunctional polymer

In an embodiment there is provided a polymer comprising a segment of Formula (I):

wherein,
R is either absent or a linking group, n is an integer greater than 0 and m is 4.

In another embodiment there is provided a polymer that consists of terminal groups and a segment of Formula (I):

wherein,
R is either absent or a linking group, n is an integer of at least 6 and m is 4.

In another embodiment there is provided a polymer that consists of terminal groups and a segment of Formula (I):

wherein,
R is 1-(ethylenethio)-1,3-propanediyl, n is an integer of at least 6 and m is 4.

In another embodiment there is provided a polymer that consists of terminal groups and a segment of Formula (I):

wherein,
R is 1-(ethylenethio)-1,3-propanediyl, n is an integer of at least 6 and m is 4, and wherein the polymer is on the surface of, optionally coats the surface of, a nanomaterial comprising at least one magnetic nanoparticle.

In another embodiment there is provided a polymer that consists of terminal groups and a segment of Formula (I):

wherein,
R is 1-(ethylenethio)-1,3-propanediyl, n is an integer of at least 6 and m is 4, and wherein the polymer coats the surface of a nanomaterial comprising at least one magnetic nanoparticle, which nanomaterial is suitable for use as a positive T1contrast agent in magnetic resonance imaging.

In another embodiment there is provided a polymer that consists of terminal groups and a segment of Formula (I):

wherein,
R is 1-(ethylenethio)-1,3-propanediyl, n is an integer of at least 6 and m is 4, and wherein the polymer coats the surface of a nanomaterial comprising at least one magnetic nanoparticle and one or more quantum dots.

In another embodiment there is provided a polymer that consists of terminal groups and a segment of Formula (I):

wherein,
R is 1-(ethylenethio)-1,3-propanediyl, n is an integer of at least 6 and m is 4, and wherein the polymer coats the surface of a nanomaterial comprising at least one magnetic nanoparticle and one or more quantum dots, which nanomaterial is suitable for use for labelling a biological molecule.

An example of the synthesis of the polymer is as follows:

a) providing a malic acid monomer and polyhydric alcohol monomer of Formula (II):

wherein m is an integer from 1 to 6;

b) combining the malic acid monomer and the polyhydric alcohol monomer, optionally at a temperature above the melting points of both the malic acid monomer and the polyhydric alcohol monomer, to form a homogeneous solution;

c) adding a lipase to the homogeneous solution so as to catalyse polycondensation of the malic acid monomer and the polyhydric alcohol monomer to form a hydroxyl-functionalised polymer;

d) reacting the pendant hydroxyl group of the malic acid monomer residue of the hydroxyl-functionalised polymer with an allyl compound comprising a leaving group to form an allyl-ether-substituted polymer; and

e) reacting the allyl group of the allyl-ether-substituted polymer with an amino-functionalised compound to form an amino-functionalised polymer.

Another example of the synthesis of the polymer is as follows:

a) providing a malic acid monomer and sorbitol;

b) combining the malic acid monomer and the sorbitol at a temperature above the melting points of both the malic acid monomer and the sorbitol, to form a homogeneous solution;

c) adding a lipase such asCandida antarcticalipase B to the homogeneous solution so as to catalyse polycondensation of the malic acid monomer and the sorbitol to form a hydroxyl-functionalised polymer;

d) reacting the pendant hydroxyl group of the malic acid monomer residue of the hydroxyl-functionalised polymer with an allyl compound comprising a leaving group to form an allyl-ether-substituted polymer; and

e) reacting the allyl group of the allyl-ether-substituted polymer with 3-aminoethanethiol in the presence of a free radical source to form the polymer.

The polymer may be used for coating the surface of a material. The material may be rendered hydrophilic by the polymer. The material may be rendered biocompatible by the polymer.

According to a third aspect of the invention there is provided a nanomaterial comprising at least one magnetic nanoparticle and the polymer of the invention at least partly coating the surface of said at least one magnetic nanoparticle. The at least one magnetic nanoparticle may be a γ-Fe2O3nanoparticle. The nanomaterial may have a saturation magnetisation of greater than about 20 emu/g. The nanomaterial may have a saturation magnetisation of between about 25 and about 35 emu/g. The nanomaterial may have a diameter between about 1 and about 20 nm. The nanomaterial may have a diameter between about 5 and about 15 nm. The nanomaterial may comprise one or more quantum dots. The one or more quantum dots may be CdSe/ZnS core-shell particles.

The nanomaterial may be used as a positive T1contrast agent in magnetic resonance imaging.

Where the nanomaterial comprises quantum dots, the nanomaterial may be used for labelling a biological molecule. The biological molecule may be a cancer cell. The cancer cell may be a Hep G2 human liver cancer cell.

According to a fourth aspect of the invention there is provided a nanomaterial comprising at least one, optionally a plurality of, nanoparticle(s) and the polymer of the invention at least partly coating the surface of said at least one nanoparticle.

According to a fifth aspect of the invention there is provided a nanoparticulate substance comprising a plurality of the nanomaterials of the third and fourth aspects described above.

DETAILED DESCRIPTION

As used herein the term “diameter” when used in relation to a non-spherical object means the equivalent spherical diameter of the object (i.e. the diameter of a sphere of equivalent volume to the object).

As used herein the term “plurality” means two or more.

As used herein the terms “polymer”, “polymeric” and related terms includes reference to dimeric, trimeric and oligomeric compounds.

As used herein the term “nanomaterial” means a material having particles having a dimension of between about 1 and about 1000 nm in at least one dimension.

The present invention provides biocompatible polymers and nanomaterials. The polymer of the invention may be used for example in taste masking and/or for surface coating of materials. The nanomaterials of the invention may be used as contrast agents in magnetic resonance imaging and for labelling of cells.

Polymer

The present invention provides a polymer comprising a segment of Formula (I):

wherein, R is either absent or a linking group, n is an integer greater than 0; and m is an integer from 1 to 6.

Where R comprises a heterocyclene, the heterocyclene may be monocyclic or polycyclic. The heterocyclene may include one or more carbon-carbon double bonds. The heterocyclene may have at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. The heterocyclene may have from 3 to 6, 10, 15 or 20 carbon atoms; from 6 to 10, 15 or 20 carbon atoms; from 10 to 15 or 20 carbon atoms; or from 15 to 20 carbon atoms. The heterocyclene may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. The heterocyclene may have one or more heteroatoms. The heterocyclene may have 1, 2 or 3 heteroatoms. The heterocyclene may have a heteroatom that is selected from the group consisting of N, O, S and P or may have more than one of these.

Where R comprises one or more alkylene or alkenylene, these groups may be branched or unbranched.

Where R comprises one or more ether, thioether and/or amine, these groups cannot be bonded directly to the oxygen or amine of the —O—R—NH2group.

The polymer of the invention may comprise a segment of Formula (I) selected from the group consisting of:

The polymer of the invention may be terminated with any suitable group. The polymer of the invention may be hydroxyl, amine, thiol, carboxyl, aldehyde, amide, acetylene or alkenyl terminated. It may have hydroxyl groups at both termini of the polymer. It may have a hydroxyl group at one end of the polymer. The polymer may be a straight chain polymer.

The polymer of the invention may consist of a segment of Formula (I) together with terminal groups. The polymer of the invention may comprise a plurality of segments of Formula (I), wherein R and n are individually defined for each segment of Formula (I) according to the above definitions of these variables, together with terminal groups. The polymer of the invention may comprise one or more other segments having a different structure to Formula (I).

The polymer of the invention may consist of a single hydroxyl-terminated segment of formula:

wherein n is at least 6.

The average molecular weight of the polymer of the invention may be greater than about 500, 1000, 2500, 5000, 10000, 15000, 25000, 50000, 100000, 250000, 500000 or 1000000 Da. The average molecular weight of the polymer of the invention may be between about 500 and about 1000, 2500, 5000, 10000, 15000, 25000, 50000, 100000, 250000, 500000 or 1000000 Da; between about 1000 and about, 2500, 5000, 10000, 15000, 25000, 50000, 100000, 250000, 500000 or 1000000 Da; between about 2500 and about 5000, 10000, 15000, 25000, 50000, 100000, 250000, 500000 or 1000000 Da; between about 5000 and about 10000, 15000, 25000, 50000, 100000, 250000, 500000 or 1000000 Da; between about 10000 and about 15000, 25000, 50000, 100000, 250000, 500000 or 1000000 Da; between about 15000 and about 25000, 50000, 100000, 250000, 500000 or 1000000 Da; between about 25000 and about, 50000, 100000, 250000, 500000 or 1000000 Da; between about 50000 and about 100000, 250000, 500000 or 1000000 Da; between about 100000 and about 250000, 500000 or 1000000 Da; between about 250000 and about 500000 or 1000000 Da; or between about 500000 and 1000000 Da. The average molecular weight of the polymer of the invention may be about 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000 or 1000000 Da. The average molecular weight of the polymer may be between about 15000 and 15500 Da. The average referred to above may be a number average or may be a weight average or may be a z-average molecular weight. The polymer may be substantially monodispersed. It may have a narrow molecular weight distribution or may have a broad molecular weight distribution. It may have a polydispersity (defined as weight average molecular weight divided by number average molecular weight) of about 1 to about 10, or about 1 to 5, 1 to 3, 1 to 2, 1 to 1.5, 1 to 1.2, 1.5 to 10, 2 to 10, 3 to 10, 5 to 10, 1.5 to 5, 1.5 to 3 or 2 to 5, e.g. about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10).

The polymer of the invention may be soluble in water. The polymer of the invention may be biocompatible. The polymer of the invention may be biodegradable. The polymer of the invention may be biodegradable in vivo into species that are non-toxic to humans.

Polymer Synthesis

The polymer of the invention may be prepared via an enzyme-catalysed condensation. Enzyme-catalysed reactions are advantageous due the non-toxic nature of enzyme catalysts. The enzyme catalyst may be any suitable lipase. The enzyme catalyst may be for exampleCandida antarcticalipase B (triacylglycerol hydrolase, EC 3.1.1.3; Novozym® 435). The enzyme may be a supported enzyme. It may be supported on a solid substrate e.g. a resin substrate. It may be supported on a particulate acrylic resin substrate. This may facilitate separation, and optionally reuse, of the enzyme.

The polymer of the invention may be prepared via enzyme-catalysed condensation of a polyhydric alcohol monomer and a dicarboxylic acid having a pendant hydroxyl group to form a hydroxyl-functionalised polymer intermediate.

The polymer of the invention may be prepared from naturally occurring monomers. Naturally occurring monomers may be inexpensive compared to synthetic monomers. Naturally occurring monomers may be non-toxic to humans. Naturally occurring monomers may be biodegradable. They may be biodegradable to non-toxic products.

The polymer of the invention may comprise a segment of Formula I which is derived from a sorbitol monomer. The polymer of the invention may comprise a segment of Formula I which is derived from a malic acid monomer. The polymer of the invention may comprise a segment of Formula I which is derived from a sorbitol monomer and a malic acid monomer. The polymer of the invention may comprise a segment of Formula I which is derived from enzyme-catalysed condensation of a sorbitol monomer and a malic acid monomer.

Sorbitol is used extensively in the food industry as it is highly water-soluble and free of discernible toxicity. Sorbitol-based transporters have been recently developed for intracellular drug delivery applications [Maiti, K. K.; Lee, W. S.; Takeuchi, T.; Watkins, C.; Fretz, M.; Kim, D.-C.; Futaki, S.; Jones, A.; Kim, K.-T.; Chung, S.-K.Angew. Chem. Int. Ed.2007, 46, 5880-5884]. The sorbitol monomer may be D-sorbitol. The sorbitol monomer may be L-sorbitol. The polymer of the invention may comprise a segment of Formula I derived from D-sorbitol and a segment of formula I derived from L-sorbitol. The sorbitol may be obtained from a natural source. For example, the sorbitol may be obtained from the fruit of a plant. The sorbitol may be obtained from the fruit of a plant of the Rosaceae family. The sorbitol may be obtained from apples, grapes, cherries or apricots. The sorbitol may be synthesised. The sorbitol may be synthesised by reducing D-glucose. The sorbitol may be synthesised by reducing D-glucono-1,4-lactone.

Malic acid is also used extensively in the food industry because of its high water-solubility and lack of discernable toxicity. The malic acid monomer may be L-malic acid. The malic acid monomer may be D-malic acid. The polymer of the invention may comprise a segment of Formula I derived from L-malic acid and a segment of Formula I derived from D-malic acid. The malic acid may be obtained from a natural source. The malic acid may be obtained from apples. The malic acid may be synthesised.

Where the polymer of the invention is prepared via a hydroxyl-functionalised polymer intermediate, the hydroxyl-functionalised polymer intermediate may be reacted to form an alkenyl-ether-substituted polymer. For example, the hydroxyl-functionalised polymer intermediate may be reacted with a suitable compound to form an allyl-ether-substituted polymer. The allyl-ether-substituted polymer may be formed by any suitable means. Methods for forming allyl-ethers are well known to those in the art. For example, the hydroxyl-functionalised polymer intermediate may be reacted with an allyl compound comprising a leaving group under basic conditions to form an allyl-ether-substituted polymer. The leaving group may be any suitable group. For example, the allyl compound comprising a leaving group may be an allyl-halogen, such as allyl chloride or allyl bromide.

The allyl group of the allyl-ether-substituted polymer may be reacted with an amino-functionalised compound to form an amino-functionalised polymer according to the invention. The amino-functionalised compound may be any suitable amino-functionalised compound. For example, the amino-functionalised compound may be an amino-alkyl-thiol, such as aminoethanethiol.

The polymer of the invention may be prepared by a process comprising the steps of:

a) providing a malic acid monomer and polyhydric alcohol monomer of Formula (II):

wherein m is an integer from 1 to 6;

b) combining said malic acid monomer and said polyhydric alcohol monomer at a temperature above the melting point of said malic acid monomer and said polyhydric alcohol monomer to form a homogeneous solution;

c) adding a lipase to said homogeneous solution so as to catalyse polycondensation of said malic acid monomer and said polyhydric alcohol monomer to form a hydroxyl-functionalised polymer;

d) reacting the pendant hydroxyl group of the malic acid monomer residue of the hydroxyl-functionalised polymer with an allyl compound comprising a leaving group to form an allyl-ether-substituted polymer; and

e) reacting the allyl group of the allyl-ether-substituted polymer with an amino-functionalised compound to form an amino-functionalised polymer.

Polymer Applications

The polymer of the invention may be used as a taste masking agent. For example, the polymer of the invention may be used as a taste-masking coating on a material. The polymer of the invention may be used to nano-encapsulate a material to mask its taste. The polymer of the invention may be used to micro-encapsulate a material to mask its taste. A nano-encapsulated material or micro-encapsulated material may be incorporated into a formulation such as a tablet, capsule, powder or dispersion. The polymer of the invention may be used as a coating on a capsule or tablet to mask its taste. A nano-encapsulated material or micro-encapsulated material may be incorporated into a food or beverage. The taste-masked material may, for example, be a pharmaceutical or a food or a drug or a neutraceutical or a herbal preparation or may be a combination of these.

The polymer of the invention may be able to bind to a biological material. The polymer may be able to bind to a biological material such as a cancer cell. The polymer of the invention may be able to bind to a biological material via the amino group of the polymer.

The polymer of the invention may be able to adsorb to a surface of a material. The polymer may be able to adsorb to the surface of a nanomaterial. The polymer may be able to adsorb to a surface of a magnetic nanoparticle. The polymer may be able to adsorb to the surface of a quantum dot. The polymer may be able to adsorb to a surface via the hydroxyl group of the polymer. Where the polymer comprises a thio group (including a thiol or thioether), the polymer may be able to adsorb to a surface via the thio group. The polymer may be able adsorbed to a surface of a material. The polymer may be adsorbed to the surface of a nanomaterial. The polymer may be adsorbed to a surface of a magnetic nanoparticle. The polymer may be adsorbed to the surface of a quantum dot. The polymer may be adsorbed to a surface via the hydroxyl group of the polymer. Where the polymer of the invention comprises a thio group, the polymer may be adsorbed to a surface via the thio group.

Adsorption of the polymer to the surface of a material may render the material hydrophilic. The polymer may be used to render a nanomaterial hydrophilic. The polymer may be used to render a magnetic nanoparticle hydrophilic The polymer may be used to render a Fe2O3magnetic nanoparticle hydrophilic. The polymer may be used to render a quantum dot hydrophilic. The polymer may be used to render a CdSe/ZnS quantum dot hydrophilic. The polymer may be used to render a nanomaterial comprising one or more magnetic nanoparticles and one or more quantum dots hydrophilic. The polymer may be used to render a nanomaterial comprising one or more Fe2O3magnetic nanoparticles and one or more CdSe/ZnS quantum dots hydrophilic.

Adsorption of the polymer to the surface of a material may render the material biocompatible. The polymer may be used to render a nanomaterial biocompatible. The polymer may be used to render a magnetic nanoparticle biocompatible. The polymer may be used to render a Fe2O3magnetic nanoparticle biocompatible. The polymer may be used to render a quantum dot biocompatible. The polymer may be used to render a CdSe/ZnS quantum dot biocompatible. The polymer may be used to render a nanomaterial comprising one or more magnetic nanoparticles and one or more quantum dots biocompatible. The polymer may be used to render a nanomaterial comprising one or more Fe2O3magnetic nanoparticles and one or more CdSe/ZnS quantum dots biocompatible.

Adsorption of the polymer to the surfaces of a plurality of particles may inhibit aggregation of the particles when dispersed in a continuous phase. Adsorption of the polymer to the surfaces of a plurality of nanomaterials may inhibit aggregation of the nanomaterials when dispersed in a continuous phase. Adsorption of the polymer to the surface of a plurality of magnetic nanoparticles may inhibit aggregation of the magnetic nanoparticles when dispersed in a continuous phase. Adsorption of the polymer to the surface of a plurality of Fe2O3magnetic nanoparticles may inhibit aggregation of the Fe2O3magnetic nanoparticles when dispersed in a continuous phase. Adsorption of the polymer to the surface of a plurality of quantum dots may inhibit aggregation of the quantum dots when dispersed in a continuous phase. Adsorption of the polymer to the surface of a plurality of CdSe/ZnS quantum dots may inhibit aggregation of the CdSe/ZnS quantum dots when dispersed in a continuous phase. Adsorption of the polymer to the surface of a plurality of nanomaterials comprising one or more magnetic nanoparticles and one or more quantum dots may inhibit aggregation of the nanomaterials when dispersed in a continuous phase. Adsorption of the polymer to the surface of a plurality of nanomaterials comprising one or more Fe2O3magnetic nanoparticles and one or more CdSe/ZnS quantum dots may inhibit aggregation of the nanomaterials when dispersed in a continuous phase.

The present invention also provides a nanomaterial comprising at least one magnetic nanoparticle having adsorbed to the surface thereof a biocompatible hydrophilic polymer, wherein said biocompatible hydrophilic polymer comprises an amino group and a hydroxyl group

The polymer adsorbed to the one or more magnetic nanoparticles may be any suitable polymer. The polymer adsorbed to the one or more magnetic nanoparticles may further comprise a thio group. The polymer adsorbed to the one or more magnetic nanoparticles may be the polymer of the invention as herein described.

A magnetic nanoparticle may comprise any suitable magnetic material. For example, a magnetic nanoparticle may comprise Fe2O3. A magnetic nanoparticle may comprise α-Fe2O3. A magnetic nanoparticle may comprise β-Fe2O3. A magnetic nanoparticle may comprise γ-Fe2O3. A magnetic nanoparticle may comprise ε-Fe2O3. A magnetic nanoparticle may comprise amorphous. Fe2O3. A magnetic nanoparticle may comprise Fe3O4(magnetite). A magnetic nanoparticle may comprise gadolinium oxide. Fe3O4. A magnetic nanoparticle may comprise a heterobimetallic cluster. A magnetic nanoparticle may comprise a cobalt compound. A magnetic nanoparticle may comprise FePt.

The nanomaterial may further comprise one or more quantum dots. The polymer may be adsorbed to the quantum dots. Where the polymer comprises a thio group, the polymer may be adsorbed to a quantum dot via the thio group. The one or more quantum dots may be any suitable quantum dots. The one or more quantum dots may be CdSe/ZnS core-shell particles. The one or more quantum dots may be CdSe/CdS core-shell particles. The one or more quantum dots may be CdS/CdSe core-shell particles. The one or more quantum dots may be ZnSe/ZnS core-shell particles. The one or more quantum dots may be PbSe/PbS core-shell particles. Mixtures of these may in some cases be used.

The nanomaterial may comprise one or more Fe2O3magnetic nanoparticles and a polymer comprising a hydroxyl terminated segment of formula:

wherein n is between about 45 and 55.

The nanomaterial may comprise one or more Fe2O3magnetic nanoparticles, one or more CdSe/ZnS quantum dots and a polymer comprising, optionally consisting of, a hydroxyl terminated segment of formula:

wherein n is between about 45 and 55.

A plurality of the nanomaterials of the invention may form a multiphase system. The multiphase system may be a dispersion. The dispersion may be a dispersion of nanomaterials in a gas continuous phase. For example, the gas continuous phase may be air. The dispersion may be a dispersion of nanomaterials in a liquid continuous phase. For example, the liquid continuous phase may be aqueous. The liquid continuous phase may be water. The liquid continuous phase may be a buffer solution. The multiphase system may be a gel. The multiphase system may be a foam. Aggregation of the plurality of nanomaterials in a multiphase system may be inhibited by the polymer adsorbed to the nanoparticles.

A plurality of the nanomaterials of the invention may form a solid material. The solid material may be a crystal. The solid material may be a glass.

The distribution of diameters of the plurality nanomaterials may be monomodal. The distribution of diameters of the plurality nanomaterials may be multimodal. For example, the distribution of diameters of the plurality nanomaterials may be bimodal.

Preparation of Nanomaterial

The nanomaterial of the invention may be prepared by any suitable means. The nanomaterial of the invention may be prepared by coating the at least one magnetic nanoparticle with the biocompatible hydrophilic polymer in the aqueous domain of a reverse micelle. Where the nanomaterial of the invention comprises one or more quantum dots, the nanomaterial may be prepared by coating the at least one magnetic nanoparticle and the one or more quantum dots with the biocompatible hydrophilic polymer in the aqueous domain of a reverse micelle.

The reverse micelles may comprise any suitable surfactant. For example, the reverse micelles may comprise tert-octylphenoxy poly(oxyethylene)ethanol (Igepal®) or polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (Triton X-100).

The nanomaterials may be separated from the organic synthesis solvent by centrifugation or precipitation. The hydrophilic polymer may allow for the transfer of the nanomaterial from the organic synthesis solvent to an aqueous medium.

Application of Nanomaterial to MRI

Contrast agents for MIR are generally classified into two types: (a) positive and (b) negative contrast agents. Positive contrast agents are characterised by the shortening of longitudinal relaxation time T1, resulting in the brightening of magnetic resonance images. Negative contrast agents are characterised by the shortening of transversal relaxation time T2, resulting in the darkening of magnetic resonance images.

In recent years, several groups have used MPs (Fe2O3, Fe3O4and MFe2O4, where M=Ni, Co, Mn, Fe) as negative T2contrast agents for MRI. Gadolinium-based paramagnetic chelates (e.g. Gd-DTPA (DTPA=diethylene triamine pentaacetic acid)) have been used as T1contrast agents in most studies. Luminescent gadolinium oxide (Gd2O3) hybrid nanoparticles have been used as T1contrast agents for both in vivo fluorescence and MRI.

Longitudinal relaxivity is dependent on core size, particle concentration and coating material. Recently, it has been shown for gadolinium oxide nanoparticles that the longitudinal relaxivity per particle increased with core size. The gadolinium oxide core induced an enhancement of the positive contrast of magnetic resonance images as compared to the widely used contrast agents in clinical MRI. However, introducing multiple functionalities, such as fluorescence and drug targeting moiety, onto Gd-DTPA has been problematic.

The shortening of the relaxation time of water protons in the tissues dictate the utility and effectiveness of contrast agents. The effectiveness of contrast agents are expressed as relaxivities, r1=1/T1and r2=1/T2. According to a recent report [Bridot, J.-L.; Faure, A.-C.; Laurent, S.; Riviere, C.; Billotey, C.; Hiba, B.; Janier, M.; Josserand, V.; Coll, J.-L.; Elst, L. V.; Muller, R.; Roux, S.; Tillement, O.J. Am. Chem. Soc.2007, 129, 5076-5084] the ratio of r2/r1should range between 1 and 2 for particles to be used as effective positive T1contrast agents. The present contrast agents may therefore meet this criterion.

The nanomaterial of the invention may exhibit an r2/r1value less than 2 at a frequency of 200 MHz. The nanomaterial of the invention may exhibit an r2/r1value less than 1 at a frequency of 200 MHz. The nanomaterial of the invention may exhibit an r2/r1value between about 1 and about 2 at a frequency of 200 MHz. Thus, the nanomaterial of the invention may possess the characteristics of a T1contrast agent.

The nanomaterial of the invention may be used as a T1contrast agent for magnetic resonance imaging. When used as a T1contrast agent for magnetic resonance imaging, the nanomaterial may be administered to a subject by any suitable means. The nanomaterial may administered by intravascular injection. The nanomaterial may administered by intravenous injection.

Application of Nanomaterial to Biolabelling

Where the nanomaterial of the invention comprises quantum dots, the nanomaterial of the invention may be used for labelling biological molecules. The nanomaterial of the invention may be used for labelling cancer cells. For example, the nanomaterial of the invention may be used for labelling a Hep G2 human liver cancer cell or a human breast cancer cell or some other type of cancer cell (e.g. human cancer cell) It may be used for labelling a non-human cancer cell i.e. a cancer cell of a non-human animal. The nanomaterials and/or quantum dots of the invention may be used for diagnosis of cancer in a human patient. They may be used for diagnosis of cancer in a non-human animal, e.g. a non-human primate or other mammal.

When used for labelling biological molecules, the nanomaterial may be administered to a subject by any suitable means. The nanomaterial may administered by intravascular injection. The nanomaterial may administered by intravenous injection.

Where the nanomaterial of the invention comprises quantum dots, the size of the quantum dots may be chosen so as to impart a particular excitation wavelength (i.e. colour) to the nanomaterial. For example, a 3 nm quantum dot emits in green and a 5 nm quantum dot emits in red.

The following examples are provided for the purpose of illustration only and are not intended to limit the scope of the present invention in any way.

EXAMPLES

Iron stearate (Fe(St)2, 3.73 g), octadecyl amine (ODA, 1.61 g), methylmorpholine N-oxide (MNO, 1.61 g) and octadecene (ODE, 90 mL) were charged into a 250-mL 3-necked round-bottom flask connected to a Schlenk line. First, the mixture was pumped under vacuum, and purged with argon for 15-30 min. It was then heated under argon to 300° C., and kept for 15 min. The heating mantle was removed and the brownish black solution was cooled to 30-40° C. Finally, the particles were purified with a mixture of cyclohexane/acetone (volume ratio=1:5) three times by centrifugation-redispersion cycles. The wet precipitate was stored in a glove box. The yield of dried magnetic nanoparticles was 2.03 g.

Synthesis of CdSe/ZnS Quantum Dots (QDs)

In a typical synthesis, CdO (0.05 g, 0.39 mmol) and SA (1.3 g, 4.57 mmol) were loaded into a three-necked flask and pumped under vacuum for 20 min. The mixture was heated under argon to ˜200° C. to form cadmium stearate, resulting in a clear solution. After being cooled to room temperature, TDPA (0.16 g, 0.57 mmol) and TOPO (7 g, 18.1 mmol) were charged into the flask, and heated to 280° C. Se (0.32 g, 4 mmol) dissolved in trioctyl phosphine (TOP) (8 g, 21.6 mmol) was injected swiftly and held at that temperature for 20 sec. The reaction was then cooled to 190° C., and a mixture of 6 mL of diethyl zinc (Zn(Et)2, 1.1 M in toluene) dissolved in 8 mL of TOP and 1.5 mL of hexamethyl disilathiane was gradually injected over 10 min. The reaction was allowed to proceed for 1 h at 180° C. Finally, the heating mantle was removed, and the reaction was cooled to 40-50° C.; 10 mL of chloroform was added to avoid the solidification of TOPO. Excess capping agents and decomposition products were removed by precipitation and re-dispersion cycles with methanol and chloroform, respectively.

Synthesis of Sorbitol-Malic Acid Polymer

A flame dried 250-mL round-bottom flask was charged with sorbitol (18.2 g, 100 mmol) and malic acid (13.4 g, 100 mmol), and heated at 95° C. on a heating block with stirring under argon atmosphere. The solid starting materials were melted to form a homogeneous solution within 10 min.

Immobilised Novozym 435 (3 g, immobilised on acrylic resin) was added, and stirring was continued for 30 min under argon. The flask was connected to a vacuum line (15 mm) to remove the condensed water. The reaction mixture was heated continuously at 95° C. for 48 h under vacuum. It was cooled to room temperature, and methanol was added and sonicated to dissolve the polymer. The enzyme catalyst was removed by filtration on a sintered funnel. The resulting filtrate was purified by extensive washing with water in an Amicon filtration system using a polyethersulfone (PES) membrane, and the residue was lyophilised to get the pure polymer as a white solid (24 g). The polymer was characterised by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies, and its molecular weight and polydispersity were measured by gel permeation chromatography (GPC).

FIGS. 1 and 2illustrate the cytotoxicity of the sorbitol-malic acid polymer on NIH3T3 and Hep G2 cells respectively. It can be seen that the IC50of the sorbitol-malic acid polymer is more than 2000 mg/mL by in vitro MTT assay.

Synthesis of Thiol and Amine-Functionalised Sorbitol-Malic Acid Polymer

The sorbitol-malic acid polymer was reacted with allyl bromide under basic conditions to produce an allyl-substituted polymer. A flame dried 100-mL round-bottom flask was charged with the allyl-substituted polymer (10 mmol based on allyl group) and degassed water (10 mL) under argon atmosphere. Freshly distilled aminoethanethiol (10 mmol) was added, followed by a catalytic amount (˜50 mg) of azobisisobutyronitrile (AIBN). Next, the reaction mixture was heated at 80° C. for 56 h under a constant flush of argon.

The reaction mixture was cooled to room temperature, and the product was purified by exhaustive filtration using an Amicon filtration system. The residue obtained was lyophilised to obtain a sticky white solid. The product was characterised by NMR spectroscopy.

Polymer Coating of Magnetic Nanoparticles

The polymer coating of MPs was done in Igepal reverse micelles. Fe2O3magnetic nanoparticles (50 μL, 5 mg/mL), prepared according to the method of Example 1, were added to the reverse micelles, and stirred for 30 min. They would remain in the oil phase, cyclohexane. Next, sorbitol-malic acid polyester aqueous polymer (100 μL, 250 mg/mL), prepared according to the method of Example 3, was added and stirred for 18-24 h. The interaction of polymers with magnetic nanoparticles allowed for the encapsulation to occur within the aqueous domains of the reverse micelles. The sorbitol-malic acid polyester coated Fe2O3(SorbMal/Fe2O3) magnetic nanoparticles were purified by several rounds of centrifugation in ethanol before they were dispersed in water or buffer solutions.

Polymer Coating of Magnetic Nanoparticle-Quantum Dot Nanocomposites (MQDs)

A schematic representation of the preparation of polymer-coated MQDs (SorbMal/Fe2O3—CdSe@ZnS) is shown inFIG. 3. The polymer coating of MPs and QDs was conducted in Igepal reverse micelles. TOPO-capped CdSe/ZnS QDs (100 μL, 2 mg/mL of chloroform) were first added to the micelles, and stirred for 30 min. QDs would remain in the oil phase, cyclohexane. Next, thiol and amine-functionalized sorbitol-malic acid polyester aqueous solution (100 μL, 250 mg/mL) was added, and stirred for 1 h to form the microemulsion. Fe2O3MPs (50 μL, 5 mg/mL) were then introduced and stirred for 18-24 h. The interaction of polymers with QDs and MPs allowed for the encapsulation within the aqueous domains of the reverse microemulsion. The Fe2O3and CdSe/ZnS MQDs coated with thiol and amine-functionalised sorbitol-malic acid polyester (SorbMal/Fe2O3—CdSe@ZnS) were purified by several rounds of centrifugation in ethanol before these nanocomposite particles were dispersed in water or buffer solutions.

Analysis of MPs

Transmission electron microscopy (TEM) illustrate that MPs coated with sorbitol-malic acid polymer prepared as described in Example 5 were not aggregated, with a size of ˜6 nm (FIG. 4). The polymer-coated MPs were highly stable in water (seeFIG. 4(inset)).

Table 1 summarises the average particle diameter and saturation magnetisation (Ms) of SorbMal/Fe2O3solutions deduced from TEM, magnetometry, relaxometry and photo-correlation spectroscopy. The average particle diameters were estimated to be 7.3 nm and 7.2 nm from magnetometry and relaxometry, respectively. The values deduced from TEM and photo-correlation spectroscopy were 6.0 nm and 13.0 nm, respectively. The latter included the MP core and the polymer shell.

Relaxometric measurements of water protons were conducted to determine whether the SorbMal/Fe2O3MPs could be used as conventional T2contrast agents in MRI, or whether they would be useful as positive T1contrast agents in place of Gd-based chelates or oxides. The proton longitudinal relaxation rates of polymer-coated MPs dispersed in aqueous solution were measured at 37° C. between 0.01 and 300 MHz. The1H nuclear magnetic resonance dispersion (NMRD) profile of SorbMal/Fe2O3MPs is shown inFIG. 6. In the range of low magnetic fields, the longitudinal relaxivity decreased, as compared to those of silica-coated MPs [Lee, J.; Lee, Y.; Youn, J. K.; Na, H. B.; Yu, T.; Kim, H.; Lee, S.-M.; Koo, Y.-M.; Kwak, J. H.; Park, H. G.; Chang, H. N.; Hwang, M.; Park, J.-G.; Kim, J.; Hyeon, T.Small,2008, 4, 143-152].

The relaxometric data shows that polymer-coated MPs could be used as potential positive T1contrast agents in place of Gd-based chelates. Table 2 shows the relaxivity values obtained for polymer-coated MP solutions at a neutral pH and 37° C. The effectiveness of contrast agents are expressed as relaxivities, r1=1/T1and r21/T2. The polymer-coated MPs of the invention exhibited r2/r1values of 1.81 and 2.57 at 20 MHz and 60 MHz, respectively. The r2/r1values of 6-nm polymer-coated MPs fell within the range of values obtained for Gd2O3nanoparticles of 1.3-3.8 nm [Bridot, J.-L.; Faure, A.-C.; Laurent, S.; Riviere, C.; Billotey, C.; Hiba, B.; Janier, M.; Josserand, V.; Coll, J.-L.; Elst, L. V.; Muller, R.; Roux, S.; Tillement, O.J. Am. Chem. Soc.2007, 129, 5076-5084].

TABLE 2Relaxivity Values of SorbMal/Fe2O3Particles at Neutral pH and 37° C.Frequency (MHz)r1(mM−1s−1)r2(mM−1s−1)r2/r1204.918.891.81604.4811.512.57

Use of MQDs for Biolabelling

FIG. 7shows the live cell imaging of Hep G2 human liver cancer cells stained with SorbMal/Fe2O3—CdSe/ZnS particles prepared as described in Example 6. The SorbMal/Fe2O3—CdSe/ZnS particles effectively labelled the cell membranes. As seen in the images, the conjugates of polymer and red-emitting quantum dots appeared to accumulate in vesicles within the cells, suggesting endocytotic uptake. This example demonstrates the efficacy of the polymer-coated magnetic nanoparticle-quantum dot composites for the labelling of live cancer cells.