Aldehyde-imidazole polymers

Technical Field

The present invention relates to polymers which may be made from aldehydes and imidazoles.

Priority

The present application claims priority from SG Patent Application No. 201002173- 1 which was filed on 29 March 2010, the entire contents of which are incorporated herein by cross-reference.

Background of the Invention

Colloidal nanoparticles (NPs) such as noble metal nanocrystals and semiconductor quantum dots (QDs) have been widely used in biological detection and imaging. The coating on nanocrystals plays a critical role on their colloidal stability and functionalization in biological applications. The most common coating method is the addition of layers of polymer or silica on surfactant-capped nanoparticles. However, colloidal stability of these coated nanoparticles may decrease significantly as their overall size can increase by over 4 nm upon ligand addition. A hydrodynamic particle size of < 6 nm for nanoparticles would be highly desired for in vivo studies.

To minimize the coating thickness, nanoparticles have been coated by direct capping with polymerized ligands, such as cross-linked glutathione, poly-cysteine or hexahistidine-tagged peptides. Thiols and imidazoles may bind to nanoparticles tightly due to their high affinity to heavy metals. Two imidazole-based copolymer ligands have been prepared very recently to coat QDs and various nanoparticles. The disadvantages of these reported imidazole-based copblymers include the use of complicated monomers, the use of a tedious copolymerization process and the production of a thick coating. Imidazole-capped nanoparticles show higher long-term stability than thiol-capped nanoparticles as the formation of disulfide bonds by slow oxidation of the latter can result in aggregation of nanoparticles.

Object of the Invention

It is an object of the present invention to substantially overcome or at least ameliorate one or more of the above disadvantages.

Summary of the Invention

In a broad aspect of the invention there is provided a polymer obtained by condensation of a hydroxyalkyl functional imidazole which is unsubstituted on the imidazole nitrogens. The imidazole may be generated, separately or in situ, by reaction of a secondary amino functional imidazole with an aldehyde. The secondary amino functional imidazole may in turn be generated separately or in situ, for example by reaction of an imidazole which is unsubstituted at positions 1, 3 and 5 and has a primary aminoalkyl group at position 4 with a second aldehyde (which may be the same as the aldehyde which is reacted with the secondary amino functional imidazole or it may be different). Suitable imidazoles for reaction with the second aldehyde include histidine. Thus the invention encompasses a polymer obtained by reaction (e.g. condensation) of an imidazole which is unsubstituted at positions 1, 3 and 5 and has a primary aminoalkyl group at position 4 (e.g. histidine) with an aldehyde (e.g. formaldehyde).

In a first aspect of the invention there is provided an oligomer or polymer comprising monomer unit (I)

(I)

wherein:

m is an integer from 1 to 4

R is hydrogen or an optionally substituted alkyl, aryl or heteroaryl group; and

R' is hydrogen or an optionally substituted alkyl, aryl or heteroaryl group or a functional group;

wherein the oligomer or polymer comprises at least two monomer units (I).

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

m may be 1.

R may be hydrogen, or may be -CHO, -RCHO (where R is CI to C6) or optionally substituted C2-C6 alkyl or optionally substituted aryl.

R' may be -C0 ₂ H or -C0 ₂ ^" , or may be, hydrogen.

The degree of polymerisation, or the number of monomer units (I) in the oligomer or polymer, may be from 2 to about 60.

The oligomer or polymer may substantially coat the surface of a nanoparticle. The nanoparticle may comprise a metal. The metal may be for example gold, platinum, iron, zinc, cadmium, nickel and mixtures or an alloy of any two or more of these. The nanoparticle may comprise iron oxide. The nanoparticle may be magnetic. It may be a quantum dot.

The oligomer or polymer may be conjugated to a dye or a biologically active compound. The biologically active molecule may be an anti-cancer drug.

In an embodiment there is provided an oligomer of structure (I) in which rh is 1, there are 2 to about 60 monomer units (I) per molecule, R is H and R' is -C0 ₂ H or -C0 ₂ \

In a second aspect of the invention there is provided a nanoparticle, or a plurality of nanoparticles, coated by an oligomer or polymer comprising monomer unit (I) as described in the first aspect.

The following options may be used in conjunction with the second aspect, either individually or in any suitable combination. The nanoparticle may comprise a metal. The metal may be for example gold, platinum, iron, zinc, cadmium, nickel and mixtures or an alloy of any two or more of these. The nanoparticle may comprise iron oxide. It may be a quantum dot. It may be magnetic.

In a third aspect of the invention there is provided a process for preparing an oligomer or polymer according to the first aspect, said process comprising reacting an imidazole of structure (II)

(Π)

with more than 2 mole equivalents of an aldehyde of structure R-CHO, wherein:

m is an integer from 1 to 4;

R is hydrogen or an optionally substituted alkyl, aryl or heteroaryl group; and

R' is hydrogen or an optionally substituted alkyl, aryl or heteroaryl group or a functional group.

At least 3 mole equivalents (optionally about 3 mole equivalents) of the aldehyde may be used.

In a fourth aspect of the invention there is provided a process for preparing an oligomer or polymer according to the first aspect, said process comprising reacting an imidazole of structure (III)

(in) with an aldehyde of structure R-CHO, wherein:

m is an integer from 1 to 4;

R is hydrogen or an optionally substituted alkyl, aryl or heteroaryl group; and

R' is hydrogen or an optionally substituted alkyl, aryl or heteroaryl group or a functional group.

The following options may be used in conjunction with either the third or the fourth aspect, and may be used either individually or in any suitable combination,

m may be 1.

The aldehyde may be formaldehyde or may be glyoxal, glutaraldehyde or an optionally substituted C2-C6 alkyl group having at least one aldehyde functional group. The R group of the aldehyde may be the same as the R group of the compound (III) however in some instances it may be different.

R ¹ may be -C0 ₂ H or -C0 ₂ ^" , or may be hydrogen.

. The imidazole of structure II may be histidine.

The process may be conducted under basic conditions.

The process may comprise the step of reacting an imidazole of structure II with 1 mole of the aldehyde of structure R-CHO, or with at least two or with about 2 moles thereof, so as to produce the imidazole of structure (III).

The process may be conducted in the presence of at least one substance which is a compound, oligomer or polymer with molecular weight less than 600. The substance may have two functional groups, each independently selected from the group consisting of - NH ₂ , -OH, -SH and -COOH. The substance may also comprise further functional groups, which may or may not be -NH ₂ , -OH, -SH or -COOH. The substance may be for example melamine, glucose, cysteine, tris(hydroxymethyl)aminomethane (TRIS) or a mixture of any two or more thereof. It may be melamine. It may be polyethylene glycol. This substance may copolymerise with imidazole (II) or (III).

The reacting may be conducted in the presence of nanoparticles. In this case process may produce a coating of the oligomer or polymer on the surface of said nanoparticles.

The process may comprise purifying the oligomer or polymer, preparing a solution of the oligomer or polymer in a solvent and generating nanoparticles in said solution. In this case the oligomer or polymer may form a coating on the surface of the nanoparticles.

The process of may comprise purifying the oligomer or polymer and exposing the oligomer or polymer to a suspension of nanoparticles in a liquid, said nanoparticles having a stabilising ligand on the surface thereof. In this case the oligomer or polymer may replace the stabilising ligand on the surface of the nanoparticles so as to form a coating on said surface of the nanoparticles.

The process may comprise exposing the oligomer or polymer to a dye or a biologically active molecule so as to conjugate said dye or a biologically active molecule to the oligomer or polymer.

In an embodiment of the invention there is provided there is provided a process for preparing a polymer according to the first aspect, said process comprising reacting histidine with more than 2 mole equivalents, optionally about 3 mole equivalents, of formaldehyde under basic conditions.

In another embodiment there is provided a process for preparing an oligomer or polymer according to the first aspect, said process comprising reacting an imidazole of structure III with formaldehyde, optionally about 2 mole equivalents thereof, under basic conditions, wherein m is 1, R is hydrogen or an optionally substituted alkyl, aryl or heteroaryl group and R' is -C0 ₂ H or -C0 ₂ ^" . In a particular form of this embodiment R is hydrogen.

The invention also encompasses an oligomer or polymer made by the process of either the third or the fourth aspect.

In a fifth aspect of the invention there is provided a process for making nanoparticles coated with the oligomer or polymer of the first aspect, said process comprising preparing the oligomer or polymer according to the process of the third or the fourth aspect (as described above) in the presence of the nanoparticles. The process may comprise the step of generating the nanoparticles prior to said preparing. It may for example comprise preparing the nanoparticles in the presence of a compound of structure II or III (as described above) and adding an aldehyde of structure R-CHO as described above.

In a sixth aspect of the invention there is provided a process for making nanoparticles coated with the oligomer or polymer of the first aspect, said process comprising generating said nanoparticles in a solution of the oligomer or polymer. The process may comprise generating said solution according to the process of the third or the fourth aspect.

In a seventh aspect of the invention there is provided a process for making nanoparticles coated with the oligomer or polymer of the first aspect, said process comprising exposing the oligomer or polymer to nanoparticles suspended in a liquid, said nanoparticles having a stabilising ligand on the surface thereof so as to replace the 0 stabilising ligand on the surface of the nanoparticles so as to form a coating on said surface of the nanoparticles. The stabilising ligand may be for example tetramethylammonium hydroxide.

The fifth to the seventh aspects may produce the nanoparticles of the second aspect.

In an eighth aspect of the invention there is provided a method for removing a metal from water, said method comprising exposing water containing said metal to an oligomer or polymer according to the first aspect, or nanoparticles according to the second aspect, or to an oligomer or polymer made by the process of the third or fourth aspect, or to nanoparticles made by the fifth, sixth or seventh aspect.

In a ninth aspect of the invention there is provided use of an oligomer or polymer according to the first aspect, or nanoparticles according to the second aspect, or an oligomer or polymer made by the process of the third or fourth aspect, or nanoparticles made by the fifth, sixth or seventh aspect, for removing a metal from water.

In _. the method of the eighth aspect or the use of the ninth aspect the metal may be for example lead, copper, gold, platinum, iron, zinc, cadmium, nickel, mercury or may be a mixture of any two or more thereof.

In a tenth aspect of the invention there is provided a method for treating a condition in a patient comprising administering to said patient a therapeutic quantity of an oligomer or polymer according to the first aspect, or nanoparticles according to the second aspect, or an oligomer or polymer made by the process of the third or fourth aspect, or nanoparticles made by the fifth, sixth or seventh aspect in which the oligomer or polymer has a biologically active compound conjugated thereto, wherein said biologically active compound is a drug which is indicated for treatment of said condition. The patient may be a human patient, or may be a non-human patient. The patient may be a mammal (e.g. a non-human mammal), a primate (e.g. a non-human primate), a domestic animal (e.g. a horse, a cow, a pig, a dog, a cat etc.), a bird or some other type of patient.

In an eleventh aspect of the invention there is provided a method of imaging cells, said method comprising exposing the cells to a plurality of nanoparticles according to the second aspect, or nanoparticles made by the fifth, sixth or seventh aspect, and imaging the nanoparticles, wherein the nanoparticles are imagable and the polymer of the nanoparticles is conjugated to a substance capable of binding to DNA. The imaging may be for example fluorescence imaging. The nanoparticles may be quantum dots. The method may be for the purpose of diagnosis of a condition in a subject or it may be for a non-diagnostic purpose. The subject may be a human subject, or may be a non-human / subject. The subject may be a mammal (e.g. a non-human mammal), a primate (e.g. a non-human primate), a domestic animal (e.g. a horse, a cow, a pig, a dog, a cat etc.), a bird or some other type of subject. The invention also extends to the use of nanoparticles as described above for the manufacture of a preparation for imaging cells. The imaging may be in vitro or it may be in vivo.

In a twelfth aspect of the invention there is provided use of an oligomer or polymer according to the first aspect, or nanoparticles according to the second aspect, or an oligomer or polymer made by the process of the third or fourth aspect, or nanoparticles made by the fifth, sixth or seventh aspect for the manufacture of a medicament for the treatment of a condition, wherein the oligomer or polymer has a biologically active compound conjugated thereto and wherein said biologically active compound is a drug which is indicated for treatment of said condition.

In a thirteenth aspect of the invention there is provided use of an oligomer or polymer according to the first aspect, or nanoparticles according to the second aspect, or an oligomer or polymer made by the process of the third or fourth aspect, or nanoparticles made by the fifth, sixth or seventh aspect in therapy, wherein the oligomer or polymer has a biologically active compound conjugated thereto and wherein said biologically active compound is a drug.

Brief Description of the Drawings

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings wherein:

Figure 1 shows (a) 1H NMR spectra of (i) His, and polymer derived at a HCHO:His molar ratio of (ii) 1 :1 and (iii) 2:1, and (iv) purified pHF (polymerized histidine- formaldehyde); (b) absorption spectra of polymer derived with [His] = 100 μΜ at different HCHO:His molar ratios: (i) 0:1, (ii) 1:1, (iii) 2:1, (iv) 3:1, (v) 4:1, (vi) 5:1, and (vii) 6:1. Inset shows the absorbances at 275 and 345 nm for the polymers derived at the different HCHO:His molar ratios, (c) M _n (measured by GPC using PEG standards and a UV detector) for the polymers derived at the different HCHO:His molar ratios. Note that MWs are not absolute but are PEG equivalent MW.

Figure 2 shows TEM micrographs of pHF-coated (a) Pt (2 nm), (b) Fe ₃ 0 ₄ (12 nm), (c) Au (6 nm), and (d) CdSe@ZnS (5 nm). Inset: photographs of the coated nanoparticles (100 ppm in solutions indicated) after 24 h incubation.

Figure 3 shows (a) absorption spectra of purified FITC-pHF (i), doxorubicin-pHF (ii), 100 μΜ of pHF (iii), 4 μΜ of free FITC (iv) and 4 μΜ of free doxorubicin (v). (b) fluorescence image of 4T1 live cells treated with doxorubicin-pHF-QDs. Fluorescence emission wavelength of QDs = 590 nm.

Figure 4 is a scheme showing formation of a coating of polymer on a nanoparticle.

Figure 5 shows (a) absorption spectra of polymer derived after 24 h of heating at 90°C with [His] = 100 μΜ at different HCHO:His molar ratios: (i) 0:1, (ii) 1 :1, (iii) 2:1, (iv) 3:1, (v) 4:1, (vi) 5:1 and (vii) 6:1. (b) absorbances at 275 nm (upper trace) and 345 nm (lower trace) for the polymers derived in (a) at the different HCHO.His molar ratios.) (c) absorption spectra of polymer derived after (i) 0 min, (ii) 10 min, (iii) 1 h, (iv) 2 h, (v) 3 h, (vi) 4 h, (vii) 5 h, (viii) 6 h, (ix) 7 h, (x) 8 h and (xi) 24 h of heating at 90°C with [His] = 500 μΜ at HCHO:His molar ratio = 4:1. (d) absorbance at 275 nm (upper trace) and 345 nm (lower trace) for the polymers derived in (c) after the different heating periods.

Figure 6 shows mass spectra of (a) His, and polymers derived at HCHO:His molar ratios of (b) 1:1, (c) 2:1, (d) 4:1 and (e) 8:1. The MS peaks are assigned to the chemical structures at the top of the Figure 6.

Figure 7 is an GPC elution profile of as-prepared pHF (— ), and standard PEG samples of the specified molecular weights (in dashed lines).

Figure 8 is DLS hydrodynamic size distributions of (a) pHF-Pt, (b) pHF-Fe ₃ 0 ₄ , (c) pHF-Au and (d) pHF-CdSe@ZnS nanoparticles in aqueous solution.

Detailed Description

The present invention relates to a novel oligomer or polymer in which the monomer unit is:

(I)

In this structure, m is an integer from 1 to 4, commonly 1 or 2. It may be 1 , 2, 3 or 4. The degree of polymerisation, or the number of monomer units (I) is greater than 1, and may commonly be about 2 to about 100, or about 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 5 to 100, 10 to 100, 20 to 100, 50 to 100, 5 to 50, 5 to 20, 10 to 50, 10 to 30 or 20 to 50, e.g. about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100, although it may be at times greater than about 100. In one commonly used definition, an oligomer has degree of polymerisation 2 to 10 and a polymer has degree of polymerisation greater than 10. This definition is used herein. The term "monomer unit" as used herein refers to a repeat unit in the oligomer or polymer. The term "monomer unit (I) as used herein refers to a monomer unit having structure (I) shown above.

In referring to the ring system of the monomer unit, the following numbering is used:

3

In the monomer unit of the polymer, each R may be the same, although in some embodiments one or more may be different. Suitable examples for R include H, alkyl, aryl or heteroaryl, each optionally being substituted.

In the present specification the following apply:

Alkyl: these may be linear, branched, cyclic or may have a combination of any two or all of these (provided that sufficient carbon atoms are present in the alkyl group). They may also have unsaturation in the form of one or more double and/or triple bonds. They may be CI to C12 or greater than C12, or CI to C6, CI to C4, C2 to C12, C6 to C12 or C3 to C6, e.g. CI, C2, C3, C4, C5, C6, C7, C8, C9, CIO, CI 1 or C12.

Aryl: these may be monocyclic bicyclic or polycyclic. If they have more than one ring, the rings may be fused or may be linked. They may have 1, 2, 3, 4, 5 or 6 rings or more than 6 rings.

Heteroaryl: these may be monocyclic bicyclic or polycyclic ring systems. If they have more than one ring, the rings may be fused or may be linked. They may have 1 , 2, 3, 4, 5 or 6 rings or more than 6 rings. They may comprise fused or linked aryl groups as defined above. The heteroatom(s) of the heteroaryl rings may, independently, be N, O, S or some other heteroatom. Each heteroaryl ring may be, independently, 5, 6 or 7 membered.

Substituents: where groups are stated as being substituted, the substituents, if present, may be alkyl or may be aryl or may be heteroaryl or may be more than one of these. The substituents themselves may also, optionally, be substituted. The substituents may where appropriate be functional groups, such as halide (e.g. CI, Br), nitro, carboxyl, alkyl carbonyl, formyl, sulfonate, phosphate etc. In the monomer unit defined above, R' may be an alkyl group or may be an aryl group or may be heteroaryl group or may be a functional group, e.g -CO ₂ H or -CO ₂ ^" , halide (e.g. CI, Br), nitro, phosphate, aldehyde etc.

In some forms of the invention the oligomer or polymer may be crosslinked, e.g. through the hydroxyl group on the carbon attached to C2 of monomer unit (I). In such forms, the oligomer or polymer may comprise crosslinking units in which the monomer unit (I) has the CH ₂ OH group on C2 replaced by CH ₂ X, in which X is a crosslink. In some forms of the invention the oligomer or polymer is a homooligomer or homopolymer, i.e. the only monomer units present are of structure (I). In some forms of the invention the oligomer or polymer comprises other monomer units than that shown above. In this case the oligomer or polymer may be a cooligomer or copolymer. Other monomer units that may be present may be crosslinking monomer units or may be non- crosslinking monomer units. Suitable other monomer units include those derived from melamine, glucose, cysteine, TRIS, polyethylene glycol etc. More than one such other monomer unit may be present. Commonly monomer units derived from compounds comprising more than two groups selected from -NH ₂ , -O¾ -SH and -COOH will be crosslinking units. Monomer units other than that of monomer structure (I) may be present in the polymer. The ratio of monomer units (I) to the other monomer units may be about 2 to about 20 (i.e. about 2:1 to about 20:1), or about 2 to 10, 2 to 5, 5 to 20, 10 to 20, 3 to 10, 3 to 5 or 3 to 4, e.g. about 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

The polymer of the present invention may be disposed on the surface of a nanoparticle. The nanoparticle may be, or may comprise, a metal, a metal oxide or some other type of nanoparticle. It may comprise a quantum dot. The polymer may entirely coat the nanoparticle, or substantially entirely coat the nanoparticle, or may only partially coat the nanoparticle. The nanoparticle may have a diameter of less than about 1 OOnm, or less than about 50, 20 10 or 5nm, or about 1 to about lOOnm, or about 10 to 100, 50 to 100, 1 to 50, 1 to 20, 1 to 10 or 10 to 50nm, e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 1 OOnm.

The polymer may have a molecular weight average (number average or weight average) of about 1 to about lOkDa, or about 1 to 5, 1 to 2, 2 to 10, 5 to 10 or 2 to 5kDa, e.g. about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or lOkDa, or may in some instances be greater than lOkDa. The molecular weight distribution may be narrow or may be broad. This molecular weight may be an absolute molecular weight, or may a molecular weight relative to polyethylene glycol standards measured by GPC.

The polymer may have terminal -CHR(OH) groups either at one end or at both ends. In some instances these may be reacted for example by esterification or etherification. The end group on the imidazole N end may be H. The end group on the other end may be -CHR(OH).

The polymer of the invention may be conjugated to a dye or to a biological molecule, e.g. a drug, or to some other substance. In the present context, the term "conjugated" indicates as association, which may comprise covalent bonding, ionic bonding, electrostatic attraction or some other form of association, whether reversible or irreversible. In some embodiments of the invention the polymer is disposed on the surface of a nanoparticle and is also conjugated to a dye or to a biological molecule or to some other substance. When the polymer is conjugated to a biological molecule, it may be useful for delivery of said biological molecule to a biological system e.g. to a cell or it may be useful for delivering the nanoparticle to the biological system, e.g. to the cell. Suitable biological molecules include drugs, proteins, peptides, oligopeptides, polypeptides, saccharides, enzymes, genes, gene fragments etc.

The polymers of the present invention may be conveniently prepared by reaction of appropriately substituted imidazoles with aldehydes. Thus reaction of imidazole II with an aldehyde R-CHO (where R, R' and m are as defined earlier) leads to the polymer of the invention.

The ratio of aldehyde to imidazole may be greater than 2 (i.e. 2:1), commonly greater than 3. It may be about 3 to about 10, or about 3 to 5, 3 to 4, 4 to 10 or 4 to 6, e.g, about 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10. As noted above, the polymer of the invention may comprise other monomer units than TV. A second monomer (i.e. a comonomer) may be used in combination with monomer II. Suitable second monomers may be for example melamine, glucose, cysteine, TRIS, polyethylene glycol or other compounds comprising two or more groups selected from -NH ₂ , -OH, -SH and -COOH. A mixture of such monomers may be used. The ratio of monomer (II) to such second monomers may be from about 2 to about 20 (i.e. about 2:1 to about 20:1) on a mole or weight basis), or about 2 to 10, 2 to 5, 5 to 20, 10 to 20, 3 to 10, 3 to 5 or 3 to 4, e.g. about 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

The reaction is commonly conducted under basic conditions. It may be conducted at pH between about 7 and about 11, or about 7 to 9, 9 to 11 or 8 to 10, e.g. about 7.5, 8, 8.5, 9, 9.5, 10, 10.5 or 11. It may be conducted at elevated temperature, e.g. about 60 to about 120°C, or about 60 to 100, 60 to 80, 80 to 120, 100 to 120 or 80 to 100°C, or at about 60, 70, 80, 90, 100, 110 or 120°C. It may be conducted in solution. It may be conducted in aqueous solution, or in an alcoholic solution (e.g. ethanolic, methanolic etc.) or it may be conducted in aqueous alcoholic solution. It may be conducted in solution in some other solvent. The reaction may be conducted for sufficient time to generate the polymer in an acceptable yield. It may be conducted for at least about 6 hours, or at least about 9, 12, 15, 18 or 24 hours, or for about 6, 9, 12, 15, 18 or 24 hours. The yield of the reaction is commonly over 50%, and may be over 60, 70 or 80%. The resulting polymer may be partially, purified by dialysis, in order to remove starting materials and low molecular weight oligomers/polymers. Conveniently, the reaction may be conducted under an air atmosphere.

It is thought that the reaction of monomer (II) with R-CHO might proceed by formation of an intermediate adduct (III). Such structures may form on reaction of histidine with formaldehyde. Thus an alternative route to the polymer of the present invention is to react adduct (III) with an aldehyde R-CHO. In this case, of course, the ratio of adduct (III) to R-CHO may be less than the ratio required if starting with monomer (II). Thus the ratio may be from about 1 to about 5 (i.e. about about 1 : 1 to about 5:1), or about 2 to 5, 3 to 5, 1 to 3 or 2 to 3, e.g. about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5. Similar reaction conditions to those above may otherwise apply. Again, a second monomer (as described above and in the proportions described above) may also be included in the reaction.

The polymer of the present invention may be coated onto nanoparticles. There are three separate ways that this may be achieved:

1) prepare the polymer in the presence of a suspension of the nanoparticles. Thus for example, the nanoparticles may be prepared in suspension (e.g. by known methods such as reduction of Au ³⁺ ). Following this the reagents for forming the polymer may be added sequentially to the suspension of nanoparticles in order to form the polymer on the surface of the nanoparticles. In a variation, the preparation of the nanoparticles may be conducted in the presence of monomer II and/or III (and optionally one or more second monomers) and, following this preparation, aldehyde R-CHO added in order to form the polymer on the surface of the nanoparticles. In a further variation, the monomer II and/or III may be present as a ligand, optionally a stablising ligand, on the surface of the nanoparticles prior to forming the polymer. In this instance the ligands may be introduced onto the surface of the nanoparticles by ligand exchange.

2) prepare the nanoparticles in the presence of the polymer. Thus for example Pt ⁴⁺ may be reduced in the presence of the polymer to form platinum nanoparticles, which are subsequently, in situ, coated with the nanoparticles. In such examples, commonly the polymer will be combined in solution with the Pt ⁴⁺ or some other nanoparticle precursor, and the reducing agent added to the solution.

3) prepare nanoparticles stabilised by a stabiliser other than the polymer of the invention, and replace the stabiliser by the polymer. Thus for example, iron oxide magnetic nanoparticles may be generated by known methods and stabilised using tetraalkylammonium hydroxide. When the stabilised nanoparticles are exposed to the polymer in solution, the tetraalkylammonium hydroxide may be replaced by the polymer so as to form polymer coated nanoparticles.

Suitable nanoparticles which may be coated by the polymer of the invention include metal nanoparticles (e.g. gold, platinum, palladium etc.), core shell nanoparticles (e.g. quantum dots, CdSe/ZnS, InAs/InP), metal oxide nanoparticles (e.g. iron oxide, optionally magnetic iron oxide), etc.

The invention therefore also encompasses such nanoparticles coated with the polymer of the invention. The coated nanoparticles (and independently the uncoated nanoparticles from which they are made) may have a mean diameter of about 1 to about 50nm, or about 1 to 20, 1 to 10, 1 to 5, 1 to 2, 2 to 50, 5 to 50, 10 to 50, 20 to 50, 2 to 20, 2 to 10, 5 to 10 or 10 to 20nm, e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50nm. They may be substantially monodispersed or may be polydispersed. They may be substantially round, or may be some other shape. The polymer may Completely coat the nanoparticles or may partially coat the nanoparticles. It may cover at least about 50% of the surface area of the particles, or at least 60, 70, 80, 90, 95 or 99% thereof, or it may coat about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.9 or 100% thereof. It is thought that hydroxyl groups of the polymer may associate with the surface of nanoparticles in order to couple the polymer to the nanoparticles.

Additionally or alternatively, the polymer of the invention may be conjugated with various substances such as dyes, biologically active molecules, catalytic species and the like. The resulting conjugates may be used for transporting the conjugated substance. Thus the invention also encompasses both substance-polymer conjugates and polymer coated nanoparticles in which the polymer is conjugated with the substance. In order to conjugate a substance with the polymer, the polymer (optionally coated on nanoparticles), may be simply exposed to the substance in solution or suspension. This may be conducted at any convenient temperature, e.g. room temperature, and may be conducted for sufficient time to obtain an acceptable degree of conjugation. Suitable times are from about 1 to about 24 hours, or about 1 to 18, 1 to 12, 1 to 6, 6 to 24, 12 to 24, 12 to 18 or 18 to 24 hours, e.g. about 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21 or 24 hours. Separation of unconjugated substance may be conveniently performed using dialysis with a membrane which allows the substance but not the polymer to pass.

As noted above, the polymer of the invention may have an affinity for particular metal species. Thus the polymer may be used to remove such species from water. The polymer may for example be loaded onto nanoparticles and the resulting coated nanoparticles mixed with the water and then removed. The removal may be for example by centrifugation, filtration (e.g. microfiltration, ultrafiltration etc.), magnetic separation (in the case that the nanoparticles are magnetic), or some other suitable method. In some cases the polymer may be coated onto the surface of particles which are then used in a packed bed system for removal of metals from water by passing the water through the packed bed.

When the polymer is conjugated to certain substances, particularly those which can bind to DNA, the polymer may be used to transport nanoparticles into live cells. Thus exposure of live cells to nanoparticles coated with ^" the polymer of the invention conjugated to a suitable substance (e.g. doxorubicin) results in passage of the nanoparticles into the cells. In cases where the nanoparticles are visualisable, e.g. quantum dots, this may enable visualisation of the cells.

Description of Particular Embodiments

In certain embodiments of the invention, the present invention represents a simple and effective strategy to form polyimidazole coatings on the surface of nanoparticles by polymerizing histidine (His) with formaldehyde at a basic pH. Unlike poly-histidine peptides, the carboxylate groups in polymerized histidine-formaldehyde (pHF) were not involved in the polymerization. Therefore, these pHF-coated nanoparticles exhibited excellent colloidal stability at physiological conditions, and could be conjugated with small bioprobes for various applications. The final product of the reaction between His and formaldehyde is largely dependent on the precursor molar ratio. At the stoichiometric ratio, formaldehyde immediately reacts with the a-amino group of His to form a highly reactive hydroxymethyl group, which spontaneously attacks the active C ₄ -H of the imidazole side chain to create a stable six-membered ring structure (Scheme 1). This product (ii), spinacine (4,5,6,7-tetrahydro-lH-imidazo[4,5-c]pyridine-6-carboxylic acid), had been identified in literature (Remelli, M.; Pulidori, F.; Guerrini, R.; Bertolasi, V. J. Chem. Cryst. 1997, 27, 507), and confirmed by nuclear magnetic resonance (NMR) spectra and mass spectrometry (MS) data (Figures la and 6).

Scheme 1. Synthesis of polymerized His-formaldehyde.

It was thought that with two or more equivalents of formaldehyde, N^ ₎ and C ₂ on the imidazole ring and N ₅ on the pyridine ring of spinacine might continue to react with formaldehyde to form hydroxymethyl adducts (iii). It was further thought that continuous condensation would lead to a pHF connected by a methylene bridge as shown in Scheme 1. This appeared to be the case. Under optimal conditions (i.e. in a molar ratio of His:HCHO=l :3), the number-average molecular weight (M _n ) of the as-prepared pHF was determined to be about 4,000 g/mol by gel permeation chromatography (GPC). After dialysing against an ultrafiltration membrane (molecular weight cut-off (MWCO) = 1 kDa) to remove unreacted formaldehyde and salts, the purified pHF was vaccum dried and characterized by MS and NMR spectroscopy. Both C ₂ -H and C ₄ -H peaks of the imidazole were missing in the Ή NMR spectrum of the purified pHF (Figure la), confirming that substitution had occurred at these two positions. The peaks "m", "h" and "p" had been assigned to methylene bridge (-N-CH ₂ -imidazole), hydroxymethyl (-CH ₂ - OH) and polymer bridge (-imidazole-CH ₂ -imidazole), respectively. Fig. lb shows the UV spectra of polymers made at different formaldehyde to histidine ratio. The inset to Fig. lb illustrates the variation in the absorption at two different wavelengths as a function of this ratio. It can be seen that once the ratio exceeds about 3, the changes to the UY spectrum are much less. Fig. lc shows the molecular weight as a function of this ratio. It can be seen that the maximum molecular weight is obtained when the ratio is about 3.

Based on the molecular weight measurement, it was calculated that there were about 20 His units in each pHF polymer, and the full length of each molecule was about 10 nm, based on a monomer unit of about 5 Angstrom length. With multiple imidazole and carboxylate groups, pHF is able to bind tightly to most heavy metals, including Zn, Fe, Au, Cd and Pt. Four different approaches were pursued to prepare pHF-coated nanoparticles (see Figure 2): (a) direct aqueous synthesis with pHF as stabilizer (e.g. Pt nanoparticles), (b) ligand exchange of surfactant-capped nanoparticles with pHF (e.g. Fe ₃ 04 nanoparticles), (c) direct aqueous synthesis with His as stabilizer, followed by in situ polymerization with formaldehyde (e.g. Au nanoparticles), and (d) ligand exchange of surfactant-capped nanoparticles with His, followed by in situ polymerization with formaldehyde (e.g. CdSe@ZnS quantum dots - QDs). The CdSe@ZnS quantum dots had a core of CdSe and a shell of ZnS, and were about 5nm crystal size. Compared with the surfactant-capped nanoparticles, the fluorescence properties of pHF-QDs and the magnetic properties of pHF-Fe ₃ 0 ₄ were largely unaffected. The hydrodynamic sizes of these pHF-nanoparticles were determined by dynamic light scattering (DLS) as 3.5 nm for Pt, 15 nm for Fe ₃ 0 ₄ , 7 nm for Au, and 6 nm for CdSe@ZnS. Only the pHF-Fe ₃ 0 ₄ nanoparticles were slightly aggregated, apparently due to the crosslinking effect of direct ligand exchange. The carboxylate groups along with hydroxymethyl groups also greatly enhanced the colloidal stability of pHF-coated nanoparticles over a broad range of pH's and under various physiological conditions. The colloidal stability of the particles was tested in phosphate-buffered saline (PBS), RPMI 1640 (Roswell Park Memorial Institute medium 1640) full cell culture medium and 10 raM of l-ethyl-3-[3- dimemylaminopropyl]carbodiimide/N-hydroxysuccinimide (EDC/NHS). No precipitation was observed after 24 h of incubation in PBS and EDC/NHS solution, as shown in Figure 2. However, in full medium, pHF-Fe ₃ 0 ₄ nanoparticles precipitated slowly, probably due to their relatively larger hydrodynamic size as compared to the other three nanoparticle systems. As controls, these four nanoparticles were coated with either His or spinacine; none of the resulting coated nanoparticles were stable in PBS for more than 30 min. Unlike the poly-histidine peptides, the synthesis of pHF did not involve amide bond formation, so all of its carboxylate groups were free and available for conjugation with bioprobes. Furthermore, the amino or hydroxymethyl groups on one terminal of pHF are available for direct conjugation with pre-activated probes such as fluorescein isothiocyanate (FITC), or amine-containing compound such as doxorubicin. A fluorescent dye (i.e. FITC), and an anti-cancer drug (i.e. doxorubicin) have been attached to pHF by direct reaction with excess FITC and doxorubicin, respectively. After dialysis against ultrafiltration membrane to collect the dye-conjugated pHF polymer, the absorption spectra of FITC-pHF and doxorubicin-pHF (Figure 3a) were measured. The molar ratio of dye (FITC or doxorubicin) to His was determined to be 1:25 by absorbance measurement (Figure 3 a). Taking the M _n of pHF (4,000 g/mol) into consideration, there was about 1 dye molecule attached to each pHF chain. The quantitative, direct conjugation between doxorubicin and pHF demonstrated that hydroxymethyl groups on the freshly prepared pHF were reactive. Through a similar method, direct conjugation with other amino-containing compounds has been achieved. Furthermore, with the activation of carboxylate groups by the EDC/NHS method, up to 5 doxorubicin molecules have been attached on each pHF chain. Doxorubicin-conjugated pHF-QDs were prepared by this EDC/NHS method. Doxorubicin could bind tightly to DNA and deliver nanoparticles into the nuclei of live cells. Live 4T1 cells have been treatedwith doxorubicin-conjugated pHF-QDs. After 4 h of incubation, the doxorubicin-pHF-QDs were well distributed inside the cytoplasmic region of live cells as shown in Figure 3b. Fig. 4 illustrates the coating of a nanoparticle with polymer, in which monomer is polymerised and then forms a coating on the nanoparticle.

Fig. 5 shows the effect of different formaldehyde to histidine ratio on (a) the UV absorption of the polymer, and (b) the absorption at 275nm (upper trace) and 345nm (lower trace), illustrating more significant changes up to a ratio of about 3.5 to 4 to 1 and lesser changes thereabove. Figs. 5c and 5d illustrate the effect of different reaction times in forming the polymer, showing that a heating time of about 5 hours is sufficient for conversion to polymer.

Fig. 7 shows a GPC trace of a polymer according to the invention together with different molecular weights of polyethylene glycol, indicating that the polymer has a hydrodynamic volume approximately the same as PEG4000 (i.e. 4000 Da PEG).

In conclusion, a simple and novel imidazole-based polymer has been synthesised via a facile His-formaldehyde thermal condensation reaction. With its binding affinity to most transition metals, pHF can help stabilize a great variety of nanoparticles. The versatile functionality of pHF-coated nanoparticles is suitable for ease of bioconjugation in biological applications. In the future, pHF may also facilitate synthesis of nanocomposite materials or core-shell nanostructures in aqueous phase. Thus a novel polymerized histidine-formaldehyde (pHF) has been synthesized by simple thermal condensation reaction between histidine and formaldehyde at a basic pH. Due to its strong binding affinity to heavy metals, pHF can be used to stabilize various nanoparticles including quantum dots, noble metals and metal oxides. pFIF-coated nanoparticles have been prepared via direct synthesis or ligand exchange. These pHF-nanoparticles exhibited excellent colloidal stability under various physiological conditions. With the carboxylate and amino surface groups, the pHF-nanoparticles could be easily conjugated with biomolecules for biological applications.

Experimental

All chemicals were purchased in high purity from Merck or Sigma- Aldrich, and used without further purification.

Synthesis of pHF

1.55 g (10 mrnol) of L-histidine and 400 mg (10 mmol) of sodium hydroxide were dissolved in 20 ml of deionized (DI) water, and 3 ml of 37% formaldehyde solution (40 mmol) was added. The mixture was stirred and heated overnight at 90°C. The solution changed from colorless to light yellow. After evaporation under vacuum, the dry weight of the final pale yellow powder was 2.5 g, corresponding to a loss of 36 mmol (650 mg) of water during the condensation reaction. The as-prepared pHF was purified with membrane dialysis (MWCO = 1 kDa) to remove unreacted formaldehyde and sodium ions. The dry weight of the purified pHF was 1.2 g, corresponding to a yield of 60%; 40% of pHF (with molecular weight of < 1000) was removed.

Synthesis of pHF-Coated Nanoparticles

pHF-Pt Nanoparticles by direct aqueous synthesis. 10 ml of K ₂ PtCl ₆ solution (1 mM) was mixed with 1 ml of pHF (20 mM), and the pH was adjusted to 10. 1 ml of NaBFL; (100 mM) was added with stirring, and then heated to 100°C for 1 h. After cooling to room temperature, pHF-Pt nanoparticles were precipitated and washed with methanol. The pellet was resuspended in water, and filtered with a 0.2-μπι syringe filter. pHF-Au Nanoparticles by two-step aqueous synthesis. 10 ml of HAuCl ₄ (1 mM) and 10 ml of L-histidine solution (5 mM) were mixed, heated to 100°C, adjusted to pH 9.5, and then mixed with 1 ml of freshly prepared D-glucose solution (100 mM). After 10 min of heating, the color changed from pale yellow to red. 1 ml of 37% formaldehyde solution was added and refluxed at 90°C for 8 h. After cooling down to room temperature, pHF-Au nanoparticles were precipitated and washed with methanol. The pellet was resuspended in water, and filtered with a 0.2-μηι syringe filter.

pHF—Fe30 ₄ Nanoparticles by ligand-exchange synthesis. The Fe ₃ 0 ₄ nanoparticles were synthesized by thermal decomposition of iron oleate complex following the published procedures [references 1—4 below]. Ethanol was added to a hexane dispersion of Fe ₃ 0 nanoparticles, and the solution was magnetically decanted. 0.2 M of tetramethylammonium hydroxide (TMAH) was added to this precipitate, and shaken for 5 min. 2-propanol was then added, and the solution was magnetically decanted. After washing with acetone, the product was resuspended in water. 10 ml of TMAH-solubilized Fe ₃ 0 ₄ nanoparticles (1 mg/ml) was mixed with 1 ml of pHF (20 mg/ml) under stirring, followed by sonication for 1 h. pHF-coated Fe ₃ 0 ₄ nanoparticles were precipitated with 2- propanol, resuspended in water, and filtered with a 0.2-μπι syringe filter.

pHF-QDs by ligand exchange and in-situ polymerization. Trioctylphosphine oxide-CdSe@ZnS QDs were prepared in organometallic synthesis and purified with a published method [reference 5 below]. 1.55 g of L-histidine and 600 mg of sodium hydroxide were dissolved in 18 ml of methanol and 2 ml of water, and mixed rapidly with 10 ml of purified TOPO-CdSe@ZnS QD solution in chloroform (10 mg/ml) under vigorous stirring. After evaporating both chloroform and methanol, 50 ml of water was added to resuspend the QDs. His-QDs were filtered with a 0.2-μιη membrane filter, and the concentration of QDs was 2 mg/ml. 3 ml of 37% formaldehyde solution was added with stirring, and reacted overnight at 70°C. Next, pHF-QDs were precipitated with 100 ml of acetone, and centrifuged. The supernatant was decanted, and the pellet was resuspended in water, followed by membrane ultrafiltration (MWCO = 50 kDa). Purified pHF-QDs were resuspended in 50 ml of tris(hydroxymethyl)methylamine (Tris) buffer (pH 8.0), and filtered with a 0.2-μιτι syringe filter. A product yield of over 90% was achieved.

Fig. 8 shows the particle size distribution of the various nanoparticles coated with the polymer of the invention.

Conjugation with pHF and pHF-coated Nanoparticles

FITC-pHF conjugation. 5 mg of FITC was dissolved in 1 ml of DM SO, and mixed with 10 ml of pHF (1 mg/ml) in 50 mM of carbonate buffer (pH 9.5). After stirring at room temperature overnight, FITC-conjugated pHF was purified by membrane dialysis (MWCO = 5 kDa).

Doxorubicin-pHF conjugation. 5 mg of doxorubicin was dissolved in 1 ml of water, and mixed with 10 ml of freshly prepared pHF (1 mg/ml). After stirring at room temperature overnight, doxorubicin-conjugated pHF was purified by membrane dialysis (MWCO = 5 kDa).

Doxorubicin-conjugated pHF-coated CdSe@ZnS QDs. 1 ml of purified pHF-QD solution (1 mg/ml) was diluted to 20 ml with 100 mM of borate buffer (pH 8.0). 10 mg of NHS and 20 mg of EDC were freshly dissolved in 2 ml of borate buffer (100 mM), and immediately added to the QD solution with stirring. After 10 min, 1 ml of doxorubicin dissolved in borate buffer (0.1 mg/ml) was added. After incubation overnight, the system was quenched with 50 mM of glycine buffer (pH 7.5). Doxorubicin-conjugated QDs were purified by membrane ultrafiltration (MWCO = 50 kDa).

Materials Characterization

Absorption spectra of all samples were recorded at room temperature on an Agilent® 8453 UV-visible spectrometer. TEM was performed on FEI Tecnai® TF-20 field emission high-resolution transmission electron microscope (200 kV). DLS of nanoparticles in aqueous solution was conducted with a BI-200SM® laser light scattering system (Brookhaven Instruments Corporation). Cell imaging was performed using an Olympus® microscope 1X81 with DP70 digital camera. All proton NMR spectra were obtained with Bruker® spectrometer (400 MHz). Liquid chromatography-MS (LC-MS) spectra were collected with a Shimadzu® LCMS-2010EV spectrometer.

References

[1] Jana, N. R.; Chen, Y.; Peng, X. Chem. Mater. 2004, 16, 3931.

[2] Park, J.; An, K.; Hwang, Y.; Park, J.; Noh, H.; Kim, J.; Park, J.; Hwang, N.; Hyeon, . Nat. Mater. 2004, 3, 891.

[3] Kovalenko, M. V.; Bodnarchuk, M. I.; Lechner, R. T.; Hesser, G.;

Schaffler, F.; Heiss, W. J. Am. Chem. Soc. 2007, 29, 6352.

[4] Jiang, J.; Gu, H.; Shao, H.; Devlin, E.; Papaefthymiou, G. C; Ying, J. Y. Adv. Mater. 2008, 20, 4403.

[5] Zheng, Y.; Li, Y.; Yang, Z.; Ying, J. Y. Adv. Mater, 2008, 20, 3410. The present technology relates to the development of polymerized histidine- formaldehyde (pHF). Features of the present technology include:

polymerized histidine-formaldehyde (pHF);

a method of forming pHF via thermal condensation under basic conditions;

nanoparticles (such as quantum dots) coated with pHF.

In particular, the present technology relates to a method of preparing imidazole- containing polymers with at least the following starting materials :- a) Monomer 1 - an imidazole-containing moiety

Preferred embodiment: histidine

Other embodiments include but are not limited to substituted imidazoles (preferably containing at least one amine group on the substituent).

b) Monomer 2 - an aldehyde-containing moiety

Preferred embodiment: formaldehyde

Other embodiments include but are not limited to glutaraldehyde, glyoxal, and substituted C2-C6 alkyl with at least one aldehyde functional group.

The preparation of the polymers may comprise an additional starting material. The additional starting material may be for example melamine. Other additional starting materials include but are not limited to glucose, cysteine, tris(hydroxymethyl)aminomethane (TRIS), polyethylene glycol (PEG) as well as molecules/polymers with molecular weight <600 having at least one of the following functional groups: -N¾, -OH, -SH, -COOH or amino acid.

Representative conditions are as follows :- a) Monomer 1 : monomer 2 = 2:1 - 20: 1 , preferred range: 3:1 - 4:1

b) pH>7, preferably about 9

Applications of these polymers include but are not limited to:

a) Coating for nanoparticles such as quantum dots

b) Drug/gene delivery

c) Chelation of heavy metal ions in wastewater

In a broad form, the present invention provides a process for preparing a polymer having at least one imidazole group wherein at least imidazole containing moiety is reacted with at least aldehyde containing moiety. The imidazole containing moiety may be histidine or an optionally substituted imidazole. In particular it may be histidine. The optionally substituted imidazole may have at least one amino group on the substituent. The aldehyde containing moiety may be formaldehyde, glyoxal, glutaraldehyde or optionally substituted C2-C6 alkyl with at least one aldehyde functional group. In particular, it may beformaldehyde. The process may further comprise the addition of at least one moiety comprising molecules or polymers with molecular weight <600 having at least one of the following functional groups: -N¾, -OH, -SH, -COOH or amino acid. This moiety may be selected from melamine, glucose, cysteine, tris(hydroxymethyl)aminomethane (TRIS) or mixtures thereof. In particular, it may be melamine. It may be polyethylene glycol. The invention also encompasses a polymer obtained from the process described above.

The invention also encompasses a polymer having the following formula:

n may be between 1 to 30. It may be about 25.

The invention also encompasses a composition comprising at least one nanoparticle coated with the polymer described above. The nanoparticle may comprise at least one metal, including but not limited to gold, platinum, iron, zinc, cadmium and nickel. The nanoparticle may be a quantum dot. The nanoparticle may comprise iron oxide.

The invention also encompasses a composition comprising at least one dye molecule conjugated to the polymer described above. The dye molecule may be for example fluorescein isothiocyanate.

The invention also encompasses a composition comprising at least one drug conjugated to the polymer described above. The drug may be for example doxorubicin.

The invention also encompasses a use of the polymer described above for removal of metals from water. The metals may be lead, copper, gold, platinum, iron, zinc, cadmium, nickel, mercury or mixtures thereof.