WO2005065724A1 - Formulations of paramagnetic ion complexes - Google Patents

Abstract

The present invention is directed to the field of delivery of paramagnetic metal ions via polymeric hydrophilic articles, and the use thereof as radiotherapeutics or magnetic resonance imaging contrast agents in mammals. The articles, preferably nanoarticles, are comprised of complexes comprising a paramagnetic ion and a chelating agent that is known to chelate the paramagnetic ion (“paramagnetic ion-complexes”) incorporated into a polymeric scaffold comprised of crosslinked hydrophilic building blocks.

Description

Formulations of Paramagnetic Ion Complexes

FIELD OF THE INVENTION The present invention is directed to the field of delivery of paramagnetic ion complexes via hydrophilic polymeric articles, preferably nanoarticles, and the use thereof as contrast agents in magnetic resonance imaging.

BACKGROUND OF THE INVENTION

Magnetic resonance imaging (MRI) is in common use today in medical practice. It has been shown to be superior in certain respects to other competitive imaging modalities

(e.g., CT scanning and ultrasound), and it does not use ionizing radiation and is thus free of the potential hazards of X-rays. However, the contrast available with MRI alone is not ideal, and contrast agents are commonly used to enhance the MR image. Paramagnetic contrast agents enhance MR imaging because they can be induced by an external magnetic field to produce an additive magnetic field by a process of magnetic susceptibility. Some of the best contrast agents are molecules containing atoms of the lanthanide metal gadolinium ("Gd"). Gadolinium enhances MRI contrast because it helps water molecules to relax. The MRI signal comes from water molecules that have been stimulated into an excited state by radio waves. The quicker the water molecules return to their normal state, the stronger the signal. Gadolinium assists the water molecules to return to that state more quickly. Because of toxicity of the metal, gadolinium has been complexed with chelates or other carriers, such as, for example, diethylenetriamine penta-acetic acid (DTPA), DOTA, and expanded porphyrins such as texaphyrin.

SUMMARY OF THE INVENTION This invention is directed to articles comprising paramagnetic ion complexes, where the paramagnetic metal ion is selected from the group consisting of Mn(ll), Mn(lll), Fe(lll) and all trivalent lanthanide metals other than La(lll), Lu(lll) and Pm(lll). More particularly, the invention is directed to a hydrophilic polymeric article comprised of "paramagnetic ion-complexes" (that is, complexes that comprise a paramagnetic ion and a chelating agent or moiety that is known to chelate said paramagnetic ion) incorporated into a scaffold comprised of crosslinked hydrophilic, preferably carbohydrate-based, building blocks. Paramagnetic ion chelating agents are known in the art. The paramagnetic ion-complex, via the chelating agent, is covalently attached to the article scaffold. The nanoarticle of the invention takes the form of a hydrogel that incorporates, and in a preferred embodiment at least partially encapsulates, the paramagnetic ions while allowing movement of water molecules into and out of the article scaffold. The article of the invention comprises at least one paramagnetic ion and may comprise from 1 to about 1000 or more paramagnetic ions. The articles of the invention may optionally further comprise one or more recognition elements (REs) to facilitate targeting and/or delivery. The articles may also optionally comprise polyethylene glycol (PEG)-based molecules. The PEG chains may be used to extend the circulation time of the article in vivo, or they may serve as linkers or tethers, with one end attached to the article scaffold and the other end functionalized with a recognition element. The invention is further directed to methods of synthesizing these paramagnetic ion- containing polymeric articles and to their use in various research, diagnostic and therapeutic applications via imaging, such as magnetic resonance imaging (MRI), or radiology. The invention is also directed to a pharmaceutical preparation comprising these paramagnetic ion-containing polymeric articles, which are effective magnetic resonance imaging contrast agents or radiotherapeutics for use in mammals. While nanoscopic-sized articles are presently preferred, larger-sized articles may be prepared according to the teachings herein and are included within the scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The terms "a" and "an" mean "one or more" when used herein, unless otherwise specifically indicated. By "water-soluble" is meant, herein and in the appended claims, having a solubility in water of greater that 10 mg/mL, and preferably greater than 50 mg/mL. This invention provides a hydrophilic article comprised of paramagnetic ion material. More particularly, the article of the invention comprises one or more paramagnetic ion- complexes (i.e., a paramagnetic ion and a chelating agent or moiety) covalently attached to and incorporated into a hydrogel scaffold comprising crosslinked hydrophilic building blocks. Paramagnetic ions are present in an individual article in an amount of from one to about

1000 or more ions. The number of chelating moieties in a single article is equal to or greater than the number of paramagnetic ions. The structure of the scaffold permits movement of water molecules into and out of the article, allowing contact between the paramagnetic ions and the water molecules. The articles of the invention act as effective magnetic resonance imaging contrast agents or as radiotherapeutics for use in mammals. The paramagnetic ion is selected from the group consisting of Mn(ll), Mn(lll), Fe(lll), and all trivalent lanthanide metals other than La(lll), Lu(lll) and Pm(lll). In a presently preferred embodiment, the paramagnetic ion is Mn(ll), Gd(lll) or Dy(lll); more preferably, the paramagnetic ion is Gd(lll) (gadolinium or "Gd"). As used herein, the terms "nanoarticle scaffold", "hydrogel scaffold" and "scaffold" are used interchangeably and refer to the portion of the nanoarticle (the polymeric matrix structure) that incorporates the paramagnetic ion-complex. The scaffold of the present invention is a chemically crosslinked, preferably nanoscopic hydrogel structure. That is, the article scaffolds are comprised of crosslinked hydrophilic building blocks. In one method of fabrication, the building blocks are crosslinked in the dispersed aqueous phase of a reverse microemulsion. Carbohydrates and carbohydrate derivatives are preferably used as polymeric building blocks. In one embodiment of the invention, paramagnetic ion chelating agents are included as scaffold components. While the articles of the invention may be larger in size, they are preferably from about 5 nm to about 1000 nm, more preferably from about 5 nm to about 500 nm, in diameter. Because of their size and hydrogel structure, the nanoarticles may circulate in the blood stream without being eliminated by the kidney or taken up by the RE system, and can localize in the leaky vasculature of certain pathological tissues. This localization in pathological tissues can be further enhanced through the use of recognition elements as described below. The nanoarticle scaffolds are chosen to be readily synthesized, to be degradable within the human or animal body on a desired time scale, to be nontoxic, and to allow facile functionalization with recognition elements and/or PEG molecules. The nanoarticle scaffolds further comprise a chelating agent that is known to chelate paramagnetic ions. Any paramagnetic ion chelating agent now known or later discovered may be used in the articles of the present invention. Examples of paramagnetic metal ion (e.g., Gd) chelating agents include, but are not limited to, expanded porphyrins and porphyrin-like derivatives, DOTA, DTPA, AngioMARK™ (a backbone-functionalized DTPA chelate), DTPA-BMA (a neutral bis-methyl amide derivative of DTPA), and HP-DO3A (a DOTA-like macrocyclic compound wherein one chelate arm is replaced with a hydroxylpropyl group). Mn chelates include, but are not limited to, DPDP (TeslaScan™). Hydrophilic building blocks with reactive groups are employed to form stable nanoarticle scaffolds. At least some of the building blocks are comprised of carbohydrate or derivatized carbohydrate (referred to herein as "carbohydrate-based" building blocks). For example, the carbohydrate region may be derived from simple sugars, such as N- acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, neuraminic acid, galacturonic acid, glucuronic acid, ioduronic acid, glucose, ribose, arabinose, xylose, lyxose, allose, altrose, apiose, mannose, gulose, idose, galactose, fucose, fructose, fructofuranose, rhamnose, arabinofuranose, and talose; a disaccharide, such as maltose, sucrose, lactose, or trehalose; a trisaccharide; a polysaccharide, such as cellulose, starch, glycogen, alginates, inulin, pullulan, dextran, dextran sulfate, chitosan, glycosaminoglycans, heparin, heparin sulfate, hyaluronates, tragacanth gums, xanthan, other carboxylic acid-containing carbohydrates, uronic acid-containing carbohydrates, lactulose, arabinogalactan, and their derivatives, and mixtures of any of these; or modified polysaccharides. Other representative carbohydrates include sorbitan, sorbitol, chitosan and glucosamine. The carboxyl, amine and hydroxyl groups of the carbohydrates can be modified, or replaced, to include crosslinking groups, other functionalities, or combinations thereof, by methods generally known to those skilled in the art. Carbohydrate-based building blocks may be prepared from the carbohydrate precursor (e.g., sucrose, inulin, dextran, pullulan, etc.) by coupling technologies known in the art of bioorganic chemistry (see, for example, G Hermanson, Bioconjugation Techniques, Academic Press, San Diego, 1996, pp 27-40,155, 183-185, 615-617; and S. Hanesian, Preparative Carbohydrate Chemistry, Marcel Dekker, New York, 1997). For example, a crosslinkable group may be attached to a carbohydrate via the dropwise addition of acryloyl chloride to an amine-functionalized sugar. Amine-functionalized sugars can be prepared by the reaction of ethylene diamine (or other amines) with 1 ,1'- carbonyldiimidazole-activated sugars. Ester-linked reactive groups can be synthesized through the reaction of acrylic or methacrylic anhydrides with the hydroxyl group of a carbohydrate such as inulin in pyridine. Aldehyde- and ketone-functionalized carbohydrates can be obtained by selective reduction of the sugar backbone or addition of a carbonyl- containing moiety. Other reactions that introduce an amine on the carbohydrate may also be used, many of which are outlined in Bioconjugation Techniques (supra). Carbohydrate-based building blocks may also be prepared by the partial (or complete) functionalization of the carbohydrate with moieties that are known to polymerize under free radical conditions. For example, methacrylic esters may be placed on a carbohydrate at varying substitution levels by the reaction of the carbohydrate with methacrylic anhydride or glycidyl methacrylate (Vervoort, L.; Van den Mooter, G.; Augustijins, P.; Kinget, R. International Journal of Pharmaceutics, 1998, 172, 127-135). Carbohydrate-based building blocks may also be prepared by chemoenzymatic methods (Martin, B. D. et. al., Macromolecules, 1992, 25, 7081), for example in which Pseudomonas cepacia catalyzes the transesterification of monosaccharides with vinyl acrylate in pyridine or by the direct addition of an acrylate (Piletsky, S., Andersson, H., Nicholls, Macromolecules, 1999, 32, 633-636). Other functional groups may be present, as numerous derivatized carbohydrates are known to those skilled in the art of carbohydrate chemistry. The carbohydrate structures are chosen in part for their hydrophilicity. Nano-articles that incorporate insoluble metal ions, such as the paramagnetic ions, must possess highly hydrophilic scaffolds in order that high water solubility is maintained after functionalization with the metal. Nanoarticles of the invention in one embodiment have a high water content for high water solubility. "High water content", as used herein, means an article comprised of about 65 to about 98 wt% water, more preferably about 75 to about 98 wt% water, and most preferably about 80 to 98 wt% water. Thus, the amount of breakdown products is less than articles with a higher polymer concentration. The high water content scaffolds also can reduce immunogenicity, because there are fewer surfaces for immune system components to interact with. Besides carbohydrate-based building blocks, other examples of acrylate- or acrylamide-derivatized polymeric building blocks include polyethylene glycol-based molecules, such as polyethyleneglycol diacrylate and polyethyleneglycol diacrylamide. In one embodiment of the invention, to facilitate metabolism of the hydrogel scaffold, degradable linkages are included within the crosslinked scaffold. Degradable linkages can be included through the use of polylactide, polyglycolide, poly(lactide-co-glycolide), polyphosphazine, polyphosphate, polycarbonate, polyamino acid, polyanhydride, and polyorthoester - based building blocks, among others. Additionally, degradable linkages may be used to attach polymerizable moieties to carbohydrates. For instance, inulin multi- methacrylate (IMMA) contains ester moieties that connect the inulin carbohydrate backbone to the alkyl chain that is formed upon free radical polymerization used to generate the scaffold of the present invention. Additionally, small molecule crosslinking agents containing similar hydrolyzable moieties as the polymers such as carbonates, esters, urethanes, orthoesters, amides, imides, imidoxy, hydrazides, thiocarbazides, and phosphates may be used as building blocks. To function as degradable components in the hydrogel scaffold, these building blocks must be functionalized with two or more polymerizable moieties. For example, polyglycolide diacrylate, polyorthoester diacrylate and acrylate-substituted polyphosphazine, acrylate-substituted polyamino acid, or acrylate-substituted polyphosphate polymers can be used as degradable building blocks. Methacrylate or acrylamide moieties can be employed instead of acrylate moieties in the above examples. Similarly, small molecules containing a hydrolyzable segment and two or more acrylates, methacrylates, or acrylamides may be used. Such degradable polymers and small molecule building blocks may be functionalized with acrylate, methacrylate, acrylamide or similar moieties by methods known in the art. The nanoarticle scaffolds and the scaffold breakdown products of this invention are designed to be non-toxic and eliminated from the body. They may have degradable, preferably carbohydrate-based, polyamino acid-based, polyester-based, or PEG-based scaffolds, with the rate of degradation controlled by the identity of the sugar, crosslink density, and other features. Thus, the articles can be metabolized in the body, preventing undesirable accumulation in the body. In one embodiment, the building blocks are crosslinked in the dispersed aqueous phase of reverse microemulsions. The number of polymerizable groups attached to one single building block can range, for example, from about one to three for low molecular weight building blocks, to ten or more for polymeric building blocks. Building blocks that contain more than one polymerizable group can act as crosslinking agents and enable the formation of a hydrogel network. Using different amounts and proportions of building blocks from a set of building blocks with one, two, or more polymerizable groups allows formation of polymer networks of different compliancy upon polymerization. Exemplary crosslinkable groups include, but are not limited to, acrylate, methacrylate, acrylamide, methacrylamide, vinyl ether, styryl, epoxide, maleic acid derivative, diene, substituted diene, thiol, alcohol, amine, hydroxyamine, carboxylic acid, carboxylic anhydride, carboxylic acid halide, aldehyde, ketone, isocyanate, succinimide, carboxylic acid hydrazide, glycidyl ether, siloxane, alkoxysilane, alkyne, azide, 2'- pyridyldithiol, phenylglyoxal, iodo, maleimide, imidoester, dibromopropionate, and halo acetals, such as bromoacetate. Carbohydrates may be derivatized with allyl functionalities, including acrylates, methacrylates, acrylamides and methacrylamides to produce compounds such as inulin multi-methacrylate (IMMA). In a presently preferred embodiment, inulin with an average degree of polymerization (DOP) of about 10 to about 20 is used. The extent to which inulin is functionalized with methacrylate moieties, that is, the number of hydroxyl moieties on inulin that are converted to methacrylic esters to produce IMMA, is a statistical process governed by the concentrations and weight ratios of inulin and methacrylic anhydride starting material. The extent of functionalization may range from one methacrylate for every 1 to 100 monosaccharide repeat units, more preferably one methacrylate for every 3 to 20 monosaccharide repeat units. The number of monosaccharide repeat units in the IMMA may be from about 1 to about 100 or more, and is preferably from about 5 to about 50. The ester linkage to inulin may advantageously function as a site of degradation in vivo, allowing the article to degrade and be cleared from the body. Dextran multimethacrylamide and pullulan multimethacrylamide are additional preferred building blocks that may be prepared using methods similar to those for preparing IMMA. Nanoarticle hydrogels may also be formed using inulin multibenzaldehyde or oxidized dextran, each of which may be synthesized by methods known in the art. Other agents can also be incorporated into the polymer matrix. These agents or "functional building blocks" have reactive groups, and such functional building blocks include, but are not limited to, N,N'-cystinebisacrylamide (CiBA), sodium acrylate (NaA), N- (3-aminopropyl)methacrylamide hydrochloride (APMA), N[ethylamino]-3-amino- propylmethacrylamide hydrochloride, polyethylene imine (PEI), polylysine, polyamido- acrylamide derivatives, and protamine sulfate. The composition of the nanoarticles can be manipulated using functional building blocks to produce articles with a desirable characteristic, such as charge (positive, negative or neutral) or degree of crosslinking. Additionally, functional building blocks may be chosen to achieve a desired content of certain functionalities in the article scaffold. Such functionalities can improve solubility and may also be used as points of attachment for REs or PEG chains. For instance, APMA may be used to introduce amines, sodium acrylate may be used to introduce carboxylates, and diacetone acrylamide (DAA) may be used to incorporate ketones. The disulfide linkage of the CiBA monomer, which has the following formula I:

provides, after reduction, free thiols for linker attachment. CiBA may be prepared by reacting L-cystine (II) with two equivalents of acryloyl chloride (III), according to the following reaction scheme:

II III Preferably, the scaffolds of the present invention comprise from about 50% to about 100% carbohydrate-based building blocks and from 0% to about 50% functional building blocks. An embodiment of the invention provides a method for the controlled delivery of gadolinium molecules to the vicinity of the targeted cell or tissue type. The nanoarticles of this embodiment are comprised of three types of molecular structures: a nanoarticle scaffold formed of building blocks, recognition elements (REs) with high affinity to proteins expressed on certain cells or in certain tissues, and complexed paramagnetic ions covalently attached to the scaffold. REs serve to bind the nanoarticle of the invention to desired biomolecules overexpressed or otherwise found on certain cell surfaces or in certain tissues. The REs can target a multitude of disease-associated biomolecules. Tumor- associated targets include folate receptors, transferrin receptors, erbB1 , erbB2, erbB3, erbB4, CMET, CEA, EphA2, carcinoembryonic (CEA) antigen, mucin antigens including Muc-1 , cellular adhesion of the cluster differentiation (CD) antigen family. Vascular targets associated with multiple pathologies, including cancer, include VEGFR-1 , VEGFR-2, integrins, including integrin αvβ3, and integrin αvβl . Additional targets are extracellular proteins such as matrix metalloproteinases (MMPs), the collagen family, and fibrin, as well as others that are known or become known in the art. The REs may be any small or large molecular structure that provides the desired binding interaction(s) with the cell surface receptors of the targeted molecule. The number of REs per nanoarticle can range from 2 to about 1000, preferably from 2 to 500, and most preferably from 2 to 100. The nanoarticles may optionally further be comprised of more than one type of RE. As used herein, a RE "type" is defined as an RE of a specific molecular structure. An additional advantage of the present invention is that multiple RE types with complementary features may be incorporated into a single nanoarticle. Thus, when administered as an imaging agent, the nanoarticles containing the gadolinium will concentrate in tissue or on cell surfaces expressing a targeted protein. REs may consist of peptides. Peptides used as REs according to this invention will generally possess dissociation constants between 10^"4 and 10^"9 M or better. Such REs may be comprised of known peptide ligands. For instance, Phoenix Peptides' peptide ligand- receptor library (www.phoenixpeptide.com/Peptidelibrarylist) contains thousands of known peptide ligands to receptors of potential therapeutic value. The peptides may be natural peptides such as, for example, lactams, dalargin and other enkaphalins, endorphins, angiotensin II, gonadotropin releasing hormone, thrombin receptor fragment, myelin, and antigenic peptides. Particular peptides of interest are comprised of the amino acid sequence YCPIWKFPDEECY, or other sequences found in Greene, et.al. (J. Biol. Chem., 2002, 277(31), 28330-28339) that bind to erbB1 ; peptides comprised of the amino acid sequence CdFCDGFdYACYMDV, where dF and dY represent the D isomer of the amino acid residues or other sequences delineated in Murali, J. Med. Chem., 2001 , 44, 2565 - 2574 as REs; peptides disclosed in PCT WO 01/74849 that bind to CEA; and peptides comprised of the amino acid sequence ATWLPPR, as described in Demangel, et.al., EMBO J., 2000, 19(7), 1525-1533. Peptides useful in this invention may be discovered via high throughput screening of peptide libraries (e.g. phage display libraries or libraries of linear sequences displayed on beads) to a protein of interest. Such screening methods are known in the art (for example, see OF. Barbas, D. R. Burton, J. K. Scott, G. J. Silverman, Phage Display, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). The high affinity peptides may be comprised of modified amino acids or completely synthetic amino acids. The length of the recognition portion of the peptide can vary from about 3 to about 100 amino acids.

Preferably, the recognition portion of the peptide ranges from about 3 to about 15 amino acids. Shorter sequences are preferred because peptides of less than 15 amino acids may be less immunogenic compared to longer peptide sequences. Small peptides have the additional advantage that their libraries can be rapidly screened. Also, they may be more easily synthesized using solid-state techniques. REs may be comprised of a variety of other molecular structures, including vitamins such as folate, folate derivatives, growth factors such as EGF, proteins such as transferrin, antibodies, antibody fragments, lectins, nucleic acids, and other receptor ligands. Humanized or fully human antibodies, and humanized or fully human antibody fragments are preferred for use in the present invention. Additionally, it will be possible to design other non-protein compounds to be employed as the binding moiety, using techniques known to those working in the area of drug design. Such methods include, but are not limited to, self-consistent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode dynamics computer programs, all of which are well described in the scientific literature. See Rein et al.,

Computer-Assisted Modeling of Receptor-Ligand Interactions, Alan Liss, New York (1989). Preparation of non-protein compounds and moieties will depend on their structure and other characteristics and may normally be achieved by standard chemical synthesis techniques. See, for example, Methods in Carbohydrate Chemistry, Vols. I-VII; Analysis and Preparation of Sugars, Whistler et al., Eds., Academic Press, Inc., Orlando (1962), the disclosures of which are incorporated herein by reference. The use of multiple RE molecules of the same molecular structure or of different molecular structure to make up the nanoarticle can increase the avidity of the nanoarticle. As used in the present invention, "high affinity" means a binding of a single RE to a single target molecule with a binding constant stronger than 10^"4 M, while "avidity" means the binding of two or more such RE units to two or more target molecules on a cell or molecular complex. Tumor-associated targets include folate receptors, transferrin receptors, erbB1 , erbB2, erbB3, erbB4, CMET, CEA, EphA2, carcinoembryonic (CEA) antigen, mucin antigens, including Muc-1 , cellular adhesion proteins, and the cluster differentiation (CD) antigen family, including CD-9, CD-20, CD-30, CD-33, CD-40, CD-44, CD-53, CD-56, CD- 70, and CD-71. Vascular targets associated with multiple pathologies, including cancer, include VEGFR-1 , VEGFR-2, integrins (including integrin αvβ3 and integrin αvβ5), and aminopeptidase-N (also denoted as CD-13). Additional targets are extracellular proteins such as matrix metalloproteinases (MMPs), the collagen family, and fibrin. Several non-limiting schemes and examples outlining the synthesis of these paramagnetic ion-containing nanoarticles are presented below. In cases where a recognition element is used, the final structure may place the targeting ligand exposed to the exterior of the nanoarticles or entrapped within the scaffold to some degree. Reagents and starting materials in some embodiments can be obtained commercially. For example, amino acids can be purchased from chemical distributors such as Sigma-Aldrich (Milwaukee, Wl) and Pierce Chemical Company (Rockford, IL). Additionally, chemical product directories and resources, such as http://pubs.acs.org/chemcy, may be used to locate starting materials. Peptides to be used as high affinity binders can be purchased from many sources, one being Peptide Biosynthesis (www.peptidebiosynthesis.com). Articles of the present invention are fabricated by forming nanoscopic hydrogel scaffolds through the crosslinking of hydrophilic building blocks solubilized in the dispersed water phase of a reverse microemulsion ("RM"). The organic solvent and non-reactive surfactants are removed after polymerization to yield crosslinked, water-soluble, hydrophilic nanoscopic articles. In one embodiment of fabricating articles of the invention, metal chelators, such as DTPA or DOTA, are included with the hydrophilic building blocks in the aqueous phase of the RM and are covalently attached to and incorporated into the resulting nanoarticle scaffold. The paramagnetic ion chelator includes one or more appropriate functional groups (which may be added by derivatization of the original chelator) for reaction with the other building blocks to form the scaffold. After the chelator-containing nanoarticle scaffold is formed, and either before or after polymerization (crosslinking) of the scaffold building blocks, the scaffold is exposed to a solution containing paramagnetic metal, for example Gd⁺³, ions. The chelating agents within the article scaffold will take up the, e.g., Gd ions to form a Gd-complex covalently attached to and at least partially encapsulated in the nanoarticle. In another embodiment of the invention, paramagnetic ion-complexes (such as a chelated Gd) are reacted with reactive sites on the final nanoarticle scaffold (which scaffold does not include chelating agents as building blocks) to covalently attach and at least partially encapsulate the paramagnetic ions. Reverse microemulsions for scaffold fabrication are formed by combining aqueous buffer or water, building blocks, organic solvent, surfactants, initiators and, optionally, suitable paramagnetic ion chelating agents in the appropriate ratios to yield a stable phase of surfactant-stabilized aqueous nanodroplets dispersed in a continuous oil phase. Stable reverse microemulsion formulations can be found using known methods by those skilled in the art. They are discussed, for example, in Microemulsion Systems, edited by H. L. Rosano and M. Clausse, New York, N.Y.: M. Dekker, 1987; and in Handbook of Microemulsion Science and Technology, edited by P. Kumar and K.L. Mittel, New York, N.Y.: M. Dekker, 1999. In this invention, an aqueous phase with solubilized hydrophilic building blocks is added to an organic solvent containing one or more solubilized surfactants to form a reverse microemulsion. The dispersed aqueous phase contains hydrophilic building blocks solubilized at about 5 to about 65 wt%, preferably about 5 to about 25 wt%, most preferably about 10 to about 20 wt%. While not wishing to be bound by theory, the use of high water-content hydrogel scaffolds also may reduce immunogenicity in end uses, because there is less foreign surface for immune system components to recognize. The high water content also provides compliancy through a more flexible scaffold. Thus, when attaching to cell surface receptors, the articles are able to conform to the cell surface, allowing more surface receptors to be bound. Binding more receptors may allow the article to better function as an antagonist. Additionally, while not wishing to be bound by theory, it is believed that article cell surface coverage can inhibit other cell signaling pathways. Polymerization of the building blocks in the nanodroplets of the dispersed aqueous phase of the reverse microemulsion follows procedures known to those skilled in the art (see, for example, Odian G.G.; Principles of Polymerization, 3^rd Ed., Wiley, New York, 1991; L.H. Sperling, Introduction to Physical Polymer Science, Chapter 1 , pp. 1-21 , John Wiley and Sons, New York, 1986; and R.B. Seymour and C.E. Carraher, Polymer Chemistry,

Chapters 7-11 , pp. 193-356, Dekker, New York, 1981). Polymerization has been performed in the dispersed phase of microemulsions and reverse microemulsions (for a review, see Antonietti, M.; and Basten, R., Macromol. Chem. Phys. 1995, 196, 441 ; for a study of the polymerization of a hydrophilic monomer in the dispersed aqueous phase of a reverse microemulsion, see Holtzscherer, C; and Candau, F., Colloids and Surfaces, 1988, 29, 411). Such polymerization may yield articles in the 5 nm to 50 nm size range. The size of the nanodroplets of the dispersed aqueous phase is determined by the relative amounts of water, surfactant and oil phases employed. Surfactants are utilized to stabilize the reverse microemulsion. These surfactants do not include crosslinkable moieties; they are not building blocks. Surfactants that may be used include commercially available surfactants such as Aerosol OT (AOT), polyethyleneoxy(n)nonylphenol (Igepal™,

Rhodia Inc. Surfactants and Specialties, Cranbrook, NJ), sorbitan esters including sorbitan monooleate (Span^® 80), sorbitan monolaurate (Span^® 20), sorbitan monopalmitate (Span^® 40), sorbitan monostearate (Span^® 60), sorbitan trioleate (Span^® 85), and sorbitan tristearate (Span^® 65), which are available, for example, from Sigma (St Louis, MO). Sorbitan sesquioleate (Span^® 83) is available from Aldrich Chemical Co., Inc. (Milwaukee,

Wl). Other surfactants that may be used include polyoxyethylenesorbitan (Tween^®) compounds. Exemplary cosurfactants include polyoxyethylenesorbitan monolaurate (Tween^® 20 and Tween^® 21), polyoxyethylenesorbitan monooleate (Tween^® 80 and Tween^® 80R), polyoxyethylenesorbitan monopalmitate (Tween^® 40), polyoxyethylenesorbitan monostearate (Tween^® 60 and Tween^® 61), polyoxyethylenesorbitan trioleate (Tween^® 85), and polyoxyethylenesorbitan tristearate (Tween^® 65), which are available, for example, from Sigma (St Louis, MO). Other exemplary commercially available surfactants include polyethyleneoxy(40)-sorbitol hexaoleate ester (Atlas G-1086, ICI Specialties, Wilmington DE), hexadecyltrimethylammonium bromide (CTAB, Aldrich), and linear alkylbenzene sulfonates (LAS, Ashland Chemical Co., Columbus, OH). Other exemplary surfactants include fatty acid soaps, alkyl phosphates and dialkylphosphates, alkyl sulfates, alkyl sulfonates, primary amine salts, secondary amine salts, tertiary amine salts, quaternary amine salts, n-alkyl xanthates, n-alkyl ethoxylated sulfates, dialkyl sulfosuccinate salts, n-alkyl dimethyl betaines, n-alkyl phenyl polyoxyethylene ethers, n-alkyl polyoxyethylene ethers, sorbitan esters, polyethyleneoxy sorbitan esters, sorbitol esters and polyethyleneoxy sorbitol esters. Other surfactants include lipids, such as phospholipids, glycolipids, cholesterol and cholesterol derivatives. Exemplary lipids include fatty acids or molecules comprising fatty acids, wherein the fatty acids include, for example, palmitate, oleate, laurate, myristate, stearate, arachidate, behenate, lignocerate, palmitoleate, linoleate, linolenate, and arachidonate, and salts thereof such as sodium salts. The fatty acids may be modified, for example, by conversion of the acid functionality to a sulfonate by a coupling reaction to a small molecule containing that moiety, or by other functional group conversions known to those skilled in the art. Additionally, polyvinyl alcohol (PVA), polyvinylpirolidone (PVP), starch and their derivatives may find use as surfactants in the present invention. Cationic lipids may be used as cosurfactants, such as cetyl trimethylammonium bromide/chloride (CTAB/CTAC), dioctadecyl dimethyl ammonium bromide/chloride (DODAB/DODAC), 1 ,2-diacyl-3-trimethylammonium propane (DOTAP), 1 ,2-diacyl-3- dimethyl ammonium propane (DODAP), [2,3-bis(oleoyl)propyl] trimethyl ammonium chloride (DOTMA), and [N-(N'-dimethylaminoethane)-carbamoyl]cholesterol, dioleoyl) (DC-Choi).

Alcohols may also be used as cosurfactants, such as propanol, butanol, pentanol, hexanol, heptanol and octanol. Other alcohols with longer carbon chains may also be used. After the assembled building blocks are crosslinked to form the hydrogel scaffold and either before or after the paramagnetic ion-complexes are incorporated into the scaffold, the article surface may be functionalized with REs. The REs can be linked either directly or through a linker molecule to the surface of the nanoarticle. In a linker configuration, part or all of the REs are "displayed" at the end terminus of the tether. Therefore, in one application of the invention, the articles consist of REs displayed on a hydrogel scaffold. In another embodiment of the invention, the articles consist of an RE, such as a high affinity peptide, linked to the surface of the article scaffold via a linker molecule, the linker comprising preferentially polyethylene glycol (PEG). For each of these embodiments, it is possible to functionalize the articles with several coupling strategies, varying both the order of addition of the different components and the reactive chemical moieties used for the coupling. The components may be attached to one another in the following sequences. The hydrogel scaffold is first reacted with a di-functional PEG-containing tether, followed by functionalization of the free terminus of a portion of the PEG chain with a RE. Alternatively, the RE is coupled first to the PEG-containing tether, followed by the attachment of the other PEG terminus to the scaffold. Several combinations of reactive moieties can be chosen to attach the RE to the tether and to react the tether with the scaffold. In using a series of orthogonal reaction sets, varying some of the scaffold building blocks and/or tethering arms, it is also possible to attach REs with different molecular structures that bind to different receptors, onto the same article scaffold in well-controlled proportions. Reactions using orthogonal reactive pairs can be done simultaneously or sequentially. As far as reaction conditions are concerned, it is preferable to functionalize the articles in an aqueous system. The surfactants and the oil phase, residual from the synthesis of the hydrogel scaffold, can be removed through the use (singularly or in combination) of solvent washing, for instance using ethanol to solubilize the surfactant and oil while precipitating the articles; surfactant-adsorbing beads; dialysis; or the use of aqueous systems such as 4M urea. Methods for surfactant removal are known in the art. The RE must contain a functionality that allows its attachment to the article. Preferentially, although not necessarily, this functionality is one member of a pair of chemoselective reagents selected to aid the coupling reaction (Lemieux, G., Bertozzi, O, Trends in Biotechnology, 1998, 16, 506-513). For example, when the article surface (and/or linkers grafted to its surface) displays a halo acetal, a peptide RE may be attached through a sulfhydryl moiety. A sulfhydryl moiety in the RE structure can be accomplished through inclusion of a cysteine residue. Coupling is also possible between a primary amine on the article or the linker terminus and a carboxylic acid on the RE. A carboxylate in the peptide structure can be found either on its terminal amino acid, for linear peptides, or through the inclusion of aspartic or glutamic acid residues. The opposite configuration, where the carboxylic acid is on the article and a primary amine belongs to the peptide, is also easily accessible. Many polymerizable building blocks contain acidic moieties, which are accessible at the surface of the beads after their polymerization. As for poly(amino acid)-based REs, a primary amine function can be found either at its N-terminus (if it is linear) and/or via introduction of a lysine residue. Another example of reactive chemical pairs consists of the coupling of a sulfhydryl with a halo acetal or maleimide moiety. The maleimide function can be easily introduced, either on a peptide, a linker, or the surface of the articles, by reacting other common functionalities (such as carboxylic acids, amines, thiols or alcohols) with linkers through methods known to one of skill in the art, such as described for example by G. T. Hermanson in Bioconjugate Techniques, Academic Press Ed., 1996. In a preferred embodiment, the inclusion of CiBA, or other disulfide-containing building blocks, in the scaffold facilitates the attachment of REs through thiol reactive moieties. After scaffold formation, reduction of the disulfide linkage in CiBA produces free thiols. Linker molecules containing groups that are reactive with thiol, such as bromoacetamide or maleimide, are added to the reduced therapeutic agent-containing article to attach the linker to the article scaffold. REs are then added, which react with the free terminus of the linker molecules to give RE-functionalized articles. Alternatively, the RE may be attached to one end of the linker molecule prior to attachment of the linker molecule to the reduced article. Peptides can also be coupled to the article and/or the tether with a reaction between an amino-oxy function and an aldehyde or ketone moiety. The amino-oxy moiety (either on the articles or in the peptide) can be introduced, starting from other common functionalities (such as amines for example), by a series of transformations known to those skilled in the art. In the same way, aldehyde- or ketone-containing articles and aldehyde-containing peptides are readily synthesized by known methods. The resulting RE-functionalized, paramagnetic ion-containing articles may be used immediately, may be stored as a liquid solution, or may be lyophilized for long-term storage. Further aspects of the present invention include the use of a paramagnetic metal complex-containing nanoarticle as described herein in an imaging method comprising administering to a subject (which can be a human or an animal) an amount of paramagnetic ion-containing article, said amount being effective as a contrast or image-brightening agent, and imaging the subject using a magnetic resonance device. Such imaging methods, which take advantage of the high relaxivity of these compounds, can include, but are not limited to: i) a method of enhancement of relaxivity comprising the administration of said nanoarticle; ii) a method of magnetic resonance image enhancement comprising administering to a subject an effective amount of said nanoarticle; iii) a method of detection of neoplastic tissue in a patient comprising the steps of administering to a patient said nanoarticle in an amount effective to enhance a magnetic resonance image and detecting neoplastic tissue by magnetic resonance imaging of said patient; and iv) a method of imaging an organ in a patient comprising administering to a patient said nanoarticle in an amount effective to enhance a magnetic resonance image of the organ and detecting the organ by magnetic resonance imaging of said patient. For the above-described uses, the nanoarticles of the invention are provided as pharmaceutical preparations. A pharmaceutical preparation of a paramagnetic metal- containing nanoarticle may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. The pharmaceutical compositions formed by combining a nanoarticle of the present invention and the pharmaceutically acceptable carriers are then easily administered in a variety of dosage forms such as injectable solutions. For parenteral administration, solutions of the nanoarticle in aqueous propylene glycol or in sterile aqueous solution may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic using, for example, saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy use with a syringe exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars such as mannitol or dextrose or sodium chloride. A more preferable isotonic agent is a mannitol solution of about 2-8% concentration, and, most preferably, of about 5% concentration. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the nanoarticle components, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The following non-limiting examples are provided to further describe how the invention may be practiced. While gadolinium is used in the below examples to exemplify the invention, it is understood that any of Mn(ll), Mn(lll), Fe(lll), and all trivalent lanthanide metals other than La(III), Lu(lll) and Pm(lll) may be used as the paramagnetic metal ion and are encompassed within the present invention.

EXAMPLES

Example 1 : Fabrication of Gd-Loaded Nanoarticles An aqueous phase was prepared comprised of inulin multi-methacrylate ("IMMA"), cystine bisacrylamide ("CiBA"), aminopropyl methacrylamide ("APMA"), and α-(2-methoxy-5- isothiocyanatophenyl)-1 ,4,7,10-tetraazacyclodecane-1 ,4,7,10-tetraacetic acid ("MeODOTA- NCS/HCI" or "MeODOTA"; Dow ref # 199801357-4, sample # XUR-YM-2003-138134) in 10mM of pH 7.2 phosphate buffer saline ("PBS") as follows: 3.3mg of APMA was dissolved in 0.5g PBS solution. 15.9mg of MeODOTA was added to the APMA solution and slowly whirled for 10 minutes. 172.4mg of IMMA and 13.8mg of CiBA were added to the APMA-

MeODOTA solution and placed on a shaker for 5 minutes. The aqueous phase was added to 5g of oil phase, which includes cyclohexane and the surfactants Igepal CO-210 and CO-720, in a Schlenk tube to produce a water-in-oil reverse microemulsion. 12.5μL of 25% sodium persulfate ("NaPS") solution and 12.5μL of 25% N,N,N',N'-tetramethylethylenediamine ("TMEDA") solution were added to the microemulsion. The reverse microemulsion was cooled in an ice bath and degassed using a water pump aspirator. The solution was allowed to react for 16 hours at room temperature. Once the polymerization was complete, the resulting nanoarticles were precipitated by adding pure ethanol directly to the solution, followed by centrifugation. The nanoarticle-containing pellet was resuspended in deionized ("Dl") water. Residual surfactants and solvents were removed by ion exchange beads. The DOTA-nanoarticle solution was filtered through a 0.2μm filter and lyophilized overnight. One mole equivalent of gadolinium chloride (GdCI₃) was added directly to the DOTA- containing nanoarticles in Dl water. The Gd was allowed to chelate at 50°C for either 4 hours or 16 hours. Unreacted Gd was removed by ultrafiltration. The Gd-DOTA-containing nanoarticles were lyophilized to determine the yield and particle size distribution. Gadolinium loading in particles was 1.3 wt % as determined by elemental analysis.

Example 2: Ligand Functionalization 460 Mg of IMMA/CiBA/APMA (ratio 14:2:4) nanoparticles loaded w/ gadolinium and made according to Example 1 , were dissolved in 7 mL PBS, pH 7.2. 216 Mg of dithiothreitol (DTT) were added to reduce the dithiols in CiBA, followed by stirring for 2 hours. The DTT was then removed from the nanoparticles using desalting columns equilibrated with PBS on the FPLC. 10 Equivalents (relative to CiBA) of PEG₄₀₀ dibromoacetate, 754 mg, were added directly to the nanoparticles and allowed to react for 2 hours. Unreacted PEG was then removed from the nanoparticles using desalting columns equilibrated with PBS on the

FPLC. The PEGylated nanoparticles were reacted with 1 equivalent (relative to CiBA) of cyclic-RGD peptide, 66 mg. After reacting for 2 hours, any unreacted bromoacetate was capped with 1 + equivalents of cysteine, 7 mg. Unreacted peptide and cysteine was removed from the nanoparticles using desalting columns equilibrated with deionized water on the FPLC. The nanoparticles were collected and lyophilized with one weight equivalent of inulin (DP10) as an excipient.

Example 3: Fabrication of Gd-Loaded Nanoarticles An aqueous phase was prepared comprised of IMMA, CiBA, APMA, DOTA and Gd in PBS as follows: 3.3mg of APMA was dissolved in 0.5g PBS solution. 15.9mg of

MeODOTA was added to the APMA solution and slowly whirled for 10 minutes. 20μL of GdCI₃ solution (370mg/mL Dl water) was added to the MeODOTA-APMA solution and slowly whirled for 10 minutes. 172.4mg of IMMA and 13.8mg of CiBA were added to the APMA-Gd-DOTA solution and placed on a shaker for 5 minutes. The aqueous phase was added to 5g of oil phase in a Schlenk tube to produce a water-in-oil reverse microemulsion. 12.5μL of 25% NaPS solution and 12.5μL of 25% TMEDA solution were added to the microemulsion. The reverse microemulsion was cooled in an ice bath and degassed using a water pump aspirator. The solution was allowed to react for 16 hours at room temperature. Once the polymerization was complete, the nanoparticles were precipitated by adding pure ethanol directly to the solution, followed by centrifugation. The nanoparticle-containing pellet was resuspended in deionized water. Residual surfactants and solvents were removed by ion exchange beads. The Gd-DOTA- containing nanoarticle solution was filtered through a 0.2μm filter and lyophilized overnight. Unreacted Gd was removed by ultrafiltration. The Gd-DOTA-containing nanoarticles were lyophilized to determine the yield, particle size distribution and to proceed with ligand attachment, if desired.

Example 4: Fabrication of Gd-Loaded Nanoarticles Using reverse microemulsion techniques, nanoarticles were made with APMA containing free amines that can react with the isothiocyanate moiety of the MeODOTA chelator and/or of fluorescein isothiocyanate (FITC). These APMA-containing articles (comprising IMMA/APMA/NaA in the ratio 14:2:1) were dissolved in 0.1 M bicarbonate buffer, pH 9.4 at 65 mg/mL. A two-fold excess of MeODOTA-NCS/HCI and 1 mg of FITC were added and allowed to react for 1.5 hours. Unreacted MeODOTA and FITC were removed by size exclusion chromatography (PD-10 column equilibrated with water). One equivalent of gadolinium chloride (GdCI₃) was added directly to the DOTA-containing articles in water and was allowed to chelate for 4 hours at 50°C. Unchelated gadolinium was removed by size exclusion chromatography. The FITC-labeled Gd-DOTA-containing articles were lyophilized to quantitate yield. Ligand attachment, if desired, follows.

Example 5: Tumor Imaging Using Gd-Loaded Nanoarticles Nanoparticles were made following procedures described in Examples 1-4. For magnetic resonance imaging studies in M21 melanoma-bearing mice, the following nanoparticles ("NPs") were used: Gd-DOTA-NP-PEG₄₀₀-cRGD (40.4 nmol Gd/mg NP preparation) ("targeted Gd-NP"), Gd-DOTA-NP-PEG₄₀₀ (40.8 nmol Gd/mg NP preparation)

("non-targeted GD-NP"), and DOTA-NP-PEG₄₀₀-cRGD (without Gd loading) ("targeted DOTA-NP"). All particles were labeled with FITC to provide an additional label for histological analysis. The average particle loading was determined to be 1.3 wt% gadolinium by elemental analysis. Peptide analysis of the cRGD-functionalized particles yielded 34.3 μg peptide/mg NP. The scaffold for all particles was composed of IMMA, CiBA, and APMA at a ratio of 14:2:4. Initially, M21 melanoma-bearing mice were treated with targeted nanoarticles (Gd- DOTA-NP-PEG400-cRGD) and images were taken at times ranging from 0-24 hours post injection. A 4.7 Telsa Bruker Advance MR scanner was used. The strongest contrast enhancement from the tumor tissue appeared two hours after injection and remained strong until four hours after injection. At the 6-hour time point the signal returned to baseline levels. Images were also taken after eight hours and 24 hours. In the next step, images were recorded with non-targeted nanoparticles (Gd-DOTA-NP-PEG400) and no contrast enhancement was observed in the tumors, indicating that the targeted nanoparticles had indeed localized in the melanoma.

Example 6: Tumor Imaging Using Gd-Loaded Nanoarticles Gd-loaded nanoparticles were made as described in Example 5. For the magnetic resonance imaging of colon carcinoma xenografts, C57BL/6 mice were injected with 1 million MC38 cells (human colon carcinoma). Tumors were allowed to grow to 1 cm in diameter prior to injection with targeted Gd-NP, non-targeted Gd-NP, or targeted DOTA-NP into the lateral tail vein under inhaled anesthesia. Imaging was performed with 4.7 Tesla Bruker Advance MR Scanner by T1 -weighted multiple slice sequences. The optimal time for greatest tumor accumulation/contrast enhancement had to be determined in the MC38 carcinoma. Measurements were done at baseline, 2, 4, 6, 8, 12, and 24 hours post- injection. For the MC38 tumor model the optimal time for tumor imaging depended on the concentration of nanoparticles. Mice tolerated an intermediate concentration of nanoparticles (25 mg/500 mg or 0.05 mmol/kg Dl water) with an optimal time of 4 hours. About a 10% signal enhancement of targeted versus non-targeted nanoparticles was achieved at 4 hours, which can be further enhanced by increasing the Gd loading of the nanoparticles.

Example 7: Histology of Tumor Sections Using Fluorescent-Labeled Gd-Loaded Nanoarticles After their use for in vivo magnetic resonance imaging of tumors, the treated mice from Example 5 were used for histological analysis. The M21 melanoma-bearing mice treated with targeted and non-targeted particles were sacrificed 6 hours after injection and tissue sections of resected tumors were prepared following standard techniques known to those skilled in the art and analyzed using confocal fluorescence microscopy. With targeted particles, more intense fluorescence signals could be seen along the lining of blood vessels within the tumor tissue. For non-targeted particles, fluorescence was thinly and diffusively spread across the tissue section. The histological data indicate that nanoparticles with their small size of 20-30 nm in diameter easily extravasated from the leaky tumor vasculature into the interstitial space of the surrounding tumor tissue. Without being bound by cellular receptors, the particles moved through the interstitial space and spread out evenly. The targeted nanoparticles bound to the endothelial lining of the tumor vasculature, which is rich in cells overexpressing Integrin α_vβ₃ receptors. Tumor vasculature is known to be of chaotic architecture and to consist of a mosaic of both endothelial vessel cells and tumor cells. M21 cells themselves overexpress Integrin α_vβ₃, thereby contributing to the massive accumulation of receptors along the tumor blood vessel walls.

Example 8: Histology of Tumor Sections Using Fluorescent-Labeled Gd-Loaded Nanoarticles Using nanoparticles described in Example 6, conventional fluorescence and fluorescence confocal microscopy were employed to analyze tissue sections of M38 colon carcinoma in resected tumors from C57BL/6 mice, since all imaging particles were also labeled with FITC. The mice were injected with an amount of FITC-labeled Gd-Ioaded nanoarticles equivalent to 0.05 mmol of gadolinium per kg (of animal) and measurements were done at baseline, 2, 4, 6, 8, 12, and 24 hours post-injection. For histological evaluation, mice were injected with Rhodamine Ricinus Communis Agglutinin I 15 minutes prior to sacrifice to provide better contrasting visualization of blood vessels in the histological sections. Fluorescence accumulated along the blood vessels in tumors from animals injected with targeted NPs. The histological data using optical imaging were suggestive of differential targeting.

Claims

WHAT IS CLAIMED IS:

1. A hydrogel article comprising i) one or more paramagnetic ion-complexes, comprised of a paramagnetic metal ion and a chelating agent, covalently attached to ii) a polymeric scaffold comprising crosslinked hydrophilic building blocks, at least some of which building blocks are carbohydrate-based monomers or polymers.

2. A hydrogel article according to claim 1 wherein the paramagnetic metal ion is gadolinium.

3. A hydrogel article according to claim 1 or 2 wherein the carbohydrate-based building blocks are selected from the group consisting of inulin multi methacrylate (IMMA), pullulan multimethacrylate, dextran multimethacrylate, and oxidized dextran, and mixtures thereof.

4. A hydrogel article according to claim 1 , 2 or 3 wherein the scaffold further comprises one or more functional building blocks selected to introduce a desired characteristic or functionality into the scaffold.

5. A hydrogel article according to claim 4 wherein the functional building blocks are selected from N,N'-cystinebisacrylamide (CiBA), sodium acrylate (NaA), N-(3- aminopropyl)methacrylamide hydrochloride (APMA), N[ethylamino]-3-amino- propylmethacrylamide hydrochloride, polyethylene imine (PEI), polylysine, polyamido- acrylamide derivatives, and protamine sulfate, and mixtures thereof.

6. A hydrogel article according to any of claims 1 to 5 wherein the scaffold comprises at least some degradable covalent linkages.

7. A hydrogel article according to any of claims 1 to 6 wherein the hydrophilic building blocks further comprise small molecule crosslinking agents.

8. A hydrogel article according to any of claims 1 to 7 which further comprises two or more recognition elements covalently attached to the polymeric scaffold, the recognition elements having binding affinity to biomolecular structures expressed on certain cells or in certain tissues.

9. A hydrogel article according to any of claims 1 to 8 which further comprises at least one polyethylene glycol molecule covalently attached to the polymeric matrix.

10. A hydrogel article according to claim 1 wherein the scaffold comprises inulin multimethacrylate (IMMA), N,N'-cystinebisacrylamide (CiBA), and N-(3-aminopropyl)- methacrylamide hydrochloride (APMA) building blocks.

11. A method of magnetic resonance image (MRI) enhancement in a subject, the method comprising: administering to the subject an effective amount of an MRI contrast agent; and imaging the subject using a magnetic resonance device; wherein the MRI contrast agent comprises i) one or more paramagnetic ion-complexes, comprised of a paramagnetic metal ion and a chelating agent, covalently attached to ii) a polymeric scaffold comprising crosslinked hydrophilic building blocks, at least some of which building blocks are carbohydrate-based monomers or polymers.

12. A method according to claim 11 wherein the scaffold further comprises one or more functional building blocks selected to introduce a desired characteristic or functionality into the scaffold.

13. A method according to claim 11 or 12 wherein the scaffold comprises at least some degradable covalent linkages.

14. A method according to claim 11 , 12 or 13 wherein the hydrophilic building blocks further comprise small molecule crosslinking agents.

15. A method according to any of claims 11 to 14 wherein the contrast agent further comprises two or more recognition elements covalently attached to the polymeric scaffold, the recognition elements having binding affinity to biomolecular structures expressed on certain cells or in certain tissues.

16. A method according to any of claims 11 to 15 wherein the contrast agent further comprises at least one polyethylene glycol molecule covalently attached to the polymeric matrix.

17. A method according to any of claims 11 to 16 wherein the paramagnetic metal ion is gadolinium.

18. A method for synthesizing a paramagnetic metal ion-containing hydrogel article, the method comprising: adding an aqueous phase containing hydrophilic building blocks and one or more paramagnetic metal ion chelating agents to an organic solvent comprising at least one surfactant, at least some of which building blocks are carbohydrate- based monomers or polymers; polymerizing the building blocks to covalently crosslink the building blocks to give a hydrophilic polymeric nanoarticle having paramagnetic metal ion chelating agents covalently attached thereto; and exposing the nanoarticle to paramagnetic metal ions; to give a hydrogel article comprising i) one or more paramagnetic metal ion-complexes, comprised of a paramagnetic metal ion and a chelating agent, covalently attached to ii) a polymeric scaffold comprising crosslinked hydrophilic building blocks, at least some of which building blocks are carbohydrate-based monomers or polymers.

19. A method according to claim 15 which comprises the further steps of: adding recognition elements to a solution containing the nanoarticle, the recognition elements comprising a functionality for attachment to the nanoarticle; and reacting the recognition elements and the nanoarticle to covalently bond the recognition element and nanoarticle.