EPOXIDE BASED LINKERS

BACKGROUND OF THE INVENTION

This application claims benefit of priority to U.S. Provisional Application Serial No. 61/410,588, filed November 5, 2010, the entire contents of which are hereby incorporated by reference.

I. Field of the Invention

The present invention relates to the fields of medicinal chemistry, biology and molecular imaging. More particular, the invention relates to novel linkers for use in joining biological and non-biological molecules, as well as methods of synthesis and use therefor.

II. Related Art

The integration of nanotechnology with molecular imaging in the past decade has made a remarkable impact on biomedical research. In particular, the development of the iron oxide nanopaiticles exemplifies this noteworthy advancement. Given their non-toxic, biocompatible, biodegradable characteristics, and their size-dependent design for in vivo studies, they have been employed widely in drug delivery (Dobson, 2006; Sonvico et al, 2005), bio-sensing (Xu and Sun, 2007), and other biomedical applications such as bio-separation (Gu et al, 2003a; Gu et al, 2003b) and magnetic resonance imaging (MRI) contrast enhancement (Grimm et al, 2004; Medarova et al, 2007; Medarova et al, 2006; Perez et al, 2008).

All of these scientific achievements, which distinguish nanopaiticles from their respective adversaries, are based on the availability of the particles multivalency. In fact, one functionalized particle can carry multiple copies of an identical biological targeting agent or a mixture of different targeting imaging agents. At present, the amine group appears to be an ideal chemical moiety for the functionalization of iron nanopaiticles, this is due to its strong nucleophilicity and compatibility with a wide range of available amine activated biological materials.

On the other hand, the functionalization of nanopaiticles with carboxylic acid groups enables the conjugation of native peptides, proteins, or antibodies. A number of techniques have been developed for the functionalization of iron oxide nanopaiticles, including the in situ conversion of dextran hydroxyl groups into amines, which is typically used for the encapsulation of iron core (Lewin et al., 2000). In recent work, Young et al. (2009) coated iron nanoparticles with amine groups using a tripartite component consisting of dopamine, polyethylene glycol (PEG), and trichloro-s-triazine. Another approach to fabricating the surface of iron nanoparticles with amine groups uses glycol chitosan (Hwang et al., 2008). Additional examples include the coating of the iron core with οςω-dicarboxyl-teiminated PEG or poly(acrylic acid) to afford the free surface carbonyl groups necessary for further covalent bonding with the targeted molecules (Ke et al, 2010; Liu et al, 2009).

Despite all of these achievements, the development of fiinctionalized nanoparticles for targeting imaging still remains a significant problem. Preparation of such probes requires synthetic steps, which make it difficult to analyze the final products. Therefore, improved methods and compositions for preparing functionalized nanoparticles are required.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided a linker having the formula of:

TsO^N-X-epoxide

wherein X is C1-C2 substituted or unsubstituted alkanediyl, C1-C20 substituted or unsubstituted alkoxyl, C1-C20 substituted or unsubstituted alkenediyl, C1-C20 substituted or unsubstituted alkynediyl, C1-C10 substituted or unsubstituted cycloakyl, or a substituted or unsubstituted aromatic ring

Also provided is a method of making an epoxide linker comprising (a) providing an alkenylamine; (b) converting the alkenylamine to an alkenylammonium salt; and (c) reacting the alkenylammonium salt with dimethyldioxirane to produce a compound having the formula of:

TsO^aN-X-epoxide

wherein X is Q-C20 alkanediyl substituted or unsubstituted, C1-C20 alkoxyl substituted or unsubstituted, C1-C20 alkenediyl substituted or unsubstituted, C1-C20 alkynediyl substituted or unsubstituted, C1-C10 cycloakyl substituted or unsubstituted, or a substituted or unsubstituted aromatic ring, or a salt thereof.

Another embodiment comprises a method of orthogonal linkage a first molecule with a high reactive and chemically strain epoxide group and a second molecule with amine reactive groups comprising (a) providing a compound having the formula of: H2N-X-epoxide

wherein X is C1-C20 substituted or unsubstituted alkanediyl, C1-C20 substituted or unsubstituted alkoxyl, C1-C20 substituted or unsubstituted alkenediyi, C1-C20 substituted or unsubstituted alkynediyl, C]-Cio substituted or unsubstituted cycloakyl, or a substituted or unsubstituted aromatic ring, or a salt thereof; (b) contacting said compound with the first molecule under conditions permitting reaction of an active moiety on the first molecule with the epoxide, thereby forming a first complex; and (c) contacting said first complex with the second molecule under conditions permitting reaction of an active moeity on the second molecule with H ₂ N group of the compound, thereby forming a second complex.

The first molecule may be a molecule disposed on a surface, such as the surface of a chip, a wafer, a glass slide, a bead, a plate, a membrane, a nanotube or a nanoparticle. The first or second molecule may be a peptide, a protein, a DNA, a dye that may include fluorescent and non-fluorescent dye, or an imaging or contrast agents, such as one useful for MRI, nuclear imaging (PET, SPECT), optical, imaging.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

These, and other, embodiments of the invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and/or rearrangements. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-B. Functionalization of dextran-coated iron oxide nanoparticles with epoxide amine linker. The process resulted in approximately 40-60 amines on the surface (FIG. 1 A). The multivalency of aminated nanoparticles was demonstrated by incorporation of multiple numbers of different active biomaterials on the surface (FIG. IB).

FIGS. 2A-B. The specificity of the dual-labeled iron oxide nanoparticles in cell-based optical imaging. Folate receptor positive (293) and negative cells (A549) were incubated with the probe and imaged using white light (FIG. 2A) and fluorescence imaging (FIG. 2B).

FIG. 3. The folate receptor positive cells (293) were incubated with either aminated iron oxide nanoparticles (-Folate) or the dual-labeled probe (+Folate). The resulted cells were dispensed in gelatin-containing phantom tubes for MR imaging.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This document reports the synthesis and characterization of a versatile linker for the surface modification of nanoparticles, as well as biological application of the linker in biomedical research. The underlying principle of this work is to generate a read-and-aasy* to-use and water-soluble linker equipped with a highly reactive functional group such as an epoxide. The approach is exemplified using dextran-coated nanoparticles developed in the inventors' laboratory as a template or such modifications (Kobukai et al., 2010). Since hydroxyl groups on dextran are weak nucleophiles, the use of an epoxide guarantees a neat reaction without the use of a catalyst to enhance the simple reaction work-up. To the inventors' knowledge, this is the first report to describe the synthesis of an epoxide amine linker to generate aminated iron oxide nanoparticles. These and other aspects of the invention are described in detail below.

I. Chemical Definitions

An "epoxide" is a cyclic ether with three-member ring. Simple epoxides are named from the parent compound ethylene oxide or oxirane, such as in chloromethyloxirane. As a functional group, epoxides feature the epoxy prefix such as in the compound 1,2- epoxycycloheptane which can also be called cycloheptene epoxide, or simply cycloheptene oxide.

When used in the context of a chemical group, "hydrogen" means -H; "hydroxy" means -OH; "oxo" means =0; "halo" means independently -F, -CI, -Br or -I; "amino" means -NH ₂ (see below for definitions of groups containing the term amino, e.g., alkylamino); "hydroxyamino" means -NHOH; "nitro" means -N0 ₂ ; imino means =NH (see below for definitions of groups containing the term imino, e.g., alkylimino); "cyano" means -CN; "azido" means -N ₃ ; in a monovalent context "phosphate" means -OP(0)(OH) ₂ or a deprotonated form thereof; in a divalent context "phosphate" means -OP(0)(OH)0- or a deprotonated form thereof; "mercapto" means -SH; "thio" means =S; "thioether" means -S-; "sulfonamido" means -NHS(0) ₂ - (see below for definitions of groups contdning the term sulfonamido, e.g., alkylsulfonamido); "sulfonyl" means -S(0) ₂ - (see below for definitions of groups containing the term sulfonyl, e.g., alkylsulfonyl); "sulfinyl" means -S(O)- (see below for definitions of groups containing the term sulfinyl, e,g., alkylsulfinyl); and "silyl" means -SiH ₃ (see below for definitions of group(s) containing the term silyl, e.g., alkylsilyl).

The term "amido" (acylamino), when used without the "substituted" modifier, refers to the group -NHR, in which R is acyl, as that term is defined above. A non-limiting example of an acylamino group is -NHC(0)CH ₃ . When the term amido is used with the "substituted" modifier, it refers to groups, defined as -NHR, in which R is substituted acyl, as that term is defined above. The groups -NHC(0)OCH ₃ and -NHC(0)NHCH ₃ are non- limiting examples of substituted amido groups.

The term "alkyl" when used without the "substituted" modifier refers to a non- aromatic monovalent group with a saturated carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups, -CH3 (Me), -CH ₂ CH ₃ (Et), -CH ₂ CH ₂ CH ₃ (rt-Pr), -CH(CH ₃ ) ₂ (/so-Pr), -CH(CH ₂ ) ₂ (cyclopropyl), -CH ₂ CH ₂ CH ₂ CH ₃ («- Bu), -CH(CH ₃ )CH ₂ CH ₃ (sec-butyl), -CH ₂ CH(CH ₃ ) ₂ (iso-butyl), -C(CH ₃ ) ₃ (teri-butyl), -C¾C(CH ₃ ) ₃ (neo-pentyl), cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl are non-limiting examples of alkyl groups. The term "substituted alkyl" refers to a non-aromatic monovalent group with a saturated carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and at and S. The following groups are non-limiting examples of substituted alkyl groups: -CH ₂ OH, -CH2CI, -CH ₂ Br, -CH ₂ SH, -CF ₃ , -CH ₂ CN, -CH ₂ C(0)H, -CH ₂ C(0)OH, -CH ₂ C(0)OCH ₃ , -CH ₂ C(0)NH ₂ , -CH ₂ C(0)NHCH ₃ , -CH ₂ C(0)CH ₃ , -CH2OCH3, -CH ₂ OCH ₂ CF ₃ , -CH ₂ OC(0)CH ₃ , -CH ₂ NH ₂ , -CH ₂ NHCH ₃₎ -CH ₂ N(CH ₃ ) ₂ , -CH ₂ CH ₂ C1, -C¾CH ₂ OH, -CH ₂ CF ₃ , -CH ₂ CH ₂ OC(0)CH ₃ , -CH ₂ CH ₂ NHC0 ₂ C(CH ₃ ) ₃ , and -CH ₂ Si(CH ₃ ) ₃ .

The term "alkenyl" when used without the "substituted" modifier refers to a monovalent group with a nonaromatic carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non- limiting examples of alkenyl groups include: -CH=CH ₂ (vinyl), -CH=CHCH ₃ , -CH=CHCH ₂ CH ₃ , -C¾CH=CH ₂ (allyl), -CH ₂ CH=CHCH ₃ , and -CH=CH-C ₆ H ₅ . The term "substituted alkenyl" refers to a monovalent group with a nonaromatic carbon atom as the point of attachment, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, a linear or branched, cyclo, cyclic or acyclic structure, and at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S. The groups, -CH=CHF, -CH=CHC1 and -CH=CHBr, are non-limiting examples of substituted alkenyl groups.

The term "alkynyl" when used without the "substituted" modifier refers to a monovalent group with a nonaromatic carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups, -C sCH, -C≡€CH ₃ , C E-CC ₆ H ₅ and -CH ₂ C ^CH ₃ , are non-limiting examples of alkynyl groups. The term "substituted alkynyl" refers to a monovalent group with a nonaromatic carbon atom as the point of attachment and at least one carbon-carbon triple bond, a linear or branched, cyclo, cyclic or acyclic structure, and at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S. The group, -C ^ !Si(CH ₃ ) ₃ , is a non-limiting example of a substituted alkynyl group.

The term "alkoxy" when used without the "substituted" modifier refers to the group -OR, in which R is an alkyl, as that term is defined above. Non-limiting examples of alkoxy groups include: -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ CH ₃ , -OCH(CH ₃ ) _2> -OCH(CH ₂ ) ₂ , -O-cyclopentyl, and -O-cyclohexyl. The term "substituted alkoxy" refers to the group -OR, in which R is a substituted alkyl, as that term is defined above. For example, -OCH ₂ CF ₃ is a substituted alkoxy group. II. Linkers and Methods of Synthesis

The bifuncitonal linkers of the present invention were designed to minimize chemistry effort for deprotection, thus enables large number of end users . In organic synthesis, one would think of protecting the amine groups with Fmoc, Boc or CbZ groups during the oxidation process to synthesis the epoxy moiety on the other end of the linker. However, deprotection of these groups (Fmoc, Boc, CbZ) requires the reaction to be carried out by a skillful chemist with extensive purification effort. Additionally, the process can be achieve in organic solvent and thus unsuited in biological environment.

To tackle this problem, the inventors protected the amine group as an alkenylammonium salt before making the epoxy moiety. The advantage of the design is three-fold. First, the alkenylammonium is effective to prevent amine being oxidation while exposed the molecule in oxidation agents. Second, the alkenylammonium salt is water soluble, thus serves well for biomedical applications where most of the work occur in aqueous condition. Third, this design enables retrieval of active amine simply by just exposure of the alkenylammonium salt in mild basic condition such as sodium carbonate buffer.

Another new chemistry in the synthesis of this linker is use of dioxirane (see compound 7 in Scheme I, below). During the course of work, the inventors discovered that conventional oxidizing agents such as mCPBA or hydrogen peroxide could not convert an olefin into epoxide. They synthesized dioxirane is stable and easy to handle. This oxidation compound is preferred to convert the olefin into an epoxide molecule.

B. Synthetic Methods

Synthesis of the linkers of the present invention are generally depicted by the schemes and reactions that follow. These methods are examplary and may be modified or substituted based on general chemical synthetic reactions and the knowledge of those chemist skilled in the art.

O Oxone rP {0.1 M In acetone)

A NaHC0 ₃ , H ₂ 0

6

T _S O ^" H ₂ N^^1

SCHEME I

Pent-4-en-l-amine (Compound 4 in Scheme I). Under an atmosphere of argon, potassium phthalimide (17.21 g, 92.9 mmol) was combined with anhydrous DMF (180 mL). Afterward, 5-bromopent-l-ene (10.0 mL, 84.4 mmol) was added slowly, and the resulting mixture was heated to 60 °C for 16 hours. The reaction was then cooled to room temperature and added to a solution of 90% NaCl water (400 mL). The resulting solution was extracted with diethyl ether (3 x 200 mL). The combined organic extracts were washed with brine (1 x 200 mL), dried over magnesium sulfate, filtered and concentrated via rotary evaporation to give a light yellow oil. Prolonged high vacuum pumping yielded a semi-solid (18.17 g, quant.), which was combined with ethanol (200 proof, 125 mL) in a 250 mL round-bottom flask equipped with a reflux condenser. To the heavily stirring solution, hydrazine hydrate (64% solution in water, 6.4 mL, 84.4 mmol) was added slowly. The mixture was then heated to 60 °C for 16 hours, during which time the reaction mixture formed a large amount of white precipitant. Concentrated hydrochloric acid (30 mL) was added slowly after cooling the reaction to room temperature. The reaction was heated to reflux for 3 hours and then cooled to room temperature, after which the solids were filtered out and washed with ethanol (200 proof, 100 mL) and dichloromethane (50 mL). The solvent was removed by rotary evaporation to reveal a white solid. This solid was then taken up in water (80 mL) and made tn 1 c/tTitti ^e extracted with diethyl ether (4 x 50 mL). The combined extracts were concentrated on a rotary evaporator at a temperature of 0 °C to reveal a light yellow oil, which was purified by short-path distillation. The product distilled over at 95-100 °C (1.463 g, 20%). Ή M (300 MHz, CDCb): d 5.84-5.71 (m, 1H), 5.02-4.89 (m, 2H), 4.75 (s, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.06 (q, J = 7.1 Hz, 2H), 1.50 (p, J = 7.4 Hz, 2H). HRMS-ESI m/z: 86.0962 (C ₅ H _n N + H requires 86.0964).

Pent-4-en-l-aminium 4-methylbenzenesulfonate (Compound 5 in scheme). To a solution of pent-4-en-l -amine (1.463 g, 17.2 mmol) in dry diethyl ether (10.0 mL) at 0 °C was slowly added a solution of p-toluenesulfonic acid (3.277 g, 17.2 mmol) in dry diethyl ether (12.0 mL). This produced a white precipitant, which was filtered out and rinsed with additional cold diethyl ether (10 mL). Drying under high vacuum yielded the title compound as a white solid (2.8894 g, 65%). Ή NMR (300 MHz, D ₂ 0): d 7.64 (ΑΑ'ΧΧ', J = 8.2 Hz, 2H), 7.28 (AA'XX ^* . J = 8.1 Hz, 2H), 5.84-5.71 (m, 1H), 5.07-4.98 (m, 2H), 2.90 (t, J = 7.6 Hz, 2H), 2.31 (s, 3H), 2.05 (q, J = 7.1 Hz, 2H), 1.66 (p, J = 7.5 Hz, 2H); ^l3 C NMR (75 MHz, D ₂ 0) d 142.3, 139.4, 137.2, 129.3, 125.2, 115.6, 38.9, 29.6, 25.7, 20.4. HRMS-ESI m/z: 258.1164 (C12H20NO3S requires 258.1164).

3-(oxiran-2-yl)propan-l-aminium 4-methylbenzenesulfonate (Compound 8 in scheme). A fresh solution of dimethyldioxirane was prepared as follows: Acetone (13.0 mL, 177 mmol), sodium bicarbonate (12.0 g, 143 mmol), and water (20 mL) were added to a 100 mL three-neck round bottom flask, which was fitted with a solid addition funnel, argon inlet and Vigreux column. Attached to the Vigreux column was a 50 mL two-neck round bottom flask equipped with a dry-ice condenser, which as then attached to a house vacuum line equipped with an inline vacuum trap. The entire setup was purged of air using a steady stream of argon. Oxone® (25.09 g, 41 mmol) was added slowly to the reaction mixture via the solid addition funnel over a period of 1.5 hours while stirring constantly and ensuring a steady flow of argon. Once the Oxone® addition was complete, the entire system was put under the house vacuum for a period of 1 hour. Dimethyldioxirane in acetone (20 mL, 0.099 M) was trapped in the two-neck round bottom flask, which was subsequently sealed and stored in a freezer until needed.

To a dry 25 mL round bottom flask was added Pent-4-en-l-aminium 4- methylbenzene-sulfonate (0.103 g, 0.40 mmol) and dry CH2CI2 (3.0 mL), which was then cooled to 0 °C under an atmosphere of argon. The freshly prepared dimethyldioxirane solution (8.0 mL, 0.84 mmol) was added slowly, and the resultant clear reaction mixture was stirred at 0 °C for a period of 3 hours. Dry diethyl ether (10 mL) was then added, which produced a white precipitant. The precipitant was filtered out and rinsed with additional cold diethyl ether (10 mL) then dried under high vacuum to afford the final product as a white solid (0.109 mg, quant.). ^! H NMR (300 MHz, CDC1 ₃ ): d 7.72 (ΑΑ'ΧΧ', J = 8.0 Hz, 2H), 7.64 (bs, 3H), 7.14 (ΑΑ'ΧΧ', J = 7.9 Hz, 2H), 2.83 (t, J = 7.5 Hz, 2H), 2.71 (m, 1H), 2.59 (t, J = 4.4 Hz, 1H), 2.31 (m, 4H), 1.65 (p, J = 7.6 Hz, 2H), 1.49 (m, 1H), 1.30 (m, 1H).

III. Methods of Linking Molecules

The epoxy amine linkers were designed to enhance the reactivity thus enble for labeling a wide range of chemical entities including hydroxy.-, amine-, or thiol-containing bioactive molecules. As mentioned elsewhere, the linkers also were developed to minimize efforts that require intensive chemistry maneuvers. The reaction of the bioactive materials with the epoxy end in the linker are carried out first, given that this is a very robust and highly reactive step which can happen in neutral condition. After removal of the unreactive materials, the mixture can be treated with alkaline solution such as sodium carbonate to reveal the amino groups on the other end of the linker. Since the linker is designed as a salt, all conjugation steps can be performed in aqueous environment. rv. Substrates for Linking

The linkers of the present invention may be used, advantageously, in a variety of different contexts. As discussed above, a major application is the linking of active (diagnostic; therapeutic; targeting) agents to particles, such as beads, nanoparticles, carbon nanotubes, quantum dots, liposomes, biochips microarrays. One may also link two active molecules, including peptides, polypeptides, nucleic acids and drugs.

A. Microspheres ("Beads")

Microsphere is a term used for small spherical particles, with diameters in the micrometer range (typically 1 μχα to 1000 μπι). Microspheres are sometimes referred to as microparticles. Microspheres vary widely in quality, sphericity, uniformity, particle size and particle size distribution. The appropriate microsphere needs to be chosen for each unique application.

Microspheres can be manufactured from various natural and synthetic materials. Glass microspheres, polymer microspheres and ceramic microspheres are commercially available. Solid and hollow microspheres vary widely in density and, therefore, are used for different applications. Hollow microspheres are typically used as additives to lower the density of a material. Solid microspheres have numerous applications depending on what material they are constructed of and what size they are. Polyethylene and polystyrene microspheres are two most common types of polymer microspheres.

Polystyrene microspheres are typically used in biomedical applications due to their ability to facilitate procedures such as cell sorting and immunio precipitation. Proteins and ligands adsorb onto polystyrene readily and permanently, which makes polystyrene microspheres suitable for medical research and biological laboratory experiments.

Polyethylene microspheres are commonly used as a permanent or temporary filler.

Lower melting temperature enables polyethylene microspheres to create porous structures in ceramics and other materials. High sphericity of polyethylene microspheres, as well as availability of colored and fluorescent microspheres, makes them highly desirable for flow visualization and fluid flow analysis, microscopy techniques, health sciences, process troubleshooting and numerous research applications. Charged polyethylene microspheres are also used in electronic paper digital displays.

Glass microspheres are primarily used as a filler and volumizer for weight reduction, retro-reflector for highway safety, additive for cosmetics and adhesives, with limited applications in medical technology.

B. Nanoparticles

In nanotechnology, a particle is defined as a small object that behaves as a whole unit in terms of its transport and properties. It is further classified according to size: in terms of diameter, fine particles cover a range between 100 and 2500 nanometers, while ultrafine particles, on the other hand, are sized between 1 and 100 nanometers. Similar to ultrafine particles, nanoparticles are sized between 1 and 100 nanometers. Nanoparticles may or may not exhibit size-related properties that differ significantly from those observed in fine particles or bulk materials. Although the size of most molecules would fit into the above outline, individual molecules are usually not referred to as nanoparticles.

Nanoclusters have at least one dimension between 1 and 10 nanometers and a narrow size distribution. Nanopowders are agglomerates of ultrafine particles, nanoparticles, or nanoclusters. Nanometer-sized single crystals, or single-domain ultrafine particles, are often referred to as nanocrystals. Nanoparticle research is currently an area of intense scientific interest due to a wide variety of potential applications in biomedical, optical and electronic fields.

Nanoparticles are of great scientific interest as they are effectively a bridge between bulk materials and atomic or molecular structures. A bulk material should have constant physical properties regardless of its size, but at the nano-scale size-dependent properties are often observed. Thus, the properties of materials change as their size approaches the nanoscale and as the percentage of atoms at the surface of a material becomes significant. For bulk materials larger than one micrometer (or micron), the percentage of atoms at the surface is insignificant in relation to the number of atoms in the bulk of the material. The interesting and sometimes unexpected properties of nanoparticles are therefore largely due to the large surface area of the material, which dominates the contributions made by the small bulk of the material.

For example, nanoparticles of usually yellow gold and gray silicon are red in color; gold nanoparticles melt at much lower temperatures (-300 °C for 2.5 nm size) than the gold slabs (1064 °C); and absorption of solar radiation in photovoltaic cells is much higher in materials composed of nanoparticles than it is in thin films of continuous sheets of material - the smaller the particles, the greater the solar absorption.

Other size-dependent property changes include quantum confinement in semiconductor particles, surface plasmon resonance in some metal particles and superparamagnetism in magnetic materials. Ironically, the changes in physical properties are not always desirable. Ferromagnetic materials smaller than 10 nm can switch their magnetisation direction using room temperature thermal energy, thus making them unsuitable for memory storage.

Suspensions of nanoparticles are possible since the interaction of the particle surface with the solvent is strong enough to overcome density differences, which otherwise usually result in a material either sinking or floating in a liquid. Nanoparticles also often possess unexpected optical properties as they are small enough to confine their electrons and produce quantum effects. For example gold nanoparticles appear deep red to black in solution.

The high surface area to volume ratio of nanoparticles provides a tremendous driving force for diffusion, especially at elevated temperatures. Sintering can take place at lower temperatures, over shorter time scales than for larger particles. This theoretically does not affect the density of the final product, though flow difficulties and the tendency of nanoparticles to agglomerate complicates matters. Moreover, nanoparticles have been found tn imn ^g rl «\m<- f»vtra T%rrmi»rti<»e tn vnrimie Hav tn Hnv nrnHiirte Pnr pvamnlp. the nrp¾ftnr.p. nf titanium dioxide nanoparticles imparts what we call the self-cleaning effect, and the size being nanorange, the particles can not be observed. Zinc oxide particles have been found to have superior UV blocking properties compared to its bulk substitute.

Clay nanoparticles when incorporated into polymer matrices increase reinforcement, leading to stronger plastics, verifiable by a higher glass transition temperature and other mechanical property tests. These nanoparticles are hard, and impart their properties to the polymer (plastic). Nanoparticles have also been attached to textile fibers in order to create smart and functional clothing.

Metal, dielectric, and semiconductor nanoparticles have been formed, as well as hybrid structures (e.g., core-shell nanoparticles). Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically sub 10 ran) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents.

Semi-solid and soft nanoparticles have been manufactured. A prototype nanoparticle of semi-solid nature is the liposome. Various types of liposome nanoparticles are currently used clinically as delivery systems for anticancer drugs and vaccines.

Nanoparticles with one half hydrophilic and the other half hydrophobic are termed Janus particles and are particularly effective for stabilizing emulsions. They can self- assemble at water/oil interfaces and act as solid surfactants.

C. Carbon Nanotubes

Carbon nanotubes (CNTs; also known as buckytubes) are allotropes of carbon with a cylindrical nanostructure. Nanotubes have been constructed with length-to-diameter ratio of up to 132,000,000:1, which is significantly larger than any other material. These cylindrical carbon molecules have novel properties which make them potentially useful in many applications in nanotechnology, electronics, optics, and other fields of materials science, as well as potential uses in architectural fields. They exhibit extraordinary strength and unique electrical properties, and are efficient thermal conductors.

Nanotubes are members of the fullerene structural family, which also includes the spherical buckyballs. The ends of a nanotube may be capped with a hemisphere of the buckyball structure. Their name is derived from their size, since the diameter of a nanotube is on the order of a few nanometers (approximately 1 /50,000th of the width of a human hair), while they can be up to 18 centimeters in length. Nanotubes are categorized as single-walled tiotinhiUos tmA miilfi.u/iilloil nansvhikac /fWWTVi Chemical bonding in nanotubes is described by applied quantum chemistry, specifically, orbital hybridization. The chemical bonding of nanotubes is composed entirely of sp ¹ bonds, similar to those of graphite. These bonds, which are stronger than the sp ³ bonds found in diamonds, provide nanotubules with their unique strength. Moreover, nanotubes naturally align themselves into "ropes" held together by Van der Waals forces.

D. Quantum Dots

A quantum dot is a semiconductor whose excitons are confined in all three spatial dimensions. As a result, they have properties that are between those of bulk semiconductors and those of discrete molecules. Researchers have studied quantum dots in transistors, solar cells, LEDs, and diode lasers. They have also investigated quantum dots as agents for medical imaging and hope to use them as qubits.

Quantum dots are semiconductors whose conducting characteristics are closely related to the size and shape of the individual crystal. Generally, the smaller the size of the crystal, the larger the band gap, the greater the difference in energy between the highest valence band and the lowest conduction band becomes, therefore more energy is needed to excite the dot, and concurrently, more energy is released when the crystal returns to its resting state. For example, in fluorescent dye applications, this equates to higher frequencies of light emitted after excitation of the dot as the crystal size grows smaller, resulting in a color shift from red to blue in the light emitted. The main advantages in using quantum dots is that because of the high level of control possible over the size of the crystals produced, it is possible to have very precise control over the conductive properties of the material.

E. Biochips/Microarrays

The development of biochips is a major thrust of the rapidly growing biotechnology industry, which encompasses a very diverse range of research efforts including genomics, proteomics, and pharmaceuticals, among other activities. Advances in these areas are giving scientists new methods for unravelling the complex biochemical processes occurring inside cells, with the larger goal of understanding and treating human diseases. At the same time, the semiconductor industry has been steadily perfecting the science of micro-miniaturization. The merging of these two fields in recent years has enabled biotechnologists to begin packing their traditionally bulky sensing tools into smaller and smaller spaces, onto so-called biochips. These chips are essentially miniaturized laboratories that can perform hundreds or screen large numbers of biological analytes for a variety of purposes, from disease diagnosis to detection of bio terrorism agents.

The microarray - dense, two-dimensional grid of biosensors - is the critical component of a biochip platform. Typically, the sensors are deposited on a flat substrate, which may either be passive (e.g., silicon or glass) or active, the latter consisting of integrated electronics or micromechanical devices that perform or assist signal transduction. Surface chemistry is used to covalently bind the sensor molecules to the substrate medium. The fabrication of microarrays is non-trivial and is a major economic and technological hurdle that may ultimately decide the success of future biochip platforms. The primary manufacturing challenge is the process of placing each sensor at a specific position (typically on a Cartesian grid) on the substrate. Various means exist to achieve the placement, but typically robotic micro-pipetting or micro-printing systems are used to place tiny spots of sensor material on the chip surface. Because each sensor is unique, only a few spots can be placed at a time. The low-throughput nature of this process results in high manufacturing costs.

Fodor and colleagues developed a unique fabrication process (later used by Affymetrix) in which a series of micro lithography steps is used to combinatorially synthesize hundreds of thousands of unique, single-stranded DNA sensors on a substrate one nucleotide at a time. One lithography step is needed per base type; thus, a total of four steps is required per nucleotide level. Although this technique is very powerful in that many sensors can be created simultaneously, it is currently only feasible for creating short DNA strands (15-25 nucleotides). Reliability and cost factors limit the number of photolithography steps that can be done. Furthermore, light-directed combinatorial synthesis techniques are not currently possible for proteins or other sensing molecules.

As noted above, most microarrays consist of a Cartesian grid of sensors. This approach is used chiefly to map or "encode" the coordinate of each sensor to its function. Sensors in these arrays typically use a universal signalling technique (e.g., fluorescence), thus making coordinates their only identifying feature. These arrays must be made using a serial process (i.e., requiring multiple, sequential steps) to ensure that each sensor is placed at the correct position.

"Random" fabrication, in which the sensors are placed at arbitrary positions on the chip, is an alternative to the serial method. The tedious and expensive positioning process is not required, enabling the use of parallelized self-assembly techniques. In this approach, large katrh ^p e nf ift/ ^» nttrnl cpncnrs an t* nrrvliifwl- eensrirs ftrim pa .h hatr.h am thfirl r.nmhitlfiH and assembled into an array. Λ non-coordinate based encoding scheme must be used to identify each sensor. As the figure shows, such a design was first demonstrated (and later commercialized by Illumina) using functionalized beads placed randomly in the wells of an etched fiber optic cable. Each bead was uniquely encoded with a fluorescent signature. However, this encoding scheme is limited in the number of unique dye combinations that can be used and successfully differentiated.

Microarrays are not limited to DNA analysis; protein microarrays, antibody microarray, chemical compound microarray can also be produced using biochips. Biochips can be used to simultaneously analyze a panel of related tests in a single sample, producing a patient profile. The patient profile can be used in disease screening, diagnosis, monitoring disease progression or monitoring treatment. Performing multiple analyses simultaneously, described as multiplexing, allows a significant reduction in processing time and the amount of patient sample required. Biochip Array Technology is a novel application of a familiar methodology, using sandwich, competitive and antibody-capture immunoassays. The difference from conventional immunoassays is that the capture ligands are covalently attached to the surface of the biochip in an ordered array rather than in solution.

In sandwich assays an enzyme-labelled antibody is used; in competitive assays an enzyme-labelled antigen is used. On antibody-antigen binding a chemiluminescence reaction produces light. Detection is by a charge-coupled device (CCD) camera. The CCD camera is a sensitive and high-resolution sensor able to accurately detect and quantify very low levels of light. The test regions are located using a grid partem then the chemiluminescence signals are analysed by imaging software to rapidly and simultaneously quantify the individual analytes.

V. Active Agents

The present invention envisions a variety of different "active" agents for linking to substrates. The following is a general discussion of such agents, which include agents that target tissues and cells in a subject, that are therapeutic, and that can be used diagnostically.

A. Targeting Agents

1. Antibodies

Antibodies (also known as immunoglobulins, abbreviated Ig) are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. They α ^η » nrop hpaw r.hains anH twn small light chains— to form, for example, monomers with one unit, dimers with two units or pentamefs with five units. Antibodies are produced by a kind of white blood cell called a plasma cell. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.

Though the general structure of all antibodies is very similar, a small region at the tip of the protein is extremely variable, allowing millions of antibodies with slightly different tip structures, or antigen binding sites, to exist. This region is known as the hypervariable region. Each of these variants can bind to a different target, known as an antigen. This huge diversity of antibodies allows the immune system to recognize an equally wide variety of antigens, and also makes them powerful tools in molecular biology.

The unique part of the antigen recognized by an antibody is called the epitope. These epitopes bind with their antibody in a highly specific interaction, called induced fit, that allows antibodies to identify and bind only their unique antigen in the midst of the millions of different molecules that make up an organism. Recognition of an antigen by an antibody tags it for attack by other parts of the immune system, but at a more general level, it permits the antibody, and anything that might be attached thereto, to "home" in on the antigen in vivo. Antibodies can also neutralize targets directly by, for example, binding to a part of a pathogen that it needs to cause an infection.

The large and diverse population of antibodies is generated by random combinations of a set of gene segments that encode different antigen binding sites (or paratopes), followed by random mutations in this area of the antibody gene, which create further diversity. Antibody genes also re-organize in a process called class switching that changes the base of the heavy chain to another, creating a different isotype of the antibody that retains the antigen specific variable region. This allows a single antibody to be used by several different parts of the immune system. Production of antibodies is the main function of the humoral immune system.

2. Receptor-Binding Molecules

A receptor is a protein molecule, embedded in either the plasma membrane or the cytoplasm of a cell, to which one or more signaling molecules, or receptor-binding mnlfViiilpB mav atfar.h A mnlppiil hirti ViinHs tn a rficmtnr is alsn nailed a lifranfL and mav be a peptide or other small molecule, such as a neurotransmitter, a hormone, a pharmaceutical drug, or a toxin. Each kind of receptor can bind only certain ligand shapes, and thus the ligand "targets" the appropriate receptor in a selective fashion.

Ligand binding stabilizes a certain receptor conformation (the three-dimensional shape of the receptor protein, with no change in sequence). This is often associated with gain of or loss of protein activity, ordinarily leading to some sort of cellular response. However, some ligands (e.g., antagonists) merely block receptors without inducing any response. Biologists often attempt to hijack such ligands to target other agents, such as diagnostics or therapeutics, to the appropriate cells and tissues. Ligand-induced changes in receptors result in cellular changes which constitute the biological activity of the ligands. Many functions of the human body are regulated by these receptors responding uniquely to specific molecules like this.

3. Peptides and Peptidomimetics

Peptides are short proteins, around 30 residues or less, that often represent portions of larger proteinaceous molecules. Peptides are useful in a variety of ways, many of which involve binding to a target - as a ligand for a non-immunologic receptor, as a substrate for an enzyme, or as an antigen for a T- or B-cell receptor. Examples of non-immunologic receptors include tumor-homing peptides and peptide hormones

B. Therapeutic Agents

1. Antibiotics

Classes of antibiotics that may be used in conjunction with the present invention include, but are not limited to, macrolides (i.e., erythromycin), penicillins (i.e., nafeillin), cephalosporins (i.e., cefazolin), carbepenems (i.e., imipenem, aztreonam), other fteio-lactam antibiotics, beta-lactam inhibitors (i.e., sulbactam), oxalines (i.e., linezolid), aminoglycosides (i.e., gentamicin), chloramphenicol, sufonamides (i.e., sulfamethoxazole), glycopeptides (i.e., vancomycin), quinolones (i.e., ciprofloxacin), tetracyclines (i.e., minocycline), fusidic acid, trimethoprim, metronidazole, clindamycin, mupirocin, polyenes (i.e., amphotericin B), rifamycins (i.e., rifampin), and azoles (i.e., fluconazole).

Examples of specific antibiotics that may be used include, but are not limited to, nafeillin, methicillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, erythromycin, nafeillin, cefazolin, iminPTipm artrfvmam amtamirin <!iilftimethnxii7nlft vanoomvfiin cinrofloxflcin. trimethoprim, rifampin, metronidazole, clindamycin, teicoplanin, mupirocin, azithromycin, clarithromycin, ofloxacin, lomefloxacin, levofioxacin, grepafioxacin, norfloxacin, nalidixic acid, sparfloxacin, pefloxacin, amifloxacin, enoxacin, fleroxacin, minocycline, linezolid, temafloxacin, tosufloxacin, clinafloxacin, sulbactam, clavulanic acid, amphotericin B, fluconazole, itraconazole, ketoconazole, and nystatin.

2. Chemo herapeutics

Chemotherapeutics include agents that directly cross-link nucleic acids, specifically DNA. Agents such as cisplatin, and other DNA alkylating agents may be used. Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg m ² for 5 days every three weeks for a total of three courses. Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.

Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg m ² at 21 day intervals for adriamycin, to 35-50 mg m ² for etoposide intravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acid precursors and subunits also lead to DNA damage. As such a number of nucleic acid precursors have been developed. Particularly useful are agents that have undergone extensive testing and are readily available. As such, agents such as 5-fluorouracil (5-FU), are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells. Although quite toxic, 5-FU, is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg kg day being commonly used. 3. Therapeutic Radionuclides

Radioactive isotopes include astatine ²¹¹ , ¹ carbon, ^5l chromium, ³⁶ chlorine, "cobalt, ⁵⁸ cobalt, copper ⁶⁷ , ¹⁵² Eu, gallium ⁶⁷ , ³ hydrogen, iodine ¹²³ , iodine ¹²⁵ , iodine ¹³¹ , indium ¹¹¹ , ^S9 iron, ³² phosphorus, rhenium ¹⁸⁶ , rhenium ¹⁸⁸ , "selenium, ³⁵ sulphur, technicium ^99m and/or and/or indium" ¹ are also often preferred due to their low energy and suitability for long range detection.

C. Diagnostic Agents

Diagnostic agents, and described herein, are a diverse class of molecules that can be detected by virtue of some intrinsic property. They are useful for both quantitative and qualitative purposes, including in vivo imaging, in vitro screening assays, and ex vivo histochemistry.

The imaging moieties used can be paramagnetic ions; radioactive isotopes; fluorochromes; MR-detectable substances; X-ray imaging agents. Other agents include enzymes, haptens, fluorescent labels, phosphorescent molecules, chemilluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin. 1. Optical Fluorescent Dyes

Cyanine dyes are a large family of dyes that, in general, contain an unsaturated carbon chain linked by heterocyclic rings such as, but not limited to indole, quinoline, isoquinoline, benzothiazole, and benzooxazole. These ring systems play an important role in the final characteristics of the dyes. Additionally, these rings serve as a critical framework to fine-tune the photophysical properties of the dyes. Modification of the rings enhances the feasibility of the dyes for biological use by helping tune the emission wavelength of the dye into the NIR region. The color of the dye is generated by electron propagation along the polymethine moieties bridging the heterocyclic ring system. The quaternary amines on the rings act as electron sinks. What makes cyanine dyes unique compared to other dyes is their flexible chemistry that allows for modifications at many possible positions on the carbon backbone.

Rhodamines and oxazines have honeycomb-like structures since they are architectural clusters of hexagonal frameworks formed by conjugated π-systems. Because of their structure, this super family of dyes exhibits notable photostability and photophysical properties when compared to other families of dyes. Many rhodamines found use in a number of biological studies even before the arrival of molecular imaging such as in microcopy, histology, and as molecular switches. Some well known versions have common names like rhodamine 6G, rhodamine B, Alexa dyes, TAMRA, and Texas Red. Specific fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6- JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.

2. Bioliiminescent Compounds

Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. One famous example is the firefly luciferase from Pkotinus pyralis. "Firefly luciferase" as a laboratory reagent usually refers to P. pyralis luciferase, although recombinant luciferases from several other species of fireflies are also commercially available.

In luminescent reactions, light is produced by the oxidation of a luciferin (a pigment):

luciferin + 0 ₂ → oxyluciferin + light

The most common luminescent reactions release CO2 as a product. The rates of this reaction between luciferin and oxygen are extremely slow until they are catalyzed by luciferase, sometimes mediated by the presence of cofactors such as calcium ions or ATP. The reaction catalyzed by firefly luciferase takes place in two steps:

luciferin + ATP→ luciferyl adenylate + PPj luciferyl adenylate + O2→ oxyluciferin + AMP + light The reaction is very energetically efficient: nearly all of the energy input into the reaction is transformed into light. As a comparison, the incandescent light bulb loses about 90% of its energy to heat. Photon emission can be detected by light sensitive apparatus such as a luminometer or modified optical microscopes. This allows observation of biological processes.

A subtle structural difference in luciferase has been discovered to be the cause of the change in bioluminescence emission color from a yellow-green to red. The structure of wild- type luciferase and red mutant (S286N) luciferase from the Japanese Genji-Botaru Luciola cruciata) in complex with an intermediate analogue 5'-0-[N-(dehydroluciferyl)-sulfamoyl] adenosine (DLSA) was examined and studies showed that the wild-type luciferase complexed with DLSA exhibited a 'closed form' of the active site, where the side chain of amino acid isoleucine 288 moved towards the benzothiazole ring of DLSA, creating a rigid hydrophobic nocket. The 'closed form' wild-tvoe luciferase bound the excited state of oxvluciferin in a highly rigid and nonpolar microenvironrnent, minimizing energy loss before emitting yellow- green light. The S286N luciferase complexed with DLSA exhibited an Open form' of the active site, where the amino acid side chain of isoleucine 288 did not move towards the benzothiazole ring of DLSA, creating a less rigid and less hydrophobic microenvironrnent. The 'open form' S286N luciferase had a less rigid microenvironrnent allowing some energy loss from the excited state of oxyluciferin, which resulted in the emission of low-energy red light.

A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. The most famous are the fireflies, although the enzyme exists in organisms as different as the Jack-O-Lantem mushroom (Omphalotus olearius) and many marine creatures. In fireflies, the oxygen required is supplied through a tube in the abdomen called the abdominal trachea. The luciferases of fireflies, of which there are over 2000 species, and of the Elateroidea (fireflies, click beetles and relatives) in general, are diverse enough to be useful in molecular phylogeny. The most thoroughly studied luciferase is that of the Photinini firefly Photin s pyralis, which has an optimum pH of 7.8.

Also well studied is the luciferase from Renilla reniformis. In this organism, the luciferase is closely associated with a luciferin-binding protein as well as a green fluorescent protein (GFP). Calcium triggers release of the luciferin (coelenterazine) from the luciferin binding protein. The substrate is then available for oxidation by the luciferase, where it is degraded to coelenteramide with a resultant release of energy. In the absence of GFP, this energy would be released as a photon of blue light (peak emission wavelength 482 nm). However, due to the closely associated GFP, the energy released by the luciferase is instead coupled through resonance energy transfer to the fluorophore of the GFP, and is subsequently released as a photon of green light (peak emission wavelength 510 nm). The catalyzed reaction is:

coelenterazine + 0 ₂ → coelenteramide + C0 ₂ + photon of light

Newer luciferases have recently been identified that, unlike Renilla or Firefly luciferase, are naturally secreted molecules. One such example is the Metridia luciferase (MetLuc) that is derived from the marine copepod Metridia longa. The M. longa secreted luciferase gene encodes a 24 kDa protein containing an N-terminal secretory signal peptide of 17 amino acid residues. The sensitivity and high signal intensity of this luciferase molecule proves advantageous in many reporter studies. Some of the benefits of using a secreted reporter molecule like MetLuc is its no-lysis protocol that allows one to be able to conduct live cell assays and multiple assays on the same cell.

Gaussia luciferase (GLuc) is a 20 kDa luciferase from the marine cocepod Gaussia princeps. This luciferase, which does not require ATP, catalyzes the oxidation of the substrate coelenterazine in a reaction that produces light (480 rim), and has considerable advantages over other luminescent reporter genes. It is normally secreted from the cells and its secretion signal also functions very efficiently in mammalian cells. GLuc offers the advantage of a greatly increased bioluminescent signal relative to the commonly used firefly (Flue) and Renilla luciferases (RLuc). GLuc was determined to emit light with a specific activity of 4.2 x 10 ²⁴ photons s/mol, the highest reported activity for any characterized luciferase.

Cypridina luciferase (CLuc) is isolated from the marine ostracod Cypridina noctiluca and is efficiently secreted from mammalian cells. CLuc differs from Flue in the form of luciferin it uses as a substrate (Cypridina luciferin), it its independence from ATP to achieve light emission. CLuc does not react with coelenterazine, a common substrate of marine luciferases. This allows the simultaneous detection of both of FLuc, Rluc, or GLuc with CLuc expressed from the same cells provided the cell-derived samples can be divided and independently analyzed for each enzymatic activity, or if a method for sequentially measuring individual enzymatic activities from the same cell-derived sample is in place.

3. Diagnostic Radioactive Compounds

Radioactive isotopes include astatine ²¹¹ , ¹⁴ carbon, ^5, chromium, ³⁶ chlorine, ⁵⁷ cobalt, ⁵⁸ cobalt, copper ⁶⁷ , ¹⁵² Eu, gallium ⁶⁷ , ³ hydrogen, iodine ¹²³ , iodine ¹²⁵ , iodine ¹³¹ , indium" ¹ , ⁵⁹ iron, ³² phosphorus, rhenium ¹⁸⁶ , rhenium ¹⁸⁸ , ⁷⁵ selenium, ^3S sulphur, technicium ⁹⁹ " ¹ and/or yttrium ⁹⁰ . ¹²⁵ I is often being preferred for use in certain embodiments, and technicium ⁹⁹ " ¹ and/or indium ¹¹¹ are also often preferred due to their low energy and suitability for long range detection.

4. Paramagnetic Ions

Paramagnetic ions one might mention by way of example include chromium (HI), manganese (H), iron (III), iron (H), cobalt (II), nickel (II), copper (II), neodymium (IH), samarium (ΠΙ), ytterbium (ΙΠ), gadolinium (ΠΙ), vanadium (II), terbium (III), dysprosium (HI), holmium (ΙΠ) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (ΙΠ), gold (ΠΙ), lead (II), and especially bismuth (III).

5. Enzyme Tags/Binding Agents

Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Particular secondary binding ligands are biotin and/or avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.

VI. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1: Materials & Methods

Reagents. Bovine serum albumin (BSA), folic acid, dicyclohexylcarbodiimide (DCC), fluorescein isothiocyanate (FITC), Fluorescamine, and reaction solvents were obtained from Sigma Chemical Company (St Louis, MO) and used without further purification. Human 293 and A549 cells were obtained from the laboratories of Dr. Vito Quaranta and Dr. Dennis Hallahan, respectively, at Vanderbilt University. RPMI-1640 medium without folic acid was obtained from Invitrogen.

Developing superparamagnetic iron oxide (SPIO) nanoparticles. Dextran-coated iron oxide nanoparticles were fabricated in our laboratory using the reported protocol, but with some modifications ( obukai et al., 2010). In brief, a saturated mixture of dextran and ferric chloride was coalesced with a ferrous chloride solution at room temperature. This was followed by elevating the pH to 10 via the dropwise addition of an ammonium hydroxide solution. The resulting solution was stirred for several hours. The synthesized particles were separated from the starting materials using deionized water, and then dialyzed into buffer representing the normalphysiological concentrations of sodium citrate and sodium chloride. The concentration of iron in the solution was determined by inductively coupled plasma mass spectrometry (ICP-MS). To obtain the desired concentration of SPIO nanoparticles for analysis, a stock solution of SPIO particles was diluted 1:10,000 in 1% nitric acid solution. Each experiment was performed in duplicate.

Synthesis of an epoxide amine linkers. Pent- -en-l-amine. Under an atmosphere of argon, potassium phthalimide (17.21 g, 92.9 rnmol) was combined with anhydrous DMF (180 mL). 5-bromopent-l-ene (10.0 mL, 84.4 mmol) was then slowly added and the resulting mixture was heated to 60 °C for a period of 16 hours. The reaction was then cooled to room temperature and added to a solution of 3:1 brine to water (400 mL). The resulting solution was then extracted with diethyl ether (3 x 200 mL). The combined organic extracts were then washed with brine (1 x 200 mL), dried over magnesium sulfate, filtered and concentrated via rotary evaporation to give a light yellow oil. Prolonged high vacuum pumping gave a semisolid (18.17 g, quant.). This material was then combined with ethanol (200 proof, 125 mL) in a 250 mL round-bottom flask equipped with a reflux condenser. To the heavily stirring solution, hydrazine hydrate (64% solution in water, 6.4 mL, 84.4 mmol) was slowly added. The mixture was then heated to 60 °C for a period of 16 hours, during which time the reaction mixture formed a large amount of white precipitant. The reaction was then cooled to room temperature and concentrated hydrochloric acid (30 mL) was slowly added. The reaction was then heated to reflux for a period of 3 hours, cooled to room temperature and the solids were filtered out and washed with ethanol (200 proof, 100 mL) and dichloromethane (50 mL). The solvent was then removed by rotary evaporation to reveal a white solid. This solid was then taken up in water (80 mL) and made basic, to litmus, by the addition of potassium hydroxide pellets. The resulting solution was then extracted with diethyl ether (4 x 50 mL). The combined extracts were then concentrated on a rotary evaporator at a temperature of 0 °C toreveal a light yellow oil. This was purified by short-path distillation. Product distilled over at 95-100 °C (1.463 g, 20%). Ή NMR (300 MHz, CDC13): d 5.84-5.71 (m, 1H), 5.02-4.89 (m, 2H), 4.75 (s, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.06 (q, J = 7.1 Hz, 2H), 1.50 (p, J = 7.4 Hz, 2H). HRMS-ESI m/z 86.0962 (C ₅ H _U N + H requires 86.0964). Pent-4-en-l-aminium 4-methylbenzenesulfonate. To a solution of pent-4-en-l- amine (1.463 g, 17.2 mmol) in dry diethyl ether (10.0 mL) at 0 °C was slowly added a solution of p-toluenesulfonic acid (3.277 g, 17.2 mmol) in dry diethyl ether (12.0 mL). This produced a white precipitant, which was filtered out and rinsed with additional cold diethyl ether (10 mL). Drying under high vacuum yielded the title compound as a white solid (2.8894 g, 65%). Ή NMR (300 MHz, D20): d 7.64 (AA'XX*, J = 8.2 Hz, 2H), 7.28 (ΑΑ'ΧΧ', J = 8.1 Hz, 2H), 5.84-5.71 (m, 1H), 5.07-4.98 (m, 2H), 2.90 (t, J = 7.6 Hz, 2H), 2.31 (s, 3H), 2.05 (q, J = 7.1 Hz, 2H), 1.66 (p, J = 7.5 Hz, 2H); 13C NMR (75 MHz, D20) d 142.3, 139.4, 137.2, 129.3, 125.2, 115.6, 38.9, 29.6, 25.7, 20.4. HRMS-ESI m/z: 258.1164 (Ci ₂ H ₂ 0NO ₃ S requires 258.1164).

3-(oxiran-2-yl)propan-l-aminium 4-methylbenzenesulfonate. A fresh solution of dimethyldioxirane was prepared as follows. To a 100 mL 3-neck round bottom flask, fitted with a solid addition funnel, argon inlet and vigreux column, was added acetone (13.0 mL, 177 mmol), sodium bicarbonate (12.002 g, 143 mmol), and water (20 mL). Attached to the vigreux column was a 50 mL 2-neck round bottom flask equipped with a dry-ice condenser, which as then attached to a house vacuum line with an inline vacuum trap. The entire setup was purged of air with a steady stream of argon. To the solid addition funnel was added Oxone® (25.09 g, 41 mmol) which was slowly added to the reaction mixture over a period of

1.5 hrs with constant stirring and a steady flow of argon. Once the Oxone® addition was complete, the entire system was put under house vacuum for a period of 1 hour.

Dimethyldioxirane in acetone (20 mL, 0.099 M) was trapped in the 2-neck round bottom flask, which was subsequently sealed and stored in a freezer until needed. To a dry 25 mL round bottom flask was added pent-4-en-l-aminium 4- methylbenzene-sulfonate (0.103 g, 0.40 mmol) and dry CH2CI2 (3.0 mL), which was then cooled to 0 °C under an atmosphere of argon. The freshly prepared dimethyldioxirane solution (8.0 mL, 0.84 mmol) was then slowly added and the now clear reaction mixture was stirred at 0 °C for a period of 3 hours. Dry diethyl ether (10 mL) was then added, which produced a white precipitant. The precipitant was then filtered out and rinsed with additional cold diethyl ether (10 mL) then dried under high vacuum to afford the final product as a white solid (0.109 mg, quant.). Ή NMR (300 MHz, CDCI ₃ ): d 7.72 (ΑΑ'ΧΧ', J = 8.0 Hz, 2H), 7.64 (bs, 3H), 7.14 (ΑΑ'ΧΧ', J = 7.9 Hz, 2H), 2.83 (t, J = 7.5 Hz, 2H), 2.71 (m, 1H), 2.59 (t, J = 4.4 Hz, 1H), 2.31 (m, 4H), 1.65 (p, J =

7.6 Hz, 2H), 1.49 (m, 1H), 1.30 (m, 1H). Coupling epoxide amine linker with the nanoparticles. The nanoparticles were prepared as described above and resuspended in PBS at 11 mg/mL of iron. The epoxide amine linker was dissolved in a stock solution at a concentration of 100 mg mL. By controlling the nanoparticle concentration at 2 or 5 mg mL, 500x and 2000x excess of the linkers (the number of molecules vs. particles) were mixed, with the reaction performed overnight, at room temperature, with gentle stirring. The free epoxide amine linker was removed from the nanoparticles by Zeba Spin Columns (Thermo Scientific, Rockford, DL) following the manufacturer's recommended procedure.

Quantification of epoxide amine linkers on the surface of nanoparticles. The copy numbers of coupled epoxide amine linkers on the surface of each nanoparticle were determined by measuring the amine group with Fluorescamine (Udenfriend et ah, 1972), and free epoxide amine linkers with a known amount were used as the standard. Aliquots of the control (uncoupled) and coupled nanoparticles were both pretreated with a 0.1 X 0.1 M borate buffer (pH 9.5). A Fluorescamine stock solution was prepared in acetone at 40 mg mL. For each tested sample and blank, Fluorescamine was added to a final concentration of 1 mg/mL. Following the addition of Fluorescamine, the samples were mixed and left to stand for several minutes. The fluorescence was then determined with a 400 nm excitation and a 460 nm emission.

Conjugation of folate and/or FITC to epoxide linker-coupled nanoparticles via the amino groups. NHS-folate and FITC were dissolved in dimethyl sulfoxide at a stock concentration of 12.5 or 100 mgmL, respectively. The pH of the epoxide linker-coupled nanoparticles was exchanged to 9.5 borate buffer using Zeba Spin Columns, and the iron concentration was adjusted to 2 mg mL. An aliquot of 1 mL (2 mg) of nanoparticle stock solution was double labeled with FITC and folic acid, by first adding 5 μΐ, of FITC solution and allowing the mixture to stir at room temperature for 45 minutes prior to subsequent addition of 80 of the NHS-folate solution. For the FITC or folic acid only labeled nanoparticles, only one reactant was added. All the reactions were performed overnight, after which the conjugated nanoparticles were separated from the unreacted folic acid and FITC by passing the reaction mixture through Zeba Spin Columns. The suspension was concentrated to 5 mgmL iron by centricon for future use.

Quantification of conjugated FITC and folic acid. The copy number of FITC in each nanoparticle was estimated simply by measuring the fluorescence strength of the labeled particles against the free FITC with a known concentration. The extent of folate conjugation was determined by spectrophotometric analysis at an absorbance of 358 nm (ε = 8643.5 M-l cm-1) based on a reported procedure (Zhang et al, 2004).

Cell culture and uptake studies. Human kidney derived 293 (folate receptor- positive) and alveolar basal epithelial derived A549 cells (lung carcinoma, folate receptor negative) were cultured in an PMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco BRL) under standard culture conditions in a humidified incubator maintained at 5% C0 ₂ at 37 °C. Cell uptake studies were performed using both cell lines with folate-conjugated or folate-free nanoparticles. A total of 1 x 10 ⁶ cells were seeded in each well of a 6-well plate. The cells were then washed with PBS, and 1.2 mL (2 mg mL of iron) of FITC labeled folate- conjugated or folate-free nanoparticle suspensions in folate-free RPMI-1640 were added to each well. After 2 hours of incubation at 37 °C, the cells were washed four times with pre- cooled PBS before they were collected by scraping with a small amount of PBS. Half of the cells (0.5 million) were pelleted and examined for fluorescence by IVIS (IVIS 200, Xenogen). Images were acquired using the IVIS imaging system with excitation set at 490 nm. The fluorescence was collected through a long-pass filter and analyzed using Living Image® software version 3.0. Half of the cells were resuspended with PBS and mixed with an equal volume of 12% gelatin to produce the middle layer for a phantom comprised of 3 layers, with 12% gelatin on the top and bottom, and a 6% gelatin and cell mixture sandwiched in the middle (all gelatin solutions were prepared in PBS).

Magnetic resonance imaging. Phantom MR imaging was performed on a 4.7 T Varian Inova scanner. First, labeled DCs were embedded in 6% gelatin, and MR imaging was performed using a single slice, T2-weighted, spin-echo sequence (TR = 5 sec, TE arrayed with values of 15, 55, 95, 135, 175 and 215 ms, FOV = 40 x 40 mm ² , acquisition matrix = 256 x 256 and slice thickness = 1.0 mm).

EXAMPLE 2 - Results and Discussion

The incorporation of targeting active biomolecules onto the surface of iron nanoparticles for imaging and therapy is indispensable in medical diagnostics (Gumbleton and Stephens, 2005). Indeed, the many useful applications for functionalized iron nanoparticles, which have been conceived over the years, have made novel chemical modification of the particles even more important The iron nanoparticles used in this study are encapsulated in dextran to enhance their size improving blood pool distribution. This coating material can render them sufficiently biocompatible and biodegradable to allow suitable in vivo imaging. The hydroxyl groups on the surface of theparticles, as a result of this fabrication will be converted into stronger nucleophiles by employing a novel bifunctional linker. Two criteria are taken into account in designing this linker. First, it must possess a highly reactive functional group to ensure reactions with hydroxyl groups. The electrophilic group on the linker should react with the hydroxyl groups under the simplest of conditions without the use of a catalyst. Second, the other end of the linker must have a strong nucleophilic moiety to support bioconjugation studies. Epoxide reactive agents are considered ideal for this application given their stability and reactivity. Epoxides have been used widely as a suitable functionality for the development of affinity probes for protein labeling (Chen et al, 2003).

As shown in Scheme I (above), synthesis of the epoxy-amine linker 8 begins with the Gabriel synthesis, enabling for the conversion of 5-bromopent-l-ene 1 into the corresponding alkenylamine 4. Protection of the newly formed amine group of compound 4 was found to be necessary prior to the formation of an epoxide, due to the fact that amines can be oxidized faster than olefins once exposed to oxidizing agents. Furthermore, the final linker product must have the amine groups blocked, since the existence of free amines would undoubtedly cause an opening of the epoxide ring via nucleophilic attack. Currently, the protection of amino groups in alkenylamines can be achieved using well known chemical reagents, such as Fmoc, Boc, or Cbz groups (Albeck and Persky, 1994; Jenmalra et al, 1994) Wilson et al, 1995). However, deprotection of these groups requires an extensive amount of work, which was not within the scope of our objectives. Instead, the inventors used a method reported by Asensio et al. (1 95) to protect compound 4 as an alkenylammonium salt 5 (Asensio et al, 1995). In this approach, the protonation of amine groups is efficient enough to prevent amine oxidation by a strong electrophilic O-transfer reagent. Another unique design of this salt is that the product is compatible with the aqueous condition of nanoparticles.

Surprisingly, when using conventional reagents, such as mCPBA or hydrogen peroxide, the olefin oxidation did not progress smoothly. It is noteworthy that Asensio also made similar observation in some of the compounds. Although further investigation is needed, the inventors cannot exclude the possible interference of the bulkycounterion tosylate group. In light of this, they synthesized dioxirane 7 using a previously reported procedure (Adam et al, 1987). Compound 7 converts olefin 5 into the desired epoxide linker 8 with 90% yield. To test the feasibility of functionalizing iron nanoparticles with the newly developed linker, the inventors treated iron nanoparticles with excess epoxide linker 8 in PBS, at physiological buffer. The conjugation was completed in an overnight reaction. To quantify the number of amines on the surface of the particles, the inventors used the spiro dye Fluorescamine, which only reacts with free amines converting the non-fluorescent Fluorescamine to a fluorescent product, the intensity of which is proportional to the quantity of free amines. While the inventors did observe, residual fluorescence in the control group, where they incubated Flurosecamine with dextran-coated iron nanoparticles, this value was accounted for by subtraction from all the measured samples. This assay allowed the inventors to estimate that approximately 40 to 60 copies of epoxy amine linkers were conjugated to each nanoparticle (FIG. 1 A). The abundant number of reactive amino groups on each particle renders the nanoparticles very useful for carrying a multiple copies of homogenous or heterogeneous targeting molecules for imaging or therapeutic applications. To address that notion, the inventors labeled the aminated nanoparticles with both folic acid and FITC, sequentially. The former was activated as a succinimide ester while the latter has isothiocyanate as an amine reactive group. The extent of folate and FITC conjugation of epoxyl-conjugated nanoparticles was evaluated by spectrophotometric or fluorometry analysis. The inventors found that about 20 folic acid molecules were conjugated onto the surface of one nanoparticle. While there were approximately 3 FITC molecules per nanoparticle, the dual-labeled probe remained stable for several months while stored at 4 °C (FIG. IB).

To test the specificity of the probe, folate receptor positive (293) and negative (A549) cells were incubated with the dual-labeled probe for 2 h at 37 °C. After incubation, both cells were washed extensively with cold PBS to remove the unassociated nanoparticles. The cells were then collected and confirmed by optical or MR imaging. As shown in FIG. 2B, the fluorescence intensity in the folate receptor (FR)-positive 293 cells is stronger than that in the FR negative A549 cells. With folate-free nanoparticles, the 293 and A549 cells have similar and weak fluorescence, which suggests some nonspecific association or endocytosis (data not shown). To prove further that the fluorescence signal emanated from the folate fluorescein conjugated nanoparticles, and not from potential residual free fluorescein or cell auto- fluorescence, aliquoted cells were also assessed for iron content via MR imaging, as shown in FIG. 3. It was observed that the 293 cells incubated with the folate conjugated nanoparticles have greater amount of iron, evidenced by decreased signal intensity. This data suggest that fhfi imtalfft rif FTTC-lahfilfiH TMvntmstrtMcji hv W¾ r.f»11s r>nr.iirs via folate i«centni\ Taken all together, this study is designed to provide proof of principle that the iron oxide nanoparticles could be efficiently functionalized with a linker in a mild reaction condition. The invnentors believe that this study establishes a platform that impacts not only iron nanoparticles, but also other types of nanoparticles or colloids such as quantum dots, which will facilitate the effort to integrate nanotechnology with molecular imaging for biomedical applications.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing f om the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

VII. References

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

U.S. Patent 3,817,837

U.S. Patent 3,850,752

U.S. Patent 3,939,350

U.S. Patent 3,996,345

U.S. Patent 4,277,437

U.S. Patent 4,275,149

U.S. Patent 4,366,241

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