WO2008140624A2 - Methods and compositions related to hybird nanoparticles - Google Patents

Abstract

Embodiments of the invention include a hybrid nanoparticle comprising an optically interrogatable component and a magnetically interrogatable component. Particularly, a hybrid nanoparticle coupled with a targeting moiety that selectively associates the nanoparticle with a particular biological location, cell, tissue, or organ.

Description

DESCRIPTION METHODS AND COMPOSITIONS RELATED TO HYBRID NANOPARTICLES

[0001] This application claims priority to U.S. Provisional Patent application serial number 60/871,569 filed December 22, 2006, which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under grants R01-CA103830 and ROl-CAl 19387 awarded by National Cancer Institute and grant DMR-0605828 awarded by the National Science Foundation. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

I. FIELD OF THE INVENTION

[0003] Embodiments of the invention relate generally to the field of medical imaging. More particularly, embodiments of the invention relate to a hybrid nanoparticle comprising an optically interrogatable component and a magnetically interrogatable component. In a particular aspect, the hybrid nanoparticle is coupled with a targeting moiety that selectively associates the nanoparticle with a particular biological location, cell, tissue, or organ.

II. BACKGROUND

[0004] Exogenous contrast agents are widely used to increase signal intensity and specificity during optical interrogation of biological materials. Organic fluorescent dyes are traditional contrast enhancing molecules for in vitro and in vivo optical imaging (Giepmans et al, 2006; Bornhop et al, 2001; Malicka et al, 2003; Ntziachristos et al, 2002). Recent advances in nanotechnology have led to the development of novel bright contrast agents, including quantum dots (Gao et al, 2004; Chan and Me, 1998; Gao et al, 2005; Michalet et al, 2005; Alivisatos et al, 2005) and plasmonic nanoparticles (Yguerabide and Yguerabide, 2001; Oldenburg et al, 1998; Sokolov et al, 2003; Loo et al, 2005; El-Sayed et al, 2005; Sokolov et al, 2003). Progress in nanomaterial chemistry has allowed synthesis of semiconductor quantum dots with increased fluorescence efficiencies (Kershaw et al, 1999), tunable emission bands (Han et al, 2001), and relatively slow photobleaching rates (Chan and Nie, 1998). Plasmonic metal nanoparticles offer additional advantages over luminescent quantum dots including significantly larger optical cross sections, complete resistance to photobleaching, and non-toxic constituent materials (Yguerabide and Yguerabide, 2001; Oldenburg et al, 1998; Sokolov et al, 2003; Loo et al, 2005; El-Sayed et al, 2005; Sokolov et al, 2003; Alivisatos, 2004).

[0005] The use of nanoparticle technology affords a flexible platform for interrogation of biological systems at the molecular level. Remaining barriers exist towards realizing a robust and generalized tool set that could potentially be used in molecular biology and healthcare settings. While issues such as biocompatibility and toxicity are of paramount importance, the ability of nanoparticle-based exogenous contrast agents to generate strong easily detectable signals which are above the endogenous background are still not realized. For example, in fluorescence imaging techniques, background autofluorescence can present a difficult barrier to overcome (Gao et al, 2004). Further, photobleaching can drastically reduce the ability to monitor longer-term molecular processes or response to therapies. High background scattering from various tissue architecture can make the isolation of molecular specific signals difficult in reflected/scattered light imaging modalities such as reflectance confocal microscopy and OCT. Therefore, the development of multi-faceted approaches will lead to improvement in the sensitivity of molecular imaging techniques.

SUMMARY OF THE INVENTION

[0006] Robust molecular imaging of cells, tumors, metastases, micro-metastases and the like typically comprises two components: a molecular-specific source of signal (typically provided through a contrast agent) and an imaging system to detect this signal. In recent years, great advances have been made in molecular imaging in small animals using MRI and optical technology to assess novel molecular contrast agents. Each of these techniques has certain advantages and constraints. While MRI provides exceptional anatomic information and depth of imaging, it suffers from limited spatial resolution. Optical imaging yields unprecedented spatial resolution (less than 1 micron) and is inexpensive, robust, and portable, but does not provide the penetration depth, field of view, or anatomic detail achievable with MRI. Thus, it is contemplated that a combination of MRI and optical techniques will address the limitations of both methods, providing an unprecedented range of imaging resolution and penetration depth.

[0007] Embodiments of the invention include approaches for molecular specific optical imaging that combines the advantages of molecularly targeted plasmonic nanoparticles and the ability to magnetically actuate a superparamagnetic particle. This combination is achieved through synthesis of hybrid nanoparticles with a superparamagnetic core surrounded by a optically interrogatable layer, e.g., metal or gold layer. The nanoparticles may be conjugated or operatively coupled with a targeting moiety, such as monoclonal antibodies, antibodies, peptides, small molecules, aptamers and the like, for molecular recognition. The hybrid nature of these particles provides for optical contrast enhancement. The addition of the gold layer provides for (1) initially strong optical signal that facilitates detection and digital processing; (2) tunable optical resonances; and (3) a convenient surface for conjugation of probe or targeting molecules or moieties. The core provides a magnetically susceptible component, e.g., iron oxide, that can be exploited to periodically actuate cells in the field of view and, therefore, allows use of an external magnetic field for modulation of the optical signal. This approach can increase sensitivity of optical imaging by orders of magnitude. In certain embodiments the methods can be used, but not limited to early cancer detection and detection of atherosclerotic plagues using optical imaging and spectroscopy.

[0008] These multicomponent, bifunctional, or hybrid nanoparticles comprise a core component and a shell component (alternatively an intermediate component located between the core and the shell, e.g., a silica layer) and an optional targeting component. The core component is typically a superparamagnetic core. Superparamagnetism occurs when the material is composed of very small crystallites (1-10 nm). Even when the temperature is below the Curie or Neel temperature (and hence the thermal energy is not sufficient to overcome the coupling forces between neighboring atoms), the thermal energy is sufficient to change the direction of magnetization of the entire crystallite. The resulting fluctuations in the direction of magnetization cause the magnetic field to average to zero. Thus the material behaves in a manner similar to paramagnetism, except that instead of each individual atom being independently influenced by an external magnetic field, the magnetic moment of the entire crystallite tends to align with the magnetic field. The energy required to change the direction of magnetization of a crystallite is called the crystalline anisotropy energy and depends both on the material properties and the crystallite size. As the crystallite size decreases, so does the crystalline anisotropy energy, resulting in a decrease in the temperature at which the material becomes superparamagnetic. The core component will be magnetically interrogatable and may comprise various metal oxides and other forms of cobalt, iron, manganese or gadolinium and the like. In certain aspects, the core component comprises an iron oxide. [0009] The shell component comprises one or more components that can be interrogated optically. Metal nanoshells are a core coating with one or more metallic (for instance, gold) layers. The shell layer is formed of a metal or metal-like material that preferably conducts electricity. Metals include gold, silver, copper, platinum, palladium, lead, iron, and the like. In certain aspects the metal is gold. Gold nanoshells possess physical properties similar to gold colloid, in particular, a strong optical absorption due to the collective electronic response of the metal to light. The optical absorption of gold colloid yields a brilliant red color which has been of considerable utility in consumer-related medical products, such as home pregnancy tests. In contrast, the optical response of gold nanoshells depends dramatically on the relative size of the nanoparticle and the thickness of the gold shell (Neeves & Birnboim, 1989; Kreibig and Vollmer, 1995). By varying the relative core and shell thicknesses, the color of gold nanoshells can be varied across a broad range of the optical spectrum that spans the visible and the near infrared spectral regions. Gold nanoshells can be made to either preferentially absorb or scatter light by varying the size of the particle relative to the wavelength of the light at their optical resonance. The shell layer and core can be linked, for example, through ionic bonds, lone -pair interactions, hydrogen bonds, or Van der Waals interaction. An exemplary linker is aminopropyltriethoxysilane.

[0010] Other materials may also be used. Organic conducting materials such as polyacetylene and doped polyanaline can be used. Additional layers, such as a nonconducting layer, a conducting layer, or a sequence of such layers, such as an alternating sequence of conducting and non-conducting layers, can be bound to the shell layer. In certain aspects, an intermediate or insulating layer may be present and is typically positioned between the core and an outer shell layer. This intermediate layer can be a polymer, an insulator, or a semiconducting material, including but not limited to silica, a dielectric material or semiconductor material, such as silicon dioxide, titanium dioxide, polymethyl methacrylate (PMMA), polystyrene, gold sulfide, and macromolecules such as dendrimers. Exemplary semiconductive materials include CdSe, CdS, and GaAs.

[0011] In certain aspects, a hybrid nanoparticle includes a targeting moiety or agent that provides for molecular recognition of biological location, such as a cell, a tissue, an organ, a biological location (e.g., tumor), or cell type of interest including a cancer cell. A targeting agent may be any molecule that has specific affinity for a molecule on the surface of the targeted cell. A variety of cell features and/or components can be utilized to selectively or specifically target a cell, such as cell surface receptors, lipids, oligosaccharides or essentially any feature or component that is identified to be present on a cell of interest and reduced or absent on non-targeted cells.

[0012] Hybrid nanoparticle composition may have hybrid nanoparticle of an average diameter of at least about, at most about, or about 10, 20, 30, 50, 60, 70, 80, 90, 100, 200, 500 or more nm, including all ranges and integers there between, as well as an associated deviation from the mean of 10, 20, 30 40, 50 nm or more.

[0013] In particular aspects of the invention hybrid nanoparticles can be used to increase the molecular-specific contrast in optical imaging of cancer cells. Cell surface receptors that are expressed or overexpressed in cancer cells and not expressed or expressed at intermediate to low levels can be targeted by the present invention. For example, epidermal growth factor receptor (EGFR) - one of the hallmarks of carcinogenesis - has been found to be over- expressed in many types of cancers including lung, breast, bladder, cervix, and oral cavity (Hanahan and Weinberg, 2000) and can be targeted by a targeting moiety that selectively or specifically bind to the EGFR. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include EGFR, carcino embryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.

[0014] A targeting agent can be coupled to a spacer. A spacer should be long enough to prevent or reduce steric hindrance between the interaction of the targeting agent and the target molecule on the surface of a cell. Carbon or other known spacers are commonly available (such as from Sigma- Aldrich). Suitable spacers typically contain between 3, 4, 5, 6 to 10, 20, 30, 50, 100 or more atoms (including all ranges and integers there between, e.g., carbon atoms. In a preferred embodiment, the spacer contains 14 carbon atoms.

[0015] Embodiments of the invention provide a platform for detection of metastatic cells and monitoring of molecular therapeutics based on molecular specific hybrid nanoparticles, small endoscopic optical spectroscopic/imaging probes, and/or MRI. Aspects of the invention provide the capabilities and methods to assess mechanisms of action of molecular therapies by enabling simultaneous imaging of whole body biodistribution of molecular therapeutics and high resolution monitoring of drug mediated molecular response. The combination of MRI and optical imaging also provides additional compositions and methods for whole body imaging of the extent of primary tumor and metastasis and optical monitoring of microscopic metastatic disease; intra-operative completeness of tumor removal; and local response to therapy based on cancer related biomarkers. The diagnostic platform can be extended to diagnostic and therapeutic approaches targeting specific cells, tumors, tissues, or organs by combining imaging methods with gold nanoshell mediated therapy.

[0016] The combination of plasmon resonance scattering inherent in gold nanoparticles with magnetic actuation may result in about a 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more fold intensity ratio between the labeled and unlabeled cells under white light illumination. This may also be seen a 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold increase over imaging with gold nanoparticles alone. The hybrid magnetic gold nanoparticles can be easily distinguished from pure gold nanoparticles using magnetic actuation. In certain embodiments, a multiplexing approach can be used that comprises combinations of magnetic and non-magnetic gold particles in molecularly labeling distinct sub-populations of cells.

[0017] In certain aspects, methods of imaging can comprise one or more of (a) labeling cells of interest via biomarkers of interest (e.g., epidermal growth factor receptor on cancer cells) with hybrid gold shell/iron oxide core nanoparticles; (b) applying an external magnetic field at a predetermined frequency; (c) the external field causes displacement of labeled cells at the same frequency; (d) detecting the oscillating labeled cells using image analysis that is sensitive to periodic movements such as Fourier image analysis techniques.

[0018] In a further aspect of the invention, optical properties (e.g., absorption) of nanoparticles can be altered or modulated when in closely spaced assemblies (Larson et al, 2007). Such methods can include, but are not limited to photothermal treatment of cancer cells comprising one or more of: (a) preparing gold shell/iron oxide core nanoparticles with absorption maximum, for example, in the green optical region (between 530 and 600 nm); (b) conjugating the nanoparticles with target cell selective or specific moiety (e.g., anti-EGFR antibodies or an antibody specific for another cancer biomarker); (c) contacting cancer cells expressing a molecule that binds with the targeting moiety (e.g., cells overexpressing EGFR or another growth factor receptor). When nanoparticles interact with closely spaced EGFR molecules on the surface of cancer cells they form closely spaced assemblies. Nanoparticles in closely spaced assemblies interact with each other through a process called "plasmon resonance coupling." Plasmon resonance coupling between nanoparticles results in a strong red shift (up to 100 nm) and broadening of their absorption spectrum. Therefore, the nanoparticles in closely spaced assemblies can absorb red and near-infrared light that individual or isolated nanoparticles do not absorb. This effect allows selective targeting of nanoparticle assemblies that are formed on the surface of cancer cells. Therefore, it increases selectivity of photothermal destruction of cancer cells. This approach is different from using nanoshells that are initially optimized for a wavelength of light that is used for photothermal treatment.

In a further aspect, a hybrid nanoparticle, for example one with an absorption maximum between 530 and 600nm, can be coupled to a targeting moiety that is specific for cancer cells. The targeting moiety can be an antibody that binds a cancer biomarker, such as a growth factor receptor, e.g., an anti-EGFR antibody.

[0019] Nanoparticles from form closely spaced assemblies after interaction with cancer cells undergo a red shift and broadening of their absorption spectrum due to plasmon resonance coupling. The closely spaced assemblies are then illuminated with red or near- infrared light (above 600 nm) for photothermal treatment of cancer cells.

[0020] In still further aspects, the methods of the invention can include calculating a thermal dose for photoablation therapy. This calculation is helpful in the planning of photoablation therapy.

[0021] In a further aspect of the invention, methods of imaging can include one or more of (a) contacting a target location with a hybrid nanoparticle coupled to a moiety that binds to a biomarker of interest present in the target location; (b) applying an external magnetic field; and (c) detecting oscillating hybrid nanoparticles using image analysis that is sensitive to periodic movements. Image analysis can include, but is not limited to Fourier image analysis. In certain aspects the biomarker is epidermal growth factor receptor, or other growth factor receptor associated with cancer.

[0022] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. The embodiments in the Example section are understood to be embodiments of the invention that are applicable to all aspects of the invention.

[0023] The terms "inhibiting," "reducing," or "prevention," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.

[0024] The term "selectively" is used to describe a particle or targeting moiety that associates or binds a target cell or cell type (cancer or pathologic cell) more frequently or more strongly than it associates or binds to other cells or tissues (a non-cancerous, non- pathologic, or "normal" cell). That is some, binding or association with non-target cells or tissue can be detected, but is distinguishable in magnitude or other quantitative or qualitative aspects.

[0025] The term "specifically" is used describe a particle or targeting moiety that associates or binds a cell or cell type and does not significantly or substantially or detectably associate or bind to other cells or tissues above background levels, e.g. in regard to an imaging agent, any biding to non-target cells or tissue does not result in a substantial increase in background.

[0026] 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."

[0027] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0028] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

[0029] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0030] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DESCRIPTION OF THE DRAWINGS

[0031] 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.

[0032] FIGs. 1A-1B (FIG. IA) shows a TEM image of magnetic gold nanoparticle solution. The scale bar is approximately 100 nm. FIG. IB shows an absorbance spectra of nanoparticles before (blue) and after (red) conjugation with antibodies.

[0033] FIGs. 2A-2E shows photothermal therapy with anti-EGFR hybrid nanoparticles. (FIG. 2A) Absorbance spectra of unlabelled cells in the presence of PEGylated hybrid nanoparticles and of labeled cells. The total amount of nanoparticles is the same in both cell suspensions. Fluorescence images of cells labeled with anti-EGFR hybrid nanoparticles (FIG. 2B) and cells pre-exposed to PEG-coated particles (FIG. 2C) after one 7 ns, 400 mJ cm^"2 laser pulse at 700 nm. Calcein AM live stain fluoresces green and indicates cellular survival. (FIG. 2D) Dark-field reflectance image of the irradiated labeled cells at the boundary of the laser beam spot; the cells that fluoresce green are located outside of the laser beam and are associated with fluorescence of the live-cell stain. (FIG. 2E) Fluorescence image of cells pre-exposed to PEG-coated hybrid nanoparticles after six hundred 7 ns, 400 mJ cm^"2 pulses at 700 nm. Fluorescence images were obtained using a 10^χ objective and a 475 nm/510 nm excitation/emission filter cube and the dark-field imaging was carried out with 20 x dark- field objective and a Xe lamp illumination. The scale bars are approximately 100 μm in fluorescence images and approximately 50 μm in the dark-field image.

[0034] FIG. 3 shows a scattering spectra of cells labeled with 50 nm hybrid nanoparticles (red line), 40nm pure gold nanoparticles (green line) and of unlabeled cells (blue line). A fluorescent tag (AlexaFluor 488, Molecular Probes) was attached to the pure gold-antibody conjugates in order to differentiate between the two types of labeled cells. Darkfield images of a 1 :1 :1 mixture of A-431 cells labeled with 40 nm anti-EGFR gold nanoparticles, 50 nm anti-EGFR gold/iron oxide nanoparticles, and unlabeled cells were acquired using: white light illumination; and a 630±15 nm bandpass filter. Images were acquired with a 2Ox darkfield/brightfield objective with a 0.5 collection NA.

[0035] FIGs. 4A-4B Monochrome images of a 1 :1 :1 mixture of A-431 cells labeled with 40nm anti-EGFR gold nanoparticles, 50 nm anti-EGFR gold/iron oxide nanoparticles, and unlabeled cells were acquired that were magnetically actuated at 0.9Hz and 1.9Hz before application of a digital frequency filter. The images were obtained using a 635/15 nm bandpass filter. Sections (FIG. 4A) and (FIG. 4B) show power spectra that are taken from the time-domain Fourier transform in the region containing a cell labeled with 50 nm gold/iron oxide particles (red), 40nm pure gold particles (green) and an unlabeled cell (blue). Note the prominent peaks in the magnetically-labeled cells' frequency spectra that correspond to the translation stage oscillation frequencies of 0.9Hz and 1.9Hz. Other images show the same fields after digital filtering at 0.9Hz and 1.9Hz using the Hanning function implementation. Only magnetically labeled cells are visible in such images.

[0036] FIGs. 5A-5C show (FIG. 5A), pixel intensity profiles are shown for the three cell types: 50nm gold/iron oxide labeled (red line), 40nm pure gold labeled (green line), and unlabeled (blue line). Profiles are drawn for the same three cells captured using white light illumination, 635/15nm bandpass illumination, as well as bandpass plus magnetic actuation and digital frequency filtering. In (FIG. 5B) and (FIG. 5C), the relative average pixel intensity from n>10 cells in each cell population and illumination/acquisition condition is compared. Asterisks and brackets in (FIG. 5B) and (FIG. 5C) indicate a statistical significant difference of the average signal values with p<10^"4.

[0037] FIG. 6 shows a scheme for synthesis of hybrid nanoparticles with an intermediate layer of silica.

[0038] FIG. 7 shows a FTIR spectrum of PEG-coated hybrid nanoparticles.

[0039] FIGs. 8A-8B (FIG. 8A) shows a real-time absorption spectra of SPIO-Au nanoparticles at different times after the addition of K-gold solution and formaldehyde into SPIO-silica nanoparticles with gold nanoseeds. (FIG. 8B) shows absorbance of SPIO-Au nanoparticles measured at 800 nm as a function of reaction time after the addition of K-gold solution and formaldehyde.

[0040] FIG. 9 shows x-ray diffraction (XRD) of SiO₂ (silica) coated γ-Fe₂O₃ NPs.

[0041] FIG. 10 shows the field cooled (FC) and zero-field-cooled (ZFC) magnetization M(T) curves for the uncoated and Au/SiO₂ doubly coated Y-Fe₂O₃ NPs.

[0042] FIG. 11 shows the magnetic hysterysis M(H) loops of both the uncoated and coated γ-Fe₂O₃ NPs, measured up to 50 kOe and at different temperatures from 5 K to 300 K. [0043] FIG. 12 shows the hysteresis loops of FIG. 8 in the zoomed region between H = - 2 k Oe and 2 kOe to see more clearly the irreversibility in this region

[0044] FIG. 13 shows the Tl/2-dependence of HC is slightly deviated from linearity, such deviation from linearity has been previously observed for SiO₂-coated γ-Fe₂θ3 NPs.

[0045] FIG. 14 shows a representative T2* MRI images and the MR temperature map of a subcutaneous A431 tumor injected with SPIO@AuNS in the tumor at a dose of 1 x 10¹¹ particles/mL. (FIG. 14A) before NS injection. (FIG. 14B) after NS injection. (FIG. 14C) Temperature map acquired after 3 min exposure to near-infrared laser light at 808 nm and output energy of 4W/cm2. (FIG. 14D) Temperature elevation ΔT vs. time curve in selected area (square). These data shows that SPIO@AuNS can be visualized with T2* MRI both in tumor xenografts. Treatment with near-infrared laser induced significant temperature elevation and tumor necrosis.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The increasing availability of nanostructures with highly controlled optical properties in the nanometer size range has created widespread interest in their use in biological systems for diagnostic and therapeutic applications (Wickline and Lanza, 2003). Nanoparticles derived from gold is an attractive nanoparticulate system for optical imaging owing to their ease of preparation, ready bioconjugation, good biocompatibility, and unique optical properties (Sokolov et al, 2003; El-Sayed et al, 2005; Loo et al, 2004). In particular, gold nanoshells (NSs) exhibit strong absorbance with wavelength tunable in the near-infrared (NIR) region of the electromagnetic spectrum. These particles not only can be used in biomedical imaging applications, but more importantly, they are potential candidates for localized hyperthermia (photothermal ablation) therapy because they mediate strong plasmon-induced surface heat flux upon absorption of NIR light (Loo et al, 2004; Hirsch et al, 2003).

[0047] Another type of inorganic nanoparticles that has showed great promise in magnetic resonance imaging (MRI) is super-paramagnetic iron oxide nanopaticles (SPIO) (Bulte and Kraitchman, 2004). SPIO have a high transverse relaxivity r₂, which result in negative contrast in T₂-weighted images. To date, SPIO-enhanced MRI has become an established technique for imaging of macrophage activity, in particular for tumor staging of the liver and detection of lymph node metastasis, with several products either approved or in clinical trials (Bulte and Kraitchman, 2004; Tanimoto and Kuribayashi, 2006). SPIO nanoparticles are being investigated for imaging atherosclerotic lesions (Schmitz et al, 2000) and monitoring the in vivo distribution of cellular trafficking (Frank et al, 2004). In addition, magnetic nanoparticles are studied as a carrier for enhanced delivery of therapeutic agents in the presence of external magnetic field (Mondalek et al, 2006; Jain et al, 2005; Morishita et al, 2005). In these systems, therapeutics {e.g., drugs or genes) are attached to the magnetic particles and injected near the target site. A magnetic field is then applied to the site externally in order to concentrate the particles at the target site.

[0048] By combining the attractive photo-thermal property of gold NSs and magnetic property of SPIO, the resulting multifunctional nanoparticles can be used in photothermal therapy with targeting potential mediated through external magnetic field and imaging capability with MRI.

[0049] Successful integration of magnetic resonance and optical modalities to image the same biological structures requires development of bi-functional contrast agents that provide bright signal in both MRI and optical imaging. This application describes the development of multimodal contrast agents for molecular specific imaging (imaging based on the selective or specific association of a contrast agent with a molecular component of a target), as well as methods using such a particle with MRI and/or optical imaging. These contrast agents are based on a combination of an optical component {e.g., gold nanocrystals) with a magnetic component {e.g., iron oxide particles) and optionally a cancer specific probe or targeting molecules or moieties {e.g., monoclonal antibodies and aptamers). For example, agents containing iron oxide provide contrast enhancement in MRI; gold nanoparticles resonantly scatter visible and near-infrared light providing bright optical signals; and monoclonal antibodies and aptamers provide specific targeting of the contrast agents to cancer related biomarkers. Such bifunctional MRI/optical molecular contrast agents, imaging systems, and protocols can be used to visualize pathological conditions in vivo. These agents, systems, and protocols can be optimized to facilitate minimally invasive, accurate, and in real-time identification of microscopic metastases.

I. HYBRID NANOPARTICLES

[0050] Bifunctional molecular contrast agents for MRI and optical imaging include, but are not limited to nanoparticles composed of a paramagnetic core, an optional thin insulator shell or intermediate layer (e.g., silica), a gold layer, and optional molecular targeting moieties attached to the gold surface and other related contrast agents.

A. Synthesis of Hybrid Nanoparticles

[0051] Typically, the synthesis of molecular specific paramagnetic gold nanoparticles comprise one or more of the following steps: (1) synthesis of the paramagnetic core; (2) deposition of the silica layer; (3) formation of the gold shell; and (4) conjugation of the nanoparticles with monoclonal antibodies for molecular specific targeting. Magnetically susceptible plasmonic nanoparticles (hybrid nanoparticles) can be synthesized using methods such as those described in Lyon et al. (2004).

[0052] Synthesis of the Paramagnetic Core. Particles can be formed via co-reduction of the core components, e.g., FeCl₂ and FeCl₃, in an aqueous NaOH solution. The cores, e.g., Fe₃O₄, can then be oxidized (Fe₃O₄ primarily oxidized to Fe₂O₃) by boiling in an acidic solution. The paramagnetic core particles can vary in composition, including, but not limited to Fe, Fe₂O₃, FePt, CoPt, Co and Ni. The general approach is to thermally decompose (often with the aid of a reducing agent like a polyalcohol) an organometallic precursor to a metal in the presence of organic molecules that bind to the surface of the particles and stabilize their size and prevent aggregation. Typically, this process is performed in an inert atmosphere on a Schlenk line. Protocols are available for the synthesis of sterically-stabilized nanocrystals with narrow size distributions.

[0053] Deposition of the Silica Layer. Once sterically-stabilized nanocrystals are synthesized, a SiO₂ shell can be grown or deposited on the surface. The protocols for silica deposition on the surface of gold, silver, and iron oxide nanoparticles have been previously described (Liz-Marzan et al., 1996; Gerion et al., 2001; Sokolov et al., 1998). The procedure involves the exchange of the capping ligands with a siloxane molecule that can serve as the seed for the SiO₂ layer growth. Once this chemical shell is made on the nanocrystals, a Stober process can be used to grow the shell to the desired thickness.

[0054] In certain aspects, an excess of the mercaptopropyltriethoxysilane (MPS) is added to the sterically-stabilized magnetic nanocrystals in methanol and the suspension is adjusted to pH 9 using tetramethylammonium hydroxide (THMA). The mixture is allowed to interact for approximately 45-60 minutes. During this reaction the capping ligands on the surface of the magnetic nanocrystals will be replaced with MPS molecules that will provide siloxane molecules for the deposition of silica layer. Then, the suspension is dialyzed in a 10,000 (molecular weight cut off) MWCO dialyzing tubing against isopropyl alcohol to remove the unreacted MPS molecules. Subsequently, the suspension can be diluted to a desired volume using about 1% THMA and about 1% H₂O in isopropyl alcohol and is heated to 40⁰C. Then, fresh tetraethylortosilicate (TEOS) can be added and the reaction carried out to completion (e.g., overnight). The thickness of the silica layer will be controlled by the amount of the TEOS added.

[0055] Formation of the Gold Shell. Subsequent to core synthesis and/or intermediate layer deposition, about a 20 nm thick plasmonic shell, e.g. , gold shell, can then be deposited, in some aspects using a hydroxylamine seeding method (Brown and Natan, 1998). For example, the deposition of a gold shell involves sequential additions of HAuCl₃ in the presence of citrate and hydroxylamine. Hydroxylamine confines the reduction OfAu³⁺ ions to the pre-existing surface of iron oxide particles, thereby largely preventing the nucleation of pure gold particles in solution. Numerical codes will be developed and used to predict the optical properties of the dielectric core/gold shell nanoparticles and to design nanoparticles with different optical properties; this can be achieved by changing the ratio of the dielectric core and the gold shell. Particles of different colors can be used and conjugated to different probe molecules for imaging of multiple targets.

[0056] Conjugation with Monoclonal Antibodies and Aptamers. There is a variety of possible strategies for preparation of conjugates of gold particles with molecule, cell, tissue, or organ specific probe molecules (e.g., cancer specific probes). Well characterized conjugation protocols have been developed to prepare gold immunostains for electron microscopy (Horisberger, 1981; Geoghegan and Ackerman, 1977). Also a variety of thiol terminated heterogeneous cross-linkers can be used to covalently attach monoclonal antibodies to a gold surface. This approach is preferable for in vivo applications because it allows co-adsorption of other thiol terminated molecules such as polyethylene glycol (PEG) which is important to increase the circulation time of the contrast agents in the body after systemic delivery.

[0057] Smaller aptamer molecules can not be directly adsorbed on the gold surface because that could significantly change their conformation and lead to loss of binding properties. Thus, in some aspects, aptamers with thiol terminated alkyl chains can be directly attached to the surface of gold particles similar to the procedures described in Elghanian et al, (1997). [0058] For vital imaging with contrast agents based on metal nanoparticles bioconjugates will be developed that have very low nonspecific binding and are not accumulated by the reticuloendothelial system (RES), namely the liver and spleen. Hybrid conjugates are prepared by co-adsorbing PEG and probe (antibodies, peptides) molecules on the surface of nanoparticles. This strategy has been recently demonstrated in studies involving in vivo molecular specific imaging of embryogenesis using quantum dots (Dubertret et al. , 2002) and in colloidal gold drug delivery system in live mice (Paciotti et al., 2001). In certain aspects, a commercially available thiol terminated PEG from Nektar Inc., AL can be used.

[0059] In an alternative synthesis scheme, silica coated SPIO (Fe₂Os) nanoparticles are synthesized based on the well-known Stόber process (Stόber et al., 1968). A commercially available SPIO (5 - 15 nm) nanoparticles stabilized with oleic acid in water can also be used as the core component. The magnetic nanoparticles are easily coated with amorphous silica via the sol-gel process because the iron oxide surface has a strong affinity toward silica. No primer is typically needed to promote the deposition and adhesion of silica. The thickness of the silica sphere could be tuned from about 2 to about 100 nm by simply changing the concentration of the sol-gel precursor, tetraethylorthosilicate (TEOS).

[0060] Next, the surface of the silica shell is functionalized with amine groups by treating with NH₄OH and APTMS. To form the gold- she lied nanoparticles, gold nanocrystal seeds (approximately 2 to 3 nm) were first attached onto the amino groups on silica sphere by reduction of chloroauric acid (HAuCl₄) with tetrakishydroxymethylphosphonium (THPC) (Duff et al., 1993). Since the THPC gold nanocrystal have net negative surface charges, they could firmly attach to the amino group terminated silica surface which is positively charged in acidic pH values. Finally, the attached gold nanocrystals are used to nucleate the growth of the gold overlayer on the silica surface to form uniform gold nanoshell. The NSs may be coated with polyethylene glycol because of its known high biocompatibility. Whereas bare NSs without a coating generally form aggregates in water, PEG coated NSs typically result in stable monodisperse nanoparticles.

[0061] In this synthesis scheme, the silica layer serves the same role as the silica core in conventional gold NSs in providing a dielectric interface for shifting the plasma resonance to NIR wavelength region. In addition, the outer surface of the silica layer functionalized with free amine groups facilitates the initial growth of the gold nanocrystals which facilitates subsequent growth of the outermost gold shell. [0062] In certain embodiments, the core component of the hybrid nanoparticle can comprise, for example, yttrium-iron garnet YsFeSO₁₂ and γ-Fe₂θ3. Different dopants can be added to the material forming the core component.

B. Characterization of the hybrid particles

[0063] Typically, the properties of the nanoparticles will be characterized using standard UV-Vis and NIR spectroscopy and by polarized reflectance spectroscopy (Sokolov et ah, 1999). Sizes are typically determined using transmittance electron microscopy (TEM).

[0064] The process of plasmonic shell deposition may be monitored using a spectrophotometer. Before the addition of the gold shell, the extinction properties of the superparamagnetic particles are consistent with sub-wavelength sized dielectric spheres. However, upon addition of the plasmonic layer onto the core, the extinction spectrum changes markedly, displaying a characteristic plasmon resonance peak.

[0065] The core and hybrid iron oxide/gold nanoparticles can be characterized using a transmission electron microscope (TEM) equipped with a digital camera detector. The average diameter of the hybrid nanoparticles can be at least about, at most about or about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 nm with a standard deviation of 5, 10, 15, 20 nm or so.

[0066] The signal strength and binding efficiency of these bifunctional contrast agents can be assessed in a variety of ways, including assessment in cell suspensions and three- dimensional tissue cultures, for example. The excitation wavelengths used in optical imaging and MRI detection schemes will be optimized to increase contrast between cells labeled with the specifically targeted contrast agents and cells incubated with non-specific contrast agents.

[0067] In certain aspects, bifunctional contrast agents can be assessed and validated for, but not limited to delivery of contrast agents in vivo; for combined MRI and optical imaging; and for determination of the range of concentrations that can be used to identify micro- metastasis in vivo.

C. Targeting Moiety Conjugation

[0068] Hybrid nanoparticles can be conjugated to a variety of targeting moieties, such as small molecules, peptides, antibodies (including monoclonal and recombinant antibodies and antibody fragments), nucleic acids, and aptamers. Targeting moieties can be attached or coupled covalently or non-covalently, and can be attached or coupled via a conjugation linker. Some linkers include polyethylene glycol (PEG) chains. In certain aspects, PEG chains may be terminated at one end by a hydrazide moiety and/or at the other end by two thiol groups. In the case of antibodies and other amino acid containing moieties, the protein can be exposed to NaIO₄, thereby oxidizing the hydroxyl moieties on the polypeptide to aldehyde groups. The formation of the aldehyde groups can be colorimetrically confirmed using a standard assay with an alkaline Purpald® solution (Sigma). Excess linker can be added to the oxidized polypeptide. The linker interacts with aldehyde groups on the polypeptide to form a stable linkage. Unreacted linker can then be removed by filtration. After purification, the modified polypeptides can be mixed with gold nanoparticles in a buffer. During this step a stable bond is formed between the surface and a linker thiol group. Monofunctional PEG-thiol molecules can be added to passivate the remaining nanoparticle surface. The conjugates can be centrifuged and resuspended in an appropriate buffer.

[0069] Humanized antibodies, where a mouse antibody-binding site is transferred to a human antibody gene, are much less immunogenic in humans and many humanized antibodies are currently in clinical trials. Since 1997, the FDA has approved more than 10 monoclonal antibody based drugs, including Herceptin for metastatic breast cancer therapy.

[0070] In certain aspects, contrast agents for two hallmarks of cancer associated with metastatic tumors are contemplated, (1) uncoupling of a cell's growth program from environmental signals - such as epidermal growth factor receptor (EGFR) and (2) sustained angiogenesis - vasculature targets, such as VEGF receptor). For example, a contrast agent specific or selective for phosphorylated and dephosphorylated epidermal growth factor receptor (EGFR) are contemplated, as well as contrast agents for vasculature targets, such as VEGF receptor. In addition, contrast agents are contemplated that assess phospatidyl serine (PS) levels to monitor apoptotic response of cancer cells to molecular therapeutics.

[0071] A variety of cell specific or cell selective agents are contemplated as targeting agents to image specific cells. These targeting agents include, but are not limited to monoclonal antibodies and/or aptamers. Contrast agents with different sources of MRI contrast will allow simultaneous imaging of 1, 2, 3, 4, or more molecular targets. Additionally, the tunability of optical resonances of gold nanoparticles can be used to create nanoparticles for simultaneous detection of multiple targets using optical imaging.

II. IMAGING USING HYBRID NANOPARTICLES

[0072] Embodiments of the present invention include a novel approach to in situ imaging, diagnostics, and therapeutics. In certain embodiments the hybrid nanoparticle can be used in the detection of metastatic cells and imaging of molecular therapy which combines the high resolution detection of phenotypic characteristics by MRI, with molecular detection capacity of optical imaging. For example, these methods can be verified using non-small cell lung cancer (NSCLC) and pulmonary metastases from non-NSCLC tumors as a model system.

A. MRI Imaging

[0073] Magnetic resonance imaging (MRI) is a non-invasive method for generating high- resolution images of biological samples with excellent intrinsic soft tissue contrast. High resolution images provide information regarding tumor extent, while functional magnetic resonance imaging techniques can be used to quantify tissue characteristics, or in surgical planning, to identify nearby critical structures that must be avoided during surgical treatment. MRI is also routinely applied in a post-treatment setting in order to gauge success or monitor for recurrence. In addition to the demonstrated necessity of MRI in staging and postoperative assessment, there are a number of prospective uses under consideration. A primary objective is the ability to measure anatomic, functional, or macroscopic characteristics of tumor tissue in hope of finding early indications of response to therapy (Hoskin et al, 1999; Baba et al, 1997). Recent studies have also shown that sufficient reason exists to employ MRI in a more proactive manner, as a screening tool for high-risk populations (Warner et al, 2001).

[0074] Magnetic resonance imaging (MRI) provides detailed images of the body in any plane. MRI has much greater soft tissue contrast than CT making it especially useful in neurological, musculoskeletal, cardiovascular and oncolological diseases. Unlike CT it uses no ionizing radiation. The scanner creates a powerful magnetic field which aligns the magnetization of hydrogen atoms in the body. Radio waves are used to alter the alignment of this magnetization. This causes the hydrogen atoms to emit a weak radio signal which is amplified by the scanner. This signal can be manipulated by additional magnetic fields to build up enough information to reconstruct an image of the body.

[0075] Magnetic gradients are generated by three orthogonal coils, oriented in the x, y and z directions of the scanner. These are usually resistive electromagnets powered by sophisticated amplifiers which permit rapid and precise adjustments to their field strength and direction. Typical gradient systems are capable of producing gradients from 20 mT/m to 100 mT/m. It is the magnetic gradients that determine the plane of imaging - because the orthogonal gradients can be combined freely, any plane can be selected for imaging. [0076] In order to understand MRI contrast, it is important to understand the time constants involved in relaxation processes that establish equilibrium following RF excitation. As the high-energy nuclei relax and realign they emit energy at rates which are recorded to provide information about their location. The realignment of nuclear spins with the magnetic field is termed longitudinal relaxation and the time required for a certain percentage of the tissue's nuclei to realign is termed "Time 1" or Tl, which is typically about 1 second. T2- weighted imaging relies upon local dephasing of spins following the application of the transverse energy pulse; the transverse relaxation time is termed "Time 2" or T2, typically < 100 ms for tissue. A subtle but important variant of the T2 technique is called T2* imaging. T2 imaging employs a spin echo technique, in which spins are refocused to compensate for local magnetic field inhomogeneities. T2* imaging is performed without refocusing. This sacrifices some image integrity (resolution) but provides additional sensitivity to relaxation processes that cause incoherence of transverse magnetization. Applications of T2* imaging include functional MRI (fMRI) or evaluation of baseline vascular perfusion (e.g., cerebral blood flow (CBF)) and cerebral blood volume (CBV) using injected agents. Because T2*- weighted sequences are sensitive to magnetic inhomogeneity (as can be caused by deposition of iron-containing blood-degradation products), such sequences are utilized to detect subtle areas of recent or chronic intra cranial hemorrhage ("Heme sequence").

[0077] Image contrast is created by using a selection of image acquisition parameters that weights signal by Tl, T2 or T2*, or no relaxation time ("proton-density images"). In the brain, Tl -weighting causes the nerve connections of white matter to appear white, and the congregations of neurons of gray matter to appear gray, while cerebrospinal fluid appears dark. The contrast of "white matter," "gray matter'" and "cerebrospinal fluid" is reversed using T2 or T2* imaging, whereas proton- weighted imaging provides little contrast in normal subjects. Additionally, functional information (CBF, CBV, blood oxygenation) can be encoded within Tl, T2, or T2*.

[0078] Both Tl -weighted and T2-weighted images are acquired for most medical examinations. However they do not always adequately show the anatomy or pathology. The first option is to use a more sophisticated image acquisition technique. The other is to administer a contrast agent (e.g., hybrid nanoparticles) to delineate areas of interest.

[0079] A contrast agent may be as simple as water, taken orally, for imaging the stomach and small bowel although substances with specific magnetic properties may be used. Most commonly, a paramagnetic contrast agent (usually a gadolinium compound) is given. Gadolinium-enhanced tissues and fluids appear extremely bright on Tl -weighted images. This provides high sensitivity for detection of vascular tissues (e.g., tumors) and permits assessment of brain perfusion (e.g., in stroke).

[0080] More recently, superparamagnetic contrast agents (e.g., iron oxide nanoparticles) have become available. These agents appear very dark on T2* -weighted images and may be used for liver imaging - normal liver tissue retains the agent, but abnormal areas (e.g. scars, tumors) do not. They can also be taken orally, to improve visualization of the gastrointestinal tract, and to prevent water in the gastrointestinal tract from obscuring other organs.

[0081] MRI technologies are in development for small animal imaging using a 4.7T Bruker Biospec experimental MRI system. Up to now, well over 2200 imaging experiments have been carried out on the 4.7T MRI in the Small Animal Cancer Imaging Facility, a core institutional resource providing imaging instrumentation, expertise, and support to researchers in the medical community. Studies conducted using this system range from simple anatomic characterizations, to evaluation of the functional or physiological state of tissue. Here, fast MR imaging techniques are employed that take advantage of the signal attenuation of normal lung tissue in order to enhance the visibility of solid tumor tissue. As shown in the figure, the tumor boundaries are much more easily identified in MR images than CT, particularly when tumor is adjacent to pleura or atelactesis.

[0082] The diagnostic and therapeutic application of related nanotechnologies in oncology have been studied. Physiologically relevant concentrations of gold nanoparticles alone have been shown to provide no artifact or contrast in MR images (Hirsch et al, 2003). In addition, systemic injection of Feridex, a commercially available iron-oxide contrast agent, is distributed to and remains clearly visible in T2* -weighted images of lung tissue one hour after administration.

B. Optical Imaging

[0083] The contrast agents described can be interrogated using known optical imaging methodologies. Optical technologies offer the ability to image tissue with unprecedented spatial and temporal resolution using low cost, portable devices; thus, they represent an ideal approach to image early neoplasia. Multiple in vivo optical modalities, including multi- spectral fluorescence imaging (Lam et al, 1998; Lam et al, 1993), multi-spectral reflectance imaging with unpolarized (Pogue et al, 1998) and polarized (Jacques et al, 2000) light, confocal microscopy (Smithpeter et al, 1998) and reflectance (Perelman et al, 1998; Bigio et ah, 1994; Bigio et ah, 1995; Sokolov et ah, 1999) and fluorescence (Lam et ah, 1993; Alfano et ah, 1987; Cothren et ah, 1990; Cothren et ah, 1995; Svanberg et a 1995; Vo-Dinh et ah, 1995) spectroscopy, have recently been explored as diagnostic tools in medicine. While optical spectroscopy provides information about both tissue biochemical and morphological structure from a point interrogated using a fiber optic probe, the number of natural chromophores that can be probed is somewhat limited. Development of molecular contrast agents like those described herein, has the potential to dramatically expand the potential of in vivo diagnosis.

[0084] Because of their very strong scattering characteristics, contrast agents based on gold nanoparticles can be measured using reflectance based optical imaging and spectroscopy. Here, using both imaging and spectroscopy for screening of metastatic cells is contemplated, particularly in the lung. Reflectance spectroscopic evaluation of a whole lymph node can be obtained using a very small (< 1 mm) diameter optical probe. The wavelength of illumination can be selected to maximize backscattering from the metastatic cells labeled with the contrast agent and to minimize scattering from unlabeled cells and unbound contrast agents.

[0085] Alternatively, reflectance based imaging can be used to map out the three- dimensional distribution of contrast agents within the node with high spatial resolution. Tools for in vivo confocal imaging have been developed that can non-invasively image epithelial cells using reflected light. In concept, confocal imaging is similar to histologic analysis of biopsies, except that 3D resolution is achieved without removing tissue. Confocal images can localize reflected light in opaque tissues like epithelium or lymph nodes with enough resolution to image individual cells and intra-cellular structure. However, in epithelial cells, native contrast is limited. The addition of the contrast agents described in this application has the potential to dramatically increase signal strength and provide molecular specific as well as microanatomic information.

[0086] Optical imaging includes a variety of techniques that use visible or infrared light, such as confocal microscopy. Confocal microscopy is an optical imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional images by eliminating out-of-focus light or flare in specimens that are thicker than the focal plane. In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded in light from a light source. Due to the conservation of light intensity transportation, all parts of specimen throughout the optical path will be excited and the fluorescence detected by a photodetector or a camera. In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information. Only the light within the focal plane can be detected, so the image quality is much better than that of wide-field images. As only one point is illuminated at a time in confocal microscopy, 2D or 3D imaging requires scanning over a regular raster (i.e., a rectangular pattern of parallel scanning lines) in the specimen. The thickness of the focal plane is defined mostly by the square of the numerical aperture of the objective lens, and also by the optical properties of the specimen and the ambient index of refraction.

[0087] A real-time reflectance based confocal microscope has been developed to assess cell morphology and tissue architecture in vivo. In epithelial tissue, 1 μm resolution has been achieved with a 200-400 μm field of view and penetration depth up to 500 μm (Collier et al, 2002; Collier et al, 2000; Drezek et al, 2000; Gonzalez et al, 1999; Gonzalez et al, 1999; Rajadhyaksha et al, 1995; Rajadhyaksha et al, 2001; Rajadhyaksha et al, 2001; Selkin et al, 2001; White et al, 1999; Delaney and Harris, 1995).

[0088] Typically, fiber optic confocal microscopes are needed to obtain images clinically. Miniature confocal microscopes (Minsky, 1961) allow confocal imaging in vivo. These instruments are designed to be inserted through a speculum, catheter, large-bore needle, or biopsy channel of an endoscope.

[0089] Dr. Richards-Kortum, in collaboration with Dr. Descour from The University of Arizona, has been developing designs for a miniature injection-molded, plastic microscope objective. The objective follows previous development of miniature microscope optics (Liang et al, 2002). Injection-molded lenses can be made rapidly and in large quantities at moderate cost. The injection-molded objective consists of five lenses, has a numerical aperture of NA = 1 (water immersion), and a magnification of m = -3.3 (3.3X / 1.0 NA). Injection-molded, plastic microscope objectives are a realistic, near-term, low-cost, and disposable option for confocal microendoscope devices. Endoscopic miniature optical probes can be optimized to detect the contrast agents described in this application using reflectance imaging and spectroscopy.

C. Combination of MRI/Optical imaging.

[0090] Recently, it has been recognized that combination of MRI and optical imaging can lead to development of new approaches that will bridge gaps in resolution and depth of imaging between these two modalities (Huber et al, 1998; McDonald and Choyke, 2003) and at the same time will provide complimentary anatomic, functional, and molecular information (Josephson et al, 2002; Ntziachristos et al, 2002). Combination of MRI with near-infrared (NIR) diffuse optical spectroscopy (DOS) was evaluated in tumor animal models (Merritt et al, 2003; Gulsen et al, 2002) and in detection of breast cancer in human pilot clinical studies (Ntziachristos et al, 2002, Ntziachristos et al, 2000). In these experiments, MRI was used to obtain precise anatomic information on location of tissue structures that were probed optically. DOS provided quantitative measurements of oxy- and deoxyhemoglobin content in tissue. MRI and DOS co-registration measurements yielded functional information about tissue blood supply in precise anatomic context of examined biological structures.

[0091] The potential benefits of combined MRI and optical imaging stimulated the development of hybrid magnetic-fluorescent probes (Josephson et al, 2002; Huber et al, 1998). In Huber et al (1998), polymers possessing metal chelators with bound gadolinium (Gd) and covalently attached fluorescent dyes were synthesized. The complexes were injected into a single cell to follow development of Xenopus laevis embryos. It was shown that confocal optical microscopy can be used to image superficial labeled cells while MRI can provide information about cells deeper in the embryo. Hybrid contrast agents that can be activated by a reducing environment or by trypsin were developed using a combination of superparamagnetic iron oxide nanoparticles and NIR fluorescent dye Cy5.5 -peptide conjugates (Josephson et al, 2002). Initially, the fluorescence of these probes was quenched due to dye-dye and dye -iron oxide interactions. Exposure of the contrast agents to a reducing agent or trypsin led to detachment of the fluorophores from the complex and activation of fluorescence. The authors suggested that these contrast agents can be used to probe the microenvironment in a living organism using optical imaging with spatial localization using MRI.

[0092] Here, embodiments of the invention extend and add to these research activities by developing molecular targeted nanoparticles with bright MR and optical signals and applying these agents to the important clinical problems, such as detecting metastatic lung cancer.

D. Diagnosis of Cancer

[0093] Cancer is a major public health problem. Worldwide, more than 6 million people die from cancer each year and more than 10 million new cases are detected. In developed countries, cancer is the second leading cause of death (World Health Organization, Cancer Control. 1996). Over 80% of all malignancies are derived from epithelial tissue, including breast, prostate, colon, lung, and head and neck carcinoma. Over 170,000 cases of lung cancer alone are diagnosed annually in the United States, and about 160,000 patients die from lung cancer each year. Accurate and sensitive methods to stage disease are needed to optimize therapy for these patients. Thus, new technologies for less invasive, quick and accurate identification of metastases will significantly improve public health and can reduce medical costs.

[0094] Patients diagnosed with cancer are staged, or classified, according to the anatomic extent of their tumor. Staging is used to select therapy, to estimate prognosis and to facilitate communication to other clinicians and scientists. Staging in patients with solid tumors consists of determining: (1) the anatomic extension of the primary tumor (T), (2) the presence and location of metastases to regional lymph nodes (N), and (3) the presence and location of metastases to distant organs (M) (Zuluaga et al., 1998). Current methods to detect and diagnose regional and distant metastases lack sufficient sensitivity and specificity to optimize therapy. Many patients with undetected micrometastases are surely being undertreated, whereas other patients who fall into "high risk" groups are given aggressive systemic therapy without ever confirming whether or not their tumor has spread.

[0095] The presence of regional metastases is a major determinant of survival for patients with early stage epithelial tumors (Zuluaga et al., 1998; Grandi, et al., 1985; Fisher et al., 1983; Balch, et al., 1992). In many types of neoplasms, including lung, colon, breast, and head and neck squamous cell carcinoma (HNSCC) and melanoma, the identification of cancer cells within regional lymph nodes changes the accepted therapeutic approach. Breast cancer patients with axillary nodal metastases are treated initially with systemic chemotherapy; those without regional disease usually have surgery with or without radiation. Melanoma patients with regional metastases are enrolled in clinical trials using vaccines, chemotherapy, or immunotherapy, or take interferon for a year. Non-small cell lung cancer patients with mediastinal nodal metastases may not be offered surgery, and instead are treated with radiation and chemotherapy. Thus, determination of whether or not an individual patient has developed regional metastases is a crucial component required for clinicians to select the most appropriate therapy.

[0096] Current methods to detect regional metastases in most cancers are insufficient. Physical examination using manual palpation of enlarged nodes, and radiographic imaging (CT, MRI, ultrasound or PET) are used to determine whether or not a patient's cancer has spread to regional lymph nodes. However, these diagnostic methods suffer from low sensitivity and specificity (Yoon et al, 2003; Van der Hoeven et al, 2002; Hannah et al, 2002; MacDonald and Hansel, 2003; Stuckensen et al, 2000; Hoffman et al, 2000; Weng et al, 2000; Atula et al, 1997). For example, in NSCLC, determination of whether or not a patient has metastatic spread to mediastinal LNs is a critical branch point in the treatment algorithm; patients with N2 disease (ipsilateral mediastinal LN metastases) are often recommended for neoadjuvant chemotherapy, whereas those with N3 disease (contralateral mediastinal LN metastases) are deemed unresectable. However, roughly 23% of NSCLC patients are overstaged and 19% are understaged using conventional CT scans (Lewis et al 1990). Mediastinoscopy is considered the gold standard for mediastinal staging of NSCLC with sensitivities from 70 - 92%, but this also suffers from several limitations. These include need for general anesthesia, complication rates of 2.5 - 9%, and inadequate visualization of all LN levels. Other endoscopic procedures including extended cervical mediastinoscopy, anterior mediastinotomy, and video-assisted thoracotmy are being evaluated to improve staging of regional metastases in NSCLC patients.

[0097] CT and MRI use lymph node size as a major criterion for diagnosing metastases. This lowers specificity by misclassifying enlarged benign inflammatory nodes, and limits detection thresholds for most anatomic sites to nodes greater than 1 cm. PET scans, while more sensitive than many other radiographic modalities, also suffer from lack of sensitivity to smaller metastatic nodal deposits. A quantitative assessment of detection limits of PET for detecting metastatic melanoma found that the sensitivity of PET is dependent on the volume of disease, and can only reliably detect metastatic foci > 80 mm³ (Wagner et al, 2001). A prospective study of FDG PET in breast cancer patients found that the overall sensitivity of PET was 25%, and the specificity was 97%. Importantly, PET failed to detect all positive sentinel node specimens, indicating that the sensitivity of PET for detecting microscopic axillary nodal metastases is quite low (Van der Hoeven et al, 2002).

[0098] Movement toward a molecular characterization of cancer would have important clinical benefits, including (1) detecting cancer earlier based on molecular characterization, (2) predicting the risk of precancerous lesion progression, (3) detecting margins in the operating room in real time, (4) selecting molecular therapy rationally, and (5) monitoring response to therapy in real time at a molecular level. While cancer markers can be visualized in vitro using complex protocols, there is an important need to image the molecular features of cancer in vivo. This requires molecular-specific contrast agents that can be safely be used in vivo, as well as systems to rapidly and non-invasively image these agents. [0099] This application describes the development of molecular specific bi-functional contrast agents that can be interrogated using MRI and optical techniques, to address a clinically important problem of detection and molecular assessment of metastatic disease. Contrast agents include, but is not limited nanoparticles composed of a paramagnetic core, an insulating shell (e.g. , silica shell), a gold layer, and molecular targeting moieties attached to the gold surface.

[00100] One of skill in the art will appreciate the various applications that can utilize the contrast agents described in this application. In one aspect, contrast agents described herein are used in the diagnosis of cancer.

[00101] Mortality from epithelial tumors is largely due to dissemination of cancer cells. Micrometastases or occult metastases are usually defined as deposits of cancer cells less than 2 mm in size. Detection of micrometastases has important clinical implications for prognosis and therapy planning, yet thresholds for detection of metastatic disease by conventional imaging modalities are only 5-10 mm in size at best. Furthermore, conventional radiographic techniques, such as CT, MRI, and ultrasound, while providing excellent anatomic information, rely on non-specific phenotypic characteristics of tissue, such as size and vascularity, to distinguish metastastases from normal tissue, and are unable to provide information about the molecular makeup of the tissue. For example, if a patient with lung or head and neck cancer is noted by CT scan to have an enlarged node in the mediastinum, an invasive procedure is required to remove tissue from this node (such as an ultrasound or CT- guided needle biopsy, or mediastinoscopy directed biopsy) for further analysis. The specimen will be reviewed using standard light microscopy for presence of cancer cells. Traditionally, these lymph nodes were bisected, and each half was stained and visually scanned for the presence of metastatic cells. Recently, reports have documented that serial sectioning of the tissue, and immunohistochemical staining of specific molecules, can greatly increase the sensitivity for detecting occult metastases (Vollmer et al, 2003). Furthermore, evidence is emerging that the detection of micrometastases in this manner has prognostic significance (Hashimoto et al, 2000).

[00102] Currently, molecular detection of occult tumor cells can only be accomplished in excised tissue or fluid, not in a living patient. While immunohistochemical and PCR analyses of tumor-specific markers in resected tissue and fluids has provided important data about the metastatic spread of cancer cells, many of the technically complicated assays are not used routinely in clinical care. PCR-based techniques have slow turnaround times, many are limited to research facilities, and most rely on conventional diagnostic tests or random screening to select specimens for analysis (Ghossein et al, 1999). Despite these limitations, molecular classification and staging is rapidly being incorporated into clinical cancer staging systems and therapeutic paradigms. The ability to directly image this molecular information non-invasively and in real time in living human patients is needed to translate the great advances in molecular detection and classification of human tumors into the clinical arena.

[00103] In cancer patients, determination of whether a malignancy has spread is the single most important factor used to develop a therapeutic plan and predict prognosis. In general, once a solid tumor has spread through lymphatics to regional lymph nodes (LN), the patient's prognosis falls by 50%; with hematogenous or distant metastases (DM), survival rates drop precipitously and systemic therapy is advocated. However, oncologists are limited in their ability to detect metastases, despite technical innovations in radiographic imaging over recent decades. CT and MRI provide high resolution images of regional lymph nodes and pulmonary nodules, these modalities still use phenotypic characteristics such as size, location, and water content for metastases detection. PET scans provide functional information on metabolic rates which helps to differentiate scar tissue from tumor, but lack sufficient sensitivity to detect lesions less than 1 cm in size. As we move towards pathologic molecular staging, minimally invasive surgery and targeted therapy of cancer patients, there is an urgent need to develop non-invasive imaging modalities to provide accurate and sensitive information down to the molecular level for cancer detection, staging, and monitoring of therapy.

[00104] A variety of imaging know in the art can be used in conjunction with the compositions and method described herein. For example, samples can be imaged using an upright microscope in epi-illuminated darkfield mode. A number of light sources can be used, such as a 75W Xenon light source. Images are collected through an objective, and signals can be detected using a CCD camera. Hyperspectral imaging may also be used to measure the spectral differences between labeled and unlabeled cells. Typically, a hyperspectral imaging system incorporates a slit and a prism dispersion configuration. In this scheme, the sample is laterally scanned using a piezoelectric stage, with the slit allowing a portion of the image through the imaging system. Each line of the image is spectrally dispersed via the prism and projected onto a two dimensional CCD detector. The device allows for a spectral range of approximately 350-850 nm, and 1 nm spectral resolution. A microscopically clean aluminum mirror can be used to collect the spectral profile of the light source, which was used to normalize the spectra recorded from cells. Fluorescence imaging can be performed on number of different imaging method, devices, etc. of which one of ordinary skill can operated and modify as needed.

[00105] In certain embodiments, the hybrid nanoparticles are interrogated magnetically. For example, a solenoid electromagnet with a cone shaped ferrite core can be used to magnetically actuate the samples. The field strength at the tip of the magnet can be determined, e.g., about 0.7 T, and the field gradient in z-direction from the tip of the core extending outward can be about 220 T/m (Oh et ah, 2006). The electromagnet can be attached to a motorized translation stage and driven by a programmable controller that permits sinusoidal movement with a user determined frequency and amplitude. The motion amplitude can be adjusted to approximately one full field of view. Care should be taken to ensure that the moving stage is mechanically-isolated from the microscope and its vibration isolation table. Any sample movements due to vibrations caused by the moving stage can be minimized.

[00106] To increase contrast between labeled and unlabeled cells, the magnetic component of the hybrid contrast agents can be exploited. Cells labeled with hybrid nanoparticles can be easily discriminated from both the unlabeled cells and cells labeled with pure gold particles. The magnetic actuation of labeled cells is based on the following principles. The force exerted on the nanoparticles is proportional to the gradient of the square of the magnetic field magnitude (Oldenburg et ah, 2005), and acts in the direction of increasing gradient; thus the iron oxide nanoparticles tend to move towards the ferrite tip in the solenoid when current is applied. By oscillating the tip of the solenoid spatially in a sinusoidal fashion, the changing direction of force exerted on the nanoparticle-labeled cells causes an oscillating displacement in the cells' position.

[00107] High field experimental MRI performed at 4.7T and above is an invaluable tool in pre-clinical oncology. Compared to common clinical field strengths (1.5T), high field scanners provide more than three times the spectral resolution in spectroscopy and spectroscopic imaging applications. In addition, higher field strength offers an increased signal-to-noise ratio (SNR) that allows the acquisition of much higher resolution images, approaching the same anatomically relative resolution in small animal models of disease as are routinely achieved in a clinical setting. Recent advances in experimental high field MRI scanners like the 4.7T Bruker Biospec 47/40USR, available in the Small Animal Cancer Imaging Research Facility (SACIRF) at UTMDACC, enable a wide variety of anatomic, functional, and quantitative imaging techniques to be applied in an experimental setting. It is highly desirable to develop technologies and techniques using such capable instruments in biologically relevant disease models, with the eventual goal of translating these findings into clinical application. Animal models provide a consistent 'patient' population on which early toxicity and dose response, or functional or molecular studies can be completed. Contrast agents and imaging probes can be fully characterized and their ability to successfully attach to their targets verified.

[00108] Currently, MRI is not the modality of choice for detection and monitoring of lung cancers, due to the low density of lung tissue and myriad of air/tissue boundaries that cause very fast T2* signal decay. Computed tomography (CT) is generally the preferred modality for imaging of lung tissue, despite relatively poor soft tissue contrast and difficulty in identifying tumor boundaries when tumor tissue abuts the pleura. Recent studies indicate that some fast MR imaging methods can provide excellent contrast between normal lung tissue and tumor, with clearly identifiable boundaries between tumor and pleura (Bankson et al., 2005).

[00109] Small animal models of head, neck, and lung cancers provide an important opportunity for researchers to study next generation diagnostic and therapeutic agents that enhance detection and monitoring of the disease. Novel imaging probes paired with new imaging methods seeking quantification of cellular and molecular events allow an unprecedented view of the tumor microenvironment alongside high resolution anatomic imaging. For example, Wunderbaldinger et al. have used iron oxide nanoparticles for strong negative enhancement to identify metastatic tissue in lymph nodes (Wunderbaldinger et al. , 2002; Josephson et al., 2002). Targeted gadolinium proteins have been used to visualize distribution of HER-2/neu receptor in a sub-cutaneous tumor model (Artemov et al., 2003), and targeted iron oxide nanoparticles have been used to visualize overexpression of transferrin in murine 9L gliosarcoma xenografts (Hogemann-Savellano et al., 2003). Others have shown that iron oxide particles can provide sufficient negative enhancement for the identification and tracking towards the single-cellular level (Stroh et al., 2005).

E. Other Uses of Hybrid Nanoparticles

[00110] MRI/optical contrast agents can also be used for a variety of other imaging/therapeutic methods, including but not limited to (1) monitoring of delivery and pharmacokinetics of nanoparticle -mediate molecular therapeutics; (2) simultaneous monitoring of delivery of molecular therapy and the earliest molecular response or the interactions of the therapeutic agent with its ligand; and/or (3) imaging of biomarkers associated with delayed response to molecular therapeutics.

[00111] As an example, the small tyrosine kinase inhibitor - FUSl peptide can be used in demonstrating the delivery of therapeutics to a target. Dephosphorylated EGFR and FUSl- EGFR complex can be used as molecular targets for the first events associated with FUSl treatment. The contrast agents can also be assessed for molecular imaging of phospatidyl serine (PS). MRI can be used to monitor distribution of the contrast agents in the whole animal. Simultaneously, interactions of the contrast agents in a tumor can be imaged with high resolution using a MRI compatible endoscopic optical system, an example of which is described herein. In certain aspects, simultaneous monitoring of the therapeutics and molecular processes which they modulate will carried out.

[00112] Other embodiments include the development of an MRI compatible endoscopic digital optical imaging systems for detection of a contrast agent in target sites in organs of interest. Two needle optical fiber probes are contemplated with one optical fiber for reflectance spectroscopy and a second optical fiber for high resolution optical imaging.

[00113] Design and synthesis of the bifunctional contrast agents will be used in combination with, but not limited to computational strategies for the optimization of optical properties of the contrast agents; a needle biopsy system to image deep within tumors with high spatial resolution; strategies for optical imaging, characterization, and bioconjugation of optical contrast agents providing a common platform for development of multi-targeted molecular specific contrast agents for high resolution optical imaging. Furthermore, additional methods for gold nanoparticles that exhibit MRI contrast will be develop related to Gd-polymer gold nanoshells for applications such as thermal treatment of cancer. The contrast agents will be applied to determine the pharmacokinetics and to monitor the therapeutic effects of nanoparticle-mediated therapeutics. In vivo evaluation of the technology will be carried out using the animal models.

[00114] These bifunctional contrast agents which will make possible many new, clinically significant applications that can take advantage of the exceptional anatomic information and penetration depth provided by MRI and the sub-cellular spatial resolution, portability, low cost and molecular specificity of optical imaging. Some of these potential applications include sentinel lymph node imaging for detection of regional micrometastatic disease by using direct injection of the contrast agent into or in the proximity of a breast, skin, colon head and neck, and other tumors; brain tumor surgery where the contrast agent is injected intravenously, particularly into the tumor vasculature; monitoring the response of therapy where the contrast agent is injected intravenously or locally; or staging and determination of resectability in ovarian, pancreatic, lung and other cancers by intraperitoneal, intrapleural, or intravenous injection. Other methods include, but are not limited to real time determination of brain tumor location and margins, and surveillance for recurrence of micrometastatic disease in ovarian cancer.

III. TREATMENT USING HYBRID NANOPARTICLES

[00115] In certain embodiments, the present invention involves the treatment of hyperproliferative cells. It is contemplated that a wide variety of cells may be treated using the methods and compositions of the invention, including gliomas, sarcomas, lung, ovary, breast, cervix, pancreas, stomach, colon, skin, larynx, bladder, prostate, and/or brain metastases and metastases of other cancers, as well as metastases and micro-metastases, precancerous cells, metaplasias, dysplasias, or hyperplasia.

[00116] Various embodiments of the present invention deal with the treatment of disease states comprised of cells that express or comprise a molecular entity that can be targeted by a bifunctional contrast agent, in some aspects cell surface receptors.

[00117] In many contexts, it is not necessary that a target cell be killed or induced to undergo cell death or "apoptosis." In fact, some aspects of the invention are related to imaging the cells to facilitate some other therapeutic procedure or method, such as surgery. To accomplish a meaningful treatment, all that is required is that the tumor or cancer cell growth be slowed to some degree. It may be that the cell's growth is completely blocked or that some tumor regression is achieved. Clinical terms such as "remission" and "reduction of tumor" burden also are contemplated given their normal usage.

[00118] The term "therapeutic benefit" refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of his/her condition, which includes treatment of metastases, micro-metastases, pre-cancer, cancer, and hyperproliferative diseases. A list of non-exhaustive examples of this includes extension of the subject's life by any period of time, decrease or delay in the neoplastic development of the disease, decrease in hyperproliferation, reduction in tumor growth, delay of metastases, reduction in cancer cell or tumor cell proliferation rate, and a decrease in pain to the subject that can be attributed to the subject's condition.

A. Bifunctional Contrast Agent Therapies

[00119] Those of skill in the art are well aware of how to apply and deliver nanoparticles generally to in vivo and ex vivo situations. For therapeutic or contrast agents, one generally will prepare a stock. Depending on the kind of virus and the titer attainable, one will deliver 1 to 100, 10 to 50, 100-1000, or up to 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x 10¹⁰, 1 x 10¹¹, or 1 x 10¹² or more particles to a patient in a pharmaceutically acceptable composition as discussed below. One may also deliver 0.01, 0.1, 0.5, 1, 5, 10, 50, 100, 500 or more fg, pg, ng, or mg of a hybrid nanoparticle to a cell, a subject, an organ, a region, or a location, particular those in, on, or a part of subject to be assessed or treated. For therapeutic purposes hybrid nanoparticles can be coupled to a therapeutic agent that is localize with the hybrid nanoparticles and/or released or sequestered at a target site for therapy.

[00120] Various routes are contemplated for various procedures and methods. Where discrete tumor mass, or solid tumor, may be identified, a variety of direct, local, and regional approaches may be taken. For example, the tumor may be directly injected with the particles. A tumor bed may be treated prior to, during or after resection and/or other treatment(s). Following resection or other treatment(s), one generally will deliver the particles by a catheter or needle having access to the tumor or the residual tumor site following surgery. One may utilize the tumor vasculature to introduce the particles into the tumor by injecting a supporting vein or artery. A more distal blood supply route also may be utilized.

[00121] The method of treating cancer includes treatment of a tumor as well as treatment of the region near or around the tumor. In this application, the term "residual tumor site" indicates an area that is adjacent to a tumor. This area may include body cavities in which the tumor lies, as well as cells and tissue that are next to the tumor.

B. Formulations and Routes of Administration to Patients

[00122] Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. [00123] One will generally desire to employ appropriate salts and buffers to render delivery solutions stable. Aqueous compositions of the present invention comprise an effective amount of the particle to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. 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 know in the art. Except insofar as any conventional media or agent is incompatible with the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

[00124] The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue or cell is available via that route. The routes of administration will vary, naturally, with the location and nature of the lesion, and include, e.g., intradermal, transdermal, parenteral, intracranial, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration and formulation. Certain embodiments include intracranial or intravenous administration. Such compositions would normally be administered as pharmaceutically acceptable compositions.

[00125] An effective amount of the agent is determined based on the intended goal, for example, imaging or elimination of tumor cells. The term "unit dose" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the composition calculated to produce the desired responses, discussed above, in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of administrations and unit dose, depends on the subject, the state of the subject and the result desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. The bifunctional contrast agents of the present invention may be administered directly into a subject, or alternatively, administered to cells that are subsequently administered to subject.

[00126] As used herein, the term in vitro administration refers to manipulations performed on cells removed from a subject, including, but not limited to, cells in culture. The term ex vzVo administration refers to cells that have been manipulated in vitro, and are subsequently administered to a subject. The term in vivo administration includes all manipulations performed on cells within a subject. In certain aspects of the present invention, the compositions may be administered either in vitro, ex vivo, or in vivo. An example of in vivo administration includes direct injection of tumors with the instant compositions by intracranial administration.

[00127] Intratumoral injection or injection into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors including tumor exposed during surgery. Local, regional, or systemic administration also may be appropriate.

[00128] In the case of surgical intervention, the present invention may be used preoperatively, e.g., to render an inoperable tumor subject to resection or in the planning and assessment of the removal of such a tumor. Alternatively, the present invention may be used at the time of surgery, and/or thereafter, to treat or identify residual or metastatic disease. For example, a resected tumor bed may be injected or perfused with a formulation comprising the contrast agent. Cancer cells remaining are then subjected to identification by various imaging procedures. Periodic post-surgical treatment or assessment also is envisioned.

[00129] Treatment regimens may vary as well, and often depend on tumor type, tumor location, disease progression, and health and age of the patient. Obviously, certain types of tumor will require more aggressive treatment, while at the same time, certain patients cannot tolerate more taxing protocols. The clinician will be best suited to make such decisions based on the known efficacy and toxicity (if any) of the therapeutic formulations.

[00130] The contrast agent may be administered parenterally or intraperitoneally, or in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride or Ringer's dextrose. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters. When the route is topical, the form may be a cream, ointment, or salve.

C. Combination Therapy

[00131] Tumor cell resistance to various therapies represents a major problem in clinical oncology. One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy, as well as other conventional cancer therapies. One way is by combining such traditional therapies with hybrid nanoparticle therapy. Traditional therapy to treat cancers may include removal of all or part of the affected organ, external beam irradiation, xenon arc and argon laser photocoagulation, cryotherapy, immunotherapy and/or chemotherapy. The choice of treatment is dependent on multiple factors, such as, (1) multifocal or unifocal disease, (2) site and size of the tumor, (3) metastasis of the disease, (4) age of the patient or (5) histopathologic findings (The Genetic Basis of Human Cancer, 1998).

[00132] In treating a hyperplasia, neoplasia, pre-cancer, cancer, or metastatic cancer according to the invention, one would contact the cells of a lesion or tumor with an agent in addition to a hybrid or bifunctional nanoparticle. In the context of the present invention, it is contemplated that nanoparticle therapy could be used in conjunction with anti-cancer agents, including chemo- or radiotherapeutic intervention, photothermal ablation therapy, as well as radiodiagnositc techniques. In certain aspects, nanoparticle administration is used in conjunction with photothermal ablation therapy.

[00133] A "target" cell contacted with a nanoparticle and optionally at least one other agent may kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce a hyperproliferative phenotype of target cells. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the target cell. This process may involve contacting the cells with a nanoparticle and an agent(s) or factor(s) at the same or different times. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, wherein one composition includes a nanoparticle and the other includes a second agent.

[00134] Alternatively, a nanoparticle treatment may precede or follow the second agent or treatment by intervals ranging from minutes to weeks. In embodiments where the second agent and nanoparticle are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the times of each delivery, such that the second agent and nanoparticle would still be able to exert a combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hr of each other and, or within about 6-12 hr of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[00135] It also is conceivable that more than one administration of either a hybrid nanoparticle and/or the second agent will be desired. In certain embodiments, the second agent can be associated with the hybrid nanoparticle. Various combinations may be employed, where a nanoparticle is "A" and the other agent is "B", as exemplified below:

[00136] A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B [00137] A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A [00138] A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

[00139] Other combinations are contemplated. Again, to achieve cell killing or inhibition of proliferation, both agents are delivered to a cell in a combined amount sufficient to affect the cell.

[00140] Agents or factors suitable for use in a combined therapy are any anti- angiogenic agent and/or any chemical compound or treatment method with anticancer activity; therefore, the term "anticancer agent" that is used throughout this application refers to an agent or a method with anticancer activity. These compounds or methods include alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, DNA antimetabolites, antimitotic agents, as well as DNA damaging agents, which induce DNA damage when applied to a cell.

[00141] Examples of chemotherapy drugs and pro-drugs include, CPTI l, temozolomide, platin compounds and pro-drugs such as 5 -FC. Examples of alkylating agents include, inter alia, chloroambucil, cis-platinum, cyclodisone, flurodopan, methyl CCNU, piperazinedione, teroxirone. Topoisomerase I inhibitors encompass compounds such as camptothecin and camptothecin derivatives, as well as morpholinodoxorubicin. Doxorubicin, pyrazoloacridine, mitoxantrone, and rubidazone are illustrations of topoisomerase II inhibitors. RNA/DNA antimetabolites include L-alanosine, 5-fluoraouracil, aminopterin derivatives, methotrexate, and pyrazofurin; while the DNA antimetabolite group encompasses, for example, ara-C, guanozole, hydroxyurea, thiopurine. Typical antimitotic agents are colchicine, rhizoxin, taxol, and vinblastine sulfate. Each of these drugs can be associated with and localized with a hybrid nanoparticle of the invention.

[00142] Other agents and factors include radiation and other forms of energy that induce DNA damage or nanoparticle heating such as, visible light, ultraviolet light, infrared light, γ- irradiation, X-rays, microwaves, electronic emissions, sound and the like. A variety of anticancer agents, also described as "chemotherapeutic agents," function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. Chemotherapeutic agents contemplated to be of use, include, e.g., adriamycin, bleomycin, 5- fluorouracil (5-FU), etoposide (VP- 16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), podophyllotoxin, verapamil, and even hydrogen peroxide. The invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.

[00143] Examples of anti-angiogenesis agents include, but are not limited to, retinoid acid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN® protein, ENDOSTATIN® protein, suramin, squalamine, tissue inhibitor of metalloproteinase-I, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor- 1, plasminogen activator inhibitor-2, cartilage-derived inhibitor, paclitaxel, platelet factor 4, protamine sulphate (clupeine), sulphated chitin derivatives (prepared from queen crab shells), sulphated polysaccharide peptidoglycan complex (sp-pg), staurosporine, modulators of matrix metabolism, including for example, proline analogs ((l-azetidine-2-carboxylic acid (LACA), cishydroxyproline, d,l- 3,4-dehydroproline, thiaproline], α, α-dipyridyl, β-aminopropionitrile fumarate, 4-propyl-5- (4- pyridinyl)-2(3h)-oxazolone; methotrexate, mitoxantrone, heparin, interferons, 2 macroglobulin-serum, chimp-3, chymostatin, beta.- cyclodextrin tetradecasulfate, eponemycin; fumagillin, gold sodium thiomalate, d-penicillamine (CDPT), β-1- anticollagenase-serum, α- 2-antiplasmin, bisantrene, lobenzarit disodium, n-(2- carboxyphenyl-4- chloroanthronilic acid disodium or "CCA", thalidomide; angostatic steroid, cargboxynaminolmidazole; metalloproteinase inhibitors such as BB94. Other anti- angiogenesis agents include antibodies, preferably monoclonal antibodies against these angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF isoforms, VEGF-C, HGF/SF and Ang-l/Ang-2. (Ferrara and Alitalo (1999) Nature Medicine 5:1359-1364. Calbiochem (San Diego, Ca) carries a variety of angiogensis inhibitors including (catalog number/product name) 658553/AG 1433; 129876/Amiloride, Hydrochloride; 164602/ Aminopeptidase N Inhibitor; 175580/Angiogenesis Inhibitor; 175602/Angiogenin (108-123); 175610/Angiogenin Inhibitor; 176600/ Angiopoietin-2, His»Tag®, Human, Recombinant, Mouse, Biotin Conjugate; 176705/Angiostatin Kl-3, Human; 176706/ Angiostatin Kl-5, Human; 176700/Angiostatin® Protein, Human; 178278/Apigenin; 189400/Aurintricarboxylic Acid; 199500/Benzopurpurin B; 211875/Captopril; 218775/Castanospermine, Castanospermum australe; 251400/D609, Potassium Salt; 251600 Daidzein; 288500/DL-a- Difluoromethylornithine, Hydrochloride; 324743/Endostatin™ Protein, His^»Tag®, Mouse, Recombinant, Spodoptera frugiperda; 324746/Endostatin™ Protein, Human, Recombinant, Pichia pastoris; 324733/Endostatin™ Protein, Mouse, Recombinant, Pichia pastoris; 329740 Eriochrome® Black T Reagent; 344845 Fumagillin, Aspergillus fumigatus; 345834 Genistein; 375670/Herbimycin A, Streptomyces sp.; 390900/4-Hydroxyphenylretinamide; 407293/a-Interferon, Mouse, Recombinant, E. coli; 407306/g-Interferon, Human, Recombinant, E. coli; 05-23-3700/Laminin Pentapeptide; 05-23-3701/Laminin Pentapeptide Amide; 428150/Lavendustin A; 454180/2-Methoxyestradiol; 475838/Mifepristone; 475843/Minocycline, Hydrochloride; 4801/Neomycin Sulfate; 521726/Platelet Factor 4, Human Platelets; 553400/Radicicol, Diheterospora chlamydosporia; 554994/RHC-80267; 565850/Shikonin; 573117/SMC Proliferation Inhibitor-2w; 572888/SU1498; 572632/SU5614; 574625/Suramin, Sodium Salt; 608050/TAS-301; 585970/(±)-Thalidomide; 605225/Thrombospondin, Human Platelets; 616400/Tranilast; 654100/TSRI265; 676496/VEGF Inhibitor, CBO-PI l; 676493/VEGF Inhibitor, Flt2-l l; 676494 VEGF Inhibitor, Je-11; 676495 VEGF Inhibitor, Vl; 676480/VEGF Receptor 2 Kinase Inhibitor I; 676485/VEGF Receptor 2 Kinase Inhibitor II; 676475/VEGF Receptor Tyrosine Kinase Inhibitor, and other such agents known to those of ordinary skill in the medical arts.

[00144] Immunotherapy may be used as part of a combined therapy, in conjunction with nanoparticle therapy and in some aspects associated with a hybrid nanoparticle. Generally, the tumor cell must bear some marker that is amenable to targeting for immunotherapy, i.e., is not present on the majority of other cells or the combination of markers is not present on a majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include EGFR, carcino embryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.

[00145] The skilled artisan is directed to "Remington's Pharmaceutical Sciences" 15th

Edition, 1980. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[00146] In addition to combining nanoparticle therapies with chemo- and radiotherapies, it also is contemplated that combination with other gene therapies will be advantageous. For example, targeting of a nanoparticle in combination with providing a therapeutic nucleic acid that is therapeutic upon transcription from the vector and/or upon translation by the target or neighboring cell(s). In certain aspect the nanoparticle can be use to image the location or effect of a gene therapy.

[00147] It is further contemplated that the therapies and procedures described above may be implemented in combination with all types of surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. These types of surgery may be used in conjunction with, either therapeutically or diagnostically, bifunctional or hybrid nanoparticles. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal or destruction of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[00148] It also should be pointed out that any of the foregoing therapies or procedures may prove useful by themselves. In this regard, reference to chemotherapeutics and nanoparticle therapy in combination also should be read as contemplation that these approaches may be employed separately.

D. Target Cells, Tissues, or Organs

[00149] In some methods of the invention, the target cell expresses molecular target, particularly on its surface, which may be assessed using standard immunological and gene expression assessment techniques know in the art. Furthermore, the cell may be administered compositions of the invention in vitro, in vivo, or ex vivo. Thus, the cancer cell may be in a patient. The patient may have a solid tumor. In such cases, embodiments may further involve performing surgery on the patient, such as by resecting all or part of the tumor. Nanoparticle compositions may be administered to the patient before, after, or at the same time as surgery. In additional embodiments, patients may also be administered nanoparticles endoscopically, intratracheally, intratumorally, intravenously, intralesionally, intramuscularly, intraperitoneally, regionally, percutaneously, topically, intrarterially, intravesically, or subcutaneously.

[00150] In some embodiments, the cancer cell that is administered nanoparticle compositions may be a neuronal, glial, bladder, blood, bone, bone marrow, brain, spinal, breast, colorectal, esophagus, gastrointestine, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testicular, tongue, or uterus cell.

[00151] Cancers that may be evaluated by methods and compositions of the invention include cancer cells and their metastases from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadeno carcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. IV. KITS

[00152] Any of the compositions described herein may be comprised in a kit. In a non- limiting example, reagents for synthesizing components of a nanoparticle, labeling a nanoparticle, using a nanoparticle, and/or evaluating a nanoparticle can be included in a kit, as well reagents and devices for performing methods related to nanoparticle compositions. The kit may further include reagents for creating or synthesizing probes and targeted nanoparticles. In other aspects, the kit may include various reagents for coupling probes and/or nanoparticles. It may also include one or more buffers, such as administration buffer, reaction buffer, labeling buffer, washing buffer, or a hybridization buffer, and compounds for preparing nanoparticles. Other kits of the invention may include components for detecting or imaging nanoparticles of the invention.

[00153] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial or vials. The kits of the present invention also will typically include a means for containing nanoparticles or nanoparticle components, targeting agent, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[00154] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.

[00155] However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[00156] A kit will also include instructions for employing the kit components as well the use of any other reagent or device not included in the kit. Instructions may include variations that can be implemented. [00157] It is contemplated that such reagents are embodiments of kits of the invention. Such kits, however, are not limited to the particular items identified above and may include any reagent used for the manipulation, characterization, or use of bifunctional nanoparticles.

V. EXAMPLES

[00158] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

EXAMPLE 1

HYBRID NANOPARTICLES FOR MRMMAGING AND PHOTOTHERMAL THERAPY

[00159] MA TERIAL AND METHODS [00160] Nanoparticle synthesis and conjugation

[00161] Core/shell γ -Fe2θ3/Au nanoparticles were synthesized as previously reported (Lyon et al, 2004). First, iron oxide seeds were synthesized by hydroxide oxidation of Fe(II) and Fe(III) ions in aqueous solution with bubbled N2 to deoxygenate the solution. The resulting Fe3θ4 particles were then boiled in 0.1 M nitric acid to oxidize the particles to γ- Fe2θ3. This step provides a better surface for deposition of gold layers. The iron oxide seeds were washed in water via centrifugation and resuspended in 0.1 M tetramethyl ammonium hydroxide to stabilize the particles. The seed suspension can be stored for several months.

[00162] A gold layer was reduced onto the surface using an iterative hydroxylamine seeding process (Brown and Natan, 1998). Iron oxide seeds were first mixed with citrate, and then alternating aliquots of hydroxylamine and HAuCU were added to the stirred solution. The color changed from the light brown of iron seeds through blue and eventually became red as the gold layer grew thicker. The process was monitored using ultraviolet-visible (UV-vis) spectroscopy and electronmicroscopy after each iteration. The gold deposition process does not coat all of the iron oxide cores; therefore, the magnetic gold was separated from an excess of bare iron oxide cores using centrifugation (Jeong et al., 2006).

[00163] The gold surface of the nanoparticles was functionalized as reported in (Kumar et al., 2007, which is incorporated herein by reference in its entirety). Anti-EGFR Neomarker clone 225 antibodies (Sigma) were purified using a 100k MW filter from Centricon and then mixed with 0.1 M sodium periodate. This results in oxidation of carbohydrate moieties on the antibody's Fc region to aldehydes. The reaction was quenched with phosphate buffered saline (PBS) and then a hydrazidepolyethylene glycol-thiol heterobifunctional linker molecule (Sensopath Technologies, Inc., SPT-OO 14B) was mixed with the antibodies for 20 min. During this step the hydrazide portion of the polyethylene glycol (PEG) linker interacts with aldehyde groups on the antibodies to form a covalent bond. One more filtration step was used to remove excess linker molecules.

[00164] The antibody/linker solution was diluted in the organic buffer 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid (HEPES), pH 8.75, to 0.05 mg/ml and mixed with the gold/iron oxide particle solution in a 1 :1 volume ratio for particle functionalization via gold- thiol interactions. The mixture was agitated for 30 min at room temperature and then a small amount of 10 ⁵ M 2 kD PEG-thiol was added to coat any remaining bare gold surface. After thirty minutes 2% 18 kD PEG in PBS was added and the particles were centrifuged at 3800 rpm for 30 min and resuspended in 1% PEG in Ix PBS.

[00165] Cell culture and phantom preparation

[00166] MDA-MB-468 cells were used as a cancer model to demonstrate molecular specific cellular imaging. Cells were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Before labeling, cells were harvested, centrifuged, and resuspended in complete MEM. Then, cells were mixed with contrast agent solution in a 1 :1 volume ratio and allowed to interact for 30 min at room temperature. After labeling, the cells were centrifuged to remove unbound particles and resuspended in 1 x PBS for optical imaging or in a buffered (pH 7.4) collagen I solution for MRI imaging.

[00167] The collagen gels create a matrix that prevents the cells from settling over time. Two-hundred microlitres of cell suspensions in buffered collagen at different cell concentrations were pipetted into NMR tubes (Wilmad Glass, 300 MHz) and allowed to gel in an incubator (37°C, 5% CCh) for 30 min. Two samples were prepared with a layer of collagen on the bottom and a second layer of labeled or unlabelled cells. A third phantom type was prepared with a series of layers containing a dilution sequence of cells labeled with the hybrid contrast agents, also with a bottom layer of pure collagen. Each layer was allowed to gel before addition of the next collagen solution. Medium was added to the top of each tube after the final layer of collagen had gelled.

[00168] Nanoparticle characterization

[00169] Mass spectrometry. During the synthesis of the iron oxide seeds there are several centrifugation steps that are not 100% efficient. In order to quantify the exact iron concentration in the final iron oxide seed solution and the final hybrid gold/iron oxide nanoparticles, mass spectrometry was performed using an Agilent ICP-MS system.

[00170] Stock iron seeds, Feridex (Bayer), and gold-coated iron oxide were diluted 10x in ultra-pure water and dissolved in an 11 N sulfuric, 6 N hydrochloric acid solution and then further diluted 10 000 ^χ in 0.2 M nitric acid to provide the necessary points to quantify the iron concentration in the colloid suspension.

[00171] In order to calibrate the system, samples were compared against a standard dilution series of iron chloride that was dissolved in deionized water. A commercial iron oxide colloid suspension, Feridex, was measured to ensure that the protocol was correctly measuring the mass of iron in iron oxide nanoparticles. The iron content of the iron oxide seed solution and the stock gold-coated solution were measured separately and compared to ensure that the gold coating was not inhibiting dissolution of the iron in the colloid suspension. Finally, a sample of purified hybrid nanoparticles, with excess iron oxide seeds removed via centrifugation, was dissolved in the sulfuric/hydrochloric acid and measured to determine how much iron had been coated by gold.

[00172] Electron microscopy. Stock iron oxide seeds and magnetic gold nanoparticle solutions were characterized with transmission electronmicroscopy to determine the morphology and size distribution. Particle suspensions were evaporated on 200 μm copper grids with a Formvar coating and imaged on a Philips EM208 with 80 kV accelerating voltage. The resulting images were analyzed using NIH ImageJ to determine the size distribution.

[00173] UV-vis spectroscopy. Three hundred micro litres of stock and functionalized gold/iron oxide nanoparticles as well as dilute cell suspensions, both unlabelled and labeled with targeted hybrid nanoparticles, were placed in a 96-well plate for absorbance measurements using a BioTek Synergy HT microtitre plate reader. [00174] To determine particle uptake by labeled cells, the cells were first mixed with anti- EGFR hybrid nanoparticles and, after a 30 min incubation period, were centrifuged to remove unbound particles. The difference in the optical density of the supernatant and the optical density of the original labeling solution was used to determine the average number of nanoparticles bound per cell (Sokolov et ah, 2003).

[00175] Optical imaging

[00176] Samples for optical imaging were prepared in the same manner as the cell samples for MRI. Aliquots of collagen/cell solutions were placed on microscope slides. Confocal microscopy was done on a Leica SP2 AOBS confocal microscope using 594 nm laser excitation and a 20^χ dry objective. Images of labeled and unlabelled cells were acquired under identical conditions. Dark-field microscopy was carried out on a Leica 6000 DM upright microscope using a 20^χ objective (0.5 NA) with a 75 W Xe illumination source. Images were acquired with a Q-Imaging Retiga EXi ultra-sensitive 12-bit CCD camera at 85 ms exposure for all samples. Images were analyzed using NIH ImageJ and MATLAB (The Math Works, Natick, MA, USA) code to subtract the background and then determine the average signal intensity. The intensity ratio between labeled and unlabelled samples was calculated by averaging the intensity across all pixels which contained a cell, defined as any pixel with an intensity value above background. The background signal was determined by averaging the signal intensity at a blank spot in each image. This value was subtracted from each image and all pixels above 0 were then averaged and compared.

[00177] Magnetic Resonance Imaging (MRI)

[00178] All data and images were acquired using a 4.7 T biospec experimental MR system (Bruker Biospin MRI, Billerica, MA, USA). The signal decay time constants Ti (spin-lattice relaxation time) and Ti (spin-spin relaxation time) of each sample were measured using a spin-echo saturation-recovery (time to echo (TE) = 10.5 ms; repetition time (TR) = 2500, 1500, 1000, 500, and 200 ms) and a Carr-Purcell-Meiboom-Gill spin-echo train (TE = 15- 360 ms in 15 ms increments; TR = 1100 ms) respectively. All images were acquired over identical geometries (FOV 32 mm x 32 mm; 1 mm slice; 64 x 64 matrix). Images were analyzed using ParaVision 3.0.2® and rate constants were fitted to the Solomon- Bloembergen-Morgan model for relaxation (Bloembergen and Morgan, 1961; Solomon, 1955) using MATLAB. J₂ and J₂* are time constants that describe the persistence of observable signals in MRI. J₂ is largely a function of the intrinsic properties of tissues and fluids, and Z₂* accounts for additional signal damping due to magnetic field inhomogeneities. The relaxivities r₂ and r₂* are the reciprocals of J₂ and J₂*, respectively.

[00179] Relaxivity values (V₁, r₂, and r2*) for the nanoparticles were calculated using measurements done with a series dilution of each colloid suspension. Nanoparticle solutions were pipetted into 5 mm NMR tubes which were then submerged in water with added gadolinium in a 50 ml centrifuge tube, all of which were aligned along the magnetic field of the MRI system (BO) to minimize any influence from susceptibility mismatch. Samples were diluted to provide total iron concentration ranging from 1.15 to 23 μg/ml. The inverse J₁, J₂, and J₂* values were plotted as a function of iron concentration in mM Fe, a linear fit was applied, and the slope of each fitted curve provided the respective relaxivities for the solution. Cell samples were imaged with the same pulse sequence used to obtain data on the colloid suspensions.

[00180] Photothermal therapy

[00181] MDA-MB-468 cells were grown to confluence on 40 mm diameter circular coverslips. The confluent monolayers were incubated with anti-EGFR hybrid nanoparticles or nontargeted nanoparticles for 30 min in medium. The non-targeted nanoparticles were prepared by exposing hybrid nanoparticles to short polyethylene glycol (PEG) molecules terminated at one end by a thiol group. Optical densities of labeling solutions were compared before and after incubation to determine the extent of particle loading. An OPOTEK tunable pulsed laser was used as the irradiation source. Labeled and unlabelled monolayers were irradiated with 7 ns pulses at 700 nm and 10 Hz repetition rate.

[00182] After irradiation the cells were incubated for 30 min in medium, and then calcein AM (Molecular Probes) was added as a live cell stain. The dye permeates cell membranes and is activated by esterases inside the cytoplasm, at which point the dye becomes membrane impermeable. The cells were then imaged under a Leica 6000 DM microscope using a 10^χ, 0.25 NA objective and a 475 nm/510 nm excitation/emission filter cube from Chroma.

[00183] RESULTS

[00184] Nanoparticle characterization

[00185] Core/shell γ-Fe2θ3/Au nanoparticles were synthesized with a final size of 45 nm ± 14 nm (FIG. IA). The hybrid nanoparticles have a plasmon peak centred at 540 nm, slightly red- shifted from the peak at approximately 535 nm for pure gold nanoparticles of comparable size. This shift can be accounted for by the core-shell structure of the nanoparticles. The gold-coated particles had 101 μg Au/ml and 18.5 /Jg Fe/ml after centrifugation to remove excess iron seeds; these values were determined by mass spectrometry. FIG. IB shows the normalized absorbance spectra taken from nanoparticle suspensions before and after conjugating antibodies to the nanoparticles. Attachment of antibodies results in a 5 nm red shift in the spectra that is attributed to an increased index of refraction surrounding the nanoparticles due to the protein coating (Sokolov et al., 2003). The ratio of iron to gold was expected to have a more significant impact on the optical properties of the particles, but spectra from the colloid suspensions are largely consistent with those from pure gold nanoparticles; the iron oxide does not appear to have a significant impact on the optical properties.

[00186] The r\ values were negligible compared to r₂ and r₂* for the solutions tested; this is in agreement with previously published data for iron oxide based MRI contrast agents (Billotay et al., 2003). The functionalized magnetic gold nanoparticles had an r₂ of 23.5 mM/Fe/sec and an r₂* of 68.8, which is in good agreement with the previously published r₂ value of 28.2 mM/Fe/sec for bare iron core/gold shell nanoparticles synthesized in an inverse micelle reaction (Cho et al., 2006a/2006b). There is a decrease by a factor of 4.3 times and by 5.5 times in r₂ and r₂* values, respectively, for the anti-EGFR hybrid nanoparticles as compared to pure iron oxide seeds. A similar effect was observed by Moffat et al where polyacrylamide-coated iron oxide nanoparticles were conjugated with PEG molecules of different sizes (Moffat et al., 2003). The authors demonstrated that the r₂ values generally went down with increasing PEG size. The observed decrease can be accounted for by the fact that the r₂ effect is dependent on water molecules diffusing past an iron oxide contrast agent and experiencing an altered magnetic field; any coating that increases the hydrodynamic radius could reduce the volume of water molecules diffusing through the altered magnetic field surrounding each nanoparticle, thus decreasing the observed contrast.

[00187] Optical imaging

[00188] Confocal reflectance and dark-field images of labeled and unlabelled MDA-MB- 468 breast cancer cells were acquired. The confocal images are false-colored greyscale images, while the dark-field images were white-balanced using a Spectralon (Lab Sphere) white standard. The acquisition settings were identical for labeled and unlabelled cells and were optimized such that the unlabelled cells were visible without causing the labeled sample to saturate the detector. The confocal reflectance image of labeled cells shows bright rings around each cell, which is consistent with labeling of an extracellular membrane bound receptor such as EGFR. Labeled cells appear yellow-orange in the dark-field reflectance images. Individual hybrid particles scatter in the green optical region, but in closely spaced assemblies the adjacent particles exhibit plasmon resonance coupling that results in a strong red shift in their scattering spectra (Sokolov et al., 2003; Aaron et al, 2006; Soennichsen et al., 2005; Rechberger et al., 2003). It has been recently demonstrated that EGFR labeling with gold nanoparticles mediates the formation of closely spaced assemblies of nanoparticles on cellular surface that can account for the observed orange color (Aaron et al., 2007).

[00189] The intensity ratio between labeled and unlabelled cells was 17-fold in confocal reflectance imaging and the difference in dark-field images was dependent on which channel (red, green, or blue) was compared. The ratio was 10-fold and 4.7- fold in the red and the green channels, respectively, and there was no contrast in the blue channel. The integrated intensity difference between labeled and unlabelled samples was 4.5- fold.

[00190] MRI measurements

[00191] Tissue phantoms were prepared using labeled and unlabelled MDA-MB -468 cells suspended in a collagen matrix, and were imaged to demonstrate the potential of these nanoparticles as molecular- specific MRI contrast agents. MRI tubes were filled with alternating layers of collagen gels containing either unlabelled cells or different concentrations of cells labeled with antibody-conjugated magnetic gold nanoparticles. The concentration of labeled cells was varied from 1.75^χ10⁶ to 1.4*10⁷ cells/ml. Dark-field reflectance images were acquired using small aliquots of solution taken from each of the suspensions of cells in the MRI tubes. Labeled cells appear orange-yellow and unlabelled cells exhibit characteristic endogenous blue scattering in the dark-field imaging. The labeled cells show a strong negative contrast in J₂ which decreases with decrease in cell density. Unlabelled cells are indistinguishable from pure collagen, and no significant T₁ effects were observed in any cell sample.

[00192] Preliminary calculations were done to estimate the range of the hybrid contrast agents and cellular concentrations that will be detectable using MRI. The criterion for minimum detection was defined to be that contrast-enhanced tissue compartments must exhibit a net change in signal level that exceeds three times the standard deviation of noise characteristics associated with its baseline intensity. This condition may be equivalently expressed as a minimal contrast to-noise ratio (CNR) of 3. The minimum detection threshold was calculated in terms of cells per voxel as a function of the initial signal-to-noise (SNR) ratio of the image. A theoretical detection limit for cells labeled with magnetic gold nanoparticle was calculated. An average cell is assumed to have 2.76 ^χ 10 ⁶ μg Fe based on the average particle loading of cells and that the particles would still have the same relaxivity as that measured for colloid suspensions.

[00193] A signal-to-noise ratio greater or equal to 30 for J₂- weighted acquisitions with a resolution of 0.15 mm x 0.15 mm x 1 mm can be easily achieved with the 4.7 T system used in this study. With this baseline SNR it was estimated that it would be possible to detect as little as about 30-40 labeled cells per 0.0225 mm³ voxel, which corresponds to about 0.1% of the cells present in this volume in a typical human tissue and to sub-nanomolar concentrations of the contrast agent.

[00194] Photothermal therapy

[00195] FIG. 2A shows the effect of receptor-mediated aggregation of anti-EGFR hybrid nanoparticles on the absorbance of labeled cancer cells. It is well known that EGFR function is associated with dimerization and clustering on the cytoplasmic membrane (Orth et al.., 2006). This receptor clustering leads to molecular specific aggregation of anti-EGFR hybrid nanoparticles and results in a marked increase in absorbance of the nanoparticles in the red and near-infrared (NIR) optical regions. This phenomenon was used for highly selective destruction of cancer cells using NIR light.

[00196] The effect of treatment of labeled and unlabelled cells with a 700 nm pulsed laser is shown in FIGs. 2B, 2C and 2E. A pulse energy was used at which a few labeled cells in the illumination spot consistently survived after a single pulse. One 400 mJ/cm pulse at 700 nm results in nearly complete death of all labeled cells with no effect on cells pre-exposed to non-targeted PEGylated hybrid nanoparticles (FIGs. 2B and 2C respectively). FIG. 5E shows that even after 600 pulses most of the unlabelled cells survive the exposure. FIG. 2D shows a dark- field reflectance image of the treated labeled cells at the laser beam spot boundary. Both the cells exposed to the laser beam and the cells which were not exposed show bright orange- yellow spots that correlate with labeling with the hybrid nanoparticles. Cells outside of the illumination spot exhibit green fluorescence from calcein AM, which indicates survival.

[00197] The energy used is slightly under reported energies required to cause bubble formation around single nanoparticles (Pitsillides et al, 2003; Zharov et al, 2005), but the effect of nanoparticle clustering greatly reduces the energy requirement to produce bubble formation (Khlebtsov et al., 2006). The 700 nm laser light is predominantly absorbed by the aggregates of molecular specific hybrid nanoparticles on the surface of labeled cancer cells. The selective excitation of the aggregates could account for the over 500 times energy difference observed in the sensitivity of labeled and unlabelled cells to the photothermal treatment.

EXAMPLE 2

INCREASED OPTICAL CONTRAST IN IMAGING OF EPIDERMAL GROWTH

FACTOR RECEPTOR USING MAGNETICALLY ACTUATED HYBRID

GOLD/IRON OXIDE NANOPARTICLES

[00198] METHODS

[00199] Iron oxide/gold hybrid nanoparticles

[00200] Magnetically susceptible plasmonic nanoparticles were synthesized using the method described in (Lyon et al., 2004). Briefly, 9 nm magnetite (Fe3θ4) particles were formed via co-reduction of FeCb and FeCb in an aqueous NaOH solution. The Fe3θ4 cores were oxidized to primarily Fe2θ3 by boiling in a 0.01M FINCh solution. X-ray diffraction measurements (not shown) of the prepared magnetic cores were characteristic for maghemite, or γ-Fe2θ3. Subsequently, a ca. 20 nm thick gold shell was deposited using the hydroxylamine seeding method (Brown and Natan, 1998). This procedure involves sequential additions of HAuCb in the presence of citrate and hydroxylamine. It was shown that hydroxylamine confines the reduction of Au3+ ions to the pre-existing surface of iron oxide particles, thereby largely preventing the nucleation of pure gold particles in solution. The iron seeds and hybrid iron oxide/gold nanoparticles were characterized using a Philips EM 208 Transmission Electron Microscope (TEM) equipped with an AMT Advantage HR 1 MB digital camera detector. The addition of gold results in an approximately 5 -fold increase in particle diameter. The average diameter of the iron oxide/gold nanoparticles was 50 nm with a standard deviation of 14 nm. The cause for the relatively large size distribution of the resulting nanoparticles is not very well understood (Oh et al. , 2006) and it presents a technical challenge that remains to be fully addressed. Possible aggregation of the iron oxide core particles before gold deposition might be part of the problem. The process of gold deposition was also monitored using an UV-Vis spectrophotometer (BioTek Synergy HT micro-titer plate spectrometer). Before the addition of the gold shell, the extinction properties of the superparamagnetic particles are consistent with subwavelength sized dielectric spheres. However, upon addition of the gold layer onto the iron oxide core, the extinction spectrum changes markedly, displaying a plasmon resonance peak at 540nm.

[00201] Theoretical simulations were run to model the scattering and absorption properties of the gold/iron oxide nanoparticles. Core-shell composite particle simulations were implemented using custom Matlab/C++ codes based on the equations from (Aden and Kerker, 1951). Dielectric functions used are based on the experimental data from (Johnson and Christy, 1972), with corrections for the effect of particle size as detailed in (Kreibig, 1986). Results from these codes have been extensively compared to composite particle simulations in the literature. In order to simulate the effects of a statistical distribution of particle sizes, the output from the cross section codes was further integrated using a Gauss-Lobatto adaptive quadrature algorithm. The simulated extinction spectrum is in excellent agreement with the measurements. The simulations showed that the peak absorption, extinction, and scattering wavelengths are 534nm, 537nm, and 550nm, respectively. The total scattering from the hybrid nanoparticles represents about 20% of the total integrated extinction.

[00202] Antibody conjugation

[00203] The hybrid nanoparticles were conjugated to anti-EGFR monoclonal antibodies (clone 29.1.1, Sigma) for molecular specific imaging. Antibodies were attached to gold nanoparticles via a conjugation linker that consists of a short polyethylene glycol (PEG) chain terminated at one end by a hydrazide moiety, and at the other end by two thiol groups. First, antibodies at a concentration of 1 mg/mL were exposed to 10 mM NaI(Mn a 40 mM HEPES pH 7.4 solution for 30-40 minutes at room temperature, thereby oxidizing the hydroxyl moieties on the antibodies' Fc region to aldehyde groups. The formation of the aldehyde groups was colorimetrically confirmed using a standard assay with an alkaline Purpald® solution (Sigma). Then, excess hydrazide-PEG-thiol linker was added to the oxidized antibodies and allowed to react for 20 minutes. The hydrazide portion of the PEG linker interacts with aldehyde groups on the antibodies to form a stable linkage. In this procedure a potential loss of antibody function is avoided because the linker can not interact with the antibody's target-binding region, which contains no glycosylation. The unreacted linker was removed by filtration through a 100,000 MWCO filter (Millipore). After purification, the modified antibodies were mixed with gold nanoparticles in 40 mM HEPES (pH 7.4) for 20 minutes at room temperature. During this step a stable bond is formed between the gold surface and the linker's thiol groups. Afterward, mono functional PEG-thiol molecules were added to passivate the remaining nanoparticle surface. Finally, the conjugates were centrifuged at 2800 rcf for 45 minutes and resuspended in Ix PBS.

[00204] Cell culture model

[00205] EGFR over-expressing A-431 cells (Lidke et ah, 2004) were used to demonstrate molecular specific imaging with hybrid iron oxide/gold nanoparticles. Cells were cultured in DMEM plus 10% FBS at 37°C in a 5% CCh environment. For labeling experiments, the cells were suspended in phenol-free DMEM, mixed with the nanoparticle-antibody conjugates, and allowed to react for 20-30 minutes under mild agitation at room temperature. Typically, 200- 300 μL of a cell suspension (~10⁵ cells/mL) were mixed with an equal volume of nanoparticles suspended at approximately 10¹⁰ particles/mL. The labeled cells were washed in phenol-free DMEM and resuspended in an isotonic 1% gelatin solution. The gelatin provides a viscous environment that is more similar to in vivo conditions than pure tissue culture media and also prevents cells from electrostatically adhering to the glass coverslip during imaging. In addition to cells labeled with hybrid nanoparticles two internal negative controls were included: unlabeled A-431 cells and cells labeled with 40 nm pure gold nanoparticles. Because of relatively small optical property differences between 40 nm pure gold and 50 nm magnetic gold nanoparticles (only ca. 10 nm separation in extinction spectra maxima) pure gold particles were conjugated with fluorescently labeled anti-EGFR monoclonal antibodies. Therefore, pure-gold particles exhibited a strong fluorescence signal, while the gold/iron oxide particles did not, allowing easy discrimination between the two populations of labeled cells. AlexaFluor 488 was used as the fluorescent tag and a standard labeling kit available from Molecular Probes to fluorescently label antibodies. The controls were prepared in the same manner as cells labeled with hybrid nanoparticles and all three cell types were mixed together in 1 :1 :1 ratio. An aliquot of this mixture was placed on a microscope slide for optical measurements.

[00206] Imaging system

[00207] Samples were imaged using a Leica DM 6000 upright microscope in epi- illuminated darkfield mode. A 75W Xenon light source was used for illumination. Images were collected through a 2Ox darkfield/brightfield objective with a 0.5 collection NA, and detected using a Q-Imaging Retiga EXi ultra-sensitive 12-bit CCD camera. Time-course images of magnetically actuated cells were taken in monochrome mode at approximately 10 frames per second. Hyperspectral imaging was used to measure the spectral differences between labeled and unlabeled cells. The hyperspectral imaging system (PARISS, LightForm, Inc.) incorporates a slit and a prism dispersion configuration. In this scheme, the sample is laterally scanned using a piezoelectric stage, with the slit allowing a ~lμm wide portion of the image through the imaging system. Each line of the image is spectrally dispersed via the prism and projected onto a two dimensional CCD detector. The device allows for a spectral range of approximately 350-850nm, and 1 nm spectral resolution. A microscopically clean aluminum mirror was used to collect the spectral profile of the light source, which was used to normalize the spectra recorded from cells. Fluorescence imaging was performed in epi-mode using a 490 nm excitation/510 nm emission fluorescence filter cube (Chroma).

[00208] Statistical Image Analysis

[00209] For each type of cell (magnetically labeled, pure gold labeled, and unlabeled), as well as for each illumination condition (white light and 635 nm band-pass illumination) and for magnetically actuated and un-actuated, 10 cells or more were analyzed. To calculate average signal intensities each cell was manually segmented from the image, the signal background subtracted, and the average non-zero pixel intensity values were calculated. Then an average signal and standard deviation were determined for each cell type and illumination condition. A one-tailed, paired t-test (assuming unequal variances) was performed among the three cell populations. Then, the resulting T statistic then was used to calculate a p-value. Calculations were repeated in both Matlab and Excel for confirmation.

[00210] Magnetic actuation

[00211] A solenoid electromagnet (Ledex 6EC) with a cone shaped ferrite core was used to magnetically actuate the samples. The electromagnet was driven by a power supply and current amplifier, which delivered up to 960W to the coil. The field strength at the tip of the magnet was 0.7 T and the field gradient in z-direction from the tip of the core extending 1 mm outward was 220 T/m. The electromagnet was attached to a motorized translation stage (Aerotech) and driven by a programmable controller that permitted sinusoidal movement with a user determined frequency and amplitude. The motion amplitude was adjusted to approximately one full field of view. Care was taken to ensure that the moving stage was mechanically-isolated from the microscope and its vibration isolation table. Any sample movements due to vibrations caused by the moving stage were minimized. [00212] RESULTS

[00213] A color dark-field reflectance image of the A-431 cell mixture was acquired that consists of unlabeled cells, cells labeled with 50 nm gold/iron oxide nanoparticles and with 40 nm pure gold nanoparticles. Labeled cell types were differentiated from one another using a fluorescent tag (AlexaFluor 488, Molecular Probes) that was attached to the monoclonal antibodies conjugated with 40 nm pure gold nanoparticles and was absent on the hybrid nanoparticles. The unlabeled cells appear blue due to the characteristic intrinsic cellular scattering. The labeled cells exhibit dim green regions and bright easily identifiable regions with different shades of orange. The green tinge is the color of the isolated nanoparticles and corresponds to regions with low density of the contrast agents. The orange color corresponds to the closely spaced assemblies of anti-EGFR gold conjugates which interact with EGFR receptors on the cytoplasmic membrane of A-431 cells. The intensity difference between the labeled and unlabeled cells which is achieved under white light illumination can be additionally improved if a 635 ± 15nm band-pass filter is placed into the illumination path. This is possible because the endogenous scattering of cells in FIG. 3, blue line, is significantly reduced in the red optical region (Backman et ah, 2001). In addition, cells labeled with 50 nm hybrid particles display a prominent scattering peak in the red region at approximately 690 nm, shown in FIG. 3, red line. Similar behavior is observed with cells labeled with 40 nm pure gold nanoparticles seen in FIG. 3, green line.

[00214] Despite the unique contrast-enhancing mechanism afforded by the plasmon resonance coupling of gold nanoparticles, unlabeled cells can still be detected in images obtained using both white light and red band-pass illumination. To further increase contrast between labeled and unlabeled cells, the magnetic component of the hybrid contrast agents was explored. It was demonstrated that cells labeled with magnetic gold nanoparticles can be easily discriminated from both the unlabeled cells and cells labeled with pure gold particles. The experimental set-up for magnetic actuation of labeled cells is based on the following principles. The force exerted on the nanoparticles is proportional to the gradient of the square of the magnetic field magnitude (Oldenberg et ah, 2005), and acts in the direction of increasing gradient; thus the iron oxide nanoparticles tend to move towards the ferrite tip in the solenoid when current is applied. By oscillating the tip of the solenoid in a sinusoidal fashion in the horizontal or x-direction, the changing direction of force exerted on the nanoparticle-labeled cells causes an oscillating displacement in the cells' position. The magnitude of the oscillation was approximately 1 field of view, or 500 microns. Magnetically induced movement of cells labeled with iron-oxide/gold nanoparticles can be visualized.

[00215] For example, images were collected and replayed at 10 frames per second. The horizontal translation of the solenoid tip causes horizontal fluctuations in the cell position due to interaction between the electromagnet and the magnetic nanoparticles which are attached to EGFR molecules on cellular surface. In addition, however, there also exists a y-component of the magnetic force due to the fact that the ferrite tip is not positioned directly beneath the cell labeled with hybrid nanoparticles. Cells that are not perfectly aligned in z-direction with the solenoid tip experience a non-oscillatory component in the y-direction. It is also important to note that the solenoid exerts the bulk of its force in the z-direction, parallel to the microscope's optical axis. While cell translation is confined in this direction due to the presence of the microscope slide and coverslip, it may produce an overall torque on cells that have an uneven distribution on particles on their surfaces. This torque will result in signal fluctuations at the same frequency as the solenoid oscillation, and thus will contribute to the overall magnetic actuation effect. Any significant signal fluctuations at the modulation frequency of the solenoid are absent in the case of unlabeled cells and cells labeled with pure gold nanoparticles or background.

[00216] To analyze the specific frequency components of the acquired signals, a fast Fourier transform (FFT) was performed at each pixel of the acquired images in the time domain and power spectra were calculated at each pixel position. Oscillation frequencies of the magnet and the total number of acquired images were chosen to avoid any aliasing effects. The precise sampling frequency was calculated via a time stamp generated in each image file that is accurate to 0.001 seconds. Monochrome darkfield images of a mixture of cells labeled with gold/iron oxide particles, with pure gold particles, as well as unlabeled cells was acquired. The samples were subjected to a magnetic field oscillation with frequencies of 0.9Hz and 1.9Hz. After data acquisition, images were analyzed in Matlab. FIGs. 4A and 4B show examples of frequency spectra from magnetic gold labeled (red line), pure gold labeled (green line), and unlabeled cells (blue line), for magnetic oscillations with frequencies 0.9 and 1.9 Hz, respectively. Frequency spectra of signals recorded from cells labeled with the magnetic/gold nanoparticles display a prominent peak at the corresponding stage oscillation frequency. Such a peak is not apparent in the case of unlabeled cells or cells labeled with pure gold nanoparticles, indicating that these cells are not displaced by the spatiotemporally oscillating magnetic field. [00217] These results also suggest that secondary effects such as localized temperature- induced convection currents within the gelatin matrix are minimal. Time- varying signal intensities are predominant in only those regions of interest which contain the magnetically labeled cells. To isolate magnetically modulated components in the acquired images, a Hanning window method implemented in Fourier space was used (Harris, 1978). First, image series were subjected to the appropriate window function and, then, to an inverse Fourier transform at each pixel in the time-dimension. Finally, images were rescaled via a simple linear multiplier to maximize the pixel intensity range. Presented images were not subjected to any thresholding procedure, which would artificially distort image contrast. Digital filtering can be applied. As a result of this treatment, signals associated with unlabeled cells, and pure gold-labeled cells are no longer apparent in images filtered at both 0.9Hz and 1.9Hz.

[00218] Implementation of frequency domain filtering results in greater contrast enhancement as compared to purely optical methods. To demonstrate this contrast enhancement, FIG. 5A shows pixel intensity profiles that were drawn across images of magnetic gold-labeled (red line), pure-gold labeled (green line) and unlabeled cells (blue line) which were obtained under different illumination conditions and with the combination of the 635 nm illumination and frequency domain filtering. The average signal intensities were calculated for each of the three cell populations in images that were acquired under four different acquisition conditions: (1) white light illumination, (2) 635/15 nm bandpass illumination, (3) white light illumination followed by magnetic actuation and frequency domain filtering, and (4) 635/15 nm bandpass illumination followed by magnetic actuation and frequency domain filtering.

[00219] Results of this analysis are shown in FIG. 5B. Under white light illumination, signal from cells labeled with magnetic and non-magnetic gold particles are statistically identical and the unlabeled cells are on average 2.5 times dimmer. Addition of the 635/15 nm bandpass filter increases the intensity difference between labeled and unlabeled cells to approximately 4, as indicated in FIG. 5C. Interestingly, implementation of magnetic actuation and frequency domain filtering leads to statistically the same results independent of which illumination condition is used, as indicated in FIG. 5C. The average signal intensity ratio between gold/iron oxide and pure gold labeled cells increases from approximately 1 in the case of no magnetic actuation to about 3 with the magnetic actuation. At the same time, the intensity ratio between gold/iron oxide labeled cells and unlabeled cells increases from approximately 2.5-3 to ca. 10 under both illumination conditions. Asterisks in FIGs. 5B and 5C indicate a statistically significant difference in the average signal values with p<10^~4 between the three cell types within each acquisition condition. These results demonstrate that frequency domain filtering is very sensitive to the magnetically controlled movement of cells.

EXAMPLE 3

HYBRID NANOPARTICLES WITH INTERMEDIATE LAYER FOR MRI/IMAGING

AND PHOTO-THERMOTHERAPY

[00220] Materials. Tetraethylorthosilicate (TEOS), 3-aminopropyltrimethoxysilane (APTMS), ammonia solution (30 wt %, 7 mL), tetrakis(hydroxymethyl)phosphonium chloride (THPC), chloroauric acid (HAuCU), potassium carbonate (K2CO3), and formaldehyde (37%) were purchased from Sigma-Aldrich (St. Louis, MO). Water-based super paramagnetic iron oxide (SPIO, γ-Fe2θ3) particles (EMG 304) were purchased from Ferrotech (Nashua, NH). Purified water (18 MΩ) was obtained from a Milli-Q Synthesis system (Millipore, Billerica, MA).

[00221] Synthesis of SPIO-Au Nanoshells. In a typical procedure, 0.2 mL of water-based SPIO (EMG 304) was diluted with 6 mL of 18 MΩ water and 80 mL of absolute ethanol. An aqueous ammonia solution (30 wt %, 7 mL) and TEOS (0.5 mL) were consecutively added into the SPIO solution at room temperature under continuous mechanical stirring. The clear solution became a murky suspension in less than 30 min, indicating the formation of silica nanoparticles. The reaction was allowed to proceed at room temperature overnight. The functional groups at the surface of these unmodified silica nanoparticles are predominantly silanol (Si-OH) or ethoxy (Si-OEt) groups (Badley et al, 1990). These silica nanoparticles were then treated with 0.04 mL of 3-aminopropyltrimethoxysilane for 6 h to introduce the amino-terminated silica surface, and then the reaction mixture was refluxed for 30 min to complete the reaction (Badley et al, 1990). After the reaction mixture had cooled, the SPIO- embedded silica was separated from the reaction medium by centrifugation at 4000 rpm and redispersed in 100 mL of absolute ethanol. Addition of 1 mL of SPIO embedded silica solution to 5 mL of undiluted THPC gold solution led to the attachment of THPC gold nanocrystals onto the silica surface. This mixture was stored at 4⁰C overnight to maximize the surface coverage of the THPC gold nanoseeds. The preparation of THPC gold solution involved the reduction of chloroauric acid (HAuCU) with THPC, which affords relatively small gold particles {e.g., 2 nm) with a net negative interfacial charge (Grabar et al, 1996). Finally, the gold nanoshells were prepared by reduction of K-gold solution with formaldehyde (37%) in the presence of SPIO-embedded silica nanoparticles covered with gold nanoseeds. To prepare K-gold solution, 2 rnL of 1 wt % HAuCU was added to 100 mL of water containing 0.025 g of K2CO3 under magnetic stirring. UV-vis absorption spectra were measured 30 min after reaction to verify the formation of nanoshells, which exhibit significant absorbance in the near infrared region.

[00222] Characterization Methods.

[00223] Transmission Electron Microscopy (TEM). To prepare each TEM sample, a small drop of solution was transferred to the top surface of a carbon-film supported Cu grid (previously glow-discharged to achieve better dispersion) and left until dried. The TEM work was carried out in a microscope (JEOL 2010, JEOL Ltd., Tokyo, Japan) at a working voltage of 200 kV. All imaging magnifications were calibrated using standards of SiC lattice fringes (for high magnifications) and cross-line grating replica (for low magnifications). The size measurements were based on a sufficient number of samples, typically over 30.

[00224] Energy-Dispersive Spectroscopy (EDS). EDS was performed using a spectrometer (INCA, Oxford Instruments, Oxon, U.K.) with an ultrathin-window Si-Li detector (capable of detecting elements with Z > 5) that was attached to a JEOL 2010 microscope.

[00225] UV- Vis Spectroscopy. UV-vis spectra of the nanoshells were recorded on a Beckman Coutler DU-800 UV-vis spectrometer (Beckman Coutler, Fullerton, CA) with a quartz cuvette of 10-mm optical path length.

[00226] Magnetization Measurements. Magnetization measurements were carried at 5 K to 300 K in a magnetic field (H) of up to 50 kOe with a superconducting quantum- interference device magnetometer (model MPMS, Quantum Design, San Diego, CA) that can measure magnetic moments as low as 10^"7 emu. In the magnetization measurement, both the uncoated and Au/Siθ2 coated Fe2θ3 NPs were in the form of dried powders. Gelatin capsule (from Capsuline.com) and water-resistive polycarbonate capsules (from Unipec Inc., Rockville, MD) were used as the sample containers for the uncoated and Au/Siθ2 coated Fe2θ3 NPs, respectively. Since both the amount of the Au/Siθ2 coated Fe2θ3 particles (0.6 mg) and the volume fraction of the magnetic Fe2θ3 cores in the sample were very small, the magnetic moment of the polycarbonate capsule was measured for background subtraction. The background magnetic moments from the sample capsules (container) were found to be negligible (~10^~5 emu). [00227] Fourier Transform Infrared (FTIR) Spectroscopy. PEG coating was investigated using a Perkin-Elmer (Wellesley, MA) Spectrum GX system.

[00228] Relaxivity. Samples prepared in water at different concentrations (C = 0.00446- 0.223 mM) were poured into 4-mm diameter nuclear magnetic resonance sample tubes (SP Industries, Inc., Warminster, PA). Axial images were obtained using a 4.7-T, 40-cm MR scanner (Bruker Biospin Corp., Billerica, MA) with a 950-mT/m, 5.7-cm-inner-diameter actively shielded gradient coil system [19000 mT/(m s) slew rate] and a 3.5 -cm-inner- diameter volume radiofrequency coil. Longitudinal and transverse relaxation studies were performed on axial images acquired with variable-TR (10 TR values ranging from 25 to 3000 ms and TE = 10.5 ms) and variable-TE (24 TE values ranging from 15 to 360 ms and TR = 1000 ms) multislice-multiecho sequences, respectively; other imaging parameters include a 3.2-cm field of view, a 1.0-mm slice thickness, and a 64 x 64 matrix. Regions of interest were used to calculate the signal intensity (SI) at each sample concentration. Plots of TR vs SI and TE vs SI were fit to exponential curves using a custom software program in IDL (ITT Industries, Inc.; Boulder, CO) to provide T\ and J₂ relaxation constants at each concentration. Then, relaxivities ^₁ and i?₂ (mM ¹ s^"1) were obtained from linear fitting of the \IT_\ vs C and l/r₂vs C plots.

[00229] Temperature Elevation Induced by NIR Laser Irradiation The laser used was a continuous-wave GCSLX-05-1600 m^"1 fiber-coupled diode laser (DHC, China Daheng Group, Inc., Beijing, China) with a center wavelength of 808 +/- 10 nm. It was powered by a DH 1715A-5 dual-regulated power supply (DHC, China Daheng Group, Inc.). A BioTex LCM-011 optical fiber (5 m in length) was used to transfer laser power from the laser unit to the target. This fiber had a lens mounting at the output that allowed the laser spot size to be changed by changing the distance from the output to the target. The output power was independently calibrated using a handheld optical power meter (Newport model 840-C) and was found to be 1 W for a spot diameter of 3.5 mm and a 2-A supply current. The end of the optical fiber was attached to a retort stand by a movable clamp and positioned directly above the sample cell.

[00230] X-ray diffraction (XRD) and X-ray absorption spectroscopy (XAS). The x-ray diffraction (XRD) measurement was performed at room temperature using a Rigaku 2005 x- ray diffractometer with Cu Ka radiation. The Fe AT-edge x-ray absorption spectroscopy (XAS) data were taken in the fluorescence mode at room temperature at the beam line X-19A at the National Synchrotron Light Source (NSLS) at Brookhaven National Lab. A double-crystal Si (111) monochromator was used for energy selection, which was detuned by reducing the incident photon flux about 20% from its maximum value in order to suppress contamination from harmonics. The energy resolution (ΔE/E) of the X-19A beam line was 2x10 ⁴, corresponding to about 1.4 Ev at the edge energy of Fe K edge. The energy calibration of the spectra was made by simultaneously measuring the spectrum of a FeO slide as reference. The XAS spectra were background subtracted and normalized to unity in the continuum region.

[00231] Synthesis and PEG Coating of SPIO-Au Nanoshells. The synthesis of the SPIO-Au nanoshells was a multistep procedure (FIG. 6). First, silica-coated SPIO (Fe2θ3) nanoparticles were synthesized according to the well-known Stober process (Stober et al, 1968). Commercially available SPIO (average diameter, 10 nm) nanoparticles stabilized with oleic acid in water were used. The magnetic nanoparticles were easily coated with amorphous silica via the sol-gel process because the iron oxide surface has a strong affinity for silica. No primer was needed to promote the deposition and adhesion of silica. The thickness of the silica sphere could be tuned from 2 to 100 nm simply by changing the concentration of the sol-gel precursor, TEOS (Lu et al, 2002). Next, the surface of the silica shell was functionalized with amine groups by treatment with NFUOH and 3- aminopropyltrimethoxysilane. Then, gold nanocrystal seeds (2-3 nm) were attached to the amino groups on the silica sphere by reduction of chloroauric acid (HAuCU) with THPC (Duff et al, 1993). Because the gold nanoseeds had net negative surface charges, they firmly attached to the amino groups on the silica sphere, which were positively charged at acidic pH. Finally, the attached gold nanoseeds were used to nucleate the growth of a gold overlayer on the silica surface to form a gold nanoshell. The nanoshells were isolated by centrifugation and washed with deionized water. The nanoshells were then resuspended in sodium citrate buffer (33 mM) to stabilize the particle solution. To control the thickness of the gold shells, the concentration of gold-seeded, SPIOcontaining silica particles were varied while keeping the concentration of gold precursor (HAuCU in 2.5% K2CO3 solution) constant.

[00232] In this synthesis scheme, the silica layer served the same role as the silica core in conventional gold nanoshells: it provided a dielectric interface for shifting the plasma resonance to the NIR wavelength region. In addition, functionalizing the outer surface of the silica layer with free amine groups facilitated the initial growth of the gold nanoseeds, which, in turn, facilitated the subsequent growth of the outermost gold shell.

[00233] In the last step, the nanoshells were coated with polyethylene glycol (PEG) because of its known high biocompatibility. PEG coating was achieved by treating the SPIO- Au nanoshells with mono functional PEG precursor MeO-PEG-SH (Mv ) 5000), resulting in stable nanoparticles. The PEG coating was confirmed using Fourier transform infrared spectroscopy, which showed peaks characteristic of PEG around 2918, 1453, and 1026 cm^"1 corresponding to the C-H stretching, C-H bending, and C-O stretching vibrations, respectively (FIG. 7). The PEG coating was found to afforded nanoshells good temporal stability. Whereas uncoated nanoshells exhibited a strong tendency toward aggregation (the dispersion became cloudy and began to show signs of settling in minutes), PEG-coated SPIOAu nanoshells showed no visible signs of aggregation over several weeks of storage at 4 ⁰C.

[00234] Characterization of SPIO-Au Nanoshells. Transmission electron microscopy images of nanoparticles at different stages of SPIO-Au nanoshell synthesis were acquired. After synthesis of silica-coated SPIO nanoparticles, SPIO nanoparticles were clearly observed inside the spherical silica. Most of these particles contained one to four single SPIO nanoparticles or clusters of SPIO nanoparticles. The mean diameter of the silica-coated SPIO nanoparticles was estimated to be 66.0 +/- 9.5 nm. After treatment with HAuCU and THPC, gold nanoseeds on the silica surface were clearly observed. The SPIO-Au nanoshells had an average diameter of 82.2 +/- 9.7 nm, and the gold shell had a thickness of ~8 nm. The gold coating was not continuous, with topographical roughness on the nanometer scale. Energy- dispersive spectroscopy of the PEG-coated SPIO-Au nanoshells across the entire image area showed the presence of C, O, Si, Fe, and Au. Absorption spectra of the SPIO-Au nanoshells at different stage of preparation are shown in FIG. 8. As the coverage of gold on the SPIO- embedded silica nanoparticles increased, the surface plasmon resonance peak became more prominent and red-shifted toward the NIR region (FIG. 8A). The absorbance at 800 nm increased with reaction time and reached a plateau by 10 min after the addition of K-gold solution (FIG. 8B). Theoretical calculations have shown that the plasmon resonance of noble metal nanoshells can vary over hundreds of nanometers, as the position of the resonance is dictated by both the shell thickness and the size of the dielectric core (Averott et a;/. 1997). The broad absorbance between 700 and 900 nm suggests that the SPIO-Au nanoshells would be suitable for photothermal therapy with light in the NIR region, where there is minimum light absorption in tissues.

[00235] Magnetic Properties of SPIO-Au Nanoshells. To determine whether the SPIO- Au nanoshells exhibited magnetic properties that could potentially be used for magnetic-field guided targeting, the SPIO-Au nanoshells were dispersed in pure water. A Nd-Fe-B magnet (~0.3 T) was then used to separate the nanoshells. The solution of SPIO-Au nanoshells, which was initially dark, was almost transparent after 15 min of exposure to the magnetic field. The particles were precipitated to one side of the vial wall. UV-vis spectroscopy revealed that more than 85% of the nanoshells were removed from the solution. Magnetization curve of the SPIO-Au nanoshells were obtained using a superconducting quantum-interference device. For comparison, the magnetization curve of the commercial SPIO nanoparticles (average diameter, 10 nm) used in the preparation of the SPIOAu nanoshells was also obtained. Plots of M(H) in the region between -2 and 2 kOe were produced. The M(H) hysteresis loop for the SPIO-Au nanoshells was almost completely reversible, indicating that the SPIO-Au nanoshells exhibit superparamagnetic characteristics. The saturation moment per unit mass, M, for the SPIO-Au nanoshells was 3.5 emu/g at 20 kOe, which is about 5.9% of the M. for SPIO. The observed M. value for the SPIO-Au nanoshells agrees well with previously reported data for silica coated iron oxide nanoparticles (3.6 emu/g)(Yu et al., 2003). The magnetic moment of Au-silica nanoshells without SPIO at 1000 Oe was measured, and the results showed that Au-silica nanoshells without SPIO exhibit diamagnetic characteristics with a magnetic moment no more than 0.3% of that of SPIO-Au nanoshells. Taking these results together, it is concluded that the decrease in M is most likely the result of a large volume of silica/ Au in the coated nanoparticles and that the gold and silica layers in SPIO-Au nanoshells do not affect the magnetization saturation of SPIO nanoparticles.

[00236] Magnetic Resonance (MR) Relaxation Study of SPIOAu Nanoshells. Because SPIO nanoparticles are effective J₂ MRI contrast agents (Bulte and Kraitchman, 2004; Wang et al. 2001) the J₂ and T₁ relaxations of SPIO-Au nanoshells were evaluated. The relaxivities of SPIO-Au nanoshells for Fe2θ3 concentrations in the range of 0.00446-0.223 mM measured at 4.7 T at room temperature were determined. Table 1 compares the relaxivities measured for SPIO and SPIO-Au nanoparticles. One of the interesting features observed for the SPIO- Au nanoshells is that their i?₂ relaxivity exhibited bilinear behavior, with a 32-fold increase in i?₂ at lower concentrations (0.00446-0.0446 mM) compared to that at higher concentrations (0.0446-0.223 mM). In comparison, Kim et al. (2006) recently reported linear i?₂ relaxivity for a superparamagnetic gold nanoshell sample in which iron oxide nanoparticles were introduced at the interface between the silica and gold layers. The observation of bilinear relaxivity for the SPIO-Au nanoshells might originate from the clusters of SPIO nanoparticles within the silica core of the SPIO-Au nanoshells. A similar bilinear relaxivity behavior has been observed with GcbN clusters encapsulated in Cso fullerene (Fatouros et al., 2006). Compared to the SPIO precursor used in the preparation of the SPIO-Au nanoshells, the SPIO-Au nanoshells had a significantly increased i?₂ relaxivity at lower concentrations but an almost completed suppressed longitudinal relaxavity (K₁) (Table 1). The amount of Fe2θ3 in the SPIO-Au nanoshells was estimated from the amount of SPIO initially added into the silica sol-gel solution, assuming a 100% yield of incorporation into the SPIO-Au nanoshells. Therefore, the actual i?₂ value might be higher than that reported because of the possible overestimation of Fe2θ3 concentrations. The increased i?₂ relaxivity and large R2/R1 ratio indicate that the SPIO-Au nanoshells can be used as a contrast agent for obtaining T₂- weighted images at reduced concentrations.

T ABLE 1: Reisxhifes of Various Nsisopaitiεles

SPlO - Aiϊ ?g;O5 + ^sJics M9 11 4 C O: ^■■- 10000 si tow

[00237] Photothermal Study of SPIO-Au Nanoshells. An important feature of nanoshells is NIR light-induced thermal effect, which could be used for selective treatment of solid tumors (Loo et al., 2004; Hirsh et al., 2003; O'Neal et al., 2004). To investigate temperature elevation induced by NIR laser irradiation in the presence of SPIO-Au nanoshells, a continuous-wave fiber-coupled diode laser with a center wavelength of 808 +/- 10 nm was used. Studies in aqueous solution showed that the temperature increased with increasing exposure time and plateaued after about 5 min of light exposure. At a concentration of 7.5 x 10¹² particles/mL, an elevation of 16.3⁰C was achieved at a power output of 1 W. In comparison, no significant temperature change was observed when pure water or an SPIO silica dispersion at a concentration of 1.0 x 10¹³ particles/mL was exposed to the laser light. These data indicate that the SPIO-Au nanoshells acted as an efficient photothermal mediator. It should be noted that the temperature increased with increasing nanoshell concentration at nanoshell concentrations of <5.0 x 10¹² particles/mL. At higher concentrations (>5.0 x 10¹² particles/mL), no further increase in temperature was observed when the concentration was increased, indicating that scattering of light by the nanoshells became a dominant factor at higher concentrations.

[00238] XRD and XAS. To make sure the commercial iron oxide nanoparticles (IONPs) are in γ-Fe2θ3 phases, XRD and XAS measurements were made on them. FIG. 9 shows the powder XRD patterns for the commercial IONPs, the SiCh coated IONPs, and the Au/SiCte doubly coated IONPs, with each pattern normalized to its maximum intensity. All of the peaks in the patterns of the commercial IONPs can be indexed with the cubic structure corresponding to either γ-Fe2θ3 or Fe3θ4 phase. Since magnetite (Fe3θ4) has a similar cubic structure and lattice constant as the maghemite (γ-Fe2θ3), it is quite difficult to distinguish γ- Fe2θ3 from Fe3θ4 using only XRD data. Thus, the existence of Fe3θ4 in the commercial IONPs cannot be excluded based only on the XRD data. On the other hand, the x-ray near- edge structure (XANES) of XAS offers a powerful mean to unambiguously distinguish iron oxide species with different formal valences, because iron oxides with different formal valence could have different edge-energy and spectral features in their Fe AT-edge spectra. When the Fe K-edge spectra of the commercial IONPs is compared with the spectra of the powder of four reference compounds: micron-size γ-Fe203, α-Fe2θ3, Fe3θ4, and FeO it can be seen that both the shape and edge energy (defined as the energy at absorption coefficient μ = 0.5) for the spectrum of the commercial IONPs are very close to those for the micron-size γ-Fe2θ3 but very different from the rest of the spectra, clearly indicating that the commercial IONPs used in this study are in γ-Fe2θ3 phase.

[00239] FIG. 9 shows that for the Siθ2 (silica) coated γ-Fe2θ3 NPs, the XRD pattern are very similar to the uncoated γ-Fe2θ3 NPs. This means that all of the coated silica are in amorphous form before the step of mixing the γ-Fe2θ3-embedded silica solution with the THPC gold solution (obtained by reduction of HAuCk with THPC) during the synthesis process. For the Au and silica doubly coated γ-Fe2θ3 particles, the XRD pattern in FIG. 9 displays three wide peaks which can be identified as the (111), (200), and (220) reflection lines of the Au cubic phase, indicating that the gold particles are crystallized. In addition, there are four narrow peaks in the pattern which can be attributed to the crystallized Siθ2 phase, indicating that certain portion of the silica is crystallized during the steps of coating Au on the surface of the γ-Fe2θ3-embedded silica NPs. At present, while the mechanism of the crystallization of silica due to the coating of the Au particles is unclear, some recent research indicates that Au nanoparticles on silica spheres could induce crystallization of silica at low temperatures (Perkas et al, 2006). The background of this pattern is very similar to the XRD pattern of amorphous Siθ2, which decreases rapidly with the increase of 2Θ from about 23° to 40° and then slowly beyond 40°. Considering the fact that most of these coated particles are spherical (See TEM result below) and the observation that usually spherical Siθ2 particles are in the amorphous form, it is believed that only a small fraction of the silica is crystallized and most of the silica nanoshells are in the amorphous phase.

[00240] Interestingly, the Fe2θ3 peaks appearing in the pattern of the uncoated Fe2θ particles can not be seen in the pattern of the coated particles. This disappearance of the Fe2θ3 peaks can be explained by the small volume fraction of the Fe2θ3 core and the large thickness of the Au/SiCh shell. First, since our TEM result (see below) shows that the average diameter of the Fe2θ3 core is about 12 nm and the average diameter of the coated particles is 82 nm, the volume fraction of the Fe2θ3 core in the whole coated particle is merely about (12 nm/82nm) = 0.3%, which could cause the intensity of the Fe2θ3 peaks washed out by the background noise. Second, since on the average each Fe2θ3 core was surrounded by a Au/SiCte shells of thickness of 35 nm, the absorption and scattering of the x-ray by the shells could also weaken the peak intensity of the Fe2θ3 cores substantially, as clearly observed in SiCh coated γ-Fe2θ3 nanoparticle systems (Yu et al, 2003).

[00241] TEM and EDS. An image of the pure or uncoated Fe2θ3 (SPIO) particles in a lower magnification was acquired. The average size of the particles is measured as Davg = 12.4 nm with a standard deviation of 4.5 nm, based on the measurements of 412 particles. This value of average size is slightly larger than the nominal size of 10 nm given by the vendor (Ferrotech). The EDS confirmed that the particle composition is Fe and O without any other impurities. A Cu signal was derived from Cu grids, and C signal was from the support film of the Cu grids.

[00242] A typical TEM image for the Au and silica doubly coated Fe2θ3 nanoparticles contains large spherical particles that are identified as the silica spheres. The average size of these particles is measured as Davg = 81.5 nm with a standard deviation of 17.0 nm, based on the measurements of 60 particles. A smaller dark particles distributed on the surfaces of these silica spheres are Au nanoparticles. The average diameter of these Au particles is measured as Davg = 6.0 nm with a standard deviation of 1.6 nm, based on the measurements of 365 particles. The coated Au only forms dispersed nanoparticles on the surface of silica spheres, rather than continuous Au layer. In the core region of this coated nanoparticle, lattice fringes of the Fe2θ3 particle are visible (in phase contrast). The Fe2θ3 particles are larger than the Au nanoparticles, with an average size around 12 nm. These Fe2θ3 particles are embedded inside the silica spheres. [00243] Because the mass of Fe2θ3 is much lighter than Au, the image contrast (scattering- absorption contrast) of Fe2θ3 is very weak, almost invisible in a BF image. However, a central dark-filed (CDF) image contrast is formed by diffraction (diffraction contrast), so both Fe2θ3 and Au particles show high contrast. However, it should be pointed out that only those particles that have reflections along a tilted incident beam direction are imaged, i.e., only part of Fe2θ3 and Au particles rather than all show up in a CDF image. Basically, it can be recognized that larger Fe2θ3 particles are at the center of the silica spheres. X-ray mapping of Fe shows the intensity distribution of Fe signals along the silica spheres, with intensity maxima in the core areas. Maps of Si and O are similar, along the silica spheres. The EDS spectrum taken over this entire area, which clearly shows the evidence of O, Si, Fe and Au (the signal of Cu is from the Cu grids). A quantitative analysis yields a composition of 62.6% O, 26.4% Si, 1.7% Fe, and 9.3% Au (all in atomic percent). The very low concentration of Fe confirms that only small amount of Fe2θ3 particles present inside the silica.

[00244] Magnetic Properties. FIG. 10 presents the field cooled (FC) and zero-fϊeld- cooled (ZFC) magnetization M(T) curves for the uncoated and Au/SiCh doubly coated γ- Fe2θ3 NPs. The M(T) curves were measured in a temperature range between 5 and 300 K and at two applied fields: 10 Oe and 500 Oe for the uncoated particles and at 500 Oe for the coated ones. FIG. 1OB shows that the 500 Oe ZFC and FC curves for the γ-Fe2θ3 NPs (with Davg^s 12 nm)are irreversible below the irreversible temperature Tm~ 300 K, and the blocking temperature 7B, defined as the temperature at the maximum of the ZFC curve (Sohn and Cohen, 1998; Banerjee et al, 2000; Jeong et al, 2006), is 7B ~ 160 K. Such observed reversibility between the ZFC and FC curves and a maximum in the ZFC curve are typical for an assembly of superparamagnetic nanoparticles (SPNP). It was noticed that the values of Tm and 7B observed here for our 12 nm γ-Fe2θ3NPs are higher than the values Tm= 175 K and 7B ~ 120 K measured at the same field (500 Oe) by Jeong et al for their γ-Fe2θ3 particles with smaller particle size of 5-8 nm. This particle size caused change in 7B can be explained by the theoretical formula (Banerjee et al, 2000; Jeong et al, 2004):

where V(=l/6 πD³ _avg) is the average volume of the NPs, K is the uniaxial anisotropic constant and kn the Boltzmann constant. Indeed, such increase of 7B due to the increase of the size of has been also observed previously for γ-Fe2θ3 NPs with average diameter Z)avg in the range between 5 nm and 13 nm; the observed rate for ΔJΕ/ΔDavg is between 15 K/nm and 80 K/nm (Jeong et al, 2004; Mukadam et al, 2004; Martinez-Boubeta et al, 2006). The 80 K difference in J¹B observed between our ~12 nm NPs and the ~8 nm NPs of Jeong et al. falls into this range of

[00245] A comparison between the curves shown in FIG. 1OA and 1OB indicates that with the decrease of the applied field H from 500 Oe to a much lower value 10 Oe, the Tm and 7B are both increased to above 300 K. Similar high 7m- and revalues (measured at the same field H = IO Oe) are also observed by other group (Banerjee et al, 2000) for γ-Fe2θ3 with similar particle size Davg = 11.1 nm. The observed increase of 7B with decreasing applied field was also previously observed for γ-Fe2θ3 NPs of other particle sizes (Mukadam et al., 2004; Datta et al., 2004) and it can be explained by the theoretical formula (El-Ηilo et al., 1992; Dormann et al, 1987):

TB=TBO(1-CΗ²/T_BO), (2)

where 7BO is the blocking temperature at zero field and C a field-independent parameter.

[00246] By comparing the ZFC curve in FIG. 1OC with that in FIG. 1OB, it can be seen that the blocking temperature 7B decreases from 160 K to about 80 K with the double coating of Au/Siθ2 on the γ-Fe2θ3 NPs. The 80 K decrease of the 7B due to the Au/SiO coating can be attributed to the following two factors: (i) the reduction of the average effective volume of the γ-Fe2θ3 cores and (ii) the decrease of the strength of the dipole-dipole interactions between the γ-Fe2θ3 cores. First, let us discuss the average effective volume of the γ-Fe2θ3 cores and see how it affects 7B. The TEM result above has shown that the Siθ2 nanoshells (about 35 nm thick) is coated on γ-Fe2θ3 spheres and the Au particles (Davg ~ 6 nm) are only dispersed in a thin layer near the outer surface of the Siθ2 nanoshells. This result means that the Fe ions located near surface of the γ-Fe2θ3 cores can only interact with the Siθ2 (silica) near the γ- Fe2θ3/Siθ2 interface, not with the Au particles. Such an interaction between Fe ions and Siθ2 could produce a thin layer of misaligned or disordered Fe spins near the surface of the spherical γ-Fe2θ3 cores. The spins in this magnetically disordered layer should have negligible contribution to the measure total magnetization M for the sample and thus can be excluded from the particle volume V m Equation (1). Thus, if the average thickness of the spin disordered layers is t, an average effective volume can be defined, Ves (Rosa et al. , 2005), for the γ-Fe2θ3 cores in the Au/Siθ2 coated NPs:

V_eff = π(D_avg - 2t)³/6 (3) [00247] Then, Equ. (1) should be replaced by

T_B=KV_eff/25£_B (4)

[00248] Combining Equ. (3) and (4), we have:

T_B(t)-T_B(0)(l-(2t/D_avg))³ (5)

Recently, Rosa et al measured the value of t for γ-Fe2θ3 NPs embedded in an amorphous SiCh matrix using a Faraday rotation technique. They found that t is 1.25 ± 0.07 nm and is almost unchanged for all γ-Fe2θ3 NPs with the average diameter in the range of 6.2 nm ≤ Davg ≤ 21.8 nm. For the above described γ-Fe2θ3 NPs, Davg = 12.4 nm falls into this range and thus t = 1.25 nm can be used for estimating 7B. Using Equation (5) with IB(O) = 160 K, Davg =12.4 nm, and t = 1.25 nm, the 7B for the Au/SiCte coated γ-Fe2θ3 NPs is estimated to be 8 IK, which is in excellent agreement with the experimental value 80 K.

[00249] The second factor which could contribute to the decrease of 7B is the reduction of the strength of magnetic dipole-dipole interaction due to the Au/SiCte coating. The dipole interaction between magnetic nanoparticles 1 and 2 is given by

[00250] Where μ_\ and μ₂ are the magnetic moments of these two nanoparticles separated by distance r₁₂. By Monte Carlo simulation (Carcia-Otero et al., 2000), it has been proven that blocking temperature decreases with the decrease of the strength of Uu. Equation (6) indicates that the strength of Un decreases with the decrease of the magnitude of the magnetic moments and increase of the separation r₁₂. For the Au/SiCte doubly coated particles, the Au/SiCte coating decreases the strength of Un by (1) the decrease of the magnetic moments of the γ-Fe2θ3 NPs due to the reduction of the effective volume VeS and (2) the increase of r₁₂ due to the separation of the neighboring γ-Fe2θ3 NPs by the coated Au/SiCte shells.

[00251] FIG. 11 shows the magnetic hysterysis M(H) loops of both the uncoated and coated γ- Fe2θ3NPs, measured up to 50 kOe and at different temperatures from 5 K to 300 K. In FIG. 12, these hysteresis loops in the zoomed region between H = -2 k Oe and 2 kOe are shown to see more clearly the irreversibility in this region. For fields greater than 2 kOe, all the M(H) hysteresis loops for both the uncoated and coated Fe2θ3 particles are reversible at all temperatures. For field less than 2 kθe, FIG. 12 shows that for the uncoated particles, the M(H) hysteresis loops are completely reversible or superparamagnetic only for in the temperature range T ≥ 100 K. For the coated particles, the irreversibility is seen in FIG. 12 at all temperatures. Even at 300 K, there is a very small but noticeable irreversibility within ± 50 Oe. Thus, it seems the Au/SiCh coating extends the irreversaibility to higher temperatures. Since the irreversibility of the hysteresis loop for the coated particle is extremely small in the temperature range 100 ≤ T ≤ 300 K, it can be said that the Au/SiCh coated γ-Fe2θ3 NPs are almost superparamagnetic in this temperature range. Another effect of Au/SiCh coating on the M(H) loops is the decrease of the Hm, which is defined as the field at which the irreversibility occurs. For example, from FIG. 12, it is seen that at 5 K, Hm is about 1 kOe for the Au/SiCh coated γ-Fe2θ3 NPs but about 2 kOe for the uncoated γ-Fe2θ3

[00252] From FIG. 11 it can be seen that the saturation moment per gram, Msat, is about 73 emu/g at 5 K and 50 kOe for the uncoated γ-Fe2θ3 NPs s, corresponding to 2.07 μβ per formula unit (f.u.), or 1.03 μβ/Fe3+. This value is about 83% of the resultant moment (1.25 μβ/Fe3+) of the bulk ferrimagnetic maghemite (γ-Fe2θ3). With the increase of temperature from 5 K to 300 K, Msat decreases monotonically from 73 emu/g to 61 emu/g. This Msat value (61 emu/g) is in excellent agreement with that reported by some groups on their uncoated γ- Fe2θ3 NPs with similar size.

[00253] For the Au/SiCh coated γ-Fe2θ3 NPs, the Mat value at 300 K and 50 kOe is about 5.6 emu/g, which is about 7.7% of the Msat value (73 emu/g) for the uncoated γ-Fe2θ3 NPs. This decrease in Msat is due the increase of the mass per γ-Fe2θ3 NP by coating Au/SiCh on the γ- Fe2θ3 NPs. A simple calculation using the values of the mass density and measured Davg for γ-Fe2θ3 core, Au particles, and SiCh matrix can confirm that such decrease of Msat requires an average mass density of 0.2 g/cm³ for the Au/SiCh nanoshells, which corresponds to 99.53% volume fraction of SiCh and 0.07% volume fraction of Au with the use of mass density 0.11 g/cm³ for SiCh and 19.3 g/cm³ for Au. The observed Msat value (5.6 emu/g) for the coated particles is in accordance with previously reported data for silica coated γ-Fe2Ch NPs which have similar sizes. From FIG. 12, it can be see that Msat for the coated NPs does not vary monotonically with the decrease of temperature. The Msat at 50 kOe has about 15% variation in the temperature range from 300 K to 16 K, but it decreases substantially when temperature changes from 16 K to 5 K. At present the origins of such a sharp decrease of Msat at 5 K is unclear. [00254] It is well-known that the coercivity, Hc, for superparamagnetic systems varies with temperature according to the well-known expression (Tartaj et al., 2004; Jeong et al., 2004):

Η_c/Ηco = l-(T/T_B)^1/2 (7)

where Hco is the coercivity at OK. Thus, in FIG. 10 it is shown that the Hc vs. Tm plots below the blocking temperature 7B for both the coated and uncoated γ-Fe2θ3 NPs, with the values of Hc obtained from the hysteresis loops shown in FIG. 12. The data in FIG. 13 show that the 7V₂- dependance of Hc is slightly deviated from linearity, such deviation from linearity has been previously observed for SiCh-coated γ-Fe2θ3 NPs (Tartaj et al., 2004). From the least square fitting (the straight lines in FIG. 13) of the data by Equation 7, it is found that Hco = 239 Oe and TB = 139.3 K for the uncoated γ-Fe2θs NPs, and Hco = 263 Oe and TB = 113.4 K for the Au/Siθ2 coated γ-Fe2θ3 NPs. This fitting result shows that Au/Siθ2 coating increases Hc but decreases 7B. The decrease of 7B by coating is consistent with the 7B results obtained from the ZFC M(T) curves in FIG. 7. However, the data points in FIG. 13 indicate that Hc is increased by coating only at low temperatures, i.e., below 5OK. Above 50 K, the values of Hc for the coated NPs are actually slightly smaller than that for the uncoated NPs.

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Claims

1. A hybrid nanoparticle comprising:

(a) a superparamagnetic core; and

(b) an optically interrogatable shell.

2. The nanoparticle of claim 1 operatively coupled to a targeting moiety.

3. The nanoparticle of claim 2, wherein the targeting moiety is an antibody, a monoclonal antibody, peptide, small molecule, or a nucleic acid.

4. The nanoparticle of claim 1, wherein the optically interrogatable shell is a metal layer.

5. The nanoparticle of claim 4, wherein the optically interrogatable shell is gold, silver, copper, platinum, palladium, lead, or iron.

6. The nanoparticle of claim 5, where in the optically interrogatable shell is gold.

7. The nanoparticle of claim 1, wherein the core is iron oxide

8. The nanoparticle of claim 1 , further comprising an intermediate layer between the core and the shell.

9. The nanoparticle of claim 8, wherein the intermediate layer is a non-conducting or conducting layer.

10. The nanoparticle of claim 8, wherein the intermediate layer is comprised of a polymer, an insulator, or a semiconducting material.

11. The nanoparticle of claim 10, wherein the intermediate layer is comprised of silica, a dielectric, silicon dioxide, titanium dioxide, polymethyl methacrylate, poystyrene, gold sulfide, CdSe, CdS, or GaAs.

12. A method of photoablation therapy comprising:

(a) administering a hybrid nanoparticle composition of claim 1 to a patient in need of cancer therapy; and

(b) subjecting subject to phototherapy.

13. The method of claim 12, wherein the hybrid naoparticle is localized to a location by application of a magnetic field.

14. The method of claim 13, wherein the localized hybrid nanoparticles are illuminated.

15. The method of claim 12, wherein hybrid nanoparticle distribution are imaged non- invasively with T2 and T2* weighted magnetic resonance imaging.

16. The method of claim 15, further comprising calculating a thermal dose for photoablation therapy.

17. The method of claim 12, wherein the hybrid nanoparticles have an absorption maximum between 530 and 600 nm.

18. The method of claim 12, wherein the hybrid nanoparticle is coupled to a targeting moiety that binds a cancer cell.

19. The method of claim 18, wherein the targeting moiety is an antibody that binds a cancer biomarker.

20. The method of claim 19, wherein the antibody is an anti-EGFR antibody.

21. The method of claim 12 wherein the nanoparticles form a nanoparticle assembly on the surface of a target cell.

22. A method of imaging comprising:

(a) contacting a target location with a hybrid nanoparticle coupled to a moiety that binds to a biomarker of interest present in the target location;

(b) applying an external magnetic field; and

(c) detecting oscillating hybrid nanoparticles using image analysis that is sensitive to periodic movements.

23. The method of claim 22, wherein image analysis is Fourier image analysis.

24. The method of claim 22, wherein the biomarker is epidermal growth factor receptor.