WO2016007194A1 - Companion nanoparticles for theranosis of macrophage-dependent diseases - Google Patents

Abstract

Many important chronic diseases are composed, in part, of activated macrophages which participate in the perpetuation of the disease but also can be used to target diagnostic and therapeutic nanoparticles. After co-localization of iron oxide and therapeutic nanoparticles in the activated macrophages, the nanoparticles can be locally heated by diathermic devices resulting in hyperthermic consequences and release of the therapeutic. This local release creates effective treatments without or with low systemic toxicity. The surprising result is companion nanoparticles theranosis for macrophage-dependent diseases.

Description

COMPANION NANOPARTICLES FOR THERANOSIS

OF MACROPHAGE-DEPENDENT DISEASES

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date, under 35 U.S.C. § 119(e), of U.S. Provisional Application No. 61/998,854, filed on July 10, 2014, the entire contents of which are incorporated herein by reference.

This application is also related to but does not claim priority to U.S. Provisional Application No. 61/690,005, filed on June 18, 2012; U.S. Provisional Application No.

61/690,006, filed on June 18, 2012; U.S. Provisional Application No. 61/742,382, filed on August 9, 2012; U.S. Provisional Application No. 61/743,428, filed on September 4, 2012; U.S. Provisional Application No. 61/797,757, filed on December 14, 2012; U.S. Provisional Application No. 61/779,123, filed on March 13, 2013, U.S.S.N. 13/920,694, filed on June 18, 2013 and published as US-2013-0336897-A1 on December 19, 2013, and its continuation application U.S.S.N. 14/202,044, filed on March 10, 2014, and published as US-2014- 0249413-A1 on September 4, 2014. The entire content of each of these applications, including drawings, are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Medical imaging is the technique and process for creating images of the live human body or parts thereof for clinical purposes, most notably for diagnostic uses or for medical science research. Over the years, numerous medical imaging devices and technologies have been developed based on distinct scientific principles that incorporate radiology, nuclear medicine, investigative radiological sciences, endoscopy, medical thermography, medical photography, and microscopy. To date, the most widely used medical imaging technologies utilize the effect of tissues upon the transmission, scattering, or absorption of energy. The energy can take the form of ionizing radiation, as used for numerous x-ray techniques, such as plain films; or computed tomography (CT scan, or Computed Axial Tomography or CAT scan); or nuclear medicine, techniques where the radiation is emitted by radioactive isotopes as used in scintigraphy, Single Photon Emission Tomography (SPECT), or Positron Emission Tomography (PET). However, these forms of radiation do carry a potential burden of causing adverse effects when their energetic photons damage the body's genes. In addition, macrophages do not have special properties that make them visible with these forms of radiation, and there are currently no good biomarkers that can be used to selectively image macrophages with this form of radiation.

Some less harmful energy forms may be used to image the macrophages.

Magnetic Resonance Imaging (MRI), also known as nuclear magnetic resonance imaging (NMRI), or magnetic resonance tomography (MRT) is a medical imaging technique used to visualize internal structures of the body. Body tissue contains large amounts of water (H₂0), and hence protons (1H nuclei) since each water molecule has two hydrogen nuclei (protons). The protons can be aligned in a strong magnetic field generated by the powerful magnet of an MRI scanner. When a person is inside this magnetic field, the average magnetic moment of many protons becomes aligned with the direction of the field. A radio frequency current is briefly turned on, producing a varying electromagnetic field. This electromagnetic field has just the right frequency, known as the resonance frequency, to be absorbed and flip the spin of the protons in the magnetic field. After the electromagnetic field is turned off, the spins of the protons return to thermodynamic equilibrium and the bulk magnetization becomes re-aligned with the static magnetic field. During this relaxation, a radio frequency signal (electromagnetic radiation in the RF range) is generated, which can be recorded and measured with receiver coils of the MRI scanner. The recorded information is used to construct an image of the scanned area of the body. The energy absorbed by the body during MRI is small, and it is thought that MRI can be used safely and repeatedly for diagnosis.

MRI provides good contrast between the different soft tissues of the body and can be used to image every part of the body, particularly for tissues with many hydrogen nuclei, such as the brain, muscle, connective tissue and most tumors, because the composition of these tissues influences the relaxation of the protons which are imaged. However, macrophages look quite similar to surrounding tissues when imaged with MRI.

To enhance the appearance of blood vessels, tumors or inflammation in MRI, certain MRI contrast agents may be injected intravenously. MRI contrast agents are a group of contrast media or biomarkers used to improve the visibility of internal body structures in MRI. They alter the relaxation times of nearby atoms within body tissues wherever they are present after administration.

Gadolinium (III) containing contrast agents are the most commonly used MRI biomarkers for enhancement of vessels in MR angiography or for brain tumor enhancement associated with the degradation of the blood-brain barrier. Without attachment to large targeting molecules, these common MR contrast agents do not accumulate in cells, including macrophages.

The other type of MRI biomarker is iron oxide based contrasting agents, which include superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO). These contrast agents consist of suspended colloids of magnetizable iron oxide nanoparticles, and, when injected, increase the relaxation of nearby protons in ways that are clearly imageable by the scanner and its programs.

MRI contrast agents may be administered by injection into the blood stream, interstitially or per os. Oral administration is well suited to G.I. tract scans, while

intravascular administration may be more useful for most other scans. Interstitial

administration is useful for lymphatic imaging.

Medical ultrasonography (or simply "ultrasound") uses high frequency broadband sound waves in the megahertz range that are reflected by tissue to varying degrees to produce up to 4D images. Ultrasound is an oscillating sound pressure wave with a frequency greater than the upper limit of the human hearing range. Thus ultrasound is not separated from audible sound based on differences in physical properties, but only on the fact that humans cannot hear it. Although this limit varies from person to person, it is about 20 kilohertz (20,000 Hz) in healthy, young adults.

Ultrasound devices operate with frequencies from 20 kHz (2 x 10⁴ Hz) up to several gigahertz (1 x 10⁹ Hz). Ultrasonic imaging typically uses frequencies of 2 megahertz (2 x 10⁶ Hz) and higher - the shorter wavelength allows resolution of small internal details in structures and tissues. A 3 GHz sound wave can produce an image resolution comparable to that of an optical image. The power density is generally less than 1 watt per square centimeter, in order to avoid heating and cavitation effects in the object under examination. Thus, ultrasound used for imaging is considered quite safe.

Ultrasound imaging is commonly associated with imaging the fetus in pregnant women, but is also broadly used in, for example, imaging the abdominal organs, heart, breast, muscles, tendons, arteries and veins. While it may provide less anatomical detail than techniques such as CT or MRI, it has several advantages which make it ideal in numerous situations, in particular that it studies the function of moving structures in real-time, and emits no ionizing radiation, and contains speckle that can be used in elastography (a non-invasive method in which stiffness or strain images of soft tissue are used to detect or classify tumors, based on the fact that a tumor or a suspicious cancerous growth is normally 5-28 times stiffer than the background of normal soft tissue. Thus when a mechanical compression or vibration is applied, the tumor deforms less than the surrounding tissue).

Ultrasound is relatively inexpensive and quick to perform. Ultrasound scanners are portable and can be taken to the patient's location. Although there are ultrasound contrast agents composed of gas bubbles that are readily visualized, these agents are relatively large with sizes hundreds of times larger than USPIOs and their persistence time is measure in minutes. They are not currently useful for imaging activated macrophages.

Optical Coherence Tomography, or "OCT," is a technique for obtaining sub-surface images of translucent or opaque materials at a resolution equivalent to a low-power microscope. It images light wave reflections from within tissue to provide cross-sectional images.

During OCT, an optical beam is directed at the tissue, and a small portion of this light that reflects from sub-surface features is collected. Most light, however, is not reflected but scatters off at large angles. In conventional imaging, this diffusely scattered light contributes background noise that obscures an image. However, in OCT, a technique called

interferometry, is used to record the optical path length of received photons allowing rejection of most photons that scatter multiple times before detection. Thus OCT can build up clear 3D images of thick samples by rejecting background signal while collecting light directly reflected from surfaces of interest. The technique, however, is limited to imaging 1 to 2 mm below the surface in biological tissue, because at greater depths, the proportion of light that escapes without scattering is too small to be detected. In order to distinguish tissues by means other than their location, special fluorescent chemicals or photodynamic agents must be given.

Cellular phagocytosis is an ancient capability for some cells, perhaps appearing in evolution as early as the amoeba. Common to more advanced species, macrophages have evolved numerous receptors on their cell membranes to discriminate what materials constitute a threat to the organism and should be ingested. In this way, macrophages serve as an internal defense against pathogens. Macrophages also endocytose a wide variety of nanoparticles, where they are aggregated and stored in lysosomes until released or metabolized.

Macrophages can be divided into normal or inflammatory macrophages. The former are also consider fixed or sessile, and are normally present within the body organs where they have been named Kuppfer cells (liver), histiocytes (muscle), dendritic cells (skin), or just identified by location (e.g. , spleen, lymph node, alveoli, adrenal, or bone marrow macrophages). Thus such macrophages are also referred to as "tissue macrophages."

On the other hand, inflammation is characterized by local accumulation of cells from the blood, including monocytes, the precursors of macrophages, and inflammatory macrophages (also known as activated macrophages, or Inflammation-Associated

Macrophages (IAMs)) invade diseased tissues in response to a "danger signal." When the disease is neoplastic, the activated macrophages are called Tumor-Associated macrophages (TAMs). Under normal circumstances, inflammatory reactions are part of the host defense, playing a critical role in the eradication of infectious agents and removing debris. However, inflammatory processes are intrinsically destructive to normal tissues, and they can in certain circumstances do far more harm than good. Thus, activated macrophages play a key pathophysiologic role in many common inflammatory diseases, disorders or conditions, including primary and metastatic tumors.

SUMMARY OF THE INVENTION

Accurate diagnosis and selective therapy for macrophage-dependent or associated diseases, such as inflammatory diseases, primary and metastatic cancer, vulnerable arterial plaque, chronic obstructive pulmonary disease, periodontal disease, rheumatoid arthritis, etc., have not previously been achieved with a theranostic approach. Yet, these diseases share certain pathophysiologic processes which can be imaged and subsequently treated based upon the activities of macrophages that are key cellular components for such diseases.

Thus a class of diagnostic agents useful for the instant invention is iron oxide nanoparticles, which are avidly accumulated from presenting fluids. The nanoparticles are phagocytosed or engulfed by receptor-mediated endocytosis, with each nanoparticle being separately captured. This process takes some time, and, when the number of presented nanoparticles is large, it may require many hours to aggregate them. This slow process, however, creates very large aggregates of nanoparticles, with resulting large increases in scattering and absorption of impinging electromagnetic fields. This in turn provides tissue differences which can be used for imaging, such as imaging by MRI, OCT, or US, or for hyperthermia with electromagnetic wave lengths from laser, microwave, radiofrequency, or high intensity ultrasound, which heat the lysosomes more effectively than surrounding biological fluids and create heat sinks within the inflamed tissues. In similar manner, phagophilic nanoparticulate therapeutic agents (or "phagophilic nanotherapeutics") can also be captured by macrophages, including both normal and activated macrophages. The bulk of systemically-administered therapeutic nanoparticles accumulates in liver and spleen. Phagocytosis of such nanotherapeutics is receptor-based, as described for USPIOs. In order to provide sufficient time for presentation to macrophages, these nanotherapeutics are often coated with materials that provide for a long blood half-life, and they are sometimes called "stealth" nanoparticles. Once internalized into the

macrophages, this pool of nanotherapeutics is slowly released and/or metabolized. When given in the same time frame as the USPIO nanoparticles, the phagophilic nanotherapeutics are also contained or co-localized within the lysosome. The co-localized nanotherapeutics are said to be "companions" to the USPIO nanoparticles - hence "companion

nanotherapeutics" or "companion nanoparticles."

To achieve the subject "companion (nanoparticle) theranosis" (diagnosis and therapy) with the two classes of nanoparticles described above - one (the USPIO nanoparticle) a macrophage imaging agent and the other (the companion nanoparticles) a therapeutic effective for the macrophage-dependent disease, each must be internally present in the appropriate amounts at around the same approximate time. The subject companion theranosis is the composite result of imaging the USPIO nanoparticles, and releasing the companion nanotherapeutics by rapid, local heating of the nanoparticle-loaded macrophage within the diseased tissue. The temperature created within the macrophages, and the period for which it is sustained, influence the therapeutic response of the macrophage-dependent disease. Upon release, the same pathophysiology that led to retention of the nanoparticles upon presentation to the macrophage, will retard clearance of the nanotherapeutics and facilitate the desired response.

Controlled local release, along with delayed clearance in the inflamed tissue, create high local drug concentrations and result in much improved therapeutic ratios, since the nanoparticles in other macrophages are not influenced by focal heating.

Both Rayleigh and Mie scattering emphasize the dependence on the ratio between particle size and the imaging wavelength. A single or only a few USPIO nanoparticles of about 30 nm would be relatively inefficient contrast agents at wavelengths used for ultrasonic imaging devices or for optical coherent tomography devices. In either case, within the sensitive scattering region for both devices, scattering increases dramatically with increased effective particle size, such as the enlarged size (typically > 100 nm, or more than 500 nm, or up to about 2 μηι) of the USPIO aggregates formed inside the lysosomal / endosomal compartment of the macrophage that has phagocytized the nanoparticles. While not wishing to be bound by any particular theory, Applicants believe that other mechanisms, such as acoustic or optical mismatches, may further contribute to detectable changes in

electromagnetic wave interactions within the loaded macrophages, since cells do not normally contain large amounts of inorganic particles.

While not wishing to be bound by any particular theory, Applicant believes that other mechanisms, such as acoustic or optical mismatches, may further contribute to detectable changes in electromagnetic wave interactions within the loaded macrophages.

Since cells do not normally contain large amounts of nanoparticle aggregates, they can be more effectively heated with diathermic devices (devices for therapeutic generation of local heat in body tissues), and the heat absorption will conform to the distribution of the activated macrophages, which closely mirrors the geometry of the tissue to be treated.

Heating of tissues, or diathermy, can be accomplished with light from lasers, microwaves, radiofrequency wave, or focused ultrasound. Normally it is basic tissue constituents which absorb the energy and create heat. However, when the target tissue to be heated contains nanoparticles-loaded macrophages, heating is much more efficient. This is true for all of the listed diathermia energies. However, USPIOs can also generate heat when in a strong magnetic field when the particles are rapidly oscillated, producing heat, a technique known as "magnetic fluid hyperthermia." The utility of focally heating iron oxide nanoparticles in macrophage-dependent diseases has been explored in prior patent applications referenced in the first paragraph that are incorporated herein.

Thus one aspect of the invention provides a method for detecting activated macrophages in a subject, and for treating a macrophage-dependent disease or condition associated with said activated macrophages, the method comprising: (a) administering to the subject a formulation of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (which may aggregate in the lysosomes of said activated macrophages); (b) administering a nanoparticulate therapeutic agent (nanotherapeutic) that is phagocytosed by said activated macrophages, wherein said nanoparticulate therapeutic agent is effective in treating said disease or condition; (c) waiting for a pre-determined time to allow the USPIO nanoparticles to accumulate as USPIO aggregates inside said activated macrophages, and to allow the nanoparticulate therapeutic agent to accumulate inside said activated macrophages;

(d) visualizing the USPIO within the activated macrophages with a medical imaging device sensitive to the presence of the USPIO aggregates to identify size, shape, and/or location of a tissue affected by the disease or condition; and, (e) locally raising the temperature of (e.g., heating) the activated macrophages containing the nanoparticulate therapeutic agent, by directing an energy that is absorbed by the USPIO aggregates, and sustaining the locally elevated temperature for a time sufficient to release the nanoparticulate therapeutic agent within or from the activated macrophages in tissues affected by the disease or condition. In certain embodiments, the methods of the invention result in treatment of the macrophage dependent disease, e.g. , improving at least one symptom of the disease, inhibiting or slowing down the progression of the disease, etc.

In certain embodiments, step (d) is carried out using any suitable medical imaging device including MRI. In certain embodiments, in step (d), the medical imaging device produces an image based at least in part on Rayleigh and/or Mie scattering by the aggregates.

In certain embodiments, the method further comprises evaluating the USPIO- enhanced image, and optionally the clinical circumstances, to determine (1) the need for treatment, (2) potential treatment options using the co-administration of appropriate nanotherapeutics for accumulation within the activated macrophages for treating the disease existing at one or more sites harboring said activated macrophages, at the time of evaluation, and/or (3) determining the need for further USPIO dosing regimens to make such treatments feasible. In certain embodiments, step (d) is carried out using any suitable medical imaging device including MRI. In certain embodiments, in step (d), the medical imaging device produces an image based at least in part on Rayleigh and/or Mie scattering by the aggregates.

In certain embodiments, step (e) is carried out by exposing the activated macrophages to a diathermic device utilizing electromagnetic energy (e.g. , light, microwave,

radiofrequency, or ultrasound frequencies emitted by those medical devices designed for that purpose).

In certain embodiments, the energy is high intensity ultrasound energy (e.g. , one delivered by a high intensity ultrasound energy device operating with frequencies between 2 and 60 MHz).

In certain embodiments, the energy is radiofrequency (RF) energy (e.g. , one delivered by a (medically) approved device utilizing radiofrequency wavelengths with a frequency within the ISM bands for such devices). In certain embodiments, the energy is laser energy (e.g. , one delivered by a laser approved for medical treatments and operating within the ultraviolet, visible, or infrared frequencies).

In certain embodiments, the energy is microwave energy (e.g. , one delivered by an approved microwave device with a frequency within the ISM bands for such devices).

In certain embodiments, the diathermy is performed within a strong magnetic field with alternating magnetic fields to cause rotation of the magnetic dipoles within the iron oxide nanoparticulate.

In certain embodiments, the method further comprises assessing macrophage density during therapeutic intervention in order to determine the desirable continuation, change, or cessation of a particular therapy.

In certain embodiments, the USPIO nanoparticles and/or the nanoparticulate therapeutic agent have an average hydrodynamic particle size of about 15-800 nm as determined by dynamic light scattering. In certain embodiments, the USPIO nanoparticles or the nanoparticulate therapeutic agents have an average size of about 15-250 nm, about 20-125 nm, or about 22-40 nm.

In certain embodiments, the USPIO nanoparticles and/or the nanoparticulate therapeutic agent are sufficiently small to extravasate and diffuse through restricted extracellular matrix surrounding said activated macrophages.

In certain embodiments, the formulation of USPIO nanoparticles is administered to the subject percutaneously, intravenously, by inhalation or ingestion, or otherwise into a body cavity connected to the outside (e.g. , rectal or oral, or spraying into, for example, the nostrils or respiratory tract).

In certain embodiments, the formulation of USPIO nanoparticles is administered to the subject: (1) intravenously at a dose of about 0.5 to 20 mg/kg, optionally repeated (as necessary) with a waiting time between 12 and 144 hours or longer; (2) interstitially (for nodal enhancement) at a dose of about 0.01-2 mg/kg, with stimulation of lymphatic uptake as feasible, and, optionally repeated (as necessary) with a waiting time of between 30 min- 14 days; or, (3) intracavitarily at a dose of about 0.05-2 mg/kg (in an appropriate suspension), and waiting 30 min-14 days before imaging.

As used herein, "nanoparticulate/nanoparticle therapeutic agents," "companion (nano)therapeutic agents," or simply "nanotherapeutics," are used interchangeably, and generally have two parts: the active pharmaceutical ingredient (API) as the core, and a nanocarrier coating. The coating may be a biocompatible and/or biodegradable common coat, which may include synthetic polymers or self-assembled lipids (such as liposomes, dendrimers, micelles, etc.). The core API can be any of many drugs known to be effective to treat the subject macrophage-dependent diseases, such as an antineoplastic (e.g. , doxorubicin or cisplatin), an anti-infective (e.g. , antimicrobial or antiviral), or an anti-inflammatory agent (e.g. , prednisone, a cytokine blocking agent against TNF-alpha, or a siRNA preventing the formation of inflammatory chemo/cytokines), just to name a few.

For use in macrophage-dependent diseases, the nanotherapeutics have a long blood half-life (e.g. , measured in hours), which may be facilitated by a size larger than 15 nm (beneficial for escaping renal clearance), and by a size smaller than about 800 nm (to allow extravasation in the presence of leaky vasculature, such as those found in the subject macrophage-dependent diseases). In certain embodiments, the nanotherapeutics of the invention is in the size range of about 25- 100 nm, and are delivered to the macrophage via the EPR phenomenon. Such smaller sizes are believed to be beneficial to facilitate diffusion through the extracelluar space to the activated macrophage.

The coating of the nanotherapeutics may comprise a material that can bind to common macrophage receptors, or may contain a ligand that serves the same purpose.

Since it is the purpose of the invention to load the activated macrophages with nanoparticles, the parameters of effective phagocytosis are similar for both USPIO

formulations and nanotherapeutic formulations. Thus, understanding, teaching, or improvements identified with either might apply to the other. This applies with respect to dose/time, phagophilicity of coating, size, etc.

In certain embodiments, the nanoparticulate therapeutic agent is administered systemically, interstitially, or per os.

In certain embodiments, the macrophage-associated disease or condition comprises: primary or metastatic cancer, vulnerable plaque, rheumatoid arthritis, inflammatory bowel disease, chronic obstructive pulmonary disease, bronchial asthma, periodontal disease, or transplant rejection.

In certain embodiments, the macrophage-associated disease or condition is an "inflammatory disease, disorder, or otherwise abnormal condition," which may include disorders associated with inflammation or have an inflammation component, such as, but are not limited to: acne vulgaris, asthma, autoimmune diseases, celiac disease, prostatitis, glomerulonephritis, hypersensitivities, Crohn' s disease, ulcerative colitis, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, vasculitis, interstitial cystitis, atherosclerosis, allergies (type 1, 2, and 3 hypersensitivity, hay fever), inflammatory myopathies, as systemic sclerosis, and include dermatomyositis, polymyositis, inclusion body myositis, Chediak-Higashi syndrome, chronic granulomatous disease, Vitamin A deficiency, periodontitis, Granulomatous inflammation (tuberculosis, leprosy, and syphilis), fibrinous inflammation, purulent inflammation, serous inflammation, ulcerative inflammation, and ischemic heart disease, type I diabetes, and diabetic nephropathy.

In certain embodiments, the inflammatory disease, disorder, or otherwise abnormal condition includes many autoimmune diseases or disorders that are associated with inflammation or have an inflammation component, e.g. , corresponding to one or more types of hypersensitivity. Exemplary autoimmune diseases or disorders that correspond to one or more types of hypersensitivity include: atopic allergy, atopic dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune polyendocrine syndrome, autoimmune urticaria, celiac disease, cold agglutinin disease, contact dermatitis, Crohn' s disease, diabetes mellitus type 1, discoid lupus erythematosus, erythroblastosis fetalis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's encephalopathy, Hashimoto' s thyroiditis, idiopathic thrombocytopenic purpura, autoimmune

thrombocytopenic purpura, IgA nephropathy, lupus erythematosus, Meniere's disease, multiple sclerosis, myasthenia gravis, narcolepsy, neuromyelitis optica, Devic' s disease, neuromyotonia, ocular cicatricial pemphigoid, opsoclonus myoclonus syndrome, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus),

paraneoplastic cerebellar degeneration, pemphigus vulgaris, pernicious anaemia, psoriasis, psoriatic arthritis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, subacute bacterial endocarditis (SBE), systemic lupus erythematosis, lupus erythematosis, temporal arteritis (also known as "giant cell arteritis"), thrombocytopenia, ulcerative colitis, undifferentiated connective tissue disease, urticarial vasculitis, and vasculitis.

In certain embodiments, the pre-determined time (e.g., 30 min to 14 days) is sufficient to allow the USPIO nanoparticles to accumulate as aggregates of at least about 100 nm in size, or 1 μιη in size, or 1.5 μιη in size, or 2 μιη in size, preferably occupying 2-80% of the macrophage cell volume. In certain embodiments, the method further comprises controlling the size of the aggregates by dose, administration route, waiting time, and frequency of the USPIO and/or nanoparticulate therapeutic agent formulation administered to said subject.

In certain embodiments, the active agent (iron oxide or nanotherapeutic) of the nanoparticles is coated by a biocompatible polymer (e.g., PEG) or contained within a liposome or a micelle.

In certain embodiments, the medical imaging device is MRI (e.g., Macrophage- Enhanced MEMRI), ultrasound (e.g. , Macrophage-Enhanced ultrasound - MEUS), or optical imaging (e.g. , Macrophage-Enhanced Optical - MEOCT).

In certain embodiments, the macrophage-associated disease or condition comprises primary or metastatic cancer, and the interventional device is positioned based on MEUS or MEOCT images.

In certain embodiments, the macrophage-associated disease or condition comprises primary or metastatic cancer, and biopsy or surgical intervention (e.g. , surgical intervention with minimally invasive devices) is performed based on the MEUS or MEOCT images.

In certain embodiments, the temperature in the activated macrophage in tissues affected by the disease or condition is raised sufficiently to effect at least one of the following: (a) increased cell membrane permeability within the activated macrophage loaded with the nanoparticulate therapeutic agent; (b) apoptosis of the activated macrophage loaded with the nanoparticulate therapeutic agent; (c) rapid necrosis of the activated macrophage loaded with the nanoparticulate therapeutic agent; and, (d) increased cell permeability, apoptosis, or necrosis of adjacent cells (e.g., due to diffusion of a heat sink within the adjacent cells, such as macrophage).

For example, for (a), an induced temperature of 43-48°C for 1- 10 minutes, or an induced temperature of 44°C for 5 minutes may be sufficient. For (b), an induced

temperature of 45-60°C for 1- 10 minutes, or 50°C for 5 minutes may be sufficient. For (c), an induced temperature of 60-90°C for 1-10 minutes, or 70°C for 5 minutes may be sufficient.

In certain embodiments, the nanoparticulate therapeutic agent is phagophilic (e.g. , due to its size, shape, coating or bonding to other phagophilic carriers). In certain embodiments, release of the nanop articulate therapeutic agent causes killing of the activated macrophage, or suppression of cytokine or chemokine production by the activated macrophage.

In certain embodiments, the method leads to amelioration of the macrophage- dependent disease or condition.

Unless specified otherwise, doses of the USPIO nanoparticles used herein are expressed as "mg Fe/kg," or "mg/kg" for short.

It should be understood that any embodiments described herein, including those described under different aspects of the invention, in different sections of the specification including the Examples, can all be combined with any other embodiments of the invention whenever appropriate.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

The presentation of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles or companion nanotherapeutics to macrophages depends upon their size, their route of administration, and the location of the challenged macrophage.

USPIO particles have originally been developed as contrast agents or biomarkers for MRI. Due to their nature, in general, these particles consist of a solid core of magnetizable iron oxides, and a coating material suitable for the intended use. They can range in size up to 200 nm, but usually are 15-45 nm as measured by in vitro light scattering techniques.

The challenged macrophages may be those normal to a tissue such as liver, spleen, lymph node, lung, intestine, adrenal cortex or bone marrow, or those not normally present in a tissue but representing a part of the pathophysiologic response occurring in that tissue. In both cases, the sequestered nanoparticles increase scattering of ultrasonic and optical wavelengths from the locally loaded macrophages, thus providing the medical utility of, for example, identifying and/or assessing the local macrophage populations associated with the disease, disorder, or condition. Medical imaging devices that are developed to use radiofrequency or microwave would also be affected by the aggregated nanoparticles.

The route of nanoparticle administration also relates to the presentation to the macrophages. When the nanoparticles are presented in the blood, liver and splenic macrophages can remove them directly as these macrophages line the respective organ blood sinusoids. Lymph nodes, bone marrow, and adrenal macrophages are extravascular, and the nanoparticle must first extravasate through their fenestrated capillaries. Nanoparticles administered by intracavitary application are taken up by macrophages that have ready access to the adjacent lumen, being separated by only surface coatings. Nanoparticles given interstitially gain access to the terminal lymphatics at that location. These nanoparticles can be effective at larger sizes and provide bigger payloads.

Under normal circumstances, for fixed macrophages, larger nanoparticles such as ferumoxides (e.g. , Feridex) are rapidly removed by liver and spleen, and their short circulation time and larger size minimize uptake in the other fixed and inflammatory macrophages. Smaller USPIOs such as ferumoxytol and ferumoxytran- 10, with a median size of about 30 nm, when combined with a longer circulation time, do get presented to fixed and inflammatory macrophages where they are readily ingested.

Many diseases, including cancer, elicit chronic inflammation in the body. Despite the great diversity of pathological events that elicit the process, there are common inflammatory responses in the affected tissues. Following local incitement, the affected tissues are invaded by monocytes, perhaps in response to a "danger signal," and these monocytes then differentiate into activated macrophages. Much of the subsequent inflammatory response is due to the activity of these macrophages. For example, in their activated state, the macrophages release cytokines and chemokines that increase vascular permeability. The presence as well as the changes the activated macrophages induce in the inflamed tissue can be identified and monitored by MRI through the use of biomarkers which assess the resulting increase in local vascular permeability and excess phagocytosis.

One such class of biomarkers, ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs), is able to report both angiogenesis and macrophage infiltration common to such inflammatory diseases. For example, increased vascular permeability, as a result of chemokines produced by the activated macrophages, facilitates escape of the small USPIO nanoparticles from the blood. Once USPIOs are in the extracellular space, interstitial diffusion presents them to the activated macrophages and uptake via phagocytosis is rapid. During phagocytosis, the USPIO particles are transported and sequestered in the lysosomes of the macrophage. There, they remain until metabolized. The loading and retention within the lysosomes create tightly packed aggregates of the ingested USPIOs. The resulting aggregated nanoparticles achieve a size many times larger than the individual nanoparticles - often in the range of hundreds or thousands of nanometers. These lysosomes may contain thousands to millions of the original nanoparticles. This aggregation changes the scattering and absorption of electromagnetic waves.

For example, in a macrophage with phagocytosed USPIO nanoparticles, as visualized with TEM, many lysosomes are seen to contain aggregated USPIO nanoparticles (not shown). At higher power, the photomicrograph discerns the tight packing of USPIO nanoparticles, appearing to occupy about 80% of the imaged lysosome.

This basic physiology extends the Enhanced Permeation and Retention (EPR) effect to nanoparticles; however, the retention of USPIO at the inflammatory site is both active and long as contrasted with the EPR benefits for most small chemotherapeutics. The USPIO labeling of the chronic inflammatory site is highly local - usually within several cell diameters of the permeable capillary. This proximity is sometimes referred to as

"microscopically intimate." So long as the activated macrophages are present, extravasated USPIOs will accumulate there in close proximity to the tissues that are the source of the danger signal, and this process will continue until the inciting circumstances are eliminated. This is a goal of the treatment.

Imaging these common responses with non-invasive MRI and other suitable medical imagining devices can be used to evaluate the seriousness of the disease, its extent, the selection of treatment, the response of the disease to a current treatment regimen, or the recurrence of the disease. The safety of noninvasive imaging, such as MRI, ultrasound or optical imaging, is useful for the many times a patient with macrophage-dependent inflammation could benefit from serial measurement of the extent and activity of his/her disease.

It is important to note that USPIOs generally do not accumulate in non-phagocytic cells within the inflammatory disease unless targeted by added ligands to specific receptors on those cells. In general, targeting diagnostic or therapeutic agents to particular cells has proven difficult and often the targeting ligand, if highly specific, limits the applicability to small cohorts of patients.

The biological conundrum for macrophage-dependent or associated diseases is that particles must be adequately small to efficiently extravasate from heterogeneously enhanced vascular permeability, but not too small or they will be rapidly cleared from the blood by the kidney with a resulting inadequate blood life. Outside the capillary, small size facilitates diffusion through the extracellular space, which has little fluid flow and even reduced diffusion within the increased tissue matrix. And then to appreciably change scattering, they must eventually become quite large and adequately concentrated. Thus, the full participation of activated macrophages is required in order to create the enhanced permeability, and then to sequester and aggregate the small particles sufficiently to effectively scatter energy of various wavelengths. For biological safety, it may also be useful to have the nanoparticles composed of an essential element such as iron.

These separate observations lead to Applicant's surprising finding that administered USPIOs are cleared from their biologic fluids by macrophages, wherein their sequestration and aggregation in lysosomes increases both particle size and volume fraction sufficiently to be visualized with current medical imaging devices (including MRI medical ultrasound or optical coherence devices, or MEMRI, MEUS and MEOC, respectively). The resulting USPIO aggregates increase scattering and absorption of a wide range of probing

wavelengths, including those of light, radiofrequency, microwave and ultrasound. The accumulation of USPIOs in the tissue macrophages has great medical utility because of the wide variety of diseases that displace fixed macrophages or are associated with local accumulation of activated macrophages.

Substantially the same process described for macrophage accumulation and retention of USPIO occurs with any phagophilic nanoparticulate agent, which can be administered separately and independently, for example, as a nanoparticulate therapeutic agent. If phagophilic, this nanotherapeutic will show the same biodistribution properties described above for USPIOs. Such nanoparticulate therapeutic agent can be formulated separately from the USPIO formulation, be stored, transported, and/or administered separately (via same or different administration routes), and can be formulated in easily adjustable doses or dosage forms. Such one or more nanoparticulate therapeutic agents may be used with the USPIO nanoparticles so long as they are all taken in by the same activated macrophage. It is the ability of activated macrophages to acquire both USPIO and suitable nanotherapeutics that provides the ability to image the diseased tissue using the USPIO aggregates, and the ability to locally heat the USPIO aggregates to control the release of the co-localized nanoparticulate therapeutic agent in the activated macrophages that is the basis for the instant invention - increasing the effectiveness of local hyperthermic treatments.

A noted, vida supra, there are many nanotherapeutic formulations that may be suitable for this platform of hyperthermia, USPIO, and phagophilic drug effective for a particular macrophage-dependent disease treatable by the methods of the invention. For example, an extensive library of nanotherapeutics for cancer is readily available, including doxorubicin, Cisplatin, Cisplatin analog, osaliplatin, vincristin, annamycin, Paclitaxel, itoxantrone, CKC 602, Cyclodextrin, Camptothecin, PEG-polyaspartate, Polymeric micelles, Docetaxel, etc.

Hyperthermia can potentially damage all cells, and the degree of damage is typically related to the temperature-time exposure, and this relationship can be quite non-linear. Cell damage usually begins at temperatures above 43°C. When this elevated temperature is sustained for only a few minutes, cells may temporarily show increased cell membrane permeability, alterations in the cell nucleus and reduced ability to proliferate. When the temperature is increased and/or sustained for longer periods of time, apoptosis, related in part to activation of caspase, ensues and this process is irreversible, but evolves over a period of many minutes to hours. If the temperature is raised further or sustained longer, rapid necrosis of the cell results, and is likely due to melting of the cell membrane and protein denaturation. The apoptosis or necrosis due to heating is sometimes characterized as thermal ablation.

When tissue regions containing nanoparticle-loaded macrophages are heated by energy absorption, the nanoparticles are the dominant heat sink. But the resulting heat diffuses from this source into the surrounding biological environment. Thus, the responses described above can be experienced, first throughout the macrophage, and then into adjacent cells. A gradient is thereby established with, for example, necrosis of the loaded

macrophage, necrosis or apoptosis of adjacent non-phagocytic cells, and permeabilization of slightly more distant cell membranes where cells are exposed to lower hyperthermia time- temperature histories.

The energy absorption and heat production by iron oxide nanoparticles is dose responsive, both large aggregates and increased volume fraction of phagocytosed

nanoparticles affect the temperature achieved within the lysosomes where the iron oxide nanoparticles are sequestered.

The dose-responsive nanoparticle heating has two benefits. First, better loading of the macrophages means that heating is more efficient. Second, the clearance of iron oxide nanoparticles from the loaded macrophage takes time - on the scale of days. Thus, as long as the iron oxide nanoparticles remain in sufficient concentration, the heating regimen can be repeated without additional administration and the thermal prescription can be modified to input more electromagnetic energy or increase the time exposure to get the desired result.

Another very useful result from heating nanoparticle-loaded macrophages is that the distribution of heating conforms to the macrophage-dependent tissue whereas absent the nanoparticles, the heated zone tends to be spherical or oblate. Because there are no useful differences in heat absorption absent the nanoparticles, it is common in current hyperthermic therapy to oversize the heated zone in order to include all the target tissues, some of which would fall outside a smaller region of effective heating. This, of course, heats some normal tissue that will also undergo hyperthermic consequences of cell damage.

Cancer response to chemotherapeutic is enhanced in the presence of hyperthermia, and this synergistic effect can be quite large. However, the synergistic effect of the subject companion theranosis with an effective (preferably phagophilic) nanotherapeutic and iron oxide nanoparticles has additional benefits. In the case of cancer foci, the diffusion of heat from the heat sink may be sufficient to cause death of the adjacent neoplastic cells. In addition, the neoplastic cells not directly killed by hyperthermia may have increased cell permeability that would allow the uptake of the companion nanotherapeutic (nanoparticulate therapeutic agent) upon release from the nearby macrophage. Finally, the EPR physiology that sustained the accumulation of nanoparticles in the macrophages is also beneficial in concentrating the released nanotherapeutic - the slow clearance and restricted diffusion of larger therapeutics within the macrophage-dependent disease process facilitates better exposure of the diseased tissues to the effective nanotherapeutic.

It is the surprising result of co-localized iron oxide nanoparticles and nanotherapeutic s in the macrophages associated with the macrophage-dependent disease that leads to much more effective treatments. The required heating can be reduced by the synergistic

nanotherapeutic while similarly the required dose of nanotherapeutic can be reduced by the heating. Finally, the major benefit of focal therapy for macrophage-dependent diseases is the sparing of adverse effects upon other macrophage -rich tissues which are not diseased.

Although iron oxide nanoparticles accumulate in other macrophages, they are not subject to the heating. Similarly, although the nanotherapeutic is expected to be widely distributed, it will not be released or enhanced by heating. Much better therapeutic ratios and lower systemic toxicity results from the focal hyperthermic intervention.

In certain embodiments, the nanoparticulate therapeutic agent is phagophilic (e.g. , due to its size, shape, coating or bonding to other phagophilic carriers).

As used herein, "phagophilic" is characterized by the ability or tendency to be engulfed by macrophages (e.g., through phagocytosis or receptor-based endocytosis), such as by activated macrophages that accumulates USPIO aggregates. For example, in order to co- localize within the macrophage lysosomes where the USPIO iron oxide nanoparticles are stored, the companion nanotherapeutic (nanoparticulate therapeutic agent) must possess certain attributes, such as receptor-based endocytosis, the size that facilitates vascular egress via enhanced permeability, as well as extravascular retention prior to phagocytosis and after subsequent release during focal hyperthermia.

A particulate nanotherapeutic agent of the invention can be evaluated for its phagophilicity in vitro using, for example, cultured murine or even human macrophages.

The medical literature documents a large number of investigative methods for evaluating nanoparticles in murine or human macrophages. Often, the phagocytic uptake of a nanotherapeutic is known from preclinical or clinical studies. A biodistribution pattern that includes liver and splenic uptake strongly suggests in vivo phagocytosis. However, some important parameters of macrophage sequestration may remain and can be evaluated with one or more of these macrophage culture methods. Once sufficient phagophilicity is established, the culture method can be extended to provide evaluation of competitive uptake in the presence of USPIO, thermal response to a particular diathermy method, or stability of the nanotherapeutic to heating within the lysosomal environment or upon release.

In certain embodiments, the method of the invention is evaluated using the steps (vida infra) below in sequence to evaluate a new or existing nanotherapeutic. Specifically, in a murine macrophage culture, e.g. J774A.1 or RAW 264.7, plated at about 30-70%

macrophage density,

1. Add USPIO at desired concentration;

2. Add experimental nanotherapeutic s at desired concentration;

3. Incubate for 8-24 hours;

4. Expose to radiofrequency ablation at about 50°C for 5 minutes;

5. Incubate for 8-24 hours;

6. Measure the macrophage response, including one or more of:

a. Cell viability;

b. Cell permeability; and,

c. Cytokine production.

In certain embodiments, the above general experiment is modified to evaluate other responses, e.g. :

1. The cell culture can include nonphagocytic cells, and the response of the

macrophages is compared to that of the other cells; 2. Two similar macrophage cultures can be used - one receiving only USPIO and the other only experimental nanoparticles.

Other useful variations will be apparent to those skilled in the art.

In certain embodiments, similar comparisons can be made in suitable animal tumor models with tumor-associated macrophages as described in Step 2 (vide infra). The same matrix of variables described for macrophage cultures can be compared.

In certain embodiments, the device used to generate the heating in culture or tumor model can be varied to compare laser wavelengths, approved microwave or radiofrequency wave lengths, focused ultrasound, etc. In these embodiments, the hyperthermia prescription can vary with respect to exposure temperature and time.

There are also a wide variety of animal models of inflammatory diseases, including neoplasia with varying amounts of tumor- associated macrophages, or other inflammatory states such as inflammatory bowel disease, arthritis, vulnerable plaque, etc. These models can be directly used or adapted to evaluate (1) uptake of the subject nanoparticulate therapeutic agent or companion nanoparticle with USPIO iron oxide nanoparticles, (2) heating response to a particular diathermic regimen, (3) repeated nanoparticle dosing and diathermia, and (4) parameters of toxicity or safety.

After suitable evaluations described above, clinical trials may be conducted to further evaluate the administration of one of more than one (not yet marketing approved)

nanoparticulate therapeutic agents. This may include evaluating the co-localization of the nanoparticles using imaging and histology techniques, and/or the response to a particular diathermic regimen.

As used herein, "diathermy" refers to "heating through" (Greek), and has been used extensively for physical therapy. Heating of tissues requires the absorption of energy, and this absorption depends upon tissue responses to the applied electromagnetic energy. This energy can be applied externally or interstitially, with the use of probes, antennae, wave guides, etc. In the treatment of cancer, the energy sources may include light via lasers, microwaves, radiofrequency waves, focused ultrasound waves, or magnetized particle rotation within magnetic fields. The parameters of the exposed tissue responsible for energy absorption by each of these sources are understood.

The energy can be delivered with devices approved for that purpose. Each of the listed forms of energy delivery generates higher temperatures in USPIO loaded macrophages. When the delivered energy is partially focused on the macrophage-dependent disease process to be heated, this enhanced energy absorption allows better conformation to the disease distribution, faster generation of the desired temperature, and realization of the therapeutic intent, which may include increased cell permeability, apoptosis, or necrosis within the target. During diathermic therapy, it is usually possible to monitor temperature response at one or more informative tissue sites and the ability to predict the biothermal response will likely result in better treatment prescriptions. It is the surprising realization that both iron oxide nanoparticles and phagophilic nanotherapeutic s can be separately administered and will co- localize in the activated macrophages present in macrophage-dependent diseases, thereby resulting in better treatments.

Macrophages have cell membrane receptors that bind a wide variety of ligands, which receptors evolved from the responsibility of macrophages to clear pathogens and debris from apoptotic cells. Some of these receptors are avid for nanoparticles, including USPIO, liposomes, micelles, and certain polymers. Those binding to such receptors are internalized one nanoparticle per receptor. Subsequent release into the intracellular milieu is followed by encapsulation into lysosomes. These receptors then return to the cell membrane to bind another nanoparticle present in the adjacent extracellular space. Thus, endocytosis takes a finite amount of time and a sustained supply of nanotherapeutic in the adjacent extracellular space facilitates nanotherapeutic loading of the activated macrophage. For example, in macrophage culture it has been found that it takes 100 minutes to internalize one million USPIO nanoparticles. In vivo, presentation of the nanoparticle to the activated macrophages must incorporate perfusion and diffusion parameters. Although the body has abundant other macrophage populations that will also acquire some of the nanotherapeutic, these other macrophages will not be subject to focal heating. Thus these other macrophages will release the therapeutic moiety more slowly than the rapid release of the therapeutic from the focally heated macrophages. In as much as macrophage heating is required, the nanotherapeutic must be resistant to the applied hyperthermic temperatures.

Following intravascular administration, the nanoparticulates must reach the activated macrophage by escaping through the leaky vasculature perfusing the macrophage-dependent disease (Enhanced Permeability). The vascular escape route is thought to occur between endothelial fenestrations and the caliber of this pathway may vary depending upon the disease, and may vary both temporally and anatomically. Smaller size of the nanoparticle facilitates extravasation although large size may carry bigger payloads. For intravascular administration, a size of <200 nm seems optimal and this size may also be more favorable for receptor capture and transport. When the nanotherapeutic is administered by another route, larger size nanoparticles may be suitable.

Inasmuch as receptor-based endocytosis transports a single nanoparticle and receptor recycling takes time, longer blood life and administration of a large number of particles is beneficial. For example, current USPIO formulations that contain 30 mg Fe/ml contain more than 10¹⁹ nanoparticles per ml and have blood T ½ of more than 15 hours.

Diffusion of the nanoparticle within the extracellular space is slow, and smaller particles can traverse the distances more rapidly. Diffusion distances are also important when the nanotherapeutic is released from the activated macrophage.

In certain embodiments, the nanoparticulate therapeutic agent (nanoparticle) facilitates receptor-mediated endocytosis, and is preferably resistant to heating.

Receptor-mediated endocytosis into activated macrophages may be facilitated by nanoparticles with average size of <400 nm, preferably <200 nm for intravascular administration.

Receptor-mediated endocytosis into activated macrophages may also be facilitated by including receptor avid ligands displayed on the surface of the nanoparticulate therapeutic agent (nanoparticle).

In certain embodiments, carrier materials, such as liposomes, micelles, or polymers that provide long intravascular blood life (e.g., 2-48 hours, preferably longer than 8 hours) may be included in the nanoparticulate therapeutic agent (nanoparticle) formulation.

In certain embodiments, the nanoparticulate therapeutic agent (nanoparticle) is effective for treating the macrophage-dependent disease, and/or is resistant to inactivation by temperatures above 44°C that are sustained for 0.5-30 minutes (preferably for at least 10 minutes).

In certain embodiments, release of the nanoparticulate therapeutic agent causes suppression of cytokine/chemokine production, or irreversible damage to the activated macrophage.

A partial list of these inflammatory or macrophage-dependent diseases that could be responsive to the subject method includes: cancer (primary and metastatic), vulnerable plaque, arthritis (Rheumatoid, Psoriatic, Osteoarthritis), Inflammatory Bowel Diseases, psoriasis, type I diabetes, COPD, periodontal disease, etc. The activated macrophages for each of these diseases may produce different cytokines or chemokines. Where it is desirable to eradicate the inflammatory focus, effective therapy should kill the participating macrophages and the nearby disease process. For example, primary or metastatic cancers are aggressive in proportion to the activity of tumor- associated macrophages. These same macrophages can accumulate the subject nanoparticulate therapeutic agent that provides for diagnosis (e.g. , presence, size, stage, anatomic features that influences therapeutic intervention, especially hyperthermic prescription) and treatment.

Macrophages normally have a lifetime of many days to a few weeks, but do not otherwise reproduce. Thus, in some inflammatory processes, it may suffice to simply turn off the production of chemokine/cytokines that are causing the symptoms. Thus in certain embodiments, the methods of the invention explores interventions that occur within the activated macrophage without killing it. For example, this can be achieved by turning off the synthesis of the offending cytokine with siRNA / miRNA / shRNA constructs or other therapeutic agents. The continued presence of such therapeutically-impaired macrophages at the site might inhibit the recruitment of new macrophages that have not experienced the genetic inhibition.

In certain embodiments, the subject method with the nanoparticulate therapeutic agent is carried out in an individual who has previously failed to respond to, has responded inadequately, or has stopped responding to another treatment. For example, an aggressive primary or metastatic cancer might benefit from the subject method in which the

nanoparticulate therapeutic agent possesses cytotoxic potency against the cancer.

In another example, an arthritic condition might benefit from the subject method in which the nanoparticulate therapeutic agent blocks the activity of a chemokine/cytokines secreted by the activated macrophages in the affected joint. In this case, administrative options might include systemic administration of all nanoparticles, direct administration into the affected joint space of either USPIO or nanotherapeutic, or systemic administration of one and direct injection of the other.

Alternatively or in addition, the nanoparticulate therapeutic agent may block the production of symptomatic cytokine/chemokine by the activated macrophage by an RNAi reagent (e.g. , siRNA, miRNA, shRNA etc.) against the secreted cytokine/chemokine. As above, the administrative route can be selected from systemic and direct injections into the affected joint(s). In certain embodiments, the nanoparticulate therapeutic agent may have unacceptable systemic side effects, and such nanoparticulate therapeutic agent can be injected directly into an inflamed region. The nanoparticulate therapeutic agent might be an anti-infective agent for periodontal disease, an anti-inflammatory agent for arthritis, or a chemotherapeutic agent for brain cancer. Other examples will be apparent to those skilled in the art.

With the inventions generally described, the sections below provide further detailed description for the various aspects of the invention, which aspects should be viewed as parts of the invention as a whole.

2. USPIOs and Formulations

The general structure of iron oxide nanoparticles (e.g., USPIO nanoparticles) useful for this invention consist of a magnetite/hemagnetite (e.g., the ferrous/ferric) core that is usually less than 10-20 nm in size. Typically, about 5-20k iron atoms reside within a crystalline core that has a single magnetic domain. The core is typically coated with a biologically compatible polymer (e.g. sucrose, dextran, or other synthetic carbohydrate) that creates a nanoparticle between 10 and 80 nm in size. These USPIO nanoparticles have been developed to serve as contrast agents or biomarkers for MRI, and are widely used for diagnostic applications both in vitro and in vivo, and, in one instance, for the treatment of iron deficiency anemia.

The macrophage-targeting iron oxide nanoparticle of the invention are small enough to slowly escape from the capillaries in or near the macrophage-dependent or -associated disease, yet remains in the circulation for a sufficient time to allow this process to be efficient. Examples of nanoparticles with these characteristics include ferumoxytol and ferumoxtran-10.

In certain embodiment, a coating is additionally added to the nanoparticles to allow safer administration of the iron oxide nanoparticulate parenterally, and to increases the blood half-life of the nanoparticles.

There are a large number of USPIO formulations suitable for the instant invention. For example, MRI contrasting agents SPIOs and USPIOs have been approved for commercial use. They include any of the following: Feridex® I.V. (also known as Endorem® and ferumoxides), Resovist® (also known as Cliavist®, approved for the European market in 2001), and Feraheme® (ferumoxytol). For a review of USPIO formulations, see Corot (Adv. Drug Del. Rev., 58: 1471-1504, 2006). As long as the size requirement is met, the USPIO nanoparticles or functionalized derivatives thereof (see below) mentioned in the above literature can all be used in the instant invention.

Change in the quantity or distribution of loaded macrophages may also be used to assess therapeutic response or lack thereof in that subject.

Table 1 Selected Parameters of Iron Oxide Nanoparticles

Ferumoxytol is one of the USPIO nanoparticles suitable for assessing local vascularity and macrophage uptake, due to its intermediate pharmacokinetics and the exceptional clinical experience of its use in treating iron-deficient anemia. Ferumoxytol, as a representative USPIO, has potent superparamagnetic properties that make it ideal for use as a MR contrast agent. Due to its slow blood clearance, ferumoxytol remains largely in the vascular space at early time points (minutes to hours after intravenous administration) where it can be used to quantify vascular perfusion and blood volume in regions of interest using appropriate MRI techniques. For macrophage-dependent diseases, this provides the opportunity to identify the location of the associated angiogenesis. Ferumoxytol is then removed from the blood entirely by macrophages, where it is aggregated in their lysosomes. USPIOs remain effective as phagocytic biomarkers until the nanoparticle is degraded in macrophages.

In certain embodiments, the subject nanoparticles are surface protected and/or functionalized by hydrophilic groups, such as biocompatible polymers. Such hydrophilic "shell" improves the stability in aqueous systems and may extend serum half-life.

In certain embodiments, the subject nanoparticles are surface functionalized by polymeric micellar structures based on amphiphilic block copolymers maleimide-terminated poly- (ethylene glycol)-block-poly(D,L-lactide) copolymer (MAL-PEGPLA) and methoxy- terminated poly(ethylene glycol)-block-poly-(D,L-lactide) copolymer (MPEG-PLA). In certain embodiments, the subject nanoparticles are surface functionalized by biodegradable and biocompatible poly(acrylic acid). See, for example, Santra et al. {Small Weinheim an der Bergstrasse, Germany) 5(16): 1862- 1868, 2009), for the use of

biodegradable and biocompatible poly(acrylic acid)-iron oxide nanoparticles (PAAIONPs) functionalized with anticancer drug, Taxol, by a solvent-diffusion method.

In certain embodiments, the subject nanoparticles are surface functionalized by N- phosphonomethyl iminodiacetic acid (PMIDA). See, for example, Das et al. (Small

Weinheim an der Bergstrasse, Germany) 5:2883-2893, 2009).

In certain embodiments, the subject nanoparticles are surface functionalized by phosphonate groups, or carboxylate groups. Compared to phosphonate groups, bonds formed through the carboxylate groups have decreased thermal stability and increased susceptibility to enzymatic degradation. Das (supra).

In certain embodiments, the subject nanoparticles are surface functionalized by monodisperse, discrete mesoporous silica materials. Various agents can be encapsulated within the silica matrix. The surface of the nanoparticles may be further functionalized with PEG groups. See, for example, Kim et al. (Angew. Chem., Int. Ed. 47:8438-8441, 2008)

In certain embodiments, the subject nanoparticles are surface functionalized by, for example, a cloaking agent such as poly(ethylene glycol) (PEG).

In certain embodiments, the subject nanoparticles are surface functionalized by targeting ligands (e.g., by using any of the above surface functionalization) that direct the nanoparticles to or near disease-associated macrophages. The targeting ligands may include those that target surface ligands or receptors of disease associated macrophages or diseased cells (such as cancer cells) surrounding the disease associated macrophages. Exemplary targeting ligands may bind to various cell surface receptors, such as transferin receptor (e.g., bound by holo-transferin ligand), folate receptor (e.g., bound by a-folate receptor-targeting folic acid groups), and human/epidermal growth factor receptor 2, upregulated on the surface of various cancer cells. Exemplary targeting ligands may include, without limitation, biotin, avidin, antibody, monoclonal antibody, phage, folate, aptamer, protein or a binding fragment thereof.

Introduction of targeting ligands in general may help to further increase the target-to- background contrast in imaging and improve the local concentration of the USPIO aggregates at the target of interest, with the goal of reducing systemic toxicity. Although this may not be essential where only disease-associated macrophages phagocytize the subject nanoparticles. In certain embodiments, the subject iron oxide nanoparticles may be functionalized or coated with a biocompatible polymer, such as PEG, PEI, etc.

In certain embodiments, the subject iron oxide nanoparticles may be functionalized with targeting ligands that are covalently attached to the dextran-coated monocrystalline iron oxide (Tf-MION), or cross-linked iron oxide (Tf-CFIO). The functionalized nanoparticles can easily be taken up by macrophages via receptor- mediated endocytosis or phagocytosis. Such covalent linking methods have been described in, for example, Ichikawa et al. ("MRI of transgene expression: Correlation to therapeutic gene expression" Neoplasia (New York, NY, United States) 4:523-530, 2002); Moore et al. ("Human transferrin receptor gene as a marker gene for MR imaging," Radiology (Oak Brook, IL, United States) 221:244-250, 2001); and Weissleder et al. ("In vivo magnetic resonance imaging of transgene expression," Nat. Med., (New York) 6:351-354, 2000). All are incorporated herein by reference.

In certain embodiments, the subject iron oxide nanoparticles may be functionalized with targeting ligands and/or therapeutic reagents that are covalently attached to or associated with Poly(ethyleneimine) (e.g., branched PEI, MW10 kDa). PEI is positively charged, and may permit negatively charged therapeutic agents to be associated with the nanoparticle. Upon entry of a low pH environment, the therapeutic agent may be protonated and lose its negative charge, thus dissociating from the PEI coating and be released. PEI coating of the nanoparticle is known in the art. See, for example, Park et al. (Biomaterials, 29:724-732, 2008, incorporated herein by reference).

In certain embodiments, the subject iron oxide nanoparticles may be coated by dextran.

In certain embodiments, the subject iron oxide nanoparticles may be coated by linear cyclodextrin-containing polycations (CDPs). See, for example, Hu-Lieskovan et al,

("Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma," Cancer Res. 65:8984-8992, 2005), and Pun et al. ("Targeted delivery of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles," Cancer Biol. Titer., 3:641-650, 2004). Iron oxide nanoparticles so coated may be

systemically injected into a subject (e.g., via i.v. injection).

In certain embodiments, the calcium phosphate nanoparticles may be further functionalized by amine carboxylate- and/or poly(ethylene glycol)-groups. See Kesteret et al. ("Calcium phosphate nanocomposite particles for in vitro imaging and encapsulated chemotherapeutic drug delivery to cancer cells," Nano. Lett., 8:4116-4121, 2008).

In certain embodiments, the CPNP particles may be further functionalized with PEG groups or other protein agents such as antibodies. See, Barth et al. (supra).

In certain embodiments, the subject iron oxide nanoparticles may be coated by poly(ethylene oxide) (PEO) -modified poly(P-amino ester) (PbAE)-based materials, or PEO- poly(caprolactam) (PCL)-based materials. Solid unprotonated PbAEs are insoluble at physiological pH. However, the solubility increases upon protonation of the amines along the backbone.

3. Dose, Administration Route, Treatment Regimen

Macrophages can sequester intravenous doses that are many times larger than those required for MRI (usually 0.5-7 mg/kg), and these sequestered particles retain their superparamagnetic properties (indicating persistent nanoparticulate form) for weeks.

Preclinical safety studies with a representative USPIO show that doses 200 times the imaging dose can be administered and removed from body fluids by macrophages. See Bourrinet (Invest. Radiol., 41:313-324, 2006). Clearly, the capacity of phagocytosis of USPIOs is very large.

As the USPIO dose increases, the size of nanoparticulate aggregates in macrophage lysosomes increases along with the volume fraction of aggregated nanoparticles. The effective amount / dose of USPIO, the route of administration, and the delay time for selective accumulation in macrophages can all be used to control or optimize the imaging and/or treatment process. It is the selective nature of aggregation in tissue macrophages that provides a utility of the invention.

The intravenous dose for MRI can vary based in part on the route of administration, the magnetic field strength, and the structures that need to be separately visualized. Table 1 shows the most common doses for MRI utility. However, macrophage uptake depends upon both the presenting fluid for the macrophages, the time to accumulate and then metabolize the ingested USPIOs, and the purpose of the administration - whether for diagnosis or therapy.

For intravenous loading of TAMs or IAMs, the following regimens may be used: a dose of about 0.5 to 20 mg/kg, about 1 to 10 mg/kg, or about 2 to 5 mg/kg.

In certain embodiments, the lower limit of the dose may be about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1.0 mg/kg, about 1.2 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, or about 2.5 mg/kg.

In certain embodiments, the upper limit of the dose may be about 100 mg/kg, about 50 mg/kg, about 30 mg/kg, about 20 mg/kg, about 18 mg/kg, about 16 mg/kg, about 15 mg/kg, about 14 mg/kg, about 12 mg/kg, about 10 mg/kg, about 9 mg/kg, about 8 mg/kg, about 7 mg/kg, about 6 mg/kg, or about 5 mg/kg.

In certain embodiments, the lower limit of the dose is chosen from any one of: about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1.0 mg/kg, about 1.2 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, or about 2.5 mg/kg, and the upper limit is chosen from any one of about 100 mg/kg, about 50 mg/kg, about 30 mg/kg, about 20 mg/kg, about 18 mg/kg, about 16 mg/kg, about 15 mg/kg, about 14 mg/kg, about 12 mg/kg, about 10 mg/kg, about 9 mg/kg, about 8 mg/kg, about 7 mg/kg, about 6 mg/kg, or about 5 mg/kg.

In certain embodiments, any of the doses may be repeated as necessary. The repeat dose may be the same or different from the previous dose. In certain embodiments, each repeat has about the same dose.

Optionally, the waiting time between the repeats may be between 12 (0.5 day) and 144 hours (12 days) or longer, or between 1-10 days, or between 2-10 days, between 3- 10 days, between 4- 10 days, between 5-10 days.

In certain embodiments, the waiting time between the repeats may be at least about 1, 2, 3, 4, 5, 6, or 7 days.

In certain embodiments, the waiting time between the repeats may be up to 20, 18, 16, 15, 14, 12, 10, 9. 8, 7, 6, or 5 days.

In certain embodiments, the USPIO nanoparticles . nanotherapeutics or formulations thereof may be administered interstitially, e.g. , for nodal enhancement. Suitable doses for this administration route may be a dose of about 0.01-2 mg/kg, about 0.02- 1.5 mg/kg, about 0.03- 1 mg/kg, about 0.05- 1 mg/kg, about 0.1-1 mg/kg, about 0.2-0.5 mg/kg.

In certain embodiments, the lower range of the interstitial dose may be any one of: about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1.0 mg/kg, about 1.2 mg/kg, or about 1.5 mg/kg.

In certain embodiments, the upper limit of the interstitial dose may be about 10 mg/kg, about 8 mg/kg, about 6 mg/kg, about 4 mg/kg, about 2 mg/kg, about 1.9 mg/kg, about 1.8 mg/kg, about 1.6 mg/kg, about 1.5 mg/kg, about 1.4 mg/kg, about 1.2 mg/kg, or about 1.0 mg/kg.

In certain embodiments, the lower limit of the wait time is about 10 min, 20 min, 30 min, 40 min, 50 min, 1 hr, 1.5 hr, 2 hr, 3 hr, 6 hr, 12 hr, 18 hr, 1 day, 2 days, 3 days, 5 days, or 7 days, and the upper limit of the wait time is about 20 days, 18 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, or 8 days. However, when vascular imaging is desirable, the wait time may be from a few seconds to several hours.

In certain embodiments, the administration is carried out with stimulation of lymphatic uptake as feasible.

The administration may be repeated as necessary, with wait time between repeats between 30 min to 14 days.

In certain embodiments, the USPIO nanoparticles / nanotherapeutics or formulations are administered intracavitarily. In certain embodiments, the lower limit of the wait time is about 10 min, 20 min, 30 min, 40 min, 50 min, 1 hr, 1.5 hr, 2 hr, 3 hr, 6 hr, 12 hr, 18 hr, 1 day, 2 days, 3 days, 5 days, or 7 days.

In certain embodiments, the upper limit of the wait time is about 20 days, 18 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, or 8 days.

In certain embodiments, the lower limit of the wait time is about 10 min, 20 min, 30 min, 40 min, 50 min, 1 hr, 1.5 hr, 2 hr, 3 hr, 6 hr, 12 hr, 18 hr, 1 day, 2 days, 3 days, 5 days, or 7 days, and the upper limit of the wait time is about 20 days, 18 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, or 8 days.

Suitable dose includes 0.05-2 mg/kg, preferably in an appropriate suspension. The pre-determined wait time is about 30 min- 14 days before imaging.

In certain embodiments, the lower limit of the dose is about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1.0 mg/kg, about 1.2 mg/kg, about 1.4 mg/kg, or about 1.5 mg/kg.

In certain embodiments, the upper limit of the dose is about 10 mg/kg, about 8 mg/kg, about 6 mg/kg, about 5 mg/kg, about 4 mg/kg, about 3 mg/kg, about 2 mg/kg, about 1.9 mg/kg, about 1.8 mg/kg, about 1.7 mg/kg, about 1.6 mg/kg, about 1.5 mg/kg, about 1.4 mg/kg, about 1.3 mg/kg, about 1.2 mg/kg, about 1.1 mg/kg, or about 1.0 mg/kg.

In certain embodiments, the lower limit of the pre-determined wait time is about 10 min, 20 min, 30 min, 40 min, 50 min, 1 hr, 1.5 hr, 2 hr, 3 hr, 6 hr, 12 hr, 18 hr, 1 day, 2 days, 3 days, 5 days, or 7 days.

In certain embodiments, the upper limit of the pre-determined wait time is about 20 days, 18 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, or 8 days.

In certain embodiments, the lower limit of the pre-determined wait time is about 10 min, 20 min, 30 min, 40 min, 50 min, 1 hr, 1.5 hr, 2 hr, 3 hr, 6 hr, 12 hr, 18 hr, 1 day, 2 days, 3 days, 5 days, or 7 days, and the pre-determined upper limit of the wait time is about 20 days, 18 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, or 8 days.

4. Heating of Aggregates

The USPIO aggregates within the activated macrophages can be selectively heated by a variety of energy sources, such as light (laser), radiofrequency, microwave, and ultrasound. There are no effective barriers to the transmission of the nanoparticle heat within the macrophage and thence to immediately adjacent structures, including other pathologic targets. Depending upon the degree and duration of the heating, thermo therapy or thermoablation of the heated cells or thermo release of drug can be accomplished.

The intermediate wavelengths that characterize radiofrequency and microwave have already been used for heating tissues. Indeed, all of these wavelengths can be associated with scattering and energy absorption by USPIO nanoparticles, and the efficiency of their size follows the scattering theories listed herein (Rayleigh and Mie scattering). An additional factor to consider is the volume fraction of the tissue region containing aggregated nanoparticles.

In the graphic solution of the two scattering domains, the relation between

nanoparticle size and probing wavelength is based upon Rayleigh and Mie scattering. The values for x are the ratios between particle size and wavelength. The illustration also shows that many common particulates scatter electromagnetic waves in proportion to their size. This is true across the electromagnetic spectrum. In as much as all tissues can be damaged or killed by sustained heating, selective heating of the target tissue is desirable.

The scattering of impinging electromagnetic waves is followed by absorption of the delivered energy, in turn leading to increase in temperature near the location where this absorption occurs. This heat is then conducted to surrounding tissues through thermal conduction. When the local temperature reaches certain value, cell damage begins in proportion to the increase in temperature and its duration.

Serving as a heat sink, the activated macrophages having the large USPIO aggregates essentially become the target for thermal ablation. Cells heated above 42°C begin to show signs of apoptosis. Temperatures above 50°C are less associated with apoptosis and more with frank necrosis. These outcomes are time and temperature dependent.

The USPIO heating is particularly effective when the particles are targeted so that the heating is focused on the target tissue. For USPIOs, the incremental heating could be accomplished by increasing the effective particle size and/or their fractional volume within the targeted tissue macrophages. In activated macrophages, increasing the size of the lysosomal USPIO aggregates increases the scattering efficiency, thus producing an improved selective heating and its consequences upon the cells in the surrounding tissue.

In certain embodiments, the size of the aggregates or the loading of the macrophages is controlled by the dose and the time these particles are available in the blood, tissue fluid or lumen. The regimens offered for imaging may be appropriate for thermal therapy as well, but the energy source selected for the particular heating regimen is influenced by the local nanoparticle size that is best suited for the thermal therapy.

Figure 1 of the incorporated US-2013-0336897- A 1 lists the effective particle sizes for Rayleigh and Mie scattering, and it is clear that lower macrophage USPIO uptake is required for laser devices operating in the ultraviolet, visible, and thermal infrared

wavelengths. Any laser system designed for photodynamic therapy in that anatomic region will be able to efficiently heat USPIO-loaded macrophages in that region.

Microwave and radiofrequency devices are limited by ISM bands which restrict frequency of use in hospitals to designated frequencies - microwave (ISM bands of 433, 975, or 2450 MHz), radiofrequency (ISM bands of 13.5, 27, and 40 GHz). Any radiofrequency or microwave system that is designed to produce hyperthermia by frictional stimulation of water will also be more efficient when heating USPIO enhanced macrophages in the same region.

Ultrasound energy absorption with nanoparticles must be experimentally measured as described in the examples, but imaging frequencies range from 2-60 MHz where higher frequencies have less penetration depth. Therapeutic ultrasound will have the same depth limitations, but will require less power than used for high intensity focused ultrasound with USPIOs.

A strength of USPIO theranosis is that the USPIO content at the proposed treatment site can be estimated from imaging studies, and the location and access for the heating devices combined with their heating efficiency for the existing macrophage loading can be derived from the imaging information, and details about the efficiency of heating for a given hyperthermia plan. The duration of heating and the power applied to the heating device are controllable - the resulting local temperatures can be determined during treatment via probes or MR thermal imaging, and eventually biothermal simulations will provide useful treatment plans. Such simulations have evolved for hyperthermia with high intensity focused ultrasound. See Chopra (Int. J. Hyperthermia, 26:804, 2010). The examples include experiments that can measure conversion of electromagnetic energy into heat when the anatomic region includes UPSIO-loaded macrophages.

The capacity of body macrophages to sequester USPIOs appears to be quite large. Ferumoxytol doses of 1 gram do not appear to alter blood clearance by this widely distributed cell population. Indeed, USPIOs appear to occupy only two body compartments - initially the blood following intravenous administration, and then the macrophages. Some

macrophages such as those in the liver or spleen can ingest the USPIOs directly from blood. Due to their size, in other tissues they leak from the blood very slowly. Upon exit from the blood in these tissues, the nanoparticles are quickly phagocytized by local macrophages where they appear to remain until metabolized. These same macrophages, in disease states, facilitate the leakage of the USPIOs through angiogenic cytokines. Many important disease states contain activated macrophages within responding tissues. These macrophages can be related to cancer (tumor-associated macrophages, TAMs) or any macrophage-dependent inflammatory process (inflammation-associated macrophages or IAMs).

When it is desirable to selectively ablate tissue containing macrophages by diathermy regimens, these macrophages can be loaded with USPIO nanoparticles whose size and volume fraction are increased by the lysosomal aggregation of the administered USPIOs within the local macrophages.

The amount and distribution of the administered UPSIOs can be determined with imaging devices sensitive to the altered electromagnetic scattering or change in local tissue relaxivity. The duration of effective amounts of phagocytosed USPIOs is dependent upon their metabolism, but may be sustained for a period of days and weeks - this allows repeated hyperthermic dosing over time or in multiple macrophage-rich body regions. This would also facilitate the co-administration of chemotherapy or radiotherapy.

Delivering the effective amount of electromagnetic energy to achieve the desired increase in local temperature and its duration will depend upon the hearing device used and the local USPIO content, but in every case the efficiency of local tissue heating near the USPIO-containing macrophages will be enhanced. A wide variety of electromagnetic wavelength could be used to heat the USPIO-loaded macrophages, including light (laser), RF, microwave, and ultrasound. This process provides a selective local heat sink. Due to their proximity to the inflammatory pathophysiology of interest, the heat absorbed by the USPIO- loaded macrophages is thermally conducted to the targeted tissues. At times, the necessary destruction of the loaded macrophages is desirable as they may be sustaining the

inflammatory disease process.

5. Diseases, Disorders, or Conditions

Although the inflammatory disease process may be widely distributed, the uptake of USPIOs is very focal. Indeed, many inflammatory diseases that create significant morbidity are focal in their expression; e.g., atherosclerosis creating vulnerable plaque is limited to local arterial sites, although there may be many such sites. The focal nature of the activated macrophage accumulation of USPIOs provides exceptional opportunities for imaging and selectively treating these diseases. Other examples of the highly focal diseases of interest include inflammatory bowel diseases (IBD) and primary cancers. Even when cancer disseminates, the cancer cells are all close to a capillary, and, if aggressive, intimately enmeshed with tumor-associated macrophages. Thus, in macrophage-dependent or - associated diseases, the active process is always adjacent to a capillary, the increased capillary permeability provides a source of USPIOs from blood, and the activated

macrophages accumulate virtually the entire local nanoparticle load. One basis of the invention is that the USPIOs accumulated in activated macrophages are not only markers of the disease, but can also be utilized to both identify the location and extent of disease and, surprisingly, to treat the disease focally with theranostic devices combining detection and treatment.

In certain embodiments, the macrophage-associated disease or condition is an "inflammatory disease, disorder, or otherwise abnormal condition," which may include disorders associated with inflammation or have an inflammation component, such as, but are not limited to: acne vulgaris, asthma, COPD, autoimmune diseases, celiac disease, chronic (plaque) prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases (IBD, Crohn' s disease, ulcerative colitis), pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis, interstitial cystitis, atherosclerosis, allergies (type 1, 2, and 3 hypersensitivity, hay fever), inflammatory myopathies, as systemic sclerosis, and include dermatomyositis, polymyositis, inclusion body myositis, Chediak-Higashi syndrome, chronic granulomatous disease, vitamin A deficiency, cancer (solid tumor, gallbladder carcinoma), periodontitis, granulomatous inflammation (tuberculosis, leprosy, sarcoidosis, and syphilis), fibrinous inflammation, purulent inflammation, serous inflammation, ulcerative inflammation, and ischaemic heart disease, type I diabetes, and diabetic nephropathy.

In certain embodiments, the inflammatory disease, disorder, or otherwise abnormal condition includes many autoimmune diseases or disorders that are associated with inflammation or have an inflammation component, e.g. , corresponding to one or more types of hypersensitivity. Exemplary autoimmune diseases or disorders that correspond to one or more types of hypersensitivity include: atopic allergy, atopic dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune polyendocrine syndrome, autoimmune urticaria, celiac disease, cold agglutinin disease, contact dermatitis, Crohn' s disease, diabetes mellitus type 1, discoid lupus erythematosus, erythroblastosis fetalis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's encephalopathy, Hashimoto' s thyroiditis, idiopathic thrombocytopenic purpura, autoimmune

thrombocytopenic purpura, IgA nephropathy, lupus erythematosus, Meniere's disease, multiple sclerosis, myasthenia gravis, narcolepsy, neuromyelitis optica, Devic' s disease, neuromyotonia, ocular cicatricial pemphigoid, opsoclonus myoclonus syndrome, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, pemphigus vulgaris, pernicious anaemia, psoriasis, psoriatic arthritis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, subacute bacterial endocarditis (SBE), systemic lupus erythematosis, lupus erythematosis, temporal arteritis (also known as "giant cell arteritis"), thrombocytopenia, ulcerative colitis, undifferentiated connective tissue disease, urticarial vasculitis, and vasculitis.

This section further describes several representative diseases, disorders, or conditions that can be imaged and/or treated with the methods of the invention. The diseases, disorders, or conditions listed herein are for illustrative purpose only, and are not limiting.

Breast Cancer

Aggressive local breast cancer can be managed by the inventions described herein. The identification and staging of the cancer utilizes imaging of the TAMs with USPIO. In addition to staging, the suitability of location for hyperthermia can be evaluated. In the event this is feasible, the effective nanotherapeutic regimen is administered and hyperthermic treatment provided when the companion nanotherapeutic s are present in the proper amounts in the cancer. The steps of treatment are as described.

Following treatment, the result can be reevaluated and potentially repeated. In some circumstances, the amount of USPIO and nanotherapeutic remaining in the tumor will allow serial treatments.

Care of the subject can be personalized with the use of Companion Nanoparticle Theranosis.

Other Cancers with Macrophage-Dependent Components

The methods described above can be adopted as clinically desirable for a wide variety of other cancers. The absence or low level of macrophage involvement may indicate a less aggressive cancer such as ductal carcinoma in situ (DCIS) or low grade prostatic cancer where gentle treatments or even watchful waiting is appropriate.

However, there are many cancers with aggressive TAM (tumor associated

macrophages) components where this invention is useful. Glioblastoma, GU, GI, lung, sarcoma, thyroid, and salivary gland tumors are a few that frequently are aggressive and associated with prominent TAMs. The teaching of this invention can be used to determine which cancers could usefully have USPIO directed theranosis. Macrophage-Dependent Inflammatory Diseases

1 ) Vulnerable Plaque

a. Imaging USPIO-enhanced plaques

Using an acceptable model of vulnerable plaque such as the rabbit or pig, the accumulation of detectable USPIO-loaded macrophages may be assessed with catheter-based imaging techniques such as virtual histology ultrasound (VH-US) or optical coherence tomography (OCT).

Macrophage loading can be varied using post injection time and dose as appropriate. The VH-US and OCT imaging devices are used for detection of the loaded macrophages in the plaque. The accuracy and sensitivity of the imaging performance is compared with vascular histopathology.

b. Treating USPIO-enhanced plaques

All of the therapeutic methods elucidated above can be adopted for treatment of the vulnerable plaque with its macrophage-enhanced USPIO content and an effective nanotherapeutic.

2 ) Arthritis with IAMs

Many forms of arthritis have a prominent population of activated macrophages (Inflammation- associated macrophages). These macrophages are intimately involved in producing the joint pathology and symptoms. Fortunately, the presence, activity, and accessibility of this IAMs can be determined by MEMRI or MEUS. Often, it may be useful to determine the vascularity of the joint pathology immediately after administering the USPIO.

The development of promising companion nanoparticles theranostics follows the sequences listed below, including assessing macrophage loading with suitable macrophage cultures, determining the thermal response of the loaded macrophages using various diathemia energies, and using established animal models of rheumatoid arthritis,

osteoarthritis, etc. There are suitable arthritic models available, and selection may be based upon expectant treatment options. However, inflamed joints offer the opportunity to administer the USPIO and/or nanotherapeutic either systemically or directly into the affected joints. These alternatives have clinical implications.

Inflammatory diseases with activated macrophages offer the chance to simply "quiet the macrophage," i.e., blocking the formation and/or secretion of the cyto/chemokines responsible for the pain, destruction, and deformity. In this regard, intracellular control of, for example, RNA-based synthesis, with siRNA nanotherapuetics, may be used. The pharmacologic suppressing might involve one or more synthetic pathways, for example, controlling IL2, IL15, and/or TNFa. Alternatively, the nanoparticulate therapeutic, upon release, may block the action of such chemokines.

There are also a greater variety of diathermic devices and thermal prescriptions for arthritis. This is still a focal disease requiring focal thermal therapy after loading of the activated macrophages.

Interstitial diathermia is feasible with laser and ultrasound probes directly inserted into the target joint. When the joint is small, for example, the proximal interphalangial joint, external application of laser or ultrasound energies can be effective. For larger joints, or for treating several adjacent joints, external plates or wrapped antennae can be used to apply RF or microwave diathermy.

Finally, the thermal prescription may tend to favor lower temperatures and altered exposure times so that the nanotherapeutic is released intracellularly and reaches the RNA target by diffusing through more permeable internal cell membranes.

A particular advantage of noninvasive diathermy is that it can be repeatedly applied by the patient at home or in outpatient clinics.

3) Periodontal Disease

When the local macrophages are unable to reverse the pathology leading to periodontal disease, they become part of the ongoing destruction. The methods described herein can be used for theranosis. For USPIO imaging, both ultrasound and optical devices are feasible to relate macrophage activity to gum scores. Heating probes included in mouthpieces similar to those used for teeth whitening or ultrasound/optical delivery devices might be used to take advantage of companion nanoparticles theranosis. A unique advantage of purely local nanotherapeutic administration is that drugs that would have systemic toxicity can be utilized - such as powerful anti-infectives or anti-inflammatories.

4) Focal macrophage-dependent inflammatory diseases

Those skilled in the art will be able to adopt companion nanoparticle theranosis to other macrophage-dependent inflammatory diseases, such as COPD, bronchial asthma, psoriasis, transplant rejection and vascular access stenosis. 6. Imaging Devices

Ultrasmall superparamagnetic iron oxide particles (USPIOs, size 15-50 nm) have been developed to serve as contrast agents or biomarkers for MRI. Applicant realized that the introduction of such particles into tissue affects the transmission of many wavelengths from the electromagnetic spectrum, which in turn can be used in medical applications such as imaging or even therapy. In as much as the useful USPIOs are small nanoparticles, they fit the domain of particle scattering as described by the Rayleigh or Mie theories and scattering increases with the effective particle size . Thus some of the imaging devices suitable for practicing the methods of the invention include those that utilize electromagnetic

wavelengths that are responsive to Rayleigh or Mie scattering.

The invention also provides the use of theranostic devices constructed to detect the aggregated nanoparticles with scattered ultrasound or light, and then interactively or sequentially heat the nanoparticles locally with a proper energy source, such as microwave, radiofrequency, high intensity ultrasound, or lasers.

The theranosis device may comprise an imaging device that cause and detects Rayleigh and Mie scattering from the USPIO aggregates, and an energy source that emits an energy suitable to raise the temperature of the USPIO aggregates. The device may a) visualize USPIO-loaded, activated macrophages in macrophage-dependent disease, and, b) locally heat the disease-responsive macrophages with absorbed energy from radiofrequency, high intensity ultrasound, or laser sources.

In certain embodiment, both the imaging and heating capabilities are contained on the same device / probe.

In certain embodiment, the device is designed for intracavitary, intravascular or interstitial insertion in order to improve proximity to the inflamed site.

In certain embodiment, the imaging and therapy utilities of the device are used simultaneous or sequentially to accomplish controlled thermal ablation of the focal process containing the identified macrophages.

In certain embodiments, the device may comprise additional elements, or be used with such additional elements, such as those that couple the device to the tissue and/or monitor local tissue temperature.

Suitable sheaths may be required to couple the device to the tissue containing the macrophage-loaded targets. Locally targeted thermotherapy is achieved by enhanced heating of the aggregated nanoparticles within the activated macrophages, creating heat sinks that lead to ablation of tissues within the effectively heated region. The region containing the enhanced macrophages may have an irregular shape, and heating the aggregated USPIO particles allows the therapy to geometrically fit the focal disease.

In some embodiments, thermal sensing technology may be useful.

The invention contemplates combinations selecting one element from each of the following categories:

Macrophage Detection Heat delivery Access for Device

Ultrasound Focused Ultrasound Endoscope

Optical coherence Radiofrequency Catheter

MRI Microwave Trocar

Laser External

Magnetic Fluid

Scattering and absorption of the wavelength utilized for either imaging or heating is increased by nanoparticle aggregation in the macrophages, and the amount of scattering and absorption is influenced by the effective aggregated nanoparticle sizes and volume fraction in the enhanced macrophages. Thus, the sensitivity for image detection and focal heating will depend upon the device combination selected. Similarly, penetration distances and energy delivered per photon absorbed will depend upon the devices used for this invention.

The various combinations of the imaging device and energy sources of the diathermy device and uses thereof are described above and will be apparent to those skilled in the art.

In certain embodiments following co-localization of the companion nanoparticles, under external ultrasound guidance, a trocar is advanced into the region containing the tumor- associated macrophages. The diathermy device is then inserted and advanced into the enhanced TAMs within the effective heating zone of said device, and this is confirmed by the interstitial ultrasound imaging capability. Heating is then delivered to that zone. Where necessary, the probe can then be repositioned to continue therapeutic heating.

For thermal therapy of breast cancer, radiofrequency and microwave probes, such those radiofrequency ablation systems marketed under the brand name STARBURST^® (AngioDynamics, Latham, NY), or microwave heating devices such as the microwave tissue ablation (MTA) system marketed under the brand name ACCULIS^® (Microsulis Medical Limited, Hampshire, UK), or similar devices may be selected, and the features of imaging guidance and temperature monitoring may be desirable. Similar devices may be used to treat other macrophage associated inflammatory disease according to the methods disclosed. These may include, but are not limited to, interstitial thermal therapy for primary or metastatic brain cancer, lung cancer, renal cancer, pancreatic cancer, prostate cancer, sarcoma as well as metastases associated with the primary cancer.

Diathermic devices used for the invention can also be used to treat ulcerative colitis of the rectosigmoid colon. The distribution of the ulcerative colitis in the rectosigmoid region is first imaged to confirm suitability for theranostic intervention. A suitable endoscope is advanced into the inflamed region and the theranostic device is deployed through side channels in the endoscope. In this example, the imaging of the USPIO-enhanced

macrophages may be accomplished, for example, by endoscopic ultrasound and the thermal therapy is performed using a radiofrequency probe. The ultrasound confirms that that the USPIO distribution within the inflamed colon is within the heating region for the

radiofrequency probe. Following application of the thermal therapy to the macrophage loaded nanoparticles within that region, the diathermy device and endoscope are repositioned for another thermal application. In this manner, the entire inflamed colon is locally treated.

For this clinical scenario, many endoscopically-guided theranostic combinations are feasible: endoscopic ultrasound plus NIR laser; endoscopic optical coherence tomography plus NIR laser, and HIFU with a suitable sheath.

The diathermy device may be further suitable for treating intracavitary macrophage- associated diseases accessed via endoscopy after nanoparticles loading, which include but are not limited to cancers of the throat, larynx, esophagus, stomach, duodenum, colon and anus, bronchus, etc., and inflammations of the bronchus, GI tract, joints, etc.

Similar devices may be deployed to treat other anatomic regions with vulnerable plaque in like manner. Suitable lesions in the carotid, aorta, renal artery, and peripheral arteries may be subject to effective intravascular thermal therapy.

The range of chemicals that can be useful for this invention can be generally described as those materials which can be formulated in phagophilic nanoparticles and have the desired effect upon the activated macrophage or the diseased tissue nearby.

Experiment steps evaluate companion nanoparticles theranostics may include:

1. In vitro macrophage culture: As noted earlier, there are a wide variety of

macrophage cell cultures that can be used to measure the cyto/chemokine production, increased membrane permeability, or cell viability that are important responses to companion nanoparticles theranosis. In these cultures the three experimental variables of USPIO, nanotherapeutic, and diathermy can be evaluated alone or in combination. For the nanoparticles, evaluating dose, exposure time, and interaction, especially competition for macrophage uptake, can be derived. The cultures can also be exposed to electromagnetic energies (laser, microwave, radiofrequency, ultrasound) to determine the time and temperature responses required to achieve the measured outcomes. Such experiments allow important interactions to be measured and estimates can be made of what limitations might exist.

In vivo - appropriate animal model: Several important variables cannot be measured in culture, especially the PK/PD of the nanoparticles and the interaction of biology with the measured response. In addition, there can be advantages and disadvantages to any aspect of the theranostic response appreciated when an appropriate animal model is used. In general, animal models need to be evaluated for their ability to represent human disease states. Nonetheless, all of the variables listed above can be evaluated for the same outcome measures, but now some administration variables can be studied. As a generality, administration can be systemic or local and the route can be the same or different for the USPIO or the nanotherapeutic(s).

Diathermy device details become important here as well. Phase 0 Clinical Trial: Phase 0 trials are the first- in-human trials. Single

subtherapeutic doses of the study drugs or treatment are given to a small number of subjects (10 to 15) to gather preliminary data on the agents pharmacodynamics (what the drug does to the body) and pharmacokinetics (what the body does to the drugs). For a test drug, the trial documents the absorption, metabolism, and removal

(excretion) of the drug, and the drug's interactions within the body, to confirm that these appear to be as expected. Phase 0 clinical studies provide this opportunity. This can include evaluating the co-localization of the nanoparticles using imaging and histology techniques. These small studies are conducted to demonstrate accumulation of companion nanoparticles. The subjects should have documented macrophage- dependent disease with uptake of USPIO. The experimental nanotherapeutic(s) can be given by the desired route -usually systemic or local. Confirmation of adequate PK/PD may include blood or tissue measurements of the nanotherapeutic in target and not target (if noninvasive determination is available) tissues.

One or more of these evaluative steps can provide go/no go direction for further clinical trials. It may well be that the nanotherapeutic is already approved for another indication and there is a USPIO, ferumoxytol (various trade names in different countries) that is approved for treatment of iron-deficiency anemia, facilitating its incorporation into such trials.

7. Exemplary Utility

The methods and systems of the invention provide useful information (e.g. , medical imaging) about the presence or absence of diseases in the interrogated tissues, as well as the specific location, shape and size of the affected tissues. Such information in turn provides basis for further medical analysis, such as disease diagnosis, staging (e.g., disease severity and extent), prognosis, treatment options, the response of the disease to a current treatment regimen, or the recurrence of the disease, etc.

For example, although the macrophage-dependent inflammatory changes are similar in general, cytokine and other cellular responses can differ among individuals or with particular inflammatory diseases. These differences can be in the nature of the disease or in the nature of the response to the disease. Such considerations have led to the postulation of the need for personalized medicine, i.e., treating a particular disease in a particular patient by a regimen that is tailored to that diseased patient. For instance, identifying the actual genetic aberration in a patient's cancer and treating that abnormality with a treatment uniquely successful for the identified abnormality is one form of personalized medicine. However, personalized medicine might encompass assessing the pathophysiology that accompanies a disease process in an individual patient. The deranged pathophysiology might enable individualized therapeutic regimens.

The invention provided herein allows personalized assessment of inflammatory diseases, and enables medical assessment of the distribution and severity of the disease or inflammation, the currently available treatments for the specific patient (this includes the underlying disease as well as the response to prior therapy or the concerns about potential contraindications due to other comorbidities), efficacy of the treatment (e.g., repeated assessments of the accompanying pathophysiology to see if a treatment responses is or is not as expected), and the current best estimate of outcome from the current treatment - a key prediction that can influence patient lifestyle choices.

The safety of non-invasive imaging is useful for the numerous times that a patient with inflammation could benefit from serial measurement of the extent and activity of his/her disease. Macrophage-dependent diseases, whether inflammation or neoplasia, can require repeated imaging and repeated exposure to ionizing radiation for diagnostic or evaluative purposes should be minimized.

Following intravenous administration of USPIOs, there is a biodistribution and pharmacokinetic response that is similar for many macrophage-dependent disease. The blood half-life and macrophage accumulation/retention times can differ. Since USPIOs are MR- imageable, they provide the ability to assess locally increased vascularity accompanying the macrophage-initiated angiogenesis and the locally increased phagocytosis of USPIO nanoparticles in IAMs can be used to address the questions posed above for all forms of chronic inflammation that are macrophage-dependent. These properties - MR visibility and the ability to assess both vascularity and macrophage activity - provide the surprising ability to characterize a diversity of inflammatory diseases following a single administration of the USPIO.

If the recommended steps of macrophage culture, response in appropriate animal models, and phase 0 clinical studies are supportive, then there is sufficient information to proceed into the usual clinical trials to gain approval for the companion nanoparticles theranostics.

BACKGROUND LITERATURE

Patent Citations

1. US 2014-0212503 Al, Lee, Hyukjin et al., "Delivery System."

2. US 8,367,113 B2, Gu, Frank X et a., "Polymers for functional particles."

3. US 2014-0170075 Al, Drummond et al., "Non-Invasive Imaging Methods for Patient Selection for Treatment with Nanoparticulate Therapeutic Agents.".

4. WO 2014/113167 Al, Drummond et al., "Noninvasive Imaging Methods for Patient Selection for Treatment with Nanoparticulate Therapeutic Agents." Other Publications

Novobrantseva, T.I. et al., "Systemic RNAi-mediated gene silencing in nonhuman primate and rodent myeloid cells" Mol. Ther. Nucleic Acids, l:e4 (2012).

Leuschner, F. et al., "Therapeutic siRNA silencing in monocytes in mice," Natl.

Biotechnol., 29: 1005-1010 (2011)

Lee, J. et al., "Molecular Self-Assembled Nucleic Acid Nanoparticles for Targeted In Vivo siRNA Delivery," Nat. Nanotechnol., 7:389-393 (2012).

Wang, Y. et al., "Nanoparticle-based delivery system for application of siRNA in vivo " Curr. Drug Metab., 11: 182-196 (2010).

Ponnappa, BC, "siRNA for inflammatory diseases," Curr. Opin. Investig. Drugs, 10:418- 24 (2009).

Tiz, NM, Torchilin, VP, "siRNA delivery: from basics to therapeutic applications," Front Biosci Landmark, 18:58-79 (2013).

Xie, J. et al., "Nanoparticle-based theranostic agents," Adv. Drug Rev., 62: 1064-1079 (2010).

Zhu, L., Torchilin, VP, "Stimulus-responsive nanopreparations for tumor targeting," Integr. Biol. (Camb.) 5:96-107 (2013).

Alexis, F. et al., "Nanoparticle technologies for cancer therapy," Handb. Esp.

Pharmacol., 197:55-86 (2010) (includes Langer, Farokhzad).

Davignon, J-L et al., "Targeting monocytes/macrophages in the treatment of rheumatoid arthritis," Rheumatology, 52:590-598 (2013) .

Bertrand, N. et al., "Cancer Nanotherapeutics: the impact of passive and active targeting the era of modern cancer biology," Adv. Drug Del. Rev., 66:2-26 (2014).

Cotran, RS, "Delayed and prolonged vascular leakage in inflammation. 3. Immediate and delayer vascular reactions in skeletal muscle," Esp. Mol. Pathol., 6: 143-155 (1967). Amocozgar, Z, Goldberg, MS, "Targeting myeloid cells using nanoparticles to improve cancer immunotherapy," Adv. Drug Deliv. Rev., Epub: pii: S0169-409X(14)00205-1 (2014).Koo, H et al., "In vivo targeted deliver of nanoparticles for theranosis," Acc. Chem. Rev., 44: 1018- 1028 (2011).

Lunov, O et al., "Modeling receptor-mediated endocytosis of polymer-functionalized iron oxide nanoparticles by human macrophages," Biomaterials, 32:547-555 (2011).

Laurent, S, Mahmoudi, M, "Superparamagnetic iron oxide nanoparticles: promises for diagnosis and treatment of cancer," Int. J. Mol. Epidemiol. Genet, 2:367-390 (2011), Cherukuri, P, Glazer, ES, Curley, SA, "Targeted hyperthermia using metal

nanoparticles," Adv. Drug Del. Ref., 62:339-345 (2010).

Xu, S et al., "Targeting receptor-mediated endocytotic pathways with nanoparticles: rationale and advances," Adv. Drug Del. Rev., 65: 121-138 (2013).

Sapareto, SA, Dewey, WC, "Thermal dose determination in cancer therapy," Int. J.

Radiat. Oncol. Biol. Phys., 10:787-800 (1984).

Ding, H-m, Ma, Y-q, "Role of physicochemical properties of coating ligands in receptor- mediated endocytosis of nanoparticles," Biomaterials, 33:5798-5802 (2012).

Song, AS et al., "Thermally induced apoptosis necrosis, and heat shock protein expression in three-dimensional culture," J. Biomech. Eng., Epub 136(7), 071006, 10 pages (2014).

Ahsan, F et al., "Targeting to macrophages: role of physicochemical properties on particular carriers— liposomes and microspheres— on the phagocytosis by macrophages," J. Control. Rel, 79:29-40 (2002).

Rahmathulla, G et al., "MRI-guided laser interstitial thermal therapy in neuro-oncology: a review of its current clinical applications," Oncol., 87:67-82 (2014).

Michl J., "Receptor-mediated endocytosis," Am. J. Clin. Nutr., 33:2462-2471 (1980). Ahmed M, Brace CL, Lee FT, "Goldberg SN. Principles of and advances in percutaneous ablation," Radiology, 258:351-369 (2011) .

Duivenvoorden, R, et al., "A statin-loaded reconstituted high-density lipoprotein nanoparticles inhibits atherosclerotic plaque inflammation," Nat. Commun., 5:3065-3083 (2014).

Leuschner, F et al., Silencing of CCR2 in myocarditis, Eur. Heart J., Epub pii: ehu225, URL: http://dx.doi.org/10.1093/eurheartj/ehu225 (2014)

Leuschner, F et al., "Therapeutic siRNA silencing in inflammatory monocytes in mice," Nat BiotechnoL, 29(11): 105-1010 (2011).

Bertrand, N et al., "Cancer nanotechnology. The impact of passive and active targeting in the era of modern cancer biology," Adv. Drug Delivery Rev., 66:2-25 (2014).

Shu, Y et al., "Stable RNA nanoparticles as potential new drugs for cancer therapy," Adv. Drug Delivery Rev., 68:74-89 (2014).

Abraham, J, Armstrong, AJ, "Oncology drugs in the pipeline," HemOnc Today, ( 2014), URL: http://www.healio.com/hematology-oncology/news/print/hemonc- today/ 7Bd377e341-5891-4ffe-b27e-l lecfe56c752 7D/2014-oncology-drugs-in-the- pipeline Biswas, S, Torchilin, VP, "Nanopreparations for organelle-specific delivery in cancer," Adv. Drug Delivery Rev., 66:26-41 (2014).

Lu, X et al., "Nanoparticles containing anti-inflammatory agents as chemotherapy adjuvants: optimization and in vitro characterization," APPS. J., 10: 133-140 (2008).

All references cited are incorporated by reference herein.

Claims

A method for detecting activated macrophages in a subject, and/or for treating a macrophage-dependent disease or condition associated with said activated

macrophages, the method comprising:

(a) administering to the subject a formulation of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (which may aggregate in the lysosomes of said activated macrophages);

(b) administering a nanoparticulate therapeutic agent that is phagocytosed by said activated macrophages, wherein said nanoparticulate therapeutic agent is effective in treating said disease or condition;

(c) waiting for a pre-determined time to allow the USPIO nanoparticles to

accumulate as USPIO aggregates inside said activated macrophages, and to allow the nanoparticulate therapeutic agent to accumulate inside said activated macrophages;

(d) visualizing the USPIO within the activated macrophages with a medical

imaging device sensitive to the aggregated USPIOs to identify size, shape, and/or location of a tissue affected by the disease or condition; and,

(e) locally raising the temperature of (e.g. , heating) the activated macrophages containing the nanoparticulate therapeutic agent, by directing an energy that is absorbed by the USPIO aggregates, and sustaining the locally elevated temperature for a time sufficient to release the nanoparticulate therapeutic agent within the activated macrophages in tissues affected by the disease or condition.

The method of claim 1, wherein the USPIO nanoparticles have an average size of about 10-250 nm, about 20-125 nm, or about 22-40 nm.

The method of claim 1, where said nanoparticulate therapeutic agent is administered systemically, interstitially, or per os.

The method of claim 1, wherein step (e) is carried out by exposing the activated macrophages to an electromagnetic wave (e.g., light, microwave, radiofrequency, or ultrasound frequencies emitted by specialized medical devices designed for that purpose).

5. The method of claim 4, where the temperature in the activated macrophage in tissues affected by the disease or condition is raised sufficiently to effect at least one of the following:

(a) increased cell membrane permeability within the activated macrophage loaded with the nanoparticulate therapeutic agent (e.g. , the step is carried out by an induced temperature of 43-48°C for 1-10 minutes, or by an induced temperature of 44°C for 5 minutes);

(b) apoptosis of the activated macrophage loaded with the nanoparticulate

therapeutic agent (e.g. , the step is carried out by an induced temperature of 45- 60°C for 1- 10 minutes, or by an induced temperature of 50°C for 5 minutes);

(c) rapid necrosis of the activated macrophage loaded with the nanoparticulate therapeutic agent (e.g. , the step is carried out by an induced temperature of 60- 90°C for 1- 10 minutes, or by an induced temperature of 70°C for 5 minutes); and,

(d) increased cell permeability, apoptosis, or necrosis of adjacent cells (e.g. , due to thermal diffusion from a heat sink within the adjacent cells, such as an activated and nanoparticle-loaded macrophage).

6. The method of claim 1, wherein the nanoparticulate therapeutic agent is phagophilic (e.g. , due to its size, shape, coating or bonding to other phagophilic carriers).

7. The method of claim 1, wherein the nanoparticulate therapeutic agent facilitates

receptor mediated endocytosis, and is resistant to heating .

8. The method of claim 7, wherein the nanoparticulate therapeutic agent has an average nanoparticle size of <400 nm, preferably <200 nm for intravascular administration.

9. The method of claim 7, wherein the nanoparticulate therapeutic agent displays

receptor-avid ligands on the surface.

10. The method of claim 7, wherein the nanoparticulate therapeutic agent comprises carrier materials (such as liposomes, micelles, polymers) that provide long

intravascular blood half life (e.g. , 2-48 hours, preferably longer than 8 hours).

11. The method of claim 7, wherein the nanoparticulate therapeutic agent is resistant to inactivation by temperatures above 44-99°C that are sustained for 0.5-30 minutes (preferably for at least 10 minutes). The method of claim 6, wherein release of the nanoparticulate therapeutic agent causes suppression of cytokine/chemokine production by the activated macrophages.

The method of any one of claims 1-12, wherein the method leads to amelioration of the macrophage-dependent disease or condition.