Mapping of Nanoparticles

Abstract

A nanoparticle material suitable for administration to a subject, comprising: (a) a copolymeric steric stabilizer that promotes dispersion of the nanoparticle material in a liquid, the copolymeric steric stabilizer comprising: (i) an immobilized polymer segment having one or more binding groups that attach the copolymeric steric stabilizer to the nanoparticle material, and (ii) a steric stabilizing polymer segment that is different from the immobilized polymer segment; and (b) a copolymeric mapping portion comprising: (i) an immobilized polymer segment having one or more binding groups that attach the copolymeric mapping portion to the nanoparticle material, (ii) one or more mapping groups that comprise an agent that specifically binds to fibroblast activation protein (FAP), and (iii) a coupling polymer segment that is different from the immobilized polymer segment, and that attaches the immobilized polymer segment to the one or more mapping groups.

Description

Technical Field

[0001] The present application generally relates to nanoparticles, compositions containing them, and their use in mapping, diagnostic and therapeutic applications.

Background Art

[0002] Surgical tumor resection is the standard treatment for many cancers, especially when the disease is localized to a single solid tumor. However, surgery is invasive and, if residual neoplastic tissue cannot be identified, can result in a positive resection margin, which is associated with local recurrence and poor patient outcomes. Furthermore, accurate identification of the tumor margin is important for the effectiveness of non-surgical treatment modalities, such as external beam radiation therapy, brachytherapy, and focal therapy, each of which requires the best possible characterization of the location and extent (e.g., volume) of the tumor.

[0003] For example, radical prostatectomy is the standard treatment for aggressive and intermediate prostate cancer. However, after this surgical intervention, approximately 20% of patients experience urinary incontinence, and approximately 70% of patients experience erectile dysfunction. Due to these serious side effects, men with limited life expectancy and slow-progressing, low-risk diseases are often recommended to "watch and wait" before undergoing surgery. Alternatively, some patients are offered treatments such as external beam radiation therapy, brachytherapy, and focal therapy before radical prostatectomy. Such treatments are associated with a reduction in side effects but are not as effective as surgical partial or complete resection of the tumor. The factor contributing to the reduced effectiveness of these alternative treatment modalities is the limitation of existing medical images that cannot provide sufficient spatial resolution to identify the boundaries and margins of the primary tumor. For example, prostate-specific membrane antigen positron emission tomography / computed tomography (PET-PSMA) may underestimate the tumor volume by 9-15%, and multi-parametric magnetic resonance imaging (mpMRI) may underestimate the tumor volume by 11-20%. Due to the drawbacks in the images, the guidelines for focal therapy require expanding the margins of the resection area surrounding the identified lesion by up to 10 mm. However, even with the expansion of the resection area, a stump recurrence rate of 20-40% has been reported. Similar problems occur in other cancers, including but not limited to glioblastoma and pancreatic cancer, where organ preservation and limitation of normal tissue toxicity are important.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Therefore, in order to improve surgical resection by providing improved preoperative and / or intraoperative guidance, or to provide a less invasive treatment modality, such as focal therapies like cryoablation, focused laser ablation, and high-frequency ultrasound ablation, photodynamic therapy, high-dose rate and low-dose rate brachytherapy, particle beam therapies such as proton and carbon ion beam therapy, and external beam radiation therapies such as intensity-modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), hypofractionated radiation therapy, and very-high-dose rate radiation therapy, there remains a need to develop new materials and compositions that can more accurately identify the margins of solid tumors such that the entire volume of the tumor can be more accurately targeted and exposure of non-target tissue can be reduced.

Means for Solving the Problems

[0005] Surprisingly, it has been found that the nanoparticle material according to the present invention selectively accumulates in the tumor microenvironment, thereby mapping the margins of solid tumors. Without wishing to be bound by theory, by combining the functions of the copolymer steric stabilizer and the copolymer mapping moiety, the nanoparticle materials described herein can be at least partially enabled to selectively bind to fibroblast activation protein (FAP) expressed by cells in the tumor microenvironment, such as tumor-associated stromal cells.

[0006] Thus, in one aspect described herein, a Magnetic nanoparticle material suitable for administration to a subject, having on its surface (a) a copolymer steric stabilizer that promotes dispersion of the Magnetic nanoparticle material in a liquid, the copolymer steric stabilizer including (i) an immobilized polymer segment having one or more linking groups for linking the copolymer steric stabilizer to the Magnetic nanoparticle material and (ii) a steric stabilizing polymer segment different from the immobilized polymer segment, and (b) a copolymer mapping moiety, the copolymer mapping moiety including (i) the MagneticAn immobilized polymer segment having one or more binding groups for binding to a nanoparticle material, (ii) one or more mapping groups comprising an agent that specifically binds to fibroblast activation protein (FAP), and (iii) a coupling polymer segment different from the immobilized polymer segment, the coupling polymer segment coupling the immobilized polymer segment to the one or more mapping groups, and a copolymer mapping portion comprising the coupling polymer segment are bonded together. Magnetic A nanoparticle material is provided.

[0007] According to the present invention Magnetic The nanoparticle material advantageously exhibits improved blood half-life circulation to support systemic injection and / or does not substantially degrade in vivo. In terms of cost, the "magnetic nanoparticle material" according to the present invention may also be simply referred to as "nanoparticle material" in this specification when relevant.

[0008] While not wishing to be bound by theory, it is believed that the nanoparticle material exhibits such advantageous properties at least through the copolymer components bound to its surface. These copolymer components include copolymer steric stabilizers used in combination with the copolymer mapping portion. Both the copolymer steric stabilizer and the copolymer mapping portion include an immobilized polymer segment that binds their respective entities to the nanoparticle material. The immobilized polymer segment has been found to be very effective, for example, in maintaining both the copolymer mapping portion and the copolymer steric stabilizer immobilized on the nanoparticle material when in a biological liquid environment. This facilitates maintaining the nanoparticle material in a dispersed form in a biological liquid environment. One of ordinary skill in the art will understand that aggregation of the nanoparticle material in a biological liquid environment can be detrimental in diagnostic and therapeutic applications.

[0009] Although not wishing to be limited by theory, it is believed that the copolymer mapping portion, by acting synergistically with the effect of imparting improved dispersion in a biological fluid environment or the like, enables the accumulation of the particulate material in the tumor microenvironment to be improved. Depending on the intended use, the nanoparticle material, by binding to the copolymer mapping portion, similarly accumulates essentially within the tumor microenvironment but can be advantageously selected for performing a given task. For example, the nanoparticle material can be provided in the form of a magnetic nanoparticle material for use in applications such as magnetic particle imaging (MPI) as well as nuclear magnetic resonance imaging (MRI), such as low-field MRI, MRI-guided external beam radiation therapy, MRI-guided focal ablation, MRI / ultrasound fusion focal ablation, MRI-guided biopsy, MRI / ultrasound fusion-guided biopsy, MRI-guided surgery, MRI-guided sealed brachytherapy, MRI-guided infrared camera-guided biopsy or treatment, and photoacoustic-guided biopsy or treatment.

[0010] Thus, it has been found that the nanoparticle material according to the present invention is particularly effective in improving conventional methods and treatment applications for mapping the tumor margin of a subject and determining the location and extent (e.g., volume) of a tumor lesion.

[0011] Those skilled in the art will understand that the application of a targeted nanoparticle material in vivo can be subject to adverse effects due to non-specific protein adsorption, resulting in the so-called protein corona, which can lead to a decrease in the binding efficiency at the target site. Such protein adsorption can not only potentially reduce the binding efficiency but also, as a result, reduce the accumulation of the nanoparticle material at the target site. Surprisingly, it has been found that the combination of a copolymer steric stabilizer and copolymer mapping can also advantageously reduce the effect of harmful protein adsorption, thereby improving the accumulation efficiency of the nanoparticle material within the tumor microenvironment.

[0012] In one embodiment, the nanoparticle material has attached to its surface a (c) copolymer luminescent moiety comprising: (i) an immobilized polymer segment having one or more linking groups that link the polymer luminescent moiety to the nanoparticle material; (ii) one or more luminescent groups that emit light or an acoustic signal responsive to light to enable visualization of the in vivo location of the nanoparticle material; and (iii) a coupling polymer segment different from the immobilized polymer segment that couples the immobilized polymer segment to the one or more luminescent groups.

[0013] In one embodiment, the sterically stabilizing polymer segment comprises a polyacrylamide-co-polyalkylene oxide block copolymer.

[0014] In another embodiment, the coupling polymer segment is composed of polyacrylamide.

[0015] In a further embodiment, the sterically stabilizing polymer segment comprises a polyacrylamide-co-polyalkylene oxide block copolymer and the coupling polymer segment is composed of polyacrylamide.

[0016] In one embodiment, the sterically stabilizing polymer segment has 10 to 70 polymerized monomer residue units.

[0017] In another embodiment, the coupling polymer segment has 15 to 100 polymerized monomer residue units.

[0018] In a further embodiment, the sterically stabilizing polymer segment has 10 to 70 polymerized monomer residue units and the coupling polymer segment has 15 to 100 polymerized monomer residue units.

[0019] In one embodiment, the nanoparticle material is a magnetic nanoparticle material.

[0020] As described elsewhere in this specification, the nanoparticle material selectively binds to fibroblast activation protein (FAP) expressed by cells within the tumor microenvironment.

[0021] In one embodiment, the cells within the tumor microenvironment are tumor-associated stromal cells.

[0022] In a further embodiment, the tumor-associated stromal cells are selected from fibroblasts, pericytes, adipocytes, mesenchymal stromal cells (MSCs), endothelial cells, and combinations thereof. In another embodiment, the tumor-associated stromal cells are selected from pericytes, endothelial cells, and combinations thereof.

[0023] In one embodiment, the agent that specifically binds to FAP is selected from small molecule inhibitors and antibodies or antigen-binding fragments thereof. In another embodiment, the small molecule inhibitor is an FAP inhibitor.

[0024] In a further embodiment, the one or more luminescent groups are selected from chemiluminescent groups, electroluminescent groups, photoluminescence groups, radioluminescence groups, and thermoluminescence groups.

[0025] In another aspect disclosed herein, there is provided a composition suitable for administration to a subject, the composition comprising a Magnetic nanoparticle material according to the present invention.

[0026] In one embodiment, the composition comprises a pharmaceutically acceptable liquid carrier.

[0027] In another aspect disclosed herein, there is provided the use of a Magnetic nanoparticle material or composition according to the present invention in performing a therapeutic or diagnostic application on a subject.

[0028] As examples of suitable therapeutic or diagnostic applications, magnetic particle imaging (MPI), magnetic resonance imaging (MRI), MRI-guided external beam radiotherapy, MRI-guided focal ablation, MRI / ultrasound fusion focal ablation, MRI-guided biopsy, MRI / ultrasound fusion-guided biopsy, MRI-guided surgery, MRI-guided sealed brachytherapy, MRI-guided infrared camera-guided biopsy or treatment, and photoacoustic-guided biopsy or treatment are exemplified.

[0029] The nanoparticle materials or compositions according to the present invention can be used in combination with in vivo imaging diagnostic techniques including, but not limited to, ultrasound, MRI / ultrasound, X-ray, optical imaging, computed tomography (CT), single photon emission computed tomography (SPECT), positron emission tomography (PET), fluorescence resonance energy transfer (FRET), and magnetic resonance imaging (MRI).

[0030] In another aspect disclosed herein, there is provided the use of the nanoparticle materials or compositions according to the present invention for in vivo imaging. Magnetic

[0031] In another aspect disclosed herein, there is provided the nanoparticle materials or compositions according to the present invention for use in in vivo imaging.

[0032] In another aspect disclosed herein, there is provided the use of the nanoparticle materials or compositions according to the present invention for detecting cancer.

[0033] In another aspect disclosed herein, there is provided the nanoparticle materials or compositions according to the present invention for use in detecting cancer.

[0034] In one embodiment, the cancer is selected from prostate cancer, glioblastoma multiforme, glioma, pancreatic cancer, colorectal cancer, breast cancer, head and neck cancer, gastric cancer, esophageal cancer, ovarian cancer, sarcoma, and lung cancer. In another embodiment, the cancer is prostate cancer.

[0035] ​In another aspect disclosed herein, there is provided the use of a nanoparticle material or composition according to the present invention for mapping the tumor microenvironment. Magnetic The use of a nanoparticle material or composition is provided.

[0036] In another aspect disclosed herein, there is provided a nanoparticle material or composition according to the present invention for use in mapping the tumor microenvironment. Magnetic The nanoparticle material or composition is provided.

[0037] In one aspect disclosed herein, a. administering a nanoparticle material or composition according to the present invention to a subject; Magnetic and b. Magnetic detecting the nanoparticle material, wherein the method for mapping the tumor margin of the subject comprises: Magnetic the nanoparticle material accumulates in the tumor microenvironment, thereby mapping the tumor margin. A method is provided.

[0038] In one embodiment, the tumor margin is mapped in situ. The tumor can be mapped in situ before or after tumor tissue resection.

[0039] In one embodiment, the method further comprises determining a clinical target volume (CTV) and / or a gross target volume (GTV) before treatment.

[0040] In one aspect disclosed herein, there is provided a method for treating cancer in a subject in need thereof, the method comprising: a. administering a nanoparticle material or composition according to the present invention to the subject; Magnetic and b. Magnetic detecting the site where the nanoparticle material accumulates; and Magnetic c. administering an effective amount of the cancer treatment to the site of nanoparticle material detection in step (b). A method is provided.

[0041] In one embodiment, the treatment is selected from surgery, radiotherapy, brachytherapy, photodynamic therapy, photothermal therapy, focal ablation therapy (such as cryoablation, focused laser ablation, and high-frequency ultrasound ablation), chemotherapy, immunotherapy, and combinations thereof.

[0042] In one embodiment, the nanoparticle material is detected using an imaging diagnostic technique selected from magnetic resonance imaging (MRI), ultrasound, X-ray, optical imaging, fluorescence imaging, computed tomography (CT), single photon emission computed tomography (SPECT), positron emission tomography (PET), and fluorescence resonance energy transfer (FRET).

[0043] In one embodiment, the method further includes determining a clinical target volume (CTV) and / or a gross target volume (GTV) before performing the treatment according to step (c).

[0044] In one aspect disclosed herein, a. administering a nanoparticle material or composition according to the present invention to a subject; Magnetic and b. Magnetic detecting the nanoparticle material, wherein the method for diagnosing cancer comprises: detecting the nanoparticle material accumulated in the tissue (such as vascular tissue) of the subject, Magnetic wherein the detection of the nanoparticle material indicates that the subject has cancer.

[0045] Further aspects and embodiments of the present invention are outlined and discussed in more detail below.

[0046] Embodiments of the present disclosure are described with reference to the accompanying non-limiting figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0047]

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DETAILED DESCRIPTION OF THE INVENTION

[0048] In this specification, some terms well known to those skilled in the art are used. However, for clarity, some of the terms are defined.

[0049] The term "subject" means either an animal or a human subject. "Animal" means primates, domestic animals (such as cows, horses, sheep, pigs and goats), companion animals (such as dogs, cats, rabbits and guinea pigs) and captive wild animals (including those kept in a zoo environment). Laboratory animals such as rabbits, mice, rats, guinea pigs and hamsters are also contemplated as they can provide useful test systems. In some embodiments, the subject is a human subject.

[0050] For a nanoparticle material or composition according to the present invention to be "suitable" for administration to a subject means that administration of the composition to the subject does not result in unacceptable toxicities such as allergic reactions and disease states.

[0051] "Administration" of a nanoparticle material or composition to a subject means that the nanoparticle material or composition is provided such that the nanoparticle material can be transferred to the subject. There is no particular limitation on the mode of administration, but this generally includes oral, parenteral (such as subcutaneous, intradermal, intramuscular, intravenous, intracerebral, intranasal, intrathecal, and intraspinal), inhalation (such as spraying), topical, rectal, and vaginal modes. The nanoparticle material or composition may also be directly administered into the tumor and / or into the tissue adjacent to one or more segments of the tumor, or may be directly administered intravascularly.

[0052] "Pharmacologically acceptable" means suitable for administration to a subject. That is, administration of the relevant substance to a subject should not result in unacceptable toxicities such as allergic reactions and disease states.

[0053] Merely as a guideline, those skilled in the art may consider "pharmacologically acceptable" as an entity approved by a regulatory agency of the federal or state government, or an entity listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, more specifically in humans.

[0054] However, those skilled in the art will understand that the suitability of administration of the nanoparticle material or composition according to the present invention to a subject, and whether or not it or its components are considered pharmacologically acceptable, depends to some extent on the selected mode of administration. Therefore, when evaluating whether a composition or its components are suitable for administration to a subject or are pharmacologically acceptable, it may be necessary to consider the mode of administration.

[0055] When a nanoparticle material is "dispersed throughout" a liquid carrier, it means that the nanoparticle material exists as a dispersed phase throughout the liquid carrier that exists as a continuous liquid medium or phase with respect to the particle material itself. That is, the composition according to the present invention can be described as including a suspension or dispersion of the nanoparticle material throughout the liquid carrier.

[0056] As used herein, the term "liquid" in the context of a liquid carrier is intended to mean a medium in which the nanoparticle material is fully dispersed and which is at least in a liquid state at the temperature of the intended use of the composition according to the present invention. Generally, a liquid carrier is considered to be in a "liquid" state if, in the absence of a stabilizer, the particulate material dispersed throughout the carrier can flocculate or sediment from the carrier to form a precipitate. That is, it is considered "liquid" if the particulate material can move relatively freely within the medium.

[0057] The liquid carrier used according to the present invention may be composed of one or more different liquids. Suitable pharmaceutically acceptable liquid carriers are described in Martin, "Remington’s Pharmaceutical Sciences", 18th Edition, Mack Publishing Co., Easton, Pennsylvania (1990), and include, but are not limited to, sterilizable liquids such as water and oils of petroleum, animal, vegetable, mineral or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil. Other liquid carriers include methylene glycol, propylene glycol, polyethylene glycol, polypropylene glycol, ethanol, isopropyl alcohol, benzyl alcohol. Water or soluble saline and aqueous dextrose and glycerol solutions are preferably used as liquid carriers, particularly for injection solutions.

[0058] The composition according to the present invention may contain one or more pharmaceutically acceptable additives known to those skilled in the art. For example, the liquid carrier may contain one or more additives such as wetting agents, defoaming agents, surfactants, buffering agents, electrolytes, preservatives, colorants, flavorings and sweeteners.

[0059] The specific nature of the liquid carrier and any additives (if present) depends in part on the intended use of the composition. A person skilled in the art will be able to select a liquid carrier and additives (if present) suitable for the intended use of the composition.

[0060] The nanoparticle material can be administered, where appropriate, in a therapeutically or diagnostically effective amount. A therapeutically or diagnostically effective amount, when administered according to a desired dosing schedule, is an amount that achieves one or more of the desired therapeutic or diagnostic effects, such as alleviation of the symptoms, prevention or delay of the onset, inhibition or delay of the progression, diagnosis, or arrest or reversal of the particular condition being treated and / or evaluated, e.g., the onset or progression of the particular condition being treated and / or evaluated.

[0061] The appropriate dosage and dosing schedule to achieve this can be determined by the attending physician and may depend on the particular condition being treated or diagnosed, the severity of the condition, as well as the general age, health status and weight of the subject.

[0062] Administration can be carried out at intervals of minutes, hours, days, weeks, months or years, or continuously over any one of these periods. The appropriate dosage of the particle material itself can be in the range of about 0.1 ng per kg of body weight per single administration to 1 g per kg of body weight. The dosage can be in the range of 1 μg to 1 g per kg of body weight per single administration, e.g., in the range of 1 mg to 1 g per kg of body weight per single administration. In one embodiment, the dosage can be in the range of 1 mg to 500 mg per kg of body weight per single administration. In another embodiment, the dosage can be in the range of 1 mg to 250 mg per kg of body weight per single administration. In yet another embodiment, the dosage can be in the range of 1 mg to 100 mg per kg of body weight per single administration, e.g., up to 50 mg per kg of body weight per single administration.

[0063] The nanoparticle material or composition according to the present invention can be administered by single administration or continuous administration.

[0064] When the nanoparticle materials or compositions according to the present invention are suitable for parenteral administration, they are generally in the form of an aqueous or non-aqueous isotonic sterile injection solution which may contain one or more of an antioxidant, a buffer, a bactericide or a solute that makes the composition isotonic with the intended subject's blood. Such compositions may be provided in sealed containers for single or multiple administrations, such as ampoules and vials.

[0065] Upon administration, the nanoparticle materials or compositions according to the present invention may be diluted in vivo. For example, dilution may occur when administered orally or parenterally. In that case, since the liquid carrier of the composition can be very well diluted in vivo, the surrounding liquid environment in which the particle material is dispersed throughout becomes more reflective of the in vivo liquid (i.e., the biological liquid / fluid within the subject) than the original liquid carrier. For example, when administered parenterally, it would be more appropriate to describe that the nanoparticle material disperses throughout the whole blood rather than the original liquid carrier of the composition. Under such circumstances, it may be convenient to refer to the nanoparticle material as being dispersed throughout the in vivo liquid carrier (i.e., the biological liquid / fluid within the subject). Except for the difference in composition between the liquid carrier of the composition according to the present invention and the in vivo liquid carrier, the matters described herein regarding the liquid carrier of the composition generally also apply to the in vivo liquid carrier.

[0066] As used herein, the expression "tumor microenvironment" refers to a heterogeneous population of non-cancerous cells surrounding and / or infiltrating a tumor, which is very important for the function, physiology and metastasis of the tumor. One of ordinary skill in the art will understand that the tumor microenvironment can include various different cell types that can vary based on the size, location, type and stage of the tumor, and examples that are useful for its description include fibroblasts, pericytes, adipocytes, mesenchymal stromal cells (MSCs), cancer cells and endothelial cells. The cells of the tumor microenvironment are non-cancerous, but the tumor mobilizes and / or controls such cells to provide an environment that is conducive to promoting cancer growth. Thus, the cells contained within the tumor microenvironment are sometimes referred to as "cancer-related" or "tumor-related".

[0067] In one embodiment, the nanoparticle material disclosed herein selectively binds to fibroblast activation protein (FAP) expressed by cells in the tumor microenvironment.

[0068] In one embodiment, the cells in the tumor microenvironment are tumor-associated stromal cells.

[0069] In one embodiment, the tumor-associated stromal cells are selected from fibroblasts, pericytes, adipocytes, mesenchymal stromal cells (MSCs), cancer cells, endothelial cells, and combinations thereof. In another embodiment, the tumor-associated stromal cells are selected from pericytes, endothelial cells, and combinations thereof.

[0070] The term "about" or "approximately" means within an acceptable range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 20% of a given value, preferably within 10% of the given value, more preferably within 5% of the given value, and even more preferably within 1% of the given value. Alternatively, especially with respect to biological systems or processes, the term can mean within 10-fold of a value, preferably within 5-fold of the value, more preferably within 2-fold of the value. Unless otherwise specified, the term "about" means within an acceptable error range for a particular value, e.g., ±1 to 20%, preferably ±1 to 10%, more preferably ±1 to 5%.

[0071] When ranges of values are provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated value or intervening value within the stated range, is included within the disclosure. The upper and lower limits of these narrower ranges can independently be included within the narrower ranges and can also be included within the disclosure and be subject to any specifically excluded limits within the stated range. When the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the disclosure.

[0072] The phrase "at least one" in a list of items refers to any combination of those items and includes a single member. As an example, "at least one of a, b, or c" is intended to include a, b, c, a - b, a - c, b - c, and a - b - c.

[0073] To assist in the description of the nanoparticle material according to the present invention, reference is made to FIG. 1. FIG. 1 is a schematic diagram of a nanoparticle material (10) according to the present invention. The nanoparticle material itself (20) is represented by an iron oxide nanoparticle material. On the surface of the nanoparticle material (20), (i) a copolymer mapping portion generically represented by forms (30), (40) and (50) and (ii) a copolymer steric stabilizer generically represented by forms (30) and (60) are bonded. The copolymer mapping portion includes an immobilized polymer segment (30) and a coupling polymer segment (40), and the immobilized polymer segment and the coupling polymer segment are distinct. The coupling polymer segment (40) has one or more mapping groups (50) coupled thereto. The copolymer steric stabilizer includes an immobilized polymer segment (30) bonded to a steric stabilizing polymer segment (60), and the immobilized polymer segment (30) is distinct from the steric stabilizing polymer segment (60). The coupling polymer segment (40) contains more polymerised monomer residue units than the steric stabilizing polymer segment (60), thereby enabling one or more mapping groups (50) to extend a longer distance from the surface of the nanoparticle material (20) compared to the steric stabilizing polymer segment (60).

[0074] For a particle material to be a "nano" particle material means that at least one of its dimensions is less than 100 nm, or less than about 75 nm, or less than about 50 nm, or less than about 30 nm. In one embodiment, all dimensions of the nanoparticle material are less than 100 nm, or less than about 75 nm, or less than about 50 nm, or less than about 30 nm.

[0075] The nanoparticle material can be in the form of primary particles or in the form of aggregates of primary particles. In one embodiment, the nanoparticle material is in the form of primary particles.

[0076] For the sake of avoiding misunderstanding, it should be noted that references to the "size" of the nanoparticle material herein are intended to indicate the average size of the particles (at least about 50% on a number basis) based on the maximum dimension of a given particle.

[0077] The size of the nanoparticle material itself is determined herein by transmission electron microscopy (TEM).

[0078] For the sake of avoiding misunderstanding, it should be noted that when the nanoparticle material is in the form of aggregates of primary particles, references to the size of such a material are intended to be references to the maximum dimension of the aggregates, rather than to the primary particles forming the aggregates.

[0079] In certain embodiments, the nanoparticle material has a size of less than about 50 nm in at least one dimension or in all dimensions. In certain embodiments, the nanoparticle material has a size in the range of about 5 nm to about 30 nm, or about 5 nm to about 20 nm, or about 8 nm to about 15 nm in at least one dimension or in all dimensions.

[0080] In one embodiment, the nanoparticle material has a size of about 6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm.

[0081] Nanoparticle materials generally have at least an outer surface that is solid at the temperatures at which they are normally exposed in their intended use. Considering the temperatures to which the nanoparticle material or composition may be exposed during use and during storage prior to use in the intended application, at least the outer surface of the nanoparticle material is generally in a solid state at at least about 40 °C or less, preferably about 50 °C or less. The nanoparticle material can, of course, have such a solid-state composition throughout, and in some embodiments, has such a solid-state composition throughout (i.e., is a solid nanoparticle material).

[0082] According to the present invention, the nanoparticle material is a magnetic nanoparticle material. In this specification, may have an organic composition, an inorganic composition, or a combination thereof as well as and may be selected from, or include, pharmaceutically active compounds (e.g., drugs), metals, alloys, metal salts, metal complexes, metal oxides, radioisotopes, luminescent compounds or groups, and / or combinations thereof the nanoparticle material is also described .

[0083] the above Nanoparticle materials can include gold, silver and their salts, their complexes or oxides, calcium carbonate, barium sulfate, bismuth sulfide, iron, iron oxide, chromium oxide, cobalt oxide, manganese oxide, iron oxyhydroxide, chromium oxyhydroxide, cobalt oxyhydroxide, manganese oxyhydroxide, chromium dioxide, other transition metal oxides, radioisotopes (selected from Auger electron emitters, alpha emitters, positron emitters, and beta emitters), and combinations thereof.

[0084] Examples of Auger electron emitters include 51 Cr, 67 Ga, 71 Ge, 75 Se, 77 Br, 80m Br, 99m Tc, 103 Pd, 103m Rh, 111 In, 113m In, 115m In, 117m Sn, 119 Sb,123 I、 125 I、 131 Cs、 161 Ho、 165 Er、 193m Pt、 195m Pt、 201 Tl and 203 Pb are exemplified.

[0085] Examples of alpha emitters include 211 At and 213 Bi are exemplified.

[0086] Examples of beta emitters include 191 Os, 35 S, 33 P, 45 Ca, 199 Au, 169 Er, 67 Cu, 47 Sc, 177 Lu, 161 Tb and 105 Rh and other low-energy beta emitters; 131 I, 153 Sm, 77 As, 143 Pr, 198 Au, 159 Gd, 109 Pd, 186 Re, 111 Ag and 149 Pm and other medium-energy beta emitters; and 165 Dy, 89 Sr, 32 P, 166 Ho, 188 Re, 114m In, 142 Pr, 90 Y and 76 As and other high-energy beta emitters are exemplified.

[0087] Examples of radioisotopes that can be used in radiotherapy include 32 P, 153m S, 90 Y, 125 I, 192 Ir, 103 Pd, 111 In,166 Ho and 213 Bi are exemplified.

[0088] Examples of radioisotopes that can be used as diagnostic agents include 99m Tc, 67 Ga, 64 Cu, 89 Zr and 18 F are exemplified.

[0089] Examples of positron emitters that can be used as diagnostic agents include gallium 68, copper 64, zirconium 89, yttrium 86, rubidium 82, scandium 44, multiple isotopes of copper, terbium 182, gallium 66, and cobalt 55.

[0090] When a radioisotope is used, the radionuclide may be used as the nanoparticle material itself or may be combined with one or more other suitable nanoparticle materials. In other words, the nanoparticle material may contain one or more radioisotopes. For example, 67 Ga may be used in a form combined with an iron oxide particle material.

[0091] used by the present invention Magnetic nanoparticle materials can exhibit ferromagnetic, ferrimagnetic, or superparamagnetic properties.

[0092] In one embodiment, the nanoparticle material exhibits superparamagnetism.

[0093] The nanoparticle material may be made from a magnetic material or may contain a magnetic material.

[0094] Examples of suitable magnetic materials include, but are not limited to, iron, nickel, chromium, cobalt, gadolinium, manganese, any of the foregoing oxides or oxyhydroxides, and any mixture of the foregoing. In certain embodiments, the magnetic nanoparticles contain iron and / or its oxide or oxyhydroxide. Suitable iron oxide magnetic materials include maghemite (γ-Fe2O3) and magnetite (Fe3O4).

[0095] In one embodiment, the magnetic nanoparticle material comprises one or more of iron, nickel, chromium, cobalt, gadolinium, manganese, and their oxides or oxyhydroxides.

[0096] In another embodiment, the magnetic nanoparticle material comprises iron (Fe), maghemite (γ-Fe2O3), magnetite (Fe3O4), or combinations thereof.

[0097] In some embodiments, the magnetic nanoparticle material is or comprises magnetite (Fe3O4) or maghemite (γ-Fe2O3) having a particle size of less than about 30 nm, such as from about 1 nm to about 20 nm.

[0098] The magnetic nanoparticle material may be in the form of a metal such as iron surrounded by a magnetic metal oxide shell such as a maghemite (γ-Fe2O3) shell around the core material.

[0099] In some embodiments, the magnetic nanoparticle material is a ferrite of the general formula MO·Fe2O3, where M is a divalent metal such as Fe, Co, Ni, Mn, Be, Mg, Ca, Ba, Sr, Cu, Zn, Pt, Gd, or mixtures thereof, or a magnetoplumbite-type oxide of the general formula MO·6Fe2O3, where M is a large divalent ion, metallic iron, cobalt, or nickel, or includes them. The magnetic nanoparticle material may be composed of Fe, Zn, Ni, Cr, Co, or Gd, or their oxides or oxyhydroxides. Alternatively, the magnetic nanoparticle material can be a mixture of any of these.

[0100] In some applications, it may be desirable to use a magnetic nanoparticle material that exhibits superparamagnetism. As used herein, the term "superparamagnetism" is intended to mean a magnetic material having the following properties: (i) coercivity, (ii) residual magnetism, or (iii) no hysteresis loop when the rate of change of the applied magnetic field is quasistatic.

[0101] In some embodiments, the nanoparticle material is a luminescent material or includes a luminescent material.

[0102] Such luminescent materials can be chemiluminescent (e.g., bioluminescent, electrochemiluminescent, pyroluminescent, solvatochromic luminescent), electroluminescent, photoluminescent (e.g., fluorescent or phosphorescent), radioluminescent, or thermoluminescent. Examples of such luminescent materials include the luminescent groups described herein.

[0103] The luminescent material can form all or part of the nano particle material (nano particula te material). For example, the luminescent material may be encapsulated within another material so as to form the nanoparticle material.

[0104] The nanoparticle material according to the present invention has a copolymer steric stabilizer bound to its surface that promotes the dispersion of the nanoparticle material in a liquid. The liquid can be the carrier liquid or the in vivo liquid described herein. "Promote" in this context means that in the absence of the copolymer steric stabilizer, the nanoparticle material flocculates, aggregates, or precipitates out of the liquid as a sediment. That is, the copolymer steric stabilizer functions to maintain the nanoparticle material in a dispersed state within the liquid.

[0105] The copolymer "steric" stabilizer means that the dispersion of the nanoparticle material in the liquid occurs as a result of steric repulsion forces. That being said, the copolymer steric stabilizer can exhibit electrostatic repulsion forces that similarly promote the stabilization of the nanoparticle material. However, those skilled in the art will understand that such electrostatic forces provide little stabilization function in liquids having a relatively high ionic strength. Thus, the steric stabilization function of the copolymer steric stabilizer used in accordance with the present invention plays an important role in enabling the nanoparticle material to be maintained in a dispersed or stable state in such liquids.

[0106] The copolymeric steric stabilizer used in accordance with the present invention has been found to be particularly effective in promoting the dispersion of nanoparticle materials in a biological fluid environment.

[0107] As used herein, terms such as "polymer" or "polymeric segment" are intended to refer to a polymer chain resulting from the polymerization of monomers. Thus, a polymeric component or polymeric segment comprises, or is composed of, polymerized monomer residue units. Such polymeric components or polymeric segments can be prepared by any suitable polymerization technique. In one embodiment, the polymeric segments described herein (e.g., immobilized, sterically stabilized, and coupled) are prepared by the polymerization of ethylenically unsaturated monomers. The polymer chains can have non-polymeric components covalently attached thereto, such as mapping groups or luminescent groups, (and a portion of the polymer chain has a covalently attached non-polymeric component). As used herein, the term "copolymer" is intended to mean a polymer chain comprising two different polymeric segments of different compositions.

[0108] The copolymeric steric stabilizer used in accordance with the present invention comprises a sterically stabilized polymeric segment.

[0109] One skilled in the art would understand the various polymers that can be used as sterically stabilized polymer segments with respect to the monomers that can be polymerized to form such polymers. The sterically stabilized polymer segments can include polyacrylamide (PA), polyvinyl alcohol (PVA), polyalkylene oxides (e.g., polyethylene oxide (PEO), polypropylene oxide (PPO)), polyoxamers, polyhydroxyethyl acrylate, poly-N-isopropylacrylamide, polydimethylamino-ethyl methacrylate, polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polymethacrylamide, polyvinyl esters, polyvinylamides, polysulfonated divinylbenzene, poly-L-lysine, polyaspartate, polylactic acid, polyethyleneimine, polyalkyl cyanoacrylate, polyaspartate, polymaleic anhydride, polymaleic acid, or can include two or more of the above copolymers, or can be composed of them. Accordingly, suitable monomers that can be used to form sterically stabilized polymer segments include acrylamide, vinyl alcohol, alkylene oxides (e.g., ethylene oxide, propylene oxide), hydroxyethyl acrylate, N-isopropylacrylamide, dimethylamino-ethyl methacrylate, vinylpyrrolidone, acrylic acid, methacrylamide, vinyl esters, vinylamides, sulfonated divinylbenzene, L-lysine, aspartate, lactic acid, ethyleneimine, alkyl cyanoacrylate, aspartate, maleic anhydride, maleic acid, or two or more of the above copolymers, but are not limited thereto.

[0110] When the sterically stabilized polymer segment contains a polyalkylene oxide, the polyalkylene oxide can be selected from polyethylene glycol, polypropylene glycol, and their derivatives. The polyalkylene oxide polymer may be end-capped with an alkyl group. The alkyl group may be an alkyl group having 1 to 6 carbon atoms, for example, a methyl group, an ethyl group, a propyl group, or an isopropyl group.

[0111] Considering that the sterically stabilizing polymer segment forms only a part of the copolymer steric stabilizer, it may be useful to refer to the number of polymer monomer units that collectively form the segment rather than defining the sterically stabilizing polymer segment in terms of its number average molecular weight. There is no particular limitation on the number of such units that can collectively form the sterically stabilizing polymer segment, but in some embodiments of the present invention, it may be desirable for the sterically stabilizing polymer segment to contain less than about 70 polymer monomer residue units that make up the entire polymer segment, and in certain embodiments, the polymer segment has about 40 to about 60 polymer monomer residue units, for example about 50 polymer monomer residue units, that make up the entire polymer segment.

[0112] In some embodiments, the sterically stabilizing polymer segment contains about 10 to about 70 polymer monomer residue units.

[0113] The sterically stabilizing polymer segment may be a homopolymer or a copolymer.

[0114] In one embodiment, the sterically stabilizing polymer segment comprises or consists of a polyacrylamide-co-polyalkylene oxide block copolymer. The block copolymer may contain or consist of about 8 to about 60 polymerized acrylamide units and about 2 to about 10 polymerized alkylene oxide units.

[0115] In another embodiment, the sterically stabilizing polymer segment contains about 10 to about 13 polymerized alkylene oxide units.

[0116] One of ordinary skill in the art will understand that the polymerized alkylene oxide units give rise to a polyalkylene oxide.

[0117] The polymer steric stabilizer, polymer mapping moiety, and polymer light-emitting moiety used in accordance with the present invention each contain an immobilized polymer segment.

[0118] The "immobilized polymer segment" is a polymer chain that has an affinity for the surface of the nanoparticle material and functions to immobilize a given entity to the nanoparticle material via one or more linking groups. It refers to a segment or region of a given polymer entity (i.e., a polymer steric stabilizer, a polymer mapping moiety, and a polymer luminescent moiety). The one or more linking groups may form part of the polymer chain backbone or may exist as side chains from the polymer chain backbone. The linking group can be any element or molecule having a binding affinity for the nanoparticle material. For example, the linking group can be any element or molecule having a binding affinity for iron or iron oxide. Suitable linking groups that can be used include groups containing one or more phosphorus (P) atoms, groups containing one or more oxygen (O) atoms, groups containing one or more sulfur (S) atoms, groups containing one or more nitrogen (N) atoms, and groups containing two or more of any of the aforementioned atoms.

[0119] In one embodiment, the immobilized polymer segment comprises one or more linking groups selected from phosphate groups, phosphonate groups, dimercaptosuccinic acid (DMSA) groups, sulfate groups, sulfonic acid groups, catechol groups, carboxylate groups, amine groups, and silane groups.

[0120] By being a polymer segment, it will be understood that the immobilized polymer segment contains polymerization monomer residues. In particular, the segment contains polymerization monomer residues that give rise to the required binding affinity for the nanoparticle material. The polymerization monomer residues constituting the immobilized polymer segment may be the same or different.

[0121] The ability of the immobilized polymer segment to provide multiple sites for binding interaction with the nanoparticle material is thought to at least partially give rise to the excellent stabilization properties provided by the copolymer steric stabilizer.

[0122] The immobilized polymer segment can have at least 2 polymer monomer residues each providing a site for binding to magnetic nanoparticles, or at least 3 such polymer monomer residues, or at least 5, or at least 7, or at least 10. It is not necessary that all of the polymer monomer residues constituting the immobilized polymer segment result in binding interactions with the nanoparticle material, but generally it is preferred that most, if not all, of the polymer monomer residues constituting the immobilized polymer segment result in binding interactions with the nanoparticle material.

[0123] Thus, the immobilized polymer segment can be described as having multiple sites for collectively immobilizing or binding a given entity to the nanoparticle material.

[0124] To achieve the desired immobilization effect, the immobilized polymer segment has a binding affinity for the nanoparticle material. The manner in which the immobilized polymer segment binds to the nanoparticle material will be by electrostatic forces, hydrogen bonding, ionic charges, van der Waals forces or any combination thereof. A particular advantage provided by the immobilized polymer segment is that it can provide multiple sites for binding interactions with the nanoparticles. Thus, even if only a given binding site produces a relatively weak interaction with the nanoparticle material, the presence of multiple such sites within the segment enables reliable binding to the nanoparticle material as a whole.

[0125] In one embodiment, the immobilized polymer segment is not covalently bound to the nanoparticle material.

[0126] The required immobilized polymer segment is generally determined by the nature of the nanoparticle material to which it binds. One of ordinary skill in the art would be able to select an appropriate immobilized polymer segment that binds to the surface of a given nanoparticle material.

[0127] When explaining the interaction between an immobilized polymer segment and a nanoparticle material, it can be convenient to refer to the hydrophilicity and hydrophobicity of the segment and the particle material. Thus, generally, when the segment and the particle material have similar hydrophilicity or hydrophobicity, appropriate binding interactions occur. For example, when the nanoparticle material has a relatively hydrophilic surface (e.g., its surface can be wetted by an aqueous solution), good binding can be achieved by using a hydrophilic immobilized polymer segment (e.g., in its isolated form, the segment is soluble in an aqueous medium). Such an example could be realized when the particle material is of the type that can form a charge on its surface. In that case, it may be desirable for the segment to contain a polymerization residue of a monomer that can also form a charge (e.g., a polymerization residue of an ionizable monomer) to facilitate an ionic bond between the segment and the particle material. The promotion of the formation of such charged species could be facilitated by adjusting the pH of the liquid carrier in which the stabilizer and the particle material are present.

[0128] The expression "ionizable monomer" means that the monomer contains a functional group that can be ionized in solution to form a cationic or anionic group. Such functional groups can generally be ionized by the loss or acceptance of a proton under acidic or basic conditions. Generally, the functional group is an acidic or basic group (i.e., a group that can donate or accept an H atom, respectively). For example, a carboxylic acid functional group can form a carboxylate anion under basic conditions, and an amine functional group can form a quaternary ammonium cation under acidic conditions. The functional group may also be ionizable by an ion exchange process.

[0129] One skilled in the art would understand the various polymers that can be used as immobilized polymer segments with respect to the monomers that can be polymerized to form such polymers. For example, suitable polymers include, but are not limited to, polyacrylic acid, polymethacrylic acid, polystyrene, polyitaconic acid, poly-p-styrenecarboxylic acid, poly-p-styrenesulfonic acid, polyvinylsulfonic acid, polyvinylphosphonic acid, polymonoacryloxyethyl phosphate, polymonoacryloxyethyl phosphonic acid, poly-2-(methacryloyloxy)ethyl phosphate, poly-2-(methacryloyloxy)ethyl phosphonic acid, polyethylacrylic acid, poly-α-chloroacrylic acid, polycrotonic acid, polyfumaric acid, polycitraconic acid, polymesaconic acid, polymaleic acid, poly-2-(dimethylamino)ethyl and propyl acrylate and methacrylate, the corresponding poly-3-(diethylamino)ethyl and propyl acrylate and methacrylate, polydimethylaminoethyl methacrylate, and their copolymers. Thus, suitable monomers that can be used to form immobilized polymer segments include, but are not limited to, acrylic acid, methacrylic acid, itaconic acid, p-styrenecarboxylic acid, p-styrenesulfonic acid, vinylsulfonic acid, vinylphosphonic acid, monoacryloxyethyl phosphate, monoacryloxyethyl phosphonic acid, 2-(methacryloyloxy)ethyl phosphate, 2-(methacryloyloxy)ethyl phosphonic acid, ethylacrylic acid, α-chloroacrylic acid, crotonic acid, fumaric acid, citraconic acid, mesaconic acid, maleic acid, 2-(dimethylamino)ethyl and propyl acrylate and methacrylate, the corresponding 3-(diethylamino)ethyl and propyl acrylate and methacrylate, dimethylaminoethyl methacrylate, and combinations thereof.

[0130] The immobilized polymer segment can contain from about 1 to about 20 phosphonate groups, such as 1 phosphonate group, 2 phosphonate groups, 3 phosphonate groups, 4 phosphonate groups, 5 phosphonate groups, 6 phosphonate groups, 7 phosphonate groups, 8 phosphonate groups, 9 phosphonate groups or 10 phosphonate groups, 11 phosphonate groups, 12 phosphonate groups, 13 phosphonate groups, 14 phosphonate groups, 15 phosphonate groups, 16 phosphonate groups, 17 phosphonate groups, 18 phosphonate groups, 19 phosphonate groups or 20 phosphonate groups. In some embodiments, the immobilized polymer segment can contain more than 20 phosphonate groups. In certain embodiments, the immobilized polymer segment contains 5 phosphonate groups.

[0131] The immobilized polymer segment can be formed by the polymerization of one type of monomer or a combination of two or more different monomers. Thus, the immobilized polymer segment can be a homopolymer segment or a copolymer segment.

[0132] There is no particular limitation on the number of polymerized monomer units that collectively form the immobilized polymer segment, but in some embodiments of the present invention, it may be desirable for the immobilized polymer segment to have a relatively low number average molecular weight. The immobilized polymer segment can contain less than about 50, or less than about 40, or less than about 30, or from about 5 to about 25, or from about 5 to about 15 polymerized monomer residue units (constituting the entire segment).

[0133] In one embodiment, the immobilized polymer segment contains from 1 to about 30 polymerized monomer residue units.

[0134] In one embodiment, the immobilized polymer segment is composed of polymerization residues of one or more ethylenically unsaturated monomers.

[0135] The immobilized polymer segment is covalently bonded to either a sterically stabilizing polymer segment or a coupling polymer segment so as to form a copolymer steric stabilizer, a copolymer mapping moiety or a copolymer luminescent moiety.

[0136] The immobilized polymer segment is different from either the sterically stabilizing polymer segment or the coupling polymer segment, whereby the copolymer properties of the copolymer steric stabilizer, the copolymer mapping moiety and the copolymer luminescent moiety are obtained.

[0137] The polymer mapping moiety and the polymer luminescent moiety (when used) include a coupling polymer segment. The coupling polymer segment is covalently coupled to the immobilized polymer segment. A "coupling" polymer segment means, as described herein, a polymer chain that links or binds the immobilized polymer segment to either a mapping group or a luminescent group. Thus, these mapping groups or luminescent groups are generally covalently coupled to the coupling polymer segment. The coupling polymer segment also serves to move the mapping group and the luminescent group away from the nanoparticle material surface, thereby making the mapping group and the luminescent group more functional, for example when the mapping group is made available by a receptor on a target site.

[0138] One skilled in the art would understand the various polymers that can be used as coupling polymer segments with respect to the monomers that can be polymerized to form such polymers. For example, suitable polymers include polyacrylamide (PA), polyvinyl alcohol (PVA), polyalkylene oxides (e.g., polyethylene oxide (PEO) and polypropylene oxide (PPO)), polyoxamers, polyhydroxyethyl acrylate, poly-N-isopropylacrylamide, polydimethylamino-ethyl methacrylate, polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polymethacrylamide, polyvinyl esters, polyvinylamides, polysulfonated divinylbenzene, poly-L-lysine, polyaspartate, polylactic acid, polyethyleneimine, polyalkyl cyanoacrylate, polyaspartate, polymaleic anhydride, polymaleic acid or any copolymer of the foregoing, but are not limited thereto. Accordingly, suitable monomers that can be used to form the coupling polymer segment include acrylamide, vinyl alcohol, alkylene oxides (e.g., ethylene oxide and propylene oxide), hydroxyethyl acrylate, N-isopropylacrylamide, dimethylamino-ethyl methacrylate, vinylpyrrolidone, acrylic acid, methacrylamide, vinyl esters, vinylamides, sulfonated divinylbenzene, L-lysine, aspartate, lactic acid, ethyleneimine, alkyl cyanoacrylate, aspartate, maleic anhydride, maleic acid and combinations thereof, but are not limited thereto.

[0139] In certain embodiments, the coupling polymer segment has less than about 100 polymerized monomer residue units constituting the entire polymer segment, and in certain embodiments, has from about 30 to about 80 polymerized monomer residue units constituting the entire polymer segment, or from about 50 to about 80 polymerized monomer residue units, for example, about 70 polymerized monomer residue units.

[0140] In one embodiment, the coupling polymer segment comprises polyacrylamide or is composed of polyacrylamide.

[0141] In another embodiment, the coupling polymer segment comprises from about 10 to about 100 polymerized monomer residue units or is composed of from about 10 to about 100 polymerized monomer residue units.

[0142] In a further embodiment of the coupling polymer segment of one or both of the polymer mapping portion and the light emitting portion, the coupling polymer segment has more polymerised monomer residue units than the sterically stabilizing polymer segment. For example, the coupling polymer segment may have at least 2, or at least 4, at least 6, or at least 8, at least 10, or at least 12, or at least 14, or at least 16, or at least 18, or at least 20 more polymerized monomer residue units than the sterically stabilizing polymer segment. The coupling polymer segment may have from about 5 to about 70, or from about 5 to about 60, or from about 5 to about 40, or from about 5 to about 20, or from about 40 to about 70, or from about 50 to about 70 more polymerized monomer residue units than the sterically stabilizing polymer segment.

[0143] Without wishing to be bound by theory, providing a coupling polymer segment having more polymerized monomer residue units than the sterically stabilizing polymer segment is thought to play a role in forming a surface environment in which protein adsorption and subsequent cellular uptake by macrophages are less likely to occur. As a result, it is thought to improve the accumulation of the nanoparticle material in the tumor microenvironment.

[0144] One skilled in the art would be able to select an appropriate combination of the steric stabilization polymer segment, the immobilized polymer segment, and the coupling polymer segment for use in combination with a given nanoparticle material to provide the functions required for each of these respective polymer segments.

[0145] The copolymer mapping portion and the copolymer luminescent portion each contain one or more mapping groups or one or more luminescent groups, respectively. These mapping groups and luminescent groups are generally covalently coupled to the coupling polymer segment of each respective portion.

[0146] One or more of the mapping groups described herein contain an agent that specifically binds to fibroblast activation protein (FAP).

[0147] "Fibroblast activation protein", or "FAP", is a cell surface-expressed proteolytic enzyme that acts on various hormones and extracellular matrix components. Structurally, FAP is composed of a six-amino acid cytoplasmic tail, a twenty-amino acid single transmembrane domain, and a 734-amino acid extracellular domain.

[0148] FAP is expressed during the growth phase and is very rare in healthy adult tissues. In contrast, FAP is highly upregulated in a wide variety of cancers and in cells of the tumor microenvironment.

[0149] Accordingly, one or more mapping groups containing an agent that specifically binds to FAP can selectively bind to tumor-associated stromal cells in a subject upon administration of the coated nanoparticles. Suitable mapping groups include fibroblast activation protein inhibitors, peptides, proteins, and antibodies that target FAP.

[0150] In one embodiment, the agent is selected from the group consisting of small molecule inhibitors and antibodies or antigen-binding fragments thereof.

[0151] In one embodiment, the agent is a small molecule inhibitor. In another embodiment, the agent is a FAP inhibitor.

[0152] Examples of suitable FAP inhibitors include those having the following structures.

Chemical formula

[0153] In the formula, R 1 and R 2 are the same or different and are each independently selected from the group consisting of hydrogen, halogen, and alkyl having 1 to 4 carbon atoms.

[0154] R 3 is alkyl having 1 to 4 carbon atoms, nitrile, or isonitrile.

[0155] R 4 , R 5 and R 6 are the same or different and are each independently selected from the group consisting of hydrogen, halogen, and alkyl having 1 to 4 carbon atoms.

[0156] In the formula, R 1 and R 2 are each halogen.

[0157] In the formula, each of R 1 and R 2 is fluorine.

[0158] In the formula, R 3 is nitrile.

[0159] In the formula, each of R 4 , R 5 and R 6 is hydrogen.

[0160] In one embodiment, the agent is an antibody or an antigen-binding fragment thereof.

[0161] Examples of other suitable FAP inhibitors include, but are not limited to, those described in WO 2013 / 107820, US 2020 / 0330624 A1, EP 3763726 A1, and US 7399869.

[0162] As used herein, the term "antibody" is understood to mean any antigen-binding molecule or molecular complex that specifically binds to or specifically interacts with a target antigen and that contains at least one complementarity determining region (CDR). The term "antibody" includes full-length immunoglobulin molecules comprising two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (HCVR, VH or V H (sometimes abbreviated as such) and a heavy chain constant region. The heavy chain constant region typically comprises three domains - C H 1, C H 2 and C H 3. Each light chain comprises a light chain variable region (LCVR, VL, VK, V K or V L (sometimes abbreviated as such) and a light chain constant region. The light chain constant region typically comprises one domain (C L 1). The V H and V L regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions also called framework regions (FRs). Each V H and V L typically comprises three CDRs and four FRs arranged in the following order from the amino terminus towards the carboxy terminus, i.e., FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0163] Also, as used herein, "immunoglobulin" (Ig) is defined herein as a protein belonging to class IgG, IgM, IgE, IgA, or IgD (or any subclass thereof), and includes all conventionally known antibodies and their functional fragments. A "functional fragment" of an antibody / immunoglobulin is defined as a fragment of an antibody / immunoglobulin that retains an antigen-binding region (e.g., the variable region of IgG).

[0164] The "antigen-binding region" or "antigen-binding fragment" of an antibody is typically found in one or more hypervariable regions of the antibody, i.e., the CDR-1, CDR-2, and / or CDR-3 regions. However, the variable "framework" regions can also play an important role in antigen binding, such as by providing a scaffold for the CDRs. "Functional fragments" include F(ab’)2 fragments, Fab fragments, scFv, or constructs containing a single immunoglobulin variable domain or single-domain antibody polypeptide, e.g., a single-chain variable domain or a single-light-chain variable domain. F(ab’)2 or Fab can be genetically engineered to minimize or completely remove intermolecular disulfide interactions that occur between the C H1 domain and the C L domain.

[0165] One or more luminescent groups can be any chemical entity that emits electromagnetic radiation or acoustic energy of a desired wavelength after some form of stimulation. Luminescent groups can be chemiluminescent (e.g., bioluminescent), electroluminescent, photoluminescent, radioluminescent, or thermoluminescent. In certain embodiments, the luminescent group is a photoluminescent group that emits light of a specific wavelength after absorption of a photon. Photoluminescent groups can be fluorescent or phosphorescent.

[0166] In certain embodiments, the luminescent group is a fluorescent group belonging to the group of cyanine dyes. Suitable fluorescent groups include indocyanine green (ICG; sodium = 4-[2-[(1E,3E,5E,7Z)-7-[1,1-dimethyl-3-(4-sulfonatobutyl)benzo[e]indol-2-ylidene]hepta-1,3,5-trienyl]-1,1-dimethylbenzo[e]indol-3-ium-3-yl]butane-1-sulfonate), IR dyes such as IRdye800, and sulfocyanine dyes such as sulfo-Cy3, sulfo-Cy5 and sulfo-Cy7. Suitable dyes are commercially available, for example, from Lumiprobe Corporation (Hunt Valley, Maryland, USA).

[0167] In one embodiment, the luminescent group is selected from indocyanine green, sulfo-Cy3, sulfo-Cy5 and sulfo-Cy7.

[0168] In certain embodiments, the nanoparticle material has at least one copolymer steric stabilizer and at least one copolymer mapping moiety attached to its surface. In other embodiments, the nanoparticle material has at least one copolymer steric stabilizer, at least one copolymer targeting moiety and at least one copolymer luminescent moiety attached to its surface.

[0169] As contemplated herein, the nanoparticle material or a composition comprising the nanoparticle material can be used for mapping, diagnostic and / or therapeutic applications.

[0170] Thus, in one embodiment, a. administering to a subject a nanoparticle material or composition according to the present invention; and b. detecting the nanoparticle material; A method for mapping a tumor margin in a subject, comprising: the nanoparticle material accumulates in the tumor microenvironment, thereby mapping the tumor margin, is provided.

[0171] As used herein, the terms "mapping" or "tumor mapping" refer to the determination of the margins of a cancer. Generally, tumor mapping is performed prior to cancer treatment to confirm the location and extent of the tumor.

[0172] As used herein, the terms "tumor" and "cancer" mean any condition associated with abnormal cell growth. Such conditions are known to those of ordinary skill in the art. In one embodiment, the tumor is a primary tumor. In another embodiment, the tumor is a solid tumor.

[0173] In one embodiment, the cancer is selected from the group consisting of prostate cancer, glioblastoma multiforme, glioma, pancreatic cancer, colorectal cancer, breast cancer, head and neck cancer, gastric cancer, esophageal cancer, ovarian cancer, sarcoma, and lung cancer. In another embodiment, the cancer is prostate cancer.

[0174] In one embodiment, the tumor is mapped in situ. The tumor can be mapped in situ before or after tumor tissue resection. It will be appreciated that in the situation where the tumor is mapped after tumor tissue resection, it is not uncommon for it to be confirmed, for example by conventional pathology, that not all tumor tissue has been completely removed from the subject by the tumor tissue resection. The nanoparticle material according to the present invention advantageously enables tumor mapping to be performed in situ after tumor tissue resection to confirm in real time that all tumor tissue has actually been resected without the need for conventional pathological examination.

[0175] In one embodiment, the nanoparticle material is detected using an in vivo imaging diagnostic technique selected from the group consisting of ultrasound, X-ray, optical imaging, computed tomography (CT), single photon emission computed tomography (SPECT), positron emission tomography (PET), fluorescence resonance energy transfer (FRET), and magnetic resonance imaging (MRI).

[0176] In another embodiment, the method further includes determining a clinical target volume (CTV) and / or a gross target volume (GTV) prior to treatment.

[0177] The "gross tumor volume", i.e., "GTV", refers to the location and extent of the gross tumor, i.e., the tumor mass that can be visualized, palpated, or imaged.

[0178] The "clinical target volume", i.e., "CTV", refers to the tumor volume that includes the GTV plus a margin for subclinical disease spread. It is generally recognized in the art that it is necessary to adequately treat the CTV to achieve a cure.

[0179] Methods for calculating CTV and GTV are known to those skilled in the art, and exemplary examples thereof include the methods described by Burnet et al. (2004, Cancer Imaging, 4(2):153 - 161).

[0180] In another aspect disclosed herein, a method for treating cancer in a subject in need of cancer treatment, a. administering to the subject a nanoparticle material or composition according to the present invention; b. detecting the site in the subject where the nanoparticle material accumulates; c. administering an effective amount of treatment for the cancer at the site of the nanoparticle material detected in step (b); is provided.

[0181] Treatment plans for the treatment of cancer can be determined by those skilled in the art and generally depend on factors including, but not limited to, the type, size, stage, and receptor status of the tumor, in addition to the age, weight, and general health of the subject. Another determining factor can be the risk of developing a recurrent disease. For example, for a subject identified as having a high risk or a relatively high risk or having developed a recurrent disease, a more aggressive treatment plan may be prescribed compared to a subject considered to have a low risk or a relatively low risk of developing a recurrent disease. Similarly, for a subject identified as having a more advanced stage of cancer, such as stage III or IV disease, a more aggressive treatment plan may be prescribed compared to a subject having a less advanced stage of cancer.

[0182] As used herein, the terms "treat", "treatment", and "treating" refer to any and all uses of treating, preventing, inhibiting, delaying, arresting, or reversing, in any way, the state or symptoms of cancer or other undesirable conditions or symptoms. Accordingly, terms such as "treating" should be considered in their broadest possible sense. For example, treatment does not necessarily mean treating a subject until complete recovery or cure. In a condition exhibiting or characterized by multiple symptoms, treatment does not necessarily treat, prevent, inhibit, delay, arrest, or reverse all of the symptoms, but can treat, prevent, inhibit, delay, arrest, or reverse one or more of the symptoms.

[0183] The subject to be treated for cancer is a human or a mammal having economic and / or social importance to humans, such as carnivores other than humans (e.g., cats and dogs), pigs (e.g., pigs, adult pigs, and wild boars), ruminants (e.g., cows, bulls, sheep, giraffes, deer, goats, bison, and camels), horses, and birds, such as endangered birds, birds in zoos, and birds including wild fowl, more specifically, birds that are also economically important to humans and thus may be domesticated birds, such as poultry including chickens, chickens, ducks, geese, and pigeons. The term "subject" does not indicate a specific age. Thus, it is intended to include adult, juvenile, and neonatal subjects.

[0184] The terms "subject", "individual", and "patient" are used interchangeably herein and refer to any subject to which the present disclosure may be applicable. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.

[0185] As used herein, the term "therapeutically effective amount" means an amount or degree of treatment sufficient to treat cancer, administered or applied to a subject in need of treatment for cancer, particularly a mammal such as a human. The exact amount of treatment to be administered or applied can be determined by a physician taking into account individual differences in the subject's age, weight, tumor size, extent of infection or metastasis, and condition.

[0186] In one embodiment, the treatment is selected from surgery, radiotherapy, brachytherapy, photodynamic therapy, photothermal therapy, focal ablation therapy (such as cryoablation, focused laser ablation, and high-frequency ultrasound ablation), chemotherapy, immunotherapy, and combinations thereof.

[0187] In one embodiment, the method further includes determining CTV and / or GTV prior to administering the treatment.

[0188] In another aspect disclosed herein, a. Administering a nanoparticle material or composition according to the present invention to a subject; b. Detecting the nanoparticle material in the subject; A method for diagnosing cancer, comprising: detecting a nanoparticle material accumulated in a tissue (such as vascular tissue) of a subject, wherein the detection indicates that the subject has cancer.

[0189] In one embodiment, detecting a nanoparticle material accumulated in the vascular tissue of a subject indicates that the subject has cancer.

[0190] The present invention provides a composition suitable for administration to a subject for use in mapping, diagnosis and / or treatment applications, the composition comprising a nanoparticle material according to the present invention dispersed in a pharmacologically acceptable liquid carrier.

[0191] Based on the weight ratio %, the nanoparticle material can bond various ranges of copolymer steric stabilizers and copolymer mapping moieties to its surface. For example, the nanoparticle material can bond 10% - 90% (by weight) of the copolymer steric stabilizer and 90% - 10% (by weight) of the copolymer mapping moiety to its surface. In certain embodiments, the nanoparticle material has, on its surface, 10% (by weight) of the copolymer steric stabilizer and 90% (by weight) of the copolymer mapping moiety, 15% (by weight) of the copolymer steric stabilizer and 85% (by weight) of the copolymer mapping moiety, 20% (by weight) of the copolymer steric stabilizer and 80% (by weight) of the copolymer mapping moiety, 25% (by weight) of the copolymer steric stabilizer and 75% (by weight) of the polymer mapping moiety, 30% (by weight) of the copolymer steric stabilizer and 70% (by weight) of the copolymer mapping moiety, 35% (by weight) of the copolymer steric stabilizer and 65% (by weight) of the copolymer mapping moiety, 40% (by weight) of the copolymer steric stabilizer and 60% (by weight) of the polymer mapping moiety, 45% (by weight) of the copolymer steric stabilizer and 55% (by weight) of the polymer mapping moiety, 50% (by weight) of the copolymer steric stabilizer and 50% (by weight) of the polymer mapping moiety, 55% (by weight) of the copolymer steric stabilizer and 45% (by weight) of the polymer mapping moiety, 60% (by weight) of the copolymer steric stabilizer and 40% (by weight) of the polymer mapping moiety, 65% (by weight) of the copolymer steric stabilizer and 35% (by weight) of the polymer mapping moiety, 70% (by weight) of the copolymer steric stabilizer and 30% (by weight) of the polymer mapping moiety, 75% (by weight) of the copolymer steric stabilizer and 25% (by weight) of the copolymer mapping moiety, 80% (by weight) of the copolymer steric stabilizer and 20% (by weight) of the copolymer mapping moiety, 85% (by weight) of the copolymer steric stabilizer and 15% (by weight) of the copolymer mapping moiety, or 90% (by weight) of the copolymer steric stabilizer and 10% (by weight) of the copolymer mapping moiety bonded thereto.In certain specific embodiments, the nanoparticle material can have 70% (by weight) of a copolymer steric stabilizer and 30% (by weight) of a copolymer mapping moiety bonded to its surface.

[0192] One skilled in the art will understand that the nanoparticle material according to the present invention exhibits a hydrodynamic diameter when dispersed in a liquid carrier. The hydrodynamic diameter is the distance or size derived from the nanoparticle material itself and at least the copolymer steric stabilizer and the mapping moiety associated or bonded to the nanoparticles. Thus, it can be seen that the hydrodynamic diameter of the dispersed nanoparticle material represents the diameter obtained by the combination of the nanoparticle material itself and at least the copolymer steric stabilizer and the mapping moiety. When the dispersed nanoparticle material does not have a symmetric shape, the hydrodynamic diameter is considered to be that of the largest hydrodynamic diameter presented by the dispersed nanoparticle material.

[0193] In one embodiment, the hydrodynamic diameter of the dispersed nanoparticle material is less than about 300 nm, less than about 250 nm, less than about 100 nm, less than about 50 nm, less than about 25 nm or less than about 15 nm.

[0194] In a further embodiment, the hydrodynamic diameter of the dispersed nanoparticle material is approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 nm.

[0195] For the avoidance of doubt, references herein to the "hydrodynamic diameter" of the dispersed nanoparticle material are intended to indicate the average diameter of the dispersed coated nanoparticles (at least about 50% on a number basis). The hydrodynamic diameter of the dispersed coated nanoparticles is determined herein by dynamic light scattering (DLS).

[0196] The nanoparticle material or composition according to the present invention can be used in combination with in vivo imaging diagnostic techniques including, but not limited to, ultrasound, X-ray, optical imaging, computed tomography (CT), single photon emission computed tomography (SPECT), positron emission tomography (PET), fluorescence resonance energy transfer (FRET), and magnetic resonance imaging (MRI).

[0197] In one application, the nanoparticle material comprises an FAP targeting group (e.g., inhibitor), and compositions containing them enable the detection of cells expressing FAP, such as cells within the tumor microenvironment (e.g., tumor-associated stromal cells and cancer cells) associated with solid tumors such as prostate cancer, glioblastoma, pancreatic cancer, colorectal cancer, breast cancer, and lung cancer. By specifically binding to FAP expressed by cells in the tumor microenvironment, the nanoparticle material is useful for identifying the boundaries and margins of cancer-affected tissue (i.e., tumor mapping). It is also contemplated herein that the nanoparticle material and compositions may be useful as part of cancer detection (i.e., diagnosis) or cancer treatment. For example, the nanoparticle material can be used for tumor mapping prior to the initiation of treatments such as focal therapy, radiation therapy, proton beam therapy, or brachytherapy. Furthermore, by accurately mapping the tumor, including the region of the tumor microenvironment, the surgical resection of the tumor can be performed more accurately, limiting unwanted side effects and minimizing the risk of suboptimal debulking of the tumor mass.

[0198] Unless otherwise specified, the terms "halogen" and "halo" as used herein refer to I, Br, Cl, and F.

[0199] In this specification, the term "alkyl" used alone or in compound words such as "alkenyloxyalkyl", "alkylthio", "alkylamino" and "dialkylamino" refers to linear, branched or cyclic alkyl, preferably alkyl or cycloalkyl having 1 to 20 carbon atoms.Examples of linear and branched alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl, isoamyl, sec-amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, heptyl, 5-methoxyhexyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl, 1-methylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-methyl-octyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-, 2- or 3-propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and 8-methylnonyl, 1-, 2-, 3-, 4-, 5- or 6-ethyloctyl, 1-, 2-, 3- or 4-propylheptyl, undecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-methyldecyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-ethyldecyl, 1-, 2-, 3-, 4- or 5-propylnonyl, 1-, 2- or 3-butylheptyl, 1-pentylhexyl, dodecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-ethyldecyl, 1-, 2-, 3-, 4-, 5- or 6-propylnonyl, 1-, 2- or 3-butyl octyl, 1-2-pentylheptyl, etc. Examples of cyclic alkyl groups include monocyclic or polycyclic alkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, etc.

Example

[0200] Example 1: Synthesis of Magnetic Nanoparticles Part (a): Large maghemite particles were produced using the coprecipitation method. In a typical reaction, FeCl2·4H2O (20 g) and FeCl3·6H2O (27 g) were dissolved in 0.4 M HCl (500 mL). Ammonia solution (3 M, 500 mL) was added to the iron salt solution while stirring with an overhead stirrer. The formed black magnetite nanoparticles were magnetically separated and washed with Milli-Q water. Then, they were oxidized to maghemite by heating the magnetite nanoparticles in 200 mL of iron(III) nitrate (0.34 M in 1 M nitric acid) at 100 °C for 1 hour. The formed brown maghemite nanoparticles were magnetically separated and washed with Milli-Q water. The particles were dispersed in Milli-Q water and dialyzed in Milli-Q water for 2 - 3 days using a 14,000 kDa molecular weight cut-off dialysis tube to remove impurities. When the particles were analyzed by transmission electron microscopy, it was found that they had an average diameter of 16.8 ± 3.3 nm.

[0201] Part (b): Tiny maghemite particles were produced using the coprecipitation method. In a typical reaction, FeCl2·4H2O (1.46 g) and FeCl3·6H2O (2.7 g) were dissolved in 0.4 M HCl (50 mL). While stirring with an overhead stirrer, ammonia solution (3 M, 50 mL) was added to the iron salt solution using a syringe pump. The formed black magnetite nanoparticles were magnetically separated and washed with Milli-Q water. Then, using 20 mL of iron(III) nitrate (0.34 M in 1 M nitric acid), the particles were oxidized to maghemite at 100 °C for 1 hour while stirring. The obtained brown maghemite nanoparticles were magnetically separated and washed with Milli-Q water. The particles were dispersed in Milli-Q water and dialyzed in Milli-Q water using a 14,000 kDa molecular weight cut-off dialysis tube to remove impurities. When the particles were analyzed by transmission electron microscopy, it was found that they had an average diameter of 12.4 ± 3.2 nm. When the Z-average diameter of the particles was measured by DLS, it was found to be 44.5 nm.

[0202] Part (c): Magnetite particles were produced using the coprecipitation method. In a typical reaction, FeCl2·4H2O (1.46 g) and FeCl3·6H2O (2.7 g) were dissolved in 2 M HCl (10 mL) and Milli-Q water (40 mL). While stirring with an overhead stirrer, an ammonia solution (3 M, 50 mL) was added to the iron salt solution using a syringe pump. The formed black magnetite nanoparticles were magnetically separated and washed 5 times with Milli-Q water (50 mL) until the pH of the final particle dispersion reached 8.2. The particles were analyzed by transmission electron microscopy and were found to have an average diameter of 12.7 ± 3.3 nm.

[0203] Example 2 (Comparative Example): Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 70 -(Glu-CO-Lys) polymer PSMA targeting moiety and poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 15 -block-(triethylene glycol monomethyl ether) polymer steric stabilizer conjugated magnetic nanoparticles (PSMA-targeted nanoparticles) synthesis Part (a): Synthesis of poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 70 Synthesis

[0204] 2-(((butylthio)carbonothioyl)-thio)-propanoic acid (0.5 g), acrylamide (10.4 g), 4,4’-azobis(4-cyanovaleric acid) (0.050 g), dioxane (20 g) and water (30 g) were combined in a round bottom flask. After purging the mixture with nitrogen gas, it was reacted at 70 °C for 3 hours. The solution was allowed to cool to room temperature and [2-(methacryloyloxy)-ethyl]phosphonic acid (2.0 g) and 4,4’-azobis(4-cyanovaleric acid) (0.050 g) were added. The reaction mixture was purged with nitrogen gas and heated to 70 °C for 4 hours. The resulting polymer was precipitated in acetone and recovered by vacuum filtration. The polymer was dissolved in water, precipitated in acetone, and re-purified by drying in a vacuum oven at 40 °C for 24 hours.

[0205] Part (b): Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 70 Synthesis of -(Glu-CO-Lys)

[0206] Preparation of Glu-CO-Lys. 40 mg of Glu-CO-Lys-(t-Bu)3 ester was dissolved in dichloromethane (DCM) containing 20% trifluoroacetic acid (TFA) to obtain a concentration of 20 mg / mL. Nitrogen gas was bubbled through the mixture at room temperature for 2 hours, and then the solvent was removed in vacuo. The residue was dissolved in 2 mL of 20% aqueous acetic acid, and the mixture was washed 3 times with chloroform and concentrated to dryness under high vacuum to obtain deprotected Glu-CO-Lys. The product was dissolved in glacial acetic acid and lyophilized, and stored at 4 °C until further use.

[0207] Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) of Glu-CO-Lys 70 Conjugation to. Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) of part (a) 70 (250 mg), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC·HCl, 60 mg) and N-hydroxysuccinimide (NHS, 12 mg) were dissolved in 8 mL of MES buffer (100 mM, pH 5.5 - 6.0). The mixture was sonicated in an ultrasonic bath for 10 minutes, and then the NHS-activated polymer was precipitated in 10 mL of acetone. The precipitate was collected by centrifugation at 3000×g for 5 minutes. Glu-CO-Lys (20 mg) was dissolved in 10-fold concentrated PBS buffer and added to the NHS-activated polymer. The reaction mixture was stirred at room temperature for 20 hours. The Gly-CO-Lys conjugate polymer was purified using a centrifugal filter with a molecular weight cut-off membrane of 3 kDa and washed 3 times with water. The product was diluted to a final concentration of 50 mg / mL and stored at 4 °C for further use.

[0208] Part (c): Synthesis of poly{[2-(methacryloyloxy)-ethyl]phosphonic acid}5-block-poly(acrylamide) 15 -block-(triethylene glycol monomethyl ether)

[0209] In a round-bottom flask, a solution of acrylamide (2.8 g), 4,4'-azobis(4-cyanovaleric acid) (0.050 g), methoxy triethylene glycol modified 2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic acid (1.0 g), dioxane (10 g) and water (10 g) was prepared. The mixture was purged with nitrogen gas for 15 minutes and then heated to 70 °C with stirring for 2 hours. The mixture was allowed to cool to room temperature and [2-(methacryloyloxy)-ethyl]phosphonic acid (2.6 g) and 4,4'-azobis(4-cyanovaleric acid) (0.050 g) were added. The reaction mixture was purged with nitrogen gas for 15 minutes and heated to 70 °C for 4 hours. The resulting polymer was precipitated in acetone and recovered by vacuum filtration. The polymer was dissolved in water, precipitated in acetone and re-purified by drying in a vacuum oven at 40 °C for 24 hours. The chemical structure of this polymer is shown in Figure 2.

[0210] Part (d): Particle stabilization using a mixture of poly{[2-(methacryloyloxy)-ethyl]phosphonic acid}5-block-poly(acrylamide) 70 -(Glu-CO-Lys)30% and poly{[2-(methacryloyloxy)-ethyl]phosphonic acid}5-block-poly(acrylamide) 15 -block-(triethylene glycol monomethyl ether)70%

[0211] The poly{[2-(methacryloyloxy)-ethyl]phosphonic acid}5-block-poly(acrylamide) 15 -block-(triethylene glycol monomethyl ether) (36 mg) prepared in part (c) and the poly{[2-(methacryloyloxy)-ethyl]phosphonic acid}5-block-poly(acrylamide) 70-(Glu-CO-Lys) (40 mg) was dissolved in 2 mL of water. The pH was adjusted to 4 using NaOH (0.1 M). Example 1

[0212] The magnetic particles of (7 wt% solids, 1 g) were added to the polymer mixture during sonication. After 10 minutes, the pH was adjusted to 5.5, and then after an additional 10 minutes of sonication, it was adjusted to 7.0. After continuing sonication for a total of 30 minutes, unbound polymers were removed using a centrifugal filter with a 100 kDa molecular weight cut-off membrane. The coated nanoparticles were washed three times with water and diluted with physiological saline to obtain an isotonic dispersion of 30 mg Fe / mL.

[0213] When the dispersed nanoparticles in 0.9% physiological saline as the suspension medium were measured by DLS, the z-average was 64.2 nm. The intensity distribution of the particle size is shown in Figure 3.

[0214] Example 3: Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 70 -FAPI copolymer mapping moiety and poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 15 -block-(triethylene glycol monomethyl ether) copolymer steric stabilizer-bonded magnetic nanoparticles (FAP mapping nanoparticles) synthesis. Part (a): Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 70 -FAPI synthesis.

[0215] Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) of Example 2 - Part (a) 70(50 mg), EDC·HCl (12 mg), and NHS (3 mg) were dissolved in 2 mL of MES buffer (100 mM, pH 5.5 - 6.0). After sonicating the solution in an ultrasonic bath for 10 minutes, the NHS-activated polymer was precipitated in 6 mL of acetone. The precipitate was collected by centrifugation at 3000×g for 5 minutes. FAPI (3 mg) was dissolved in DMSO (50 μL) and further diluted to a total volume of 2 mL with 10-fold concentrated PBS buffer, and then added to the NHS-activated polyacrylamide polymer. The reaction mixture was stirred at room temperature for 20 hours. The FAPI-conjugated polymer was purified using a centrifugal filter with a molecular weight cut-off membrane of 3 kDa and washed three times with water. The product was diluted to a final concentration of 50 mg / mL and stored at 4 °C for further use. The chemical structure of this polymer is shown in Figure 4.

[0216] Part (b): Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 70 -FAPI copolymer mapping part 30% and poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 15 -block-(triethylene glycol monomethyl ether) copolymer steric stabilizer 70% was used for particle stabilization

[0217] Example 2 - Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) of Part (c) 15 -block-(triethylene glycol monomethyl ether) 18 mg and poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) prepared in Part (a) 70-FAPI (20 mg) was dissolved in 2 mL of water. Using NaOH (0.1 M), the pH was adjusted to 4. The polymer mixture was added to the magnetic particles (35 mg) of Part (a) of Example 1 while sonicating. After 10 minutes, the pH was adjusted to 5.5, then further adjusted to 7.0 after sonicating for another 10 minutes. After continuing sonication for a total of 30 minutes, an unbound polymer was removed using a centrifugal filter with a molecular weight cut-off membrane of 100 kDa. The coated nanoparticles were washed three times with water and diluted with physiological saline to obtain an isotonic dispersion of 30 mg Fe / mL.

[0218] When the dispersed nanoparticles in 0.9% physiological saline as a suspension medium were measured by DLS, the z-average was 59.7 nm. The intensity distribution of the particle size is shown in Figure 5.

[0219] Part (c): Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 70 30% of the non-targeted part of the copolymer and poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 15 -block-(triethylene glycol monomethyl ether) copolymer steric stabilizer 70% was used for particle stabilization of non-targeted particles (non-targeted nanoparticles) used as a control.

[0220] Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) of Part (c) of Example 2 15 -block-(triethylene glycol monomethyl ether) 18 mg and poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) of Part (a) of Example 2 70(20 mg) was dissolved in 2 mL of water. The pH was adjusted to 4 using NaOH (0.1 M). The polymer mixture was added to the magnetic particles (35 mg) of Example 1 part (a) while sonicating. After 10 minutes, the pH was adjusted to 5.5, then to 7.0 after sonicating for an additional 10 minutes. After sonicating for a total of 30 minutes, unbound polymers were removed using a centrifugal filter with a 100 kDa molecular weight cut-off membrane. The coated nanoparticles were washed three times with water and diluted with saline to obtain an isotonic dispersion of 30 mg Fe / mL.

[0221] Example 4: Animal study using tracers for prostate tumor delineation Male NODscid gamma mice, 6 - 8 weeks old, were directly injected with human prostate cancer cells (LNCaP) into the prostate. After 4 - 6 weeks of tumor growth, the mice were injected via the tail vein with 15 mg / ml of magnetic nanoparticles with PSMA or FAP targeting moieties (prepared in Example 2 - part (d) and Example 3 - part (b), respectively), or magnetic nanoparticles without targeting moieties (prepared in Example 3 - part (c)) at 40 mg / kg. 24 hours after injection, the mice were sacrificed and tissues were harvested for analysis. The excised prostate tumors were fixed in 10% neutral buffered formalin, mounted in 1% agar gel containing 2 mM gadopentetate dimeglumine, and T2 - weighted MRI was performed using a Siemens 3.0T device (Figure 6). Analysis of the mean signal intensity of the tumors showed that FAP - mapping nanoparticles increased the contrast by 71% compared to nanoparticles without mapping moieties. PSMA - targeted nanoparticles increased the contrast by only 18% compared to nanoparticles without targeting moieties.

[0222] The resected prostate tumors fixed with 10% neutral buffered formalin were mounted in paraffin blocks. Sections with a thickness of 5 μm were cut out and stained with Prussian blue to visualize the presence of iron nanoparticles (shown by dark blue staining at a magnification of 5 times in Fig. 7). Mice injected with PSMA-targeted nanoparticles or FAP mapping nanoparticles showed increased staining for iron, in contrast to mice injected with nanoparticles without the mapping / targeting moiety, which showed minimal staining. In visual inspection, the tumors of mice injected with FAP mapping particles showed increased iron staining, in contrast to those injected with PSMA-targeted nanoparticles. It was observed that FAP mapping nanoparticles were particularly taken up near the tumor boundary and along the vasculature.

[0223] After a 24-hour nanoparticle uptake period, the mice were injected intraperitoneally with pentobarbital, fixed with 10% neutral buffered formalin, and perfused transcardially. The entire fixed mice were examined by T2-weighted MRI scanning using a Bruker 14.1T system (Fig. 6). The tumors of mice injected with FAP- or PSMA-based nanoparticles showed an increase in low signal areas / negative contrast, particularly around the prostate tumors (tumors indicated by white arrows in Fig. 8).

[0224] Example 5: Synthesis of maghemite core particles from part (b) of Example 1 using short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 30 -FAPI copolymer mapping moiety and short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 10 -block-(triethylene glycol monomethyl ether) copolymer steric stabilizer-bound maghemite nanoparticles (micromaghemite FAP mapping nanoparticles). Part (a): Synthesis of short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 30 of

[0225] 2-(((Butylthio)carbonothioyl)-thio)-propanoic acid (0.2 g), acrylamide (1.8 g), 4,4’-azobis(4-cyanovaleric acid) (0.020 g), dioxane (3.6 mL) and water (4 mL) were combined in a round-bottom flask. The mixture was purged with nitrogen gas and then reacted at 70 °C for 2.5 h. The solution was allowed to cool to room temperature, and [2-(methacryloyloxy)-ethyl]phosphonic acid (0.489 g) and 4,4’-azobis(4-cyanovaleric acid) (0.020 g) were added. The reaction mixture was purged with nitrogen gas and heated at 70 °C for 2.5 h. The resulting polymer was precipitated in acetone and recovered by centrifugation. The polymer was dissolved in water, precipitated in acetone, and re-purified by drying under vacuum for 48 h.

[0226] Part (b): Short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 30 -Synthesis of FAPI

[0227] The poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) of part (a) 30 (150 mg), EDC·HCl (72 mg) and NHS (18 mg) were dissolved in 8 mL of MES buffer (100 mM, pH 5.5 - 6.0). The solution was sonicated in an ultrasonic bath for 10 min and then the activation solution was removed using a centrifugal filter with a 1 kDa molecular weight cut-off membrane. FAPI (18 mg) was dissolved in DMSO (50 μL), further diluted to a total volume of 8 mL with 10-fold concentrated PBS buffer, and then added to the NHS-activated polyacrylamide polymer. The reaction mixture was stirred at room temperature for 20 h. The short FAPI-conjugated polymer was purified using a centrifugal filter with a 1 kDa molecular weight cut-off membrane and washed three times with water. The product was diluted to a final concentration of 50 mg / mL and stored at 4 °C for further use. The chemical structure of this polymer is shown in Figure 9.

[0228] Part (c): Short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide)10 Synthesis of -block-(triethylene glycol monomethyl ether)

[0229] In a round-bottom flask, a solution of acrylamide (0.93 g), 4,4'-azobis(4-cyanovaleric acid) (0.030 g), methoxy triethylene glycol modified 2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic acid (0.5 g), dioxane (7 mL) and water (4 mL) was prepared. After purging the mixture with nitrogen gas for 15 minutes, it was heated to 70 °C with stirring for 1.5 hours. The mixture was allowed to cool to room temperature, and [2-(methacryloyloxy)-ethyl]phosphonic acid (1.2 g) and 4,4'-azobis(4-cyanovaleric acid) (0.030 g) were added. The reaction mixture was purged with nitrogen gas for 15 minutes and heated to 70 °C for 2 hours. The resulting polymer was precipitated in acetone and recovered by centrifugation. The polymer was dried in vacuo for 48 hours. The chemical structure of this polymer is shown in Figure 10.

[0230] Part (d): Short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 30 -FAPI copolymer mapping part 30% and short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 10 Stabilization of maghemite nanoparticles (Tracer 1, 30% FAP mapping maghemite nanoparticles) using a mixture of 70% of -block-(triethylene glycol monomethyl ether) copolymer stabilizer

[0231] Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) prepared in part (c) 10 -block-(triethylene glycol monomethyl ether) 44 mg and poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) prepared in part (b) 30-FAPI (50 mg) was dissolved in 2 mL of water. Using NaOH (0.1 M), the pH was adjusted to 4. The polymer mixture was added while sonicating 60 mg of magnetic particles in water (30 mg / mL) in Part (b) of Example 1. After 10 minutes, the pH was adjusted to 5.5, then further adjusted to 7.0 after sonication for another 10 minutes. After continuing sonication for a total of 30 minutes, unbound polymers were removed using a centrifugal filter with a 10 kDa molecular weight cut-off membrane. The coated nanoparticles (Tracer 1) were washed three times with water and diluted with physiological saline to obtain an isotonic dispersion of 20 mg Fe / mL.

[0232] Part (e): Short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 30 Stabilization of maghemite nanoparticles (Tracer 2, 100% FAP mapping maghemite nanoparticles) using 100% of the FAPI polymer mapping portion.

[0233] Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) prepared in Part (c) 30 -FAPI (50 mg) was diluted with water to a total volume of 2 mL. When the pH was measured, it was 6.9. The polymer was added while sonicating 30 mg of magnetic particles in water (30 mg / mL) in Part (b) of Example 1. When the pH was measured after the sonication step, it was 5.8. Then, using NaOH (0.1 M), the pH was adjusted to 7.0 while sonicating. After continuing sonication for a total of 20 minutes, unbound polymers were removed using a centrifugal filter with a 10 kDa molecular weight cut-off membrane. The coated nanoparticles (Tracer 2) were washed three times with water and diluted with physiological saline to obtain an isotonic dispersion of 20 mg Fe / mL.

[0234] Part (f): Short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 3030% copolymer non-targeting moiety and 5-block-poly(acrylamide) of short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 10 Stabilization of non-targeting maghemite nanoparticles (Tracer 3, non-targeting maghemite nanoparticles) used as a control, using a mixture with 70% of a steric stabilizer of -block-(triethylene glycol monomethyl ether) copolymer

[0235] Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) prepared in part (c) 10 44 mg of -block-(triethylene glycol monomethyl ether) and poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) prepared in part (a) 30 (50 mg) were dissolved in 2 mL of water. The pH was adjusted to 4 using NaOH (0.1 M). The polymer mixture was added while sonicating 60 mg of magnetic particles in water (30 mg / mL) in part (b) of Example 1. After 10 minutes, the pH was adjusted to 5.5, then sonicated for an additional 10 minutes and then adjusted to 7.0. After continuing sonication for a total of 30 minutes, unbound polymers were removed using a centrifugal filter with a 10 kDa molecular weight cut-off membrane. The coated nanoparticles (Tracer 3) were washed 3 times with water and diluted with physiological saline to obtain an isotonic dispersion of 20 mg Fe / mL.

[0236] Example 6: In vitro test of binding of micro-FAP mapping maghemite nanoparticles to FAP The binding affinities of 30% FAP-mapping maghemite nanoparticles (tracer 1), 100% FAP-mapping maghemite nanoparticles (tracer 2), and non-targeted maghemite nanoparticles (tracer 3) in parts (d), (e), and (f) of Example 5 for fibroblast activation protein were evaluated in vitro by comparing their cellular uptake in FAP-expressing melanoma cell line C32. C32 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a T75 cell culture flask and maintained in a 37 °C, 5% CO2 incubator. When the confluence reached 70-80%, the cells were passaged. To measure cellular uptake and test the binding affinity of the nanoparticles, C32 cells were seeded in a 6-well plate at a density of 3×10 5 cells / well in the above complete cell culture medium. The plate was placed in a 37 °C, 5% CO2 incubator and allowed to adhere for 24 hours.

[0237] All nanoparticles were prepared at a concentration of 0.150 mg Fe·mL -1 in cell culture medium supplemented with only 1% penicillin-streptomycin. The growth cell culture medium was removed from the plate and replaced with the tracer dispersion. Each tracer was tested using three replicates. C32 cells were incubated with the tracer dispersion in a 37 °C, 5% CO2 incubator for 24 hours. The cells were washed twice with PBS, detached using trypsin, and the cell pellet was recovered by centrifugation at 500×g for 5 minutes. The cell pellet was washed two more times with PBS and finally dried overnight at 60 °C using a heating block. The dried cell pellet was digested with trace metal grade nitric acid and hydrochloric acid (volume ratio 1:1), and the sample was diluted with water to a total volume of 3 mL. The iron concentration was measured using inductively coupled plasma mass spectrometry (ICP-MS). From the results, as shown in Figure 11, it was shown that the cellular uptake in C32 cells was much less for tracer 3 (non-targeted maghemite nanoparticles) than for tracer 1 (30% FAP-mapping maghemite nanoparticles) and tracer 2 (100% FAP-mapping maghemite nanoparticles).

[0238] Example 7: In Vitro Test of Binding Activity Comparing the Binding Activity of Microcopolymer FAP-Mapping Magnetite Nanoparticles and Single Polymer Micro FAP-Mapping Magnetite Nanoparticles in the Presence or Absence of Serum The binding activities of the 30% FAP-mapping (tracer 1) and 100% FAP-mapping (tracer 2) fibroblast activation proteins from parts (d) and (e) of Example 5 were evaluated in vitro by comparing their cellular uptake into the FAP-expressing melanoma cell line C32 in the presence or absence of serum (FBS) in the cell growth medium. C32 cells were cultured as described in Example 6. C32 cells were seeded into a 6-well plate at a density of 3×10 5 cells / well in complete cell culture medium. The plate was placed in a 37 °C, 5% CO2 incubator and allowed to adhere for 24 hours.

[0239] After 24 hours, the complete cell culture medium was removed from one of the 6-well plates and replaced with medium supplemented with only 1% penicillin-streptomycin. The cell culture medium in the remaining 6-well plates was removed and replaced with medium supplemented with 1% penicillin-streptomycin and 10% FBS. The nanoparticles from parts (d) and (e) of Example 5 were added at 0.150 mg Fe·mL -1It was added to each well until the final concentration was reached. C32 cells were incubated for 24 hours in a 37 °C, 5% CO₂ incubator with the tracer dispersion. The cells were washed twice with PBS, detached using trypsin, and the cell pellet was recovered by centrifugation at 500×g for 5 minutes. The cell pellet was washed two more times with PBS and finally dried overnight at 60 °C using a heating block. The dried cell pellet was digested with trace metal grade nitric acid and hydrochloric acid (volume ratio 1:1), and the sample was diluted with water to a total volume of 3 mL. Iron concentration was measured using inductively coupled plasma mass spectrometry (ICP-MS). The obtained data were analyzed and the significance was tested using an independent samples T-test. From the results shown in Figure 12, it is shown that for both tracers, the binding in the presence of FBS was significantly lower than the binding in the absence of FBS. However, the binding reduction rate of tracer 1 (30% FAP mapping nanoparticles) was 55%, which was statistically significantly smaller than 63% of the binding reduction rate of tracer 2 (100% FAP mapping nanoparticles) (p < 0.01). No significant difference was observed between tracer 1 and tracer 2 in terms of uptake in the absence of FBS (p > 0.05).

[0240] Example 8: Short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) using the magnetite core particles from part (c) of Example 1 30 -FAPI copolymer mapping moiety and short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 10 -block-(triethylene glycol monomethyl ether) copolymer sterically stabilized magnetite nanoparticles (magnetite FAP mapping nanoparticles) synthesis. Part (a): Short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 30 -FAPI copolymer mapping moiety 30% and short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 10Stabilization of magnetite nanoparticles (Tracer 4, 30% FAP mapping magnetite nanoparticles) using a mixture of 70% of a steric stabilizer of a -block-(triethylene glycol monomethyl ether) copolymer.

[0241] Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) prepared in Example 5 part (c) 10 -block-(triethylene glycol monomethyl ether) 44 mg and poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) prepared in Example 5 part (b) 30 -FAPI (50 mg) was dissolved in 2 mL of water. Using NaOH (0.1 M), the pH was adjusted to 4. The polymer mixture was added to 50 mg of magnetic particles in water (25 mg / mL) in part (c) of Example 1 during 10 minutes of sonication. The pH of the nanoparticles was measured before adding the polymer mixture and was 7.8. When measuring the pH after the sonication step, it was 5.4. Then, using NaOH (0.1 M), the pH was adjusted to 6.0 and 7.0 while sonicating. After continuing sonication for a total of 30 minutes, an unbound polymer was removed using a centrifugal filter with a 10 kDa molecular weight cut-off membrane. The coated nanoparticles (Tracer 4) were washed 3 times with water and diluted with physiological saline to obtain an isotonic dispersion of 20 mg Fe / mL.

[0242] Part (b): Short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 30 30% of the non-targeted part of the copolymer and short poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) 10 Stabilization of magnetite nanoparticles (Tracer 5, non-targeted magnetite nanoparticles) used as a control, using a mixture of 70% of a steric stabilizer of a -block-(triethylene glycol monomethyl ether) copolymer.

[0243] Example 5 Poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) prepared in part (c) 10 -block-(triethylene glycol monomethyl ether) 44 mg and poly{[2-(methacryloyloxy)-ethyl]phosphonic acid} 5-block-poly(acrylamide) prepared in part (a) of Example 5 30 (50 mg) were dissolved in 2 mL of water. The pH was adjusted to 4 using NaOH (0.1 M). The polymer mixture was added to 50 mg of magnetic particles in water (30 mg / mL) in part (c) of Example 1 with sonication. Before adding the polymer mixture, the pH of the nanoparticles was measured and found to be 7.8. After the sonication step, the pH was measured and found to be 5.4. Then, using NaOH (0.1 M), the pH was adjusted to 6.0 and 7.0 with sonication. After continuing sonication for a total of 30 minutes, unbound polymers were removed using a centrifugal filter with a 10 kDa molecular weight cut-off membrane. The coated nanoparticles (Tracer 5) were washed three times with water and diluted with physiological saline to obtain an isotonic dispersion of 20 mg Fe / mL.

[0244] Example 9 : In vitro test of the binding of micro-FAP mapping magnetite nanoparticles to FAP. The binding affinity of the non-targeted magnetite nanoparticles (Tracer 5) in parts (a) and (b) of Example 8 to fibroblast activation protein was evaluated in vitro by comparing their uptake in melanoma cell line C32 with FAP expression in those cells to that of 30% FAP mapping magnetite nanoparticles (Tracer 4). C32 cells were cultured as described in Example 6. To measure cell uptake and test the binding affinity of the nanoparticles, C32 cells were seeded in a 6-well plate at a density of 3×10 5 cells / well in the above complete cell culture medium. The plate was placed in a 37 °C, 5% CO2 incubator and allowed to adhere for 24 hours.

[0245] All the nanoparticles were prepared at a concentration of 0.150 mg Fe·mL -1 in cell culture medium supplemented with 1% penicillin-streptomycin and 10% FBS. The cell culture medium was removed from the plates and replaced with the tracer dispersion. Each tracer was tested using four replicas. C32 cells were incubated with the tracer dispersion in a 37 °C, 5% CO₂ incubator for 24 h. The cells were washed twice with PBS, detached using trypsin, and the cell pellet was recovered by centrifugation at 500×g for 5 min. The cell pellet was washed two more times with PBS and finally dried overnight at 60 °C using a heating block. The dried cell pellet was digested with trace metal grade nitric and hydrochloric acids (volume ratio 1:1), and the sample was diluted with water to a total volume of 3 mL. Iron concentration was measured using inductively coupled plasma mass spectrometry (ICP-MS). From this result, as shown in Fig. 13, it was shown that cellular uptake in C32 cells was much less for tracer 5 (non-targeted magnetite nanoparticles) than for tracer 4 (30% FAP mapped magnetite nanoparticles).

[0246] It will be understood by those skilled in the art that the present disclosure is not limited to the specific applications described in its use. The present disclosure is also not limited with respect to the specific elements and / or features described or depicted herein in its preferred embodiments. It will be understood that the present disclosure is not limited to the one or more disclosed embodiments and that numerous rearrangements, modifications, and substitutions are possible without departing from the scope of the present disclosure as set forth and defined by the appended claims.

[0247] Throughout the following specification and the claims, unless the context requires otherwise, the terms "comprise" and "include" and variations such as "comprising" and "including" are to be understood to imply the inclusion of the stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0248] Any reference to prior art in this specification is not an admission of any kind that such prior art forms part of the common general knowledge and should not be construed as such.

Claims

**Claim 1** A magnetic nanoparticle material suitable for administration to a subject, on the surface of which there is: (a) a copolymer steric stabilizer that promotes the dispersion of the magnetic nanoparticle material in a liquid, which comprises (i) an immobilized polymer segment having one or more binding groups for binding the copolymer steric stabilizer to the magnetic nanoparticle material and (ii) a steric stabilization polymer segment comprising a polyacrylamide-co-polyalkylene oxide block copolymer, which is a steric stabilization polymer segment different from the immobilized polymer segment; and (b) a copolymer mapping portion, which comprises (i) an immobilized polymer segment having one or more binding groups for binding the copolymer mapping portion to the magnetic nanoparticle material, (ii) one or more mapping groups comprising an agent that specifically binds to fibroblast activation protein (FAP), and (iii) a coupling polymer segment composed of polyacrylamide, which is a coupling polymer segment different from the immobilized polymer segment and which binds the immobilized polymer segment to the one or more mapping groups. The magnetic nanoparticle material has the copolymer steric stabilizer and the copolymer mapping portion bound thereto. **Claim 2** The magnetic nanoparticle material according to claim 1, on the surface of which there is: (c) a copolymer luminescent portion, which comprises (i) an immobilized polymer segment having one or more binding groups for binding the copolymer luminescent portion to the magnetic nanoparticle material, (ii) one or more luminescent groups for enabling visualization of the position of the magnetic nanoparticle material in vivo or for emitting an acoustic signal in response to light, and (iii) a coupling polymer segment composed of polyacrylamide, which is a coupling polymer segment different from the immobilized polymer segment and which binds the immobilized polymer segment to the one or more luminescent groups. The magnetic nanoparticle material has the copolymer luminescent portion bound thereto. **Claim 3** The magnetic nanoparticle material according to claim 2, wherein the one or more luminescent groups are selected from indocyanine green, sulfo-Cy3, sulfo-Cy5 and sulfo-Cy7. **Claim 4** The magnetic nanoparticle material according to claim 1 or 2, wherein the polyacrylamide-co-polyalkylene oxide block copolymer contains about 8 to about 60 polymerized acrylamide units and about 2 to about 10 polymerized alkylene oxide units.

5. The magnetic nanoparticle material according to claim 1 or 2, wherein the steric stabilization polymer segment has 10 to 70 polymerized monomer residue units.

6. The magnetic nanoparticle material according to claim 1 or 2, wherein the coupling polymer segment has 15 to 100 polymerized monomer residue units.

7. The magnetic nanoparticle material according to claim 1 or 2, wherein the coupling polymer segment has more polymerized monomer residue units than the steric stabilization polymer segment.

8. The magnetic nanoparticle material includes iron (Fe), maghemite (γ-Fe ２ O ３ ), magnetite (Fe ３ O ４ ), or a combination thereof. The magnetic nanoparticle material according to claim 1 or 2.

9. The magnetic nanoparticle material according to claim 1 or 2, wherein the agent that specifically binds to fibroblast activation protein (FAP) is selected from small molecule inhibitors and antibodies or antigen-binding fragments thereof.

10. The immobilized polymer segment of the copolymer steric stabilizer, or the immobilized polymer segment of the copolymer mapping portion, or both of them, is acrylic acid, methacrylic acid, itaconic acid, p-styrenecarboxylic acid, p-styrenesulfonic acid, vinylsulfonic acid, vinylphosphonic acid, monoacryloxyethyl phosphate, monoacryloxyethyl phosphonic acid, 2-(methacryloyloxy)ethyl phosphate, 2-(methacryloyloxy)ethyl phosphonic acid, ethacrylic acid, α-chloroacrylic acid, crotonic acid, fumaric acid, citraconic acid, mesaconic acid, maleic acid, 2-(dimethylamino)ethyl and propyl acrylates and methacrylates, the corresponding 3-(diethylamino)ethyl and propyl acrylates and methacrylates, or dimethylaminoethyl methacrylate, or a polymerized residue of a combination thereof. The magnetic nanoparticle material according to claim 1 or 2.

11. A composition suitable for administration to a subject, comprising the magnetic nanoparticle material according to claim 1 or 2 in a pharmaceutically acceptable liquid carrier.

12. The magnetic nanoparticle material according to claim 1 or 2, for use in a method of mapping a tumor margin in a subject, wherein the method is a. Administering the magnetic nanoparticle material to the subject; b. Detecting the magnetic nanoparticle material; and the magnetic nanoparticle material accumulates in the tumor microenvironment, thereby mapping the tumor margin. Magnetic nanoparticle material.

13. The magnetic nanoparticle material according to claim 1 or 2 for use in a method of diagnosing cancer, wherein the method comprises: a. Administering the magnetic nanoparticle material to a subject; b. Detecting the magnetic nanoparticle material; and the detection of the magnetic nanoparticle material accumulated in the tissue of the subject indicates that the subject has cancer. Magnetic nanoparticle material.

14. The magnetic nanoparticle material according to claim 1 or 2 for use in a method of treating cancer in a subject in need of cancer treatment, wherein the method comprises: a. Administering the magnetic nanoparticle material to the subject; b. Detecting the site where the magnetic nanoparticle material accumulates in the subject; c. Administering an effective amount of treatment for the cancer to the site detected for the magnetic nanoparticle material in step (b); and Magnetic nanoparticle material.

15. A composition for use in a method of mapping the tumor margin in a subject, wherein the composition comprises the magnetic nanoparticle material according to claim 1 or 2 in a pharmaceutically acceptable liquid carrier, and the method comprises: a. Administering the magnetic nanoparticle material or the composition to the subject; b. Detecting the magnetic nanoparticle material; and the magnetic nanoparticle material accumulates in the tumor microenvironment, thereby mapping the tumor margin. Composition.

16. A composition for use in a method of diagnosing cancer, wherein the composition comprises the magnetic nanoparticle material according to claim 1 or 2 in a pharmaceutically acceptable liquid carrier, and the method comprises: a. Administering the magnetic nanoparticle material or the composition to a subject; b. Detecting the magnetic nanoparticle material; and the detection of the magnetic nanoparticle material accumulated in the tissue of the subject indicates that the subject has cancer. Composition.

17. A composition for use in a method of treating cancer in a subject in need of cancer treatment, The composition contains the magnetic nanoparticle material according to claim 1 or 2 in a pharmaceutically acceptable liquid carrier, The method is, a. administering the magnetic nanoparticle material or the composition to the subject; b. detecting the site in the subject where the magnetic nanoparticle material accumulates; c. administering an effective amount of treatment for the cancer to the site of magnetic nanoparticle material detection in step (b); and comprises Composition.

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