WO2012178184A2

WO2012178184A2 - Removable protective shell for imaging agents and bioactive substances

Info

Publication number: WO2012178184A2
Application number: PCT/US2012/044052
Authority: WO
Inventors: Travis J. Williams; Andy Y. Chang
Original assignee: Childrens Hospital Los Angeles; University of Southern California USC
Current assignee: Childrens Hospital Los Angeles; University of Southern California USC
Priority date: 2011-06-23
Filing date: 2012-06-25
Publication date: 2012-12-27
Anticipated expiration: 2013-12-23
Also published as: WO2012178184A3

Abstract

The invention provides nanoparticles for delivery of imaging agents, drugs, and other molecules, such as genetic material. The nanoparticles have a core structure comprising the imaging agent and/or drug, and a shell structure that allows for water solubility. The shell structure further provides a barrier with limited water permeability that protects the core. The nanoparticles can be induced to release their cargo by treatment with ultrasound. Methods of delivering drugs and imaging agents are also provided, whereby the nanoparticles are delivered to tissues of interest in a substantially inert form, then activated using ultrasound to release the drugs or imaging agents.

Description

REMOVABLE PROTECTIVE SHELL FOR IMAGING AGENTS

AND BIOACTIVE SUBSTANCES

CROSS-REFERENCE TO RELATED APPLICATION

[001] This application relies on the disclosure of, and claims the benefit of the filing date of, U.S. provisional patent application number 61/500,585, filed 23 June 201 1 , the entire disclosure of which is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

[002] The present invention relates to the fields of nanotechnology, medicine, diagnostics, and medical and clinical research. More specifically, the invention relates to nanoparticles for use in medical diagnostics, evaluation, and treatment of patients, and in research on diseases and disorders and on substances and techniques for detecting biological materials of interest.

Discussion of Related Art

[003] Numerous agents are known in the art for imaging of tissues and organs of animals. In addition, numerous vehicles for delivery of such agents to the tissues and organs are known in the art. Likewise, numerous bioactive agents and molecular probes are known for therapeutic or prophylactic treatment of animals suffering from, being pre-disposed to, or at risk of developing various diseases and disorders.

[004] For example, imaging agents that are detectable using X-ray technologies (e.g., X- rays, CT/CAT scans) and magnetic resonance imaging (MRI) are well known and widely used in the medical diagnostics field. Broadly speaking, the agents possess a property that can be detected by a particular detection device. When introduced into the body of a patient, the presence of the agent at a site of interest (e.g., a target tissue) allows an image of the site to be created, thus allowing the medical practitioner to view and assess the site. Use of such agents is possible in numerous diseases and disorders, and for a wide range of tissues and organs in animals.

[005] While it is possible to use such agents directly, it is common to combine the agents with other substances or complex the agents with other substances to improve the half-life of the agent in the patient or to target the agent to a particular organ, tissue, or cell type. Various designs for delivery vehicles for agents have been published and patented, many involving technologies to reduce clearance of the vehicles (and thus agents) by the liver. Many such vehicles are nanoparticles that complex the agent with molecules that sequester or otherwise protect the agent from degradation and clearance from the patient's body. For example, a publication by Parac-Vogt et al. (Parac-Vogt, T. N.; Kimpe, K.; Laurent, S.; Pierart, S.; Vander Elst, L.; Muller, R. N.; Binnemans, K. Gadolinium DTPA-Monoamide Complexes Incorporated into Mixed Micelles as Possible MRI Contrast Agents. Eur. J. Inorg. Chem. 2004, 3538-3543) discloses a hybrid particle featuring a non-covalent core composed of phospholipids and functionalized gadolinium monomers coated with a shell composed of polysorbitol-20 (Tween- 80).

[006] While there are numerous agents and delivery vehicles available for diagnostic and therapeutic uses, the present inventors have recognized that there still exists a need in the art for vehicles that can target and deliver imaging agents, bioactive agents, molecular probes, and the like to organs, tissues, and cells of animals, for both medical applications and research purposes.

SUMMARY OF THE INVENTION

[007] In general, the present invention provides a strategy for protecting a particle of interest with a coating while, optionally, eliminating the particle's surface charge (zeta potential) and maintaining its solubility or making it soluble. According to the invention, the coating can be controllably removed using an appropriate chemical agent or using an appropriate amount of energy, including but not limited to, electromagnetic energy and ultrasound. Using the strategy, a large number of particles can be coated to mask or protect them, then uncoated at a desired time by way of exposure of the coated particle to an appropriate chemical or energy.

[008] The supermolecular complex of the invention comprises an outer coating, or "shell", which comprises three portions, as depicted schematically in Figure 1 : an interior binding region for bonding to the core of the supermolecular complex; a central hydrophobic region that acts as a water-impermeable barrier; and an exterior hydrophilic region that provides water solubility for the shell, and thus the supermolecular complex. The binding region comprises any charged group or group capable of participating in hydrogen bonding, such as, but not limited to, a guanidine, amine, carboxylate, or pyrene group, or any logical derivative or combination thereof. The binding region serves to bind the shell molecules to molecules making up the core of the supermolecular complex. The hydrophobic region of the shell comprises a series of -CF₂- or -CH₂- groups, or any logical derivative or combination thereof. The hydrophilic region comprises a poly(ethylene glycol), a charged group, or any logical derivative or combination thereof. Combining shell molecules with a core molecule having a surface comprising appropriate charged groups allows for chemical bonding of the shell binding region to the charged groups on the core molecule, resulting in creation of a shell around the core, and thus creating the framework for a supermolecular complex.

[009] The strategy of the invention can be applied to the assembly of a shell around any suitable core. According to the invention, the core is or can comprise a particle (also referred to herein as a "substance" or a "cargo"). Non-limiting examples of suitable particles useful in the present invention include micelles, vesicles, living cells, non-living cells, bioactive agents, MRI contrasting agents or other detectable markers, molecular probes, covalently linked nanoparticles or microp articles, non-covalently linked nanoparticles or microparticles, fullerenes, carbon nanotubes, and polar molecules. The nature of the particle is not generally limited, with the exception that, if it serves as the core molecule, it includes one or more suitable chemical groups for bonding to the binding region of the shell molecules. Preferably, the bonding is by way of one or more hydrogen bonds.

[010] According to the invention, the term supermolecular complex is used to denote a complex comprising: a shell component; a core component; and a particle or cargo. In exemplary embodiments discussed herein, the core consists of the particle, and as such, the supermolecular complex is referred to simply as a coated particle. However, as discussed below, in some embodiments, the core is simply a structural component for delivery of a cargo. Further, in some embodiments, the cargo is present in both the core and in the inner portion (e.g. , binding region) of the shell component.

[011] In general, coated particles according to the invention include a core structure surrounded by a shell structure. The core structure is not particularly limited, but in general includes at least one cargo. In exemplary embodiments, the core structure includes a substance that is to be delivered to a site of interest within a biological system, such as a patient's body or a tissue sample in vitro. The core structure and shell structure are bonded, for example by way of hydrogen bonding or ionic bonding, to each other such that they are dissociable upon application of energy, such as ultrasound, or a chemical agent, such as urea. In exemplary embodiments, bonding is by way of one or more hydrogen bonds.

[012] In embodiments, the present invention provides a coated particle that serves as a nanoparticle delivery vehicle that can be used selectively to deliver a substance to an area of an animal's body, including a pre-selected organ, tissue, or cell type. The nanoparticle delivery vehicle is particularly well suited for delivery of imaging agents to organs, tissues, and cells of interest for diagnosis and prognosis of diseases and disorders affecting or involving such organs, tissues, and cells. The nanoparticle delivery vehicle is also particularly well suited for delivery of bioactive agents to target cells to treat or prevent diseases and disorders. As such, the nanoparticle delivery vehicle can be used as a "smart" imaging agent for in vivo and in vitro analysis of samples.

[013] Yet again, in other embodiments, the present invention provides a coated particle, including a nanoparticle delivery vehicle, that can be used as a diagnostic agent for research purposes. The diagnostic agent can be designed for, and used in, either or both in vivo and in vitro applications.

[014] In exemplary embodiments, the present invention provides a coated particle, including a nanoparticle delivery vehicle, that can be used as a fluorine contrast agent, a fluorine imaging agent, and/or a fluorine detection agent. As discussed in more detail below, the present invention provides for the use of detection of fluorine atoms that are part of the coated particle to determine the location of the coated particle within a biological system (in vivo, in vitro, in situ, or ex vivo).

[015] One noteworthy feature of coated particles according to the invention is that the bonds that adhere the core structure to the shell structure can be broken by input of relatively low amounts of energy, such as electromagnetic energy (e.g. , radio waves, microwaves) or, preferably, mechanical energy (e.g. , ultrasound), or by treatment with bond-destabilizing chemical reagents, such as urea or change in pH. As such, the core structure and shell structure can be controllably separated when properly treated with the appropriate type and level of energy or chemical reagent. This feature is particularly advantageous in situations where the coated particle is used to deliver a substance to a site within a subject's body or other situations where it is desirable to control the exposure or activation of a substance: release of the substance can be regulated by targeting of the coated particle to a site, then release of the substance at that site. Furthermore, the coated particles are designed such that the amount of energy required to separate the core from the shell is either not damaging to biological tissue or is at a level where any damage to biological tissue can be easily repaired.

[016] Yet another noteworthy feature of the coated particle design is that, when attached to the core structure, the shell structure limits or prevents interaction of the core structure with an external aqueous environment by way of sequestering the core structure within a water-resistant (i.e., semi-permeable) or water-impermeable barrier, which is provided by the hydrophobic region of the shell structure. For ease of reference, this barrier is referred to herein at times as a "hydrophobic barrier" or "raincoat region". Dissociation of all or part of the shell structure from the core structure removes or impairs the action of this hydrophobic barrier and allows the core structure to interact with the aqueous environment. As such, the core structure and any substances present in the core structure (e.g. , imaging agents, bioactive molecules) are unmasked and/or released, allowing for "activation" of the core structure and its components.

[017] The coated particles of the invention can be provided as purified (to any detectable extent), homogeneous substances in solid form (e.g., as lyophilized or otherwise dried material). However, more typically, the coated particles are provided as part of a composition that comprises at least one other substance, which is preferably compatible with the coated particles and is biologically tolerable. By compatible, it is meant that the other substances do not destabilize the coated particles such that they do not (i.e., no longer) substantially function as designed. Because the coated particles of the invention contain known chemical constituents arranged in defined order via known chemical bonding, the skilled artisan will immediately be aware of suitable other substances to include in compositions according to the invention if a composition is desired. For example, it will be immediately apparent to the skilled artisan that compositions of the invention can comprise the coated particles of the invention in an aqueous liquid at about neutral pH (e.g., pH 6.5 - pH 7.5, such as pH 6.8 - 7.2 or about pH 7.0). Buffers or other components typically used in the art for preparing liquid compositions for use in vivo and in vitro for in vivo treatments or in vitro diagnostics or research can be included in the compositions. The skilled artisan is aware of such buffers and components, such as, but not limited to, aqueous bicarbonate buffers, monovalent and divalent salts (e.g., NaCl, KC1, MgCl₂, MnCl₂), organic solvents (e.g., ethanol and other alcohols), polymers, particularly biological polymers (e.g., nucleic acids, polypeptides, polysaccharides, etc.), and substances normally found in the blood of animals. By biologically tolerable, it is meant that the substance does not cause any significant negative biological reaction if exposed to a living cell. It is to be understood that the "other substance" discussed in this passage is not to be confused with the "cargo" or "bioactive agent" discussed in other portions of this document.

[018] The present invention also provides methods of using the coated particles of the invention. In general, the methods can be any methods in which an imaging agent (used herein interchangeably with "contrasting agent"), a bioactive agent (e.g., a small bioactive molecule, a biologic), a molecular probe, or the like is used. For example, the method can be a method of delivering an imaging agent to an organ, tissue, or cell to be imaged, or a method of delivering a small molecule to an organ, tissue, or cell to be analyzed or treated. The methods of the invention can be practiced in vivo, in vitro, and ex vivo.

[019] The method, as practiced in vivo, thus can include the following steps: a) administering to an animal a coated particle according to the invention; b) allowing adequate time for the coated particle to locate to an organ, tissue, or cell of interest; and c) providing conditions under which the coated particle performs its desired activity. In some embodiments, step c) includes subjecting the coated particle to energy or a chemical reagent in an amount sufficient to break the bond between the core structure and the shell structure, causing the core structure and shell structure to dissociate. In some embodiments, the environment of the organ, tissue, or cell of interest provides the conditions under which the coated particle expresses its activity (e.g., by way of the presence of certain substances in the environment of the organ, tissue, or cell; as a result of the pH of the environment of the organ, tissue, or cell; etc.).

[020] Where desired, the in vivo method can be extended to make it a method of imaging a target organ or tissue by including in the core an imaging agent, and further providing the additional step of using an imaging device that is compatible with the imaging agent to create an image of the target organ or tissue. In embodiments, dissociation of the shell structure from the core structure does not cause or result in dissociation of the imaging agent from the core structure. In exemplary embodiments, the method is a method of diagnosing and/or monitoring vesicoureteral reflux (VU ). However, the invention is not limited to such a use. In other words, because delivery of an imaging agent can result in imaging of any number of organs, tissues, and cells, the method can be a method of diagnosing or following the progression of a disease or disorder, or following the progression of treatment of a subject suffering from, suspected of suffering from, or at risk of developing a disease or disorder.

[021] In some embodiments of the method of invention, the method can be a method of delivering a small molecule, such as a bioactive agent (e.g., a drug), to an animal organ, tissue, or cell of interest. The method thus can be practiced in vivo and can include the following steps: a) administering to an animal a coated particle according to the present invention; b) allowing adequate time for the coated particle to locate to an organ, tissue, or cell of interest; and c) providing conditions under which the coated particle performs its desired activity. In some embodiments, step c) includes subjecting the coated particle to energy or a chemical reagent in an amount sufficient to break the bond between the core structure and the shell structure, causing the core structure and shell structure to dissociate. In some embodiments, the environment of the organ, tissue, or cell of interest provides the conditions under which the coated particle expresses its activity (e.g. , by way of the presence of certain substances in the environment of the organ, tissue, or cell; as a result of the pH of the environment of the organ, tissue, or cell; etc.). In exemplary embodiments, dissociation of the core structure and the shell structure causes the bioactive agent to dissociate from both of those structures as well. The proximity of the coated particle to the organ, tissue, or cell of interest results in a relatively high concentration of the bioactive agent close to the target organ, tissue, or cell, and thus results in delivery of the bioactive agent to the organ, tissue, or cell of interest. Because delivery of a bioactive agent can cause a desired clinical effect, the method can be a method of treating a subject suffering from, suspected of suffering from, or at risk of developing a disease or disorder.

[022] In another aspect, the method can be a method of delivering a molecular probe, such as a cell-type specific labeling agent, to an animal organ, tissue, or cell. The method thus can be an in vivo method that includes the following steps: a) administering to an animal a coated particle according to the present invention; b) allowing adequate time for the coated particle to locate to an organ, tissue, or cell of interest; and c) providing conditions under which the coated particle performs its desired activity. In some embodiments, step c) includes subjecting the coated particle to energy or a chemical reagent in an amount sufficient to break the bond between the core structure and the shell structure, causing the core structure and shell structure to dissociate. In some embodiments, the environment of the organ, tissue, or cell of interest provides the conditions under which the coated particle expresses its activity (e.g., by way of the presence of certain substances in the environment of the organ, tissue, or cell; as a result of the pH of the environment of the organ, tissue, or cell; etc.). In exemplary embodiments, dissociation of the core structure and the shell structure causes the molecular probe to dissociate from both of those structures as well. The proximity of the coated particle to the organ, tissue, or cell of interest results in delivery of the molecular probe to the organ, tissue, or cell of interest.

[023] The coated particles and methods of the invention have broad applicability in in vitro applications as well. In general, in vitro methods involve: a) combining a coated particle with cell culture material in an in vitro environment; b) allowing adequate time for the coated particle to locate to a tissue or cell of interest; and c) providing conditions under which the coated particle performs its desired activity. In some embodiments, step c) includes subjecting the coated particle to energy or a chemical reagent in an amount sufficient to break the bond between the core structure and the shell structure, causing the core structure and shell structure to dissociate. In some embodiments, the environment of the tissue or cell of interest provides the conditions under which the coated particle expresses its activity (e.g., by way of the presence of certain substances in the tissue culture environment; as a result of the pH of the tissue culture environment; etc.). In exemplary embodiments, dissociation of the core structure and the shell structure causes at least the cargo in the core structure to dissociate from both of those structures.

[024] Where desired, the in vitro method can be extended to make it a method of imaging a target tissue or cell type by including in the core an imaging agent, and further providing the additional step of using an imaging device that is compatible with the imaging agent to create an image of the target tissue or cell. In embodiments, dissociation of the shell structure from the core structure does not cause or result in dissociation of the imaging agent from the core structure. In general, the in vitro method of delivering an imaging agent is a method of diagnosing and/or monitoring a disease or disorder or the progression of treatment of a subject suffering from, suspected of suffering from, or at risk of developing a disease or disorder. [025] In some embodiments of the method of invention, the method can be a method of delivering a small molecule, such as a bioactive agent (e.g., a drug), to a target tissue or cell in vitro. The method thus can be practiced in vitro and can include the following steps: a) combining a coated particle with cell culture material in an in vitro environment; b) allowing an adequate time for the coated particle to locate to a tissue or cell of interest; and c) providing conditions under which the coated particle performs its desired activity. In some embodiments, step c) includes subjecting the coated particle to energy or a chemical reagent in an amount sufficient to break the bond between the core structure and the shell structure, causing the core structure and shell structure to dissociate. In some embodiments, the tissue culture environment provides the conditions under which the coated particle expresses its activity (e.g., by way of the presence of certain substances in the tissue culture environment; as a result of the pH of the tissue culture environment; etc.). In exemplary embodiments, dissociation of the core structure and the shell structure causes the bioactive agent to dissociate from both of those structures as well. The proximity of the coated particle to the tissue or cell of interest results in a relatively high concentration of the bioactive agent close to the target tissue or cell, and thus results in delivery of the bioactive agent to the tissue or cell of interest. Because the in vitro effects of the bioactive agent can be indicative of its effects in vivo, this embodiment can be practiced as a method of discovering drugs that are effective for treatment of diseases and disorders. It likewise can be used as a method for determining the effectiveness of certain drugs for treatment of certain diseases or disorders. It likewise can provide guidance relating to toxicity and dosing of a particular bioactive agent for a particular disease or disorder.

[026] In another aspect, the method can be an in vitro method of delivering a molecular probe, such as a cell-type specific labeling agent, to a tissue or cell. The method thus can be an in vitro method that includes the following steps: a) combining a coated particle with cell culture material in an in vitro environment; b) allowing an adequate time for the coated particle to locate to a tissue or cell of interest; and c) providing conditions under which the coated particle performs its desired activity. In some embodiments, step c) includes subjecting the coated particle to energy or a chemical reagent in an amount sufficient to break the bond between the core structure and the shell structure, causing the core structure and shell structure to dissociate. In some embodiments, the environment of the tissue or cell of interest provides the conditions under which the coated particle expresses its activity (e.g., by way of the presence of certain substances in the cell culture environment; as a result of the pH of the cell culture environment; etc.). In exemplary embodiments, dissociation of the core structure and the shell structure causes the molecular probe to dissociate from both of those structures as well. The proximity of the coated particle to the organ, tissue, or cell of interest results in delivery of the molecular probe to the organ, tissue, or cell of interest. As with other embodiments of the in vivo and in vitro methods of the invention, release of the cargo in the proximity of the target cells improves the effectiveness of the cargo for its intended purpose.

[027] It is to be recognized that, although the present invention is described substantially with regard to its applicability to animals, including humans, the invention has broad

applicability to detection, imaging, and treatment of diseases and disorders relating to bacterial and viral infections. As such, it is to be understood that the target cell for in vivo methods can be a bacterial cell or a virus. It is also to be understood that the in vitro methods can be used in a clinical or research setting to determine if an animal sample (e.g. , tissue, cells) is contaminated with one or more bacterial or viral species, or with tissues or cells from another animal. For example, molecular probes (e.g., labeled nucleic acids) specific for one or more bacterial or viral species can be the cargo for the coated particles, and upon release can provide a detection scheme for determining if target bacteria or viruses are present in the animal sample. In a similar manner, delivery of bioactive agents, such as antibacterial agents, can be accomplished via the in vivo methods. Of course, these concepts apply equally in some settings to detection of target bacteria and viruses in environmental samples.

[028] Furthermore, it is to be understood that the methods of the invention can be practiced ex vivo in a manner in which an animal sample (e.g., blood) is removed from the animal, treated ex vivo with a coated particle containing a cytotoxic agent specific for a bacterium, virus, neoplastic cell, etc., then the treated sample returned to the animal.

[029] In another aspect of the invention, a method for temporarily coating a surface is provided. The method comprises combining shell molecules with a particle having a surface with an appropriate chemical moiety having at least one reactive group, and allowing the shell molecules to bind to one or more reactive groups on the particle surface. In embodiments, the method is used to temporarily mask a surface-mounted catalyst or enzyme, then activate it by removal of the shell. For example, the method can be used with a particle with a catalytic antibody, which one wants to turn on in a specific location. It can also be useful for activating an inorganic electrocatalyst, to which a shell is applied to temporarily inactivate it while it is localized to its target.

[030] The invention contemplates use of electromagnetic radiation, mechanical energy, and chemical processes to controllably separate the shell from the core at a desired time. While exemplary embodiments of methods discussed herein generally focus on the use of urea as a chemical reagent to remove the shell from the core (or "unmask" the core), it is to be understood that a wide variety of chemical agents can be used. For example, the shell can be released from the core by a change in pH (i.e., by use of H⁺ or OH^"), a change in redox potential (e.g., a potential gradient over a membrane, DTT, etc.), or the use of an aldose (e.g., glucose). In embodiments, the use of such chemical reagents could be used to differentiate living from dead cells. In one specific embodiment, the use of glucose could be used to detect diabetes in a subject. For example, if the hydrogen bonding region is an acyl hydrazine, it could rapidly and irreversibly form a hydrozone with soluble glucose. By detecting removal of the shell in vivo in the bladder, or ex vivo in urine, one could determine that there is glucose present, and a diagnosis of diabetes warranted. Likewise, the detection of cAMP or cyclocreatine can be used to determine the ischemic status of a cell or tissue.

[031] The present invention further provides methods of making the coated particles of the invention. In general, the methods include, in any order: synthesizing the shell molecules and combining them with a particle to be coated under conditions where the binding region of the shell molecules can bond with chemical groups of the particle. In preferred embodiments, the shell molecule comprises a fluorine-rich region that acts as a hydrophobic barrier and protects substances located in the core region of the coated particle. It has been found that a fluorine- containing layer provides advantages over hydrophobic layers comprising other hydrophobic materials, the combination of such advantages providing unexpectedly superior performance of the coated vehicles of the invention. Fluorine-containing chains are expected to be hydrophobic in nature, and thus to repel water or reduce infiltration of water across the fluorine-containing region. However, it has unexpectedly been found that fluorine layers comprising (CF₂) units, with as few as 2-10 units, are essentially completely impermeable to water within the context of the structure of the present covered particles. Furthermore, it now has been recognized that the fluorine molecules of the hydrophobic barrier can themselves be used as detection molecules for the particles, and thus their cargoes. The combination of relatively short fluorine-containing regions and use of the fluorine to detect the particles provides a relatively inexpensive and relatively simple molecule for detection, and thus for use as an MRI agent. Yet further, the invention provides a rapid and simple method for producing guanidinium-labeled fluorinated amphiphiles (see the Examples below), which further simplifies and reduces the cost of production of shell regions according to the present invention. These advantages were not predicted by prior work and were a surprise when discovered by the present inventors.

[032] In another aspect, the invention provides kits for practicing the methods of the invention. In general, a kit of the invention comprises a coating for a charged molecule. The coating can be provided in a single container within the kit, or in a packaged combination with other coatings and/or other substances. In some embodiments, the kit comprises a coating for a charged molecule or a coated particle according to the invention. The coating or coated particle can be provided in a single container within the kit, or in packaged combination with other coatings or coated particles and/or other substances. Within the kit, the coating or coated particle can be provided as a dried composition (preferably purified or substantially purified), or as a liquid composition comprising essentially the coating or coated particle and an aqueous composition. If provided as dried composition, the kit can further comprise an appropriate liquid to reconstitute (e.g., rehydrate) the coating or coated particle. Within the kit, some or all of the ancillary substances (i.e., reagents, hardware) for performing any of the methods disclosed herein can be included. Any combination of coating, coated particle, composition, and ancillary substances is contemplated as being disposed in a particular container within the kit. It is to be understood that the kit may comprise any number of individual containers, each having the same, different, or any combination of contents.

[033] The present invention has wide applicability and utility in the fields of medical diagnosis and treatment. Non- limiting examples include: the use in patients undergoing a Voiding Cystourethrogram (VCUG); as a non-invasive replacement for VCUG; treatment of neoplasias; imaging the selective delivery of ultrasound to living tissue or other aqueous media, either in vivo or in vitro; selective imaging and drug delivery to tumors; differentiating living from dead cells; reversibly protecting reactive substances; and detecting contaminating materials, such as bacteria and viruses, in animal or environmental samples. In general, the invention is applicable to all situations where delivery of contrast/imaging agents, therapeutic agents, or molecular probes to any tissue is desired, potentially with release of the agent(s) using externally-supplied energy, such as ultrasound, or use of chemical agents, to achieve site-specific detection and, in embodiments, delivery, of the agent(s).

[034] Further, the invention includes, but is not limited to, the following additional uses of the coated particles of the invention: providing MRI contrast in vivo by treatment of tissue containing the coated particles of the invention with ultrasound; diagnosis and surveillance of vesicoureteoral reflux disease (VUR) or other disease states; catheter-free cystography; the delivery of a drug or molecular probe to a locus selected by application of ultrasound or chemical activation.

BRIEF DESCRIPTION OF THE DRAWINGS

[035] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and/or concepts of the invention, and together with the written description, serve to explain certain principles of the invention.

[036] Figure 1 A shows a diagrammatic representation of a shell molecule of the invention.

[037] Figure IB shows an exemplary embodiment of a shell molecule of the invention.

[038] Figure 2 shows treatment of an exemplary gadolinium-containing MRI contrasting agent (Dotarem^®; Guerbet S.A. SOCIETE ANONYME FRANCE, Villepinte FRANCE) with a functionalized guanidine.

[039] Figure 3 shows the chemical formulas of phenolphthalein (phth) and a shell-alcohol molecule.

[040] Figure 4 shows a scheme for generating a covered particle according to embodiments of the invention, and shows that treatment of functionalized guanidine (1) with gadolinium-rich nanoparticles (4) increases the magnetic relaxation rate of fluorine atoms in (1). DETAILED DESCRIPTION OF THE INVENTION

[041] Reference will now be made to various exemplary embodiments of the invention, examples of which may be illustrated in the accompanying drawings. It is to be understood that the following description is provided to assist the reader in understanding certain details and features of embodiments of the invention and should not be considered as a limitation on the scope or content of the invention. For example, while the following description focuses on the use of particular core and shell molecules, the concepts and methods are equally applicable to other core and shell molecules according to the invention, as broadly stated above.

[042] Before embodiments of the present invention are described in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Further, where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Further, in accordance with generally accepted terminology, the term "nanoparticle" means particles having a size between one and one thousand nanometers (nm), whereas microp articles are on the order of up to one thousand times larger.

[043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The present disclosure is controlling to the extent it conflicts with any incorporated publication. [044] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nanoparticle" includes a plurality of such particles and reference to "an agent" includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth. Furthermore, the use of terms that can be described using equivalent terms include the use of those equivalent terms. Thus, for example, the use of the term "subject" is to be understood to include the terms "patient", "animal", "human", and other terms used in the art to indicate one who is subject to a medical treatment. As another example, the use of the term "neoplasia" is to be understood to include the terms "tumor", "cancer", "aberrant growth", and other terms used in the art to indicate cells that are replicating, proliferating, or remaining alive in an abnormal way.

[045] The present invention relates to a means to coat or protect substances of interest with a coating or shell that, when associated with the particles, protects the particles from water while at the same time renders the particles water soluble. The particle being coated can be any substance, but in non-limiting exemplary embodiments, is a contrasting agent, a bioactive agent, a molecular probe, a biological cell, or a catalyst. Broadly speaking, the coated particles of the invention comprise a shell component and a core component. The core component is the interior portion of the overall structure that binds the shell component molecules and holds them together to form the coated particle of the invention. The core component thus comprises a molecule that contains at least one reactive group that bonds with a chemically reactive group of the shell component molecules. Those of skill in the art are well aware of a wide range of suitable reactive group pairs that can be used within the concept of the present invention without the need for a listing herein. Specifically, the invention encompasses all reactive group pairs that can participate in hydrogen bonding or an analogous not-covalent interaction and can be separated by treatment with energy or chemical agents.

[046] Because the bond between the core and shell can be broken by absorption of electromagnetic or mechanical energy, the coated particles of the invention can be thought of as "activatable". For example, coated particles comprising one or more bioactive agents (e.g., drugs), agents for imaging tissues and organs (e.g., contrasting agents for MRI), or other agents having utility in medical treatments and clinical diagnostics are provided, where the coated particles have a structure in which the agents are encapsulated or coated with a shell that renders the particles inert or inactive for their intended purpose (e.g., biological activity, imaging agent). The coated particles are treated with energy, e.g., ultrasound, or a chemical reagent to expose the agents to the external environment when and where desired, thereby providing the desired activity at the desired site. It is to be understood that the step of "exposing" can be an active step in which a chemical or type of energy is supplied, or can be a passive step, which relies on one or more chemical or physical features present at the site of the particle that are not introduced by the practitioner of the invention.

[047] The invention provides a coated particle comprising a core structure that can vary widely in structure and activity. The core structure is limited only in having an appropriate reactive group that is available for interaction with a reactive group present on molecules to be used to form the shell around the core. In preferred embodiments, the reactive group binding pairs form hydrogen bonds with each other, which can be broken with, e.g., ultrasound or chemical agents. As such, in embodiments, it is not preferred to use covalent bonding to hold the core structure to the shell structure. As discussed in detail below, the core can comprise a single molecule for forming the core and binding to the shell molecules, or it can be a more complex region comprising an inner core and an outer core.

[048] As exemplified in Figure 1 A, the shell structure comprises, in sequential

arrangement: a binding region for binding to the core structure; a hydrophobic region for protection of the binding region and core structure from hydrophilic substances, and a hydrophilic region for rendering the coated particle soluble in aqueous environments.

Preferably, the hydrophobic region is also lipophobic. In the example depicted in Figure IB, the binding region includes amino groups, the hydrophobic region includes a series of CF₂ groups, and the hydrophilic region includes poly(ethylene glycol). However, it is to be understood that these exemplary functional groups can be replaced by any number of equivalent groups to provide the same function.

[049] In embodiments, the coated particles further comprise a cargo, which is not the particle that makes up the core of the coated particle. In these embodiments, the coated particle is termed a supermolecular complex. In the supermolecular complex, the cargo can be part of the core (e.g. , provide some of the bonds that hold the shell to the core), can reside within the core, can reside within the binding region of the shell, or any combination of these.

[050] The cargo can be any substance that the practitioner desires to deliver to a target within a given environment, such as the body of a patient. While not so limited, in exemplary embodiments, the cargo is a detectable agent (e.g., an MRI contrasting agent), a bioactive agent (e.g., a small molecule, a drug), a molecular probe, or a combination of two or all three of these classes of molecules. Broadly speaking, cargo according to the invention can be biologically inactive or can be bioactive. As used herein, a bioactive agent is any substance that has a biological or biochemical effect on a subject to whom it is administered. The number and identity of bioactive agents encompassed by the present invention is vast, and is not particularly limited by structure or function. Non-limiting examples include small molecule drugs (e.g., chemotherapeutic agents, anti-inflammatory agents), biologicals (e.g., therapeutic peptides, polypeptides, proteins), antibodies, antigens, cytotoxins, hormones, nucleic acids (e.g., anti-sense DNA molecules, siRNA, microRNA, protein-encoding dsDNA), and anti-viral agents. Those of skill in the medical art can envision numerous other bioactive agents, the listing of all of which would not be practical within this document. All such agents are contemplated by the present invention.

[051] The size of the core may be varied to suit particular applications of the technology. For example, for delivery of imaging agents, the core can be on the order of 1 nm to 5 nm in diameter, whereas for delivery of bioactive agents or molecular probes, the core can be on the order of 5 nm to 25 nm in diameter or greater. It is to be understood that other sizes outside of these exemplary ranges may be used as well. The particular size will be selected by the practitioner based on the intended use of the supermolecular complexes and the environment in which they are to be used (e.g., in vivo as compared to in vitro).

[052] In embodiments, the core is divided into an inner core and an outer core, where the inner core region is surrounded by the outer core region. In such embodiments, the outer core region comprises molecules that link the inner core region to the shell. The type of molecule used for the outer core region molecules is not particularly limited, with the exception that it should be able to form a sufficiently strong linkage to a metal at the inner core, to other outer core molecules, or to another substance at the inner core region to maintain the integrity of the core structure during fabrication and use. Outer core molecules also should contain reactive groups for binding to the binding region groups on shell molecules. In embodiments, the outer core molecule forms a covalent bond on one end with a metal comprising the inner core region, and forms a non-covalent bond (e.g., a hydrogen bond) on the other end with a molecule comprising the shell structure. In exemplary embodiments, the outer core region comprises organic molecules, such as phosphonic acid surfactants, which are capable of covalently bonding to a metal, such as gold, at the inner core region, and also capable of bonding to the molecules that comprise the shell (discussed in more detail below). In exemplary embodiments, the organic molecules of the outer core region and the metal of the inner core region bond as a result of a sulfhydryl group at one terminus of the organic molecules.

[053] Depending on the intended use of the cargo, it can be chemically bonded to any part of the core molecules, or can be freely associated with the core molecules. For example, where the cargo is an imaging agent that does not require release from the nanoparticle structure for activity, the imaging agent can be chemically (e.g., covalently) bound to the inner core region (e.g., gold particle). In contrast, where the cargo is a bioactive agent that functions inside a target cell, the bioactive agent can be freely associated with the outer core molecules such that, upon removal of the shell, the bioactive agent can diffuse out from the core and enter the target cell. Yet further, where the cargo is not required to be bound to a core molecule, it can be present in the bonding region of the shell portion of the supermolecular complex, or in both the core and the binding region of the shell.

[054] The size or length of the coated particle (used hereinbelow to denote both coated particles and supermolecular complexes, unless otherwise noted) will vary depending on the substance(s) serving as the core and the length of the shell molecules. In many circumstances, the size of the coated particle will be defined by the intended use of the coated particle. For example, for delivery of imaging agents to the kidney for vesicoureteral reflux, the coated particle will have an overall size/diameter of 10 nm or less. Alternatively, for delivery of certain imaging agents to other tissues or organs, the coated particle can be on the order of 5 nm to 200 nm. Yet again, for delivery of imaging agents, bioactive agents, and molecular probes by way of release of these substances from the core structure upon dissociation of the shell structure, the coated particle can be on the order of 5 nm to 200 nm or more, for example up to about 500 nm, with the understanding that as a general rule more imaging agent, bioactive agent, or molecular probe that is loaded into the core structure the larger the core will need to be. The design of the coated particle should take into account the total size of the particle and its intended use. In general, the coated particle will be on the order of 5 nm to 500 nm, such as from 10 nm to 200 nm, 50 nm to 200 nm, or 100 nm to 200 nm. Of course, as stated above, each and every specific value, and each and every specific range, encompassed by the range of 5 nm to 500 nm is inherently disclosed herein without the need for an exhaustive listing of each value and range. Omission of each and every value and possible range is made for the sake of brevity of the disclosure, and is not to be construed as a failure to disclose such values and ranges.

[055] In many embodiments, the core region also comprises a cargo. In some

embodiments, the core is the cargo, while in other embodiments, the core is a combination of two or more substances, not all of which are a cargo according to the present invention. A cargo according to the present invention is any substance that the practitioner desires to be delivered to a target site by the coated particle delivery vehicle of the invention. While not so limited in structure or function, exemplary embodiments relate to imaging or contrasting agents for clinical/medical diagnostics, which can be used either in vitro, in vivo, or both. In these embodiments, the cargo is preferably a substance that can be detected using one or more commercially available systems. It thus may be any of the commercially available imaging agents known in the art, such as those agents having paramagnetic properties. For example, the imaging agent can comprise a thiol-terminated paramagnetic substance, such as a gadolinium (e.g., Gdⁱⁿ). Other non-limiting exemplary cargoes include: an imaging contrast agent, a medicinal entity, a biologically active molecule, siR A, and a molecular probe. Yet further specific non-limiting examples of cargoes are: bioactive agents, such as cytotoxins, anti-viral agents, and anti-parasitic agents, which can be delivered to target cells to treat or prevent diseases and disorders, including infections and malignancies.

[056] One advantage provided by the coated particle delivery vehicles of the present invention is the use of imaging agents that can be used with systems that do not rely on X-rays for detection. While X-ray detection is common and widely practiced, the ability to avoid using X-rays, and to avoid the collateral damage they can cause, is a distinct advantage that is provided by this invention. [057] In addition, a key advance provided by the present invention is the creation of a fluorine contrasting agent, such as one for MRI. More specifically, prior contrasting agents rely on the use of a proton detection system to locate the particle (and cargo). In contrast, the present invention, in embodiments, detects the presence of fluorine at a target site. The presence of fluorine at the target site indicates the presence of the delivery vehicle at the site. Upon this confirmation of the presence of the vehicle at the appropriate site, energy (mechanical or chemical) can be used to detach the shell of the vehicle from the core, allowing the core and cargo to be exposed to the target environment and perform its intended activity.

[058] In embodiments where the core comprises multiple substances, and depending on the intended use of the cargo, it can be chemically bonded to other substances present in the core or can be freely associated with the other substances.

[059] As mentioned above, the core structure is surrounded by a shell structure. The shell structure comprises molecules having three distinct regions arranged in the following sequential order: a bonding region for bonding to the outer core region molecules, a hydrophobic

"raincoat" region, and a hydrophilic region. The unique design of the shell molecules provides, in a single molecule, the ability to bond to the core structure a molecule that possess a water- resistant or water impermeable layer surrounded by a water soluble layer. When bound to the core structure, the nanoparticle delivery vehicle is water soluble, yet at the same time protects its cargo from interaction with water. Such a design allows for delivery of water labile cargoes to their intended sites of action without significant degradation of the cargoes. It also allows for delivery of cargo without loss of cargo during delivery by diffusion from the delivery vehicle. Likewise, the design allows for activation of water-activated cargoes only upon removal of the shell.

[060] The hydrophobic region can comprise any atom, chemical moiety, chemical group, etc. that has the characteristic of hydrophobicity. In some embodiments, it has the characteristic of lipophilicity. In other embodiments, e.g., those that are rich in fluorine, it has the

characteristic of lipophobicity. It thus may be a series of hydrophobic amino acids, for example three to twenty residues in length. Alternatively, it may comprise hydrophobic groups commonly found in thermoplastics or thermosetting resins. Yet further, it may comprise an alkane region. Numerous atoms, moieties, groups, etc. are known in the chemical arts, and any one or combination of two or more may be used in accordance with the invention. The non- limiting exemplary embodiment depicted in the figures shows the use of fluorine atoms as the hydrophobic region of the shell molecule. As such, in embodiments the hydrophobic region of the shell structure comprises the following structure: (CF₂)_n, wherein n = 2-10, such as 3, 4, or 6.

[061] The shell structure comprises numerous shell structure molecules bound via their bonding region to the core structure. While not so limited, it is highly preferred that the shell structure comprise numerous molecules all of the same structure. Regardless of whether multiple identical molecules are used or whether molecules having different structures are used, it is important that the shell structure molecules, once bound to the core molecules, form an uninterrupted, or substantially uninterrupted, hydrophobic barrier that surrounds or encases the bonding region, the core structure, and the cargo. As such, where shell structure molecules having more than one chemical structure are used, it is important that the distance between the bond between the shell structure molecules and the hydrophobic region be engineered to be the same so that a hydrophobic barrier is created.

[062] A hydrophilic region is located adjacent the hydrophobic region of the shell structure molecule. The hydrophilic region is provided to make the coated particle soluble in aqueous environments. As with the other regions of coated particles of the invention, the hydrophilic region of the shell molecule is not particularly limited in size, length, or chemical make-up. As long as the region confers upon the resulting coated particle the property of hydrophilicity or solubility in aqueous environments (e.g., blood, insterstitial space), it satisfies the criteria. As with hydrophobic atoms, chemical groups, moieties, etc., those of skill in the chemical arts are well aware of the numerous hydrophilic substances known in the art. As with the hydrophobic substances, the practitioner is free to select an appropriate hydrophilic substance to include as part of the shell molecule. In general, the hydrophilic region can comprise one or more structures that are capable of hydrogen bonding with water, causing it to be hydrophilic. Non- limiting examples include: glycerol or glycerol-based molecules, polyethylene glycol (PEG) or PEG-based molecules, or folic acid derivatives.

[063] In embodiments, the shell further comprises one or more substances that target the nanoparticle delivery vehicle to a particular organ, tissue, or cell. Such targeting substances are well known in the art, as are the advantages to being able to specifically deliver a cargo to a chosen cell or cell type. Exemplary targeting substances for use in the present invention include, without limitation, substances such as: peptides, antibodies, ligands for cell-surface receptors, and antigens. The use of targeting substances to specifically target bioactive substances to preselected cells or cell types is well known in the medical art, and any suitable targeting substance can be used as part of the coated particles of the present invention with regard not only to delivery of bioactive agents, but to other cargoes as well. Binding of such targets to the shell structure molecules can be accomplished using standard chemical or biochemical reactions without undue or excessive experimentation.

[064] The coated particles of the invention are typically spherical, having a diameter on the order of 500 nm or less, such as 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, 25 nm, 20 nm, 15 nm, 10 nm, 5 nm, or less. In accordance with the discussion above, those of skill in the art will immediately recognize that the particles can be any particular value within this disclosed range, without the need for this document to specifically list all values individually. Those of skill in the art will immediately recognize that, for any particular preparation, the particles will be present as a population of particles, all of which might not have the exact same size. Rather, the population will have a size distribution that will cluster around a particular diameter. It is to be understood that reference herein to a particular size or diameter is intended to encompass this noted size distribution.

[065] The coated particles of the present invention have numerous uses throughout biological systems. Non-limiting exemplary uses disclosed herein focus on the use for delivering imaging agents, bioactive agents, and molecular probes. As such, the invention provides coated particles for use in delivering imaging agents to a site to be imaged. It likewise provides coated particles for use in imaging an organ, tissue, cell, or cell type of interest. Similarly, it provides coated particles for use in delivering bioactive agents to an organ, tissue, cell, or cell type of interest. Yet further, it provides coated particles for use in treating an organ, tissue, cell, or cell type of interest. The use in treating can be a use for treating a subject suffering from, susceptible to, or at risk of developing a disease or disorder involving an organ, tissue, cell, or cell type. Additionally, the coated particles may be used for delivering molecular probes to an organ, tissue, cell, or cell type of interest. [066] Target cells, tissues, and organs are not limited. Non-limiting examples include the bladder, a tumor (including both solid and non-solid tumors), a blocked or constricted blood vessel, the heart, the liver, the brain, the stomach, muscle tissue/cells, pancreatic tissue/cells, blood vessel wall tissue/cells, skin cells, neural tissues/cells, tissues and cells of the

gastrointestinal tract, bone cells and bone progenitor cells, bacterial cells, and viruses. In this regard, it is important to recognize that the coated particles of the invention, in embodiments, are capable of passing through the blood-brain barrier.

[067] Use of the coated particles of the invention can be explained in terms of methods of using them. While the present detailed description of the invention focuses on the use of the particles in vivo, it is to be understood that the particles can also be used ex vivo for therapeutic or prophylactic purposes, and can be used in vitro for research purposes.

[068] In embodiments, the method of using the particles of the invention is a method of delivering an imaging agent to an organ, tissue, cell, or cell type to be imaged. Typically, the clinician or medical practitioner practicing this aspect of the invention will be interested in using the particles for diagnosis or prognosis of a disease or disorder. Often, the disease or disorder results in or is a result of morphological changes in an organ, tissue, or cell, which can be imaged using known techniques. For example, the disease might be a tumor, and the medical practitioner is interested in determining the size and shape of the tumor to determine the best course of treatment. Alternatively, the disease or disorder might be due to limited function of an organ, and the medical practitioner is interested in determining the size and shape of the organ, or the blood flow in, around, and through the organ. There are, of course, numerous other applications of imaging technology to the medical profession. The present invention relates to all such applications.

[069] The general steps for delivering an imaging agent are discussed above and again briefly below. In certain embodiments, the coated particle comprises a targeting substance on its outer surface to specifically target the particle to a pre-selected organ, tissue, cell, or cell type. Typically, the method of delivering the imaging agent is practiced so as to image the target organ, tissue, cell, or cell type. As such, the method may include using an imaging device that is compatible with the imaging agent to create an image of the target organ, tissue, cell, or cell type. Many such devices are known in the art, and any suitable one may be used. The practitioner is fully capable of selecting compatible imaging/contrasting agents and imaging devices/systems without a lengthy discussion herein. Preferably, the imaging device is an MRI device and the shell structure of the particle is partially or completely separated from the core structure and imaging agent as a result of delivery of ultrasound energy to the area of the patient's body where the organ, tissue, cell, or cell type of interest is located.

[070] In embodiments, the method of using the particles of the invention is a method of delivering a bioactive agent to an organ, tissue, cell, or cell type of interest. Typically, the medical practitioner practicing this aspect of the invention will be interested in using the particles for therapeutic or prophylactic treatment of a patient in need, or suspected of being in need, of such treatment. In contrast to delivering an imaging agent, which results in an image being made of the target, the method of delivering a bioactive agent results in a biological effect on the organ, tissue, cell, or cell type. As with other embodiments of the method of the invention, preferably the method of delivering a bioactive agent uses a particle that comprises a specific targeting substance on its surface to target the particles to a pre-selected organ, tissue, cell, or cell type. Further, it is preferable that, once targeted to a specific organ, tissue, cell, or cell type, the particles be "activated" through removal of some or all of the shell structure molecules from the core molecules, allowing the bioactive agent to be released. It is to be recognized that complete removal of the shell structure molecules is not a required step in the method. Rather, once the particles are targeted to a chosen target, they may be partially "activated" then allowed to remain at the targeted position (e.g., bound to a target cell). Partial activation allows for slow, long-term delivery of the bioactive agent to the cell.

[071] In yet other embodiments, the method of using the particles of the invention is a method of delivering a molecular probe, such as a cell-type specific labeling agent, to an organ, tissue, cell, or cell type of interest. The method steps are those used in the method of delivering a bioactive agent, and the same considerations apply with regard to release of the probe from the nanoparticle delivery vehicle.

[072] In an exemplary embodiment, the use of the coated particle is a method of co-delivery of an imaging agent and a bioactive agent. The method steps are those discussed above with regard to delivery of imaging agents and bioactive agents: a) administering to a subject in need or suspected of being in need a coated particle according to the present invention; and b) allowing adequate time for the coated particle to locate to an organ, tissue, or cell of interest. Preferably, the method further comprises: c) subjecting the coated particle to energy in an amount sufficient to break the chemical bond between the core structure and the shell structure, causing the core structure and shell structure to dissociate. In this exemplary embodiment, dissociation of the core structure and the shell structure causes or allows the bioactive agent to dissociate from both of those structures. The proximity of the particle to the organ, tissue, cell, or cell type of interest results in delivery of the bioactive agent to the organ, tissue, cell, or cell type of interest. Further, dissociation of the shell structure and the core structure exposes the imaging agent to the aqueous environment of the body, allowing for imaging of the organ, tissue, cell, or cell type being treated with the bioactive agent. The delivery of a single particle having both an imaging agent and a bioactive agent provides a substantial advantage to the medical practitioner, as he is now able to achieve two goals with a single procedure. Doing so results in fewer procedures performed, real-time collection of data and delivery of bioactive agent, and significant cost savings for the patient. Furthermore, combined delivery of imaging agent and bioactive agent allows the medical practitioner to follow the progression of a disease under treatment and the response of a patient to a treatment regimen. For example, using the combined delivery vehicle, an oncologist can not only deliver an anti-cancer agent to a solid tumor, but can, at the same time, determine the effectiveness of the delivery of the treatment at the tumor site by imaging the tumor's size, shape, or other physical characteristics.

[073] It is important to recognize that delivery of energy to the nanoparticle delivery vehicle (regardless of the cargo that it contains), or exposing it to an appropriate chemical agent, causes the shell structure to dissociate from the core structure. However, dissociation might not be complete. For example, exposure of coated particles to ultrasound energy can, in some embodiments, result in dissociation of all of the shell structure molecules from all of the outer core molecules. However, it can also result in dissociation of fewer than all of the molecules dissociating, resulting in a partial or incomplete dissociation. In such a situation, successful use of the particles can still be achieved, as the hydrophobic barrier will still have been breached, at least to some extent, allowing water to enter the core structure and, where the cargo is not covalently bound to the inner core, diffusion of some or all of the cargo from the core structure. [074] The design of the coated particle of the present invention provides a particle that has an outer layer that is hydrophilic (thus making the particle water soluble) and a core that is protected from water by a hydrophobic barrier. Unlike other structures known in the art for similar uses, the coated particle of the present invention can have a core that is completely protected from water by the hydrophobic barrier (i.e., the hydrophobic barrier can be water impermeable). However, it is to be understood that while it is preferred that the hydrophobic barrier be water impermeable, in some embodiments, the hydrophobic barrier is not a complete barrier to water, but is rather a semi-permeable barrier. This characteristic can result from designing the shell structure molecule to include holes or breaches in the hydrophobic barrier, designing the shell structure molecule to allow for chemical or enzymatic degradation of the shell structure molecule, core molecules, or both, or as a result of normal movement of the molecules about each other within the supermolecular complex that is the coated particle. Such "leakiness" can result in pre-activation of a certain portion of the cargo or can result in loss of a certain portion of cargo prior to "activation" of the particles. Such "leakiness" can be used to the practitioner's advantage by providing a method in which the coated particle is delivered to a target, and simply allowed to remain in contact with the target while a bioactive agent (e.g., a cytotoxic agent) slowly diffuses out of the particle, thus delivering a low dose of agent to the target over an extended period of time.

[075] In contrast to particles known in the art for similar uses, the present coated particle is unique in that it includes a controllable "on/off switch" that allows activation of the particle at a time and place selected by the practitioner. It thus provides an improvement over the current art.

[076] As used herein, administering is any action that results in the particles of the invention being present within the body (including the alimentary canal). It thus can be any action commonly known in the art for delivery of bioactive agents and/or imaging agents to patients, including, but not limited to, oral ingestion, injection (of any type), infusion, via mucosal membranes, inhalation, and transdermal. In this regard, it is important to note that, in some embodiments relating to macromolecular structures, the coated particle takes the form of a pill or capsule for oral ingestion of a bioactive agent (e.g. , drug) or imaging agent. In an exemplary embodiment, the cargo is an imaging agent is used to diagnose gastroesophageal reflux disease. [077] When used to study the urinary tract, the present invention allows administration via a route other than catheterization. The amounts to be delivered will vary depending on the route chosen, and can easily be determined by the practitioner using standard medical protocols without undue or excessive experimentation.

[078] The amount of time required for delivery of the imaging agent to the target site will also vary depending on the route of administration chosen. For example, for intravenous administration of a coated particle for imaging of the urinary tract, imaging can commence approximately 45 minutes after administration. Of course, the time required for each

administration route can be determined using standard protocols without undue or excessive experimentation.

[079] Further, the amount of energy or chemical agent needed to release the shell from the core will vary depending on the type of energy or chemical agent used and the particular atoms involved in the bond. As with the other parameters, the amount of energy or reagent to be delivered can be determined for each bonding scheme without undue or excessive

experimentation. For example, the amount of energy can be on the order of 5 watts or less. However, for deep tissue or dense tissue, the amount can be higher.

EXAMPLES

[080] The invention will be further described with respect to creation and characterization of a coated particle containing a core having a gadolinium compound hydrogen bonded to a shell molecule comprising a fluorine hydrophobic barrier and a poly(ethylene glycol) hydrophilic region. Those of skill in the art will immediately recognize that the concepts disclosed are applicable to other structures, for use in numerous diseases, disorders, systems, organs, and tissues.

[081] Example 1 : Protection of a Small Molecule MRI Contrast Agent by Shell

Component

[082] To show the protective effect of a shell molecule according to the invention on Dotarem^®, an experiment was performed, and it was found that the shell molecule reduced the magnetic relaxivity of the Dotarem^® and that the relaxivity could be reversed by treatment with urea or urea plus sonication. Figure 2 illustrates a scheme for demonstrating modulation of MRI contrast ability of the small molecule agent Dotarem^®. More specifically, a complex of the two compounds depicted in Figure 2 was formed. This compound showed an attenuated contrast relative to Dotarem^® alone, as observed by a decrease in molar relaxivity, r_ls from 2.88(32) mM^" V¹ to 2.74(16) mM^'V¹. Upon addition of urea to the solution of the complex, the ri value remained constant at 2.74(18) mM^'V¹. Upon sonication of the complex in the presence of urea, the ri increased to 2.93(18) mM^'V¹. This indicates the return of the contrast by dissociation of the complex to its original components.

[083] This experiment shows, for the first time, the use of ultrasound to modulate the MRI contrast of a small molecule relaxivity agent.

[084] Example 2: Protection of Small Molecule pH Indicator by Shell Component

[085] Phenolphthalein (phth) is a chemical indicator that reacts with NaOH to produce a characteristic fuchsia colored solution. With the objective of encapsulating the dye phth in a fiuorous shell molecule (see Figure 3), two sample vials were set up. The first vial contained 4 ml of water, 2 microliters of phth, and the fiuorous shell molecule (experimental vial). The second vial contained 4 ml of water, 2 microliters of phth, but no fiuorous shell molecule (control vial). In contrast to the fiuorous shell molecules used in Example 1, which terminated in guanidine groups (for binding to core molecules or cargo molecules), the fiuorous shell molecule used in this Example was a fiuorous alcohol (i.e., the molecule terminated in an -OH group rather than a guanidine group as in Examples 1 and 2). The samples were mixed for one hour to ensure complete mixture and to provide time for the fiuorous shell molecule to bind to the phth. At one hour, 15 microliters of 0. IN NaOH was added to each vial, and the vials mixed. The control vial immediately turned a deep fuchsia color, indicating activity of the phth as a pH indicator. In contrast, the vial containing the fiuorous shell molecule did not significantly change in color, indicating a reaction of phth with the fiuorous shell molecule, and protection or sequestration of the phth from the NaOH.

[086] Both the experimental and control vials were then sonicated. After continued sonication for about 2 minutes, the experimental vial began to show a slight fuchsia color, indicating release of the shell from the phth. The control solution remained the same deep fuchsia color throughout the experiment. The results show that a fluorous shell molecule can protect a pH-sensitive dye, and thus the invention can be used to identify the pH of an environment at a pre-selected time and under pre-selected conditions. The results also show that linking of the core to the shell can potentially be accomplished via a single O-H-0 hydrogen bond.

[087] A second control experiment was performed to ensure that the difference in reaction of NaOH with indicator (phth) seen between the experimental vial and the control vial was not purely a pH interaction in which the fluorous shell molecule was acting as an acid and reacting with the added NaOH, causing no appearance of the fuchsia indicator. In this experiment, an equal amount of trifluoroethanol (pKa = 10.6, believed to be similar to pKa of the fluorous shell molecule) was added to the control sample. This experiment proceeded identically to the previous experiment until the sonication step, where no return of fuchsia color was visualized in the experimental sample. This result indicated that the fluorous shell molecule is not acting merely as an acid, but is instead binding to and protecting the phth in a manner that is reversible by input of sonication.

[088] Example 3 : Activation of a Fluorine-Based Responsive MRI Contrast Agent Based on Gadolinium and the Shell Component

[089] The shell molecules of the coated particles and supermolecular complexes of the present invention contain, in preferred embodiments, a hydrophobic barrier comprised of two or more (CF₂) units. These units provide a hydrophobic and lipophobic barrier to entry of substances into the core region of the particles and complexes. Through experimentation, it was also determined that these fluorine-containing units can act as imaging agents.

[090] Contrasting or imaging agents generally used in the art detect the position and spin of protons in samples, such as portions of a subject's body. The present invention provides a new type of contrasting agent - one that is based on detection of ¹⁹F from the hydrophobic barrier portion of the shell molecule of the present invention.

[091] Using a shell-coated, gold-centered particle, in which gadolinium-based MRI contrast agents were surrounded by and protected by shell molecules comprising a (CF₂)₄ hydrophobic barrier (shown in Figure 4), the effect on relaxation of the fluorine atoms of the hydrophobic barrier was determined. It was found that treatment of the fluorous shell (functionalized with a guanidine group for binding to a phosphonate group) with the gadolinium-containing particle increased the magnetic relaxation rate of the fluorine atoms in the shell molecule. The effect could be reversed by treating the complex with urea and sonication. More specifically, the shell molecule was dissolved in a 25 mM TRIS buffer (pH 7.6), and ¹⁹F Ti relaxation rates were measured. Upon addition of the gadolinium-containing particles derivatized with thiol phosphonic acid, the Ti relaxations were decreased, suggesting binding to the fluorous compound. Upon addition of urea, Ti relaxation times for all fluorine nuclei were significantly increased and were increased even further upon exposure to ultrasound. The Ti data are summarized in Table 1, below. The data show the development of a ¹⁹F contrast agent that can be activated upon ultrasound in the presence of urea.

[098] Table 1: Summary of ¹⁹F Tj Values

[099] It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

1. A coated particle comprising:

a) a core structure comprising a molecule that comprises a chemical group that bonds to a shell structure molecule through one or more hydrogen bonds; and

b) a shell structure comprising one or more molecules having, in sequential arrangement: a binding region for binding to the core structure, wherein the binding region comprises a molecule that comprises a chemical group that bonds to a core structure molecule through one or more hydrogen bonds,

a hydrophobic region for protection of the binding region and core structure from hydrophilic substances, wherein the hydrophobic region comprises at least two (CF₂) groups covalently linked via the carbon atoms, and

a hydrophilic region for rendering the nanoparticle soluble in hydrophilic environments;

wherein the coated particle is stable in an aqueous environment, and

wherein the core structure and the shell structure are separable by input of mechanical energy or chemical energy.

2. The coated particle of claim 1, wherein

the core structure comprises a single type of molecule that comprises a chemical group that bonds to a shell structure molecule, wherein the core structure molecule bonds to the shell structure molecule through a single hydrogen bond, and the shell structure molecule that comprises a chemical group that bonds to a shell structure molecule is of a single type, and wherein the shell structure molecule bonds to the core structure molecule through a single hydrogen bond.

3. The coated particle of either of claim 1 or claim 2, wherein

the core structure molecule that participates in hydrogen bonding with a shell structure molecule is phenolphthalein,

the shell structure molecule that participates in hydrogen bonding with a core structure molecule is a fluorous alcohol, and

wherein the coated particle is a molecular probe.

4. The coated particle of any one of claims 1-3, wherein the hydrophobic region of the shell structure comprises the following structure: (CF₂)_n, wherein n = 2-10.

5. A contrasting agent comprising the coated particle of any one of claims 1, 2, or 4, wherein the contrasting agent is based on detection of properties of one or more of the fluorine atoms of the hydrophobic region of the shell structure.

6. The coated particle or contrasting agent of any one of claims 1-5, which is a supermolecular complex that further comprises:

c) a cargo molecule located in the core structure, the binding region of the shell structure, or both.

7. The supermolecular complex of claim 6, wherein the cargo is an imaging or contrasting agent; a bioactive agent; a molecular probe; a biological cell; a virus; a biological, organic, or inorganic catalyst; or a combination of two or more of these.

8. The supermolecular complex of claim 7, wherein the cargo is an anti-cancer drug.

9. The coated particle of any one of claims 1-4, the contrasting agent of claim 5, or the supermolecular complex of any one of claims 6-8, wherein the shell further comprises a substance that targets the particle, agent, or complex to a particular tissue or cell of an animal.

10. A method of imaging animal tissue, said method comprising:

a) administering to an animal subject a coated particle comprising:

i) a core structure comprising a molecule that comprises a chemical group that bonds to a shell structure molecule through one or more hydrogen bonds; and

ii) a shell structure comprising one or more molecules having, in sequential arrangement:

a binding region for binding to the core structure, wherein the binding region comprises a molecule that comprises a chemical group that bonds to a core structure molecule through one or more hydrogen bonds,

a hydrophilic region for rendering the nanoparticle soluble in

hydrophilic environments;

wherein the coated particle is stable in an aqueous environment, and wherein the core structure and the shell structure are separable by input of mechanical energy or chemical energy;

b) allowing adequate time for the coated particle to locate to a tissue of interest;

c) subjecting the coated particle to energy or a chemical reagent in an amount sufficient to break the bond between the core structure and the shell structure, causing the core structure and shell structure to dissociate; and

d) subjecting the tissue and imaging agent to an imaging technique.

11. The method of claim 10, wherein the energy is ultrasound and the ultrasound is applied at a level that is not harmful to animal tissue.

12. The method of either of claim 10 or claim 11, wherein the coated particle further comprises a bioactive agent, and wherein dissociation of the core structure from the shell structure enables the bioactive agent to be released from the coated particle.

13. The method of claim 12, wherein the bioactive agent is a pharmaceutical.

14. The method of claim 12, wherein the bioactive agent has anti-cancer activity.

15. The method of any one of claims 10-14, wherein the shell structure further comprises a substance that targets the nanoparticle to a particular tissue or cell of an animal.

16. A method of delivering a bioactive agent to an animal tissue, said method comprising:

a) administering to an animal subject a coated particle comprising:

a hydrophilic region for rendering the nanoparticle soluble in

hydrophilic environments;

b) allowing adequate time for the coated particle to locate to a tissue of interest; and c) subjecting the coated particle to energy or a chemical reagent in an amount sufficient to break the bond between the core structure and the shell structure, causing the core structure and shell structure to dissociate.

17. The method of claim 16, wherein the energy is ultrasound and the ultrasound is applied at a level that is not harmful to animal tissue.

18. The method of either of claim 16 or claim 17, wherein dissociation of the core structure from the shell structure enables the bioactive agent to be released from the coated particle.

19. The method of any one of claims 16-18, wherein the bioactive agent is a pharmaceutical.

20. The method of claim 19, wherein the bioactive agent has anti-cancer activity.

21. The method of any one of claims 16-20, wherein the shell structure further comprises a substance that targets the coated particle to a particular tissue or cell of an animal.