WO2011143128A2

WO2011143128A2 - Multi-phasic nanostructures for imaging and therapy

Info

Publication number: WO2011143128A2
Application number: PCT/US2011/035779
Authority: WO
Inventors: Xiaohu Gao; Shang-Hsiu Hu
Original assignee: University Of Washington Through Its Center For Commercialization
Priority date: 2010-05-10
Filing date: 2011-05-09
Publication date: 2011-11-17
Also published as: WO2011143128A3

Abstract

Aspects of the disclosure are directed to nano-scale composites with spatially separated functionalities such as for imaging probe engineering and therapeutic formulations. Additional aspects of the disclosure are directed to magnetic field modulated imaging and therapies. Included in the disclosure is the use of the composites for magnetolytic therapy, whereby cell lysis is effected by impacting magnetic composites into a cell using a spinning or oscillating magnetic field.

Description

MULTI-PHASIC NANOSTRUCTURES FOR IMAGING AND THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos. 61/333244, filed May 10, 2010, and 61/333660, filed May 11, 2010, the disclosure of each is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with U.S. government support under R01CA131797 and R01CA140295 awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention.

BACKGROUND

Compact nanostructures with highly integrated functionalities are of considerable current interest to research fields such as drug delivery, multimodality imaging, and electronic devices. A key challenge, however, is how to combine individual components together without interfering or sacrificing their original properties, such as their electronic and optical properties. On the nanometer scale, a variety of heterodimer structures composed of quantum dots, magnetic nanoparticles (MNPs), and metallic NPs has been produced. However, because different components are touching each other, their signature optical, magnetic, and electronic properties are often altered or completely lost. In addition, despite the excellent similarity and compatibility in size between these heterodimer nanocrystals and biomolecules, often their small sizes also limit their response to external stimuli. For example, the magnetic domains (few nanometers) in these heterodimers do not respond to external magnetic fields efficiently (particles move slowly in magnetic fields). In parallel, the functionality interferences between different building blocks are also commonly observed on the micrometer scale. For example, doping fluorophores with MNPs in microspheres leads to significant fluorescence quenching.

One solution to this interference problem is to engineer multifunctional particles with individual building blocks spatially separated. Towards this end, microspheres with asymmetric architectures, also known as Janus particles, have been produced routinely. They offer unique features such as precisely controlled distribution of surface charges, compositions, hydrophobicity and hydrophilicity, dipole moments, combined and modular functionalities, which enable important applications unavailable to their symmetrical counterparts. For example, modulated optical nanoprobes (MOONs) based on sub-micrometer fluorescent spheres with one side capped with metal shells have been prepared that block light transmittance. Due to size-dependent rotational Brownian motion, the signature frequency of fluorescence fluctuation provides a unique mechanism for identification of the MOONs. Similarly, in device engineering, asymmetric structure also allows remote control of particle movement by electrical and magnetic fields, a promising strategy for engineering electronic paper, displays, and potentially spintronic memory devices.

Despite these recent advancements and the strong need of multifunctionalities, asymmetric particles have found limited applications in biomedical research, to a significant degree, due to the size mismatch between the current Janus microspheres and the biological systems. On the length scale of micrometers, synthetic approaches based on microfluidics, phase separation, and toposelective surface modification for making uniform Janus colloids of tunable composition are well-established. However, a key problem is that the sizes of these colloids range from 1-100 μιη, which are too big for bioapplications such as molecular imaging, drug delivery, and biomolecule labeling.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, a composite particle is provided. In one embodiment, the composite particle includes a core formed from a host polymer material and a plurality of first nanoparticles non-uniformly dispersed in the core, wherein the composite particle has a diameter of from 100 nm to 500 nm; and wherein each of the plurality of first nanoparticles has a diameter of from 1 nm to 60 nm.

In another aspect, a method for imaging a target is provided. In one embodiment, the method includes the steps of:

immobilizing a plurality of composite particles, adjacent to the target, wherein the composite particles comprises a luminophore and wherein the first nanoparticles are paramagnetic nanoparticles;

aligning the composite particles with a magnetic field in a first direction to provide the plurality of composite particles in a first orientation;

imaging the cells in the first orientation at a wavelength suitable to excite the luminophore to provide a first signal;

aligning the composite particles with a magnetic field in a second direction that is different from the first direction to provide the plurality of composite particles in a second orientation;

imaging the cells in the second orientation at a wavelength suitable to excite the luminophore to provide a second signal; and

comparing the first signal to the second signal to determine the position of at least a portion of the plurality of composite particles.

In another aspect, a method is provided for damaging a target using one or more paramagnetic composite particles. In one embodiment, the method includes the steps of: disposing the composite particles adjacent or inside the target; and

exposing the composite particles to a spinning or oscillating magnetic field of sufficient strength to cause the composite particles to impact the target. DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURES 1A-1N. FIGURE 1A is a schematic illustration of the steps involved in the synthesis of multifunctional composite particles in accordance with the provided embodiments. FIGURES IB and 1C are TEM micrographs of magneto-optical nanocomposites at magnifications of 10K and 60K. FIGURE ID graphically illustrates the hydrodynamic size (190 + 38 nm) of the nanocomposites in aqueous solution. FIGURES IE- IN illustrate composition tunability of the composite particles studied with TEM. Increasing concentrations of MNPs increase the size of the nanocomposites and their magnetic segments. Scale bars: FIGURES IE- II: 200 nm, and FIGURES 1J-1N: 20 nm.

FIGURES 2A-2D show orientation dependent fluorescence of magneto-optical nanocomposites in accordance with the provided embodiments. FIGURE 2A: Schematic of nanocomposite orientation modulation with magnetic fields. The magnetic segment blocks both excitation and fluorescence when it is facing down, but has no effect on fluorescence when facing up (not in the light path). FIGURE 2B: TEM images of the nanocomposites with the two phases of similar volume. Scale bar 100 nm. FIGURES 2C and 2D: Fluorescence imaging of nanocomposites of opposite orientations.

FIGURES 3A-3F show simultaneous imaging and treatment of cancer cells using nanocomposites in accordance with the provided embodiments. FIGURE 3A: Schematic illustration of the experimental conditions and magnetolytic therapy. Nanocomposites are first brought down to cell surface by magnetic attraction. Cells can then be examined by fluorescence microscopy or treated with oscillating magnetic fields, which result in mechanical forces to break down cell membranes. FIGURE 3B: Fluorescence imaging of cells coated with nanocomposites. FIGURES 3C-3F: Magnetolytic therapy on tumor cells. In comparison with the controls, missing either magnetic fields or nanocomposites, the viability of cells exposed to both is significantly reduced. Note the low cell density in the treatment group FIGURE 3F resulted from detaching of dead cells from cell culture dishes. The number of live cells in FIGURE 3F compared with that in FIGURE CF is reduced by 77%, whereas those in FIGURE 3D and FIGURE 3E show greater than 99.5% cell survival.

FIGURE 4: Graphically illustrates the solubility of magnetic nanoparticles and host polymers, in chloroform, as used in an exemplary embodiment. Magnetic nanoparticles start to aggregate above a concentration of 8 mg/ml, whereas the polymers remain soluble even at 50 mg/ml.

FIGURE 5A illustrates an exemplary method for forming composite particles having a compartment by way of a double-emulsion method, in accordance with embodiments provided herein. FIGURE 5B illustrates the chemical structure of an exemplary polymer host material and a diagrammatic illustration of a cross-section of a representative composite particle having a water compartment in accordance with exemplary embodiments provided herein.

FIGURE 6 illustrates an exemplary method for forming composite particles having a compartment that in turn has nanorods enclosed therein.

FIGURES 7A-7G are micrographs of gold nanorods, nano bowls, and composite particles having nanorods embedded therein, in accordance with the embodiments provided herein; particularly, with regard to the emulsion technique illustrated in

FIGURE 6.

FIGURE 8 illustrates various potential applications for composite particles having a compartment and a plurality of first nanoparticles in accordance with the embodiments provided herein.

FIGURE 9 is a photoluminescence micrograph of composite particles having the fluorescent dye FITC embedded therein, in accordance with an exemplary embodiment. FIGURE 10A is a photoluminescence micrograph of composite particles with compartments, wherein luminescent quantum dots are embedded within the body of the particle, in accordance with an exemplary embodiment. FIGURE 10B is an intensity spectrum obtained from the micrograph of FIGURE 10A.

FIGURE 11A is a photoluminescence micrograph of composite particles with compartments, wherein luminescent molecule pyrene is embedded within the body of the particle, in accordance with an exemplary embodiment. FIGURE 11B is an intensity spectrum obtained from the micrograph of FIGURE 11 A.

FIGURE 12 graphically illustrates the photoluminescence spectra used to determine encapsulation efficiency for a doxo-containing composite particle in accordance with the embodiments provided herein.

FIGURES 13A-13E graphically illustrate photoluminescence spectra of control samples and representative composite particles, each containing a specific luminescent analyte. FIGURE 13A is doxo. FIGURE 13B is FITC. FIGURE 13C is PEG-QD600. FIGURE 13D is QD600. FIGURE 13E is pyrene.

FIGURES 13F-13J are bar graphs illustrating the encapsulation efficiency of exemplary composite particles comprising luminescent materials in accordance with the embodiments provided herein. FIGURE 13F is doxo. FIGURE 13G is FITC. FIGURE 13H is PEG-QD600. FIGURE 131 is QD600. FIGURE 13J is pyrene.

FIGURE 14 graphically illustrates the percentage of doxo released from a series of representative composite particles in accordance with the embodiments provided herein.

FIGURE 15 illustrates the percentage of PEG-QD600 nanoparticles released from within composite particles in accordance with the embodiments provided herein.

FIGURE 16 is micrographs of representative composite particles formed using the amphiphilic copolymer poly(styrene-co-allyl alcohol), in accordance with the embodiments provided herein. FIGURE 17 is micrographs of representative composite particles formed using the amphiphilic copolymer poly(styrene-co-acrylic acid), in accordance with the embodiments provided herein.

FIGURE 18 is micrographs of representative composite particles formed using the amphiphilic copolymer poly(styrene-co-polyethylene glycol), in accordance with the embodiments provided herein.

FIGURE 19 is micrographs of representative composite particles formed using the amphiphilic copolymer poly(styrene-co-maleic anhydride), in accordance with the embodiments provided herein.

FIGURE 20 is a schematic diagram of a method for imaging using composite particles in accordance with the embodiments provided herein.

FIGURES 21A-21D schematically illustrate representative composite particles in accordance with the embodiments provided herein.

FIGURE 22 is a flow chart illustrating the steps of a method for imaging a target according to the embodiments provided herein.

FIGURE 23 is a flow chart illustrating the steps of a method for damaging a target by applying a magnetic field to composite particles in accordance with the embodiments provided herein. DETAILED DESCRIPTION

In one aspect, composite particles comprising a core formed from a host polymer material and a plurality of first nanoparticles non-uniformly dispersed in the core are provided. In additional aspects, method of making and using the composite particles are provided. In certain embodiments, the first nanoparticles are magnetic nanoparticles, which gives rise to methods for using the composite particles as a tool for imaging (e.g., imaging biological samples), hyperthermic therapy (killing cells using heat), and/or magnetolytic therapy, whereby targeted cells are destroyed by applying a magnetic field to composite particles adjacent or attached to the cells. In the embodiments provided herein, the hydrophobic or hydrophilic nature of the described compositions and methods is important to the composition of the composite particles. For ease of explanation, the embodiments described herein typically refer to the body of the composite particle as being hydrophobic, and further features of the embodiments (e.g., compartments within the particles, etc.) are described accordingly. However, it will be appreciated that the opposite composite particles are also contemplated.

Composite Particles

In one aspect, a composite particle is provided. The composite particle is multi- phasic, meaning that it has two or more distinct material phases. In one embodiment, the composite particle comprises a core formed from a host polymer material and a plurality of first nanoparticles non-uniformly dispersed in the core, wherein the composite particle has a diameter of from 100 nm to 500 nm; and wherein each of the plurality of first nanoparticles has a diameter of from 1 nm to 60 nm.

Because of the nanoscale of the composite particles, they are also referred to herein as "nanocomposites." The composite particle is "multi-phasic," meaning that it has two or more distinct material phases.

A representative composite particle 10 is illustrated in FIGURE 21A. The core 15 is formed from a host polymer material. A plurality of first nanoparticles 20 are non-uniformly dispersed within the core 15 to form a biphasic composite particle 10.

The core 15 has a diameter of from about 100 nm to about 500 nm. Such a size range is particularly advantageous for in vivo applications. For example, a particle 10 having a maximum diameter of 500 nm will fit within a capillary vessel. Micron- scale particles will not suffice for such applications.

The first nanoparticles 20 are disposed within the core 15 so as to be non-uniformly dispersed. For example, the first nanoparticles 20 may be dispersed such that every first nanoparticle 20 is disposed within a first hemisphere 30 of a spherical composite particle 10, as illustrated in FIGURE 21A. That is, there are no first nanoparticles 20 in the opposing second hemisphere 35, defined as being opposed across an axis 25 dividing the particle 10 into two hemispheres 30 and 35.

Alternatively, as illustrated in FIGURE 2 IB, if sufficient nanoparticles 20 are present in a particle 40, they may populate the second hemisphere 35, although the distribution is still non-uniform because a vacancy exists in the second hemisphere 35 where no nanoparticles 20 are disposed, even though the core 15 is of uniform composition throughout the particle 10.

While representative arrangements of the first nanoparticles 20 within the core 15 are described and illustrated herein, it will be appreciated that any configuration of the composite particle is contemplated as long as the first nanoparticle 20 distribution is non-uniform.

Non-uniform distribution is particularly critical if, for example, the first nanoparticles 20 are magnetic nanoparticles ("MNPs"), which can be applied for use in imaging and therapeutic methods, as disclosed below. Such methods are only possible if the embedded MNPs are dispersed such that a rotating or oscillating magnetic field will rotate and/or translate the composite particle.

However, additional embodiments disclosed herein do not require non-uniform ("asymmetric") distribution of embedded MNPs, as will be described below with regard to certain imaging and therapeutic applications. For such applications, the MNPs may be uniformly dispersed. However, the multi-phasic composite particles are still essential, for example, to allow for the separation of imaging agents from MNPs to avoid quenching issues.

In certain embodiments, the host polymer of the core 15 is hydrophobic. In certain embodiments, the host polymer is a hydrophobic block copolymer. Exemplary hydrophobic units of the block copolymer are selected from the group consisting of polystyrene, those used to form poly(lactic-co-glycolic acid) ("PLGA"), and combinations thereof. Exemplary hydrophilic units of the block copolymer are selected from the group consisting of poly(allyl alcohol), poly(acrylic acid), poly(ethylene glycol), poly(maleic anhydride), and combinations thereof. The host polymer may also be hydrophilic and/or amphiphilic.

In embodiments where the first nanoparticles are non-uniformly distributed, the first nanoparticles will phase separate in the host polymer. Typically such a phase separation results from hydrophobic-hydrophilic repulsion, but it will be appreciated that an absolute hydrophobic-hydrophilic interaction need not be present to provide phase segregation. For example, if the nanoparticles are simply more hydrophilic (or hydrophobic) than the host polymer, then the degree of difference may be sufficient to provide the necessary energetics to cause the nanoparticles to phase separate and aggregate to provide non-uniform distribution within the composite particle.

The first nanoparticles 20 have a size range of from about 1 nm to about 60 nm in diameter. The diameter of the first nanoparticle is defined in the traditional sense for spherical shapes. For non-spheres, the diameter is defined as the smallest dimension of the particle. For example, a nanorod has a diameter that is defined as the lateral cross- sectional diameter. The size of the first nanoparticles 20 is such that a plurality can fit into a single particle 10. As described above, the core 15 has a nanoscale size range, as well. The nanoscale size of both the core 15 and the first nanoparticles 20 makes formation of the composite particles 10 difficult when using traditional methods (e.g., photolithography or other "top down" approaches). Accordingly, preferred embodiments of the composite particles 10 are formed using the emulsion techniques provided herein. The emulsion formation route to forming the composite particles 10 may result in additional structural features of the particles 10 (e.g., a surfactant layer surrounding the core 15) as will be described elsewhere herein.

In certain embodiments, the first nanoparticles are selected from the group consisting of magnetic particles, quantum dots, metallic particles, and combinations thereof.

Representative magnetic particles include particles that include a suitable metal or metal oxide. Suitable metals and metal oxides include iron, nickel, cobalt, iron platinum, zinc selenide, ferrous oxide, ferric oxide, cobalt oxide, aluminum oxide, germanium oxide, tin dioxide, titanium dioxide, gadolinium oxide, indium tin oxide, cobalt iron oxide, magnesium iron oxide, manganese iron oxide, and mixtures thereof.

Typical magnetic particles (e.g., iron oxide) are prepared by means that result in hydrophobic magnetic particles, although it will be appreciated that any magnetic particle can be made more or less hydrophobic by functionalizing the surface of the magnetic particle, applying a surfactant, or the like.

Referring to FIGURE 21C, in certain embodiments, a multi-phasic composite particle 50 is provided that includes a compartment 55. The compartment 55 typically holds a material in a liquid state at room temperature (25°C) and pressure (1 atm.). The compartment 55 is typically encompassed entirely within a single hemisphere 30 of the particle 50. The material that forms the compartment 55 is typically of a different hydrophobic-hydrophilic composition than the core 15. For example, if the core 15 is hydrophobic, the compartment 55 is hydrophilic. The compartment 55 is defined by a surfactant layer 57. An exemplary surfactant layer 57 is oleic acid when the compartment 55 is aqueous.

A preferred compartment material is water. Accordingly, aqueous solutions that include hydrophilic therapeutic compounds, DNA, RNA, proteins, and/or hydrophilic nanoparticles (e.g., luminescent quantum dots, and the like) can be disposed within the compartment 55. However, it will be appreciated that if the hydrophobic and hydrophilic regions of the composite particle are reversed, the hydrophilic compounds or particles would reside in the body 15 and any hydrophobic compounds or particles would reside in the compartment 55.

The addition of the compartment 55 is useful for storing compounds (e.g., therapeutics) or particles (e.g., quantum dots for imaging) that are of a different hydrophobic/hydrophilic composition that that of the core 15. Additionally, the compartment allows for separation of the functionalities of the components of the compartment 55 and the core 15. For example, MNPs are known to quench fluorescence, and therefore a composite particle with a fluorescent imaging agent intermixed with MNPs will result in a composite particle having a reduced or otherwise quenched fluorescence signal. However, by separating the imaging agent into, for example, the compartment 55, while the MNPs are in the core 15, the special separation reduces the quenching effect and allows for each component (MNPs and fluorescent imaging agent) to perform to its maximum possible ability.

Referring still to FIGURE 21C, the plurality of first nanoparticles 20 are nonuniformly distributed throughout the particle 50, typically in such a way that the composite nanoparticles 20 abut the compartment 55. As illustrated in FIGURE 21C, in certain embodiments, the compartment 55 and the plurality of first nanoparticles 20 are both disposed within a single hemisphere 30 of the particle 50. It will be appreciated, however, if sufficient first nanoparticles 20 are disposed within the particle 50, some first nanoparticles 20 may eventually be disposed within the second hemisphere 35.

In the embodiments illustrated in FIGURES 21A-21C, the various components of the composite particles can optionally be enhanced, or otherwise contain, functional surface layers. The embodiments described below with regard to FIGURE 2 ID are applicable equally to each of FIGURES 21 A-21C. Referring to FIGURE 21D, a composite particle 60 is illustrated having a plurality of first nanoparticles 75 disposed within a core 65. An exterior functionalized surface 70 is disposed surrounding the core 65. The surface 70 may be a surfactant compound, such as polyvinyl alcohol (PVA), which is an artifact of a particle 60 made from an emulsion process, as described elsewhere herein. The surface 70 may also be a functional surface that allows the particle 60 to preferentially bind to a target. In this regard, a potential target is a cell, such as a cancer cell. The functionalized surface 70 can be bound with a binding moiety that would preferentially bind to a protein or other structure located on a surface of the target cell. Binding between a particle 60 and a target cell would occur if the two objects were placed in close proximity. Binding a particle 60 to a target cell is advantageous so as to allow for imaging purposes, magnetolytic therapy, or hypothermic therapy to be applied to the cell, in accordance with the embodiments provided herein. It will be appreciated that any binding or immobilization techniques known to those of skill in the art are contemplated in the embodiments provided herein (e.g., affinity binding, antibody- antigen binding, etc.). The primary goal of such binding or immobilization techniques is to place a composite particle 60 into close proximity with a target so as to allow for imaging, hyperthermic therapy, and/or magnetolytic therapy.

Referring still to FIGURE 21D, the first nanoparticles 75 may also include a functionalized surface 85 surrounding a core 80. The functionalized surface 85 may be useful, for example, to allow the core 80 to become soluble in a particular solution during synthesis of the composite particle 60, or for other reasons known to those of skill in the art. For example, hydrophobic quantum dots can be made hydrophilic through a PEG coating.

In representative embodiments, as described elsewhere herein, the composite particle is formed using emulsion techniques. It will be appreciated, however, that non-emulsion techniques may also be used to form the composite particle.

Representative Methods For Making the Composite Particles

Representative methods for making the composite particles provided herein include emulsion-based techniques. Such techniques are generally known to those of skill in the art.

In a "single-emulsion" technique, as illustrated in FIGURE 1A, a hydrophobic

("oil" or "organic") phase is provided that includes a plurality of first nanoparticles and the host polymer. A second phase, that is hydrophilic ("water" or "aqueous"), includes a surfactant, such as PVA. The two solutions are then emulsified, for example by ultrasonic emulsification, and then the hydrophobic phase is evaporated to provide a solution of composite particles in an aqueous phase. The composite particles include a core and a plurality of the first nanoparticles nonuniformly disposed therein. The core of the composite particle is comprised of the host polymer starting material from the hydrophobic phase, and the surfactant from the hydrophilic phase (e.g., PVA) defines the surface of the composite particle. The solution surrounding the plurality of composite particles illustrated in FIGURE 1A is the hydrophilic phase solvent.

A "double emulsion" technique can be utilized to form composite particles, such as those described with reference to FIGURE 21C, that include a compartment. Referring to FIGURE 5A, the first step of the emulsion is to form an emulsion that includes a hydrophilic phase formed as particles within form an emulsion of the hydrophilic phase material while the hydrophobic phase includes the host polymer and plurality of first nanoparticles, as well as a surfactant. The hydrophilic emulsion is then combined with a second hydrophilic phase and the combined solutions are emulsified to provide a "double emulsion" that results in the original hydrophilic phase becoming a compartment within a composite particle having a core comprising the contents of the original hydrophobic phase. The composite particles are then an emulsion within a hydrophilic solution.

The product of the double emulsion technique described with reference to FIGURE 5 A is illustrated in FIGURE 5B. Using the exemplary water and chloroform phases described in FIGURE 5A, the formed composite particle includes an outer surfactant shell (PVA), a core formed from the host polymer, polystyrene-allyl alcohol copolymer, and a water phase compartment having an oleic acid surfactant defining the compartment. If the plurality of first nanoparticles is hydrophobic iron oxide, they are disposed within the host polymer core and not within the water-phase compartment.

With the double emulsion technique, flexibility is added to form composite particles having a variety of compounds and/or structures included therein. For example, referring to FIGURE 6, nanorods, such as hydrophilic gold nanorods, disposed within the hydrophilic phase at the beginning of the double emulsion process, will then be contained within the compartment of the completed double emulsion composite particles. Similarly, chemical compounds can be integrated into the composite particles by adding the compounds to either the initial hydrophilic phase or the initial hydrophobic phase. Compounds added to the hydrophilic phase will eventually become incorporated into the compartment of the composite particle, and hydrophobic compounds will be integrated into the core of the completed composite particle. FIGURES 7A-7G are micrographs of gold nanorods, nano bowls, and composite particles having nanorods embedded therein, in accordance with the embodiments provided herein, particularly with regard to the emulsion technique illustrated in FIGURE 6.

Referring to FIGURE 8, a variety of potential strategies for incorporating multiple modalities into a composite particle is illustrated. Hydrophilic compounds can be incorporated into the composite particle and carried within the compartment. Exemplary hydrophilic compounds include therapeutic compounds, such as the chemotherapy drug doxorubicin (referred to herein as "doxo"), and imaging compounds, such as the luminescent dye fluorescein isothiocyanate ("FITC"). In certain embodiments, the plurality of first nanoparticles is entirely contained within the core. Similarly, hydrophobic second nanoparticles can be incorporated into the body of the composite particle, for example, typical quantum dots are hydrophobic in character and do not need to be further functionalized to be integrated into the core. Additionally, hydrophobic molecules, such as pyrene (a luminophore) contained within the original hydrophobic phase, will then be integrated into the core of the finished composite particle.

A plurality of third nanoparticles, having hydrophilic character, such as poly(ethylene glycol) ("PEG")-fiinctionalized quantum dots or hydrophilic compounds (e.g., therapeutic or imaging compounds), can be incorporated into the compartment.

FIGURES 9-1 IB illustrate various experimental results of the variants of the composite particles illustrated in FIGURE 8. FIGURE 9 is a photoluminescence micrograph of composite particles having the fluorescent dye FITC embedded therein, in accordance with an exemplary embodiment. FIGURE 10A is a photoluminescence micrograph of composite particles with compartments, wherein luminescent quantum dots are embedded within the body of the particle, in accordance with an exemplary embodiment. FIGURE 10B is an intensity spectrum obtained from the micrograph of FIGURE 10A, which positively identifies the quantum dots. FIGURE 11A is a photoluminescence micrograph of composite particles with compartments, wherein luminescent molecule pyrene is embedded within the body of the particle, in accordance with an exemplary embodiment. FIGURE 1 IB is an intensity spectrum obtained from the micrograph of FIGURE 11 A, which positively identifies the embedded pyrene.

The encapsulation efficiency ("EE") of the double-emulsion method quantitatively defines what percentage of the material to be encapsulated (e.g., therapeutic) is actually incorporated into formed composite particles. The EE can be calculated according to Equation 1.

Encapsulation Efficiency (EE) = (A-B)/A*100 (1) Wherein:

A. Total amount of compound

B. The amount of compound remaining in supernatant

To determine the value for A, the amount of the compound in the initial solution, prior to creation of the composite particles, is measured. Referring to FIGURE 12, an exemplary embodiment of this is the measurement of doxo in a solution ("doxo control") is graphed. The composite particles are then formed by an emulsion and the composite particles are filtered from the solution (e.g., by centrifugation or other methods). The supernatant is removed and analyzed to determine the remaining concentration of the compound of interest to determine B for Equation 1. Referring again to FIGURE 12, the "B-NP60" spectrum is the supernatant signal after representative composite particles, which have doxo incorporated into a hydrophilic compartment, are formed and removed.

As used herein, the designation B-NPXX refers to a composite particle formed using poly(styrene-co-allyl alcohol) host polymer and iron oxide as MNPs, wherein the iron oxide, as a starting material, is XX of the weight of amount of host polymer starting material. Accordingly, B-NP60 has 60 parts MNP to 100 parts host polymer, by weight, as starting components when synthesizing the composite particles. Referring again to Equation 1, and the data obtained from FIGURE 12, A is determined to be 9.1 and B is determined to be 4.3, which leads to an encapsulation efficiency of 52.7%. In other words, the forming composite particles encapsulated over 50% of the doxo from the original hydrophilic phase used to form the composite particles.

Similar analyses of the encapsulation efficiency were performed for several different hydrophilic and hydrophobic molecules, each one being tested in a variety of different compositions of composite particles.

The hydrophilic molecules (or particles) doxo, FITC, and PEG-QD600 are analyzed for encapsulation efficiency in FIGURES 13F, 13G, and 13H, respectively, based on the luminescence data provided in FIGURES 13 A, 13B, and 13C, respectively. The hydrophilic molecules (or particles) are each contained within a hydrophilic compartment of a composite particle.

Similarly, hydrophobic molecules (or particles), such as QD600 and pyrene, can be integrated into the composite particles. These hydrophobic compounds (or particles) are contained within the core/host polymer, as opposed to within the compartment. The encapsulation efficiencies of QD600 and pyrene are graphically illustrated in FIGURES 131 and 13 J, respectively, based on luminescence data illustrated in FIGURES 13D and 13E, respectively.

In addition to the first nanoparticles, additional materials can be incorporated into the composite particles. The additional materials are typically functional materials that provide a function, such as allowing the particles to be imaged (e.g., fluorescent functional materials). Another representative functional material is a therapeutic. Multiple functional materials can be incorporated into the particles. In the embodiments where a compartment is included in the particles, both hydrophobic and hydrophilic functional materials can be incorporated into the particles, as the compartment and the core are always formed from materials having opposite hydrophobic/hydrophilic character. By integrating therapeutics, such as doxo, or other nanoparticles, such as quantum dots, into the composite particles, a delivery system is formed wherein the composite particles can be disintegrated or otherwise compromised at a targeted location so as to deliver the contained compound or particle. The particles can be disintegrated through biodegradation, or triggered release, such as by heating the particle. Heating the particle can be accomplished in a number of ways.

Light can be used to heat the particle. For example, metallic nanorods can be embedded in the core (or compartment) of the particle. Metal nanorods will have an absorption wavelength defined by the size of the nanorod. Excitation at the absorption wavelength will heat the nanorod, and, therefore, will heat the particle. Sufficient heat will dissolve or otherwise compromise the particle.

Heat can also be generated by placing the particle in a high frequency (e.g., kHz or greater) oscillating magnetic field if embedded nanoparticles are magnetic. The magnetic field causes the magnetic nanoparticles to heat, and the heat can then disintegrate the composite particle.

As an example of targeted delivery of therapy using the composite particles, the chemotherapy drug doxo can be targeted and delivered to a cancel cell by functionalizing the surface of a composite particle that includes doxo in the compartment. The composite particle can be directed to and will bind to the cancer cell and decomposition of the composite particle by the body will release the doxo so as to provide specific targeted delivery of the therapeutic agent to precisely the location within the body where it is needed to perform. Referring to FIGURES 14 and 15, simulated biodegradation in buffer, and the resulting cumulative release of doxo (FIGURE 14) and hydrophilic quantum dots (FIGURE 15) in the body are graphically illustrated. It can be seen from the data in FIGURES 14 and 15 that after a matter of two days, up to 60% of a therapeutic, such as doxo, can be delivered to a subject using the composite particles provided herein. FIGURE 16 is micrographs of representative composite particles formed using the amphiphilic copolymer poly(styrene-co-allyl alcohol), in accordance with the embodiments provided herein. While the PS/PAA composition for the host polymer has been described elsewhere herein, it will be appreciated that any amphiphilic polymer can be used to form the composite particles. FIGURE 17 is micrographs of representative composite particles formed using the amphiphilic copolymer poly(styrene-co-acrylic acid), in accordance with the embodiments provided herein. FIGURE 18 is micrographs of representative composite particles formed using the amphiphilic copolymer poly(styrene-co-polyethylene glycol), in accordance with the embodiments provided herein. FIGURE 19 is micrographs of representative composite particles formed using the amphiphilic copolymer poly(styrene-co-maleic anhydride), in accordance with the embodiments provided herein.

Imaging Applications Using the Composite Particles

In the particular embodiments wherein the plurality of first nanoparticles are magneto-nanoparticles, imaging applications of the composite particles provide for enhanced imaging of biological samples. One embodiment of the imaging method is illustrated as a flow-chart in FIGURE 22.

As an exemplary embodiment, a cell will be used to describe the techniques below, although it will be appreciated that the description is equally applicable to embodiments used on targets besides cells, such as viruses and bacteria.

As an initial step, the composite particles are directed to a target area for imaging, such as a tumor or other biological structure. The targeting can be accomplished using functionalized binding agents on the surface of the composite particles, or other means known to those of skill in the art. The composite particle can be attached to the target, or can be internalized within the target. For example, if the target is a cell, the composite particle can be internalized through techniques known to those of skill in the art, such as endocytosis. When the composite particles are in close contact with the target, as illustrated in FIGURE 20, a sample volume 101 will include the target 105 and a plurality of composite particles 110. Imaging the sample 100, for example, using a photoluminescent microscope 115, will typically provide a very weak signal from luminescent materials contained within the composite particles 110. This weak signal results from the large background typically encountered in biological samples such that the single-to-noise ratio of photoluminescent imaging is very low and therefore only faint signal attributable to the luminescent particles is detectable.

The improvement of the image begins by first applying a magnet 120 to the sample 100 so as to orient the composite particles 110 as a result of the nonuniform distribution of the magnetic nanoparticles within the composite particles 110. An image is then acquired.

Next, an opposite magnetic field is applied using either a spinning, oscillating, or physically moving magnet 120 that then reorients the composite particles 110 into a different orientation than the first orientation. An image is then acquired.

Through image processing, the first image and the second image can be compared, and the different luminescent intensities of the composite particles 110 in the first and the second images can be used to isolate and pinpoint the location of the composite particles and therefore the location of the target structure 105. Such image processing techniques are known to those of skill in the art and will not be described further herein.

Still referring to FIGURE 20, further oscillations of the magnet and imaging processing may improve the signal-to-noise ratio and the final image.

In a related embodiment for improving image quality, if a magnetic field is applied of sufficient strength that the composite particles 110 translate (i.e., move) a small amount while still adjacent or bound to the target 105, the movement of the composite particles 110 can be detected using image processing software. In such imaging processing, only the moving components of a series of images is allowed to remain in the final processed image. Because the composite particles 110 are the only aspect of the images that are moving (due to translation within the oscillating magnetic field), the composite particles 110 are the only aspect of the series of images that passes through the image processing and therefore the processed image reveals only the location 135 of the composite particles in the finished image 140.

It will be appreciate that in embodiments where translation, as opposed to rotation, of the composite particles is utilized for imaging, the MNPs need not be non- uniformly dispersed in the core. The MNPs can be uniformly dispersed in the core because the composite particle need only be paramagnetic such that it moves linearly (e.g., magnetophoreses) in a magnetic field, as opposed to the rotational movement induced by an asymmetric (non-uniform) MNP distribution. However, multi-phasic composite particles can still be used in these embodiments because it may be useful, for example, to separate the MNPs and an imaging agent. For example, the MNPs may be uniformly dispersed within the core and an imaging agent dispersed within a compartment. By separating these two components, there is a reduced interaction, and less potential for quenching of the imaging agent by the MNPs.

Exemplary Experimental Embodiments

General Experimental Conditions.

Materials and instruments. Unless specified, chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Organic soluble iron oxide nanoparticles of 5 nm and 15 nm are from Oceannanotech LLC (Springdale, AR). Particles of both sizes are coated with oleic acids and their compositions are maghemite and magnetite, respectively. Detailed particle characterizations are available from the company's website. TEM images were obtained on a FEI Tecnai G2 F20 STwin TEM (Hillsboro, OR). For TEM analysis, nanoparticles were dried on 200 mesh copper grids with carbon support film. Dynamic Light Scattering (DLS) were used for characterization of particle sizes in solution (Malvern, UK). A fluorometer (Fluoromax4, Horiba Jobin Yvon, Edison, NJ) was used to characterize the emission spectra of the composite particles.

Synthesis of representative composite particles incorporating MNPs. PS16-b-PAA10 (20 mg) was first dissolved in 500 μΐ chloroform followed by addition of MNPs of various quantities. (Table 1). To prepare the aqueous phase for microemulsion, 40 mg of PVA (MW 9,000-10,000) as a polymer stabilizer was dissolved in 2 ml of D.I. water at 70°C. After PVA completely dissolved, the clear solution was cooled to room temperature. The organic phase was added to the PVA solution and emulsified for several minutes by pulsed sonication (100 watts and 22.5 kHz, MISONZX ultrasonic liquid processors, XL-2000 series). The emulsion was then stirred at room temperature for 24 hours to evaporate the organic solvent. The resulting nanocomposites were washed with D.I. water three times to remove excess PVA, unincorporated MNPs and polymers, and some ultrasmall particles. For the magneto-optical nanocomposites, the same experiment procedure was used except that a fluorescent dye, pyrene (2 mg), was added to the organic phase. The formed composite particles have a core of PS/PAA and first nanoparticles that are iron oxide MNPs. There is no compartment.

Modulation of the nanocomposites with magnetic fields. 15 nm MNPs were incorporated in the nanocomposites for quick response to magnetic fields. The magneto-optical nanocomposites were dispersed in water and spread on glass slides. A small magnet was placed above or below the glass slides to orient the MNP phase in the nanocomposites facing up or facing down. True-color fluorescence images were immediately obtained on an inverted fluorescence microscope (IX-71, Olympus) before the nanoparticles rotate into random orientations.

Cell imaging and magnetolytic cell therapy. 200 μL· of 1% nanocomposites were added to prostate tumor LNCaP cells. A magnet was placed under the cell culture dishes for three minutes to pull the nanocomposites onto cell surfaces. Free particles (unbound) were washed away. The cell culture plates were then placed in a spinning magnetic field of 50 rpm for 15 minutes. Dead cells were identified by trypan blue staining to assess cell viability.

Exemplary results. To incorporate the functionality of responsiveness to environmental stimuli, MNPs are used as one of the building blocks for remote modulation with magnetic fields. As shown in FIGURE 1A, hydrophobic MNPs (5nm) and amphiphilic block copolymer, poly(styrene-block-allyl alcohol) (PS^-b-PAA_jQ,

M.W. 2,200), are mixed in an organic solvent (chloroform) at various ratios; whereas polyvinyl alcohol (PVA, M.W. 9,000) is dissolved in water. A fluorescent dye, pyrene, is used to label the polymer phase for optical detection. PVA was selected over common small-molecule surfactants because of not only its emulsifying capability but also its outstanding biocompatibility, which is important for applications oriented towards biology and medicine. Note that due to the broad availability of NPs, polymers and fluorophores, the composition of the resulting biphasic nanocomposites is highly flexible. The combination of MNPs and pyrene labeled PS-b-PAA merely represents a proof-of-concept. Ultrasonic emulsification is used to emulsify the solution mixture, followed by slow evaporation of the organic phase, leading to formation of compact and uniform nanocomposites.

FIGURE IB shows the transmission electron microscopy (TEM) images of the nanocomposites. The particles are well dispersed and uniform with average diameter of approximately 180 nm. Some small particles are also observed under TEM, which can be readily removed by size- selective centrifugation. Regardless of the size, the volume ratios of the embedded MNPs and polymers appear to be identical. This is because the ratio is determined by the initial concentrations of MNPs and polymers in the organic phase. During emulsification, the amounts of MNPs and polymers distribute into small droplets proportional to the droplet size. TEM images of higher magnification (FIGURE 1C) clearly reveal the biphasic structure with MNPs assembled on one side and polymers on the other, resulted from evaporation-induced phase separation between the two building blocks. Note that the MNPs used in the current exemplary embodiment are superparamagnetic and do not aggregate spontaneously before solvent evaporation. When their concentration reaches saturation value, they agglomerate driven by van der Waals interaction. Under current experimental conditions, MNPs precipitate out first as the organic phase slowly dries because their solubility is significantly lower than that of the polymers (FIGURE 4). The composites phase separate rather than form core-shell structure, which results from a preference to minimize interfacial tensions.

The uniformity of the resulting biphasic nanocomposites is also confirmed by dynamic light scattering (DLS) measurements. FIGURE ID displays a hydrodynamic diameter of 190 nm for the same batch of particles, confirming excellent colloidal dispersity in aqueous solution without the need of other surfactants, stabilizers, or chemical modifications to the surface.

To probe the composition tunability of this technology the quantity of MNPs in the initial stock solutions was varied while keeping the polymer concentration constant. TEM images in FIGURES IE- IN reveal that the increasing concentrations of MNPs not only produce larger MNP phases in the composites but also increase the overall particle sizes. As the weight ratio of MNP/polymer increases from 3.7 to 60%, the size of the composite particles increases from -120 nm to 220 nm, as shown in Table 1.

Table 1. Reagents used for synthesis of the biphasic nanocomposites, and the averages particle hydrodynamic sizes.

When PVA is used as the stabilizer for emulsion of polymer/toluene solutions in water, the surface of the solidified particles has a dimple due to evaporation of a small amount of organic solvent initially trapped inside the beads.

Using the new multifunctional nanocomposites, we investigated their responses to external stimuli, which will serve as the basis for potential applications in medicine and electronics. For example, responses to spinning magnetic fields and the resulting mechanical forces could become an effective approach for cancer treatment and solve the size problem encountered by magnetic-vortex microdiscs, which are too large (i.e., micron- sized) to fit into capillaries. Similarly, quick response to magnetic fields is also desirable to fabricate smart displays. To magnetically modulate the orientation, the magneto-optical nanocomposites were placed between two glass coverslips for optical imaging and microscopy. As schematically illustrated in FIGURE 2A, a magnet was first placed immediately below the sample to make the MNP phase facing down. When placed onto the stage of an inverted microscope where both incident light and fluorescence travel through objectives on the bottom, fluorescence is significantly reduced due to blockage of the excitation and emission. Next, when the magnet is placed on top of the coverslips to switch the orientation of the magneto-optical biphasic nanocomposites, fluorescence is observed because neither excitation nor emission is blocked. For efficient light blocking, we prepared nanocomposites with two phases of approximately the same volume (hemispheres, FIGURE 2B).

FIGURES 2C and 2D show remarkable difference in fluorescence intensity with or without MNP blocking. The quenching effect is likely dominated by attenuation of both excitation light (350+20 nm) and emitted fluorescence (>420 nm) in the light path since MNPs are stronger absorbers in the UV and visible spectra. For small amount of pyrene molecules partitioned into the MNP phase or located at the boundary of the two phases, additional quenching effect such as energy transfer through dipole coupling is also possible. Regardless of the quenching mechanisms, the imaging results clearly demonstrated the responsiveness of the biphasic nanocomposites to external magnetic fields and tunable fluorescence depending on the directions of incident and emitted lights. This magnetic field modulated fluorescence is unique to the nanocomposites with spatially separated functionalities. In contrast, composites with fluorophores and MNPs mixed homogeneously should remain in the low fluorescence state at all times. It's also noteworthy that despite the efficient response to magnetic fields, the nanocomposites do not agglomerate in solution in the absence of magnetic fields, which is essential for biomedical uses. They remain single in solution because the magnetic compartment is not composed of a large ferromagnet but an ensemble of small superparamagnetic NPs whose magnetic moments average to zero collectively.

Therapy Using the Composite Particles

In accordance with certain embodiment of the present disclosure, a target, such as a cell, virus, or bacteria, can be damaged or killed. As an exemplary embodiment, a cell will be used to describe the techniques below, although it will be appreciated that the description is equally applicable to embodiments used on targets besides cells.

In one embodiment, a plurality of magnetic composite particles are disposed adjacent to or within the target, using the techniques described above with regard to imaging modalities.

In certain embodiments, the magnetic particles are the composite particles disclosed herein having a plurality of magnetic nanoparticles non-uniformly dispersed in a host core. However, such an asymmetric MNP distribution is not necessary for the therapies described herein. The asymmetric MNP distribution is only essential if the composite particles must be rotated for imaging. The high- and low-frequency therapies described herein need not incorporate imaging modalities.

FIGURE 23 illustrates one embodiment of magnetic therapies possible. The frequency of the applied magnetic field, as well as the contents of the composite particle will define the type of therapy. Low frequency oscillation will provide magnetolytic therapy. High frequency oscillation will heat the MNPs within the composite particle to provide hyperthermic therapy and harm the target through localized heating. The Hyperthermic therapy may also compromise and dissolve the composite particle to release any therapeutic stored therein (e.g., an anti-cancer therapeutic).

At low frequencies (i.e., 1-999 Hz) of magnetic field oscillation or spinning, magnetolytic therapy can be achieved to damage a target, such as a cell membrane damage through mechanical force. As schematically illustrated in FIGURE 3A, the nanocomposites can be quickly attached to cell surface using a magnetic field for both imaging and treatment. Fluorescence imaging confirms that the nanocomposites can coat on cell surface at high density within 3 minutes (FIGURE 3B). In one embodiment, the cell-particle attachment is achieved through non-specific adsorption facilitated by magnetic manipulation; however, in other embodiments, specific targeting of a certain type of cells can be accomplished by linking the nanocomposites with targeting ligands.

As illustrated in FIGURE 3A, magnet therapies can be effected in through multiple modalities using the composite particles provided herein.

First, a low frequency, 1-999 Hz, spinning or oscillating magnetic field can be applied to the composite particles. A low-frequency field will cause the particles to repeatedly impact (and retract from) the cell, thereby mechanically damaging, and eventually killing, the cell.

Second, a high frequency, kHz-MHz, magnetic field can be applied to the composite particles. The high-frequency field will cause the MNPs to heat to provide hyperthermic therapy. The heat generated by the MNPs in the magnetic field may be sufficient to kill target cells. Additionally, the composite particle can be heated by the MNPs in the magnetic field to a temperature sufficient to melt/rupture/or otherwise compromise the composite particle. If the composite particle comprises a therapeutic agent in the core and/or compartment, the therapeutic will be released and exposed to the cell. An appropriate therapeutic agent will kill the cell.

In an exemplary embodiment of low-frequency magnetic field magnetolytic therapy, cells are placed in a spinning magnetic field (50 rpm), which creates a mechanical force on cell membranes. FIGURES 3C-3F illustrate the results of magnetolytic therapy (FIGURE 3F) and control samples (FIGURES 3C-3E) on tumor cells. The sample of FIGURE 3C has no magnetic composite particles and no magnetic field applied. The sample of FIGURE 3D has no magnetic composite particles and a magnetic field applied. The sample of FIGURE 3E has magnetic composite particles and no magnetic field applied. The sample of FIGURE 3F has magnetic composite particles and magnetic field applied (i.e., magnetolytic therapy is effected). In comparison with the controls, missing either magnetic fields or nanocomposites, the viability of cells exposed to the composite particles and the magnetic field is significantly reduced. Note the low cell density in the treatment group FIGURE 3F resulted from detaching of dead cells from cell culture dishes. The number of live cells in FIGURE 3F compared with that in FIGURE CF is reduced by 77%, whereas those in FIGURE 3D and FIGURE 3E show greater than 99.5% cell survival. Accordingly, the success of magnetolytic therapy is demonstrated.

FIGURE 3F shows that after a 15-minute exposure to the spinning magnetic field, majority of the tumor cells were killed, identified by Trypan blue staining. In contrast, control experiments, where either the nanocomposites or the magnetic fields are missing, show virtually no effect on cell viability. This magnetic induction specificity provides important advantages for image-guided in vivo therapy, because NP-based therapeutics regardless of surface coating (non-fouling polymers such as polyethylene glycol or dextran) and targeting ligands (e.g., antibody and peptide), always show some degree of nonspecific uptake by the reticulo-endothelial systems. Image guided local magnetic field induction at the diseased sites could help reduce therapeutic side effects.

In one embodiment, the disclosure includes a simple, versatile, and scalable synthetic approach based on microemulsion, free of small-molecule surfactants, and controlled phase separation. A magneto-optical combination by incorporating fluorophores and MNPs in separate compartments is used to demonstrate the concept. The resulting nanocomposites share virtually all the desirable properties with the conventional Janus microspheres, but simultaneously being compact in size. Accordingly, in accordance with aspects of the disclosure, compact nanocomposites with spatially separated functionalities, uniform size, tunable composition, and efficient response to stimuli for applications in biological research are presented herein. We further demonstrate magnetic field modulated imaging and novel application of this technology in cancer cell therapeutics based on magnetically controlled mechanical forces, referred to herein as magnetolytic therapy.

In one embodiment, the disclosure is directed to a method of making and composite material having spatially separated compartments. The method includes mixing a first material and a second material in an organic solvent to form a composite mixture. The method also includes adding an amphiphilic polymer in water to the composite mixture to form a solvent mixture. In some embodiments the amphiphilic polymer is biocompatible. In a specific example, the amphiphilic polymer is polyvinyl alcohol. Following the adding step, the method includes ultrasonically emulsifying the solvent mixture and evaporating an organic phase, whereby the method provides formation of a compact and uniform-sized composite material having a first compartment including the first material and second material including the second material.

In another embodiment, the disclosure is directed to a microemulsion process. The microemulsion process includes adding a first composition mixed in an organic solvent to an amphiphilic polymer in water to form a pre-emulsion mixture. The method also includes ultrasonically emulsifying the pre-emulsion mixture to form an emulsion and slowly evaporating an organic phase from the emulsion to form a composite material. Optionally, the microemulsion process can further include, before the emulsifying step, adding a second composition. In such embodiments, the first composition can be separated spatially from the second composition, or in other embodiments, the first composition can be spatially intermixed with the second composition. The method can also include, optionally, linking targeting ligands onto a surface of the composite material. The first and/or second compositions or materials described herein can be, but are not limited to, nanoparticles, magnetic nanoparticles, polymers, pharmaceutical compositions having one or more drugs, etc. Accordingly, the disclosure is also directed to nanoparticles, drug nanoparticles, magnetic nanoparticles, drug cocktail materials, polymer materials, pharmaceutical preparations and formulations and the like, including formulations and compositions suitable for delivery in vivo for therapeutic treatment, as well as for use in in vitro.

In a further embodiment, the disclosure is directed to a method of making a composite nanoparticle having spatially separated functionalities. The method includes mixing hydrophobic magnetic nanoparticles (MNP) and polymer in an organic solvent to form an MNP mixture. The method also includes adding polyvinyl alcohol (PVA) in water to the MNP mixture to form a solvent mixture. The method further includes ultrasonically emulsifying the solvent mixture and evaporating an organic phase.

Other aspects of the disclosure are directed to methods for imaging cells or other particles. In one embodiment, a method for imaging cells includes providing a plurality of magnetic composite nanoparticles (e.g., such as MNP composite nanoparticles formed by the process described herein), wherein the individual composite nanoparticles include a fluorophore. The method also includes adsorbing the nanoparticles onto cells, and exposing the cells to a magnet in a first orientation. The method further includes exposing the cells to a magnet in a second orientation and imaging the cells in the second orientation at a wavelength suitable to excite the fluorophore.

Further aspects of the disclosure are directed to methods for providing magnetolytic therapy. In one embodiment, the method can include providing a plurality of magnetic composite nanoparticles (e.g., such as MNP composite nanoparticles formed by the process described herein), and adsorbing the nanoparticles onto living cells. The method also includes exposing the cell to a spinning or oscillating magnetic field, whereby exposing the cells causes the cells to die. For example, the spinning or oscillating magnetic field can cause mechanical disruption of cell membranes. While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composite particle comprising a core formed from a host polymer material and a plurality of first nanoparticles non-uniformly dispersed in the core, wherein the composite particle has a diameter of from 100 nm to 500 nm; and wherein each of the plurality of first nanoparticles has a diameter of from 1 nm to

60 nm.

2. The composite particle of Claim 1, wherein the host polymer is a hydrophobic polymer.

3. The composite particle of Claim 1, wherein the first nanoparticles are paramagnetic.

4. The composite particle of Claim 1, wherein the first nanoparticles are iron oxide.

5. The composite particle of Claim 1, wherein the first nanoparticles are immiscible in the host polymer.

6. The composite particle of Claim 1, wherein the first nanoparticles comprise a functionalized surface.

6. The composite particle of Claim 1 further comprising a surfactant layer surrounding the core.

7. The composite particle of Claim 1, wherein the surfactant layer is biocompatible.

8. The composite particle of Claim 1 further comprising a functional layer surrounding the core.

9. The composite particle of Claim 1, wherein the functional layer is capable of binding to a target.

10. The composite particle of Claim 9, wherein the target is selected from the group consisting of cells, bacteria, and viruses.

11. The composite particle of Claim 1, wherein the core comprises at least one second nanoparticle, wherein said second nanoparticle is different than the first nanoparticle.

12. The composite particle of Claim 11, wherein the second nanoparticle is selected from the group consisting of quantum dots, metal nanoparticles, and magnetic nanoparticles.

13. The composite particle of Claim 1 further comprising a therapeutic agent.

14. The composite particle of Claim 1 further comprising an imaging agent.

15. The composite particle of any one of Claims 1-14 further comprising a compartment within the core, wherein said compartment is disposed at a surface of the composite particle and disposed asymmetrically in contact with the core.

16. The composite particle of Claim 15, wherein the compartment comprises a compartment surfactant layer surrounding a compartment core, wherein said compartment surfactant layer segregates the compartment from the host polymer material.

17. The composite particle of Claim 15, wherein the compartment comprises a polar material and the host polymer comprises a non-polar material.

18. The composite particle of Claim 15, wherein the compartment does not contain any first nanoparticles.

19. The composite particle of Claim 15, wherein the compartment comprises at least one third nanoparticle, wherein said third nanoparticle is different than the first nanoparticle.

20. The composite particle of Claim 19, wherein the third nanoparticle is selected from the group consisting of metal nanoparticles, quantum dots, and magnetic nanoparticles.

21. The composite particle of Claim 15, wherein the compartment comprises a therapeutic agent.

22. The composite particle of Claim 15, wherein the compartment comprises an imaging agent.

23. The composite particle of Claim 15, wherein the compartment is in a liquid state and the host polymer is in a solid state at room temperature and pressure.

24. The composite particle of Claim 15, wherein the entire plurality of first nanoparticles are disposed adjacent to an interior surface of the compartment.

25. The composite particle of Claim 1, wherein the host polymer is an amphiphilic polymer.

26. A method for imaging a target, comprising the steps of:

immobilizing a plurality of composite particles according to any of Claims 1-25, adjacent to the target, wherein the composite particles comprises a luminophore and wherein the first nanoparticles are paramagnetic nanoparticles;

aligning the composite particles with a magnetic field in a first direction to provide the plurality of composite particles in a first orientation; imaging the cells in the first orientation at a wavelength suitable to excite the luminophore to provide a first signal;

27. The method of Claim 26, wherein the target is selected from the group consisting of a cell, bacteria, and a virus.

28. The method of any one of Claims 26 and 27, wherein immobilizing the plurality of composite particles to the target comprises binding the particles to the target.

29. The method of any one of Claims 26-28 wherein the composite particles translate location as a result of the difference between the first direction of the magnetic field and the second direction of the magnetic field, and wherein said translation is used to determine the position of at least a portion of the plurality of composite particles.

30. A method for damaging a target using one or more composite particles according to any one of Claims 1-25, wherein the first nanoparticles are paramagnetic nanoparticles, the method comprising the steps of:

disposing the composite particles adjacent or inside the target; and

exposing the composite particles to a spinning or oscillating magnetic field of sufficient strength to cause the composite particles to impact the target.

31. The method of Claim 30, wherein disposing the composite particles adjacent or inside the target comprises adsorbing the composite particle to the target. .

32. The method of Claim 30, wherein the composite particles comprise a targeting ligand, and wherein the adsorbing step includes specifically adsorbing the composite particle to a target having ligand receptors recognizing the targeting ligand.

33. The method of Claim 30, wherein the magnetic field spins or oscillates at a rate of 1 Hz to 999 Hz.

34. The method of Claim 30, wherein the magnetic field oscillates at a frequency sufficient to heat the magnetic nanoparticles, wherein the magnetic field oscillates at a frequency of 1 kHz or greater.

35. The method of Claim 34, wherein said heat is sufficient to kill the living cell.

36. The method of Claim 34, wherein said heat is sufficient to degrade the composite particle, wherein the composite particle comprises a therapeutic agent, and wherein degrading the composite particle releases the therapeutic agent.