WO2009154963A1

WO2009154963A1 - Composition for therapy and imaging of cancer and associated methods

Info

Publication number: WO2009154963A1
Application number: PCT/US2009/045235
Authority: WO
Inventors: Kimberly Homan; Stanislav Emelianov; Lisa Brannon-Peppas
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 2008-05-27
Filing date: 2009-05-27
Publication date: 2009-12-23

Abstract

Compositions and methods comprising an anti-cancer agent, exogenous dye, or other imaging agent disposed within a degradable matrix; a metal disposed around the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a cage; and a targeting moiety or stealthing agent or both.

Description

COMPOSITIONS FOR THERAPY AND IMAGING OF CANCER AND ASSOCIATED

METHODS

STATEMENT OF GOVERNMENT INTEREST

The present invention was made with support under Grant Number DGE-0333080 awarded by the National Science Foundation. The U.S. government has certain rights in the invention.

BACKGROUND

The present disclosure, according to certain embodiments, generally relates to compositions for imaging and drug delivery. In particular, the present disclosure provides, in certain embodiments, compositions useful in cancer imaging and therapy.

There is a definite and urgent need for targeted therapy and improved imaging techniques for cancers. Early detection, more accurate diagnosis, and effective therapies lie at the forefront of our battle with cancer. As with most chemotherapeutic and/or imaging regimens, treatment involves systemic intravenous injections. The concentrations of a therapeutic agent used per session is dose-limited by its cytotoxic effects to healthy tissue. Some hypothesize that this systemic approach to drug delivery is ineffective for most patients because drug concentrations accumulated within the tumor itself are not high enough to realize their estimated therapeutic potential. In response to this issue several solutions have surfaced. A few groups have studied the use of endoscopic ultrasound guided fine needle injections of immunotherapies or tumor necrosis factors with gemcitabine, a chemotherapeutic drug, directly into inoperable pancreatic tumors, for example. Other groups are employing different strategies altogether such as gene therapy for cancer. Yet another strategy involves encapsulation of a prodrugs or drugs in liposomes.

DRAWINGS

Some specific example embodiments of the disclosure may be understood by referring, in part, to the following description and the accompanying drawings.

Figure 1 shows a pictorial representation of an example structure of a composition of the present disclosure.

Figure 2 shows a plot of optical absorption of tissue constituents at different wavelengths of light.

Figure 3 shows a scanning electron micrograph of doxorubicin loaded PLGA nanoparticles (right). Figure 4 shows the release of doxorubicin from PLGA nanoparticles at varying pH and percent loadings of DOX.

Figure 5 shows a plot of viability of MDA-MB-231 breast cancer cells after exposure to doxorubicin-loaded particles (white), free doxorubicin (hatched), or blank nanoparticles (dotted) as a fraction of control cells in DPBS (black).

Figure 6 shows transmission and corresponding confocal microscopy images of MDA-MB-231 cells exposed to (A) DOX loaded PLGA particles and (B) free DOX at a concentration of 1.0 ug DOX /ml for 2 hrs. (C) Image along the width and depth of live cells exposed to DOX loaded particles. Figure 7 shows an image of gemcitabine loaded PLGA nanoparticles.

Figure 8 shows a reaction scheme for gemcitabine transformed to a prodrug by addition an 18 length carbon chain to its 4'-amino group.

Figure 9 shows an image of silica core particles coated with a silver cage.

Figure 10 shows a plot of the absorbance spectrum of the particles shown in Figure 9. Figure 11 shows a reaction scheme using DIC to transform the carboxyl end groups on PLGA to carboxamide end groups.

Figure 12 shows an image of silver-PLGA aggregates produced using the Tollen's reagent reduction method.

Figure 13 shows a photoreduction mechanism of silver ions in aqueous solution promoted by PVA.

Figure 14 shows a scanning electron micrograph of silver seeded PLGA nanospheres after using the photoreduction method depicted in Fig. 13.

Figure 15 shows silver seeded PLGA nanospheres (left) growing into varying morphologies of silver nanocages around the PLGA cores (right) after addition of ascorbic acid as the reducing agent.

Figure 16 shows the absorbance spectrum of silver nanocages surrounding PLGA cores.

Figure 17 shows cell viability of MDA-MB-231 breast cancer cells as determined by the MTT assay after exposure to the different morphologies of nanostructures shown at three separate DOX concentrations.

Figure 18 shows a composite light microscopy image comprised of two fluorescence and one phase contrast image of the same set of MDA-MB-231 breast cancer cells post exposure to doxorubicin loaded PLGA nanoparticles with a silver cage. (Blue - DAPI stained nuclei, Pink - DOX) The co-location of blue and pink resulting in a purple hue demonstrates that DOX was located in the nucleus where it intercalates with DNA and disrupts replication.

Figure 19 shows doxorubicin release from PLGA nanospheres and PLGA nanospheres with a pegylated silver cage. Figure 20 shows a diagram of the PAUS imaging system showing light and ultrasound delivery from the same spatial direction.

Figure 21 shows a block diagram of the PAUS imaging system showing an alternate light delivery path where light is perpendicular to ultrasound delivery.

Figure 22 shows poly(vinyl alcohol) molds housing different concentrations of silver- silica nanospheres.

Figure 23 is an imaging set-up showing the position of light and ultrasound delivery used to measure the photoacoustic signal from nanocages inside PVA molds.

Figure 24 shows a plot of photoacoustic signal divided by fluence versus nanocage concentration for the four samples shown in the inset. Inset: photoacoustic images of 0, 2-10⁷, 2-10⁸, 2-10⁹ particles per ml from left to right where the white circle outlines the boundaries of the phantom as determined by ultrasound.

Figure 25 shows ultrasound (left), photoacoustic (middle), and combined (right) images of nanocages injected directly into an ex- vivo canine pancreas. All images are 20 mm by 10.5 mm. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the figures and are described in more detail below. It should be understood, however, that the description of specific example embodiments is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as illustrated, in part, by the appended claims.

DESCRIPTION The present disclosure, according to certain embodiments, generally relates to compositions for imaging and drug delivery. In particular, the present disclosure provides, in certain embodiments, compositions useful in cancer imaging and therapy. In certain embodiments, the present disclosure provides a composition comprising a degradable matrix and a metal disposed around at least a portion of the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a cage.

In certain embodiments, the present disclosure provides a composition comprising an anti-cancer agent, exogenous dye, or other imaging agent disposed within a degradable matrix and a metal disposed around at least a portion of the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a cage.

In certain embodiments, the present disclosure provides a composition comprising an anti-cancer agent, exogenous dye, or other imaging agent disposed within a degradable matrix; a metal disposed around at least a portion of the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a cage; and a targeting moiety or stealthing agent or both.

In certain embodiments, the present disclosure provides a method comprising: providing a composition comprising: an anti-cancer agent, exogenous dye, or other imaging agent disposed within a degradable matrix; a metal disposed around the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a cage; and a targeting moiety; and introducing the composition into a subject.

In certain embodiments, the present disclosure provides a method comprising providing a composition comprising: an anti-cancer agent, exogenous dye, or other imaging agent disposed within a degradable matrix; a metal disposed around at least a portion of the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a cage; and a targeting moiety; introducing the composition into a subject; providing an imaging device; and obtaining an image of at least a portion of the subject.

Compositions of the Present Disclosure

The compositions of the present disclosure may confer a number of advantages, including, but not limited to, the ability, in certain embodiments, to concurrently treat a subject with a localized dose of an anti-cancer agent and obtain an image of a cancerous region to which the composition may be designed to target. In certain embodiments, delivering such a localized dose of an anti-cancer agent may reduce the side effects that result from systemic doses of anticancer agents. In certain embodiments, the delivery of a localized dose of an anti-cancer agent may allow the anti-cancer agent to more effectively reduce the size of a tumor, for example, to a resectable size.

In certain embodiments, the compositions of the present disclosure may be in the form of particles. While such particles, according to preferred embodiments, are substantially spherical, the particles may be of any suitable shape. Factors affecting the desired shape may include, but are not limited to, the desired delivery route and/or delivery site of the particles or the imaging contrast properties of the particles. In certain embodiments, such particles of the compositions of the present disclosure may have a largest dimension (for example, a diameter, in the embodiments in which the particles are substantially spherical) suitable for delivery to a desired site. In certain embodiments, such particles of the compositions of the present disclosure may have a largest dimension of about 5 nm to about 500 μm. In certain embodiments, such particles of the compositions of the present disclosure may have a largest dimension of less than about 5000 nm. In certain embodiments, such particles of the compositions of the present disclosure may have a largest dimension of less than about 500 nm. In certain embodiments, such particles of the compositions of the present disclosure may have a largest dimension of less than about 300 nm.

Any suitable anti-cancer agent may be used in the compositions and methods of the present disclosure. The selection of a suitable anti-cancer agent may depend upon, among other things, the type of cancer to be treated and the composition of the degradable matrix of the compositions of the present disclosure. In certain embodiments, the anti-cancer agent may be effective for treating one or more of pancreatic cancer, esophageal cancer, rectal cancer, colon cancer, prostate cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, and stomach cancer. In certain embodiments, the anti-cancer agent may be but is not limited to gemcitabine, doxorubicin, or paclitaxel. In certain embodiments, the anti-cancer may be a prodrug form of an anti-cancer agent. As used herein, the term "prodrug form" and its derivatives is used to refer to a drug that has been chemically modified to add and/or remove one or more substituents in such a manner that, upon introduction of the prodrug form into a subject, such a modification may be reversed by naturally occurring processes, thus reproducing the drug. The use of a prodrug form of an anti-cancer agent in the compositions and methods of the present disclosure, among other things, may increase the concentration of the anti-cancer agent in the compositions and methods of the present disclosure and/or decrease the solubility of the anti-cancer agent in an aqueous fluid. In certain embodiments, an anti-cancer agent may be chemically modified with an alkyl or acyl group or some form of lipid. The selection of such a chemical modification, including the substituent(s) to add and/or remove to create the prodrug, may depend upon a number of factors including, but not limited to, the particular drug and the desired properties of the prodrug. One of ordinary skill in the art, with the benefit of this disclosure, will recognize suitable chemical modifications. Any suitable exogenous dye or imaging contrast agent may be used in the compositions and methods of the present disclosure in addition to, or in place of, an anti-cancer agent. The selection of suitable agents depends on, among other things, the cancer to be treated and/or imaged, the composition of the biodegradable matrix of the compositions of the present disclosures, and which imaging modalities are chosen for multimodal detection and/or diagnosis. In certain embodiments, these other suitable agents may include, but are not limited to, exogenous dyes such as rhodamine or indocyanine green, and other suitable imaging contrast agents such as gadolinium-DTPA or small iron oxide nanoparticles.

The degradable matrices of the compositions of the present disclosure may comprise any material suitable for use in a desired environment which degrades over time when introduced into such an environment. The term "degradable matrix," as used herein, is used to refer to a degradable material in which the anti-cancer may be disposed and is not intended to imply any particular structure of properties of such a material. In certain embodiments, the degradable matrix may undergo hydrolytic degradation (i.e., the matrix may degrade when exposed to an aqueous fluid). In certain embodiments, the degradable matrix may be a polymer which undergoes hydrolytic degradation. Suitable degradable matrix materials include, but are not limited to, hydrolytically degradable polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), polyvinyl pyrollidone (PVP), and polycaprolactone (PCL). Combinations and/or derivatives of one or more materials (such as blends and copolymers, in the case of polymeric degradable matrices) may also be suitable for use as degradable matrices in the compositions and methods of the present disclosure. The term "derivative" includes any compound that is made from one of the listed compounds, for example, by replacing one atom in the listed compound with another atom or group of atoms, rearranging two or more atoms in the listed compound, ionizing one of the listed compounds, or creating a salt of one of the listed compounds. The term "derivative" also includes copolymers, terpolymers, and oligomers of the listed compound. The choice of a suitable material for the degradable matrix may depend upon, among other things, the desired degradation rate of the degradable matrix, the environment into which the degradable matrix is to be introduced, and the anti-cancer agent which is to be disposed within the degradable matrix. One of ordinary skill in the art, with the benefit of this disclosure, will recognize additional suitable materials for use as a degradable matrix. Such materials are considered to be within the spirit of the present disclosure.

The metals used in the compositions and methods of the present disclosure may be any metal suitable to be disposed around the exterior surface of the degradable matrix of the compositions and methods of the present disclosure in the form of a cage. The term "cage," as used herein, is defined to mean a porous structure, as distinguished from a continuous or solid coating of metal. In certain embodiments, such a cage structure may impart a number of benefits upon the compositions and methods of the present disclosure, including, but not limited to, allowing the anti-cancer agent to diffuse through the metal, allowing a surrounding medium to enter the degradable matrix, and/or allowing a surrounding medium to degrade the degradable matrix. In certain embodiments, the metal may be a metal suitable for imparting antibacterial properties to the compositions of the present disclosure. This metal may also enable or enhance imaging of the compositions of the present disclosure. In certain embodiments, the metal may be a metal suitable for imaging the compositions of the present disclosure by endoscopic photoacoustic and ultrasound imaging. In certain embodiments, the metal may be a metal suitable for imaging the compositions of the present disclosure by one or more of the following imaging techniques: radiographic X-ray imaging, computed tomography (CT), magnetic resonance imaging (MRI), optical imaging (such as optical coherence tomography (OCT)), and thermoacoustic imaging. In certain embodiments, the metal may be any noble metal or any iron oxide. Examples of suitable noble metals include, but are not limited to, gold and silver. The targeting moieties useful in the compositions and methods of the present disclosure include molecules that may be bound to the cage and which recognize a particular site of interest in a subject. In certain embodiments, the targeting moieties may be bound to the cage by a linking molecule. In certain embodiments, the targeting moiety may be chosen, among other things, to at least partially increase the uptake of the compositions of the present disclosure into a desired cell and/or tissue type when introduced into a subject. In certain embodiments, the targeting moiety may recognize a particular ligand or receptor present in a desired cell and/or tissue type when introduced into a subject. In certain embodiments, the targeting moiety may be an antibody that recognizes such a particular ligand or receptor. The use of antibody fragments may also be suitable in the compositions and methods of the present disclosure. The choice of a targeting moiety may depend upon, among other things, the cell and/or tissue type into which an at least partial increase in uptake of the compositions of the present disclosure is desired, as well as particular ligand(s) present in such cell and/or tissue types. In certain embodiments, the targeting moiety may be a moiety that recognizes a molecule which is present in higher amounts in an abnormal form of a tissue when compared to a normal form of the same tissue (i.e. the molecule is "up-regulated" in the abnormal form of the tissue). For example, in certain embodiments, antibodies which bind to epidermal growth factor (EGFR) may be suitable for use in the compositions and methods of the present disclosure when it is desired to at least partially increase the uptake of the compositions of the present disclosure into cancerous epithelial tissue. As a further example, antibodies such as anti-Claudin-4, anti-Mucl, or anti-EGFR may be suitable for use in the compositions and methods of the present disclosure when it is desired to at least partially increase the uptake of the compositions of the present disclosure into cancerous pancreatic tissue. One of ordinary skill in the art, with the benefit of this disclosure, will recognize other targeting moieties that may be useful in the compositions and methods of the present disclosure. Such targeting moieties are considered to be within the spirit of the present disclosure.

In certain embodiments, the targeting moieties useful in the compositions and methods of the present disclosure may be bound directly to the metal. In certain embodiments, the targeting moieties useful in the compositions and methods of the present disclosure may be bound to the metal via a linking molecule. The linking molecules useful in the compositions and methods of the present disclosure may be any molecule capable of binding to both the metals used in the compositions and methods of the present disclosure and the targeting moieties used in the compositions and methods of the present disclosure. In certain embodiments, the linking molecule may be a hydrophilic polymer. Suitable linking molecules include, but are not limited to, poly( ethylene glycol) and its derivatives, dithiol compounds, dithiol compounds with hydrazide and/or carboxylic functionality, or single thiols and/or amines or their derivatives. In certain embodiments, the linking molecule and the targeting moiety may be bound by one or more covalent bonds. In certain embodiments, the linking molecule, in addition to linking the targeting moiety and the metal, may impart certain benefits upon the compositions of the present disclosure, including, but not limited to, improved hydrophilicity, reduced immunogenic responses upon introduction of the compositions of the present disclosure into a subject, increased circulation time of the compositions of the present disclosure when introduced into the bloodstream of a subject. The choice of a linking molecule may depend upon, among other things, the targeting moiety chosen and the subject into which the compositions of the present invention are to be introduced. One of ordinary skill in the art, with the benefit of this disclosure, will recognize additional suitable linking molecules. Such linking molecules are considered to be within the spirit of the present disclosure. The stealthing agents useful in the compositions and methods of the present disclosure include molecules that may inhibit, delay, and/or prevent opsonisation (i.e., the depositing of proteins on a surface) while in the bloodstream.. By inhibiting or delaying protein adsorption to the surface of a device or nanoparticle, it is essentially "stealthed" from immune system recognition. Examples of suitable steathling agents inlcude, but are not limited to, poly(ethylene glycol) and dextran. In certain embodiments, the stealthing agents may be bound to the cage by a linking molecule. In certain embodiments, a stealthing agent useful in the compositions and methods of the present disclosure may be bound directly to the metal. In certain embodiments, a stealthing agent useful in the compositions and methods of the present disclosure may be bound to the metal via a linking molecule. The linking molecules useful in the compositions and methods of the present disclosure may be any molecule capable of binding to both the metals used in the compositions and methods of the present disclosure and the stealthing agents used in the compositions and methods of the present disclosure. In certain embodiments, the linking molecule may be a hydrophilic polymer. Suitable linking molecules include, but are not limited to, those described above.

Methods of the Present Disclosure

In certain embodiments, the present disclosure provides a method comprising: providing a composition comprising: an anti-cancer agent, exogenous dye, or other imaging agent disposed within a degradable matrix; a metal disposed around the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a porous cage; and a targeting moiety; and introducing the composition into a subject.

In certain embodiments, the present disclosure provides a method comprising providing a composition comprising: an anti-cancer agent, exogenous dye, or other imaging agent disposed within a degradable matrix; a metal disposed around the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a cage; and a targeting moiety; introducing the composition into a subject; providing an imaging device; and obtaining an image of at least a portion of the subject. A representation of one exmaple of such an embodiment is shown in Figure 1.

A variety of imaging devices may be useful in the methods of the present disclosure. The selection of a suitable imaging device may depend upon a number of factors, including, but not limited to, the portion of the subject to be imaged and the metal chosen for use in the compositions of the present disclosure. In certain embodiments, a photoacoustic imaging device may be used in the methods of the present disclosure. In certain embodiments, an endoscopic photoacoustic imaging device may be used in the methods of the present disclosure. Other suitable imaging devices include, but are not limited to, devices for one or more of the following techniques: radiographic X-ray imaging, computed tomography (CT), magnetic resonance imaging (MRI), optical imaging (such as optical coherence tomography (OCT)), and thermoacoustic imaging. Endoscopic ultrasound imaging (EUS) currently uses the acoustic contrast between normal and abnormal tissue to differentiate between cancerous and normal masses. Optical absorption of tissue is another possible contrast mechanism to detect and stage cancer. Malignant tumors are often associated with higher blood content due to enhanced microvascularization inside or around the tumor (31-34). The absorption coefficient of blood is approximately 1 to 10 cm^"1 in the near-infra-red spectral range, depending on laser wavelength and the level of oxygen saturation in hemoglobin, while background absorption of normal tissue is only about 0.03-0.05 cm^"1. The increased concentration of strongly absorbing molecules (hemoglobin and other porphyrins) was shown to yield 2-8 fold optical contrast between tumors and normal tissues (37,38). Furthermore, malignant tumors have enhanced and noticeably hypoxic blood content (35,36). In contrast, benign tumors have a normal level of blood oxygenation (39,40).

Therefore, a technique for remote measurements of tissue optical absorption would be an optimal method for staging tumors. Such a technique - named photo/opto/thermo-acoustic imaging - exists, and aims to remotely estimate optical properties of tissue at high spatial and temporal resolution (41). Specifically, during photoacoustic imaging the tissue is irradiated with pulses of low energy laser light, which may range in duration from 1 femtosecond to 1 second. The 10-30 mJ/cm² laser fluence of near-infrared irradiation will be sufficient to deliver optical energy to most desired tissues - and this laser fluence is well within the safe level of laser irradiation of tissue defined by the American National Standards and the U.S. Food and Drug Administration (FDA) (42). Therefore, a photoacoustic level of pulsed laser energy will not produce any appreciable thermal damage to the tissue and will result in a negligible temperature increase. Next, through the processes of optical absorption followed by thermoelastic expansion, broadband acoustic waves are generated within the irradiated volume. Using an ultrasound detector, these waves can be detected and spatially resolved to provide an image of the internal tissue structure. The received ultrasound signal contains information about both position (time of flight) and strength of the optical absorber (amplitude of the signal). The amplitude of the thermoelastic response of the tissue is proportional to the optical absorption, i.e., the stronger the absorption, the stronger the signal. Therefore, contrast in photoacoustic imaging is primarily determined by optical contrast of different types of tissues. For cancer imaging, the contrast mechanism in photoacoustic imaging offers the prospect of identifying both anatomical features and different functional activities of the tissue that are indistinguishable using other imaging modalities such as ultrasound, MRI, PET or CT/X-ray alone. The measurements of optical properties of many relevant tissues are limited, quite variable and offer only an approximate guide to the optical behavior of tissues. However, several observations can be made from the typical absorption spectrum of tissue. In the near- infrared (2000-3000 nm) region, water is the dominant absorber; the light penetration depth (the distance through tissue over which diffuse light decreases in fluence rate to 1/e or 37% of its initial value) varies from about 1 mm to 0.1 mm. At the other end of the spectrum, in the ultraviolet region (near and below 300 nm), the absorption depth is shallow, owing to absorption by cellular macromolecules. In the central region as shown in Figure 2, tissue absorption is modest while contrast between tissue components remains high. Within 650-1100 nm wavelengths, the average optical penetration depth is on the order of tens of millimeters - therefore, this spectral range is very suitable for photoacoustic imaging of tissues such as the pancreas.

A simple form of photoacoustic imaging (i.e., without contrast agent) may already discriminate between cancer cells and surrounding healthy tissue. However, by using near infrared laser light and the compositions of the present disclosure, photoacoustic response may be further enhanced, among other things, because healthy tissue will not absorb the near infrared light nor will they have any significant concentration of the absorber (nanoparticles). In contrast, the nanoparticles accumulated in the tumor will efficiently convert light energy into acoustic pressure waves that can provide a large signal against the tissue background. Finally, the photoacoustic imaging may be augmented by ultrasound imaging - these imaging systems are complementary. Indeed, photoacoustic imaging can be transparently integrated with ultrasound since both photoacoustic and ultrasound imaging systems can utilize the same ultrasound sensor and associated receiver electronics. The ultrasound imaging will visualize the overall anatomical features of tissue and potentially identify large tumors, while the photoacoustic imaging augmented by the compositions of the present disclosure may identify both large and small regions of cancerous cells.

The present disclosure also provides for the opportunity to enhance contrast for several different imaging modalities simultaneously. When the composition of the current disclosure comprises MRI contrast agents in the biodegradable core of the structure (such as Gd- DTPA or iron oxide particles), then both photoacoustic and MRI can be used to visualize tissue with accumulated nanoparticles. This multimodal approach can also be used at a cellular level to look at accumulated nanoparticles in cells. For instance, when the composition of the current disclosure comprises exogenous dyes in the biodegradable core of the structure (such as rhodamine or ICG), then both microscopy and OCT can be used to visualize tissue with accumulated nanoparticles, etc. The list of possible imaging modalities is not limited to those mentioned here; those skilled in the art, with the help of this disclosure will recognize other imaging modalities that could apply.

Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this disclosure as illustrated, in part, by the appended claims.

EXAMPLES

Formation of a PLGA Degradable Matrix Containing Doxorubicin Encapsulation of the hydrophobic chemotherapeutic agent doxorubicin (DOX) in

PLGA nanoparticles was achieved using a modified water-in-oil nanoprecipitation technique. Briefly, 5 mg DOX was dissolved in 1 ml of methanol and mixed with a solution of 100 mg of PLGA in 3 ml of acetone. This oil phase was then added to 10 ml of an aqueous phase containing 10 mg/ml bovine serum albumin or poly(vinyl alcohol) as a stabilizer and the system was briefly sonicated. Nanoparticles were spontaneously formed as a result of solvent migration to the aqueous phase and consequent precipitation of the polymer in the form of nanoparticles. Organic solvent was removed by stirring under vacuum at room temperature for 45 minutes. Nanoparticles were then recovered by centrifugation and washed several times to remove unencapsulated drug. Nanoparticle pellets were frozen, freeze dried, and stored at -20⁰C. Freeze dried nanoparticles were characterized with respect to size, morphology, surface charge, drug loading, drug release profile. The particles were roughly spherical with an average size after resuspension in water of 210 nm as determined by a Coulter Nanosizer. Transmission electron microscopy images of nanoparticles stained with uranyl acetate suggest that the drug is uniformly dispersed within the polymer matrix. A scanning electron micrograph of doxorubicin loaded PLGA nanoparticles is shown in Figure 3. Drug loading within the nanoparticles was assessed by dissolving the nanoparticles in a mixture of methylene chloride and methanol (60:40 v/v%), measuring the absorbance of the solution at 480 nm, and determining the drug content based on a calibration curve of doxorubicin in the same solvent system. The maximum doxorubicin loading achieved was close to 5 mg per 100 mg of nanoparticles, and the average encapsulation efficiency was 80%.

Drug release studies performed in buffered saline at pH 7.4 and 4.0 revealed that doxorubicin was released significantly faster at the lower pH, as shown in Figure 4. In fact, more than 50 and 90% of the drug was released after 1 or 12 hours, respectively. In contrast, at pH 7.4 less than 30% of the drug was released during the first hour, and less than 75% during the first day. This formulation is expected to deliver the drug in a controlled manner over extended periods of time and to deliver the drug at accelerated rates once in acidic tumor tissue or within the acidic endolysosomal compartments of target cancer cells. Possible causes for the differences in release profiles with pH are accelerated degradation of the polymeric nanoparticles, or the loss of ionic interaction between the weakly basic DOX molecule and the carboxylic acid end groups of PLGA (63).

Efficacy of PLGA Degradable Matrix Containing Doxorubicin

The therapeutic efficacy of the doxorubicin loaded PLGA nanoparticles was tested in MDA-MB-231 breast cancer cells using a colorimetric metabolism assay. Figure 5 shows the viability of cells exposed to doxorubicin loaded nanoparticles, blank nanoparticles, or free doxorubicin as a fraction of control. Blank nanoparticles did not result in lowered viability at the concentrations tested. Doxorubicin-loaded nanoparticles and free doxorubicin significantly lowered cell viability at concentrations above lOug/ml compared to control, but did not cause significantly different therapeutic effect. Importantly, the therapeutic effect observed is associated with the drug or nanoparticles that had entered the cells within the 2 hours of exposure, after which the media was replaced with complete growth media. During this time, less than 35% of the drug would have been released at pH 7.4. Thus, the anti-tumor effect of the nanoparticulate formulation is actually higher than that of the free drug that was 100% available for cell internalization during cell exposure.

Confocal microscopy studies of cell interaction revealed that nanoparticles containing doxorubicin were able to deliver their doxorubicin faster and in greater amounts to the MDA- MD-231 breast cancer cells when compared to the free drug, as can be seen in Figures 6A and 6B. High fluorescence intensity, corresponding to high doxorubicin accumulation, was observed after only 1 hour of exposure. Accumulation was observed to increase with dose and time of exposure, as expected. Interestingly, high level of nuclear drug accumulation was observed. Since nanoparticles are too large to enter the nucleus, it is probable that DOX was rapidly released upon cellular endocytosis and was then free to enter the nuclei. Figure 6C shows a picture of live cells (not fixed) imaged immediately after a 1 hour exposure to DOX loaded nanoparticles at a DOX concentration of 5.0 ug/ml along the x-y plane and the corresponding cross-section along the depth of the cells. Fluorescence along the depth confirms homogeneous drug accumulation throughout the volume of the cells. Formulation of a PLGA Degradable Matrix Containing Gemcitabine

Encapsulation of the hydrophilic chemotherapeutic agent gemcitabine in PLGA nanoparticles was achieved using a water-in-oil-in-oil double emulsion method. An inner aqueous phase was created by dissolving 24 mg of gemcitabine in 500 μl of a 10 mg/ml polyvinyl alcohol solution. This phase was slowly added onto an organic phase (inner oil) containing 100 mg of PLGA in 1.25 ml of acetone, and the system was sonicated for 1 minute. This first emulsion was poured into 35 ml of the external oil phase which contained mineral oil with dissolved lecithin (1.25 mg/ml) and sonicated again for 1 minute. The organic solvent was removed while stirring under vacuum, the particles were collected by centrifugation at 19,000 rpm for 15 min, and the supernatant was removed. Particles were washed repeatedly by suspension in hexane containing lecithin (1.25 mg/ml) to remove mineral oil. Storage of the particles entailed adding 120mg of trehalose (cryoprotectant) to the particle pellet and freezing overnight in a -80⁰C freezer. The resulting solution was then lyophilized in a Freeze Dryer 4.5 (Labconco, Kansas City, MO, USA) and the particles stored at -20⁰C. Encapsulation efficiencies of up to 70% with loadings of up to 17mg gemcitabine per lOOmg nanoparticles, as determined by spectrophotometric assay, were observed.

Formulation of a PLGA Degradable Matrix Containing a Gemcitabine Prodrug As is evident in Figure 7, a challenge associated with this method is achieving particle sizes in the nanometer range because of the high viscosity of the outer oil phase. The gemcitabine loaded particles have a large polydispersity in size, generally ranging from 200 nm- 20 um. In order for the compositions of the present disclosure to circulate in the blood stream and target the microvasculature of a tumor, particle diameters in the range of 50-350 nm are preferred. This size is well below the pore cutoff range of 380-780nm as reported for several different tumor models (62). Additionally, due to the hydrophilicity of gemcitabine, drug release from the degradable matrix pictured in Figure 7 in phosphate buffer saline solution at 37°C was almost complete within 30 minutes.

To overcome both limitations, a gemcitabine prodrug may be used in the compositions and methods of the present disclosure. In some cases, this prodrug may be considerably more hydrophobic than the original gemcitabine agent which may allow for its use in a water-in-oil encapsulation method. This water-in-oil nanoprecipitation technique is expected to produce mean particle sizes of -220 nm. Furthermore, since the gemcitabine prodrug is more hydrophobic, its release from the degradable matrix in aqueous environments, like blood, is expected to be considerably slower. This delayed gemcitabine release will allow for circulation in the bloodstream, proper targeting, and accumulation at the pancreatic tumor site prior to significant drug release.

The prodrug, shown in Figure 8, may be formed by adding a carbon chain to the A- amino group of gemcitabine. The length of the carbon chain imparts varying degrees of hydrophobicity to gemcitabine, thus extending its release profile as well as making the process of encapsulating it more efficient. The length of the carbon chain chosen here is 18 units since that length has been shown to yield 98% encapsulation efficiencies in liposomes (24) and should impart enough hydrophobicity to gemcitabine to maintain adequate release profiles through the compositions of the present disclosure. The creation of the prodrug may follow the method described by Immordino et al.

(24). First, 1.1 g of stearic anhydride may be dissolved in 16 ml of dioxane. In a separate vial, 263 mg of gemcitabine may be dissolved in 4 ml of water. The stearic anhydride solution may be then added to the gemcitabine solution and stirred for 48 hours. This room temperature reaction may be monitored by thin layer chromatography. At the conclusion, the reaction mixture may be placed under vacuum and evaporated to dryness at 60⁰C. The resulting residue, containing mostly 4-(N)-stearoyl-gemcitabine, may be purified through silica gel microcolumn flash chromatography, and this final product may be verified using nuclear magnetic resonance spectroscopy.

The procedure to encapsulate 4-(N)-stearoyl-gemcitabine in PLGA may be a modified oil-in-water nanoprecipitation technique (63). First, 6.0 mg of 4-(N)-stearoyl- gemcitabine may be dissolved in 1 ml of methanol or methylene chloride. Separately, 100 mg of PLGA may be dissolved in 3 ml of acetone. Then 1 ml of the gemcitabine solution may be added to the PLGA solution, forming the oil phase. This oil phase may then be poured into 10 ml of a 10 mg/ml poly( vinyl alcohol) solution sonicated for 30 seconds. Sonication will aid in the creation and subsequent precipitation of nanoparticles. Removal of the organic solvents may be accomplished by stirring under vacuum for 45 minutes. Finally the particles may be collected by centrifugation at a speed of 48,000 x g for 10 minutes. The particles then may be washed with deionized water for three cycles to remove any excess unencapsulated drug.

Another method which may be useful for creating such a prodrug is described by Myhren et al. (82). Briefly, 570mg of oleoyl chloride and 380mg of gemcitabine will be dissolved in 5 ml of pyridine and stirred for 2.5 hours in a nitrogen atmosphere. This room temperature reaction may be monitored by thin layer chromatography. At the conclusion, the reaction mixture may be placed under vacuum and evaporated to dryness. The resulting residue, containing Gemcitabine-N4 Oleoyl Amide, may be purified through silica gel flash chromatography using a mobile phase of 15% methanol in chloroform. The fractions collected from chromatography that contain this prodrug then may be subjected to a 50:50 etheπhexane bath under sonication in order to remove any bound pyridine or other small solvent molecules.

Production of Metal Cages In the budding field of photoacoustic contrast agents, metal nanoshells are traditionally used (64). Metal nanoshells generally have a dielectric core-shell morphology where a silica core is surrounded by a thin layer of metal. The surrounding metal could be Ag, Au, Pt, or any other bulk metal, but the Au layered shells are the most widely studied. The plasmon optical resonance peak of elemental gold or silver can be shifted from the visible to the near infrared region (NIR) by varying the core diameter and metal shell thickness (65). This shift to the NIR is critical considering that light in the NIR region can penetrate deep (2-3 cm) into biological tissue (66). Thus, metal nanoshells have the potential, once injected into the body, to respond thermally to light shone externally on the body. This property is important in photoacoustic imaging since it allows for thermoelastic expansion and a resulting pressure wave to be produced which is ultimately measured as the photoacoustic signal. Such a structure is, however, not preferred in the compositions and methods of the present disclosure. Instead, a porous cage which will allow for diffusion of a drug through the degradable matrix and the metal is preferred. Therefore, a modified stoichiometrically controlled reduction method (49) was used to add a porous silver cage onto a silica core (Figure 9). These particles have a core diameter of 520nm, but these silver cages have also been successfully produced over silica core sizes ranging from 180-520nm. Using a Shimadzu UV- vis spectrophotometer, high broad absorbance across the wavelengths of 600-1100 nm has been observed for these particles (Figure 10).

In some cases, formation of the metal cage may be facilitated by modifying the degradable matrix. In one example, a pretreatment of PLGA may be used involving converting the carboxylic acid groups that reside at the end of the lactide portion of a PLGA chain to carboxamide functional groups as depicted in Figure 11. To avoid PLGA hydrolytic degradation during this functional group conversion, the reaction may take place in organic solvents. Specifically, 100 mg of carboxylic acid end capped PLGA (MW~12kDa) may be dissolved in 20 ml of dichloromethane. Then 5 mg of diisopropyl carbodiimide (DIC) may be added to this solution and allowed to stir at room temperature for 2 hours. As shown in Figure 11, during this step DIC activates the carboxylic acid group, forming an o-Acylisourea intermediate (67). At the end of the activation, 5 ul of 28% ammonium hydroxide may be added and allowed to react for another 2 hours. During this time, an amide bond will be formed along with the byproduct diisopropylurea. To wash out this byproduct, the dichloromethane solution may be poured into 20 ml of methanol. PLGA will precipitate out since it is insoluble in methanol. The byproduct, however, is soluble in methanol and may be removed with the supernatant after centrifugation of the dichloromethane/methanol mixture. Several washes may be performed in this manner by resuspending/dissolving PLGA in dichloromethane and precipitating using methanol. The resulting carboxamine functionalized PLGA may then be used in an oil-in-water nanoprecipitation technique for formation of the degradable matrix. Additionally, the carboxamine functionalized PLGA may be verified using nuclear magnetic resonance spectroscopy.

Silver is aminophilic and will readily deposit on silica nanoparticles with amine surface chemistry (68). Therefore, pretreating the PLGA to have carboxamide groups, as described earlier, may help facilitate the deposition of a silver cage. Once the degradable matrix is built using the carboxamine functionalized PLGA, wet chemistry techniques similar to those used to create silica-silver nanocages (49) may be employed. Specifically, preparations may begin by suspending 1.2 mg of the degradable matrix in 29.6 ml of water. Then 0.6 ml of 0.15 M silver nitrate may be added along with 50 uL of a 36% glucose solution (the reducing agent). The pH of the solution may be increased to 9 by then adding 5OuL of a 3% ammonium hydroxide solution. Within 10 minutes silver may deposit on the outside of the degradable matrix in a rough, cage-like manner. The redox reaction scheme for this method is shown in the following equation (49): RCHO (aq) + 2 Ag(NH₃)₂OH (aq) → 2 Ag (s) + RCOONH₄ (aq) + 3 NH₃ (aq) where Ag(NH₃)₂OH is a silver diamine complex (commonly known as the Tollens' reagent) and RCHO represents glucose which is used to reduce the Tollens' agent to metallic silver.

Unfortunately, using this Tollen's reagent scheme to coat PLGA with silver resulted in large aggregates of PLGA and silver. The interaction between the amine groups on PLGA and the silver was so strong that as the silver seeding process started, multiple particles would be attracted to a single silver particle and as the reduction process continued, the aggregates only increased in size. A SEM micrograph of these aggregates is shown in Figure 12.

Another method, photoreduction, was more successful. Use of alcohols as photoreducing agents for the formation of silver colloids was introduced in the 1970's (83). Since then researchers have been employing this photoreduction technique to reduce Au, Ag, and Cu ions in aqueous solutions into metallic nanoparticles and crystallites of controllable size (84,85). Since PVA may be used as a surfactant during the creation of stable drug-loaded PLGA degradable matrices as described above, PVA may already be surrounding the degradable matrix. In the presence of UV light the PVA can act as a photoreducing agent, radicalizing under the influence of the light as shown in Figure 13, and transferring an electron to silver ions in solution. This photoreduction mechanism concludes via donation of one final electron and formation of an aldehyde (86).

Alcohol-mediated photoreduction has been successful in formation of more monodispersed metal colloid solutions because it acts as macroscopic support for the forming particles and limits aggregation (86). When used to form the compositions of the present disclosure, alcohol-mediated photoreduction acts in a similar way, supporting the formation of nanocrystals of silver which aggregate around the entire core of the PLGA particle. These silver domains are further stabilized on the PLGA surface via attraction of the positively charged silver to PLGA' s negatively charged carboxylic acid end groups. A scanning electron micrograph of these silver seeded PLGA nanoparticles is shown in Figure 14. Note that images of these silver nanocages are difficult to capture due to the instability of the PLGA core under the influence of the electron beam, but these images provide insight into nature of the silver coating produced by alcohol-mediated photoreduction. Specific procedures used to create silver-PLGA nanocages are described here briefly.

First the oil in water emulsion method described above was used to create the PLGA degradable matrix particles, then 1.5 mg of the particles are suspended via sonication in 30 ml of DI ultrafiltrated (DIUF) water. Next, 0.2 ml of 0.15 M silver nitrate is added and the entire mixture is placed in a wide recrystallization glass dish. Light is shone on the top of the sample as provided by an 8W UVP source (Fisher Scientific, Fair Lawn, NJ, USA). The solution is stirred at 400 rpm under the light for 30 minutes. At this point the PLGA is seeded with small silver crystals. Sometimes these crystals are evident as the solution appears slightly yellow and the absorbance spectra of the sample show small peaks at 400 nm and 800 nm. The yellow color might not always be evident, especially if silver seeds <2 nm in size are present. Next, the solution is removed from the UV-light assembly and 100 ul of 18 wt% ascorbic acid is added. This addition causes a rapid reduction of the rest of the aqueous silver ions in solution to form cage-like structures around the PLGA core nanoparticles, as shown in Figure 15. The absorbance spectra for the more confluent cage-like morphologies, loaded with doxorubicin is shown in Figure 16. Attachment of PEG and the Monoclonal Antibody Cetuximab to the Silver Cage

Poly( ethylene glycol) (PEG) chains may be attached to the surface of the silver cage of the particles using thiol-PEG chemistry (76). PEGylation of the particles in this way is known to limit protein adsorption in vivo, thus increasing nanoparticle residence time in the blood prior to recognition by the reticuloedothelial system (76,77). Heterofunctional PEG (SH-PEG- COOH) purchased from Quanta Biodesign or Layson Bio, Inc. may be attached to silver by combining a 5 μM solution of SH-PEG-COOH and 1.6xlO⁹ particles/ml (these particles may already have antibodies attached, as described below) in DIUF water for 20 min, similar to procedures described by Hirsch et al. (59). During that time, PEG will form a self assembled monolayer on the surface of the silver through silverthiolate bonds (87).

Attachment of the monoclonal antibody Cetuximab to the silver cage may be accomplished in a manner similar to PEG attachment. As previously discussed, silver is highly thiol- and aminophilic. Thiol and amine functional groups are naturally found on amino acid residues (e.g. cysteine and lysine). Since these amino acids are prevalent on the heavy chain of the anti-EGFR antibody, Cetuximab, the antibody will form covalent bonds with silver at room temperature in an aqueous environment. Similar to gold-antibody conjugation demonstrated by other researchers (58,88), the process may begin with preparation of a 2OmM HEPES buffer (pH 7.4). Two solutions constituted in this HEPES buffer will then be created: a solution of 100 μg/ml of Cetuximab, and a solution of particles having a drug disposed within a degradable matrix and a silver cage on the degradable matrix at a concentration of 1.6xlO⁹ particles/ml. The two solutions may be mixed in a 1 : 1 volume ratio and allowed to react at room temperature for 20min. The particles may be collected by centrifugation at a speed of 48,000 x g for 10 minutes in a Beckman J2-21 refrigerated centrifuge (Beckman Instruments Inc., Palo Alto, CA, USA). They may be washed with HEPES buffer for three cycles to remove any excess antibody. Thermogravimetric analysis may be used to quantify the amount of antibody bound to silver while X-ray photoelectron spectroscopy may be used to verify the nature of the bonds formed with silver (78). Alternatively, a directional conjugation of the antibody to the metal can be achieved using dithiol linker molecules as previously described (Kumar, S., J. Aaron, and K. Sokolov, Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties. Nat Protoc, 2008. 3(2): p. 314- 20).

Efficacy of PLGA Degradable Matrix Containing Doxorubicin, Silver Outer Cage, and a Stealth agent

The therapeutic efficacy of the doxorubicin loaded PLGA nanoparticles with a pegylated silver outer cage was tested in MDA-MB-231 breast cancer cells using a colorimetric metabolism assay. Figure 17 shows the viability of cells exposed to doxorubicin loaded nanoparticles, blank nanoparticles with a silver cage, doxorubicin loaded nanoparticles with a silver cage, or free doxorubicin as a fraction of control. Blank nanoparticles with a silver cage did not result in statistically significant lowered viability at the concentrations tested. Doxorubicin-loaded nanoparticles, doxorubicin loaded silver cage nanoparticles, and free doxorubicin significantly lowered cell viability at concentrations above 1.2 ug/ml compared to control, but did not cause significantly different therapeutic effect relative to each other. Importantly, the therapeutic effect observed is associated with the drug or nanoparticles that had entered the cells within the 5 hours of exposure, after which the media was replaced with complete growth media and incubated for another 2 days prior to performing the viability assay.

Phase and fluorescence microscopy studies of cell interaction with both the doxorubicin loaded in PLGA and the doxorubicin loaded in PLGA with a silver cage nanoparticles revealed that nanoparticles containing doxorubicin were able to deliver their doxorubicin faster and in greater amounts to the MDA-MD-231 breast cancer cells when compared to the free drug. The image in Figure 18 is a composite image comprised of two fluorescence and one phase contrast image of the same set of MDA-MB-231 breast cancer cells post exposure to doxorubicin loaded PLGA nanoparticles with a silver cage. High pink fluorescence intensity, corresponding to high doxorubicin accumulation, was observed after only 2 hr of exposure. These pink regions overlapped with blue regions, which are DAPI strained nuclei. This large overlap shows that doxorubicin delivered from doxorubicin loaded PLGA nanoparticles with a silver cage were reaching the nucleus and likely intercalating with DNA and thereby damaging the cells replication potential.

Doxorubicin release studies performed at 37C at a pH of 7.4 revealed that the silver cage does in fact seriously inhibit the burst release of doxorubicin from these nanostructures as shown in Figure 19. When the cage is present, less than 10% of doxorubicin was released in the critical first 5 hr, whereas >50% of the drug is released in the same time frame when no cage is present.

Imaging Systems A schematic view of a photoacoustic and ultrasound imaging system is presented in both Figures 20 and 21. To capture ultrasound and photoacoustic data, a 128 element linear array transducer operating at 5 MHz center frequency and interfaced with a 32 channel ultrasound imaging system was used. To generate photoacoustic transients at a reasonable depth, a pulsed laser (5-7 ns) operating at 800 nm or 1064 nm optical wavelength was utilized. Laser fluences of up to 30 mJ/cm² were employed in photoacoustic imaging. In preliminary experiments, the laser beam was oriented from the side of the specimen or from the bottom of the specimen, as shown in Figure 21. In clinical settings, a more practical configuration will be used where the light delivery and acoustic transducer are positioned on the same side of the specimen as pictured in Figure 20. The pulsed laser can operate at a maximum repetition rate of 20 Hz while the ultrasound imaging can be performed at higher repetition rates. Therefore, during imaging, the tissue is first irradiated with light. The photoacoustic response is received on all elements of the array and the individual signals are beamformed to produce a high quality photoacoustic image. Immediately after photoacoustic imaging, conventional ultrasound imaging is performed. Thus, both ultrasound and photoacoustic data are acquired by the same transducer in the same spatial position. In this way, both images are automatically co-registered and provide complementary information about the tissue.

Fabrication of Tissue-Mimicking Phantoms Tissue-mimicking phantoms that incorporate photoacoustic contrast agents can be produced using poly(vinyl alcohol) (PVA) or gelatin molds. Figure 22 shows an example of several molds (or plugs), that were constructed using PVA and the silver-silica photoacoustic contrast agents shown in Figure 9 at different concentrations.

Although PVA was used to mold the silver-silica photoacoustic contrast agents, gelatin may be used instead to create plugs containing the compositions of the present disclosure due to the flexibility it provides. Gelatin has favorable ultrasonic, optical and mechanical properties, it is easy to handle, and inexpensive to produce. Aqueous solutions of gelatin can be poured into a container of desired shape and size, and cooled down to crosslink. The malleable nature of gelatin allows for molds to be created which closely match the size and geometry of the pancreas. Optical properties of gelatin can also be greatly manipulated by adding dye to increase optical absorption. For ultrasound imaging, silica particles (0.4-2 vol%) may be added to act as ultrasonic scatterers. The speed of sound in human water-based tissues ranges from 1450 to 1620 m/s while the attenuation ranges from 0.1 to 1.3 dB/cm/MHz (89). The attenuation and the speed of sound in gelatin is 0.1 dB/cm/MHz (or less) and 1510 m/s, respectively. These gels maintained their size, shape, integrity, and acoustic/optical properties for several days when stored in a refrigerator.

Imaging of Tissue Phantoms (1)

To evaluate the ability of PAUS imaging to detect inclusions and to test the optical properties of the silver nanocages, imaging experiments using phantoms or molds similar to those shown in Figure 22 were performed. These silica-silver particles are expected to have similar optical absorption properties to the compositions of the present disclosure. The nanocage PVA samples were created by incorporating the 183 nm silica core, silver nanocages in PVA at concentrations of 10⁷, 10⁸, and 10⁹ particles per ml. These concentrations are similar to those reported elsewhere (59,72) for use in imaging in vivo. Phantoms of varying concentrations of nanocages were created for two reasons (1) to test the concentration dependence of the photoacoustic signal from these contrast agents and (2) to test that the PAUS imaging system can accurately detect nanocage inclusions. For background, the photoacoustic pressure (P) generated from an absorbing source immediately following a laser pulse can be defined as,

P ∞^μ_aF (1)

P where β is the thermal expansion coefficient, v is the acoustic velocity in the medium, μ_a is the absorption coefficient, F is the fluence of light reaching the absorber (absorbers are the nanocages), and C_p is the specific heat at constant pressure. The absorption coefficient can be defined as μ_a = Nσ_a, where N is the number of absorbing particles per volume and σ_a is the absorption cross-section of one absorber. Given these relationships and minimal temperature increases, a plot oiP/F versus N should be linear. To test this hypothesis, the phantoms shown in Figure 22 were imaged photoacoustically along the transverse plane. Briefly, pulsed 800 nm light, was directed at the circular end of the cylindrical sample at 4 mJ/cm² in a diffuse beam (spot size ~1 cm²) as shown in Figure 23. The 7.5 MHz, 128 elements linear array transducer was set to image a traverse plane about ~2 mm away from the cap of the cylindrical samples. Unfortunately, the set-up of the phantom imaging study did not allow for positioning of the transducer over the 0 mm plane, i.e., at the tip of the cylindrical samples. Therefore, the change in fluence of the light in the imaging plane due to absorption and scattering events was accounted for using Beer's Law: F = F_oe-^(μ"^{+ Λ )z} (2) where z is axial distance in the light path (2 mm), μ_a is the absorption coefficient of the nanocages, μ_s is the scattering coefficient (scattering was assumed here to be the same for each phantom), F₀ is the incident fluence (4 mJ/cm²), and F is the fluence at the 2 mm transverse plane for each phantom. Photoacoustic imaging of the phantoms resulted in a visible increase in signal as the concentration of nanocages increased as shown in Figure 24. To measure the changes in photoacoustic pressure quantitatively, pixels inside the white circular regions of interest labeled in Figure 24 were set into grids of 20 by 20, inside which, sets of 5 by 5 pixels were averaged. The mean of these 16 sets of 5 by 5 pixel averages were taken as the relative photoacoustic pressure for each phantom. A plot of these relative pressure values divided by the fluence (per Eq. 2) versus concentration for each phantom was linear with a R² = 0.999. This result confirms the fundamental relationship that the photoacoustic signal from nanocages increases linearly with the concentration of nanocages for a reasonable range of concentration of absorbers (nanocages),. Further examining this linear relationship in future work could lead to the quantification of nanocages accumulated inside tissue. If these nanocages were carrying drugs or other molecules of interest as per the current disclosure, performing photoacoustic imaging over time could provide noninvasive, quantifiable, image-guided therapy. Imaging of ex vivo Organs

The gelatin tissue-mimicking phantoms containing the compositions of the present disclosure may be used in conjunction with a PAUS system to demonstrate the enhanced imaging capabilities provided by the compositioins of the present disclosure ex vivo to provide a model for the function of such compositions in vivo. The set-up of the PAUS system may be as shown in Figure 20 where an 800 nm pulsed laser will interrogate either pancreatic tissue or a tissue-mimicking pancreas- shaped phantom with a built in inclusion or injection of silica-silver particles. To capture ultrasound and photoacoustic data, a 128 element linear array transducer operating at 7.5MHz center frequency and interfaced with a 32 channel ultrasound Winprobe imaging system may be employed. This PAUS system may be used to evaluate two criteria: (1) the concentration of particles required to receive a photoacoustic signal and (2) the penetration depth at which a photoacoustic signal from the particles can still be received for a given particle concentration. In other words, this may determine ex vivo how many of the particles must accumulate at the tumor site in order to maintain effective photoacoustic imaging contrast. Secondly, testing for a given particle accumulation concentration, this example may show how deep the laser will penetrate into tissue or a gelatin pancreas-mimicking phantom model and still reach the particles so that a reliable photoacoustic signal can be detected.

Shown in Figure 25 is one example of PAUS imaging results from the set-up shown in Figure 20. A canine pancreas was set in a gelatin mold and injected with silica-silver particles (core silica size 183 nm). The image on the left of Figure 25 is from ultrasound and it outlines the tissue boundaries. The photoacoustic image (middle) shows the location of optical absorbers (nanocages). The co-registered, combined PAUS image (right) provides details about the location of the particles in relation to surrounding tissues. This result strongly suggests that PAUS imaging of optically absorbing contrast agents such as silver nanocage particles can be used to detect pancreatic cancer at sub-millimeter spatial resolution.

Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this disclosure as illustrated, in part, by the appended claims. References:

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Claims

What is claimed is:

1. A composition comprising: an anti-cancer agent, exogenous dye, or other imaging agent disposed within a degradable matrix; a metal disposed around the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a cage; and and a targeting moiety or stealthing agent or both.

2. The composition of claim 1, wherein the composition is in the form of particles of a size of less than about 5000nm.

3. The composition of claim 1 wherein the anti-cancer agent is effective in treating multiple cancer types.

4. The composition of claim 1 wherein the anti-cancer agent is gemcitabine, doxorubicin, paclitaxel, or their derivatives or any other chemotherapeutic molecule which can be encapsulated in a biodegradable matrix.

5. The composition of claim 1 wherein the metal is suitable for imaging by photoacoustic imaging of any form or optical imaging of any form.

6. The composition of claim 1 wherein the metal is silver, gold or iron oxide.

7. The composition of claim 1 wherein the targeting moiety is a cancer specific monoclonal antibody such as anti-EGFR, anti-Claudin-4, anti-Mucl, and many others.

8. The composition of claim 1 wherein the targeting moiety is covalently attached to the metal.

9. The composition of claim 1 further comprising a linking molecule.

10. The composition of claim 9 wherein the linking molecule is polyethylene glycol or any bifunctional linker with ability to attach to metals via covalent linkage at one end and targeting moieties via covalent linkage on the other end.

11. The composition of claim 9 wherein the targeting moiety is covalently attached to the linking molecule.

12. The composition of claim 9 wherein the targeting moiety is covalently attached to the linking molecule and the metal.

13. A composition comprising: a degradable matrix; and a metal disposed around the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a cage.

14. The composition of claim 14 further comprising a targeting moiety.

15. A method comprising: providing a composition comprising: an anti-cancer agent, exogenous dye, or other imaging agent disposed within a degradable matrix; a metal disposed around the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a cage; and a targeting moiety; and a stealthing agent; and introducing the composition into a subject.

16. A method comprising: providing a composition comprising: an anti-cancer agent, exogenous dye, or other imaging agent disposed within a degradable matrix; a metal disposed around the exterior surface of the degradable matrix, wherein the metal is disposed in the form of a cage; and a targeting moiety; introducing the composition into a subject; providing an imaging device; and obtaining an image of at least a portion of the subject.

17. The method of claim 16 wherein the imaging device is a photoacoustic imaging device, a radiographic X-ray imaging device, a computed tomography device, magnetic resonance imaging device, an optical coherence tomography device, or a thermoacoustic imaging.

18. The method of claim 16 wherein the imaging device is an endoscopic photoacoustic and ultrasound imaging device.

19. The method of claim 16 wherein the obtaining an image of at least a portion of the subject comprises obtaining an image of the location of at least a portion of the composition in the subject.

20. The method of claim 16 wherein the portion of the subject comprises cancerous tissue.

21. The method of claim 16 further comprising allowing the anti-cancer, exogenous dye, or other imaging agent to diffuse out of the degradable matrix.