BR112015020782B1 - AGENT ATTACHED TO FLUORESCENT SILICA-BASED NANOPARTICLE, ITS USE AND PHARMACEUTICAL COMPOSITION COMPRISING IT - Google Patents

Abstract

AGENT ATTACHED TO THE FLUORESCENT SILICA-BASED NANOPARTICLE, ITS USE AND PHARMACEUTICAL COMPOSITION THAT COMPRISES IT. The present invention relates to a silica-based fluorescent nanoparticle that allows the precise detection, characterization, monitoring and treatment of a disease such as cancer. The nanoparticle has a range of diameters, has a fluorescent compound positioned within the nanoparticle, and has a higher brightness and fluorescent quantum yield than the free fluorescent compound, has high biostability and biocompatibility, can be coated with an organic polymer , such as poly(ethylene glycol) (PEG). The small nanoparticle size, silica base, and organic polymer coating minimize nanoparticle toxicity when administered in vivo. The nanoparticle can be additionally conjugated to a ligand. A therapeutic agent can be attached to the nanoparticle. Additionally, magnetic resonance imaging (MRI), radionuclides/radiometals or paramagnetic ions can be conjugated to the nanoparticle.

Description

CROSS REFERENCE TO RELATED ORDERS

[001] This application claims priority to United States Provisional Patent Application No. 61/794,414 filed March 15, 2013, and United States Patent Application No. 14/215,879 filed March 17, 2014 The description of each of the requirements identified above is incorporated by reference in its entirety.

DECLARATION REGARDING STUDY OR DEVELOPMENT SPONSORED BY THE FEDERAL GOVERNMENT

[002] This invention was made with support from the federal government under the NIH-NRRA, Clinical and Translational Science Center Grant; NIH-NCI R01CA161280-01A1, NSF STC Program Agreement No. ECS-9876771; ICMIC P50 CA86438; NIH SAIRP Grant No. R24 CA83084; and NHI Center Grant No. P30 CA08748.

FIELD OF INVENTION

[003] The present invention relates to silica-based fluorescent nanoparticles, and methods of using the nanoparticles to detect, diagnose, or treat diseases such as cancer.

BACKGROUND OF THE INVENTION

[004] Early tumor detection and treatment selection are fundamental to achieving therapeutic success and long-term survival rates. In their early stage, many cancers are localized and can be treated surgically. However, in surgical situations, assessment of spread of metastatic disease and tumor margins, particularly in areas of complex anatomy, is limited by a lack of imaging technologies. This has led to a disproportionate number of invasive biopsies. Molecularly targeted probes incorporating contrast-producing (i.e., optical, PET) tags and offering improved specificity are needed for early detection by imaging of molecular differences between normal and tumor cells, such as cancer-specific changes in receptor expression levels. When combined with higher sensitivity and higher resolution imaging tools, specific molecularly targeted probes will greatly increase the sensitivity of cancer detection, staging, and monitoring and/or treatment.

[005] The present fluorescence probes for imaging typically consist of conventional single fluorophores (e.g., organic dyes, fluorescent proteins), fluorescent proteins (e.g., GFP) and semiconductor quantum dots (Q-dots or Q-dots). Single fluorophores are generally not stable and have limited brightness for imaging. Similar to dyes, fluorescent proteins tend to present interactions in an excited state, which can lead to stochastic scintillation, extinction and photodegradation. Quantum dots (Q-dots) are generally produced from heavy metal ions such as Pb2+ or Cd2+ and are therefore toxic. Burns et al. "Fluorescent core-shell silica nanoparticles: for "Lab on a Particle" architectures for nanobiotechnology", Chem. Soc. Rev., 2006, 35, 1028-1042.

[006] Fluorescent nanoparticles having an electrically conductive shell and a silica core are known and have utility in the modulated release of a therapeutic agent. United States Patent Nos. 6,344,272, and 6,428,811. A shortcoming of existing fluorescent nanoparticles is their limited brightness and low detectability as fluorescent probes in dispersed systems.

[007] The present multifunctional silica-based fluorescent nanoparticles offer many advantages over other typically larger diameter particle probes. Nanoparticles are non-toxic, exhibit excellent photophysical properties (including fluorescent efficiency and photostability), and demonstrate increased binding affinity, potency, as well as a distinct pharmacokinetic signature -- one in which renal clearance largely predominates. without significant uptake by the reticuloendothelial system (RES). Its relatively small size and PEG surface coating facilitates excellent renal clearance. The fluorescent nanoparticles of the present invention contain a fluorescent core and silica shell. The shell and core architectures, large surface area, and diverse surface chemistry of the nanoparticle allow multiple functionalities to be delivered simultaneously to a target cell. For example, the nanoparticle may be functionalized with targeting moieties, medical imaging contrast agents, therapeutic agents, or other agents. The targeting moieties on the surface of the nanoparticle can be tumor ligands, which, when combined with nanoparticle-conjugated therapeutic agents, make the nanoparticle an ideal vehicle for targeting and potentially treating cancer. Webster et al. Optical calcium sensors: development of a generic method for their introduction to the cell using conjugated cell penetrating peptides. Analyst, 2005; 130:163-70. The silica-based nanoparticle can be labeled with contrast agents for PET, SPECT, CT, MRI, and optical imaging.

SUMMARY

[008] The present application provides a method for detecting tumor cells comprising the steps of: (a) administering to a patient a plurality of silica-based fluorescent nanoparticles at a dose ranging from about 0.01 nanomol/ kg of body weight up to about 1 nanomol/kg of body weight, the nanoparticle comprising: a silica-based core comprising a fluorescent compound positioned within the silica-based core; a silica shell surrounding at least a portion of the core; an organic polymer attached to the nanoparticle; a ligand attached to the nanoparticle and capable of binding a tumor marker; and at least one therapeutic agent; and (b) detection of nanoparticles.

[009] The nanoparticle can be administered subdermally, peritumorally, orally, intravenously, nasally, subcutaneously, intramuscularly or transdermally. A silica-based fluorescent nanoparticle comprising:

[0010] The present invention also provides a silica-based fluorescent nanoparticle comprising: a silica-based core comprising a fluorescent compound positioned within the silica-based core; a silica shell surrounding at least a portion of the core; an organic polymer attached to the nanoparticle; and a ligand attached to the nanoparticle, wherein the nanoparticle has a diameter between about 1 nm and about 15 nm, and after administration of the nanoparticle to a subject, the renal clearance of the nanoparticle varies from about 80% of the DI (initial dose) to about 100% of the ID in about 24 hours, or from about 90% of the ID to about 100% of the ID in about 24 hours.

[0011] The present invention provides a silica-based fluorescent nanoparticle comprising a silica-based core having a fluorescent compound positioned within the silica-based core; a silica shell surrounding at least a portion of the core; an organic polymer attached to the nanoparticle; from about 1 to about 30 ligands, or from about 1 to about 20 ligands attached to the nanoparticle; and a contrast agent or a chelate attached to the nanoparticle.

[0012] The diameter of the nanoparticle varies from about 1 nm to about 25 nm, or from about 1 nm to about 8 nm. Organic polymers that can be attached to the nanoparticle include poly(ethylene glycol) (PEG), polylactate, polylactic acids, sugars, lipids, polyglutamic acid (PGA), polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), Polyvinyl acetate (PVA), or combinations thereof.

[0013] The ligand may be capable of binding to at least one cellular component, such as a tumor marker. The number of ligands attached to the nanoparticle can also vary from about 1 to about 30, from about 1 to about 25, or from about 1 to about 10. Examples of the ligand include peptide, protein, biopolymer, synthetic polymer, antigen, antibody, microorganism, virus, receptor, hapten, enzyme, hormone, chemical compound, pathogen, toxin, surface modifier, or combinations thereof. Peptides such as RGD tripeptide, cRGD cyclic peptide, cRGDYC cyclic peptide, octreotate, EPPT1 and alpha-MSH peptide analogs are encompassed by the present invention. Any linear, cyclic or branched peptide containing the RGD or alpha-MSH sequence is within the scope of the present invention.

[0014] A contrast agent, such as a radionuclide including 89Zr, 64Cu, 68Ga, 86Y, 124I and 177Lu, can be attached to the nanoparticle. The nanoparticle can be attached to a chelate, for example, DFO, DOTA, TETA and DTPA, which is adapted to bind a radionuclide.

[0015] The nanoparticle of the present invention can be detected by positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), optical imaging (such such as fluorescence imaging including near-infrared fluorescence (NIRF) imaging), bioluminescence imaging, or combinations thereof.

[0016] A therapeutic agent can be attached to the nanoparticle. Therapeutic agents include antibiotics, antimicrobials, antiproliferatives, antineoplastics, antioxidants, endothelial cell growth factors, thrombin inhibitors, immunosuppressants, antiplatelet agents, collagen synthesis inhibitors, therapeutic antibodies, nitric oxide donors, antisense oligonucleotides , wound healing agents, therapeutic gene transfer constructs, extracellular matrix components, vasodilators, thrombolytics, antimetabolites, growth factor agonists, antimitotics, statins, steroids, steroidal and non-steroidal anti-inflammatory agents, inhibitors angiotensin-converting enzyme (ACE), free radical scavengers, PPAR-gamma agonists, small interfering RNA (siRNA), microRNA, and anticancer chemotherapeutic agents. The therapeutic agents encompassed by the present invention also include radionuclides, for example, 90Y, 131I and 177Lu. The therapeutic agent may be radiolabeled, such as labeled by binding to 18F radiofluorine.

[0017] After administration of the nanoparticle to a subject, the half-life of the nanoparticle in the blood can vary from about 2 hours to about 25 hours, from about 3 hours to about 15 hours, or from about 4 hours to about 10 hours. The tumor half-life of the nanoparticle after administration of the nanoparticle to a subject can vary from about 5 hours to about 5 days, from about 10 hours to about 4 days, or from about 15 hours to about 3.5 days. The ratio of the tumor half-life to the blood half-life of the nanoparticle after administration of the nanoparticle to a subject can vary from about 2 to about 30, from about 3 to about of 20, or from about 4 to about 15. Renal clearance of the nanoparticle after administration of the nanoparticle to a subject can range from about 10% of the DI (initial dose) to about 100% of the DI in about 24 hours, from about 30% DI to about 80% DI in about 24 hours, or from about 40% DI to about 70% DI in about 24 hours . In one embodiment, after the nanoparticle is administered to a subject, the blood half-life of the nanoparticle varies from about 2 hours to about 25 hours, the tumor half-life of the nanoparticle varies from from about 5 hours to about 5 days, and the renal clearance of the nanoparticle varies from about 30% DI to about 80% DI in about 24 hours.

[0018] When nanoparticles in the amount of about 100 times the equivalent human dose are administered to a subject, substantially no anemia, weight loss, agitation, increased breathing, gastrointestinal disturbance, abnormal behavior, neurological dysfunction, hematology abnormalities , abnormalities in clinical laboratory, drug-related injuries in organ pathology, mortality, or combinations thereof, is observed in the subject in about 10 to about 14 days.

[0019] The present invention also provides a silica-based fluorescent nanoparticle comprising a silica-based core comprising a fluorescent compound positioned within the silica-based core; a silica shell surrounding at least a portion of the core; an organic polymer attached to the nanoparticle; and a linker attached to the nanoparticle, wherein the nanoparticle has a diameter between about 1 nm and about 15 nm. After administration of the nanoparticle to a subject, the blood half-life of the nanoparticle may vary from about 2 hours to about 25 hours, or from about 2 hours to about 15 hours; the nanoparticle's half-life in the tumor can vary from about 5 hours to about 2 days; and renal clearance of the nanoparticle can vary from about 30% of DI to about 80% of DI in about 24 hours. The number of ligands attached to the nanoparticle can range from about 1 to about 20, or from about 1 to about 10. The diameter of the nanoparticle can be between about 1 nm and about 8 nm. A contrast agent, such as a radionuclide, can be attached to the nanoparticle. Alternatively, a chelate can be attached to the nanoparticle. The nanoparticle can be detected by PET, SPECT, CT, MRI, optical imaging, bioluminescence imaging, or combinations thereof. A therapeutic agent can be attached to the nanoparticle. After administration of the nanoparticle to a subject, the blood half-life of the nanoparticle may also vary from about 3 hours to about 15 hours, or from about 4 hours to about 10 hours. The tumor residence half-life of the nanoparticle after administration of the nanoparticle to a subject can also vary from about 10 hours to about 4 days, or from about 15 hours to about 3.5 days. The ratio of the tumor half-life to the blood half-life of the nanoparticle after administration of the nanoparticle to a subject can vary from about 2 to about 30, from about 3 to about of 20, or from about 4 to about 15. Renal clearance of the nanoparticle can also vary from about 45% DI to about 90% DI within about 24 hours after administration of the nanoparticle to a subject.

[0020] Also provided in the present invention is a silica-based fluorescent nanoparticle comprising a silica-based core comprising a fluorescent compound positioned within the silica-based core; a silica shell surrounding at least a portion of the core; an organic polymer attached to the nanoparticle; and a linker attached to the nanoparticle, wherein the nanoparticle has a diameter between about 1 nm and about 8 nm. After administration of the nanoparticle to a subject, the ratio of the tumor half-life to the blood half-life of the nanoparticle varies from about 2 to about 30, and the renal clearance of the nanoparticle varies from from about 30% of DI to about 80% of DI in about 24 hours.

[0021] The present invention further provides a method for detecting a component of a cell comprising the steps of: (a) contacting the cell with a silica-based fluorescent nanoparticle comprising a silica-based core comprising a fluorescent compound positioned within silica-based core; a silica shell surrounding at least a portion of the core; an organic polymer attached to the nanoparticle; from about 1 to about 30 ligands attached to the nanoparticle; and a contrast agent or a chelate attached to the nanoparticle; and (b) monitoring the binding of the nanoparticle to the cell or a cellular component by at least one imaging technique.

[0022] The present invention further provides a method for targeting a tumor cell comprising administering to a cancer patient an effective amount of a silica-based fluorescent nanoparticle comprising a silica-based core comprising a fluorescent compound positioned within the core. silica-based; a silica shell surrounding at least a portion of the core; an organic polymer attached to the nanoparticle; a ligand attached to the nanoparticle and capable of binding a tumor marker; and at least one therapeutic agent. The nanoparticle can be radiolabeled. The nanoparticle can be administered to the patient by the following routes, but not restricted to them: oral, intravenous, nasal, subcutaneous, local, intramuscular or transdermal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The patent or application file contains at least one drawing executed in color. Copies of this patent or this patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fees.

[0024] Figure 1a shows a dynamic light scattering (DLS) plot (average number) of particle size for uncoated silica (in gray) and PEG-coated Cy5-containing silica nanoparticles (in black).

[0025] Figure 1b shows in vivo fluorescence imaging of spectrally separated (pseudocolored) Cy5 particles superimposed on visible light imaging of nude mice 45 min after injection with uncoated silica nanoparticles.

[0026] Figure 1c shows in vivo fluorescence imaging of spectrally separated (pseudocolored) Cy5 particles superimposed on visible light imaging of nude mice 45 min after injection with PEG-uylated Cy5 nanoparticles.

[0027] Figure 1d shows an in vivo biodistribution study using co-registered PET-CT. Top row is serial co-registered PET-CT image 24 hours post 124I-labeled PEG-coated nanoparticle injection, flanked by independently acquired microCT and microPET scans. The bottom line is serial microPET imaging.

[0028] Figure 2a shows fluorescence correlation spectroscopy (FCS) data and single exponential fits for Cy5 dye (light gray), PEG-coated nanoparticles containing Cy5 of 3.3±0.06 nm in diameter (dark gray , mean ± standard deviation, n=9) and 6.0±0.1 nm in diameter (black, mean ± standard deviation, n=6) showing the differences in diffusion time resulting from the different hydrodynamic sizes of the different species.

[0029] Figure 2b shows the absorption and emission spectra of Cy5 dye (light gray), PEG-coated nanoparticles of 3.3 nm in diameter (dark gray) and 6.0 nm in diameter (in black).

[0030] Figure 2c shows the comparison of the relative brightness of the free dye (light gray) with nanoparticles of 3.3 nM (dark gray) and 6.0 nm in diameter (in black), measured as count rate per molecule / particle as determined from the FCS curves.

[0031] Figure 2d shows photodegradation data for the Cy5 dye (light gray), PEG-coated nanoparticles of 3.3 nm in diameter (dark gray), and 6.0 nm in diameter (in black) under laser excitation of ~3.5 mW.

[0032] Figure 3a shows the percentage of the initial particle dose (% of DI) retained by the blood (in black) and by the tissues: liver (light gray), lung (medium light gray), spleen (medium gray), and rim (medium dark gray) for 6.0 nm diameter nanoparticles at various time points from 10 min to 48 h post injection (n=3 mice, mean ± standard deviation).

[0033] Figure 3b shows a graph of the concentration of retained particles for nanoparticles of 3.3 nM (light gray) and 6.0 nm (in black) in diameter and the associated logarithmic decays and half-lives adjustments.

[0034] Figure 3c shows a graph of the estimated particle excretion for nanoparticles of 3.3 nM (light gray) and 6.0 nm (black) in diameter and the associated logarithmic fits and half-lives (mean ± standard deviation , n=9 (three mice per time point)).

[0035] Figure 4 shows the in vivo biodistribution of nanoparticles in non-tumor-bearing and tumor-bearing mice with subcutaneous C6 xenografts. (A) Uncoated silica particles; (B) PEGylated RGD particles.

[0036] Figure 5 shows total specific binding data for cRGD- and PEG-ylated spots (i.e., nanoparticles) using flow cytometry in the Cy5 channel as a function of time (a) and particle concentration ( B).

[0037] Figure 6 shows the multimodal C dot scheme for targeting and characterizing αvβ3-integrin.

[0038] Figure 6a. Schematic representation of 124I-cRGDY-PEG-uyl core-shell silica nanoparticle with surface carrying radiolabels and peptides and core containing reactive dye molecules (insets).

[0039] Figure 6b. FCS results and single exponential fits for measurements of Cy5 dye in solution (in black), PEG-coated (PEG-dot, red), and PEG-coated cRGDY-tagged dots (blue, red data set below) showing differences of diffusion time as a result of varying hydrodynamic sizes.

[0040] Figure 6c. Hydrodynamic sizes (mean ± standard deviation, n=15), and comparisons of relative brightness of free dye with PEG-coated dots and cRGDY-PEG dots derived from FCS curves, along with corresponding dye and particle concentrations.

[0041] Figure 7 shows the purification and quality control of 124I-RGDY-PEG-dots using size exclusion column chromatography. Radioactivity (right column) of 124I-RGD-dots and 124I-PEG-dots detected by Y count and corresponding fluorescence signal intensity (Cy5, left column) of 124I-RGDY-PEG-dots and 124I-PEG-dots in each elutriated fraction.

[0042] Figure 8 shows competitive integrin receptor binding studies with 124I-cRGDY-PEG-dots, cRGDY peptide, and anti-αvβ3 antibody using two types of cells.

[0043] Figure 8a. High affinity and specific binding of 124I-cRGDY-PEG-dots to M21 cells by Y counting. Inset shows Scatchard analysis of binding data plotting the ratio of receptor-bound radioligand concentration (B) to unbound (or free, F), or the ratio of bound to free, B/F, versus receptor-bound receptor concentration, B; the slope corresponds to the dissociation constant, Kd.

[0044] Figure 8b. Blockade of M21 cells by αvβ3-integrin receptors using flow cytometry and excess non-radiolabeled cRGD or anti-αvβ3 antibody before incubation with cRGDY-PEG-dots.

[0045] Figure 8c. Specific binding of cRGDY-PEG-dots to M21 cells as against M21L cells lacking surface integrin expression using flow cytometry.

[0046] Figure 8d. Specific binding of cRGDY-PEG-dots to HUVEC cells by flow cytometry. Each bar represents mean ± standard deviation of three replicates.

[0047] Figure 9 shows the pharmacokinetic and excretion profiles of the targeted and non-targeted particle probes.

[0048] Figure 9a. Biodistribution of 124I-cRGDY-PEG-dots in M21 tumor-bearing mice at various time points from 4 to 168 h post injection. The inset shows a representative graph of these data for blood to determine the dwell half-life (TI/2).

[0049] Figure 9b. Biodistribution of 124I-PEG-dots from 4 to 96 h post injection.

[0050] Figure 9c. Clearance profile of urine samples collected up to 168 hours after injection of non-radiolabeled cRGDY-PEG-dots (n=3 mice, mean ± standard deviation).

[0051] Figure 9d. % of corresponding cumulative DI/g for feces at intervals up to 168 hours post injection (n=4 mice). For biodistribution studies, bars represent mean ± standard deviation

[0052] Figure 10 shows the results of the acute toxicity test.

[0053] Figure 10a. Liver stained with representative H&E at 400* (upper frames) and kidneys stained at 200* (lower frames). Mice were treated with a single dose of either non-radiolabeled 127I-RGDY-PEG-dots or 127I-PEG-coated dots (vehicle control) via intravenous injection and organs were harvested 14 days later.

[0054] Figure 10b. Average daily weights for each toxicity study treatment group. The scale bar in Figure 10a corresponds to 100 μm.

[0055] Figure 11 shows serial in vivo PET imaging of tumor-selective targeting.

[0056] Figure 11a. Typical full-body coronal microPET images at 4 hours post-injection demonstrating tumor uptakes of M21 (left, arrow) and M21L (middle, arrow) of 3.6 and 0.7% ID/g, respectively, and increased M21 tumor contrast at 24 hours (right).

[0057] Figure 11b. In vivo uptake of 124I-cRGDY-PEG-dots in M21 tumors overexpressing αvβ3 integrin (black, n=7 mice) and non-expressing M21L (light gray, n=5 mice) and 124I-PEG-dots in M21 (dark gray , n=5).

[0058] Figure 11c. Tumor to muscle M21 ratios for 124I-cRGDY-PEG-dots (in black) and 124I-PEG-dots (in gray).

[0059] Figure 11d. Correlation of in vivo and ex-vivo M21 tumor uptakes of cRGDY-labeled and unlabeled probes. Each bar represents the mean ± standard deviation

[0060] Figure 12 shows a nodal mapping using multiscale near-infrared optical fluorescence imaging.

[0061] Figure 12a. Whole-body fluorescence imaging of the tumor site (T) and inguinal (ILN) and axillary (ALN) draining lymph nodes and communicating lymphatic channels (bar, LC) 1 hour post injection in a surgically exposed live animal.

[0062] Figure 12b. Corresponding high-resolution, white-light cor-registered fluorescence images (top row) and fluorescence-only images (bottom row) revealing the nodal infrastructure of local and distant lymph nodes, including high endothelial venules (HEV). The largest scale bar in (b) corresponds to 500 μm.

[0063] Figure 13a shows the experimental setup of using a spontaneous mini pig melanoma model for mapping the lymph node basins and regional lymphatic drainage of the site of a known primary melanoma tumor.

[0064] Figure 13b shows a small field of view PET image 5 minutes after subdermal injection of multi-modal particles (124I-RGD-PEG-dots) around the tumor site.

[0065] Figure 14a shows dynamic whole-body 18F-fluorodeoxyglucose (18F-FDG) PET scan demonstrating sagittal, coronal, and axial images through the site of nodal disease in the neck.

[0066] Figure 14 b shows fused 18F-FDG PET-CT scans demonstrating sagittal, coronal, and axial images through the site of nodal disease in the neck.

[0067] Figure 14c shows the image of the mini pig's entire body.

[0068] Figure 15 shows the same sets of images as in Figure 14, but at the level of the primary melanoma lesion, adjacent to the spine over the upper back.

[0069] Figure 16a shows high-resolution dynamic PET images after subdermal injection, in 4 quadrants of 124I-RGD-PEG-dots over the tumor site over a period of time of 1 hour.

[0070] Figure 16b shows PET-CT images merged after subdermal injection, in 4 quadrants of 124I-RGD-PEG-dots around the tumor site over a period of time of 1 hour.

[0071] Figure 16c shows Cy5 imaging (top image), the resected lymph node (second top image), and H&E staining (bottom two images).

[0072] Figure 17 shows a schematic for a nanoparticle with a fluorescent dye inside the core and a PEG surface coating. The nanoparticle is decorated with triple bonds for subsequent "click chemistry" with both DFO and Tyr3-octreotate functionalized with azide groups.

[0073] Figure 18 shows PEG derivative structures. Routine chemical reactions are used to produce PEG functionalized with triple bonds, which will then be covalently attached to the nanoparticle through the silane group.

[0074] Figure 19 shows structures of DFO derivatives.

[0075] Figure 20a shows structures of Tyr3-octreotate.

[0076] Figure 20b shows the synthesis of the azide-containing acid for incorporation into Tyr3-Octreotate.

[0077] Figure 21a shows a schematic of the production of functionalized nanoparticle with a near-infrared fluorescent dye inside its core, a surface coating of PEG, DFO chelates and Tyr3-octreotate.

[0078] Figure 21b shows a schematic of the production of a multimodality 89Zr-labeled nanoparticle (PET and fluorescence) decorated with Tyr3-octreotate.

[0079] Figure 21c shows the tetrazine - norbornene bond.

[0080] Figure 21d shows a schematic of the strategy for creating radiolabeled core and shell nanoparticles using the tetrazine - norbornene linkage.

[0081] Figure 21e shows a schematic of the strategy for creating peptide-targeted core-shell radiolabeled nanoparticles using the tetrazine - norbornene linkage. Both one-step (in which the pre-metalated chelator-norbornene complex is reacted with the particle) and two-step (in which the chelator is metalated after conjugation) pathways are shown.

[0082] Figure 22 shows microscopic images demonstrating the co-localization between cRGF-PEG nanoparticles and lysotracker red in the endocytotic pathway.

[0083] Figures 23a to 23i Image-guided SLN (sentinel lymph node) mapping: Pre-operative PET imaging. (a,b) Axial CT images reveal a left pelvic soft tissue mass (a, arrow) and left frank sentinel lymph node (b, arrow). (c,d) Axial 18F-FDG images show localized activity within the tumor (c, arrow) and left frank sentinel lymph node (d, arrow) after i.v. tracer injection. Co-registered PET-CT images (e ) Axial and (f) coronal 124I-cRGDY-PEG-C dots show the injection site around the pelvic lesion (e, arrow). Corresponding co-registered (g) axial and (h) coronal PET-CT images localize activity to the sentinel lymph node (g, arrow). (i) Radioactivity levels of the primary tumor, sentinel lymph node (in vivo, ex vivo), and a site remote from the primary tumor (i.e., background), using a portable gamma probe.

[0084] Figures 24a to 24q Image-guided sentinel lymph node (SLN) mapping: Real-time intraoperative optical imaging with correlative histology. Intraoperative sentinel lymph node mapping was performed on the animal shown in Figures 23a to 23i. (a-i) Near-infrared optical imaging of two exposed nodal basin channels. Local injection of Cy5.5-incorporated particles presented in a dual-channel model (a) RGB color and (b) near-infrared fluorescent channels (in white). (c-f) Lymphatic drainage distal to the injection site. Fluorescence signal within the main proximal (c, d), middle (e), and distal (f) draining lymphatic channels (arrows) extending towards the sentinel lymph node ('N'). Smaller caliber channels are also shown (arrowheads). Images of the SLN presented in the (g) color and (h) near-infrared channels. (i) Image of the exposed SLN. (j-m) SLN images in color and near-infrared channels during (j,k) and after (l,m) excision, respectively. (n) Low power view of H&E stained SLN shows cluster of pigmented cells (black box) (bar = 1 mm). (o) Higher magnification of (n) reveals round pigmented melanoma cells and melanophages (bar = 50 μm). (p) Low power view of SLN stained with HMB45 confirms the presence of metastases (black box, bar=500 μm). (q) Higher magnification in (p) reveals clusters of melanoma cells expressing HMB45+ (bar = 100 μm).

[0085] Figures 25a to 25k Discrimination of inflammation from metastatic disease: Comparison of 18F-FDG and 124I-cRGDY-PEG C dot tracers. (a-d) Imaging of inflammatory changes using 18F-FDG-PET with tissue correlation. (a) Axial CT scan from 18F-FDG-PET study shows calcification within the left posterior neck (arrows). (b) Axial fused 18F-FDG PET-CT reveals hypermetabolic activity at this same site (arrows). Increased PET signal is also seen in metabolically active bone structures (asterisks). (c) Low- and (d) higher power views of calcified tissue stained with H&E demonstrate extensive inflammatory cell infiltration. (e-k) Detection of metastatic disease after injection of 124I-cRGDY-PEG C dots around the tumor site. (e) Pre-injection axial CT scan of 124I-cRGDY-PEG-C dots shows calcified soft tissue within the posterior neck (arrows). (f) Co-registered PET-CT shows no evident activity corresponding to calcified areas (arrow), but demonstrates a hypermetabolic lymph node on the right (arrowhead). (g) Axial CT at a more superior level shows lymph nodes (arrowheads) bilaterally and a calcified focus (arrow). (h) Fused PET-CT demonstrates PET-avid lymph nodes (N) and lymphatic drainage (curved arrow). Calcification shows no activity (arrow). (i) Low- and (j) high-power views confirm the presence of nodal metastases. (k) Single frame of a three-dimensional (3D) PET image reconstruction shows multiple bilateral metastatic lymph nodes (arrowheads) and lymphatic channels (arrow). Bladder activity is seen without significant tracer accumulation in the liver. Scale bars: 500 μm (c, d); 100 μm (i, j).

[0086] Figures 26a to 26c show Integrated 3D PET-CT of 18F-FDG and 124I-cRGDY-PEG-C dots. (a-c) 3D Volume rendered images were generated from CT and PET imaging data shown in Figures 7a to 7k. (a) PET-CT fusion image (coronal view) shows no obvious nodal metastases (asterisks). Increased activity within bone structures is identified. (b,c) High-resolution PET-CT fusion images showing coronal (b) and superior (c) views of bilateral metastatic lymph nodes (open arrows) and lymphatic channels (curved arrows) within the neck after tracer injection. local particles.

[0087] Figures 27a to 27o. Assessment of treatment response after radiofrequency ablation (RFA) using 124I-cRGDY-PEG-C dots. (a-c) Location of the sentinel lymph node by single-dose particle radiotracer. (a) Basal coronal CT (white arrowhead), (b) PET (black arrowhead), and (c) fused PET-CT (white arrowhead) images after a peritumoral injection. (b–d) Tumor particle tracker activity. (b) PET avid exophytic left pelvic mass (black arrow). (c, d) Combined PET-CT images showing a hypermetabolic lesion (white arrow) and particle tracer flow within a draining lymphatic channel (asterisk) to the sentinel lymph node (curved arrow). (e,f) Pre-ablation axial CT images localize the sentinel lymph node (e, white arrowhead) before RFA electrode placement (f, arrow) within the lymph node (crosshairs below). (g) Pre-ablation fused PET-CT reveals increased sentinel lymph node activity (posterior to mira). (h) Post-ablation PET-CT scan shows mildly reduced activity at the sentinel lymph node site, anterior to the needle tip. (i) Corresponding pre-ablation H&E staining of sentinel lymph node core biopsy tissue confirms pigmented tumor infiltration (bar = 200 μm). (j) High magnification of boxed area in (i) reveals large clusters of rounded, pigmented melanoma cells (bar = 50 μm). (k) Post-ablation H&E staining shows necrotic changes within a partially tumor-infiltrated lymph node (box) and multifocal hemorrhages (bar = 500 μm). (l) High magnification of (k) reveals significant tissue necrosis (arrowheads) within the metastatic lymph node in addition to lymphoid tissue (bar = 50 μm). (m) TUNEL staining of metastatic sentinel lymph node before ablation (bar = 20 μm). (n) Post-ablation TUNEL staining demonstrating focal areas of necrosis with adjacent scattered tumor foci and normal nodal tissue (NT) (bar = 500 μm). (o) High magnification of boxed area in (n) shows positive TUNEL staining, consistent with necrosis (bar = 20 μm).

[0088] Figures 28A to 28C. Core-shell hybrid silica nanoparticle platform (dots of 124I-cRGDY-PEG-C) and study schematic overview. (A) Schematic representation of hybrid inorganic imaging probe (PET-optics) (right) showing the core containing deep red dye and polyethylene glycol (PEG) chains attached to the surface that carry cRGDY peptide ligands and radiolabels for detection of tumors expressing Human αvβ3 integrin (left). (B) Combined absorption spectra (left: red, black curves) and emission spectra (right) for free (blue curve) and encapsulated (green curve) dyes revealing enhanced fluorescence of encapsulated fluorophores. (C) Timeline of clinical trial events. Biol specs, biological samples (blood, urine).

[0089] Figures 28D to 28E. Whole-body distribution and pharmacokinetics of 124I-cRGDY-PEG-C dots. (D) Maximum intensity projection (MIP) PET images at 2 hours (left), 24 hours (middle), and 72 hours (right) post injection of 124I-cRGDY-PEG-C dots reveal activity in the bladder ( *), in the heart (yellow arrow), and in the intestine (white arrowhead). (E) The percentage of injected dose per gram corrected for decay (% ID/g) of urine and plasma collected approximately 30 min, 4 h, 24 h and 72 h after particle injection was determined by gamma ray counting; Individual graphs were generated for each patient. ROIs were drawn over the major organs for each patient's PET scans for each patient to derive standardized uptake values and % DI/g.

[0090] Figure 29 Metabolic analyzes of biological samples. (A, B) Time-dependent activity concentrations (% DI/gx100) in plasma and urine, respectively, corrected for decay for the time of injection. (C to H) RadioTLC (4:1 acetic acid : methanol as mobile phase) of plasma and urine samples (counts per minute, cpm, decay corrected). (C to E) Plasma chromatograms show a single peak at 0.5 hour (C), 3 hours (D), and 24 hours (E) post injection (F to H). Chromatograms of urine samples reveal two peaks at 0.5 hour (F), 3 hours (G), 24 hours (H) post injection. Insets (G, H) present respective data scaled to a maximum of 50 cpm. (I to K) Chromatograms of standards: injected (I), radioiodinated peptide (131I) (J) and free 131I (K). Vertical lines discriminate peaks corresponding to particle tracker (long dashes; Rf = 0.04), 131I-cRGDY (short dashes; Rf = 0.2), and 131I (dotted; Rf = 0.7).

[0091] Figure 30 Whole-body PET-CT particle imaging biodistribution and preferential tumor uptake after systemic injection of 124I-cRGDY-PEG-C dots (A) Reformatted coronal CT demonstrates a hypodense and hypodense left hepatic lobe metastasis well defined (arrowhead). (B) Coronal PET imaging at 4 hours after injection demonstrates increased activity throughout the peripheral aspect of the tumor (arrowhead), in addition to the bladder, gastrointestinal tract (stomach, intestines), gallbladder, and heart. (C) Coregistered PET-CT localizes activity to the tumor margin.

[0092] Figure 31 Multimodal particle uptake imaging in a pituitary lesion. (A to B) Multiplanar contrast-enhanced axial (A) and sagittal (B) MR images at 72 hours post injection demonstrate a subcentimeter cystic focus (arrows) within the right aspect of the anterior pituitary gland. (C to D) Co-registered axial (C) and sagittal (D) MRI-PET images reveal increased focal activity (red) localized to the site of injury. (E to F) Axial (E) and sagittal (F) PET-CT images localize activity to the right aspect of the sella. (G to I) Axial PET images at 3 hours (G), 24 hours (H) and 72 hours (I) post injection demonstrate progressive accumulation of activity (SUVmax) within the sellar region along with a corresponding decline in activity background around the lesion. (J) Ratios of tumor to brain (T/B) and tumor to liver (T/L) activity increasing as a function of post-injection times.

[0093] Figure 32. Structure of the N-Ac-Cys-(Ahx)2-D-Lys-ReCCMSH (or alpha MSH) peptide used for nanoparticle conjugation.

[0094] Figure 33. Structure of the original Re-CCMSH targeting molecule.

[0095] Figures 34A and 34B. Competitive binding studies using a melanocortin-1 receptor agonist (125I-NDP). Particles conjugated to N-Ac-Cys-(Ahx)2-D-Lys-ReCCMSH (or alpha-MSH) (Figure 34A) had greater affinity for cultured B16/F1 melanoma cells than a version of the scrambled sequence molecule ( Figure 34B).

[0096] Figures 35A and 35B. Dose-response data were obtained as a function of targeted particle concentrations (Figure 35A) and incubation times (Figure 35B) for both B16F10 and M21 melanoma cell lines.

[0097] Figure 36. Survival studies of human M21 cells, carried out over a range of particle concentrations for a fixed incubation time of 48 hours, did not demonstrate any significant loss of cell viability.

[0098] Figures 37A and 37B. 125I-Radiolabeled alpha-MSH-conjugated nanoparticles demonstrated largely renal excretion over a 24-hour period in both M21 and B16F10 murine xenograft models.

[0099] Figures 38A and 38B. Neither M21 nor B16F10 murine xenograft models showed significant accumulation of the targeted particle probe in the reticuloendothelial system (i.e., not an agent of the reticuloendothelial system) nor in the kidney.

[00100] Figure 39. Competitive binding of integrin receptors and temperature-dependent uptake using cRGDY-PEG-C dots and anti-αvβ3 antibody for 2 cell types. A. Specific binding and uptake of cRGDY-PEG-C dots in M21 cells as a function of temperature (4° C., 25° C., 37° C.) and concentration (25 nM, 100 nM) using anti-αvβ3 integrin receptor and flow cytometry. Anti-αvβ3 integrin receptor antibody concentrations were 250 times (i.e., 250x) the particle concentration. B. Uptake of cRGDY-PEG-C dots in M21 cells, as against M21L cells lacking normal surface integrin expression by flow cytometry. C. Selective particle uptake in HU-VECs cells using anti-αvββ integrin receptor antibody and flow cytometry. D. Co-localization test of cRGDY-PEG-C dots (1 μM, red) with endocytic (transferrin-Alexa-488, FITC-dextran, green) and lysosomal (LysoTracker Red) markers after 4 h particle incubation using M21 cells. Co-localized vesicles (yellow), Hoechst counterstain (blue). Scale bar = 15 μm. Each data point (A to C) represents the mean ± standard deviation of 3 replicates.

[00101] Figure 40. Expression levels of FAK, Src, MEK, Erk1/2, and phosphorylated Akt in M21 cells. A. The FAK/Src complex transduces signals from integrin cell surface receptors through activation of downstream signaling pathways (PI3K-Akt, Ras-MAPK) to elicit a range of biological responses (boxes indicate protein intermediates evaluated). B. Western blots of phosphorylated protein and total protein expression levels of key pathway intermediates after exposure (2 h, 37° C.) of G0/G1 phase-synchronized M21 cells to cRGDY-PEG-C-dots of 100 nM relative to cells in serum-deprived medium (0.2% FBS) (i.e., control). After trypsinization of the cells and resuspension of the pellet in lysis buffer, proteins were resolved by 4 to 12% gradient SDS-PAGE and analyzed by antibodies anti-FAK 397, pFAK 576/577, p-Src, pMEK, pErk1 /2, and pAkt. Antibodies against FAK, Src, MEK, Erk, and Akt were also used to detect the amount of total protein. C. Graphical summary of the percentage of signal intensity changes in phosphorylated protein to total protein (Adobe Photoshop CS2; see Methods) for particle-exposed cells versus serum-deprived cells. GF, growth factors; EC, endothelial cell; ECM, extracellular matrix.

[00102] Figure 41. Signaling induction and inhibition studies in M21 cells using PF-573228 (PF-228), a FAK inhibitor. A. Western blots of phosphorylated and total protein expression levels using the preceding procedure of Figure 2 with the addition of 250 nM or 500 nM PF-228 (0.5 h, 37° C.) to cells before exposure to particles. B. Summary of percentage changes in intensity from phosphorylated to total protein expression levels in particle-exposed cells versus control cells with and without PF-228.

[00103] Figure 42. Effect of cRGDY-PEG-C dots on M21 cell migration using time-lapse imaging. A. Time-dependent changes in cell migration using ORIS® collagen-coated plates for a range of particle concentrations (0 to 400 nM; 37° C.) in RPMI 1640 medium supplemented with 0.2% FBS, as counter medium supplemented alone (controls). Images were captured at time t = 0 (pre-migration) and at subsequent 24 h intervals after shutter removal by a Zeiss Axiovert 200M inverted microscope (5x/0.25NA objective) and a sliding scanning module (Metamorph ® Microscopy Automation & Image Analysis Software) for a total of 96 hours. B. Graphical representation of changes in mean closure area (%) as a function of concentration using ImageJ software. The average closure area represents the difference in areas demarcated by the advancing cell border (pixels) at arbitrary time points and after shutter removal (t = 0), divided by the latter area. Quadruplicate samples were statistically tested for each group using a one-tailed t-test: *, p=0.011; **, p=0.049; ***, p=0.036. Scale bars = 100 μm and 33 μm (x and xv amplified images).

[00104] Figure 43. Effect of cRGDY-PEG-C dots on HUVEC cell migration. A. Serial migration of HUVEC was assessed using the same process as in Figure 4 over a 24-hour time interval. The images presented and the closure area values indicated are representative of a single experiment. B. Average closure areas (%) were determined during this time interval for a range of particle concentrations (0 to 400 nM) using ImageJ software. Tests were performed in triplicate for each concentration and time point. One-tailed T-test *, p<0.05. Scale bar = 76 μm.

[00105] Figure 44. Inhibition of HUVEC cell migration using PF-228. A. Same process as in Figure 5, with the exception that cells were exposed to 250 nM and 500 nM PF-228 (0.5 h, 37° C.) prior to particle exposure or incubation at 0. 2% medium supplemented with FCS. B. Average area closure (%) for cells incubated under the preceding conditions. C. Tabulated p-values for each exposure condition using a one-tailed t-test. Quadruplicate samples were taken for each inhibitor concentration. Scale bar = 50 μm.

[00106] Figure 45. Modulation of the spread and adhesion of M21 cells using cRGDY-PEG-C dots. A. Time-lapse imaging showing changes in cell attachment and cell spreading. Cells were preincubated in 0.2% RPMI supplemented with FBS (0.5 h, 25° C.), without and with particles (400 nM), followed by seeding in (5 μg/ml) 96-well plates. cavities coated with fibronectin. Images were captured at t = 0, 0.5 h, 1 h, and 2 h using a Zeiss Axiovert 200M inverted microscope (20x/0.4NA objective) and a Metamorph® slide-scan module. B. Plot showing the average number of rounded and elongated cells within two groups as a function of time: not exposed to particles (elongated, graph #1) and exposed to particles (rounded, graph #2). Cells in each of three wells of a 96-well plate were counted manually in a minimum of three high-power fields (x200 magnification) and averaged. C. Absorbance values (À = 650 nm; SpectroMax M5 microplate reader) for cells fixed with 4% paraformaldehyde, exposed to medium or 400 nM cRGDY-PEG C-dots, and treated with methylene blue reagent (1 ml; 1 h, 37° C.), as a measure of cell attachment. Scale bar = 30 μm. Quadruplicate samples were taken for each group.

[00107] Figure 46. Influence of cRGDY-PEG-C dots on the cell cycle. A. Percentage (%) of viable cells in G1, S, and G2 phases of the cell cycle as a function of particle concentration (0, 100, 300 nM) added to M21 cells synchronized to G0/G1 phase incubated for a total of 96 hours. Numbers in italics above each bar represent the percentage of cells (incubated with particles, control) in S, G1 and G2 phase, determined by flow cytometry. B. Representative cell cycle histograms are shown for cells under control conditions (i.e., no particle) and after incubation with 100 and 300 nM particles. One-way ANOVA: *, p<0.05; **, p<0.005 in relation to S phase control. Inserts: Mean values per group (n = 3) ± standard deviation for each phase of the cell cycle. Experiments were performed in triplicate.

[00108] Figure 47. Dose-response effects and saturation binding kinetics using cRGDY-PEG-C dots and cells expressing αvβ3 integrin. A, B. Cellular binding/uptake as a function of particle concentration (A) and incubation time (B) by flow cytometry. Monolayers of M21 cells and HUVEC were incubated (4 h, 25° C.) with increasing concentrations of particles (5 to 600 nM) as well as over a range of incubation times (0.5 to 4 h; 100 nM) and evaluated by fluorescence-activated cell assortment (FACS) analysis. The percentage of total events detected is displayed. Each data point represents mean ± standard deviation of 3 replicates.

[00109] Figure 48. Competitive binding of integrin receptors with 124I-cRGDY-PEG-C dots and cRGDY peptide in two cell types. A. Specific binding of 124I-cRGDY-PEG-dots to M21 cells and HUVEC after incubation with excess cRGDY peptide using gamma radiation counting. Monolayers of M21 cells and HUVEC were incubated (4 h, 25° C.) with 25 nM of 124I-cRGDY-PEG-dots in the presence and absence of cRGDY peptide (85, 170 nM). Binding is expressed as a percentage of control (i.e., radioiodinated cRGDY-PEG-dots). B. Tumor-directed binding of non-radiolabeled cRGDY-PEG-C dots. M21 cells and HUVEC were incubated for 4 h at 25°C with two concentrations of targeted particles (25 nM, 100 nM) or with particle controls (PEG-C dots) and evaluated by flow cytometry.

[00110] Figure 49. Viability and proliferation of M21 and HUVEC cells as a function of particle concentration and incubation time. A, B. Absorbency (Àex=440 nm) as a measure of viability in subconfluent, G0/G1 phase synchronized M21 (A) and HUVEC (B) cells over a 24 hour period using 10% or 2% supplemented medium of FBS alone, respectively, or with added particles (25 to 200 nM). C, D. Cell proliferative activity in M21 (C) and HUVEC (D) cells over a 93 h time interval using either medium alone or particle-containing media as specified in A, B.

[00111] Figure 50. Changes in cell signaling as a function of particle incubation time in M21 cells. Western blots of selected phosphorylated protein intermediates and total protein intermediates over an 8 h time period. The normalized intensity ratios (i.e., difference of phospho-protein and total protein divided by the latter) are illustrated graphically.

[00112] Figure 51. Cell signaling modulation as a function of particle concentration in M21 cells. Western blots of selected phosphorylated protein intermediates and total protein intermediates over a range of particle concentrations (i.e., 0 to 400 nM).

[00113] Figure 52. Cell signaling inhibition studies in M21 cells. A. Western blots of selected phosphorylated protein intermediates and total protein intermediates (serum-free medium, 100 nM particles alone, or 100 nM particles after addition of 250 nM or 500 nM inhibitor). B. Relative intensities of phospho-protein and β-actin blots in A relative to control cells (0.2% FBS).

DETAILED DESCRIPTION OF THE INVENTION

[00114] The present invention provides a silica-based fluorescent nanoparticle that allows the precise detection, characterization, monitoring and treatment of a disease such as cancer. The invention also provides a method for detecting tumor cells. The method may contain the following steps: (a) administering to a patient a plurality of silica-based fluorescent nanoparticles at a dose ranging from about 0.01 nanomol/kg of body weight to about 1 nanomol/kg of body weight, from about 0.05 nanomol/kg of body weight to about 0.9 nanomol/kg of body weight, from about 0.1 nanomol/kg of body weight to about 0.9 nanomol/kg body weight, from about 0.2 nanomol/kg body weight to about 0.8 nanomol/kg body weight, from about 0.3 nanomol/kg body weight body weight to about 0.7 nanomol/kg body weight, from about 0.4 nanomol/kg body weight to about 0.6 nanomol/kg body weight, or from about 0.2 nanomol/kg body weight to about 0.5 nanomol/kg body weight, and (b) detection of the nanoparticles. In one embodiment, the nanoparticle comprises a silica-based core having a fluorescent compound positioned within the silica-based core; a silica shell surrounding at least a portion of the core; an organic polymer attached to the nanoparticle; a ligand attached to the nanoparticle and capable of binding a tumor marker; and at least one therapeutic agent.

[00115] The nanoparticle has a range of diameters including between about 0.1 nm and about 100 nm, between about 0.5 nM and about 50 nm, between about 1 nm and about 25 nm, between about from 1 nm to about 15 nm, or from about 1 nm to about 8 nm. The nanoparticle has a fluorescent compound positioned within the nanoparticle, and has a higher brightness and fluorescent quantum yield than the free fluorescent compound. The nanoparticle also has high biostability and biocompatibility. In order to facilitate efficient urinary excretion of the nanoparticle, it can be coated with an organic polymer, such as poly(ethylene glycol) (PEG). The small nanoparticle size, silica base, and organic polymer coating minimize nanoparticle toxicity when administered in vivo. In order to target a specific cell type, the nanoparticle can be additionally conjugated to a ligand, which is capable of binding to a cellular component (e.g., the cell membrane or other intracellular component) associated with the specific cell type. , such as a tumor marker or a signaling pathway intermediate. In one embodiment, a therapeutic agent can be attached to the nanoparticle. In order to allow the nanoparticle to be detectable by not only optical imaging (such as fluorescence imaging) but also by other imaging techniques such as positron emission tomography (PET), single photon emission computed tomography (SPECT) ), computed tomography (CT), and magnetic resonance imaging (MRI), the nanoparticle can also be conjugated to a contrast agent, such as a radionuclide.

[00116] The properties of nanoparticles lead to large-volume excretion through the kidneys, increased potency relative to a native peptide ligand, increased uptake, and preferential accumulation in tumors compared to normal tissues. This, along with the lack of in vivo toxicity, resulted in a unique product resulting in its translation to the clinic.

[00117] The present particles can be used to preferentially detect and localize tumors. For example, doses of nanomolar particle tracers administered in a microdosing regimen accumulate and preferentially localize to disease sites, although not optimized for targeted detection.

[00118] The present nanoparticles may exhibit distinct and reproducible human pharmacokinetic signatures in which renal clearance predominates (e.g., renal clearance of the nanoparticle is greater than about 90% of DI in about 24 hours after administration of the nanoparticle to one subject) without significant uptake by the reticuloendothelial system (e.g., less than about 10%).

[00119] The present nanoparticles are excellent diagnostic probes, presenting excellent physicochemical properties in humans that allow them to "reach the target and be removed" ("target and clear") from the body during relatively short intervals of time (e.g. example, hours, days, etc.).

[00120] The present particles carrying multiple actively targeted ligands (e.g., cRGDY and alpha-MSH) demonstrate increased binding affinity to cellular targets compared to the affinity of an isolated ligand. In some embodiments, the present particles bind to a cellular target from about 2 to about 20 times more, from about 3 to about 15 times more, from about 5 to about 10 times more. than an isolated ligand.

[00121] In certain embodiments, dual modality targeted particles specifically assess tumor burden and can discriminate metastatic tumor from chronic inflammatory disease in large animal models of metastatic melanoma.

[00122] Targeted particles can increase receptor binding affinity and avidity, increase plasma residence times, bioavailability and tumor retention, and/or promote intracellular release through internalization. The use of similar targeted probes within surgical (or medical) oncology situations may enable highly selective treatment of cancer-bearing tissues, potentially reducing rates of concomitant complications. The present particles may be advantageous over passively targeted nanocarriers, since the latter penetrate and accumulate nonspecifically within the tumor interstitium by increasing permeability and retention (EPR) effects.

[00123] Particle-based imaging systems for cancer diagnosis must be non-toxic, and selectively detect sites of primary and metastatic disease while presenting relatively rapid renal clearance. Under these conditions, the likelihood of potential toxicity will be reduced given the smaller area under the plasma concentration-time curve. In one embodiment, these renal clearance properties can be achieved by ultrasmall particle-based platforms or macromolecular systems that meet effective renal glomerular filtration size cutoffs of 10 nm or less. The absence of acute single-dose toxicity and the minimization of similar risks will also be important. A platform regimen should maximize safety through rapid whole-body clearance and the selection of biokinetic profiles that minimize nonspecific uptake in the reticuloendothelial system (RES), thereby reducing potential adverse exposures.

[00124] In one embodiment, the present particles are safe and stable in vivo. The particles exhibit distinctly unique and reproducible pharmacokinetic signatures defined by renal excretion. Together with preferential uptake and localization of the probe at sites of disease, these particles can be used in cancer diagnosis.

[00125] The nanoparticle can have both a ligand and a contrast agent. The ligand allows the nanoparticle to target a specific cell type through specific binding between the ligand and the cellular component. This targeting, combined with multimodal imaging, has multiple uses. For example, nanoparticles can be used for mapping metastatic disease, such as sentinel lymph node (SLN) mapping, as well as for identifying tumor margins or neural structures, allowing the surgeon to resect malignant lesions under direct visualization. and prevent complications during the surgical procedure. The ligand can also facilitate nanoparticle entry into the cell or barrier transport, for example, to assess the intracellular environment.

[00126] The nanoparticle can be coupled with a ligand and a therapeutic agent with or without a radiolabel. The radiotracer can additionally serve as a therapeutic agent to create a theranostic platform. This coupling allows the therapeutic particle to be released to the specific cell type through the specific binding between the ligand and the cellular component. This specific binding of the therapeutic agent ensures selective treatment of the disease site with minimal side effects.

Nanoparticle Structure

[00127] The fluorescent nanoparticle of the present invention includes a silica-based core comprising a fluorescent compound positioned within the core, and a silica shell over the core. The silica shell may surround at least a portion of the core. Alternatively, the nanoparticle may have only the core and no shell. The nanoparticle core may contain the reaction product of a reactive fluorescent compound and a co-reactive organosilane compound. In another embodiment, the nanoparticle core may contain the reaction product of a reactive fluorescent compound and a co-reactive organosilane compound, and silica. The core diameter may be from about 0.05 nm to about 100 nm, from about 0.1 nm to about 50 nm, from about 0.5 nm to about 25 nm , from about 0.8 nm to about 15 nm, or from about 1 nm to about 8 nm. The nanoparticle shell may be the reaction product of a silica-forming compound. The nanoparticle shell can have a range of layers. For example, the silica shell may have from about 1 to about 20 layers, from about 1 to about 15 layers, from about 1 to about 10 layers, or from about 1 to about 10 layers. from 1 to about 5 layers. The shell size can vary from about 0.01 nm to about 90 nm, from about 0.02 nm to about 40 nm, from about 0.05 nm to about 20 nm , from about 0.05 nm to about 10 nm, or from about 0.05 nm to about 5 nm.

[00128] The silica shell of the nanoparticle can cover only a portion of the nanoparticle or the entire particle. For example, the silica shell may cover from about 1 to about 100 percent, from about 10 to about 80 percent, from about 20 to about 60 percent, or from about from 30 to about 50 percent of the nanoparticle. The silica shell may be either solid, that is, substantially non-porous, meso-porous, such as semi-porous, or porous.

Nanoparticle Synthesis

[00129] The present fluorescent nanoparticle can be synthesized by the steps of: covalently conjugating a fluorescent compound, such as a reactive fluorescent dye, with the reactive moieties including, but not limited to, maleimide, iodoacetamide, thiosulfate, amine , N-Hydroxysuccimide ester, 4-sulfo-2,3,5,6-tetrafluorophenyl ester (STP), sulfosuccinimidyl ester, sulfodichlorophenol esters, sulfonyl chloride, hydroxyl, isothiocyanate, carboxyl, to an organocompound silane, such as a co-reactive organosilane compound, to form a fluorescent silica precursor, and reacting the fluorescent silica precursor to form a fluorescent core; covalently conjugating a fluorescent compound, such as a reactive fluorescent dye, with an organosilane compound, such as a co-reactive organosilane compound, to form a fluorescent silica precursor, and reacting the silica precursor fluorescent with a silica-forming compound, such as tetraalkoxysilane, to form a fluorescent core; and reacting the resulting core with a silica-forming compound, such as a tetraalkoxysilane, to form a silica shell over the core, to provide the fluorescent nanoparticle.

[00130] The synthesis of monodisperse fluorescent core and shell nanoparticles is based on a two-step process. First, near-infrared organic dye molecules (e.g., tetramethylrhodamine isothiocyanate (TRITC)) are covalently conjugated to a silica precursor and condensed to form a dye-rich core. Second, silica gel monomers are added to form a denser silica redd around the fluorescent core material, providing protection against solvent interactions that can be detrimental to photostability. The versatility of the preparation route allows the incorporation of different fluorescent compounds, such as dyes or fluorescent organic compounds, depending on the intended application of the nanoparticles. Fluorescent compounds that can be incorporated into the dye-rich core can cover the entire emission and absorption spectrum from UV-Vis to near-infrared. United States Patent Application Ser. Nos. 10/306,614, 10/536,569 and 11/119,969. Wiesner et al., PEG-coated Core-shell Silica Nanoparticles and Methods of Manufacture and Use, PCT/US2008/74894.

[00131] For the synthesis of the compact core-shell nanoparticle, the dye precursor is added to a reaction vessel containing appropriate amounts of ammonia, water and solvent and left to react overnight. The dye precursor is synthesized by addition reaction between a dye close to the specific red of interest and 3-aminopropyltriethoxysilane in a molar ratio of 1:50, excluding moisture. After the synthesis of the compact dye-rich core is completed, tetraethylorthosilicate (TEOS) is next added to grow the silica shell that surrounded the core.

[00132] The synthesis of the expanded core and shell nanoparticle is carried out by co-condensing TEOS with the dye precursor and allowing the mixture to react overnight. After the synthesis of the expanded core is completed, additional TEOS is added to grow the silica shell that surrounded the core.

[00133] The synthesis of homogeneous nanoparticles is carried out by co-condensing all the reagents, the dye precursor and TEOS and leaving the mixture to react overnight.

Fluorescent Compound

[00134] Nanoparticles can incorporate any known fluorescent compound, such as fluorescent organic compounds, dyes, pigments, or combinations thereof. A wide variety of suitable chemically reactive fluorescent dyes are known, see, for example, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, 6th ed., R. P. Haugland, ed. (1996). A typical fluorophore is, for example, an aromatic or heteroaromatic fluorescent compound such as a pyrene, an anthracene, a naphthalene, an acridine, a stilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine, a carbocyanine, a carbostyril, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, a coumarin (including hydroxycoumarins and aminocoumarins and fluorinated derivatives thereof), and similar compounds, see, for example, United States Patent Nos. 5,830,912, 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,433,896, 4,810,636 and 4,812,409. In one embodiment, Cy5, a near-infrared fluorescent (NIRF) dye, is positioned within the silica core of the present nanoparticle. Near-infrared emission probes show decreased attenuation and tissue autofluorescence. Burns et al. "Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine", Nano Letters, 2009, 9 (1), 442-448.

[00135] Non-limiting fluorescent compounds that can be used in the present invention include Cy5, Cy5.5 (also known as Cy5++), Cy2, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, Cy7, fluorescein (FAM ), Cy3, Cy3.5 (also known as Cy3++), Texas Red, LightCycler- Red 640, LightCycler Red 705, tetramethylrhodamine (TMR), rhodamine, rhodamine derivative (ROX), hexachlorofluorescein (HEX), rhodamine 6G (R6G) , the rhodamine derivative JA133, Alexa Fluorescent Dyes (such as Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 555, and Alexa Fluor 647), 4',6-diamidino-2-phenylindole (DAPI), Iodide of propidium, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, and fluorescent transition metal complexes such as europium. Fluorescent compounds that can be used also include fluorescent proteins such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein and derivatives (CFP, ECFP, Cerulean, CyPet) and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet). International Patent Publication No. WO2008142571, International Patent Publication No. WO2009056282, and International Patent Publication No. WO9922026.

[00136] The surface of the silica shell of the nanoparticles can be modified using known cross-linking agents to introduce surface functional groups. Crosslinking agents include, but are not limited to, divinyl benzene, ethylene glycol dimethacrylate, trimethylol propane trimethacrylate, N,N'-methylene bisacrylamide, alkyl ethers, sugars, peptides, DNA fragments, or other agents. known functionally equivalents. The ligand may be conjugated to the nanoparticle of the present invention, for example, through binding reactions using carbodiimide, carboxylates, esters, alcohols, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, halides, or any other suitable compound known in the art. art. United States Patent No. 6,268,222.

Organic Polymer

[00137] An organic polymer can be attached to the present nanoparticle, for example, attached to the surface of the nanoparticle. An organic polymer can be attached to the silica shell of the present nanoparticle. The organic polymer that can be used in the present invention includes PEG, polylactate, polylactic acids, sugars, lipids, polyglutamic acid (PGA), polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), and combinations thereof. The attachment of the organic polymer to the nanoparticle can be carried out by a covalent bond or by a non-covalent bond, such as by ionic bond, hydrogen bond, hydrophobic bond, coordination, adhesive, and physical absorption. In one embodiment, the nanoparticle is covalently conjugated with PEG, which prevents adsorption of serum proteins, facilitates efficient urinary excretion, and decreases nanoparticle aggregation. Burns et al. "Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine", Nano Letters, 2009, 9 (1), 442-448.

[00138] The surface of the nanoparticle can be modified to incorporate at least one functional group. The organic polymer (e.g., PEG) attached to the nanoparticle can be modified to incorporate at least one functional group. For example, the functional group may be a maleimide or an N-Hydroxysuccinimide (NHS) ester. The incorporation of the functional group makes it possible to attach various ligands, contrast agents and/or therapeutic agents to the nanoparticle.

Ligand

[00139] A ligand can be attached to the present nanoparticle. The ligand is capable of binding to at least one cellular component. The cellular component may be associated with specific cell types or have elevated levels in specific cell types, such as cancer cells or cells specific to particular tissues and organs. Therefore, the nanoparticle can target a specific cell type, and/or provide targeted release for the treatment and diagnosis of a disease. As used herein in this patent application, the term "ligand" refers to a molecule or entity that can be used to identify, detect, target, monitor, or modify a physical state or condition, such as a disease state or condition. . For example, a ligand can be used to detect the presence or absence of a particular receptor, the expression level of a particular receptor, or metabolic levels of a particular receptor. The ligand may be, for example, a peptide, a protein, a protein fragment, a peptide hormone, a sugar (i.e., lectins), a biopolymer, a synthetic polymer, an antigen, an antibody, an antibody fragment ( e.g., Fab, nanobodies), an aptamer, a virus or viral component, a receptor, a hapten, an enzyme, a hormone, a chemical compound, a pathogen, a microorganism or a component thereof, a toxin, a surface modifier, such as a surfactant to alter the surface properties or histocompatibility of the nanoparticle or an analyte when a nanoparticle associates with it, and combinations thereof. In one embodiment, the ligands are antibodies, such as monoclonal or polyclonal antibodies. In another embodiment, the ligands are receptor ligands. In yet another embodiment, the linker is poly-L-lysine (pLysine).

[00140] An antigen can be attached to the nanoparticle. The nanoparticle attached to the antigen can be used for vaccination.

[00141] The terms "component of a cell" or "cellular component" refer, for example, to a receptor, an antibody, a hapten, an enzyme, a hormone, a biopolymer, a antigen, a nucleic acid (DNA or RNA), a microorganism, a virus, a pathogen, a toxin, combinations thereof, and similar components. The component of a cell may be positioned on the cell (e.g., a transmembrane receptor) or within the cell. In one embodiment, the component of a cell is a tumor marker. As used herein in this patent application, the term "tumor marker" refers to a molecule, entity or substance that is expressed or overexpressed in a cancer cell but not in a normal cell. For example, overexpression of certain receptors is associated with myotropic cancers. A ligand capable of binding to a tumor marker can be conjugated to the surface of the present nanoparticle, so that the nanoparticle can specifically target the tumor cell.

[00142] A linker can be attached to the present nanoparticle directly or through a linker. The attachment of the ligand to the nanoparticle can be carried out by a covalent bond or non-covalent bond, such as by ionic bond, hydrogen bond, hydrophobic bond, coordination, adhesive, and physical absorption. The ligand can be coated onto the surface of the nanoparticle. The ligand can be embedded into the surface of the nanoparticle. The ligand can be attached to the surface of the fluorescent nanoparticle, or it can be attached to the core when the shell is porous or is covering a portion of the core. When the linker is attached to the nanoparticle via a linker, the linker can be any suitable molecules, such as a functionalized PEG. PEGs can have multiple functional groups for attachment to the nanoparticle and ligands. The particle can have different types of functionalized PEGs carrying different functional groups that can be attached to multiple ligands. This can enhance multivalency and/or contrast effects at the target site, which allow the design and optimization of a complex multimodal platform with improved targeted sensing, treatment, and in vivo detection.

[00143] A variety of different ligands can be attached to the nanoparticle. For example, the tripeptide Arg-Gly-Asp (RGD) can be attached to the nanoparticle. Alternatively, the cyclic peptide cRGD (which may contain another amino acid(s), e.g., cRGDY) may be attached to the nanoparticle. Any linear, cyclic or branched peptide containing the RGD sequence is within the scope of the present invention. RGD binds the αvβ3 integrin, which is overexpressed on the surface of activated endothelial cells during angiogenesis and in several types of tumor cells. It has been shown that αvβ3 integrin expression levels correlate well with the aggressiveness of tumors. Ruoslahti et al. New perspectives in cell adhesion: RGD and integrins. Science 1987; 238:491. Gladson et al. Glioblastoma expression of vitronectin and alpha v beta 3 integrin. Adhesion mechanism for transformed glial cells. J. Clin. Invest. 1991; 88:1924-1932. Seftor et al. Role of the alpha v beta 3 integrin in human melanoma cell invasion. Proc. Natl. Academic. Sci. 1992; 89:1557-1561.

[00144] The synthetic peptide EPPT1 can be the ligand attached to the nanoparticle. EPPT1, derived from the monoclonal antibody binding site (ASM2), targets under-glycosylated MUC1 (uMUC1). MUC1, a transmembrane receptor, is heavily glycosylated in normal tissues; however, it is overexpressed and aberrantly underglycosylated in almost all human epithelial cell adenocarcinomas, and is implicated in tumor pathogenesis. Moore et al. In vivo targeting of underglycosylated MUC-1 tumor antigen using a multimodal imaging probe. Cancer Res. 2004; 64:1821-7. Patel et al. MUC1 plays a role in tumor maintenance in aggressive thyroid carcinomas. Surgery. 2005; 138:994-1001. Specific antibodies including monoclonal antibodies against uMUC1 can alternatively be conjugated to the nanoparticle to target uMUC1.

[00145] In one embodiment, peptide analogs of α-melanotropin stimulating hormone (α-MSH) are the ligands attached to the nanoparticle. Peptide analogues of α-MSH are capable of binding to melanocortin-1 receptors (MC1R), a family of G protein-coupled receptors overexpressed in melanoma cells. Loir et al. Cell Mol. Biol. (Noisy-le-grand) 1999, 45:1083-1092.

[00146] In another embodiment, octreotate, a 14-amino acid peptide analogue of somatostatin, is the ligand attached to the nanoparticle. Octreotide, which has a longer half-life than somatostatin, is capable of binding to the somatostatin receptor (SSTR). SSTR, a member of the G protein-coupled receptor family, is overexpressed on the surface of several human tumors. Reubi et al. Distribution of Somatostatin Receptors in Normal and TumorTissue. Metab. Clinic. Exp. 1990; 39:78-81. Reubi et al. Somatostatin receptors and their subtypes in human tumors and in peritumoral vessels. Metab. Clinic. Exp. 1996; 45:39-41. Other somatostatin analogues can alternatively be conjugated to the nanoparticle to target SSTR, such as Tyr3-octreotide (Y3-OC), octreotate (TATE), Tyr3-octreotate (Y3-TATE), and 111In-DTPA-OC. These somatostatin analogues can be used for both PET diagnostic imaging and targeted radiotherapy of cancer. de Jong et al. In-ternalization of radiolabelled [DTPA0]octreotide and [DOTA0, Tyr3]octreotide: peptides for somatostatin receptor targeted scintigraphy and radionuclide therapy. Nucl. Med. Commun. 1998; 19:283-8. de Jong et al. Comparison of ^In-Labeled Somatostatin Analogues for Tumor Scintigraphy and Radionuclide Therapy. Cancer Res. 1998; 58:437-41. Lewis et al. Comparison of four 64Cu-labeled somatostatin analogues in vitro and in a tumor bearing rat model: evaluation of new derivatives for PET imaging and targeted radiotherapy. J Med Chem 1999; 42:1341-7. Krenning et al. Somatostatin Receptor Scintigraphy with Indium-111-DTPA-D-Phe-1-Octreotide in Man: Metabolism, Dosimetry and Comparison with Iodine-123-Tyr-3-Octreotide. J Nucl. Med. 1992; 33:652-8.

[00147] Various ligands can be used to map sentinel lymph nodes (SLNs). Sentinel lymph node mapping can be used in the diagnosis, staging and treatment of cancer. J591 is an anti-prostate-specific membrane antigen (i.e., anti-PSMA) monoclonal antibody. J591 has previously been used to detect and stage prostate cancer. Tagawa et al., Anti-prostatespecific membrane antigen-based radioimmunotherapy for prostate cancer, Cancer, 2010, 116(4 Suppl):1075-83. Bander et al., Targeting Metastatic Prostate Cancer with Radiolabeled Monoclonal Antibody J591 to the Extracellular Domain of Prostate Specific Membrane Antigen, The Journal of Urology 170(5), 1717-1721 (2003). Wernicke et al., (2011) Prostate-Specific Membrane Antigen as a Potential Novel Vascular Target for Treatment of Glioblastoma Multiforme, Arch Pathol Lab Med. 2011; 135:1486-1489. The F(ab')2 fragment of J591 can be used as a linker attached to the present nanoparticle. The nanoparticle can also be radiolabeled to create a dual-modality probe. In one embodiment, nanoparticles carrying the F(ab')2 fragment of J591 (e.g., HuJ591-F(ab')2 fragments, or humanized J591-F(ab')2 fragments) are used in the diagnosis of prostate cancer. or endometrial cancer (e.g. endometrial endometrioid adenocarcinoma). In another embodiment, nanoparticles carrying the F(ab')2 fragment of J591 can be used to target brain tumor neovasculature for treatment, monitoring disease progression. Brain tumor neovasculature was seen to overexpress PSMA, as shown from previous immunohistochemical evaluations of excised high-grade glioma specimens.

[00148] Cyclic peptides containing the HWGF sequence are potent and selective inhibitors of MMP-2 and MMP-9 but not several other members of the MMP family. The peptide CTTHWGFTLC inhibits the migration of human endothelial cells and tumor cells. Furthermore, it prevents tumor growth and invasion in animal models and increases the survival of mice carrying human tumors. Phage displaying CTTHWGFTLC specifically targets angiogenic blood vessels in vivo. This peptide and its extension GRENYGHCTTHWGFTLC or GRENYGHCTTHWGFTLS can be used as ligands to be attached to the present nanoparticle. Peptides can also be radiolabeled, for example radioiodinated. Koivunen et al., Tumor targeting with a selective gelatinase inhibitor, Nature Biotechnology 17, 768-774 (1999). Penate Medina et al., Liposomal tumor targeting in drug delivery utilizing MMP-2- and MMP-9-binding ligands, J. Drug Delivery, Volume 2011 (2011), Article ID 160515. Anticancer Research 21:4101-4106 (2005) . In one embodiment, nanoparticles attached to 124I-labeled matrix metalloproteinase peptide inhibitor (MMPI) are used for sentinel lymph node mapping to stage endometrioid cancer.

[00149] The number of ligands attached to the nanoparticle can vary from about 1 to about 30, from about 1 to about 20, from about 2 to about 15, from about 3 to about 10, from about 1 to about 10, or from about 1 to about 6. The small number of ligands attached to the nanoparticle helps maintain the hydrodynamic diameter of the present nanoparticle which meets the renal clearance cutoff size range. Hilderbrand et al., Near-infrared fluorescence: application to in vivo molecular imaging, Curr. Opinion. Chem. Biol., 14:71-9, 2010. The number of ligands measured may be an average number of ligands attached to more than one nanoparticle. Alternatively, a nanoparticle can be measured to determine the number of attached ligands. The number of ligands attached to the nanoparticle can be measured by any suitable methods, which may or may not be related to the properties of the ligands. For example, the number of cRGD peptides bound to the particle can be estimated using FCS-based measurements of absolute particle concentrations and the initial concentration of reactants for the cRGD peptide. The average number of RGD peptides per nanoparticle and the binding efficiency of RGD to functionalized PEG groups can be evaluated colorimetrically under alkaline conditions and Biuret spectrophotometric methods. The number of ligands attached to the nanoparticle can also be measured by other suitable methods.

Contrast Agent

[00150] A contrast agent can be attached to the present nanoparticle for medical or biological imaging. As used herein in this patent application, the term "contrast agent" refers to a substance, molecule, or compound used to enhance the visibility of structures or fluids in medical or biological imaging. The term "contrast agent" also refers to a contrast-producing molecule. The imaging techniques encompassed by the present invention include positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), optical bioluminescence imaging, optical fluorescence imaging, and combinations thereof. The contrast agent encompassed by the present invention can be any molecule, substance or compound known in the art for PET, SPECT, CT, MRI, and optical imaging. The contrast agent can be radionuclides, radiometals, positron emitters, beta ray emitters, gamma ray emitters, alpha ray emitters, paramagnetic metal ions, and supraparamagnetic metal ions. Contrast agents include, but are not limited to, iodine, fluorine, copper, zirconium, lutetium, astatine, yttrium, gallium, indium, technetium, gadolinium, dysprsio, iron, manganese, barium and barium sulfate. Radionuclides that can be used as the contrast agent attached to the nanoparticle of the present invention include, but are not limited to, 89Zr, 64Cu, 68Ga, 86Y, 124I and 177Lu.

[00151] The contrast agent can be conjugated directly to the nanoparticle. Alternatively, the contrast agent can be conjugated indirectly to the nanoparticle, by attachment to linkers or chelates. The chelate can be adapted to bind a radionuclide. Chelates that can be attached to this nanoparticle may include, but are not limited to, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), desferrioxamine ( DFO) and triethylenetetramine (TETA).

[00152] Suitable means for imaging, detecting, recording or measuring the present nanoparticles may also include, for example, a flow cytometer, a laser scanning cytometer, a fluorescence microplate reader, a fluorescence microscope, a confocal microscope, a bright field microscope, a high content scanning system, and similar devices. More than one imaging technique can be used at the same time or consecutively to detect the present nanoparticles. In one embodiment, optical imaging is used as a sensitive high-throughput screening tool to acquire multiple time points on the same subject, allowing semi-quantitative assessments of tumor marker levels. This compensates for the relatively reduced temporal resolution obtained with PET, although PET is necessary to obtain adequate depth penetration in order to acquire volumetric data, and to detect, quantify, and monitor changes in the levels of receptors and/or other cellular markers as a means assessing disease progression or improvement, as well as stratifying patients into appropriate treatment protocols.

Therapeutic Agent

[00153] A therapeutic agent can be attached to the fluorescent nanoparticle, for example, for targeted treatment of a disease. The therapeutic agent can be delivered to a diseased site in a highly specific manner or localized with release of the therapeutic agent at the disease site. Alternatively, the therapeutic agent may not be released. The ligand-conjugated fluorescent nanoparticle can be used for targeted release of a therapeutic agent at a desired location in a variety of systems, such as on, or within, a cell or a cellular component, within the body of an organism, such as like a human, or through the blood-brain barrier.

[00154] The therapeutic agent can be attached to the nanoparticle directly or indirectly. The therapeutic agent can be absorbed into the interstices or pores of the silica shell, or coated onto the silica shell of the fluorescent nanoparticle. In other embodiments where the silica shell is not covering the entire surface, the therapeutic agent may be associated with the fluorescent core, such as by physical absorption or by binding interaction. The therapeutic agent can be associated with the ligand that is attached to the fluorescent nanoparticle. The therapeutic agent can also be associated with the organic polymer or the contrast agent. For example, the therapeutic agent can be attached to the nanoparticle through PEG. PEGs can have multiple functional groups for attachment to the nanoparticle and the therapeutic agent. The particle can have different types of functionalized PEGs carrying different functional groups that can be attached to multiple therapeutic agents. The therapeutic agent can be attached to the nanoparticle covalently or non-covalently.

[00155] As used herein in this patent application, the term "therapeutic agent" refers to a substance that can be used in the diagnosis, cure, mitigation, treatment, or prevention of disease in a human or other animal. Said therapeutic agents include substances recognized in the official United States Pharmacopeia, the official Homeopathic Pharmacopeia of the United States, the official National Formulary, or any supplement thereto.

[00156] Therapeutic agents that can be incorporated with the fluorescent nanoparticles or linked fluorescent nanoparticles of the invention include nucleosides, nucleoside analogues, small interfering RNA (siRNA), microRNA, oligopeptides, polypeptides, antibodies, COX-2 inhibitors , apoptosis promoters, urinary tract agents, vaginal agents, neurodegenerative vasodilatory agents (e.g., Parkinson's disease), obesity agents, ophthalmic agents, osteoporosis agents, parasympatholytics, parasympathomimetics, antianesthetics, prostaglandids - nas, psychotherapeutic agents, respiratory agents, sedatives, hypnotics, agents for the skin and mucous membranes, antibacterials, antifungals, antineoplastics, cardioprotective agents, cardiovascular agents, antithrombotics, central nervous system stimulants, cholinesterase inhibitors, contraceptives, dopamine receptor agonists, erectile dysfunction agents, fertility agents, gastrointestinal agents, gout agents, hormones, immunomodulators, conveniently functionalized analgesics or general or local anesthetics, anticonvulsants, antidiabetic agents, antifibrotic agents, anti-infective agents, motion sickness, muscle relaxants, immunosuppressive agents, migraine agents, nonsteroidal anti-inflammatory drugs (NSAIDs), smoking cessation agents, or sympatholytics (see Physicians' Desk Reference, 55th ed., 2001, Medical Economics Company, Inc. , Montvale, N.J., pages 201-202).

[00157] Therapeutic agents that can be attached to the present nanoparticle include, but are not limited to, DNA alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducing agents, a platinum compound, an antimetabolite, vincalkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, therapeutic antibodies, tyrosine kinase inhibitors, boron radiosensitizers (i.e., velcade), and chemotherapy combination therapies.

[00158] Non-limiting examples of DNA alkylating agents are nitrogen mustards, such as Mechlorethamine, Cyclophosphamide (Ifosfamide, Trophosphamide), Chlorambucil (Melphalan, Prednimustine), Bendamustine, Uramustine and Estramustine; nitrosoureas, such as Carmustine (BCNU), Lomustine (Semustine), Fotemustine, Nimustine, Ranimustine and Streptozocin; alkyl sulfonates, such as Busulfan (Manosulfan, Treosulfan); Aziridines, such as Carboquone, ThioTEPA, Triaziquone, Triethylenemelamine; Hydrazines (Procarbazine); Triazenes such as Dacarbazine and Temozolomide; Altretamine and Mitobronitol.

[00159] Non-limiting examples of Topoisomerase I inhibitors include Campothecin derivatives including CPT-11 (irinotecan), SN-38, APC, NPC, campothecin, topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin, lurtotecan, rubitecan , silatecan, gimatecan, diphlomotecan, extatecan, BN-80927, DX-8951f, and MAG-CPT as described in Pommier Y. (2006) Nat. Rev. Cancer 6(10):789-802 and the United States Patent Publication States No. 2005/10250854; Protoberberine alkaloids and derivatives thereof including berberrubine and coralline as described in Li et al. (2000) Biochemistry 39(24):7107-7116 and Gatto et al. (1996) Cancer Res. 15(12):2795-2800; phenanthroline derivatives including Benzo[i]phenanthridine, Nitidine, and fagaronin as described in Makhey et al. (2003) Bioorg. Med. Chem. 11 (8): 1809-1820; Terbenzimidazole and derivatives thereof as described in Xu (1998) Biochemistry 37(10):3558-3566; and Anthracycline derivatives including Doxorubicin, Daunorubicin, and Mitoxantrone as described in Foglesong et al. (1992) Cancer Chemother. Pharmacol. 30(2):123-]25, Crow et al. (1994) J. Med. Chem. 37(19):31913194, and Crespi et al. (1986) Biochem. Biophys. Res. Commun. 136(2):521-8. Topoisomerase II inhibitors include, but are not limited to, Etoposide and Teniposide. Dual inhibitors of topoisomerases I and II include, but are not limited to, Saintopin and other Naphthecenediones, DACA and other Acridine-4-Carboxaminides, Intoplicin and other Benzopyridoindoles, TAS-I03 and other 7H-indeno[2,1-c]Quinoline -7-ones, Pyrazoloacridine, XR 11576 and other Benzophenazines, XR 5944 and other Dimeric compounds, 7-oxo-7H-dibenz[f,ij]Isoquinolines and 7-oxo-7H-benzo[e]Perimidines, and Conjugates of Anthracenyl-amino acids as described in Denny and Baguley (2003) Curr. Top. Med. Chem. 3(3):339-353. Some agents present Topoisomerase II and have DNA intercalation activity such as, but not limited to, Anthracyclines (Aclarrubicin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Anrubicin, Pyrrubicin, Valrubicin, Zorubicin) and Antrakenediones (Mitoxantrone and Pixantrone ).

[00160] Examples of endoplasmic reticulum stress inducing agents include, but are not limited to, dimethyl-celecoxib (DMC), nelfinavir, celecoxib, and boron radiosensitizers (i.e., velcade (Bortezomib)).

[00161] Non-limiting examples of platinum-based compounds include Carboplatin, Cisplatin, Nedaplatin, Oxaliplatin, Triplatin tetranitrate, Satraplatin, Aroplatin, Lobaplatin, and JM-216. (see McKeage et al. (1997) J. Clin. Oncol. 201:1232-1237 and generally, CHEMOTHERAPY FOR GYNECOLOGICAL NEOPLASM, CURRENT THERAPY AND NOVEL APPROACHES, in Series Basic and Clinical Oncology, Angioli et al. Eds., 2004 ).

[00162] Non-limiting examples of antimetabolite agents include folic acid-based inhibitors, that is, dihydrofolate reductase inhibitors, such as Aminopterin, Methotrexate and Pemetrexed; thymidylate synthase inhibitors such as Raltitrexed, Pemetrexed; Purine-based inhibitor, i.e., an adenosine deaminase inhibitor such as Pentostatin, a thiopurine such as Thioguanine and Mercaptopurine, a halogenated ribonucleotide reductase inhibitor such as Cladribine, Clofarabine, Fludarabine, or a guanine/ guanosine: thiopurine, such as Thioguanine; or Pyrimidine-based, i.e., cytosine/cytidine: hypomethylating agent, such as Azacitidine and Decitabine, a DNA polymerase inhibitor, such as Cytarabine, a ribonucleotide reductase inhibitor, such as Gemcitabine, or a thymine/thymidine: inhibitor of thymidylate synthase, such as Fluorouracil (5-FU). Equivalents to 5-FU include prodrugs, analogues and derivatives thereof such as 5'-deoxy-5-fluorouridine (doxyfluroidine), 1-tetrahydrofuranyl-5-fluorouracil (ftorafur), Capecitabine (Xeloda), S-I (MBMS-247616 , consisting of tegafur and two modulators, a 5-chloro-2,4-dihydroxypyridine and potassium oxonate), ralititrexed (tomudex), nolatrexed (Thymitaq, AG337), LY231514 and ZD9331, as described, for example, in Papami-cheal (1999) The Oncologist 4:478-487.

[00163] Examples of vincalkaloids include, but are not limited to Vinblastine, Vincristine, Vinflunine, Vindesine and Vinorelbine.

[00164] Examples of taxanes include, but are not limited to, docetaxel, Larotaxel, Ortataxel, Paclitaxel and Tesetaxel. An example of an epothilone is iabepilone.

[00165] Examples of enzyme inhibitors include, but are not limited to, farnesyltransferase inhibitors (Tipifamib); CDK inhibitor (Alvocidib, Seliciclib); proteasome inhibitor (Bortezomib); phosphodiesterase inhibitor (Anagrelide; rolipram); IMP dehydrogenase inhibitor (Tiazofurine); and lipoxygenase inhibitor (Masoprocol). Examples of receptor antagonists include, but are not limited to ERA (Atrasentan); retinoid X receptor (Bexarotene); and a sex steroid (Testolactone).

[00166] Examples of therapeutic antibodies include, but are not limited to anti-HER1/EGFR (Cetuximab, Panitumumab); Anti-HER2/neu (erbB2) receptor (Trastuzumab); Anti-EpCAM (Catumaxomab, Edrecolomab) Anti-VEGF-A (Bevacizumab); Anti-CD20 (Rituximab, Tositumomab, Ibritumomab); Anti-CD52 (Alemtuzumab); and Anti-CD33 (Gemtuzumab). United States Patent Nos. 5,776,427 and 7,601,355.

[00167] Examples of tyrosine kinase inhibitors include, but are not limited to ErbB: HER1/EGFR inhibitors (Erlotinib, Gefitinib, Lapatinib, Vandetanib, Sunitinib, Neratinib); HER2/neu (Lapatinib, Neratinib); Class III RTK: C-kit (Axitinib, Sunitinib, Sorafenib), FLT3 (Lestaurtinib), PDGFR (Axitinib, Sunitinib, Sorafenib); and VEGFR (Vandetanib, Semaxanib, Cediranib, Axitinib, Sorafenib); bcr-abl (Imatinib, Nilotinib, Dasatinib); Src (Bosutinib) and Janus kinase 2 (Lestaurtinib).

[00168] Chemotherapeutic agents that can be attached to the present nanoparticle can also include ansacrine, Trabectedin, retinoids (Alitretinoin, Tretinoin), arsenic trioxide, asparagine depletor Asparaginase/Pegaspargase), Celecoxib, Demecolcine, Elesclomol, Elsamitrucin, Etoglucid , Lonidamine, Lucantone, Mitoguazone, Mitotane, Oblimersen, Tensirolimus, and Vorinostat.

[00169] Examples of specific therapeutic agents that can be chained, linked, or associated with the fluorescent nanoparticles of the invention are flomoxef; fortimycin(s); gentamicin(s); solasulfone glycosulfone; gramicidin S; gramicidin(s); grepafloxacin; guamecycline; hetacillin; isepamycin; josamycin; kanamycin(s); flomoxef; fortimycin(s); gentamicin(s); solasulfone glycosulfone; gramicidin S; gramicidin(s); grepafloxacin; guamecycline; hetacillin; isepamycin; josamycin; kanamycin(s); bacitracin; bambermycin(s); bia- penem; brodimoprim; butyrosin; capreomycin; carbenicillin; carbomycin; carumonam; cefadroxil; cefamandole; cefatrizin; cefbuperazone; cefclidin; cefdinir; cefditoren; cefepime; cefetamet; cefixime; cefinenoxime; cefininox; cladribine; apalcilin; apiciclin; apramycin; arbecacin; aspoxycillin; azidanphenicol; aztreonam; cefodizime; cefonicid; cefoperazone; ceforamide; cefotaxime; cefotetan; cefotiam; cefozopran; cefpimizole; cefpyramide; cefpirome; cefprozil; cefroxadine; cefteram; ceftibuten; cefuzonam; cephalexin; cephaloglycin; cephalosporin C; cefradine; chloramphenicol; chlortetracycline; clinafloxacin; clindamycin; clomocycline; colistin; cyclacillin; dapsone; demeclocycline; diathymosulfone; dibecacin; dihydrostreptomycin; 6-mercaptopurine; thioguanine; capecitabine; docetaxel; etoposide; gemcitabine; topotecan; vinorelbine; vin-cristine; vinblastine; teniposide; melphalan; methotrexate; 2-p-sulfanilyanilineethanol; 4,4'-sulfinyldianiline; 4-sulfanilamidosalicylic acid; butorphanol; nalbuphine. streptozocin; doxorubicin; daunorubicin; plicamycin; idarubicin; mitomycin C; pentostatin; mitoxantrone; cytarabine; fludarabine phosphate; butorphanol; nalbup- fine. streptozocin; doxorubicin; daunorubicin; plicamycin; idarubicin; mitomycin C; pentostatin; mitoxantrone; cytarabine; fludarabine phosphate; acediasulfone; acetosulfone; amikacin; amphotericin B; ampicillin; atorvastatin; enalapril; ranitidine; ciprofloxacin; pravastatin; clarithromycin; cyclosporine; famotidine; leuprolide; acyclovir; paclitaxel; azithromycin; lamivudine; budesonide; albuterol; indinavir; metformin; alendronate; nizatidine; zidovudine; carboplatin; metoprolol; amoxicillin; diclofenac; lisinopril; ceftriaxone; captopril; salmeterol; xinafoate; imipenem; cilastatin; benazepril; cefaclor; ceftazidime; morphine; dopamine; bialamicol; fluvastatin; fenamidine; Podophyllinic acid 2-ethylhydrazine; acriflavine; chloroazodine; arsphenamine; amicarbilide; aminoquinuride; quinapril; oxymorphone; buprenorphine; floxuridine; dirithromycin; doxycycline; enoxacin; Sendomycin; epicillin; erythromycin; leukomycin(s); lincomycin; lomefloxacin; lucensomycin; lymecycline; meclocycline; meropenem; methacycline; micronomycin; midecamycin(s); minocycline; moxalactam; mupirocin; nadifloxacin; natamycin; neomycin; netilmicin; norfloxacin; oleandomycin; oxytetracycline; p-sulfanilylbenzylamine; panipenem; paromomycin; Pazufloxacin; penicillin N; pipacycline; pipemidic acid; polymyxin; primycin; quinacillin; ribostamycin; rifamide; rifampin; rifamycin SV; rifapentine; rifaximin; ristocetin; ritipenem; rocitamycin; rolitetracycline; rosaramycin; roxithromycin; salazosulfadimidine; sancycline; sisomycin; sparfloxacin; spectinomycin; spiramycin; streptomycin; succisulfone; sulphacrysoidine; sulphaloxic acid; sulfamidochrysoidine; sulfanilic acid; sulfoxone; teicoplanin; temafloxacin; temocillin; tetroxoprim; thiamphenicol; thiazolsulfone; thiostrepton; ticarcillin; tigemonam; tobramycin; tosu- floxacin; trimethoprim; trospectomycin; trovafloxacin; tuberactinomycin; vancomycin; azaserine; candicidin(s); chlorphenesin; dermostatin(s); Filipina; fungichromin; mepartricin; nystatin; oligomycin(s); perimycin A; tubecidin; 6-azauridine; 6-diazo-5-oxo-L-norleucine; aclacinomycin(s); ancitabine; anthramycin; azacitadine; azaserine; bleomycin(s); ethyl biscumacetate; ethylidene dicumarol; iloprost; lamifiban; taprostene; thioclomarol; Tyrofiban; amiprilose; bucillamine; gusperimo; gentisic acid; glucametacin; glycol salicylate; meclofenamic acid; mefenamic acid; mesalamine; niflumic acid; olsalazine; oxaceprol; S-enosylmethionine; salicylic acid; salsalate; sulfasalazine; tolfenamic acid; carubicin; carzinofillin A; chlorozotocin; chromomycin(s); denopterin; doxifluridine; edatrexate; eflornithine; elliptinium; enocitabine; epirubicin; manomustine; menogaril; mitobronitol; mitolactol; mopida- mol; mycophenolic acid; nogalamycin; olivomycin(s); peplomycin; pirarubicin; pyritrexim; prednimustine; procarbazine; pteropterin; puromycin; ranimustine; streptonigrin; thiamiprine; mycophenolic acid; procodazole; ruptured; sirolimus (rapamycin); tacrolimus; butethamine; phenalcomine; hydroxytetracaine; naepain; orthocaine; pyridocaine; salicylic alcohol; 3-amino-4-hydroxybutyric acid; aceclofenac; alminoprofen; amfenac; bronfenac; bromosaligenin; bumadizon; carprofen; diclofenac; diflunisal; ditazol; enfenamic acid; etodolac; etofenamate; fendosal; fepradinol; flufenamic acid; Tomudex® (N-[[5-[[(1,4-dihydro-2-methyl-4-oxo-6-quin-azolinyl)methyl]methylamino]-- 2-thienyl]carbonyl]- L-glutamic acid) , trimetrexate, tubecidin, ubenimex, vindesine, zorubicin; argatroban; cumetarol or dicumarol.

[00170] Lists of additional therapeutic agents can be found, for example, in: Physicians' Desk Reference, 55th ed., 2001, Medical Economics Company, Inc., Montvale, N.J.; USPN Dictionary of USAN and International Drug Names, 2000, The United States Pharmaceutical Convention, Inc., Rockville, Md.; and The Merck Index, 12th ed., 1996, Merck & Co., Inc., Whitehouse Station, N.J.

[00171] The therapeutic agent may also include radionuclides when the present nanoparticle is used in targeted radiotherapy. In one embodiment, low-energy beta-emitting radionuclides, such as chelated-177Lu constructs, are associated with the nanoparticle and used to treat relatively small tumor burdens or micrometastatic disease. In another embodiment, higher energy beta emitters, such as yttrium-90 (90Y), can be used to treat larger tumor burdens. Iodine-131 (131I) can also be used for radiotherapy.

[00172] The surface of the nanoparticle can be modified to incorporate at least one functional group. The organic polymer (e.g., PEG) attached to the nanoparticle can be modified to incorporate at least one functional group. For example, the functional group may be a maleimide or an N-Hydroxysuccinimide (NHS) ester. The incorporation of the functional group makes it possible to attach various ligands, contrast agents and/or therapeutic agents to the nanoparticle.

[00173] In one embodiment, a therapeutic agent is attached to the nanoparticle (to the surface or to the organic polymer coating) through an NHS ester functional group. For example, a tyrosine kinase inhibitor such as dasatinib (BMS) or a chemotherapeutic agent (e.g., taxol), can be coupled via an ester bond to the nanoparticle. This ester bond can then be cleaved in an acidic environment or enzymatically in vivo. This approach can be used to deliver a prodrug to a subject where the drug is released from the particle in vivo.

[00174] We tested the prodrug approach by coupling the small molecule inhibitor dasatinib with the nanoparticle PEG molecules. Based on biodistribution results and human drug dosage calculations, it was seen that the nanoparticle has unique biological properties, including relatively rapid clearance from blood compared to tumors and subsequent accumulation of the therapeutic agent in tumor tissue, which suggests that a pro-drug approach is viable. The functionalized nanoparticle allows drugs to be dosed multiple times, ensuring that the drug concentration in the tumor is greater than that specified by the IC-50 in tumor tissue, although it will not be dose-limiting for organs in other tissues. , such as the heart, liver or kidneys. The therapeutic agent and nanoparticle can be radiolabeled or optically labeled separately, allowing independent monitoring of the therapeutic agent and nanoparticle. In one embodiment, radiofluorinated dasatinib (i.e., 18F) is coupled with PEG-3400 moieties attached to the nanoparticle via NHS ester linkages. Radiofluoride is crucial to be able to independently monitor time-dependent changes in drug distribution and release from the radioiodinated (124I) fluorescent (Cy5) nanoparticle. This way, we can monitor the prodrug (dasatinib) and the nanoparticle separately. This allows optimization of prodrug design compared to methods in the prior art where a dual-labeling approach is not used. In another embodiment, radiotherapeutic iodine molecules (i.e., I-131), or other therapeutic gamma or alpha ray emitters, are conjugated to PEG via a maleimide functional group, where the therapeutic agent may not dissociate from the PEG in vivo. .

[00175] A generalizable approach referred to herein in this patent application as "click chemistry" is described below. In order for the present nanoparticle to readily accommodate wide ranges of ligands, contrast agents or chelates, the surface of the nanoparticle can be modified to incorporate a functional group. The nanoparticle can also be modified with organic polymers (e.g., PEGs) or chelates that can incorporate a functional group. However, the ligand, contrast agent, or therapeutic agent is modified to incorporate a functional group that is capable of reacting with the functional group on the nanoparticle, or on the PEGs or chelating agents attached to the nanoparticle under suitable conditions. Therefore, any ligand, contrast agent or therapeutic agent that has the reactive functional group is capable of being readily conjugated to the nanoparticle. This generalizable approach is referred to here in this patent application as "click chemistry", which will allow a great deal of versatility to explore multimodal applications. In "click chemistry" chemical reactions, two molecular components can be joined through a selective, fast, clean, bioorthogonal, and/or biocompatible reaction. Kolb et al., (2001) Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angewandte Chemie International Edition 40, 2004-2021. Lim et al., (2010) Bioorthogonal chemistry: recent progress and future directions. Chemical Communications 46, 1589-1600. Sletten et al., (2009) Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angewandte Chemie International Edition 48, 6973-6998.

[00176] Any suitable reaction mechanism can be adapted in the present invention to "click chemistry", as long as attachment of the ligand, contrast agent or chelate to the nanoparticle can be achieved in an easy and controlled manner. In one embodiment, a free triple bond is introduced onto PEG, which is already covalently conjugated to the nanoparticle shell. In the meantime, an azide bond is introduced onto the desired ligand (or contrast agent, chelate). When the PEGylated nanoparticle and the ligand (or contrast agent, chelate) are mixed in the presence of a copper catalyst, cycloaddition of azide will occur to the bond, resulting in the conjugation of the ligand with the nanoparticle. An example of click chemistry is the Huisgen cycloaddition between an azide and alkyne catalyzed by Cu(I) [3+2]. Moses et al., (2007) The growing applications of click chemistry. Chemical Society Reviews 36, 1249-1262. Glaser et al., (2009) 'Click labelling' in PET radiochemistry. Journal of Labeled Compounds & Radiopharmaceuticals 52, 407-414. Mindt et al., (2009) A "Click Chemistry" Approach to the Efficient Synthesis of Multiple Imaging Probes derived from a Single Precursor. Bioconjugate Chemistry 20, 1940-1949. New et al., (2009) Growing applications of "click chemistry" for bioconjugation in contemporary biomedical research. Cancer Biotherapy and Radiopharmaceuticals 24, 289-301. Wang et al., (2010) Application of "Click Chemistry" in Synthesis of Ra-diopharmaceuticals. Progress in Chemistry 22, 1591-1602. Schultz et al., (2010) Synthesis of a DOTA-Biotin Conjugate for Radionuclide Chelation via Cu-Free Click Chemistry. Organic Letters 12, 2398-2401. Martin et al., (2010) A DOTA-peptide conjugate by copper-free click chemistry. Bioorganic & Medicinal Chemistry Letters 20, 4805-4807. Lebedev et al., (2009) Clickable bifunctional radiometal chelates for peptide labeling. Chemical Communications 46, 1706-1708. Knor et al., (2007) Synthesis of novel 1,4,7,10-tetraazacyclodecane-1,4,7,10- tetraacetic acid (DOTA) derivatives for chemoselective attachment to unprotected polyfunctionalized compounds. Chemistry--a European Journal 13, 6082-6090. In a second embodiment, a maleimide functional group and a thiol group can be introduced onto the nanoparticle and the desired ligand (or contrast agent, chelate), with the nanoparticle having the maleimide functional group, the ligand (or contrast agent, chelate) having the thiol group, or vice versa. The maleimide double bond readily reacts with the thiol group to form a stable carbon-sulfur bond. In a third embodiment, an activated ester functional group, for example, a succinimidyl ester group, and an amine group can be introduced onto the nanoparticle and the desired ligand, contrast agent or chelate. The activated ester group readily reacts with the amine group to form a stable carbon-nitrogen amide bond. In a fourth embodiment, click chemistry involves the inverse electron demand Diels-Alder reaction between a tetrazine moiety and a displaced alkene (Figure 21c). Devaraj et al., (2009) Fast and Sensitive Pretargeted Labeling of Cancer Cells through a Tetrazine/trans- Cyclooctene Cycloaddition. Angewandte Chemie-International Edition 48, 7013-7016. Devaraj et al., (2008) Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging. Bioconjugate Chemistry 19, 2297-2299. Blackman et al., (2008) Tetrazine ligation: fast bioconjugation based on inverse electron demand Diels-Alder reactivity. Journal of the American Chemical Society 130, 13518-13519. Binding can be selective, rapid, biocompatible, and/or bioorhogonal. Unlike many Diels-Alder reactions, the binding is irreversible, forming some stable pyridazine products after the release of dinitrogen from retro-Diels-Alder by the reaction intermediate. A tetrazine moiety and a displaced alkene may be introduced onto the nanoparticle and the desired ligand (or contrast agent, chelate), with the nanoparticle having the tetrazine moiety, the ligand (or contrast agent, chelate) having the displaced alkene, or vice versa. A number of different tetrazinc displaced alkene pairs can be used for the reaction, including, for example, the combination of 3-(4-benzylamino)-1,2,4,5-tetrazine (Tz) and or norbornene- or trans-cyclooctene-derivatives. Schoch et al., (2010) Post-Synthetic Modification of DNA by Inverse-Electron-Demand Diels-Alder Reaction. Journal of the American Chemical Society 132, 8846-8847. Devaraj et al., (2010) Bioorthogonal Turn-On Probes for Imaging Small Molecules Inside Living Cells. Angewandte Chemie International Edition 49. Haun et al., (2010) Bioorthogonal chemistry amplifies nanoparticle binding and enhances the sensitivity of cell detection. Nature Nanotechnology 5, 660-665. Rossin et al., (2010) In vivo chemistry for pretargeted tumor imaging in live mice. Angewandte Chemie International Edition 49, 3375-3378. Li et al., (2010) Tetrazine-trans-cyclooctene ligation for the rapid construction of 18-F labeled probes. Chemical Communications 46, 8043-8045. Reiner et al., (2011) Synthesis and in vivo imaging of a 18F-labeled PARP1 inhibitor using a chemically orthogonal scavenger-assisted high-performance method. Angewandte Chemie International Edition 50, 1922-1925. For example, the surface of nanoparticles can be decorated with tetrazine moieties, which can then be conjugated to norbornene-modified ligand (or contrast agent, or chelate) (Figures 21d and 21e). In one aspect, the nanoparticles are coated with tetrazine, which can then be modified with DOTA chelates using the tetrazine-norbornene bond, and radiolabeled with 64Cu.

Length of stay/clearance in vivo

[00177] After administration of the present nanoparticle to a subject, the blood half-life of the nanoparticles may vary from about 2 hours to about 25 hours, from about 3 hours to about 20 hours , from about 3 hours to about 15 hours, from about 4 hours to about 10 hours, or from about 5 hours to about 6 hours. Longer blood half-life means longer circulation, which allows more nanoparticles to accumulate at the target site in vivo. The half-life of residence in the blood can be evaluated as follows. The nanoparticles are first administered to a subject (e.g., a mouse, a mini pig, or a human). At various time points after administration, blood samples are taken to measure nanoparticle concentrations using appropriate methods.

[00178] In one embodiment, after administration of the PEGylated (or control) nanoparticle to a subject, the blood half-life of the nanoparticle can vary from about 2 hours to about 15 hours, or from from about 4 hours to about 10 hours. The tumor half-life of the nanoparticle after administration of the nanoparticle to a subject may vary from about 5 hours to about 2 days, from about 10 hours to about 4 days, or from about 15 hours to about 3.5 days. The ratio of tumor half-life to blood half-life of the nanoparticle after administration of the nanoparticle to a subject can vary from about 2 to about 30, from about 3 to about 20, or from about 4 to about 15. Renal clearance of the nanoparticle after administration of the nanoparticle to a subject can vary from about 10% of the DI (initial dose) to about 100% of the DI at about 24 hours, from about 30% DI to about 80% DI in about 24 hours, or from about 40% DI to about 70% DI in about 24 hours. In one embodiment, after the nanoparticle is administered to a subject, the blood half-life of the nanoparticle varies from about 2 hours to about 25 hours, the tumor half-life of the nanoparticle varies from from about 5 hours to about 5 days, and renal clearance of the nanoparticle varies from about 30% of DI to about 80% of DI in about 24 hours.

[00179] After administration of the present nanoparticle to a subject, the tumor half-life of the present nanoparticles can vary from about 5 hours to about 5 days, from about 10 hours to about 4 days, from about 15 hours to about 3.5 days, from about 20 hours to about 3 days, from about 2.5 days to about 3.1 days, from about 1 day to 3 days, or about 73.5 hours.

[00180] The ratio of the half-life of residence in the tumor to the half-life of residence in the blood of the nanoparticle can vary from about 2 to about 30, from about 3 to about 20, from from about 4 to about 15, from about 4 to about 10, from about 10 to about 15, or about 13.

[00181] In one embodiment, the present invention provides a silica-based fluorescent nanoparticle comprising a silica-based core comprising a fluorescent compound positioned within the silica-based core; a silica shell surrounding at least a portion of the core; an organic polymer attached to the nanoparticle; and a linker attached to the nanoparticle, wherein the nanoparticle has a diameter between about 1 nm and about 15 nm. After administration of the PEGylated (control) nanoparticle to a subject, the blood half-life of the nanoparticle may vary from about 2 hours to about 25 hours, or from about 2 hours to about 15 hours; the nanoparticle's half-life in the tumor can vary from about 5 hours to about 2 days; and renal clearance of the nanoparticle can vary from about 30% of DI to about 80% of DI in about 24 hours. The number of ligands attached to the nanoparticle can range from about 1 to about 30, or from about 1 to about 10. The diameter of the nanoparticle can be between about 1 nm and about 8 nm. A contrast agent, such as a radionuclide, can be attached to the nanoparticle. Alternatively, a chelate can be attached to the nanoparticle. The b nanoparticle can be detected by PET, SPECT, CT, MRI, optical imaging, bioluminescence imaging, or combinations thereof. A therapeutic agent can be attached to the nanoparticle. After administration of the radiolabeled targeted nanoparticle to a subject, the blood half-life of the nanoparticle may also vary from about 3 hours to about 15 hours, or from about 4 hours to about 10 hours. The tumor residence half-life of the nanoparticle after administration of the nanoparticle to a subject can also vary from about 10 hours to about 4 days, or from about 15 hours to about 3.5 days. The ratio of the tumor half-life to the blood half-life of the targeted nanoparticle after administration of the nanoparticle to a subject can vary from about 2 to about 30, from about 3 to about 20, or from about 4 to about 15. Renal clearance of the nanoparticle can also vary from about 45% DI to about 90% DI in about 24 hours after nanoparticle administration. to a subject.

[00182] In one embodiment, in order to estimate the values of the half-life of residence (or clearance) of radiolabeled nanoparticles (TI/2) in the blood, tumor, and other larger organs/tissues, the values of the percentage of injected dose per gram (% DI/g) are measured by sacrificing groups of mice at specified times after administration of the nanoparticles. Blood, tumor, and organs are harvested, weighed, and counted in a Y scintillation counter. % ID/g values are corrected for radioactive decay at the time of injection. The resulting activity-time concentration data for each tissue are fit adjusted to a decreasing monoexponential function to estimate tissue/organ T1/2 values.

[00183] After administration of the present nanoparticle to a subject, the renal clearance of the present nanoparticles can vary from about 45% of the DI (initial dose) to more than 90% of the DI in about 24 hours, from from about 20% DI to about 90% DI in about 24 hours, from about 30% DI to about 80% DI in about 24 hours, from about 40% DI to about 70% of DI in about 24 hours, from about 40% of DI to about 60% of DI in about 24 hours, from about 40% of DI to about 50% of DI in about 24 hours, about 43% of DI in about 24 hours, from about 10% of DI to about 100% of DI in about 24 hours, from about 40% of DI to from about 100% DI in about 24 hours, from about 80% DI to about 100% DI in about 24 hours, from about 90% DI to about 95% DI in about 24 hours, from about 90% DI to about 100% DI in about 24 hours, or from about 80% DI to about 95% DI in about 24 hours . Renal clearance can be assessed as follows. The nanoparticles are first administered to a subject (e.g., a mouse, a mini pig, or a human). At various time points after administration, urine samples are collected to measure nanoparticle concentrations using appropriate methods.

[00184] In one embodiment, renal clearance (e.g., the fraction of nanoparticles excreted in urine over time) is assessed as follows. A subject is dosed with the present nanoparticles, and urine samples are collected over a certain period of time (e.g., for 168 hours). Particle concentrations at each time point are determined using fluorometric analyzes and a serial dilution calibration curve generated from measurements of background-corrected fluorescence signals from urine samples spiked with known particle concentrations (% ID). The concentration values, along with estimates of average daily mouse urine volumes, are used to compute the cumulative % DI/g of urine excreted. In another embodiment, renal clearance of radiolabeled nanoparticles is assessed by measuring the activities of urine samples (counts per minute) during similar time intervals using, for example, Y counts, and after administration of the nanoparticles to compute cumulative urine excretion. .

[00185] In a third modality, in order to evaluate cumulative fecal excretion, feces are collected in metabolic cages during similar time intervals after administration of the nanoparticles and the activities of the samples are determined using a Y-counter.

[00186] When nanoparticles in an amount of about 100 times the equivalent human dose are administered to a subject, substantially no anemia, weight loss, agitation, increased breathing, gastrointestinal disturbance, abnormal behavior, neurological dysfunction, abnormalities are observed. in hematology, abnormalities in clinical laboratory, drug-related injuries in organ pathology, mortality, or combinations thereof in about 10 to about 14 days.

[00187] When the present nanoparticle contains at least one ligand attached, the reinforcement of the multivalency nanoparticle (e.g., compared to the isolated ligand) can vary from about 1.5 times to about 10 times, from about from 2 times to about 8 times, from about 2 times to about 6 times, from about 2 times to about 4 times, or about 2 times.

[00188] The nanoparticles of the present invention present unexpected physicochemical and biological parameters in vitro and in vivo in view of the prior art. For example, the estimated blood half-life for ligand-attached nanoparticles (e.g., about 5.5 hours for cRGD-attached nanoparticles) is substantially longer than that of the corresponding ligand (e.g., about 13 minutes for cRGD). Montet et al. Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem. 49, 60876093 (2006). Prolonged blood half-lives may enhance probe bioavailability, facilitate tumor targeting, and produce greater tumor uptake over longer periods of time. In one embodiment, the tumor half-life for targeted nanoparticles (i.e., ligand-attached nanoparticles) is about 13 times greater than the blood half-life, whereas the half-life for nanoparticles is about 13 times longer than the half-life for targeted nanoparticles. in the tumor for non-targeted nanoparticles (i.e. corresponding nanoparticles not attached with ligands) is only about 5 times longer than the half-life of residence in the blood. This difference suggests substantially greater accumulation in tumor tissue of targeted nanoparticles compared to non-targeted nanoparticles. In certain embodiments, given the number of ligands attached to the nanoparticle, the present nanoparticles exhibit unexpected high-affinity binding (e.g., Kd of 0.51 nM and IC50 of 1.2 nM for nanoparticle attached to cRGD), multivalence reinforcement (e.g., example, more than 2-fold boost for nanoparticles attached to cRGD compared to cRGD peptide alone), significant differential tumor uptake (e.g., PEG nanoparticles attached to cRGD show about a 3- to 4-fold increase in differential tumor uptake relative to PEG-coated nanoparticles during 72 hours post-administration), and significant tumor versus normal muscle contrast (e.g., approximately 3- to 5-fold during 72 hours post-administration) based on tumor-to-muscle uptake ratios.

[00189] In one embodiment, three-fold increases in activity - concentration were found for ligand-attached nanoparticles in integrin-expressing tumors relative to controls (e.g., ligand-attached nanoparticles in non-integrin-expressing tumors, or corresponding nanoparticles not attached with ligands in integrin-expressing tumors) at the time of maximum tumor uptake (about 4 hours after injection of the nanoparticles). Furthermore, tumor-to-muscle uptake ratios for targeted nanoparticles (i.e., ligand-attached nanoparticles) reveal increased contrast of tumor tissue relative to normal muscle compared with reduced contrast of tumor tissue relative to normal muscle for nanoparticles. non-targeted (i.e., corresponding nanoparticles not attached with ligands), suggesting that targeted nanoparticles are tumor-selective.

[00190] In another modality, both targeted and non-targeted nanoparticles exhibit efficient renal excretion during the same period of time. Almost half of the injected dose is excreted during the first 24 hours after injection and about 72% by 96 hours, suggesting that the majority of excretion occurred within the first day after injection. By contrast, the fecal excretion profiles of the targeted nanoparticles indicate that, on average, 7% and 15% of the injected dose is eliminated over 24 and 96 hours, respectively.

[00191] The physicochemical and biological parameters of non-toxic nanoparticles, along with their multimodal imaging capabilities (e.g., PET and optical imaging), expand the range of their potential biomedical applications. Applications include (a) long-term monitoring: the extended blood circulation time and corresponding bioavailability of the nanoparticles highlight their versatility for both early and long-term monitoring of the management of various stages of disease (such as screening diagnosis, pre- -treatment, therapeutic intervention, and post-treatment monitoring) without restrictions imposed by toxicity considerations; (b) increased tumor penetration: the clearance properties of targeted nanoparticles (e.g., their renal clearance is lower than that of molecular probes in the prior art) will be useful for various types of biological applications. For example, nanoparticles will be particularly useful in cases of poorly vascularized and relatively inaccessible solid tumors in which localization of agents is typically slow after systemic administration; (c) Multimodal imaging capabilities: These modalities can be combined at multiple scales (i.e., whole-body to cellular levels) to acquire complementary, depth-sensitive biological information. For example, in sentinel lymph node mapping, deep lymph nodes can be mapped by PET in terms of their distribution and number, whereas more precise and detailed localization of superficial lymph nodes can be obtained by fluorescence imaging; and (d) targeted therapeutics: longer clearance of targeted nanoparticles from tumor compared to blood can be exploited for combined diagnostic/therapeutic applications, in which nanoparticles can serve as a radiotherapeutic or as a drug delivery vehicle.

Pharmaceutical compositions

[00192] The present invention additionally provides a pharmaceutical composition comprising the present nanoparticle. The pharmaceutical compositions of the invention can be administered orally in the form of a suitable pharmaceutical dosage unit form. The pharmaceutical compositions of the invention can be prepared in many forms which include tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow release formulations, such as shaped polymeric gels.

[00193] Suitable modes of administration for the present nanoparticle or composition include, but are not limited to, oral, intravenous, rectal, sublingual, mucosal, nasal, ophthalmic, subcutaneous, intramuscular, transdermal, intradermal, subdermal, peritumoral, spinal, intrathecal, intra-articular, intra-arterial, subarachnoid, bronchial, and lymphatic, and other dosage forms for systemic delivery of active ingredients. The present pharmaceutical composition can be administered by any method known in the art, including, without limitation, transdermal (passive via patch, gel, cream, ointment or iontophoretic); intravenous (bolus, infusion); subcutaneous (infusion, depot); transmucosal (buccal and sublingual, e.g., orodispersible tablets, wafers, film, and effervescent formulations; conjunctive (eye drops); rectal (suppository, enema)); or intradermal (bolus, infusion, depot). The composition can be released topically.

[00194] Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or another suitable vehicle before use. Said liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous carriers (which may include edible oils), or preservatives.

[00195] The nanoparticle pharmaceutical compositions of the invention may also be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes , small volume infusion containers or multi-dose containers with an added preservative. Pharmaceutical compositions may take forms such as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the pharmaceutical compositions of the invention may be in powder form, obtained by aseptic isolation of a sterile solid or by lyophilization of a solution, for constitution with a suitable vehicle, for example, sterile, pyrogen-free water, before use.

[00196] For topical administration to the epidermis, pharmaceutical compositions can be formulated as ointments, creams or lotions, or as the active ingredient of a transdermal patch. Suitable transdermal delivery systems are disclosed, for example, in A. Fisher et al. (United States Patent No. 4,788,603), or in R. Bawa et al. (United States Patent Nos. 4,931,279; 4,668,506; and 4,713,224). Ointments and creams can be formulated, for example, with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will generally also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Pharmaceutical compositions may also be released via ionophoresis, for example, as disclosed in United States Patent Nos. 4,140,122; 4,383,529; or 4,051,842.

[00197] Pharmaceutical compositions suitable for topical administration in the mouth include dosage unit forms such as lozenges comprising a pharmaceutical composition of the invention in a flavored base, generally sucrose and acacia or tragacanth; tablets comprising the pharmaceutical composition in an inert base such as gelatin and glycerin or sucrose and acacia; mucoadherent gels, and mouthwashes comprising the pharmaceutical composition in a suitable liquid carrier.

[00198] For topical administration to the eye, pharmaceutical compositions can be administered as drops, gels (S. Chrai et al., United States Patent No. 4,255,415), gums (S. L. Lin et al., United States Patent No. 4,136,177) or through an extended release ocular insert (A. S. Michaels, United States Patent No. 3,867,519 and H. M. Haddad et al., United States Patent No. 3,870,791).

[00199] When desired, the pharmaceutical compositions described above can be adapted to provide gradual release of a therapeutic compound employed, for example, by combination with certain hydrophilic polymer matrices, for example, comprising natural gels, synthetic polymer gels or mixtures of the same.

[00200] Pharmaceutical compositions suitable for rectal administration in which the support is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art, and the suppositories can conveniently be formed by mixing the pharmaceutical composition with the one or more softened or molten carriers followed by cooling and shaping in molds.

[00201] Pharmaceutical compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing, in addition to nanoparticles and the therapeutic agent, the aforementioned supports are generally known in the art.

[00202] For administration by inhalation, the pharmaceutical compositions according to the invention are conveniently released from an insufflator, a nebulizer or a pressurized package or other convenient means of releasing an aerosol spray. Pressurized packages may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to release a metered amount.

[00203] Alternatively, for administration by inhalation or insufflation, the pharmaceutical compositions of the invention may take the form of a dry powder composition, for example, a powder mixture of the pharmaceutical composition and a suitable powder base such as lactose or starch. . The powder composition may be presented in unit dosage form, for example in capsules or cartridges or, for example, gelatin or blister packs from which the powder can be administered with the aid of an inhaler. or an inflator.

[00204] For intranasal administration, the pharmaceutical compositions of the invention can be administered through a liquid spray, such as through a plastic bottle atomizer. Typical of these are the Mistometer® (Wintrop isoproterenol inhaler) and the Medihaler® (Riker isoproterenol inhaler).

[00205] Pharmaceutical compositions of the invention may also contain other adjuvants such as flavorings, colorings, antimicrobial agents, or preservatives.

[00206] It will be further recognized that the amount of pharmaceutical compositions required for use in treatment will vary not only with the therapeutic agent selected, but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and in The final analysis will be at the discretion of the clinician or attending physician. For assessments of these factors, see J. F. Brien et al., Europ. J. Clin. Pharmacol., 14, 133 (1978); and Physicians' Desk Reference, Charles E. Baker, Jr., Pub., Medical Economics Co., Oradell, N.J. (41st ed., 1987). Generally, dosages of the therapeutic agent when used in combination with the fluorescent nanoparticles of the present invention can be lower than when the therapeutic agent is administered alone or in conventional pharmaceutical dosage forms. The high specificity of the fluorescent nanoparticle for a target site, such as a receptor located on a cell surface, can provide a relatively highly localized concentration of a therapeutic agent, or alternatively, a gradual release of a therapeutic agent over an extended period of time.

[00207] The present nanoparticles or compositions can be administered to a subject. The subject may be a mammal, preferably a human. Mammals include, but are not limited to, murine, rat, rabbit, ape, bovine, sheep, swine, canine, feline, farm animals, competitive animals, pets, equines, and primates.

Uses of Nanoparticles

[00208] The present invention further provides a method for detecting a component of a cell comprising the steps of: (a) contacting the cell with a silica-based fluorescent nanoparticle comprising a silica-based core comprising a fluorescent compound positioned within silica-based core; a silica shell surrounding at least a portion of the core; an organic polymer attached to the nanoparticle; from about 1 to about 25 ligands (from about 1 to about 20 ligands, or from about 1 to about 10 ligands, or other ranges; see included discussions) attached to the nanoparticle; and a contrast agent or a chelate attached to the nanoparticle; and (b) monitoring the binding of the nanoparticle to the cell or a cellular component (and/or its potential intracellular uptake) by at least one imaging technique. The imaging technique may be PET, SPECT, CT, MRI, optical bioluminescence or fluorescence imaging, and combinations thereof.

[00209] The location of the cellular component can be detected and determined within a metabolically active whole cell, in a whole cell lysate, in a permeabilized cell, in a fixed cell, or with a partially purified cellular component in a cell-free environment. cells. The amount and duration of contact may depend, for example, on the diagnostic or therapeutic objectives of the treatment method, such as antibody-mediated or fluorescent detection of upregulated signaling pathway intermediates (i.e., Akt, NF-KB ), from disease states or conditions, from the release of a therapeutic agent, or both. The amount and duration of contact may also depend on the relative concentration of the fluorescent nanoparticle to the target analyte, the particle incubation time, and the state of the cell for treatment.

[00210] The present invention further provides a method for targeting a tumor cell comprising administering to a cancer patient an effective amount of a silica-based fluorescent nanoparticle comprising a silica-based core comprising a fluorescent compound positioned within the core. silica-based; a silica shell surrounding at least a portion of the core; an organic polymer attached to the nanoparticle; a ligand attached to the nanoparticle and capable of binding a tumor marker; and at least one therapeutic agent.

[00211] The nanoparticle can be radiolabeled. The nanoparticle can be radiolabeled using any suitable techniques. Radiomarking can be automated. In one embodiment, the nanoparticle is radiolabeled on the FASTlab (GE Healthcare) radiochemistry synthesis platform. Synthesis parameters can be optimized to achieve high reproducibility, radiochemical yield/purity, labeling efficiency, and relatively short synthesis times. In particle trackers can be accommodated in the same FASTlab module.

[00212] The nanoparticle can be administered to the patient, but not restricted, via the following routes: oral, intravenous, nasal, subcutaneous, local, intramuscular or transdermal.

[00213] In certain embodiments, it may be desirable to use a mixture of two or more types of fluorescent nanoparticles having different properties to evaluate different types of tissue.

[00214] The methods and compositions of the invention can be used to help a physician or surgeon identify and characterize areas of disease, such as cancers and inflammatory/infectious processes, including, but not restricted to, cancers of the skin (melanoma), of head and neck, prostate, brain, and intestines, to distinguish diseased and normal tissue, such as detecting tumor margins that are difficult to detect using a usual operating microscope, for example in brain surgery, to help prescribe an intervention therapeutic or surgical, for example, determining whether a lesion is cancerous and should be removed or is non-cancerous and should not be removed, or in surgical staging of a disease, for example, intraoperative lymph node staging, sentinel lymph node (SLN) mapping , for example, nerve-sparing procedures to preserve vital neural structures (intraparotid nerves).

[00215] The methods and compositions of the invention can be used in detecting metastatic disease, monitoring response to treatment, mapping/targeting sentinel lymph nodes, nerve sparing procedures, detecting residual disease, targeted release of therapeutics (diagnostic platform/combination therapeutics), in the local release of non-targeted nanoparticles carrying drugs (catheter delivery), in blood-brain barrier therapeutics, in the treatment of inflammatory/ischemic diseases (i.e., brain, cardiac, tract urinary tract, bladder), in the combined treatment and detection of disease (e.g. Ratiometric pH detection, oxygen detection), etc.

[00216] The methods and compositions of the invention can also be used in detecting, characterizing and/or determining the location of a disease, especially initial disease, the severity of a disease or a condition associated with disease, the staging of a disease, and/or monitoring a disease. The presence, absence, or level of a signal emitted may be indicative of a disease state. The methods and compositions of the invention can also be used to monitor and/or guide various therapeutic interventions, such as surgical and catheter-based procedures, and drug therapy monitoring, including cell-based therapies. The methods of the invention may also be used in the prognosis of a disease or disease condition. Inherent cellular subpopulations within or bordering the site of disease, such as stem-like cells ("cancer stem cells") and/or inflammatory/phagocytic cells can be identified and characterized using the methods and compositions of the invention. With respect to each of the foregoing, examples of similar disease or disease conditions that may be detected or monitored (before, during, or after therapy) include cancers (e.g., melanoma, thyroid, colorectal, ovarian, lung, breast , prostate, cervical, skin, cerebral, gastrointestinal, oral, renal, esophageal, bone), which can be used to identify subjects who have an increased susceptibility to developing cancer and/or malignancies, that is, are predisposed to developing cancer and/or malignancies, inflammation (e.g., inflammatory conditions induced by the presence of cancerous lesions), cardiovascular disease (e.g., atherosclerosy and inflammatory conditions of blood vessels, ischemia, stroke, thrombosis), dermatological disease (e.g., Kaposi's sarcoma, psoriasis), ophthalmic disease (e.g., macular degeneration, diabetic retinopathy), infectious disease (e.g., bacterial, viral, fungal, and parasitic infections, including Acquired Immune Deficiency Syndrome), immunological disease (e.g., a autoimmune disorder, lymphoma, multiple sclerosis, rheumatoid arthritis, diabetes mellitus), central nervous system disease (e.g., a neurodegenerative disease such as Parkinson's disease or Alzheimer's disease), hereditary diseases, metabolic diseases, environmental diseases ( e.g., lead, mercury and radioactive poisoning, skin cancer), bone-related disease (e.g., osteoporosis, primary and metastatic bone tumors, osteoarthritis) and a neurodegenerative disease.

[00217] The methods and compositions of the invention, therefore, can be used, for example, to determine the presence and/or location of tumor cells and/or co-resident stem-like cells ("cancer stem cells"), to presence and/or location of inflammatory cells, including the presence of activated macrophages, e.g., in peritumoral regions, the presence and location of vascular disease including areas at risk for acute occlusion (i.e., vulnerable plaques) in coronary arteries and peripheral, regions of expanding aneurysms, unstable plaque in carotid arteries, and ischemic areas. The methods and compositions of the invention can also be used in identifying and evaluating cell death, injury, apoptosis, necrosis, hypoxia and angiogenesis. Patent Publication No. PCT/US2006/049222.

[00218] The following examples are presented for illustration purposes only and are not limiting of the invention.

Example 1 Preparation and Characterization of PEG-Coated Nanoparticles

[00219] Nanoparticles containing a NIR emitting dye (Cy-5) were synthesized and functionalized by PEGylation according to well-established protocols as disclosed in Patent Application No. PCT/US2008/074894 and by Stober et al. Controlled growth of monodispersed silica spheres in the micron size range. Colloid Interface Sci. 1968; 26:62-69. Ohnishi et al. J. Mol. Imaging 2005, 4:172181. Cy5 malemide was reacted with a coreactive organosilane compound, (3-Mercaptopropyl)tromethoxysilane to form a fluorescent silica precursor. This fluorescent silica precursor was co-condensed with tetraethylorthosilicate to form a fluorescent silica-based core. A PEG-silane compound, with poly(ethylene glycol) chains terminated with methoxy chains (PEG, ~0.5 kDa) Methoxy(Polyethyleneoxy)Propyl]-Trimethoxysilane, was added to the fluorescent silica-based core to form a PEG coating over the core. PEG-coated nanoparticles were dialyzed into physiological saline (0.15 M NaCl in H2O) through 3500 MWCO Snakeskin Dialysis Membranes and sterile filtered. All samples were optical density matched at their peak absorption wavelength (640 nm) prior to injection. Hydrodynamic size measurements were obtained by Dynamic Light Scattering (DLS) and Fluorescence Correlation Spectroscopy (FCS). Briefly, particles dialyzed to water were measured on a Brookhaven Instruments Company 200SM static/DLS system using a HeNe laser (À = 632.8 nm). Due to the overlap of dye absorption with the excitation source, integration times of 15 min were used to obtain acceptable signal-to-noise ratios. For FCS, particles were dialyzed into water, diluted in 0.15 M NaCl, and measured on a Zeiss LSM 510 Confocor 2 FCS (633 nm HeNe excitation). The instrument was size calibrated before all measurements. Comparison of the relative brightness of PEGylated nanoparticles with free dye was determined from FCS curves, measured as count rate per molecule/particle.

Example 2 Renal Clearance of PEG-Coated Nanoparticles

[00220] Fluorescent core-shell silica nanoparticles, having a hydrodynamic radius of about 3 nm, were synthesized. These nanoparticles were considered to be in the range of 6 to 10 nm in diameter, as shown by dynamic light scattering (DLS) results (Figure 1a). In vivo whole-body near-infrared fluorescence imaging of uncoated silica nanoparticles (without PEG coating), on the order of 6 nm and 3.3 nm, in nude mice showed considerable renal clearance 45 min after injection with a significant accumulation remaining in the liver (Figure 1b). Eventual excretion into the enterohepatic circulation occurred during the following 24 h. Based on these results, the particles were covalently coated with methoxy-terminated poly(ethylene glycol) chains (PEG, ~0.5 kDa) according to the protocols in Patent Publication No. PCT/US2008/074894, in order to prevent opsonization and further enhance particle clearance while maintaining a small hydrodynamic size. This treatment decreased retention by the liver and resulted in increased renal filtration into the bladder within 45 min after injection by near-infrared fluorescence imaging (Figure 1c), with bladder fluorescence visible for up to 24 h. The probes were well tolerated, with no adverse effects or animal deaths observed during the course of the study. Serial co-registered PET-CT imaging 24 hours post injection of 124I-labeled PEG-coated nanoparticles (Figure 1d, top row) demonstrated a small amount of activity in the residual bladder, as well as overlapping activity in the liver/ gastrointestinal tract (center), flanked by independently acquired microCT and microPET scans. Serial microPET imaging confirmed the near-infrared fluorescence imaging findings. The blood half-life of 124I-labeled PEGylated nanoparticles based on time-dependent changes in blood activity over a 96-hour period was considered to be 7.3 hours. For nanoparticles linked to RGD, with a 124I label, the half-life of stay in the blood was considered to be 5.6 hours.

[00221] Based on these in vivo data, a more detailed study of biodistribution and clearance of coated nanoparticles was undertaken on two groups of particles containing PEGylated Cy5 to evaluate the effects of probe size on biodistribution. Nanoparticles with hydrodynamic diameters of 3.3 ± 0.06 and 6.0 ± 0.1 nm, as measured by fluorescence correlation spectroscopy (FCS), were generated (Figure 2a). Prior to in vivo studies, the photophysical properties of the particles were investigated to establish their performance levels versus free dye. Despite the extremely small particle size, silica-encapsulated dye molecules showed photophysical enhancements relative to the free dye that scaled with particle size, including significant increases in brightness, as determined by absorption and emission spectroscopy (Figure 2b). and FCS (Figure 2c). Compared with free dye, 3.3- and 6.0-nm diameter nanoparticles showed 2- and 3-fold increases in photodegradation half-life, respectively, when irradiated with a 635-nm high-power laser ( Figure 2d). Therefore, these nanoparticle probes were considered to be both brighter and more photostable than their free-dye counterparts.

[00222] In addition to semi-quantitative assessment of nanoparticle behavior in vivo from whole-body imaging, ex vivo analysis of tissue homogenates and fluids was performed using a fluorescence plate reader, which allowed calibrated quantification of the variations observed in near-infrared fluorescence imaging. Samples were grouped as "retained" (liver, kidney, lung, spleen homogenates, and blood) and "excreted" (urine) particle fluorescence sources, were background corrected, and were converted to percentage of initial dose (% DI ) per animal based on calibration curves. Tissue analysis showed minimal particle retention in major organs, with most of the fluorescence attributed to circulating blood (Figure 3a). Pure particle retention, calculated as the sum of “retained” components, was fitted with an exponential decay curve to determine excretion kinetics (Figure 3b). Larger particles had a longer tissue half-life (W2(3.3 nM) = 190 min, t/2(6.0 nm) = 350 min) and greater initial organic retention. After 48 h, the 6 nm particle showed minimal retention in the body (Rtotal(6.0 nm) = 2.4 ± 0.6% of DI). Urine samples collected at the time of sacrifice, together with serial dilution calibration data, were used to estimate total renal clearance based on a conservative estimate of the mean urinary volume excreted per unit time. Using this method, the % of DI excreted over time was estimated for both particle sizes (Figure 3c).

Example 3 Fluorescent Silica Nanoparticles Conjugated with αvβ3 Integrin Targeting Peptide (Melanoma Model)

[00223] In order to synthesize a multimodal nanoparticle (optical-PET) with high affinity for the tumor marker αvβ3 integrin, cyclic penta-peptide RGD (RGDYC) was conjugated to the nanoparticle through a Cys-maleimide bond. The tyrosine linker, Y, was used to subsequently attach a radiotracer. Male athymic nude mice were injected subcutaneously into their flanks with C6 rat glioma cells. At ~0.5 cm in diameter, mice were injected IV with either uncoated silica nanoparticles (Figure 4A) or PEG-ylated RGD nanoparticles (Figure 4B, ~500 nm/kg). Figure 4 shows in vivo biodistribution in non-tumor-bearing and tumor-bearing mice using whole-body optical imaging.

[00224] In vitro binding of targeted (RGD-bound) and non-targeted (PEG-coated) nanoparticles to αvβ3-integrin (M21) positive human melanoma cell lines was investigated as part of a dose response study using cytometry flow (Figures 5A, 5B). Particle binding/uptake was assessed as a function of time (Figure 5A) and concentration (Figure 5B).

Example 4 Fluorescent Silica Nanoparticles Conjugated with αvβ3 Integrin Targeting Peptide and Nodal Mapping (Melanoma Model)

[00225] We use a biocompatible material, silica, which has an architecture that can be precisely adjusted to particle sizes optimized for renal clearance. We attached small targeting peptides and a radioactive tag to the particle surface for serial PET imaging measurements in a well-characterized in vivo human melanoma model, and mapped draining lymph nodes and lymphatic channels using a near-infrared (NIR) dye. ) encapsulated and multi-scale optical fluorescence imaging methods. Ballou et al., Sentinel lymph node imaging using quantum dots in mice tumor models. Bioconjugate Chem. 18, 389-396 (2007). Kim et al., Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat. Biotechnol. 22, 93-97 (2003). Tanaka et al, Image-guided oncologic surgery using invisible light: completed pre-clinical development for sentinel lymph node mapping. J Surg Oncol. 13, 1671-1681 (2006). Toxicity testing was also performed and radiation doses of normal human organs were derived. Specifically, we synthesize core-shell silica nanoparticles encapsulating near-infrared (NIR) dye of about 7 nm in diameter, coated with PEG chains and functionalized on the surface with a small number (about 6 to 7) of targeting peptides and radiolabels.

[00226] We demonstrate that these probes are simultaneously non-toxic, exhibit high affinity/avidity binding, efficient excretion, and significant differential uptake and contrast between tumor and normal tissues using multimodal molecular imaging approaches. The sensitive detection, localization, and interrogation of lymph nodes and lymphatic channels, enabled by near-infrared dye fluorescence, highlights the distinct potential advantage of this multimodal platform for detecting and staging metastatic disease in the clinical setting, while also extending the smallest range of nodal sizes that can be detected.

Materials and methods Synthesis of Nanoparticles-cRGDY-PEG and Nanoparticles-PEG

[00227] The particles were prepared by a silica condensation of the modified Stober type as previously described. Wiesner et al., PEG-coated Core-shell Silica Nanoparticles and Methods of Manufacture and Use, PCT/US2008/74894. Larson, et al., Silica in noparticle architecture determines radiative properties of encapsulated chromophores. Chem. Mater. 20, 2677-2684 (2008). Bogush, et al., Preparation of Monodisperse Silica Particles: Control of Size and Mass Fraction. J. Non-Cryst. Solids, 104, 95-106 (1988). Sadasivan, et al., Alcoholic Solvent Effect on Silica Synthesis--NMR and DLS Investigation. J. Sol-Gel Sci. Technol. 12, 5-14 (1998). Herz, et al., Large Stokes-Shift Fluorescent Silica Nanoparticles with Enhanced Emission over Free Dye for Single Excitation Multiplexing. Macromol Rapid Commun. 30, 1907-1910 (2009). Tyrosine residues were conjugated to PEG chains for attachment of radioiodine or stable iodine moieties. Hermanson, Bioconjugate Techniques, (Academic Press, London, ed. 2, 2008). All samples were optical density matched at their peak absorption wavelength (640 nm) prior to radiolabeling. The cRGD peptides were attached to functionalized PEG chains via a cysteine-maleimide linkage, and the number of particle-bound cRGD peptides was estimated using FCS-based measurements of absolute particle concentrations and the starting concentration of reactants for the cRGD peptide.

Hydrodynamic Size Measurements and Relative Brightness Comparisons by Fluorescence Correlation Spectroscopy (FCS)

[00228] Particles dialyzed to water were diluted in physiological saline (0.15 M NaCl in H2O) and measured on a Zeiss LSM 510 Confocor 2 FCS using 633 nm HeNe excitation. The instrument was size calibrated before all measurements. Diffusion time differences were used to evaluate variations in the hydrodynamic sizes of the dye and particle species. Comparisons of the relative brightness of the free dye and PEG-nanoparticles and RGDY-PEG nanoparticles were performed using counting rates per molecule/particle.

Radiolabeling of C Dot Conjugates

[00229] Radiolabeling of cRGDY-PEG-nanoparticles and PEG-nanoparticles was performed using the IODOGEN method (Pierce, Rockford, Ill.). Piatyszek, et al., Iodine-mediated radioiodination of nucleic acids. J. Anal. Biochem. 172, 356-359 (1988). Activities were measured by gamma (Y) ray counting and fluorescence was measured using a Varian fluorometer (650 nm excitation/680 nm emission).

Cells and Cell Culture

[00230] Human melanoma cell lines M21 and M21 variants (M21-L, αv negative) were maintained in RPMI 1640 medium/10% fetal BSA, 2 mM penicillin L-glutamine, and streptomycin (Core Media Preparation Facility, Memorial Sloan Kettering Cancer Center, New York). Human umbilical cord endothelial cells (HUVECs) were cultured in M199 medium/10% fetal bovine serum, 20 μg/ml endothelial cell growth factor, 50 μg/ml heparin, penicillin, and streptomycin.

In Vitro Cellular Binding and Molecular Specificity of 124I-cRGD-PEG- Nanoparticles

[00231] In order to evaluate particle binding and specificity for M21 cells, 24-well plates were coated with 10 μg/ml type I collagen (BD Biosciences, Bedford, Mass.) in phosphate-buffered saline (PBS) and incubated (37° C., 30 min). M21 cells (3.0 to 4.0x105 cells/well) were grown to confluence and washed with RPMI 1640 medium/0.5% bovine serum albumin (BSA). 124I-cRGD-PEG-nanoparticles (0 to 4.0 ng/ml) were added to the wells and incubated cells (25° C., 4 hours), washed with RPMI 1640 medium/0.5% BSA, and dissolved in 0.2 M NaOH. Radioactivity was assessed using a 1480 Automatic Gamma Counter (Perkin Elmer) calibrated for iodine-124. Nonspecific binding was determined in the presence of a 1000-fold excess of cRGD (Peptides International, Louisville, Ky.). Scatchard plots were generated from the binding data and analyzed using linear regression analysis (Microsoft Excel 2007) to derive receptor binding parameters (Kd, Bmax, IC50).

In Vitro Cell Binding Studies Using Optical Detection Methods

[00232] Maximum differential binding of cRGDY-PEG-nanoparticles and PEG-nanoparticles to M21 cells was determined for a range of incubation times and particle concentrations using flow cytometry, with optimal values used in competitive binding and specificity studies. Cells (3.0*105 cells/well) were washed with RPMI 1640 medium/0.5% BSA, detached using 0.25% trypsin/EDTA, and pelleted in a microcentrifuge tube (5 min at 1400 rpm, 25° C.). The pellets were resuspended in BD FACSFlow solution (BD Biosciences, San Jose, CA.) and analyzed in the Cy5 channel to determine the percentage of probe bound to the particle (FACSCalibur, Becton Dickinson, Mountain View, CA.). Competitive binding studies were additionally performed after incubation of cRGDY-PEG-nanoparticles (2.5 ng/ml) with M21, M21L, and HUVEC cells in the presence of excess cRGD and/or anti-human integrin αvβ3 fluorescein-conjugated antibody. mouse monoclonal (Millipore, Temecula, CA.) and analyzed by flow cytometry. In order to evaluate the potency of the RGDY-PEG nanoparticles in relation to the cRGD peptide, anti-adhesion tests were carried out. 96-well plates were coated with vitronectin in PBS (5 μg/ml), followed by 200 μl of RPMI/0.5% BSA (1 h, 37° C.). Cells (3*104/100 μl/well) were incubated in quadruplicate (30 min, 25° C.) with various concentrations of cRGDY-PEG nanoparticles or cRGD peptide in RPMI/0.1% BSA, and added to vitronectin-coated plates (30 min, 37° C.). Wells were gently rinsed with RPMI/0.1% BSA to remove non-adherent cells; adherent cells were fixed with 4% PFA (20 min, 25° C.) and stained with methylene blue (1 h, 37° C.) to determine optical densities, measured using a Tecan Safire plate reader (Àex = 650 nm, Àem = 680 nm, bandwidth 12 nm). The multivalent boost factor was computed as the ratio of cRGD peptide to cRGDY-PEG-dot IC50 values. Montet, et al., Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem. 49, 6087-6093 (2006).

Animal Models and Tumor Inoculation

[00233] All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Memorial Sloan-Kettering Cancer Center and followed the National Institutes of Health guidelines for animal welfare. Male athymic nude/nude mice (6 to 8 weeks old, Taconic Farms Inc, Hudson, N.Y.) were provided with water containing potassium iodide solution to block uptake by the thyroid gland of any free radioiodine in vivo, and maintained in a Harlan Teklad Global Diet 2016, ad libitum, as detailed elsewhere 10. In order to generate M21 or M21L xenografts, equal volumes of cells (~5*106/100 μl) and matrigel were co-injected subcutaneously in the hind paw in different mice. Tumor sizes were regularly measured with calipers, yielding mean tumor volumes of 200 mm3.

In Vivo Pharmacokinetics and Dwell Half-Life Measurements (T112)

[00234] Time-dependent activity concentrations (% DI/g), corrected for radioactive decay at the time of injection, were measured by sacrificing groups of mice at specified times after i.v. injection of 124I-cRGDY-PEG-nanoparticles or 124I -PEG-nanoparticles (~20 μCi/mouse) and harvesting, weighing, and counting blood, tumor, and organs in a Y scintillation counter calibrated to 124I. The resultant time activity concentrations data for each tissue were fitted to a decreasing monoexponential function in order to determine the TI/2 and A values, the half-life of tissue/organic residence and zero-hour intercept, respectively, of the function .

[00235] The fraction of particles excreted in urine over time was estimated using previously described methods. Burns, et al., Fluorescent Silica Nanoparticles with Efficient Urinary Excretion for Nanomedicine, Nano Letters 9, 442-8 (2009). Briefly, mice were injected i.v. with either 200 μl of unlabeled cRGDY-PEG-nanoparticles or PEG-nanoparticles, and urine samples collected over a 168-hour period (n = 3 mice per time point). Particle concentrations at each time point were determined using fluorometric analyzes and a serial dilution calibration curve generated from background-corrected fluorescence signal measurements of urine samples spiked with known particle concentrations (% ID ). The concentration values, along with estimates of the mice's average daily urinary volumes, were then used to compute the % DI/g of cumulative urine excreted over time. In order to assess cumulative fecal excretion, metabolic cages were used to collect feces over a similar time interval after i.v. injection of 200 μl of 124I-cRGDY-PEG-nanoparticles (n = 4 mice per time point). Sample activities were measured using a Y counter calibrated to 124I.

Dosimetry

[00236] Time - activity functions derived for each tissue were analytically integrated (with inclusion of the effect of radioactive decay) to produce the corresponding cumulative activity (i.e., the total number of radioactive decays). The doses absorbed by the organs of 124I mice were then calculated by multiplying the cumulative activity by the constant 124I equilibrium dose for non-penetrating radiation (positrons), assuming complete local absorption of similar radiation and ignoring the contribution of penetrating radiation (i.e., radiation Y). Eckerman, et al., Radionuclide Data and Decay Schemes, 2nd ed. Reston, Va.: Society of Nuclear Medicine; 1989. Activities accumulated by normal mouse organs were converted to activities accumulated by normal human organs by adjusting for differences in total weight and organ masses between mice and humans (70 kg Standard Human). Cristy, et al., Specific absorbed fractions of energy at various ages from internal photon sources (I-VII). Oak Ridge National Laboratory Report ORNL/TM-8381/V1-7. Springfield, Va.: National Technical Information Service, Dept of Commerce; 1987. The calculated accumulated activities of normal human organs were introduced into the OLINDA dosimetry computing program to calculate, using the formalism of the Medical Internal Dosimetry (MIRD) Committee of the Society of Nuclear Medicine, the doses absorbed by organs by the Standard Man (Standard Man). Loevinger, et al., MIRD Primer for Absorbed Dose Calculations (Society of Nuclear Medicine, New York, 1991). Stabin, et al., OLINDA/EXM: the second-generation personal computer software for internal dose assessment in nuclear medicine. J Nucl Med. 46, 1023-1027 (2005).

Acute Toxicity and Histopathology Studies

[00237] Acute toxicity testing was performed on six groups of male and female B6D2F1 mice (7 weeks old, Jackson Laboratories, Bar Harbor, Me.). The treatment group (n = 6 males, n = 6 females) received unlabeled targeted probe (127I-RGDY-PEG-nanoparticles) and the control group (n = 6 males, n = 6 females) received iodinated PEG-nanoparticles unlabeled (vehicle, 127I-RGDY-PEG-nanoparticles) in a single i.v. injection (200 μl). Untreated controls (n = 2 males, n = 2 females) were additionally tested. Mice were observed daily for 14 days post-injection for signs of morbidity/mortality and weight changes, and autopsy, histopathology, and blood sampling for hematology and assessment of serum chemistry were performed at 7 and 14 days post-injection (Figure 10 and Table 3).

Serial PET Imaging for Tumor-Specific Targeting

[00238] Imaging was performed using a dedicated small animal PET scanner (Focus 120 microPET; Concorde Microsystems, Nashville, Tenn.). Mice carrying M21 or M21L tumors in the hind paw were maintained under anesthesia with 2% isoflurane in oxygen at 2 L/min throughout the scanning period. One-hour list-mode acquisitions were initiated at the time of i.v. injection of 200 μCi of 124I-cRGDY-PEG-nanoparticles or 124I-PEG-nanoparticles into all mice, followed by serial static imaging for 30 min during an interval of 96 hours. The image data was corrected for non-uniformity of the scanner response, counting losses due to dead time, random counts, and physical decay until the injection time. Voxel count rates in the reconstructed images were converted to activity concentrations (% DI/g) by use of a measured system calibration factor. Three-dimensional region of interest (ROI) analysis of the reconstructed images was performed using ASIPro software (Concorde Microsystems, Nashville, Tenn.) to determine the mean, maximum, and standard deviation of probe uptake in tumors. Tumor to muscle activity concentration ratios were derived by dividing the image-derived % ID/g tumor values by the Y counter values of % ID/g muscle.

Nodal Mapping Using NIR Fluorescence Imaging and Microscopy Combined

[00239] Nude mice carrying tumors in the hind paw were injected by peritumoral administration in the 4 quadrants, using equal volumes of a 50 μl sample of cRGDY-PEG-dot and were allowed to roam freely. After an interval of 30 min to 1 hour, mice were anesthetized with a 2% isofluorine/98% oxygen mixture, and a superficial paramidline incision was made vertically along the ventral aspect of the mouse in order to surgically expose the region of the hind paw to the axilla ipsilateral to the tumor. In situ optical imaging of locoregional (i.e., inguinal, axillary) and draining lymph nodes (including axillary region) was performed using a macroscopic fluorescence microscope equipped with 650 ± 20 nm NIR excitation and long-pass emission filters. 710 nm. Whole-body optical images (Cambridge Research Instruments Maestro imager) were additionally acquired and spectrally deconvolved as previously reported. Burns, et al., Fluorescent Silica Nanoparticles with Efficient Urinary Excretion for Nanomedicine, Nano Letters 9, 442-8 (2009).

Statistical Analysis

[00240] Statistical analyzes comparing groups of tumor-bearing mice receiving targeted/non-targeted probes or carrying M21/M21L tumors were performed using a one-tailed Mann-Whitney U test, with P<0.05 considered statistically significant. For biodistribution studies, the mean values of % tissue-specific DI/g of 124I-cRGDY-PEG-(n = 7 mice) and 124I-PEG-nanoparticles (control, n = 5 mice) were compared at each time point. time, with statistically significant differences in tracer activities observed in blood, tumor, and larger organs at 4 and 96 hours post-injection, as well as at 24 hours post-injection for tumor and other tissues (Table 1). For tumor targeting studies, differences in mean % DI/g values between M21 (n = 7) and M21L (n = 5) tumor-bearing mice, as well as mice receiving control probes (n = 5), were considered to be maximum at 4 hours post injection (p = 0.0015 for both controls), remaining significantly elevated at 24 hours (p = 0.0015 and p = 0.004, respectively), 48 hours (p = 0.001 and p = 0.003, respectively), 72 hours (p = 0.015 and 0.005, respectively), and 96 hours (p = 0.005 for M21- M21L). The tumor to muscle ratios for 124I-cRGDY-PEG-nanoparticles (n = 7) versus 124I-PEG-nanoparticles (n = 5) were considered to be statistically significant at 24 hours post injection (p = 0.001) and 72 hours post injection (p = 0.006), but not at 4 hours post injection (p = 0.35). The goodness of fit values (R2), along with their associated p values, were determined for the urinary calibration curve (R2 = 0.973, p = 0.01) as well as for the % ID of the urinary cumulative excretion curves. (R2>0.95, p = 0.047) and fecal (R2>0.995, p<0.002) using non-linear regression analysis (SigmaPlot, Systat, v. 11.0).

Results Scheme and Characterization of Nanoparticles

[00241] Core-shell silica nanoparticles encapsulating Cy5 dye (maximum emission >650 nm), coated with methoxy-terminated polyethylene glycol (PEG) chains (PEG ~0.5 kDa), were prepared according to previously published protocols . Burns, et al., Fluorescent Silica Nanoparticles with Efficient Urinary Excretion for Nanomedicine, Nano Letters, 9, 442-8 (2009). Ow, et al., Bright and stable core-shell fluorescent silica nanoparticles. Nano Lett. 5, 113-117 (2005). The neutral PEG coating prevented nonspecific uptake by the reticuloendothelial system (opsonization). The use of bifunctional PEGs allowed the attachment of small numbers (~6 to 7 per particle) of αvβ3 integrin-targeting cyclic arginine-glycine-aspartic acid (cRGDY) peptide ligands to maintain a small hydrodynamic size facilitating efficient renal clearance. Peptide ligands were additionally labeled with 124I through the use of a tyrosine linker to provide a signal which can be quantitatively evaluated by three-dimensional PET imaging (124I-cRGDY-PEG-dots, Figure 6A); An important practical advantage of relatively long-lived 124I (physical half-life: 4.2 days) is that sufficient signal persists long enough to permit radiodetection until at least several days post-administration, when background activity has largely cleared and tumor-to-background contrast is maximized. The purity of the radiolabeled targeted nanoparticle was >95% by radiothin layer chromatography. The stability of the non-radiolabeled targeted nanoparticle is about 1 year by FCS measurements. The particle is excreted intact in the urine by FCS analysis. As used herein in this patent application, "dot" and "nanoparticle" are used interchangeably. A PEG-coated particle containing a tyrosine residue for 124I labeling served as the control probe (124I-PEG-dots). Purification of the radiolabeled samples by size exclusion chromatography (Figure 7) resulted in radiochemical yields of >95%. Hydrodynamic diameters of ~7 nm inner diameter (i.d.) were measured for cRGDY-PEG-dots and non-radioactive PEG-dots by fluorescence correlation spectroscopy (FCS) (Figures 6B and 6C). The relative brightness of the cRGDY-PEG-dots was determined, on average, to be 200% greater than that of the free dye (Figure 6C), consistent with previous results. Burns, et al., Fluorescent Silica Nanoparticles with Efficient Urinary Excretion for Nanomedicine, Nano Letters, 9, 442-8 (2009). Larson, et al., Silica nanoparticle architecture determines radiative properties of encapsulated chromophores. Chem. Mater. 20, 2677-2684 (2008). Based on these physicochemical properties, we envision achieving a favorable balance between selective tumor uptake and retention versus renal clearance of the targeted particle, thereby maximizing localization to the target tissue while minimizing toxicity to normal tissues and radiation doses.

In Vitro Receptor Binding Studies

[00242] In order to examine the binding affinity and in vitro specificity of 124I-cRGDY-PEG-dots and 124I-PEGdots to vascular and tumor endothelial surfaces, melanoma cell lines overexpressing αvβ3 integrin (M21) were used and non-expressing αvβ3 integrin (M21L) and human umbilical vein endothelial cells (HUVECs). Highly specific linear and saturable binding of the cRGDY-PEG-dots was observed over a range of particle concentrations (0 to 8 ng/ml) and incubation times (up to 5 hours), with maximum differential binding at 4 hours and ~2. 0 ng/ml particle concentration (data not shown) using flow cytometry. The receptor binding specificity of 124I-cRGDY-PEG dots was tested using Y-radiation counting methods after initially incubating M21 cells with excess non-radiolabeled cRGD and then adding various concentrations of the radiolabeled targeted probe ( Figure 8A). Scatchard analysis of the binding data yielded an equilibrium dissociation constant, Kd, of 0.51 nM (Figure 8A, inset) and receptor concentration, Bmax, of 2.5 pM. Based on the Bmax value, the αvβ3 integrin receptor density was estimated to be 1.0x104 per M21 cell, in reasonable agreement with the previously published estimate of 5.6x104 for this cell line. Cressman, et al., Binding and uptake of RGD-containing ligands to cellular αvβ3 integrins. Int J Pept Res Ther. 15, 49-59 (2009). Incremental increases in integrin-specific M21 cell uptake were also observed over a temperature range of 4 to 37°C, suggesting that receptor-mediated cellular internalization contributed to total uptake (data not shown). Additional competitive binding studies using the targeted probe showed complete blockage of receptor-mediated binding with anti-αvβ3 integrin antibody (Figure 8B) by flow cytometry. No significant reduction in the magnitude of receptor binding (~10% of M21) was seen with M21L cells (Figure 8C) using either excess cRGDY or anti-αvβ3 integrin antibody. These results were confirmed by additional Y-counting studies, and a 50% binding inhibition concentration, IC50, of 1.2 nM was determined for 124I-cRGDY-PEG-dot. An associated multivalent enhancement factor of more than 2.0 was found for cRGDY-PEG-dot relative to the monomeric cRGD peptide using an anti-adhesion assay and M21 cells (data not shown). Montet, et al., Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem. 49, 6087-6093 (2006). Li, et al., 64Cu-labeled tetrameric and octomeric RGD peptides for small-animal PET of tumor αvβ3 integrin expression. J. Nucl Med. 48, 1162-1171 (2007). Similar to M21 cells, excess antibody effectively blocked cRGDY-PEG-dot receptor binding to HUVEC cells by flow cytometry (Figure 8D).

Biodistribution and Clearance Studies

[00243] Time-dependent biodistribution as well as renal and hepatobiliary clearance were evaluated by intravenously administering tracer doses (~0.2 nanomoles) of 124I-cRGDY-PEGdots and 124I-PEG-dots to mouse models of M21 tumor xenograft (Figure 9). Although tissue activity concentrations (percentage of injected dose per gram (% DI/g)) for the targeted probe were measured over a time interval of 196 hours post injection (pi), comparison of the 124I tracers -cRGDY-PEGdot (Figure 9A) and 124I-PEG-dot (Figure 9B) were restricted to a 96-hour window, as data for the latter were not acquired in week 1. Statistically significant differences (p< 0.05) in tracer activities were observed for blood, tumor, and major organs at 4 and 96 hours post-injection, as well as at 24 hours post-injection for tumor and several other tissues (Table 1). The targeted probe was almost entirely eliminated from the carcass within 1 week post injection (~3% of DI). The dwell half-lives (TI/2) for blood, tumor, and major organs for these tracers are shown in Table 2 (columns 2 and 5). A representative data set (blood retention) is shown in the inset of Figure 9A. A relatively long blood T1/2 value of 7.3 ± 1.2 hours was determined for 124I-PEG-dot. After fixation of the cRGDY peptide to synthesize 124I-cRGDY-PEG-dot, the T1/2 value decreased slightly to 5.6 ± 0.15 hours, but was accompanied by greater bioavailability of the probe (Table 2, column 3) . The tumor T1/2 value for 124I-cRGDY-PEG-dot was found to be about 13 times higher than that for blood, versus only a 5-fold difference for 124I-PEG-dot (Table 2 , columns 2 and 5).TABLE 1p-values from biodistribution study comparing 124I-cRGDY-PEG- and 124I-PEG-dots. T TABLE 2

[00244] By appropriate mass-adjusted translation of the preceding biodistribution data for man, radiation doses of normal human organs were derived and were found to be comparable to those of other commonly used diagnostic radiotracers (Table 2, columns 8 and 9). Along with the discovery that the targeted probe was non-toxic and did not result in tissue-specific pathological effects (i.e., no acute toxicity) (Figure 10 and Table 3), applications of targeted and non-targeted molecular imaging are planned. first time in man with these agents. TABLE 3 N: normal, U: Not available, NP: not present, 1: minimal, 2: mild F: focal, MF: multifocal

[00245] In another study to confirm that 127I-RGD-PEG dots are non-toxic after intravenous administration in mice, formal single dose toxicity testing was performed over the course of 2 weeks using 127I-RGD-PEG dots. (127I-RGD-PEG dots) at about 100 times the equivalent human dose. 127I-PEG dots served as the control particle. In summary, the procedure was as follows. Twenty-eight 8-week-old B6D2F1 mice were used in the acute toxicity study and were divided into a treatment group and a control group. The treatment group (n=6 males + 6 females) received a dose of RGD 127I-PEGkylated silica nanoparticles at a dose of 1x10-9 moles/mouse intravenously, and the control group (n=6 males + 6 females) received the same amount of vehicle. Two mice/group (one male and one female/group) were sacrificed on day 7 post dose and clinical chemical autopsy, hematology and tissue specific histopathology were performed. All remaining animals (n=5 males + 5 females/group) were observed for 14 days after treatment. Four untreated mice (two males and two females) were used as references. The conclusion of the studies was that no adverse events were observed during dosing or during the 14-day observation period. No mortality or morbidity was observed. Clinical observations included the absence of the following: anemia, weight loss, agitation, increased breathing, gastrointestinal disturbance, abnormal behavior, neurological dysfunction, hematology abnormalities, clinical chemistry abnormalities, or drug-related lesions in terms of pathology. of the bodies. Therefore, a single injection of 1x127I-PEGylated RGD silica nanoparticles and 1x9 moles/mouse, a dose equivalent to a 100-fold excess of the dose of PEGylated RGD silica nanoparticles required for Phase 0 imaging studies, is safe and not toxic in B6D2F1 mice.

[00246] Efficient renal excretion was found for targeted and non-targeted probes of ~7 nm in diameter over a time period of 168 hours by fluorometric analyzes of urine samples. Fluorescence signals were background corrected and converted to particle concentrations (% DI/μl) based on a serial dilution calibration scheme (Figure 9C, inset; Table 4, column 2). Burns, et al., Fluorescent Silica Nanoparticles with Efficient Urinary Excretion for Nanomedicine, Nano Letters, 9, 442-8 (2009). The concentration values, together with conservative age-dependent estimates of the mean urinary excretion rate, allowed the cumulative % DI excreted to be computed (Table 4, column 4). Drickamer, Rates of urine excretion by house mouse (mus domesticus): differences by age, sex, social status, and reproductive condition. J. Chem. Ecol. 21, 1481-1493 (1995). It was observed that almost half of the injected dose (about 43% of the DI) was excreted during the first 24 hours post injection and ~72% of the DI by around 96 hours, Figure 9C), suggesting that the majority of the excretion had occurred on the first day after injection. No significant particle fluorescence could be detected in urine 168 hours post injection. Fecal excretion profiles of 124I-cRGDY-PEG-dot indicated that, on average, 7% of the DI and 15% of the DI of the injected dose was eliminated during 24 and 96 hours, respectively (Figure 9D). FCS analysis of urine samples obtained at multiple time points after injection of the targeted probe revealed that the particle was excreted intact and without release of the encapsulated dye (data not shown). TABLE 4Urinary Concentration and Cumulative Excretion Data

Serial Whole Body PET Studies

[00247] PET scan imaging of integrin expression in M21 and M21L hindpaw subcutaneous xenograft mouse models was performed at multiple post-injection time points following i.v. injection of 124I-cRGDY-PEG-dots or 124I-PEG -dots (control). Representative full-body coronal microPET images at 4 hours (left: M21 tumor; middle: M21L tumor) and 24 hours (right: M21 tumor) post injection are shown in Figure 11A. The specific targeting of the M21 tumor overexpressing αvβ3 integrin is clearly visible from these images. Mean % tumor DI/g and standard deviations are shown for groups of M21 (n = 7) and M21L (control) tumors (n = 5) receiving the targeted 124I-cRGDY-PEG-dots, as well as for M21 tumor-bearing mice (n = 5) receiving non-targeted 124I-PEG-dot tracer (Figure 11B). At the time of maximum tumor uptake (~4 hours post injection), three-fold increases in activity concentration (in % DI/g) were seen in M21 tumors in relation to controls. The differences were statistically significant at all post-injection time points (p<0.05) with the exception of 1 hour (p = 0.27).

[00248] Imaging-derived tumor-to-muscle uptake ratios (% DI/g) for the 124I-cRGDY-PEG-dots revealed increased tumor contrast in the later hours (~24 to 72 hours post injection), whereas for 124I-PEG-dots declined (Figure 11C). This finding suggested that 124I-cRGDY-PEG-dots were, in fact, tumor-selective, which became more evident as activity in the blood cleared during the initial 24-hour period (compare Figure 11C with inset of Figure 9A). A statistically significant correlation was found between the values of % ID/g tissue of tumors derived by PET for both 124I-cRGDY-PEG-dots and 124I-PEG-dots, and the values of % ID/g of tumor counted by corresponding ex vivo Y radiation (correlation coefficient r=0.94, P<0.0016; Figure 11D), confirming the accuracy of PET for non-invasively derived quantitative biodistribution data.

In Vivo Near-Infrared Fluorescence Imaging and Microscopy

[00249] We perform in vivo fluorescence imaging studies using our targeted small nanoparticles for mapping local/regional lymph nodes and lymphatic channels, thereby overcoming the foregoing limitation. Importantly, the multimodal nature and small size of our targeted particle probe can be exploited to visualize a range of nodal and lymphatic branch sizes in our melanoma model after 4-quadrant peritumoral administration by simulating mapping procedures. of intraoperative human sentinel lymph node. Initially, serial near-infrared fluorescence microscopy was performed on intact mice over a 4-hour time period using either the targeted or non-targeted particle probes. Peritumoral administration of the targeted probe revealed inward drainage and persistent visualization of adjacent inguinal and popliteal lymph nodes during this interval, with smaller and/or more distant lymph nodes and lymphatics more difficult to visualize. In contrast, the untargeted probe produced shorter term (~1 hour) visualization of local lymph nodes with progressively weaker fluorescence signal observed (data not shown). After surgical exposure, this observation was considered to be the result of faster particle diffusion from the tumor site, compared to the prolonged retention observed with the targeted probe.

[00250] We next performed representative lymph node mapping over multiple spatial scales using live animal whole-body optical imaging (Figure 12A) and near-infrared fluorescence microscopy techniques (Figure 12B) to visualize lymphatic drainage from from the peritumoral region to the inguinal and axillary lymph nodes in surgically exposed live animals. Furthermore, higher resolution fluorescence images (Figure 12B, lower arrow) allowed us to visualize the intra-nodal architecture in more detail, including high endothelial venules, which facilitate the passage of circulating naïve lymphocytes into the lymph node, and the which may have important implications for nodal staging and the ability to detect micrometastases at earlier disease stages. Smaller, less intense lymphatic branches were also visualized by fluorescence microscopy in the axillary region (data not shown). Therefore, the small size of the targeted probe not only allows the first drainage (or sentinel lymph node), proximal to the tumor, to be visualized, but also allows more distant lymph nodes to be visualized and the lymphatic drainage pattern to be visualized.

Discussion

[00251] We report on non-toxic, high-affinity, and effectively cleared silica nanoparticles for tumor-selective targeting and tumor-selective nodal mapping, having successfully addressed a number of current challenges associated with other particle technologies. This is the first targeted nanoparticle that, based on its favorable properties, can be said to be clinically translatable as a combined optical-PET probe. The complementary nature of this multimodal probe, coupled with its small size (~7 nm in diameter), can facilitate clinical assessment by enabling the seamless integration of imaging data acquired at different spatial, temporal, and sensitivity scales, potentially cially providing new insights into the fundamental molecular processes that govern tumor biology.

[00252] Our in vitro results show receptor binding specificity of the ~7 nm particle probes targeted to M21 and HUVEC cells. Similar findings have been reported with receptor binding assays using the same cell types, but with the monovalent form of the peptide. Cressman, et al., Binding and uptake of RGD-containing ligands to cellular αvβ3 integrins. Int J Pept Res Ther. 15, 49-59 (2009). Importantly, the multivalence enhancement of the cRGDY-linked particle probe, along with extended blood and tumor T1/2 values, are key properties associated with the particle platform that are not found with the monovalent form of the peptide.

[00253] The relatively long blood T1/2 value of 7.3 ± 1.2 hours estimated for the 124I-PEG-dot tracer may be related to the chemically neutral PEG-coated surface, making the probe biologically inert and significantly less susceptible to phagocytosis by the reticuloendothelial system. It has been seen that a similar reduction in T1/2 values up to 5.6 ± 0.15 hours found for the 124I-cRGDY-PEG-dot tracer is most likely the result of recognition by target integrins and/or ma- more active crophages. However, it is substantially longer than published blood T1/2 values of existing cRGDY peptide tracers (~13 minutes), and results in greater probe bioavailability, facilitating tumor targeting and producing higher tumor uptakes during longer periods of time. Montet, et al., Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem. 49, 6087-6093 (2006). Furthermore, the tumor T1/2 value for 124I-cRGDY-PEG-dot was about 13 times higher than that for blood, versus only a five-fold difference for 124I-PEG-dot, suggesting localization in the substantially larger target tissue of the former than of the latter. Said mechanistic interpretations of in vivo data can be explored in order to refine clinical diagnosis, treatment planning, and treatment monitoring protocols.

[00254] The results of this study highlight the clear advantages offered by PET, a powerful, quantitative, and highly sensitive imaging tool for non-invasively extracting molecular information related to receptor expression levels, affinity binding, and specificity. The greater accumulation and slower clearance of M21 tumors, relative to surrounding normal structures, allows discrimination of specific tumor uptake mechanisms from nonspecific mechanisms (i.e., tissue perfusion, leakage) in normal tissues. A small component of M21 tumor uptake, however, can presumably be attributed to changes in vascular permeability (i.e., increased permeability and retention effects). Seymour, Passive tumor targeting of soluble macromolecules and drug conjugates. Crit. Rev. Ther. Drug Carrier Syst. 9, 135-187 (1992). This nonspecific mode of uptake reflects a relatively small portion of the total tumor uptake at early post-injection time points based on the %DI/g increases observed in mice receiving the control tracer (124I-PEG-dots, Figure 11B). At 1 hour post injection, no significant increases in % DI/g were seen in M21 tumors in relation to controls. This observation may reflect differential perfusion effects in the first hour, with tumor accumulation and retention essentially seen at later post-injection times (i.e., 24 hours). Additionally, compared to the clinically approved peptide tracer, 18F-galacto RGD, almost twice as much uptake in M21 tumors was seen for 124I-cRGDY-PEG-dots34, while additionally offering advantages of multivalent binding, blood circulation times prolonged periods, and greater renal clearance.

[00255] An advantage of a combined optical-PET probe is the ability to evaluate anatomical structures having sizes at or well below the resolution limit of the PET scanner (i.e., the so-called partial volume effect), which may compromise detection. and the quantification of activity in lesions. For example, in small animal models, assessment of metastatic disease in small local/regional lymph nodes, important clinically for staging and treatment of melanoma, may not be adequately resolved by PET scan imaging, given that the Sizes of observed lymph nodes are typically on the order of the spatial resolution of the system (1 to 2 mm). Using a second complementary and sensitive imaging modality, near-infrared (NIR) fluorescence imaging, functional maps revealing nodal disease and lymphatic drainage patterns can be obtained. Ballou, et al., Sentinel lymph node imaging using quantum dots in mice tumor models. Bioconjugate Chem. 18, 389-396 (2007). Although additional studies investigating the distribution of intra-nodal cRGDY-PEG-dot fluorescence in relation to metastatic foci are needed in order to determine whether sensitive localization of similar foci can be achieved, these results clearly demonstrate the advantages of working with a similar combined optical-PET probe.

[00256] In the clinic, the benefits of a similar combined platform for tumor staging and treatment cannot be overstated. The prolonged blood circulation time and resulting bioavailability of this nanoprobe highlights its use as a versatile tool for both early and long-term monitoring of the various stages of disease management (screening diagnosis, pre-treatment assessment, therapeutic intervention , and post-treatment monitoring) without restrictions imposed by toxicity considerations. An important additional advantage is that while rapidly cleared probes may be useful for certain applications where tissue localization itself is rapid, the localization of many agents is often poorly vascularized and otherwise relatively inaccessible solid tumors are likely to be slowed after systemic administration. Therefore, the present nanoparticle platform expands the range of applications of the aforementioned agents, since the kinetics of target tissue localization is no longer limiting. Furthermore, deep lymph nodes can be mapped by PET in terms of their distribution and number whereas more precise and detailed localization of superficial lymph nodes can be obtained by near-infrared fluorescence imaging. Finally, the relatively prolonged residence of the targeted probe in the tumor compared to that in the blood, in addition to its multivalency reinforcement, can be exploited for future theranostic applications as a radiotherapeutic or drug delivery vehicle.

Example 5 Fluorescent Silica Nanoparticles Conjugated with αvβ3 Integrin Targeting Peptide and/or uMUC1 Targeting Peptide (Thyroid Cancer and Squamous Cell Carcinoma (SCC) Models)

[00257] A cRGD peptide (Peptides International), having a cysteine end functionality, will be attached to the PEG-uylated nanoparticle through a thiol-maleimide bond. The nanoparticles can optionally be further functionalized by a synthetic peptide ligand, EPPT1. The nanoparticles will be characterized based on particle size, size distribution, and photodegradation.

Characterization of Nanoparticle-Peptide Conjugates

[00258] To evaluate photophysical properties on a per-particle basis, spectrophotometry, spectrofluorometry, and multiphoton fluorescence correlation spectroscopy (FCS) will be used to determine particle size, brightness, and size distribution. Size data will be corroborated by scanning electron microscopy and dynamic light scattering (DLS) measurements. Ow et al. Bright and stable core-shell fluorescent silica nanoparticles. Nano Letters 2005; 5, 113. The average number of RGD peptides per nanoparticle and the binding efficiency of RGD to functionalized PEG groups will be evaluated colorimetrically under alkaline conditions and Biuret spectrophotometric methods (À = 450 nm, maximum absorbance).

[00259] The nanoparticle conjugates will be iodinated through tyrosine linkers to create a radiolabeled (124I)(TI/2 ~4 days) and stable (127I) form using Iodogen (Pierce, Rockford, Ill.). The final product will be purified using size exclusion chromatography.

Assessment of In Vitro Targeting Specificity and Biodistribution Patterns of RGD- and RGD-EPPT-Nanoparticles.

[00260] The expression patterns of αvβ3 integrin and uMUC1 in thyroid and squamous cell carcinoma (SCC) cell lines will be evaluated against αvβ3 integrin-negative and αvβ3 integrin-positive controls (human melanoma cell lines M21-L and M21, respectively) and known uMUC1-negative and uMUC1-positive cell lines (U8728, H-29, respectively) using anti-integrin and anti-uMUC 1 antibodies. Cell lines highly expressing αvβ3-integrin and/or MUC1 will be selected for differential binding studies with RGD- and RGD-EPPT-nanoparticles, as well as for in vivo imaging.

[00261] Quantitative cell binding tests will evaluate the efficiency of marking tumor cells, and biodistribution studies evaluating uptake in the tumor, organs, and fluids will be carried out using radioiodinated nanoparticle conjugates (124I-RGD- nanoparticles, 124I-RGD-EPPT-nanoparticles). In order to compare PET uptake data of nanoparticle conjugates with that initially observed using NIRF optical imaging, each nanoparticle conjugate will also be iodinated to create a radiolabeled (124I) and stable (127I) form.

[00262] Fluorescence Microscopy with RGD- and RGD-EPPT-C- dots. Differential binding of RGD-nanoparticles and RGD-EPPT-nanoparticles to thyroid carcinoma/SCC cell lines highly expressing αvβ3-integrin and/or MUC1, versus control lines will be visualized by fluorescence microscopy.

[00263] Animal models. All animal experiments will be performed in accordance with protocols approved by the Institutional Animal Care and Use Committee and following NIH guidelines for animal welfare.

[00264] In vivo Biodistribution: Male athymic nude mice (6 to 8 weeks old, n = 5 per tumor) will be injected subcutaneously (s.c.) into both flanks with integrin-negative/positive or uMUC1-negative/positive tumors. different tissue origins (n = 3/each tumor). At 0.5 cm diameter (i.d.), mice will be injected intravenously (IV) with 124I-labeled nanoparticle conjugates (~500 nm/kg). Animals are sacrificed at 0.5, 1, and 24 hours later, with tumors, organs, and fluids removed for weighing and counting (gamma counter). Biodistribution results will be expressed as the percentage of the injected dose per gram of tissue.

[00265] Quantitative Cellular Connection Test. Labeling efficiency will be assessed by incubating fixed numbers of carcinoma cells highly expressing αvβ3-integrin and/or MUC1, with preselected concentrations of 124I-labeled nanoparticle conjugates for 1 hour in a humidified CO2 atmosphere at 37°C. Cells are extensively washed, lysed with 0.1% Triton X, with cell lysates counted in a gamma counter.

Assessment of Relative Differences in Tumor-Specific Targeting Using Multimodality Imaging (PET-NIRF) In Vivo.

[00266] As a high-throughput screening diagnostic tool, optical NIRF imaging can be used to assess relative differences in the biodistribution of progressively functionalized nanoparticle conjugates in vivo with increased sensitivity and temporal resolution. Semiquantitative data on tumor-specific targeting can also be derived. These preliminary studies facilitate the selection of cell lines strongly expressing markers of interest for additional detailed quantification of biodistribution and tumor-specific targeting using PET.

[00267] Whole-body NIRF and microPET® optical imaging will be performed over a 1-week period in order to assess differential uptake in flank tumors. The results of these studies will be validated with fluorescence microscopy of ex vivo tumors.

[00268] In Vivo Serial NIRF Imaging. Mice will be injected bilaterally with cells negative for αvβ3 integrin and positive for αvβ3 integrin or with cells negative for uMUC1 and positive for uMUC1 (n = 5/tumor). After tumors reach ~0.5 cm internal diameter (i.d.), iodinated and non-iodinated stable nanoparticle conjugates (RGD, 127I-RGD, RDG-EPPT, 127I-RGD-EPPT) will be injected IV. Serial imaging will be performed using the Maestro® in vivo fluorescence imaging system (Maestro® In Vivo Fluorescence Imaging System) (CRI, Woburn, Mass.) at 0, 0.5, 1, 2, 4, 6, 12, and 24 hours. At 24 hours, mice are euthanized, and larger tissues/organs are dissected, weighed, and placed in 6-well plates for ex vivo imaging. Fluorescence emission will be analyzed using regions of interest (ROIs) in relation to tumor, selected tissues, and injected reference, employing spectral unmixing algorithms to eliminate autofluorescence. Dividing the average fluorescence intensities of the tissues by the values of those injected will allow comparisons to be made between the various tissues/organs for each nanoparticle conjugate injected.

[00269] Dynamic MicroPET Imaging Acquisition and Analysis. Two groups of tumor-bearing mice (n = 5/tumor) will be injected with 124I-radiolabeled nanoparticle conjugates (radiotracers), and dynamic PET imaging will be performed for 1 hour using a Focus 120 microPET® (Concorde Microsystems, TN) . One-hour list-mode acquisitions are initiated at the time of IV injection of ~25.9 MBq (700 μCi) of radiotracers. The resulting list-mode data is reconstructed into a 128x128x96 pore filtered back-projection matrix. ROI analysis of the reconstructed images is performed using ASIPro® software (Concorde Microsystems, TN) to determine the mean radiotracer uptake (% DI/g) and standard deviation in tumors, other organs/tissues, and the left ventricle (LV ). Additional data will be obtained from still images at time points 24, 48, and 72 hours post injection. A three-compartment, four-parameter kinetic model will be used to characterize tracer behavior in vivo. For this analysis, arterial input is measured using a region of interest (ROI) placed over the left ventricle.

Example 6 Nodal Mapping in Mini Pig

[00270] Real-time intraoperative scanning of the nodal basin cannot be performed in practice at this time, as these systems are generally too expensive and complicated for use in the operating room or may not be able to provide the field of view or the necessary fabric contrast. Additionally, there are no clinically promising biostable fluorophore-containing agents offering improved photophysical characteristics and longer circulation lifetimes relative to parent dyes available to enhance tissue contrast for prolonged nodal mapping/resection procedures. With this animal study, we will show that advances in both multimodal particle probes and real-time molecular imaging device technologies can be readily translated to a variety of future human clinical trials. These transformative technologies can significantly impact routine intraoperative cancer care by providing state-of-the-art targeted visualization tools to facilitate metastatic sentinel lymph node detection and enabling accurate delineation of one or more lymph nodes from adjacent anatomy. so as to minimize the risk of injury to crucial structures. Benefits include prolonged in vivo real-time intraoperative mapping of nodal disease spread and tumor extension to the head and neck. Deep lymph nodes can be mapped by PET, while precise and detailed localization of superficial lymph nodes can be obtained by near-infrared fluorescence imaging. The small size of the particle probe may also extend the lower limit of nodal sizes that can be sensitively detected. The net effect of the proposed non-toxic multimodal platform, together with the application of combined diagnostic/treatment procedures, has important implications for disease staging, prognosis, and clinical outcome for this highly lethal disease.

Disease Target.

[00271] In addition to melanoma, a number of other tumors (i.e., breast, lung, and brain) overexpress αvβ3 integrin receptors and may serve as disease targets. Metastatic melanoma has a very poor prognosis, with a median survival of less than 1 year. Treatment success is based on early identification with adequate surgical excision of the cancer. Surgical removal of primary disease, screening, and treatment for spread to regional lymph nodes is the standard of care in the United States to accurately stage disease and personalize treatment. Recently revised staging guidelines recognize the presence of microscopic nodal metastases as a hallmark of advanced-stage disease leading to dramatically reduced survival. Knowledge of pathologic nodal status is crucial for early risk stratification, improved outcome predictions, and selection of patient subgroups likely to benefit from adjuvant treatment (therapeutic nodal dissection, chemotherapy) or clinical trials.

Sentinel Lymph Node (SLN) Mapping.

[00272] Sentinel lymph node mapping techniques, routinely used in melanoma staging, identify the one or more specific lymph nodes that are at greatest risk for tumor metastases. This procedure identifies patients harboring metastatic disease for additional treatment. Standard of care techniques rely on injection of radioactive technetium (99mTc) sulfur colloid dye around the primary tumor to localize the sentinel lymph node, followed by intraoperative use of a gamma probe to measure radioactivity in lymphatic structures within a basin. nodal exposed. Blue dye injected around the primary tumor can help delineate small sentinel lymph nodes from adjacent tissue, but the technique is unreliable and prone to complications. Current sentinel lymph node mapping and biopsy techniques have limitations, and represent higher rates of non-localization of one or more sentinel lymph nodes in the head and neck compared to other anatomical sites. The head and neck region is notorious for its unpredictable patterns of metastatic disease. The close proximity of the primary disease to nodal metastases in this region makes intraoperative use of the gamma probe difficult due to interference at the injection site. Importantly, current technology does not allow the surgeon to visualize the sentinel lymph node and reliably differentiate it from adjacent fat or other tissues, placing vital structures (i.e., nerves) at risk of injury during dissection to identify and harvest this lymph node. The small size of lymph nodes and wide variation in drainage patterns provide additional challenges, resulting in a non-localization rate of about 10%.

Nanoparticles.

[00273] Most preclinical studies have used radiotracers conjugated to peptide or RGD peptide as targeting ligands for imaging by αvβ3-integrin expression. 18F-galacto-RGD and 99mTc-NC100692 are peptide tracers that have been used successfully in patients to diagnose disease. Peptide tracers clear rapidly, which can result in reduced receptor binding and increased background signal from nonspecific tissue scatter. These properties limit the potential of peptide tracers for longer-term monitoring. In contrast, nanoparticle probes (~10 to 100 nm), which have also been used for integrin expression imaging throughout tumor neovasculature, have extended circulation half-lives to perform longer time monitoring (i.e., days ). Nanoparticles are typically larger than antibodies and radiopharmaceuticals (<10 kDa), and are associated with slower transmembrane transport, increased uptake by the reticuloendothelial system (RES), and increased nonspecific uptake due to altered tumor vascular permeability. The 7 nm diameter targeted nanoparticles used for this sentinel lymph node mapping study are roughly comparable to the average diameter of an albumin molecule and 2 to 3 times smaller than the average diameter of a typical antibody. In relation to peptide tracers, the targeted particle probe is less prone to extravasation and is associated with prolonged half-paths in the circulation that reinforce tumor targeting. Importantly, 124I-cRGDY-PEG-dots demonstrate key in vitro and in vivo properties in M21 tumors required for translation to the clinic.

Materials and methods.

[00274] Sinclair miniature pigs with spontaneous melanoma (10 to 12 kg, Sinclair Research Center, MO) were injected intravenously with 5 mCi of 18F-fluoro-deoxyglucose (18F-FDG) for whole-body screening of nodal metastases and /or organs. Mini pigs underwent 1-hour whole-body PET scanning by dynamic 18F-FDG PET using a clinical PET scanner 40 minutes after injection to screen for metastatic disease, followed by CT scan acquisition for anatomical localization. The mini pigs were then injected subdermally in a 4-quadrant pattern around the tumor site (preferably head and neck sites) with multimodal 124I-RGD-PEG-dots 48 hours after 18F-FDG PET, and a second Dynamic PET-CT scan was performed to evaluate additional nodal metastases.

[00275] The mini pigs were taken to the operating room for lymph node identification. Optical fluorescence imaging was performed using a large-field-of-view near-infrared fluorescence camera system, a modified smaller-field-of-view endoscope, and a modified stereomacroscope to obtain higher resolution within-field fluorescence images. exposed surgery.

[00276] Validation of the fluorescent signal was performed intraoperatively by counting gamma rays with a clinically approved handheld PET scan device within the operating bed to locate transdermally targeted points acquired intraoperatively from the skin and lymph nodes within and in the nodal basin.

[00277] The primary melanoma skin lesion was excised, and an incision was made to allow access to the sentinel lymph node(s). Nodal identity was confirmed using manual PET and multi-scale optical imaging systems, and the lymph nodes in question were excised. Samples were sent for histological evaluation for metastases and confocal optical microscopy to confirm the presence of both malignancy and nanoparticle fluorescence.

[00278] After harvesting the sentinel lymph nodes, the entire lymph node basin was excised and further evaluated using histological methods (with immunohistochemical markers for melanoma as needed), fluorescence microscopy, and the manual PET probe for correlative purposes. . This step helped identify any other malignant lymph nodes within the nodal basin and the number of 124I-RGD-PEG-dots present in adjacent lymph nodes by their appearance on imaging.

[00279] 124I-RGD-PEG-dots were administered subcutaneously to the animal's limbs sequentially. The transit of the 124I-RGD-PEG-dots to the inguinal/axillary lymph nodes was followed using the optical imaging system and handheld PET probes to confirm the duration of transit along the lymphatic pathways. The drainage of the nodal basins was surgically exposed and the drainage pattern of the lymph nodes was observed. The sentinel lymph node was harvested from each site to confirm the lymphatic nature of the tissue. Animals were euthanized, and any additional lesions noted on imaging were excised in the animal facility's necropsy room.

Discussion

[00280] A full-body 18F-fluorodeoxyglucose (18F-FDG) PET-CT scan revealed a primary melanomatous lesion adjacent to the spine over the upper back, as well as a single lymph node in the neck, posteriorly over the right side of the animal, which were both FDG-avid, and suspicious for metastatic disease. This finding was confirmed after subdermal injection, in 4 quadrants of 124I-RGD-PEG-dots around the tumor site, which additionally identified two more hypermetabolic lymph nodes, as well as draining lymph nodes. Interpretation of the final scan pointed to 3 potential metastatic lymph nodes. Surgical excision of the primary lesion, hypermetabolic lymph nodes, and tissue from other nodal basins in the neck was performed bilaterally after manual PET probes identified and confirmed high count rates at the location of the sentinel lymph node(s). The irregular fluorescence signal measured in excised right posterior sentinel lymph node tissue correlated with sites of melanoma metastases by histological analysis. All hypermetabolic nodal samples were pigmented black, and were seen to correlate with the presence of distinct clusters of melanoma cells. Therefore, results from surgically resected tissue subjected to pathology for H&E and staining for other known melanoma markers confirmed the multimodal imaging findings.

[00281] Figure 13a shows the experimental setup of using a spontaneous mini pig melanoma model for mapping lymph node basins and regional lymphatic drainage from the site of a known primary melanoma tumor. This intermediate-sized mini pig model is needed to simulate the application of sentinel lymph node (SLN) biopsy procedures in humans, and more accurately recapitulates human disease. Figure 13b shows small field of view PET image 5 minutes after subdermal injection of multimodal particles (124I-RGD-PEG-dots) around the tumor site. The tumor region, lymph nodes, and lymphatic drainage from the tumor site are seen as areas of increased activity (in black).

[00282] Figure 14 shows dynamic whole-body 18F-fluorodeoxyglucose (18F-FDG) PET scans (Figure 14a) and fused 18F-FDG PET-CT scans (Figure 14b) demonstrating sagittal, coronal, and axial images through the site of nodal disease in the neck. 18F-FDG PET scanning was performed to map sites of metastatic disease after intravenous administration and before administration of the radiolabeled nanoparticle probe. A single hypermetabolic lymph node is seen in the neck posteriorly on the right side of the animal (arrows, axial images, top/bottom panels), also identified on the full-body image of the mini pig (Figure 14c).

[00283] Figure 15 shows the same sets of images as in Figure 14, but at the level of the primary melanoma lesion, adjacent to the spine on the upper part of the back. The avid lesion by PET is identified (arrows, axial images, top/bottom panels) as well as on the full-body image of the mini pig (Figure 15c).

[00284] Figure 16 shows high-resolution dynamic PET images (Figure 16a) and fused PET-CT images (Figure 16b) after subdermal injection, in the 4 quadrants of 124I-RGD-PEG-dots around the tumor site, simulating the clinical protocol, for a period of time of 1 hour. Three hypermetabolic lymph nodes (arrows) were found in the neck, suggesting metastatic disease. The excised right posterior sentinel lymph node was excised and whole-body near-infrared (NIR) fluorescence imaging was performed. Cy5 fluorescence signal was detectable within the resected lymph node (Figure 16c, top, Cy5 imaging) on whole-body optical imaging. Pathologic analysis of this black-pigmented lymph node (arrow, SLN) demonstrated clusters of invasive melanoma cells on low-power (arrows) and high-power cross-sectional views of the lymph node by H&E staining (two smaller images), and we expect the specificity of the melanoma was further confirmed using special dyes (Melan A, HMB45, PNL2, and "melanoma-associated antigen" biogenex clone NKI/C3). We additionally expect particle colocalization with these metastatic clusters of cells on confocal fluorescence microscopy and high-resolution digital autoradiography, confirming detection of metastatic disease.

Example 7 Fluorescent Silica Nanoparticles Conjugated with MC1R Targeting Peptide (Melanoma Model)

[00285] For multi-modality diagnostic imaging experiments (PET-NIRF), the targeting peptide and radiotracer on the nanoparticle surface will be exchanged to determine target specificity, affinity/avidity of connection, and detection sensitivity. Subsequent therapeutic particles will also be synthesized using therapeutic radiolabels (lutetium-177, 177Lu, t1/2=6.65 days) for targeted killing of melanoma cells expressing MC1R. Combined quantitative PET and optical imaging findings will be correlated with tumor tissue autoradiography and optical imaging across spatial scales. For cellular microscopy, an in vivo confocal fluorescence scanner will be used for combined reflectance and fluorescence imaging.

Example 8 Fluorescent Nanoparticles for Targeted Radiotherapy

[00286] Dose escalation studies with 131I-RGD nanoparticles will be carried out and response to treatment will be monitored weekly, over the course of six weeks, using 18F-FDG PET. Time-dependent tumor uptake and dosimetry of the nanoparticle platform will be performed using planar gamma-ray camera imaging. In vivo imaging data will be correlated with gamma ray counts from excised tumor samples.

[00287] Male nude mice (6 to 8 weeks old, Charles River Labs, MA) will be used to generate hindpaw xenograft models after injection of M21 human melanoma cells (5x105 in PBS). Tumors will be left to grow for 10 to 14 days to 0.5 to 0.9 cm3 in size.

[00288] 131I-based targeted radiotherapy studies. The therapeutic radionuclide 131I will be used as a radiotracer for targeted radiotherapy. In estimating the highest possible dose of 131I resulting in no animal deaths and less than 20% weight loss (MTD), a dose escalation study will be carried out in tumor-bearing nude mice. For a dose of 200 rad to blood 54, an administered activity of 10 MBq is required, which will deliver a dose of 270 rad to the tumor. 4 doses of 10 MBq each will be administered to obtain a tumor dose greater than 1000 rad with dose fractionation designed to allow bone marrow repair and sparing. 131I enables planar gamma camera imaging using a pinhole collimator to measure time-dependent tumor uptake and nanoparticle dosimetry. 18F-FDG PET allows quantitative monitoring of tumor response, thus providing complementary information.

[00289] Based on these data, and in vivo data on the effect of nanoparticles loaded with paclitaxel, a therapy study with the 131I-RGD-nanoparticle conjugate will be conducted. Two groups of tumor-bearing mice (n = 10 per group) will each receive four, 10.4 MBq activities once a week for 4 weeks of nanoparticle-131I-RGD conjugates administered by i.v. or saline vehicle ( control, n = 10), and will be monitored over a period of 6 weeks. Treatment response/progression will be quantified based on tumor volume (through caliper measurements). All mice in the treatment groups will also be imaged once a week (~1 hour sessions) by SPECT imaging (Gamma Medica) over a period of 6 weeks.

[00290] 18F-FDG PET Imaging Acquisition and Analysis. Two groups of tumor-bearing mice (n = 10/group) will undergo initial PET scanning before and then on a weekly basis after treatment over a 6-week interval. The mice will be injected intravenously (i.v.) with 500 μCi of 18F-FDG and static images will be acquired by 10-minute PET scan using a Focus 120 microPET® (Concorde Microsystems, TN) before and after treatment. The acquired data will be reconstructed into a 128x128x96 matrix by filtered back-projection. Regions of interest (ROI) analyzes of the reconstructed images will be performed using ASIPro® software (Concorde Microsystems, TN) to determine the mean radiotracer uptake and standard deviation (% DI/g) in tumors. The animals will be sacrificed at the end of the study and the tumors will be excised for gamma ray counting.

Example 9 Fluorescent Nanoparticles Conjugated with Radionuclide Chelate and Targeting Peptide for MC1R

[00291] PEG-ylated nanoparticles will be conjugated with targeting peptides and macrocyclic chelates binding radiolabels with high specific activity.

[00292] Commercially available activated two-branch high purity PEGs, derivatized with NHS or maleimide esters, will be attached to the silica shell of the nanoparticle using routine procedures. Either of the two functionalized PEG groups (NHS or maleimide esters) will be available for further conjugation to either the peptide-chelate construct, cyclic peptide Re-[Cys3,4,10,D-Phe7]α-MSH3-13 ( ReCCMSH(Arg11)), or with 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) linker chelators. Covalent attachment of derivatized PEGs to the nanoparticle surface will be performed in a manner so as to expose different functional groups for binding of DOTA and peptide-chelate constructs, as discussed below.

Synthesis and Physicochemical Characterization of Functionalized Nanoparticles.

[00293] Functionalized nanoparticles will be synthesized by establishing covalent bonds of the following portions with the derivatized PEG groups:

[00294] (A) DOTA chelates for subsequent radiolabeling of high specific activity with positron-emitting radiometals (i.e., 64Cu) to enable diagnostic detection with PET imaging. DOTA will be conjugated to the functionalized PEGs using routine Fmoc chemistry, and purification of the chelated nanoparticles will be performed by chromatography. 64Cu and 177Lu will be attached to DOTA by incubating the reaction mixture at 60° C. for 30 min followed by gel filtration or purification by high pressure liquid chromatography. Alternatively, PET nuclides, such as 124I, 86Y, 68Ga and 89Zr, can be conjugated to the nanoparticle, either through PEG functionalized with DOTA (radiometals) or PEG functionalized with tyrosine (124I). The single photon emitter, 177Lu, obtained in the form of 177LuCh will be complexed with DOTA for radiotherapy.

[00295] (B) Melanoma targeting peptide analogue αMSH (ReCCMSH(Arg11)) is cyclized by rhenium. It is necessary to confirm the proportion of DOTA chelates to portions of ReC- CMSH(Arg11) on the surface of the PEG-uylated nanoparticle.

[00296] The characterization of the functionalized nanoparticle preparations will be carried out as follows:

[00297] (A) The average number of DOTA chelates per nanoparticle will be determined by routine isotopic dilution test with 64Cu. In summary, 64Cu will be added to solutions containing a known amount of ReCCMSH(Arg11)-nanoparticles. The incubated solutions will be dotted onto glass plates coated with silica gel, developed in 1:1 of 10% ammonium acetate to methanol (with EDTA), and analyzed by radio-TLC. While 64Cu-labeled ReCCMSH(Arg11) nanoparticles will remain at the origin, 64Cu bound to EDTA will migrate. The percentage labeling efficiency will be plotted against the total nanomoles of 64Cu added to the reaction mixture. The number of chelates attached per nanoparticle can be determined from the inflection point of this curve.

[00298] (B) The average number of ReCCMSH(Arg11) peptides per nanoparticle and the binding efficiency of ReCCMSH(Arg11) to the functionalized PEG groups will be evaluated using spectrophotometric methods (À = 435 nm, maximum absorbance) and the known extinction coefficient of ReCCMSH(Argll). The incorporation of rhenium offers the advantage that highly sensitive absorbance measurements of rhenium concentrations can be made on a small sample of product.

Multifunctional Nanoparticle In Vitro and In Vivo Optical-PET Imaging

[00299] Nanoparticles in Melanoma Models to Assess Tumor-Specific Targeting and Treatment Response.

[00300] 64Cu-DOTA-ReCCMSH(Arg11)-nanoparticles will be compared with the native construct 64Cu-DOTA-ReCCMSH(Arg11) to test the targeting capabilities of the nanoparticles.

[00301] Competitive binding tests. B16/F1 murine melanoma lines positive for the MC1R receptor will be used. ReCCMSH(Arg11) peptide IC50 values, the peptide concentration required to inhibit 50% of radioligand binding, will be determined using 125I-(Tyr2)-NDP7, a radioiodinated α-MSH analog with picomolar affinity for the MC1R. Single wells will be incubated at 25° C. for 3 h with approximately 50,000 cpm of 125I-(Tyr2)-NDP in 0.5 ml of binding medium with 25 mmol/L of N-(2-hydroxyethyl) acid -piperazine-N-(2-ethanesulfonic acid), 0.2% BSA and 0.3 mmol/L 1,10-phenanthroline], with (Arg11)CCMSH concentrations ranging from 10 to 13 to 10 to 5 mol/ L. Radioactivity in the cells and medium will be collected and measured separately, and the data will be processed to calculate the IC50 value of the Re(Arg11)CCMSH peptide with the Kell software package (Biosoft, MO).

[00302] Receptor Quantification Test. Aliquots of 5*105 B16/F1 cells will be added to the wells, cultured in 200 μL of RPMI medium, and incubated at 37° C. for 1.5 h in the presence of increasing concentrations of 125I-(Tyr2)-NDP (a from 2.5 to 100 nCi) in 0.5 mL of binding medium (MEM with 25 mM HEPES, pH 7.4). The cells will be washed with 0.5 mL of ice-cold, pH 7.4, 0.2% BSA/0.01 M PBS twice, and the activity level associated with the cellular fraction will be measured in a counter. Y. Nonspecific binding will be determined by incubating the cells and 125I-(Tyr2)-NDP with non-radioactive NDP at a final concentration of 10 μM. Scatchard plots will be obtained by plotting the proportion of specific binding to free 125I-(Tyr2)-NDP versus the concentration of specific binding (fmol/million cells); Bmax, the maximum number of connection sites, is the X-intercept of the linear regression line.

[00303] B16/F1 murine melanoma lines (5*105 in PBS) will be injected subcutaneously into the hind paws of male nude mice (6 to 8 weeks old). The tumors will be left to grow for 10 to 14 days to 0.5 to 0.9 cm3 in size.

[00304] Biodistribution: A small amount of the 64Cu-DOTA-ReCCMSH(Arg11)-nanoparticle conjugate (~10 μCi, 0.20 μg) will be injected intravenously into each of the mice carrying palpable B16/F1 tumors. Animals will be sacrificed at selected time points after injection (2, 4, 24, 48, 72 hours; n = 4 to 5/time point) and desired tissues will be removed, weighed, and counted for accumulated radioactivity. Additional mice (n = 5) injected with the native radiolabeled construct, 64Cu-DOTA-ReCCMSH(Arg11) (~10 μCi, 0.20 μg) will serve as the control group, and evaluated 1 h post injection. In order to examine the specificity of uptake in vivo, an additional group of mice (2 h time point) will be pre-injected with 20 μg of NDP to act as a receptor blockade immediately prior to injection of the NDP nanoparticle conjugate. 64Cu-DOTA-ReCCMSH(Arg11). Large organs and tissues will be weighed and counted in a gamma counter, and the percentage of dose injected per gram will be determined (% of DI/g).

[00305] NIRF Serial In Vivo Imaging. In parallel with the PET studies below, NIR (fluorescence tomographic imaging, FMT 225, Visen, Woburn, Mass.) will be performed using an adjustable 680 nm scanning NIR laser beam and CCD before and after injection. i.v. of tumor-bearing animals (n = 10). Mice will be maintained under continuous isoflurane anesthesia, and placed in a portable multimodal imaging cassette (compatible with both our FMT 2500 and Focus 120 microPET) for FMT scanning before and after injection (1, 2, 4, 6 , 12, 24, 48 and 72 hours). The NIR fluorescence image, measured over a period of 1 to 10 minutes, will be reconstructed using proprietary Visen software and superimposed over a normal photograph of the mouse. The imaging data is quantitative as the measured intensity is directly related to the NIR fluorophore concentration, allowing parametric maps of absolute fluorophore concentrations to be generated for co-registration with the acquired PET imaging data.

[00306] Dynamic PET Image Acquisition and Analysis. Two groups of tumor-bearing mice (n = 5/group) will be placed in the imaging cassette for PET-optical sequential co-registration studies. The mice will be injected intravenously (i.v.); one with radiolabeled 64Cu-DOTA-ReCCMSH(Arg11) nanoparticle conjugates and the second with native 64Cu-DOTA-ReCCMSH(Arg11) constructs. After injection, 1-hour dynamic PET images will be acquired using a Focus 120 microPET® (Concorde Microsystems, TN). Acquisitions for one hour in list-mode are initiated at the time of IV injection of radiolabeled probe (~1 mCi). The resulting list-mode data will be reconstructed into a 128x128x96 matrix by filtered back-projection. Regions of interest (ROI) analyzes of reconstructed images are performed using ASIPro® software (Concorde Microsystems, TN) to determine mean radiotracer uptake and standard deviation (% DI/g) in tumors, other organs/tissues , and in the left ventricle (LV). Kinetic modeling of tracer data will allow estimation of pharmacokinetic parameters, including release, clearance, and volume of distribution. As mentioned, arterial blood input is measured using a region of interest placed over the left ventricle (as a measure of blood activity). Additional data will be obtained from still images at time points 24 hr, 48 hr, 72 hr after injection.

[00307] Fluorescence microscopy and tissue autoradiography. A combination of optical imaging technologies featuring progressively smaller spatial scales (i.e., whole-body fluorescence imaging, fluorescence macroscopy, and in vivo confocal laser scanning microscopy) will be used to image tumors in animals. intact alive within 72 hours post injection. Mice will be maintained under continuous isofluorane anesthesia, thereby allowing detection and localization of the fluorescence signal from the whole animal/organ level to the cellular level over a range of magnifications. Macroscopic imaging of the whole animal will be performed with a fluorescence stereomicroscope (Visen; Nikon SMZ1500) equipped with a series of Cy5 fluorescence filters and CCD cameras. Fluorescence confocal laser scanning microscopy capabilities will be developed. The mice will then be euthanized for autoradiography in order to map tracer biodistributions in high-resolution tumor volume throughput. Tumors will be excised, snap-frozen, serially sectioned (100 μ sections), and mounted on slides, with alternating pieces placed in contact with a phosphor plate in a light-tight cassette (up to 1 week). H&E staining will be performed on the remaining consecutive sections. Autoradiographic findings will be correlated with PET imaging data and histological results.

[00308] Therapeutic radionuclides 177Lu or 90Y can alternatively be used for targeted radiotherapy. In estimating the highest possible dose of 177Lu not resulting in animal death and resulting in less than 20% weight loss (MTD), a dose escalation study will be carried out in tumor-bearing nude mice. Radiopharmaceutical doses suspected to be at (or close to) the MTD based on literature values for 177Lu will be evaluated.

Example 10

[00309] Functionalized Fluorescent Nanoparticles to Conjugate with Ligand and Contrast Agent Through "Click Chemistry"

Synthesis of Nanoparticles Containing Versatile Functional Groups for Subsequent Conjugation of Ligand (e.g., Peptides) and Contrast Agent (e.g., Radionuclides).

[00310] In order to synthesize a series of nanoparticle-peptide-chelate constructs suitable for radiolabeling of highly specific activity, a "click chemistry" approach can be used to functionalize the nanoparticle surface (Figure 17). This method is based on the copper-catalyzed cycloaddition of azide to a triple bond. This approach will allow great versatility to explore multimodal applications.

[00311] Synthesis and characterization of nanoparticles. The PEG groups that will be covalently attached will be produced following the scheme in Figure 14. PEG will be covalently attached to the nanoparticle through the silane group. Routine chemical pathways will be used to produce PEG functionalized with triple bonds.

[00312] Functionalization of nanoparticles with triple bonds. In order to synthesize bifunctionalized PEGs, the first step will employ the well-studied reaction of activated carboxylic ester with aliphatic amine (Figure 18). Alternatively, another amine carrying a suitable triple bond may be used, for example p-aminophenylacetylene. The second stage of the synthesis is also based on a general knowledge conjugation reaction.

Synthesis and Physicochemical Characterization of Functionalized Nanoparticles Conjugated with Model Peptides and Chelates.

[00313] The functionalized nanoparticle contains both (A) desferrioxamine B (DFO) for subsequent radiolabeling of highly specific activity with the positron-emitting zirconium 89 (89Zr) and (B) the SSTR targeting peptide, octreotate.

[00314] Synthesis of DFO with an azide linkage. DFO with an azide group will be produced by reacting DFO-B with benzoic Pazido acid) (Figure 19) and purified. The "click chemistry" reaction is a 1,3-dipolar cycloaddition at room temperature and the conditions are often referred to as "Huigsen Conditions". Although reactions can generally be completed at room temperature in ethanol, it may be appropriate to heat the reaction. The catalyst is often Cu(I)Br, but alternatives include Cu(I)I or Cu(II)SO4 (with a reductant). Knor et al. Synthesis of novel 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) derivatives for chemoselective attachment to unprotected polyfunctionalized compounds. Chemistry, 2007; 13:6082-90. Click reactions can also be carried out in the absence of any catalyst. Alternatively, the NH3+ group in DFO-B can be converted directly to an azide group.

[00315] Synthesis of Tyr3-octreotate with an azide. Solid phase peptide synthesis (SPPS) of Tyr3-octreotate (Figure 20A) will be performed on a peptide synthesizer. In summary, the synthesis will involve the Fmoc (9-fluorenylmethoxycarbonyl) method as previously described for this peptide. In summary, the instrument protocol requires 25 μmol of subsequent Fmoc-protected amino acids activated by a combination of 1-hydroxybenzotriazole (HOBt) and 2-(1H-benzotriazol-1-yl)-1,1,3,3- hexafluorophosphate. tetramethyluronium (HBTU). Fmoc-protected amino acids will be purchased commercially unless otherwise stated; prepackaged amino acids will be obtained from Perkin-Elmer (Norwalk, Conn.), while those unavailable in prepackaged form, such as Damino Acids and Fmoc-Cys(Acm) will be supplied by BACHEM Bioscience, Inc. (King of Prussia, Pa.) or Novabiochem (San Diego, CA.). The azide groupamenoth (for "click" chemistry) will be introduced into the peptide backbone by attaching an azide-containing acid to the N-terminus of the peptide, while the peptide is still protected and attached to the resin (Figure 20B).

[00316] Synthesis of functionalized nanoparticles. The next step will be to conjugate both DFO having an azide linkage and Tyr3-octreotate having an azide linkage (Figures 21A and B) to the nanoparticle. "Click chemistry" is highly selective, quantitative and can be carried out very quickly and using mild conditions. The number of combined azide groups of DFO and Tyr3-octreotate will be controlled so as never to exceed the number of available triple bonds; triple bonds will always be in <5% excess.

[00317] Characterization of functionalized nanoparticles. The average number of DFO chelate peptides per nanoparticle will be determined by performing a routine isotopic dilution test with 89Zr (or 68Ga). 89Zr will be produced in a cyclotron and purified. In summary, 10 concentrations of 89Zr-oxalate will be added to solutions containing a known amount of DFO-derived nanoparticles. After incubation for 30 min. at room temperature, the solutions will be dotted onto glass plates coated with silica gel, developed in 1:1 of 10% ammonium acetate to methanol (with EDTA) and analyzed by radio-TLC. Whereas nanoparticles derived from 89Zr-DFO will remain at the origin, 89Zr bound nonspecifically to EDTA will migrate. The percent labeling efficiency will be plotted as a function of the total nanomoles of 89Zr added to the reaction mixture. The number of chelates attached to the nanoparticle can then be determined from the inflection point of this curve.

[00318] The average number of Tyr3-octreotate peptide per nanoparticle will be determined by evaluating the Tyr3-octreoate disulfide bond. In summary, the disulfide bonds of Tyr3-octreotate can be quantitatively cleaved by excess sodium sulfite at pH 9.5 and room temperature. DTNB or Elman's reagent can be used to quantify thiols in proteins by absorption measurements. It readily forms a mixed disulfide with thiols, releasing the chromophore 5-merapto-2-nitrobenzoic acid (maximum absorption 410 nm). Only protein thiols that are accessible to this water-soluble reagent are modified. Alternatively, the Measure-iT® Thiol Assay Kit from Invitrogen can be used.

In Vivo Testing in Appropriate Tumor Models.

[00319] Subcutaneous xenograft models will be generated using female SCID mice bearing AR42J tumor. In summary, AR42J cells (1x107) will be injected subcutaneously into the flanks of female SCID mice. The tumors will be left to grow for 10 to 12 days to 0.5 to 0.9 cm3 in size.

[00320] Radiolabeling of DFO-nanoparticles by 89Zr is expected to proceed in <15 min. at room temperature. Nonspecifically bound 89Zr will be removed by addition of EDTA followed by a gel filtration step.

[00321] Receptor binding tests. Receptor binding tests will be performed using 89Zr-DFO-nanoparticles on membranes obtained from AR42J tumors. The competing ligands, natZr-DFO-Nanoparticles and natZr-DFO-octreotate, will be prepared by reacting high-purity natural zirconium oxalate with DFO-octreotate and DFO-Nanoparticles, respectively. The purity of the final products will be confirmed by HPLC. IC50 values will be determined according to previously published methods using the Millipore MultiScreen test system (Bedford, Mass.). Data analysis will be performed using the programs GraFit (Erithacus Software, United Kingdom), LIGAND (NIH, Bethesda, Md.), and GraphPad PRISM® (San Diego, CA.).

[00322] In vitro tests. AR42J cells will be harvested from monolayers with cell dissociation solution (Cell Dissociation Solution, from Sigma Chemical Co., St. Louis, Mo.) and resuspended in fresh DMEM medium at a concentration of 2x106 cells/ ml. An aliquot of about 0.3 pmol of 89Zr-DFO- nanoparticles will be added to 10 mL of cells, incubated at 37° C. with continuous shaking. At 1, 5, 15, 30, 45, 60 and 120 min aliquots of 200 μL in triplicate will be removed and placed on ice. The cells will be isolated immediately by centrifugation, and the % uptake of the compound into the cells will be calculated.

[00323] Biodistribution. A small amount of the 89Zr-DFO-nanoparticles (~10 μCi, 0.20 μg) will be injected intravenously into each of the mice carrying palpable AR42J-positive tumors. Animals will be sacrificed at selected time points after injection (1, 4, 24, 48, 72 hours; n = 4 to 5) and desired tissues will be removed, weighed, and counted for radioactivity accumulation. Two additional control groups will be studied at 1 h post-injections: (A) mice injected with the native radiolabeled peptide 89Zr-DFO-octreotate (~10 μCi, 0.20 μg), and (B) mice pre-injected with a Tyr3-octreotate block (150 μg) to demonstrate receptor-mediated accumulation of 89Zr-DFO-nanoparticles. Tissues including blood, lung, liver, spleen, kidney, adrenals (STTR positive) muscle, skin, fat, heart, brain, bone, pancreas (STTR positive), small intestine, large intestine, and AR42J tumor will be counted. The percentage of dose injected per gram (% of DI/g) and the percentage of dose injected per organ (% of DI/organ) will be calculated by comparison with a weighed and counted standard solution.

[00324] NIRF imaging in vivo. Serial imaging will be performed using the Maestro® In Vivo Fluorescence Imaging System (CRI, Woburn, Mass.) at 0, 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 hours. Within 72 hours, the mice will be euthanized, and the larger tissues/organs dissected, weighed, and placed in 6-well dishes for ex vivo imaging. Fluorescence emission will be analyzed using regions of interest (ROIs) on tumor, selected tissues, and reference injectates, employing spectral demixing algorithms to eliminate autofluorescence. Fluorescence intensities and standard deviations (SD) will be weighted for groups of 5 animals. Dividing the average fluorescence intensities of the tissues by the values of those injected will allow comparisons to be made between the various tissues/organs for each injected nanoparticle conjugate.

[00325] In vivo small animal PET imaging. Small animal PET imaging will be performed on a micro-PET®-FOCUS® system (Concorde Microsystems Inc, Knoxville Tenn.). Mice carrying AR42J tumors (n = 5 per group) will be anesthetized with 1 to 2% isoflurane, placed in a supine position, and immobilized on a custom-prepared support. The mice will receive 200 μCi of the 89Zr-DFO-octreotate-nanoparticle complex via the tail vein and will be studied by side-by-side imaging. Initially the animals will be imaged by acquiring multiple successive 10 minute scans continuously from the time of injection over a 1 hour time frame, followed by static data acquisitions for 10 min at 2, 4, 24, 48 and 72 hours. post injection. Standard uptake values (SUVs) will be generated from regions of interest (ROIs) drawn over the tumor and other organs of interest. Co-registration of PET scan images will be achieved in combination with a microCAT-II camera (Imtek Inc., Knoxville, Tenn.), which provides high-resolution anatomical images by X-ray CT. Image registration between microCT and PET scan images will be performed using a reference registration technique and AMIRA image display software (AMIRA, TGS Inc, San Diego, CA.). The registration method proceeds by rigid transformation of the microCT reference images provided by reference marks directly affixed to the animal's bed.

[00326] Pharmacokinetic measurements. Biodistribution and dynamic PET data will provide the temporal concentration of 89Zr-DFO-octreotate-nanoparticle in the tissue, which will allow the characterization of the agent's pharmacokinetic parameters.

[00327] Fluorescence microscopy and autoradiography of ex vivo tissues. The localization of nanoparticle conjugates in tissues on frozen sections will be carried out. MicroPET imaging will allow us to fully assess global distribution in tumors and other non-target tissues. After the acute stage of the imaging test, autoradiography will also be performed on the tumors, and this data will be correlated with both PET imaging and histological results. Consecutive sections (~10 μm) will be taken, alternating sections for autoradiography and histological analysis. These sections will also be analyzed by multichannel fluorescence microscopy in the near-infrared channel.

Example 11 Particle Internalization Studies

[00328] The goal of this study is to evaluate the binding and internalization of the present nanoparticles to evaluate their localization in subcellular organelles and exocytosis. This will help study the fate of functionalized particles with different targeting moieties and the attached therapies. For example, both diagnostic nanoparticles (e.g., non-targeting PEG-coated versus cRGD-PEG coated nanoparticles) and therapeutic nanoparticles (e.g., cRGD-PEG-iodine-attached nanoparticles for radiotherapy, attached to tyrosine inhibitors kinase, or attached to chemotherapy drugs such as Taxol.)

Materials and methods

[00329] Internalization / uptake studies. Internalization and colocalization tests were carried out to identify specific uptake pathways.

[00330] Melanoma cells, including human M21 and mouse B16 cells (~2*105 cells/well), were plated on 8-well chamber slides (1.7 cm2/well) or in 24-well plates (1.9 cm2/well) with a 12 mm round glass slide and incubated at 37° C. overnight. In order to monitor internalization of targeted nanoparticles, cells were incubated with dots of cRGD-PEG (0.075 mg/ml) for 3 hours at 37°C. In order to remove unbound particles in the medium, cells were rinsed twice with PBS. Confocal microscopy was performed on a Leica inverted confocal microscope (Leica TCS SP2 AOBS) equipped with a HCX PL APO: 63x1.2NA Water DICD objective to assess the co-localization of cRGD-PEG-dots with organelle-specific antibodies or dyes. Images were analyzed using ImageJ software version 1.37 (NIH Image; http://rsbweb.nih.gov/ij/).

[00331] Co-localization Tests / Dye-Linked Markers. In order to identify endocytic vesicles involved in C dots internalization, colocalization tests were performed in live cells using dye-linked markers. The cells were coincubated with nanoparticles and different dyes. Dyes included: 100 nM Lysotracker red for 30 min to label acidic organelles along the endosomal pathway; 2 μg/mL Alexa 488 transferrin conjugate to label recycling and sorting endosomes (clathrin-dependent pathway); 1 mg/mL70 kDa of dextran-FITC conjugate at 37° C. for 30 min to label macropinosomes.

[00332] Co-localization / Organelle-specific antibodies. Immunocytochemistry will be performed with known markers for Golgi and lysosomes. For Golgi, Giantin (Abcam, rabbit polyclonal, 1:2000) will be used for human cells; GM-130 (BD Pharmingen, 1 μg/ml) will be used for mouse cells. For Lysosomes, LC3B (Cell Signaling, rabbit polyclonal, 0.5 μg/ml) will be used.

[00333] For Giantin or LC3B staining, cells will be blocked for 30 minutes in 10% normal goat serum / 0.2% BSA in PBS. Incubation by primary antibody (rabbit polyclonal anti-Giantin antibody (Abcam catalog no. ab24586, dilution 1:2000) or LC3B (Cell Signaling, C no. 2775, 0.5 ug/ml) will be done for 3 hours, followed by 60 minutes of incubation with biotinylated goat anti-rabbit IgG (Vector labs, cat no.: PK6101) at 1:200 dilution. Detection will be performed with Secondary Antibody Blocker, Blocker D, Streptavidin-HRP D. (Ventana Medical Systems) and DAB Detection Kit (Ventana Medical Systems) according to the manufacturers' instructions.

[00334] For GM-130 staining, cells will be blocked for 30 min in Mouse IgG Blocking reagent (Vector Labs, Cat no.: MKB-2213) in PBS. The incubation of the primary antibody (anti-GM130 monoclonal, from BD Pharmingen; Cat no. 610822, concentration of 1 ug/mL) will be done for 3 hours, followed by 60 minutes of incubation with biotinylated mouse secondary antibody (Vector Labs, MOM Kit BMK-2202), diluted at 1:200. Detection will be performed with Secondary Antibody Blocker, Blocker D, Streptavidin-HRP D (Ventana Medical Systems) and DAB Detection Kit (Ventana Medical Systems) according to the manufacturers' instructions.

[00335] For temperature-dependent studies, nanoparticles will be incubated with cRGD-PEG-nanoparticles at 4° C., 25° C., and 37° C. to evaluate the fraction of surface bound versus internalized particles.

[00336] For exocytosis studies, nanoparticles (0.075 mg/ml) will be incubated for 4 hours and chamber slides washed with PBS, followed by addition of fresh medium. At time intervals of 0.5, 1.0, 1.5, 2.5, 4.5, and 8.0 hours, the cells will be washed, trypsinized, and the fluorescence signal of the cells and medium measured. by fluorometry. In dose response studies, cells will be incubated over a range of concentrations and incubation times, and evaluated using flow cytometry. In viability studies, cell viability will be measured using a trypan blue exclusion test before and after incubation to assess for toxicity. In time-lapse studies, the mechanism of nanoparticle internalization in living cells will be investigated after incubating the cells with nanoparticle conjugates at different incubation temperatures (4° C., 25° C., and 37° C.) using an inverted confocal microscope over a 12 hour period at 20 min intervals.

Discussion

[00337] It was seen that cRGD-PEG-dots and PEG-dots co-localize with Lysotracker Red in M21 and B16 cells suggesting uptake in the endosomal pathway (Figure 22). The data showed that these particles colocalize strongly with transferrin and dextran. Regardless of surface functionality and total charge, the nanoparticles (6 to 7 nm in hydrodynamic diameter) studied seemed to follow the same pathway. Time-lapse imaging in both cell types demonstrated internalization of the functionalized nanoparticles within a small fraction of the laminated cells. The particles were eventually released into vesicular structures in the perinuclear region. Colocalization tests with Giantin (or GM-130) are not expected to show fluorescent nanoparticle signal in the Golgi.

Example 12 Dual-Modality Silica Nanoparticles for Intraoperative Sentinel Lymph Node Mapping and Image-Guided Interventions Dual-Modality Silica Nanoparticles for Intraoperative Image-Guided Sentinel Lymph Node Mapping

[00338] These studies have been expanded to include optical imaging using the ArteMIS® portable fluorescence camera system, along with radiodetection using the gamma radiation probe, to perform real-time assessments of tumor lymphatic drainage and nodal metastases, as well as as an assessment of tumor burden. In a representative mini pig (Figures 23a to 23i), initial preoperative PET-CT scanning was performed using 18F-FDG and 124I-cRGDY-PEG-C dots using the preceding imaging procedure. Axial CT imaging revealed a primary pelvic tumor (Figure 23a) and draining sentinel lymph node (Figure 23b), which were seen as areas of increased activity on the corresponding 18F-FDG PET scan (Figures 23c, 23d). These findings were confirmed 2 days later by dynamic PET-CT imaging approximately 5 minutes after subdermal injection into 4 quadrants of the particle tracer around the tumor site; Co-registered axial (Figures 23e, 23g) and coronal (arrows, 23f, 23h) views demonstrate these findings. After preoperative scanning, the skin over the sentinel lymph node site was marked for intraoperative localization, and the mini pig was transported to the intraoperative suite. Baseline activity measurements, taken over the sentinel node and primary tumor sites using the portable gamma probe (Figure 23i), showed a 20-fold increase in activity within the sentinel node relative to the background signal.

[00339] For real-time optical imaging of the lymphatic system, a second subdermal injection of dots of 124I-cRGDY-PEG-C was administered around the tumor site with the skin intact, and the signal was visualized in the colored channels (Figure 24a ) and Cy5.5 fluorescent (Figure 24b). The adjacent nodal basin was exposed, and the fluorescent signal was seen in the NIR channel flowing from the injection site (Figure 24c) into the proximal (Figures 24c, 24d), merium (Figure 24e), and distal (Figure 24e) main lymphatic branches. Figure 24f), which drained to the sentinel lymph node (Figure 24f). Smaller lymphatic channels were also visualized (Figures 24d, 24e). The black-pigmented sentinel lymph node, visualized in dual-channel mode (Figures 24g, 24h), was additionally exposed (Figure 24i) before successive nodal excision (Figures 24j to 24m). The fluorescence signal within the nodal sample in situ (Figure 24k) and ex vivo (Figure 24m) was confirmed by gamma emissions using the gamma radiation probe (Figure 24i), and was seen to correspond to scattered clusters of tumor cells on views. low-power (box, Figure 24n) and high-power (Figure 24o) H&E-stained tissue sections. Positive expression of HMB45 was identified on low-power (Figure 24p) and high-power (Figure 24q) views, consistent with metastatic melanoma.

[00340] Surprisingly, and in contrast to the observed 18F-FDG findings, 124I-RGD-PEG-C dots were considered to specifically discriminate between metastatic tumor infiltration and inflammatory processes in these mini pigs. Mechanistic differences in the behavior of these agents at the cellular and subcellular levels, as well as the presence of an integrin-targeting moiety on the particle surface, may represent the observed imaging findings. In multiple mini pigs harboring pathologically proven inflammatory changes due to granulomatous disease (n = 3), 18F-FDG failed to detect metastatic disease, whereas it identified inflammatory and other metabolically active sites. These discrepant findings highlight the particle tracer's ability to selectively target, localize, and stage metastatic disease, while 18F-FDG failed in many cases to accurately stage cancer spread, rather than identifying sites of inflammation.

[00341] In a representative mini pig study illustrating these findings, initial 18F-FDG PET-CT scans showed calcification within the left posterior neck on CT (Figure 25a), corresponding to an area of intense activity in the 18F-FDG PET (Figure 25b). Low-power (Figure 25c) and high-power (Figure 25d) views of H&E-stained tissue sections revealed diffuse inflammatory changes, consistent with granulomatous disease. Intense 18F-FDG PET activity was additionally seen within the metabolically active bone marrow compartment of these young mini pigs (Figures 25a, 25b). In contrast, the particle tracer imaging study identified bilateral metastatic neck lymph nodes. A lymph node in the right neck on axial CT imaging (Figure 25e) was seen to be PET avid on co-registered PET-CT (Figure 25f); Additional bilateral lymph nodes on a more superior CT image (Figure 25g) were also hypermetabolic on fused PET-CT (Figure 25h). Additionally, calcifications in the left neck (Figures 25e, 25g) did not show any activity by PET on co-registered scans (Figure 25f, 25h). Corresponding H&E-stained SLN tissue sections revealed dark melanoma clusters in low-power (box, Figure 25i) and high-power (Figure 25j) views, seen to consist of melanoma cells and melanophages. A single frame (Figure 25k) selected from reconstructed 3D PET images again illustrated multiple bilateral PET-avid lymph nodes and associated draining lymphatic channels. Importantly, most activity was seen in the bladder 1 hour after injection without significant accumulation of tracers over the liver region.

[00342] It was seen that the above findings made better use of fusion PET-CT MIP images generated from dynamic imaging data acquired over a 1 hour period after administration of 18F-FDG (Figure 26a) or particle tracers locations (Figures 26b, 26c). For 18F-FDG, a clear absence of nodal metastases is noted, with diffusely increased activity seen within metabolically active bone structures. In contrast to these findings, 124I-cRGDY-PEG-C dots detected bilateral metastatic neck lymph nodes, along with draining lymphatic channels.

Dual-Modality Silica Nanoparticles for Image-Guided Interventions: Response to Treatment.

[00343] The ability of the particle tracer to discriminate metastatic disease from inflammatory tissue changes can potentially be explored in a variety of therapeutic situations -- whether surgically or interventionally oriented -- since assessments of treatment response are often confounded by the presence of inflammatory changes, making interpretation difficult. Image-guided interventions, such as therapeutic tumor ablations, may specifically benefit from the innovative coupling of novel particle platform and imaging device technologies to (1) enable earlier post-procedural response assessment; (2) verify complete ablation or detect residual tumor representing treatment failure, and (3) improve tumor surveillance strategies. Locally ablative therapies, including microwave ablation, cryoablation, radiofrequency ablation (RFA), and interstitial laser therapy, induce local thermal injury by inserting an energy applicator into tumors. These methods are typically employed as alternative options in patients considered ineligible for surgical excision. Additionally, patients undergoing ablative therapies are often poor surgical candidates due to co-morbidities. Widely used in clinical practice, they offer a distinct advantage, as they can be performed percutaneously as outpatient procedures with significantly less morbidity, and can improve quality of life and survival in selected patient cohorts.

[00344] Accurate post-therapy imaging, typically acquired 1 to 3 months after an ablation procedure, has traditionally utilized contrast enhancement of volumetric imaging, such as CT or MRI. These techniques suffer from a number of disadvantages. Firstly, they are limited to identifying the presence of abnormal reinforcement or growth in the size of the tumor area, considered primary indicators of residual tumor or recurrent disease. Diffuse enhancement of the rim around the ablation zone in post-procedural evaluations may be related to inflammation and hyperemia in the ablation zone, and often does not necessarily represent residual tumor. Increasing enhancement, notably irregular or nodular, is considered suspicious for tumor. However, these interpretations are controversial, as an ablation zone may appear larger than expected for several months post-procedure, and enhancement may also reflect granulation or scar tissue formation.

[00345] Functional methods, such as 18F-FDG PET, have also been used to evaluate the efficacy and effects of locally ablative procedures, but may suffer from an inability to accurately discriminate tumor from inflammatory changes. Therefore, interpretation of imaging changes (i.e., inflammation, tumor) at the tissue level in response to ablative procedures using current morphological or functional assessments, particularly at early time intervals, is an important challenge. What is needed are reliable outcomes for ablation success and unequivocal detection of residual disease in the post-ablation period.

[00346] As a precursor to performing future ablations of metastatic liver lesions, a proof-of-concept radiofrequency ablation (RFA) study of a larger (i.e., 1 to 2 cm) sentinel lymph node was performed on a mini pig with metastatic melanoma to assess response to early treatment in the presence of particle tracer. PET-CT imaging findings before and after RFA were histologically correlated. After subdermal injection of 124I-cRGDY-PEG-C dots (~0.6 mCi) around the primary left pelvic tumor, an initial basal coronal CT showed a 2.2*1.6 cm sentinel lymph node (Figure 27a ) superior to the tumor site, which was avid by PET (Figures 27b, 27c). The hypermetabolic left pelvic tumor is also shown (Figure 27b), noting additional tracer particle flow within a draining lymphatic channel on fused PET-CT images (Figure 27c, 27d). Additional serial CT scans were acquired to localize the lymph node (Figure 27e) prior to the ablation procedure and guide insertion of the RFA probe (Figure 27f) into the lymph node (level below the sights). On the corresponding pre-ablation co-registered PET-CT scan, the PET-avid SLN was seen just posterior to the sights (Figure 27g). A partial lymph node ablation was performed for 12 minutes using a 2 cm active tip RFA probe (Cool-tip “cold tip” ablation system, Covidien plc, Dublin, Ireland). Post-ablation PET-CT scan showed mildly reduced tracer activity in the ablation zone, anterior to the electrode tip (Figure 27h). Pre- and post-ablation imaging findings were confirmed histologically. H&E staining of pre-ablated sentinel lymph node core biopsy tissue confirmed diffuse metastatic tumor infiltration in low-power (Figure 27i) and high-power views (Figure 27j). Post ablation, the extent of metastatic infiltration decreased over H&E-stained nodal tissue, seen in corresponding low-power (Figure 27k) and high-power (Figure 27l) views. Coagulative necrosis and lymphoid tissue were also identified, along with multifocal hemorrhages (Figures 27k, 27l, respectively). High-power views stained with TUNEL before ablation reveal scattered neoplastic cells (Figure 27m). On post-ablation TUNEL staining, focal areas of necrosis (red) were seen in both low-power (Figure 27n) and high-power views (Figure 27o).

CONCLUSIONS

[00347] Lymph node metastases are a powerful predictor of outcome for melanoma. Early detection of micrometastases in regional lymph nodes using SLN mapping may allow timely stratification of patients to appropriate treatment arms, and may potentially improve patient outcomes. Although present standard-of-care SLN mapping and biopsy techniques are based on the use of radioactivity-based identification of SLNs, there are a number of limitations of this technology. These include low spatial resolution, reduced staging accuracy, lack of target specificity, slow tracer clearance that can obscure the surgical field, and lack of accurate intraoperative visualization to prevent injury to vital structures that lie in close proximity to sentinel lymph nodes. .

[00348] The recent introduction of newer generation biocompatible particle platforms, which can be actively tuned and refined to overcome these drawbacks according to key design criteria, while allowing selective probing of crucial cancer targets, may offer important insights into cellular and molecular processes that govern the spread of metastatic disease. Further adaptation of similar platforms for multimodality imaging can be used to leverage the surgeon or interventionalist to explore these processes in a variety of image-guided procedural situations.

[00349] A similar dual-modality platform, a clinically developed translated integrin-targeting silica nanoparticle developed for both optical and PET imaging, satisfies a number of key design criteria -- small size, superior brightness, increased tissue retention of tumor, and low background signal - - which make it an ideal agent for SLN localization and staging during SLN biopsy procedures when coupled with portable real-time optical camera systems. The ability to discriminate metastatic disease from inflammatory tissue changes in melanoma models, which are often co-existing processes, may provide a more accurate and reliable marker for assessing treatment response in the future. Further investigation into a broader group of cancer types and treatments is warranted using either surgically based or interventionally oriented therapies.

Example 13 Prostate Cancer Sentinel Lymph Node Mapping

[00350] Multimodal nanoparticles carrying HuJ591-F(ab')2 fragments are new diagnostic probes for prostate-specific membrane antigen (PSMA) binding. By synthesizing particles carrying multiple F(ab')2 fragments, we can (1) enhance affinity/binding potency due to multivalency effects and (2) alter in vivo distributions, since clearance and uptake will be dominated by the kinetic behavior of the particles, rather than the antibody itself. By maintaining particle size below or exactly at the renal cutoff of 10 nm in diameter, renal clearance is promoted. Additionally, target-to-background ratios will increase based on improvements in (1) and (2), potentially improving diagnostic specificity and disease staging.

Synthesis/Characterization of Particles linked to 124I-J591 F(Ab')2.

[00351] In order to generate PEGylated F(ab')2 constructs, 100 ug of F(ab')2 was added to an eppendorf tube in 100 μl of PBS, followed by incubation with 4 μl of 2 mg/ml of Traut's reagent for 1 h at room temperature. Maleimide-PEG (6 mg) dissolved in buffer solution was then added. The solution was incubated overnight and purified with a PD-10 column before particle attachment. F(ab')2 fragments are radiolabeled with iodine-124 (124I) to create a dual-modal particle platform, and specific activity, purity, and radiochemical yield will be derived. The size and concentration of particles will be additionally evaluated by FCS.

Example 14 Dual-Modality Silica Nanoparticles for Integrin Targeting in Melanoma in Humans

[00352] Nanomaterials, in particular nanoparticle probes, have unique physicochemical and biological properties, as well as surface versatility. By exploiting their surface versatility, biocompatible multifunctional particles can be selectively modified with molecular markers that recognize and localize key carcinogenic targets. Of increasing importance in operative situations is the need for more robust optically oriented multifunctional particle probes which can enhance targeted real-time detection of local/regional disease spread around the primary tumor site, thereby facilitating treatment. Real-time delineation of disease from critical neural and/or vascular structures in intraoperative situations will also be fundamental. Duncan, R, The dawning era of polymer therapeutics, Nat Rev Drug Discov 2, 347-360 (2003). Wagner, et al., The emerging nanomedicine landscape, Nat Biotechnol 24, 1211-1217 (2006). Scheinberg, et al., Conscripts of the infinite armada: systemic cancer therapy using nanomaterials, Nat Rev Clin Oncol 7, 266-276 (2010). Miyata et al., Polymeric micelles for nano-scale drug delivery, React. Func. Polym. 71, 227-234 (2011). Lee et al., Multifunctional mesoporous silica nanocomposite nanoparticles for theranostic applications, Acc Chem Res 44, 893-902 (2011). Schroeder et al., Treating metastatic cancer with nanotechnology, Nat Rev Cancer 12, 39-50 (2012). Rosenholm et al., Nanoparticles in targeted cancer therapy: mesoporous silica nanoparticles entering preclinical development stage, Nanomedicine (Lond) 7, 111-120 (2012). Ashley et al. The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers, Nat Mater 10, 389-397 (2011). Vivero-Escoto et al., Silica-based nanoprobes for biomedical imaging and theranostic applications, Chem Soc Rev 41, 2673-2685 (2012). Tada et al., In vivo real-time tracking of single quantum dots conjugated with monoclonal anti-HER2 antibody in tumors of mice, Cancer Res 67, 1138-1144 (2007). However, despite extensive particle developments to date, no probe for inorganic fluorescent particle imaging has successfully transitioned to the clinic as a targeted multifunctional platform technology. Altinoglu et al., Near-infrared emitting fluorophore-doped calcium phosphate nanoparticles for in vivo imaging of human breast cancer, ACS Nano 2, 2075-2084 (2008). Hilderbrand et al., Near-infrared fluorescence: application to in vivo molecular imaging, Curr Opin Chem Biol 14, 71-79 (2010). Choi et al., Design considerations for tumor-targeted nanoparticles, Nat Nanotechnol 5, 42-47 (2010). He et al., Near-infrared fluorescent nanoprobes for cancer molecular imaging: status and challenges, Trends Mol Med 16, 574-583 (2010).

[00353] Here we describe that a first ~7 nm fluorescence nanoparticle, modified as a hybrid targeted platform (optical/PET) by attaching radiolabels and cyclic arginine-glycine-aspartic acid-tyrosine (cRGDY) peptide, is not only well tolerated in patients with metastatic melanoma, but can preferentially detect and localize tumors presumed to express integrin in a microdosing regimen with high signal-to-noise ratios. No significant binding of the serum protein is observed, and the integrity of the particle and its surface components are maintained in vivo. The results on safety, pharmacokinetics, dosimetry and targeting suggest the general usefulness of this particle excreted by the kidneys in the diagnosis of cancer and for potentially guiding treatment planning. This dual modality probe constitutes a platform that may be suitable for specific tumor types which can enhance optical and/or PET-based lesion detection and cancer staging in humans, as well as drug delivery, potentially leading to improved management. of better and more personalized cancer.

[00354] We use an ultra-small (~7-nm in diameter) dual-modality (optical/PET) inorganic nanoparticle probe for targeted molecular imaging of cancers expressing αvβ3 integrin. This nanoparticle probe is the FDA's first new investigational drug in its class and with properties approved for first-in-human studies (Figure 28a). The fluorescent silica nanoparticle (Cornell, or C dots) covalently sequesters dye molecules in a core encapsulated by a silica shell to prevent dye filtration and to enhance brightness and photostability. Ow, et al. Bright and stable core-shell fluorescent silica nanoparticles, Nano Lett 5, 113-117 (2005). Burns, et al. Fluores-cent silica nanoparticles with efficient urinary excretion for nanomedicine, Nano Lett, 9, 442-448 (2009). Combined absorption spectra demonstrate this dye enhancement of brightness as differences in intensity between emissions from encapsulated and free dyes (Figure 28b). A neutral layer of surface poly(ethylene glycol) (PEG) chains allows attachment of a small number of cyclic arginine-glycine-aspartic acid-tyrosine (cRGDY) peptides for potent and selective integrin receptor binding. Benezra, et al. Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma, J Clin Invest, 121, 2768-2780 (2011). Activated integrins induce many structural and signaling changes within the cell, in addition to regulating differentiation pathways, mediating adhesion properties, and promoting cell migration, survival, and cell cycle progression. Hood et al., Role of integrins in cell invasion and migration, Nat Rev Cancer, 2, 91-100 (2002). Attachment of nuclear imaging tags, such as 124I, via tyrosine residues amplifies signal sensitivity for serial PET imaging. The final product, a highly biocompatible and biostable tumor-selective particle tracer (124I-cRGDY-PEG-C-dots), previously provided a readout of integrin receptor status by PET imaging in human melanoma xenografts, thereby defining a distinct class of renally cleared theranostic platforms for nanomedicine. Jokerst et al., Molecular imaging with theranostic nanoparticles, Acc Chem Res, 44, 1050-1060 (2011).

[00355] A first-in-human clinical trial was initiated, employing PET to quantitatively evaluate time-dependent tumor uptake and biodistribution, radiation dosimetry, and the safety of this agent in a cohort of five patients with metastatic melanoma. Implicit in the rationale for the use of PET imaging is the preclinical evidence that the particle radiotracer has no other demonstrable pharmacological, radiogenic, or biological effects. Collins, J. M. Phase 0 clinical studies in oncology, Clin Pharmacol Ther, 85, 204-207 (2009). Kummar et al., Phase 0 clinical trials: recommendations from the Task Force on Methodology for the Development of Innovative Cancer Therapies, Eur J Cancer, 45,741-746 (2009). After a single intravenous (i.v.) injection dose of approximately 185 megabequerels (MBq) (~3.4 to 6.7 nanomoles, nmol) of the 124I-cRGDY-PEG-particle tracer (specific activity range of 27.8 at 57.4 GBq/μmol) in human subjects (Figure 28a), three whole-body PET-CT scans were acquired over a 72-hour period to evaluate pharmacokinetics, in addition to analyzing metabolites in blood and urine samples during a two-week interval by gamma ray counting and radio thin layer chromatography (radioTLC).

[00356] The five patients had no adverse events and the agent was well tolerated during the study period. The pharmacokinetic behavior, expressed as the percentage of the injected dose per gram of tissue (% DI/g), versus the post-injection time and the average doses absorbed by the corresponding organs (Figure 28d), were comparable to those found for other diagnostic radiotracers commonly used. Serial PET imaging of this representative patient (Figure 28d) showed progressive loss of blood pool activity presumed by the larger organs and tissues, with no appreciable activity seen at approximately 72 hours post injection (p.i.). Whole body clearance half-lives in these patients were estimated to range from 13 to 21 hours. Interestingly, there was no notable localization in the liver, spleen, or bone marrow, in contrast to many hydrophobic molecules, proteins, and larger particle platforms (>10 nm). Although patients were pretreated with potassium iodide (KI) to block uptake by thyroid tissue, a higher mean dose absorbed by the thyroid was obtained in this patient relative to other tissues. The particles were also essentially excreted by the kidneys, with both the kidney and the bladder wall (after the thyroid and tumor, see below) demonstrating one of the highest % DI/g values around 72 hours post injection (Figure 28d); As is often the case for renally excreted radiopharmaceuticals, the bladder wall received a higher mean absorbed dose than other large organs and tissues. The detailed clinical protocol (Figure 28C) and rationale for this regimen are further described in Materials and Methods.

[00357] These findings highlight the fact that renal excretion, rather than hepatobiliary, is the predominant route of clearance from the body. Efficient renal clearance will be conditioned on the design of ultra-small particle-based platforms or macromolecular systems that are on the order of effective renal glomerular filtration size cutoffs of about 10 nm or less. Choi, et al., Targeting kidney mesangium by nanoparticles of defined size, Proc Natl Acad Sci USA, 108, 6656-6661 (2011). A small fraction of the administered activity (less than 5%) was seen as uptake in this patient's stomach and salivary glands, consistent with free iodine, which was progressively cleared during the imaging period. The particle does not present typical properties of reticuloendothelial agents (i.e., technetium-99m sulfur colloid), whose uptake reflects the function of macrophages, mainly in the liver. Based on previous data acquired for cRGD radiotracers, no unexpected foci of activity were observed. Haubner, R., et al. Synthesis and biological evaluation of a (99m)Tc-labelled cyclic RGD peptide for imaging the alphavbeta3 expression, Nuklearmedizin, 43, 26-32 (2004).

[00358] Importantly, these properties point to the somewhat unique pharmacokinetic behavior exhibited by this inorganic dual-modality imaging particle as a kidney-clearing agent. Metabolic analyzes of blood (Figure 29a) and urine (Figure 29b) samples by gamma ray counting revealed at least an order of magnitude drop in tracer activity over a 72-hour period, with essentially no activity remaining at the end of this period. interval. Particle activity was largely confined to the plasma fraction of blood without evidence of significant serum protein binding (data not shown). RadioTLC analysis of plasma samples revealed a single peak at 24 h post injection (Figures 29c to 29e), corresponding to the intact radiolabeled nanoparticle. In urine samples, two peaks, one corresponding to the intact nanoparticle and the other to a more mobile sample (identified as free iodine), were seen over a 24-hour period (Figures 29f to 29h). RadioTLC analyzes of the particle tracer (Figure 29i), radioiodinated peptide (131I-cRGDY, Figure 29j), and free radioiodine (131I, Figure 29k) confirmed that the first and second peaks in the radiochromatograms corresponded to the intact nanoparticle and free iodine. , respectively. Therefore, these findings suggested that no measurable loss of particle integrity occurred during the course of the study, even after excretion through the kidneys.

[00359] Although tumor detection was not the objective of this PET microdosing study, surprisingly, despite the low dose injected, there was evidence of tumor localization of the particle tracer. In the first case, a full-body PET-CT scan was acquired four hours after i.v. administration of 124I-cRGDY-PEG-C-dots in a patient with melanoma of the anorectal mucosa. On coronal CT imaging (Figure 30a), a large rounded area of reduced density (arrowhead) was seen in the left lower lobe of the liver, the site of a metastatic lesion known from previous fluorodeoxyglucose PET/CT imaging ( 18F-FDG) (data not shown). On coronal PET (Figure 30b) and co-registered PET and CT scans (Figure 30c), an arc of increased uptake circumscribed this lesion (Figure 30b arrowhead; Figure 30c) and subsequently cleared by the time of the PET scan of 24 hours, suggesting some preferential localization in this presumed integrin-expressing metastasis. Significant activity was seen within the bladder, gastrointestinal tract (stomach, intestines), heart, and gallbladder. In a second subject, a well-defined cystic lesion was seen in the right anterior lobe of the pituitary gland by axial (Figure 31a) and sagittal (Figure 31b) magnetic resonance imaging (MRI).

[00360] This lesion, a stable finding on previous MRI scans, was presumed to be a pituitary microadenoma, an intracranial neoplasm known to exhibit malignant properties such as neoangiogenesis and progression into peritumoral tissues. Accurate co-registration of this tracker-avid focus with multiplanar MRI (Figures 31c, 31d) and CT (Figure 31e, 31f) images confirmed its location within the anterior pituitary gland. Initially seen as a focus of intense activity, it progressively increased in intensity over a 72-hour interval (arrow, Figures 31g to 31i), accompanied by a corresponding decrease in background signal around the marrow, thereby producing greater tumor-to-background ratios. (i.e., tumor to brain (T/B) ~6) and tumor to liver (T/L) ~2)) (Figure 31j). The results of PET imaging can be explained based on findings in a previous study showing increased expression of αvβ3 integrin in the parenchyma of a subset of adenomas, as well as increased levels of integrin expression in stromal cells relative to adenomatous cells. of normal connective tissue. Farnoud, et al., Adenomatous transformation of the human anterior pituitary is associated with alterations in integrin expression, Int J Cancer, 67, 45-53 (1996).

[00361] Our initial data supports the notion that human PET scan studies with this targeted imaging vehicle are a rationale approach for detecting and localizing presumptive integrin-expressing tumors. We are devising dose-escalation methods to determine an optimal balance between safety, whole-body clearance, and tumor targeting efficiency in more advanced clinical trials. The aforementioned PET scan-guided studies can also allow precise quantification of integrin receptor expression levels in order to obtain maximum targeting efficiency, as well as detection of changes in these levels. Additionally, the use of these quantitative molecular imaging tools can produce information about time-dependent changes in particle uptake and accumulation within tumors. Kelloff, G. J., et al., The progress and promise of molecular imaging probes in oncologic drug development, Clin Cancer Res, 11, 7967-7985 (2005). For the case of pituitary adenoma, we were able to compute the cumulative uptake of particles within this lesion. Specifically, we computed the fraction of total injected activity and the number of particles that accumulated at this site during a 72-hour imaging period. Using the measured standardized maximum uptake value (SUVmax, see Methods) of the lesion (i.e., 46.5) at 72 hours post injection (almost a factor of ten greater than that in normal pituitary tissue), corrected for partial volume, as well as the approximate mass of the lesion (i.e., product of the lesion volume and an assumed density of 1 g/cm3), and the patient's body mass, we found that, relative to an injected particle load of 2x1015, roughly 1.78x1011 particles (0.01% of the injected dose, % of the DI) or 1 part per 10,000 accumulated at the site of injury. Standard uptake values (SUV) are defined as the activity per gram of tissue divided by the activity delivered per gram of body mass. Time-dependent changes in % DI/g and the dosimetry of this lesion, relative to large organs and tissues, are shown in Figures 28c and 28d.

[00362] The results suggest that the systemically injected particle tracer was well tolerated and safe. Safety measures included monitoring uptake in normal organs as well as laboratory indicators of toxicity. Secondary objectives were to estimate radiation doses and evaluate metabolic activity of plasma and urine. Safety assessments were based on dosimetry, lack of clinical symptoms, and the absence of any laboratory indications of particle (drug) toxicity.

[00363] The results obtained from this first-in-human clinical study point to a systemically injected particle tracer that showed favorable and reproducible pharmacokinetic signatures defined by renal excretion. In contrast to reticuloendothelial agents (i.e., technetium-99m sulfur colloid) and macromolecules such as antibodies, there was no appreciable particle tracer accumulation within the liver, spleen, or bone marrow. Our data clearly indicate that a large proportion of the administered activity was eliminated through the urinary system (Figures 28E, 29B); activity concentrations in the urinary bladder were up to an order of magnitude higher than those in the liver, for example. Based on the conservative hypothesis that all hepatic activity is essentially excreted via the hepatobiliary route, these data are consistent with ~90% of administered activity being excreted via the urinary system and only ~10% via the hepatobiliary route. In the remaining organs/tissues, notably at early time points (2 to 4 hours), residual activity largely reflects that of the blood pool.

[00364] Targeted detection did not serve as a study endpoint, and dose-escalation procedures to obtain maximum uptake at disease sites were therefore not incorporated into the test design (i.e., no attempt was made to adjust doses of particles to optimize tumor targeting). However, despite the low nanomolar amounts used, preferential localization and accumulation of the tracer particle occurred in several tumors. In one of the patients with a presumed pituitary adenoma, PET imaging results showed progressive pure accumulation of particle tracer activity at the lesion site. The results of this study suggest that our systemically injected particles are well tolerated and exhibit a distinctly unique "macromolecular" pharmacokinetic signature, where most renal clearance predominates without significant uptake by the reticuloendothelial system. This is in contrast to many hydrophobic molecules, proteins, and larger particle platforms (>10 nm). This feature is highly atypical for nanoparticles (which generally have diameters larger than estimated renal cutoff values, and therefore little renal excretion and slower clearance) and warrants clinical evaluation of our ultrasmall nanoparticle platform.

[00365] These results, together with essential data on safety, pharmacokinetics, and dosimetry of the dual-modality C-dot imaging platform, suggest the general utility of this human microdosing technique in terms of producing key tumor-specific readouts for the cancer diagnosis. Said estimated tumor-accumulated tracer particle loads (or concentrations), together with knowledge of the cellular inhibitory response (i.e., IC50 or 50% inhibitory concentration), can potentially be used to predict therapeutic dosage requirements for a certain drug.

Methods Synthesis and Characterization of cRGDY-PEG-C dots (cRGDY-PEG-C-dots).

[00366] For details regarding the synthesis and characterization of PEGylated and cRGDY (Peptides International, Louisville Ky.) surface-functionalized fluorescent core-shell silica nanoparticles (cRGDY-PEG-C-dots) encapsulating the organic dye, Cy5 (maximum emission ~650 nm, 2 dye equivalents within the nucleus of the core particle), see a previous publication by this group and references therein. Benezra, M., et al., Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma, J Clin Invest, 121, 2768-2780 (2011). Briefly, the particles were prepared by a modified Stober-type silica condensation. Bogush, et al., Preparation of monodisperse silica particles: Control of size and mass fraction, J Non-Cryst Solids, 104, 95-106 (1988). Herz, et al., Large stokes-shift fluorescent silica nanoparticles with enhanced emission over free dye for single excitation multiplexing, Macromol Rapid Commun, 30, 1907-1910 (2009). Sadasivan, et al., Alcoholic solvent effect on silica synthesis--NMR and DLS investigation, J Sol-Gel Sci Technol, 12, 5-14 (1998). Bifunctional PEGs were derivatized with silanes for attachment to the silica surface and for peptide binding. cRGDY peptides containing the cyclo-(Arg-Gly-Asp-Tyr) sequence and carrying cysteine residues (Peptide International) were attached to functionalized PEG chains via a cysteine-maleimide bond. The hydrodynamic radius, brightness, and spot concentrations of cRGDY-PEG-C, as well as against free Cy5 dye, were analyzed on a Zeiss LSM 510 Confocor 2 FCS using HeNe excitation at 633 nm.

Radiolabeling of cRGDY-PEG-C dots (cRGDY-PEG-C-dots).

[00367] Tyrosine residues were conjugated to PEG chains for attachment of radioiodine moieties (i.e., 124I, 131I). Hermanson, G, Bio-conjugate Techniques, (Academic Press, London, UK, 2008). Yoon, T. J., et al. Specific targeting, cell sorting, and bioimaging with smart magnetic silica core-shell nanomaterials. Small, 2, 209-215 (2006). Radiolabeling of cRGDY-PEG-C dots (cRGDY-PEG-C-dots) was performed using the IODOGEN method (Pierce). Piatyszek, et al., Iodine-Gen-mediated radioiodination of nucleic acids, Anal Biochem, 172, 356-359 (1988). The radiolabeled product was elutriated from PD-10 columns and evaluated using a dose calibrator (Capintec, Ramsey N.J.) and radioTLC; the specific activities of the 124I-bound particle fractions were on the order of 1450 millicuries (mCi)/μmol and the radiochemical purity was greater than 95%.

Patient Selection.

[00368] Subjects with metastatic melanoma with histological confirmation of the disease and harboring newly diagnosed or recurrent tumor were eligible for the study. Individuals who had a medical illness unrelated to melanoma, which would preclude administration of the particle tracer, were excluded from the study. Potassium iodide solution (SSKI, 130 mg per day) was administered 2 days before and up to 2 weeks after intravenous injection of radioiodinated particle tracer (dots of 124I-cRGDY-PEG-C, 185 MBq/5 ml) to block thyroid function. This protocol was approved by the Institutional Review Board of the Memorial Sloan Kettering Cancer Center. Patients were tested for hemotologic, renal, and hepatic function before and after PET and provided signed informed consent.

Image Acquisition and Processing.

[00369] Low-dose spiral CT scans were obtained per routine procedure, followed by the acquisition of three whole-body PET scans on a dedicated GE Discovery STE PET/CT 4 scanner, 24, and 72 hours post-dose injection. particle tracker. Positron emission data were reconstructed using the ordered subgroup expectation maximization (OSEM) algorithm. Images were corrected for attenuation using CT transmission data collected over the same region as for emission imaging, and serial data set registration was performed using CT data sets.

Imaging and Metabolic Analysis.

[00370] For pharmacokinetic and dosimetric analyses, regions of interest (ROIs) were drawn on PET imaging data (AW Workstation, GE Healthcare, Ridgewood, N.J.) in order to extract mean and maximum standard uptake values (SUVs ) for all major organs and normal tissues, including brain, lung, left ventricle, liver, spleen, intestine, kidney, bladder, muscle, breast, and tumor(s). For pharmacokinetic assessment, SUVs were converted to % ID/g values (i.e., SUV = % ID/g tissue x patient body mass / 100). Organ/tissue uptake data was supplemented by time-activity data from blood and urine. Venous blood and urine samples were collected approximately 30 min, 3 hours, 24 hours, 72 hours, and up to 2 weeks after 124I-cRGDYPEG-C dot injection. After centrifugation of the whole blood samples (4000 rpm, 10 min), the plasma supernatant, together with urine samples, were evaluated in a scintillation well counter (1480 Automatic Gamma Counter, Perkin Elmer, Shelton Conn.) calibrated to 124I. RadioTLC analyzes were additionally performed on biological samples. RadioTLC analysis of the particle tracer, 131I-labeled native peptide (cRGDY), and free iodine (131I) served as controls to facilitate interpretation. Activities (counts per minute, cpm) were converted to microcuries, corrected for decay, and adjusted for aliquoted volumes. Final values were expressed as % DI/g. Retention factor (Rf) values for the tracer were obtained and used to identify the parent compound and possible metabolites.

Radiation Dosimetry.

[00371] The routine radiation dosimetry method is an adaptation of the method promulgated by the MIRD Committee (Medical Internal Radionuclide Dosimetry), responsible for the physical properties of the administered radionuclides (124I) as well as the biological properties (pharmacokinetics and biodistribution) of the radiopharmaceutical in individual patients. The 124I emissions and their respective frequencies and energies are obtained from the MIRD Radionuclede Data and Decay Scheme publications. Serial whole body PET scans allowed derivation of normal organ absorbed dose estimates (rad and rad/mCi) using ROI-derived time - activity data. PET scans were acquired with all parameters identical, including scan time. Using the patient's total body mass (in kg) and the organ masses of the 70-kg Standard Man, total body and organ ROI data (i.e., average standard uptake values (SUVs) were converted to activities (i.e., fraction of injected dose). The preceding imaging-derived time-activity data were fit to exponential functions using a least-squares fitting algorithm and the resulting time-activity functions were integrated. analytically, incorporating the effect of the physical decay of 124I to produce the accumulated activities (or lengths of stay) in μCi hours/μCi in organs and the total body. The accumulated activities were used to calculate average doses absorbed by particles with 124I. 124I label for the organs (rad/mCi) and the effective dose (rem/mCi) for the standard 70 kg male anatomical model (Standard Man) using the OLINDA EXM MIRD program Loevinger et al., MIRD Primer for Absorbed Dose. Calculations, Society of Nuclear Medicine, New York, N.Y., 1991.

Serum Protein Binding Tests.

[00372] Whole blood samples were collected in serum separator tubes from a mini pig model of metastatic melanoma (Sinclair Research Center, MO), followed by centrifugation (4000 rpm, 10 min) in order to isolate the plasma fraction . An aliquot of serum was set aside for gamma ray counting; the remaining fraction was treated with ethanol (200 proof, Decon Labs, King of Prussia, Pa.), swirled until cloudy, and placed on dry ice (5 min) to promote precipitation of serum proteins. After centrifugation, the supernatant was collected for gamma ray counting and the pellet was repeatedly washed with phosphate-buffered saline and centrifuged (4000 rpm, 10 min) to collect 100 μL aliquots of supernatant for gamma ray counting (1480 Wizard 3 '').

Example 15 Alpha-MSH-PEG-Cy5-Particles (MSH Peptide-Bound Particles)

[00373] The N-Ac-Cys-(Ahx)2-D-Lys-ReCCMSH (or alpha MSH) peptide used for nanoparticle conjugation has the structure shown in Figure 32. It is attached to the nanoparticle through the cysteine thiol N-terminal. A spacer of two amino hexanoic acid units separates the nanoparticle attachment point from the D-Lys-ReCCMSH targeting molecule. The original ReCCMSH targeting molecule is shown in Figure 33. It was designed to target the radionuclide to melanoma tumors for imaging and therapy. The peptide analogue of MSH can be directly radiolabeled with 99mTc or 188Re (at the Re site) or through a metal chelator attached to the amino terminus. The MSH alpha peptide analog shown in Figure 32 is quite different from the original MSH analog shown in Figure 33. The original MSH molecule cannot be attached to a nanoparticle. The MSH peptide of the nanoparticle contains a free amino group containing side chain for radiolabeling. This is presently a D-Lysine but may be an amino-terminated side chain of 1 to many carbons in length.

[00374] Conjugation of N-Ac-Cys-(Ahx)2-D-Lys-ReCCMSH (or alpha-MSH) to the nanoparticle allows the nanoparticle to target and bind melanoma cells and tumors. The resulting particles, or dots of alpha-MSH-PEG-Cy5-C were about 6 to 7 nm in internal diameter (i.d.) using FCS and contained, on average, about 2.6 dyes per particle. The number of alpha-MSH ligands per particle was estimated to be <10.

[00375] Competitive binding studies using particles conjugated to N-Ac-Cys-(Ahx)2-D-Lys-ReCCMSH (or alpha-MSH): In a competitive binding test with a melanocortin-1 receptor agonist ( 125I-NDP), the alpha MSH-conjugated nanoparticles had an IC50 for cultured B16/F1 melanoma cells of 6.6x10-10 M (Figure 34A), whereas a mixed-sequence version of the molecule had an IC50 of 2. 3*10-7 M (Figure 34B). Furthermore, there was a difference in binding of 3 orders of magnitude. For reference in the same type of competitive binding test, the original DOTA-ReCCMSH had an IC50 of 2.1*10-9 M.

[00376] The N-Ac-Cys-(Ahx)2-D-Lys-ReCCMSH (or alpha-MSH) peptide-linked nanoparticle conjugate had better affinity for B16/F1 cells than the original DOTA-ReCCMSH and much higher affinity than mixed peptide nanoparticle.

[00377] Dose-response data were additionally obtained as a function of targeted particle concentrations (Figure 35A) and incubation times (Figure 35B) for both B16F10 and M21 melanoma cell lines. Saturation concentrations for these lines, based on flow cytometry studies, were considered to be on the order of ~100 nM for these two cell types, and incubation times of at least 2 hours were required to maximize binding.

[00378] Survival studies of human M21 cells, carried out over a range of particle concentrations for a fixed incubation time of 48 hours, demonstrated no significant loss of cell viability (Figure 36).

[00379] 125I-radiolabeled alpha-MSH-conjugated nanoparticles demonstrated large-volume renal excretion over a 24-hour period in both B16F10 and M21 murine xenograft models (Figures 37A, 37B); no appreciable particle tracer was measured at later time points (i.e., >24 hours). Furthermore, neither the B16F10 nor M21 xenograft models showed significant accumulation of the targeted particle probe in the reticuloendothelial system (i.e., not an agent of the RES), nor in the kidney (Figures 38A, 38B), the latter organ typically a site which accumulates alpha-MSH nonspecifically given its pure positive charge. Therefore, attachment of alpha-MSH to the particle probe significantly enhanced its renal clearance properties and eliminated accumulation within the kidneys.

Example 16 Integrin-Mediated Signaling and Modulation of Tumor Biology

[00380] Integrin signaling regulates diverse functions in tumor cells, including adhesion/spread, migration, invasion, proliferation and survival. In several types of tumors, including melanoma, the expression of particular integrins correlates with increased disease progression and decreased patient survival. Integrin-mediated patch interactions have identified novel functions of integrin in cell survival mechanisms and in the activation of divergent signaling pathways. It is generally known that the binding of peptides (or clusters of peptides), containing the RGD sequence, to integrin receptors leads to cross-linking or clustering of integrins which, in turn, modulates the above processes. However, it is unclear whether nanoparticles carrying multiple RGD peptide ligands can additionally trigger similar integrin signaling events, as this will reflect a complex dependence on multiple particle-based factors (size, charge, composition, surface chemistry, number/type of ligand), the type of tumor cells (or endothelial cells), the density of cellular receptors, and the dosage of particles. Our dose response studies demonstrate modest activation of divergent signaling pathways following cellular exposure to particle concentrations greater than 100 nM which, in turn, promote M21 and HUVEC cell migration, proliferative activity, and alter adhesion properties.

Binding of cRGDY-PEG-C-dots to M21 and HUVEC Cells

[00381] The αvβa integrin receptor plays a key role in vascular remodeling and angiogenesis, demonstrating increased expression on the surfaces of endothelial and tumor cells for a variety of tumor cell lines. This leads to the use of this receptor as a target for diagnostic and therapeutic purposes. In our case, cRGDY-PEG-C dots were tested for their ability to bind to melanoma cells (M21) or human umbilical vascular endothelial cells (HUVEC) as a function of concentration and time. Initial dose response studies showed a progressive increase in the binding of cRGDY-PEG-C dots to M21 and HUVEC cells as a function of concentration by flow cytometry (Figure 39, Figure 47A). Particle binding demonstrated saturation at about 100 nM for both cell lines, with average % blocked values of about 80% for M21 cells and 96% for HUVEC cells. Dose-response behavior was further investigated as a function of particle incubation times for both cell types after incubation with 100 nM cRGDY-PEG-C dots. Maximum binding was observed at 2 hours post-incubation, appearing relatively constant thereafter (Figure 47B).

Endocytosis and Intracellular Trafficking of cRGDY-PEG-C-dots

[00382] In order to elucidate the nature of the pathway or pathways utilized by cRGDY-PEG-C-dots after their incubation in M21 cells -- whether this is an αvβ3 integrin receptor-mediated and/or nonspecific uptake process, we examined the temperature-dependent uptake of these particles for a 4-hour incubation time at three temperatures: 4°, 25° C., and 37° C. The results, summarized in Figure 39A,C indicate an increase in cRGDY-PEG- C-dots associated with cells at 37° C. compared to 25° C. or at 4° C. in both cell lines tested. Furthermore, internalization of cRGDY-PEG-C-dots was partially blocked in the presence of excess (*250) αvβ3 receptor antibody in M21 and HUVEC cells (Figures 39A, 39C), suggesting a receptor-mediated binding component. . Additionally, uptake in αvβ3-negative M21L cells was roughly a factor of 4 to 8 times lower than that seen with αvβ3-expressing cells at both 37° C. and 25° C. (Figure 39B), respectively.

[00383] In order to further characterize the cellular compartments involved in the internalization of cRGDY-PEG-C dots, we performed colocalization tests in M21 cells with cRGDY-PEG-C dots and biomarkers from different endocytotic vesicles. Targeted particle internalization (~1 μM, red, 4 hour incubation) was sensitively detected by an inverted confocal microscope (Leica TCS SP2 AOBS) equipped with a HCX PL APO objective (63X1.2NA Water DIC D) (Figure 39D). . Using endocytotic markers LysoTracker Red (100 nM, green) (Figure 39D) and transferin-Alexa488, uptake within acidic endocytic structures was confirmed, suggesting clathrin-dependent pathway activity and gradual acidification of the vesicles. Figure 39D shows co-localization data between the particle and acidic endocytic vesicles 30 (yellow dots). Uptake was also observed within macropinocytes with 70 kDa of dextran-FITC which co-localized with dots of cRGDY-PEG-C; This finding suggested a second path of internalization. Nuclear counterstaining (blue) was performed with Hoechst. No particles entered the nucleus. Time-lapse imaging confirms particle uptake within M21 cells and colocalization with the lysosomal marker, Lamp1.

Competitive Binding of αvβ3 Integrin Receptor and Molecular Specificity

[00384] Competitive binding tests showed blocking of receptor-mediated particle binding in M21 and HUVEC cells (Figure 48A) by 80% to 85% in the former and 30 to 40% in the latter using excess (x50-x100) cRGDY peptide and counting gamma rays from the radiolabeled particle tracer (dots of 124I-PEG-cRGDY-C). In contrast to cRGDY-PEG-C dots, significant reductions in the magnitude of receptor binding were seen in M21 cells (~30% to 43%) and HUVEC (~13% to 27%) after incubation with particle probes. non-targeted cells (i.e., PEG-C dots) by flow cytometry (Figure 48B).

Influence of cRGDY-PEG-C dots on Cell Viability and Proliferation

[00385] In order to demonstrate that cRGDY-PEG-C dots did not adversely affect cell survival and proliferation, G0/G1 phase-synchronized M21 and HUVEC cells were exposed to a range of particle concentrations (25 to 200 nM of dots of cRGDY-PEG-C; 15 min or 24 hr) and incubation times (0 to 93 hours) in medium supplemented with serum (2%, 10% FBS) at 37° C., and measurements were made. time-dependent changes in absorbance using an optical plate reader (À=440 nm). Relative to controls (i.e., serum-supplemented medium), absorbance measurements were found to be relatively constant over the range of particle concentrations tested, suggesting that there was no significant loss of cell survival (Figures 49A, 49B ). Additionally, no time-dependent reductions in absorbance were seen after multi-dose addition (n = 5) of 100 nM cRGDY-PEG-C dots to M21 and HUVEC cells, suggesting that there was no change in the proliferative properties of the cells. (Figures 49C, 49D).

Activation of the FAK Pathway by cRGDY-PEG-C-dots

[00386] Binding of a ligand to the αvβ3 integrin receptor is known to initiate signal transduction pathways. After binding, integrin clumping occurs, which leads to autophosphorylation of the non-receptor FAK kinase at tyrosine 397, a binding site for Src family kinases. Recruitment of Src kinase results in the phosphorylation of FAK at tyrosine 576/577 and FAK at tyrosine 925 in the carboxyl-terminal region. Phosphorylation of FAK at tyrosine 397 is also known to activate numerous signaling cascades and intermediates of downstream pathways, such as Ras Mitogen-Activated Protein (MAPK) signaling, which induces the activation of Raf pathways. /MEK/Erk and phosphatidylinositol 3-kinase (PI3K)/AKT (AKT, mTOR, S6K) (Figure 40A).

[00387] In order to determine whether αvβ3 integrin-binding cRGDY-PEG-C-dots modulates the activity of these pathways, we treated serum-deprived M21 cells (0.2% FCS) with 100 cRGDY-PEG-C-dots. nM for 2 hours at 37° C.; Serum-deprived cells treated with 0.2% FCS alone served as controls. Western blot analysis of lysates from particle-exposed cells revealed enhanced phosphorylation levels of multiple protein intermediates: pFAK-397 and pFAK-576/577, Src, pMEK, pErk, and AkT (Figure 40B), which suggested the activation of downstream signaling pathways. These findings, represented graphically as normalized intensity ratios, were expressed relative to their respective total protein levels, and normalized to corresponding values measured under control conditions (Figure 40C). Incubation of cells with PEG-C dots did not increase phosphorylation levels of the proteins tested (data not shown).

[00388] We assessed whether the observed activation of downstream signaling pathways was dependent on the phosphorylation of FAK at tyrosine 397 by blocking this pathway with a small molecule inhibitor, PF-573228. This inhibitor interacts with FAK in the ATP-binding pocket and effectively blocks both the catalytic activity of the FAK protein and subsequent FAK phosphorylation on Tyr397. After addition of two concentrations of PF-573228 to serum-starved M21 cells previously exposed to 100 nM cRGDY-PEG-C dots, Western blot analysis revealed inhibition of FAK phosphorylation on Tyr397 and Src (Figure 41A), graphically presented in Figure 41B Inhibition of MEK or Erk phosphorylation was observed only at the highest concentration of inhibitor (Figures 41A, 41B).

Effect of cRGDY-PEG-C dots on Cell Migration

[00389] The αvβa integrin receptor, which is highly expressed on many types of tumor and angiogenic cells, is known to modulate a number of downstream biological processes, including cell migration, adhesion, proliferation, invasion, and angiogenesis. In the experiments that follow, we sought to determine whether dots of cRGDY-PEG-C alter the migration of M21 and HUVEC cells. A series of initial experiments examined time-dependent changes in M21 cell migration, as reflected in the mean closure area, using time-lapse imaging at three successively higher particle concentrations (0 nM to 400 nM; 37° C.) over a period of time of 96 hours (Figure 42A, i-xx). Statistically significant increases in mean closure area were observed over a 96-hour period, as a percentage of baseline values (t = 0), which were relatively constant for the particle concentration range used (i.e., ~87 % to 92%), compared to control samples (73%; p<0.05,) (Figure 42A, B). No significant changes were seen at earlier time intervals (Figure 42B). Incubation of HUVEC cells (37° C., 20 hours) with a range of concentrations of cRGDY-PEG-C dots (100 to 400 nM) in the presence of 0.2% FCS showed a statistically significant increase in the mean area of closure to a concentration of 400 nM (i.e., 34%) (Figures 43A, 43B), compared to 19% (p<0.05) for cells not exposed to particles. No appreciable changes in migration were observed for the lowest particle concentrations used (Figures 43A, 43B). It was seen that cells exposed to particles show higher migration rates compared to control cells.

[00390] We additionally showed that increases in cell migration rates were attenuated by the addition of the FAK inhibitor, PF-573228. Early-phase contrast images were acquired after incubating HUVEC cells with 400 nM particles over a 24-hour time interval (Figure 44A, i-viii), and the average area of closure was determined and plotted against isolated serum (Figure 44B). The percentage of change in mean closure areas relative to controls, and before and after the addition of an inhibitor, was seen to decrease from +34% to +3%. Statistically significant differences were found between values for cells exposed to particles without inhibitor (p<0.03) and particles treated with different concentrations of inhibitor (250 nM, 500 nM; p<0.001) in relation to serum-deprived controls; the differences between the first two groups were also statistically significant (Figure 44C).

Effect of cRGDY-PEG-C Dots on Cell Adhesion and Dissemination

[00391] The RGD tripeptide is known to be a component of extracellular matrix constituents, fibronectin and vitronectin. A competitive binding assay was performed using M21 cells (104 cells/well) pre-incubated (25°C, 30 minutes) with or without 400 nM cRGDY-PEG-C dots and fibronectin, after transferring the cells to cavities coated with fibronectin. About ∼60% of cells that were not preincubated with cRGDY-PEG-C-dots attached and spread onto the fibronectin-coated plates within the first 30 minutes. In contrast, a very low percentage (15%) of M21 cells preincubated with 400 nM cRGDY-PEG-C-dots were attached and spread onto the fibronectin-coated plate even 120 minutes after being seeded (Figures 45A, 45B). Average cell counts of "elongated" versus "round" spreading cells over a 2 hour period revealed that in the absence of particles (i.e., control condition), spreading cells (elongated cells) were observed in a majority of the cells. , whereas a predominantly "rounded" appearance was seen in the case of cells exposed to particles (Figure 45A). These results are graphically represented in Figures 45B and 45C, respectively. Quantification of the optical densities of cells after addition of methylene blue (absorbance) revealed that cells which adhered and spread on fibronectin (i.e., without pre-incubation of particles) absorbed twice as much methylene blue as exposed cells. to particles (Figure 45D). We observed the same phenomenon when vitronectin-coated wells were used (data not shown). Taken together, these data demonstrate that cRGDY-PEG-C-dots enhance cell migration and spreading by binding to the αvβ3 integrin receptor found on M21 and HUVEC cells.

Influence of cRGDY-PEG-C Dots on the Cell Cycle in M21 Cells

[00392] As integrin receptors are involved in cell survival and proliferation, we analyzed the effect of cRGDY-PEG-C-dots on the cell cycle. G0/G1 phase-synchronized M21 cells were incubated for 48 hours using two concentrations of cRGDY-PEG-C-dots (100 nM, 300 nM) in 0.2% FCS-supplemented medium. In this range, the percentage of cells in S phase increased by 11%, with statistical significance reached at 100 nM (p<0.05) and 300 nM (p<0.005) in relation to controls (Figures 46A, 46B). Corresponding declines of 6% and 5% were seen in the G1 and G2 phases of the cell cycle, respectively. Taken together, these data indicate an enhancement in the S phase in the presence of cRGDY-PEG-C dots.

Materials and methods Reagents, Antibodies and Chemicals.

[00393] RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin and HBSS (without calcium and magnesium containing 0.25% trypsin and 0.05% EDTA) were obtained from the Core Media Preparation Facility, Memorial Sloan Kettering Cancer Center (New York, N.Y.). Anti-rabbit polyclonal: phospho-Erk1/2 (p-Erk1/2Thr 202/Tyr204), phospho-AKT (p- Akt.sup.ser473)phospho-Src (p-Src.sup.Tyr419)phospho-p70 S6 ( p-p70S6 .sup.Thr389), phospho-MEK1/2 (p-MEK1/2 .sup.Ser217/221), phospho-FAK- .sup.Tyr397, phospho-FAK-.sup.Tyr576/577 phospho-FAK .sup.Tyr925, Erk, AKT, Src, p70 S6, MEK1/2 (47E6), and FAK were obtained from Cell Signaling Technology (Danvers, Mass.). Goat anti-rabbit IgG, goat anti-mouse IgG horseradish peroxidase (HRP) conjugates were purchased from Santa Cruz Biotechnology (Santa Cruz, CA.). Propidium iodide, RNase A, Transferrin-Alexa Fluor 488 conjugate, FITC-Dextran, Fluorescein, LysoTracker Red DND-99, LysoTracker Green DND-26, pHrodo Red Dextran, and Hoechst were obtained from Invitrogen-Life Technologies (Carlsbad, CA. ). PF-573228 was obtained from TOCRIS bioscience (Ellisville, Mo.). Cycle (Arg-Gly-Asp-D-Tyr-Cys) was from Peptides International (Louisville, Ky.).

Synthesis of cRGDY-PEG-C Dots and PEG-dots.

[00394] Fluorescent particles, containing the organic dye Cy5, were prepared by a modified Stober-type silica condensation, as previously described. Tyrosine residues were conjugated to PEG chains for radioiodine attachment. cRGDY peptides containing the sequence cyclo-(Arg-Gly-Asp-Tyr), and carrying cysteine residues (Peptide International), were attached to functionalized PEG chains via a cysteine - maleimide bond. The number of cRGD ligands per nanoparticle was calculated empirically.

PEG Attachment Mechanism to C Dot Surface.

[00395] Bifunctional PEGs, MAL-dPEG®12-NHS ester (Quanta Biodesigns, Ltd) were derivatized with silanes, specifically 3-aminopropyl triethoxysilane (Gelest), for attachment to the silica surface and for peptide binding through reactions between the sulfhydryl groups and the maleimide moieties of the derivatized PEGs. Furthermore, methoxy-terminated PEG chains were added to the surface of the particles using functional organosilicon compounds (Si compounds), specifically (MeO)3Si-PEG (Gelest), according to modified protocols. Briefly, (MeO)3Si-PEG was added, in approximately three molar excess, to the particles in a basic water/alcohol mixture (~1:5 v/v), and the mixture was stirred overnight at room temperature.

Hydrodynamic Size and Relative Brightness Comparison Measurements by Fluorescence Correlation Spectroscopy (FCS).

[00396] The hydrodynamic radius, brightness, and concentrations of cRGDY-PEG- and PEG-dots, as against free Cy5 dye, were initially determined by dialyzing these particle samples into water, diluting in physiological saline (0.15 M of NaCl in H2O), and analyzing the resulting samples on a Zeiss LSM 510 Confocor 2 FCS using HeNe excitation at 633 nm. The instrument was calibrated with respect to particle size before all measurements. Mean hydrodynamic sizes of dye and particle species were estimated based on differences in diffusion time, while relative differences in brightness were assessed using count rates per molecule/particle.

Cells and Cell Culture:

[00397] Human melanoma cell line M21 and M21 variant M21L (αv negative) were obtained from D. A. Cheresh (University of California, San Diego, CA.). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and Penicillin Streptomycin. Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from LONZA (Walkersville, Md.) cultured in EGM-2 medium containing 2% FBS, and LONZA growth factors.

In Vitro Cell Binding Studies Using Optical Detection Methods:

[00398] In order to test particle binding to M21, M21-L or HUVEC cells, 24-well plates were coated with 10 μg/ml type I collagen (BD Biosciences) in PBS, incubated at 37° C. for 30 minutes, and washed once with PBS. Cells (3.0x105 cells/well to 4.0x105 cells/well) were grown to confluence. Differential binding of cRGDY-PEG-C-dots to M21 cells or HUVEC was assessed over a range of incubation times (up to 4 hours) and particle concentrations (10 to 600 nM) using flow cytometry. After incubation, cells were washed with RPMI 1640 medium/0.5% BSA, detached using 0.25% trypsin/EDTA, pelleted in a microcentrifuge tube (5 minutes at 153 g, 25° C.), resuspended in BD FACSFlow solution (BD Biosciences), and analyzed in the Cy-5 channel to determine the percentage of probe bound to particles (FACSCalibur, Becton Dickinson, Mountain View, CA).

Internalization Study:

[00399] The experiment was carried out as above but incubated for 4 hours at three different temperatures: 4° C., 25° C. and 37° C. Co-incubation of excess (x250) of antibody conjugated to αvβ3 fluorescein anti-integrin human cRGDY (Millipore, Billerica, Mass.) with cRGDY-PEG-C-dots was used for receptor blockade in order to evaluate specificity. The tests were carried out with fixed and live cells. For fixed M21 cells, inverted confocal microscopy (Leica TCS SP2 AOBS) equipped with a HCX PL APO objective (63x1.2NA Water DIC D) was used and time-lapse imaging was used to track live M21 cells. Imaging was acquired after co-incubation of targeted (or control) particles at various concentrations with the following dye labels) for 4 hours: 70 kDa dextran conjugate -- FITC (1 mg/mL, 37°C. for 30 min; Invitrogen) to mark macropinosomes, Lysotracker Red (100 nM; Invitrogen) to mark the endocytotic pathway (i.e., acidic organelles), and transferrin Alexa488 (2 μg/mL.

Competitive Linkage Studies:

[00400] In order to evaluate specific binding, M21 cells were incubated (25° C., 4 hours) with 124I-cRGDY-PEG-C-dots (25 nM) and excess cRGDY peptide. The cells were then washed with RPMI 1640 medium/0.5% BSA, and dissolved in 0.5 ml of 0.2 M NaOH. Radioactivity was assessed using a 1480 Auto-matic Gamma Counter (Perkin Elmer ) calibrated for iodine-124.

Western Blot (WB):

[00401] M21 cells (1*106 cells/six-well plate) were cultured in six wells coated with collagen (10 mg/ml), and made quiescent by culture under serum-deprived conditions. Then the medium was changed to 0.2% FCS, and different concentrations (25 to 400 nM) of cRGDY-PEG-C-dots were added (37° C., 0.5 to 8 hours). Cells were rinsed twice in ice-cold PBS, collected by trypsinization, and the pellet was resuspended in lysis buffer (10 mM Tris, pH 8.5, 150 mM NaCl, 1 mM EDTA, 1% Triton 100; ACROS organics, NJ), 1% Na deoxycholate, 0.1% SDS, Complete® protease inhibitors (Roche, Indianapolis, Ind.), and Tablet--PhosSTOP phosphatase inhibitor cocktail (Roche, Indianapolis, Ind.), , Ind.). Lysates were centrifuged (10 min, 4° C.). Protein concentrations were determined by the bifivenine acid test (BCA, Thermo Scientific, Rockford, Ill.). A 50-μg protein aliquot from each fraction was separated by 4 to 12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Invitrogen, Carlsbad, CA.) . Membranes were blocked with 5% defatted dry milk (Bio-Rad, Hercules, CA.) in 0.1% Tris-Buffered Saline (TBS)-Tween, and the signal was visualized by ECL chemiluminescence ( Thermo Scientific, Rockford, Ill.) or Immobilon Western, (Millipore Billerica, Mass.) after application of primary (1:1000) and secondary (1:2000 to 1:5000) antibodies.

Cell Cycle Analysis:

[00402] G0/G1 phase-synchronized M21 cells were incubated for 48 hours using two concentrations (100 and 300 nM) of cRGDY-PEG-C-dots after modifying the medium to 0.2% FCS. After trypsinization, the cells were centrifuged (1200 rpm, 5 min), and the pellet was suspended in PBS, followed by fixation with 70% ethanol (4° C., 0.5 to 1 hour). Cells were successively resuspended in 1 ml of PBS containing 1% FCS and 0.1% Triton X-100, 200 μl of PBS containing 25 μg/ml propidium iodide, and 100 mg/ml of RNaseA ( 4° C., 60 minutes). Cell cycle analysis was performed using flow cytometry, FACSCalibur, (Mountain View, CA.) and Phoenix Flow MultiCycle software (Phoenix Flow Systems, Inc., San Diego, CA.).

Cell Proliferation:

[00403] Cells were divided (1x104 cells/well) in a collagen-coated 96-well plate as described in in vitro cell binding studies. Different concentrations of cRGDY-PEG-C-dots were added (25 to 200 nM) for 24 to 48 hours at 37° C. Then, 20 ml of WST-1 proliferation reagent (Roche, Indianapolis, Ind.) was added on the plate (37° C., 1 hour). To determine optical densities, we used a SpectraMax5 microplate reader (Molecular Devices, Sunnyvale, CA.). Absorbance was measured at 440 nm.

Migration Test: M21 Cells:

[00404] M21 cells were seeded (6*104 cells/well) using a migration kit (Collagen I Oris® coated plate, PLATYPUS TEC). Twenty-four hours after being seeded, the cell plugs on the plate were removed. Fresh culture medium (100 μl) supplemented with 0.2% FBS was introduced and cRGDY-PEG- C-dots were added at various concentrations: 25, 100 and 400 nM. Every 24 hours thereafter, the medium was replaced, along with new particles, for a time interval of 72 hours. Before incubating the plate at 37°C overnight, zero hour images were captured by the Axiovert 200M microscope (Carl Zeiss) using a 5* objective (0.15NA) and using a slide scan module on the Metamorph software (Molecular Devices, PA). Serial microscopy was then performed and images were captured every 24 hours for a total of 96 hours post-incubation. Data were analyzed using ImageJ software. HUVEC cells: HUVEC cells were additionally seeded (5*104 cells/well) and, 24 hours later, incubated with various concentrations of particles (100, 200, and 400 nM) after medium replacement. A similar microscopy procedure was performed as that for M21 cells, with serial imaging acquired 20 hours later.

Adhesion and Dissemination Test:

[00405] The effect of cRGDY-PEG-C-dots on the binding of M21 cells to fibronectin-coated plates was evaluated by initially coating 96-well microtiter plates with fibronectin in PBS (5 μg/ml), followed by 200 μl of RPMI/0.5% of BSA (37° C., 1 hour). Cells (1-3*104 cells/100 μl/well) were incubated (25°C, 30 minutes) with or without 400 nM cRGDY-PEG-C-dots in RPMI/0.1% BSA, and added to fibronectin-coated wells (37° C., 30 to 120 minutes). To quantify the number of attached cells, the wells were rinsed with RPMI/0.1% BSA to remove non-adherent cells. Adherent cells were fixed with 4% PFA (25° C., 20 minutes) and stained with methylene blue (37° C., 1 hour). Methylene blue was extracted from the cells by adding 200 ml of 0.1 M HCl (37° C., 1 hour). To determine optical densities we used a SpectraMax5 microplate reader and absorbance was measured at 650 nm. For dissemination test: Time lapse (37° C., 2 hours) was performed and images were captured by Axiovert 200M microscope (Carl Zeiss) using a 20* objective (0.15NA) and using a scanning module by sliding in Metamorph Software (Molecular Devices).

Quantitative Analysis:

[00406] In order to quantify differences in size and intensity between Western blot bands, we performed densitometry of phosphorylated protein intermediates and total protein using Photoshop CS2 (Adobe, San Jose, CA.). Bands were scanned at 300 dpi (Scanjet 7650, Hewlett Packard, Palo Alto, CA.), and converted to grayscale. Then the lasso tool was used to draw a region of interest (ROI) within the boundaries of each band in order to derive the following: area (number of pixels), average grayscale value within the selected area (0 to 255) and the associated standard deviation. The product of the first two values for each band was computed, and divided by the product for the initial band in each series (control band), producing an intensity value for each sample relative to the control. Finally, the ratio of phosphorylated protein to total protein and the corresponding propagated error (standard error) were computed for each sample using the relative intensities.

[00407] Phase contrast images captured for migration studies were analyzed using ImageJ 1.45s (National Institutes of Health, rsbweb.nih.gov/ij/) in order to quantify the extent of cell migration (i.e., closure of the area ) for M21 cells and HUVECs. In high-power views, a bounded area was drawn adjacent to the edge of the fixated cells seen in each image after shutter removal. The area enclosed for each image was measured (pixels) and used to calculate the percent closure relative to time zero (after particle addition and medium replacement) as follows: difference in area at a given time point (24 , 48, 72 or 96 hours) and at hour zero divided by the same area at hour zero multiplied by 100. The resulting values were weighted and a standard error was computed for each group.

Statistic:

[00408] All graphical values are plotted as mean ± standard error, except where noted. One-tailed Student's t-test was used to test the statistical significance of differences in cell migration between HUVECs or M21 cells incubated with serum alone or dots of cRGDY-PEG-C. One-way analysis of variance (ANOVA) was used to perform statistical pairwise comparisons between the percentage of M21 cells in S phase that were incubated with serum alone, 100 nM or 300 nM cRGDY-PEG-C dots. We assigned statistical importance to all tests at P<0.05.

Example 17 Sphingomyelin Liposomes for Enhancement of Tumor Release of Diagnostic/Therapeutic Silica Particles

[00409] The therapeutic potential of sub-10 nm particles (e.g., C dots) as drug delivery vehicles is under investigation. Drug-bound particles were developed by attaching drugs to particle-bound bifunctionalized PEG chains. Model drugs used for this proof-of-concept work are receptor tyrosine kinase (RTK) inhibitors. The release and accumulation of therapeutic particle payloads at the target site can be maximized by employing dose-escalation strategies to assess increased particle uptake and distribution within tumors by optical imaging and/or PET. An alternative approach to enhancing particle cargo release utilizes liposomal formulations to encapsulate therapeutic particle probes. Liposomes passively target tumors by increasing permeability and retention effect (EPR) while protecting payloads from degradation in circulation. Prior to particle encapsulation, either cRGDY or drug-linker conjugates will be attached to the particle surface as an active targeting mechanism to maximize release at the target site.

[00410] Once at the target site, particles may be released from liposomal formulations into the tumor interstitium as a result of hyperregulated enzyme systems present in tumors. The extracellular release of acid sphingomyelinase (AS-Mase, acid SMase or SMase) from tumor cells in response to cellular stress, such as X-ray irradiation and toxins, leads to rapid ceramide-mediated cell injury and, subsequently, apoptosis. Acid SMase hydrolyzes sphingomyelin, present within liposomal formulations, to ceramide. Ceramide has a role in biological systems as a secondary messenger in the apoptotic process. Ceramide has significantly different membrane properties than the oritem molecule sphingomyelin. When liposomes containing sphingomyelin are cleaved with SMase, there is a dramatic change in membrane stiffness. Sphingomyelin is a lipid suitable for liposome formulations; This is not true for ceramide, since its incorporation as a liposome building block leads to liposome leakage. This leakage will lead to the release of the liposome contents. This opens up the possibility of intracellular targeting of a large payload of silica nanoparticles, functionalized with drugs and/or cellular internalization markers (i.e., KLAKLAC peptide and small molecule inhibitors) to monitor and/or treat disease.

[00411] In order to determine whether liposome encapsulation significantly increases particle delivery to the tumor site relative to non-encapsulated particle probes, we will utilize optical and PET imaging approaches to monitor uptake from both batches of sphingomyelinase liposome-encapsulated and non-encapsulated particles in brain tumor xenograft models expressing EGFRmt+ (i.e., H1650, A431) both liposomes and particle probes will contain optical labels for visualization.

[00412] Ref: Sochanik A1, Mitrus I, Smolarczyk R, Cicho T, Snietura M, Czaja M, Szala S. Experimental anticancer therapy with vascular-disruptive peptide and liposome-entrapped chemotherapeutic agent. Arch Immunol Ther Exp (Warsz). 2010 June; 58(3):235-45.

[00413] Previous methods for detecting and targeting stressed cells involve some formulations consisting of bilayer liposomes each having an initial outer layer of liposome-forming lipids, sphingomyelin, or a mixture thereof; a second inner layer of liposome-forming lipids, sphingomyelin or a mixture thereof; the first and second layers forming a bilayer liposome and defining an interior space therein; wherein the interior space contains a silica nanoparticle. The silica nanoparticle and/or liposome will consist of optical markers and/or PET tags (dye, fluorophore, radiolabel, contrast agent), an enzymatic substrate, and/or therapeutic agents, including cytotoxic drugs, DNA segments , or a radiotracer indicator label. The marker and/or drug-labeled silica particles contained within the sphingomyelin liposomes contact a cellular sample under conditions in which sphingomyelinase is present in the cell or is released by the cell. This enzymatic contact, in turn, will hydrolyze the sphingomyelin in the liposome to release the silica nanoparticle and/or the marker tag and/or the drug through the hydrophilic (interior) or hydrophobic (bilayer) compartments of the liposome.

[00414] The scope of the present invention is not limited by what was specifically shown and described above here, in this patent application. Those skilled in the art will recognize that there are suitable alternatives to the represented examples of materials, configurations, constructions and dimensions. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein in this patent application. All references cited and discussed in this specification are incorporated herein into this patent application by reference in their entirety. Variations, modifications and other implementations of what is described herein in this patent application will occur to persons with regular knowledge of the art without departing from the spirit and scope of the invention. Although certain embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications can be made without departing from the spirit and scope of the invention. The subject matter stipulated in the foregoing description and in the attached drawings is offered by way of illustration only and not as a limitation.

Claims (15)

1. Agent attached to the fluorescent silica-based nanoparticle, characterized by the fact that it comprises: a silica-based core; a fluorescent compound within the nucleus; and a silica shell surrounding at least a portion of the core, wherein the nanoparticle is coated with an organic polymer wherein from 1 to 20 ligands are attached to the nanoparticle for binding to a tumor marker, wherein the ligands have the structure N-Ac-Cys-(Ahx)2-D-Lys-ReCCMSH, in which the nanoparticle has a diameter within a range from 1 nm to 15 nm; and wherein the nanoparticle can be excreted by renal clearance within a range from 90% of the initial dose (DI) to 100% of the DI within 24 hours after administration. 2. Agent according to claim 1, characterized in that the agent is selected from the group consisting of antibiotics, antimicrobials, antiproliferatives, antineoplastics, antioxidants, endothelial cell growth factors, thrombin inhibitors, immunosuppressants , antiplatelet agents, collagen synthesis inhibitors, therapeutic antibodies, nitric oxide donors, antisense oligonucleotides, wound healing agents, therapeutic gene transfer constructs, extracellular matrix components, vasodilators, thrombolytics, antimetabolites, growth factor agonists , antimitotics, statins, steroids, steroidal and non-steroidal anti-inflammatory agents, angiotensin-converting enzyme (ACE) inhibitors, free radical scavengers, PPAR-gamma agonists, small interfering RNAs (siRNAs), microRNA, and anticancer chemotherapeutic agents. 3. Agent according to claim 1, characterized by the fact that the agent is radiolabeled. 4. Agent according to claim 3, characterized in that the agent is linked to radiofluorine 18F. 5. Agent according to claim 1, characterized by the fact that the agent is a radionuclide. 6. Agent according to claim 5, characterized in that the radionuclide is selected from the group consisting of 90Y, 131I and 177Lu. 7. Agent according to any one of claims 1 to 6, characterized in that the organic polymer is selected from the group consisting of poly(ethylene glycol) (PEG), polylactate, polylactic acids, sugars, lipids, polyglutamic acid (PGA ), polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), Polyvinyl acetate (PVA), or combinations thereof. 8. Agent according to any one of claims 1 to 7, characterized by the fact that the number of ligands attached to the nanoparticle varies from about 1 to about 10. 9. Agent according to any one of claims 1 to 8, characterized by the fact that the nanoparticle has a diameter between about 1 nm and about 8 nm. 10. Agent according to any one of claims 1 to 9, characterized in that the fluorescent compound is Cy5 or Cy5.5. 11. Use of an agent attached to the fluorescent silica-based nanoparticle, as defined in claim 1, characterized by the fact that it is for use in the preparation of a composition or medicine. 12. Pharmaceutical composition, characterized by the fact that it comprises an agent attached to the fluorescent silica-based nanoparticle as defined in claim 1. 13. Use of an agent attached to the fluorescent silica-based nanoparticle as defined in claim 1, characterized in that it is for use in preparing a composition or medicine for detecting a tumor cell. 14. Use according to claim 13, characterized by the fact that the cellular abnormality or disease comprises cancer. 15. Use of an agent attached to the fluorescent silica-based nanoparticle as defined in claim 1, characterized by the fact that it is for use in preparing a composition or medicine for dual-channel optical imaging of a region within an individual.