US 2014024821 OA1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0248210 A1 Bradbury et al. (43) Pub. Date: Sep. 4, 2014

(54) MULTIMODAL SILICA-BASED (52) U.S. Cl. NANOPARTICLES CPC ...... A61K 51/1244 (2013.01); A61K 49/0093 (71) Applicants: CORNELL UNIVERSITY, Ithaca, NY (2013.01); A61K 47/48884 (2013.01) (US); SLOAN-KETTERING USPC ...... 424/1.29: 424/9.6; 424/9.4: 42479.3 INSTITUTE FOR CANCER RESEARCH, New York, NY (US) (57) ABSTRACT (72) Inventors: Michelle Bradbury, New York, NY The present invention provides a fluorescent silica-based (US); Ulrich Wiesner, Ithaca, NY (US); nanoparticle that allows for precise detection, characteriza Oula Penate Medina, Kiel (DE); tion, monitoring and treatment of a disease such as cancer. ity t N."NSS): The nanoparticle has a range of diameters including between SS, in on N.Y. S.Rs. about 0.1 nm and about 100 nm, between about 0.5 nm and Tom Quinn, Columbia, MO (US) s about 50 nm, between about 1 nm and about 25 nm, between about 1 nm and about 15 nm, or between about 1 nm and about (73) Assignees: CORNELL UNIVERSITY, Ithaca, NY 8 nm. The nanoparticle has a fluorescent compound posi RSSSSS tioned within the nanoparticle, and has greater brightness and RESEARCH, New York, NY (US) fluorescent quantum yield than the free fluorescent com pound. The nanoparticle also exhibits high biostability and (21) Appl. No.: 14/215,879 biocompatibility. To facilitate efficient urinary excretion of the nanoparticle, it may be coated with an organic polymer, (22)22) FileFiled: Mara 17, 2014 such as poly(ethylene glycol) (PEG). The small size of the Related U.S. Application Data nanoparticle, the silica base and the organic polymer coating (63) Continuation-in-part of application No. 13/381,209 minimizes the toxicity of the nanoparticle when administered filed on Sep. 27 PS 2 R aS application No. PCT, in vivo. In order to target a specific cell type, the nanoparticle US 10/4O994 on Jul. 2,200. may further be conjugated to a ligand, which is capable of (60) Provisional application No. 61/794,414, filed on Mar. binding to a cellular component associated with the specific 15, 2013, provisional application No. 61/222,851, cell type, such as a tumor marker. In one embodiment, a filed on Jul. 2, 2009, provisional application No. therapeutic agent may be attached to the nanoparticle. To 61/312,827, filed on Mar. 11, 2010. permit the nanoparticle to be detectable by not only optical O O fluorescence imaging, but also other imaging techniques, Publication Classification Such as positron emission tomography (PET), single photon (51) Int. Cl emission computed tomography (SPECT), computerized A6 IK5I/2 (2006.01) tomography (CT), bioluminescence imaging, and magnetic A6 IK 47/48 (2006.01) resonance imaging (MRI), radionuclides/radiometals or A6 IK 49/00 (2006.01) paramagnetic ions may be conjugated to the nanoparticle. Patent Application Publication Sep. 4, 2014 Sheet 1 of 50 US 2014/024821.0 A1

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MULTIMODAL. SILICA-BASED cent nanoparticles is their limited brightness and their low NANOPARTICLES detectability as fluorescent probes in dispersed systems. 0007. The present multifunctional fluorescent silica-based CROSS REFERENCE TO RELATED nanoparticles offer many advantages over other typically APPLICATIONS larger diameter particle probes. The nanoparticles are non 0001. This application claims priority to U.S. Provisional toxic, exhibit excellent photophysical properties (including Application No. 61/794,414 filed Mar. 15, 2013. This appli fluorescent efficiency and photostability), and demonstrate cation is also a continuation-in-part of U.S. patent application enhanced binding affinity, potency, as well as a distinct phar Ser. No. 13/381,209, which is a 371 national stage filing of macokinetic signature—one in which bulk renal clearance International Application No. PCT/US 10/40994 filed Jul. 2, predominates without significant reticuloendothelial system 2010, which claims priority to U.S. Provisional Application (RES) uptake. Their relatively small size, and surface PEG Nos. 61/222,851 (filed Jul. 2, 2009) and 61/312,827 (filed coating facilitates excellent renal clearance. The fluorescent Mar. 11, 2010). The disclosure of each of the above-identified nanoparticles of the present invention contain a fluorescent applications is incorporated by reference in their entirety. core and silica shell. The core-shell architectures, the great Surface area and diverse Surface chemistry of the nanoparticle STATEMENT REGARDING FEDERALLY permit multiple functionalities simultaneously delivered to a SPONSORED RESEARCH ORDEVELOPMENT target cell. For example, the nanoparticle can be functional ized with targeting moieties, contrast agents for medical 0002 This invention was made with government support imaging, therapeutic agents, or other agents. The targeting under NIH-NRRA, Clinical and Translational Science Center moieties on the Surface of the nanoparticle may be tumor Grant; NIH-NCI R01 CA161280-01A1, NSF STC Program ligands, which, when combined with nanoparticle-conju Agreement No. ECS-9876771; ICMIC P50 CA86438; NIH gated therapeutic agents, makes the nanoparticle an ideal SAIRP Grant No R24 CA83084; and NIH Center Grant No vehicle for targeting and potentially treating cancer. Webster P3O CAO8748. et al. Optical calcium sensors: development of a generic method for their introduction to the cell using conjugated cell FIELD OF THE INVENTION penetrating peptides. Analyst, 2005: 130:163–70. The silica 0003. The present invention relates to fluorescent silica based nanoparticle may be labeled with contrast agents for based nanoparticles, and methods of using the nanoparticles PET, SPECT, CT, MRI, and optical imaging. to detect, diagnose, or treat diseases such as cancer. SUMMARY BACKGROUND OF THE INVENTION 0008. The present application provides for a method for 0004 Early tumor detection and treatment selection is detecting tumor cells comprising the steps of: (a) administer paramount to achieving therapeutic Success and long-term ing to a patient a plurality of fluorescent silica-based nano Survival rates. At its early stage, many cancers are localized particles in a dose ranging from about 0.01 nanomole/kg body and can be treated Surgically. However, in Surgical settings, weight to about 1 nanomole/kg body weight, the nanoparticle the evaluation of metastatic disease spread and tumor mar comprising: a silica-based core comprising a fluorescent gins, particularly in areas of complex anatomy, is limited by a compound positioned within the silica-based core; a silica lack of imaging technologies. This has led to a disproportion shell Surrounding at least a portion of the core; an organic ate number of invasive biopsies. Molecularly-targeted probes polymer attached to the nanoparticle; a ligand attached to the incorporating contrast-producing (i.e., optical, PET) labels nanoparticle and capable of binding a tumor marker, and at and offering improved specificity are needed for early imag least one therapeutic agent; and (b) detecting the nanopar ing detection of molecular differences between normal and ticles. tumor cells, such as cancer-specific alterations in receptor 0009. The nanoparticle may be administered subdermally, expression levels. When combined with higher-sensitivity peritumorally, orally, intravenously, nasally, Subcutaneously, and higher-resolution imaging tools, specific molecular-tar intramuscularly or transdermally. A fluorescent silica-based geted probes will greatly improve detection sensitivity, stag nanoparticle comprising: ing, and the monitoring and/or treatment of cancer. 0005. Current fluorescence imaging probes typically con 0010. The present invention also provides for a fluorescent sist of single conventional fluorophore (e.g., organic dyes, silica-based nanoparticle comprising: a silica-based core fluorescent proteins), fluorescent proteins (e.g., GFP) and comprising a fluorescent compound positioned within the semiconductor quantum dots (Q-dots). Single fluorophores silica-based core; a silica shell Surrounding at least a portion are usually not stable and have limited brightness for imaging. of the core; an organic polymer attached to the nanoparticle: Similar to dyes, the fluorescent proteins tend to exhibit and a ligand attached to the nanoparticle, wherein the nano excited State interactions which can lead to stochastic blink particle has a diameter between about 1 nm and about 15 nm, ing, quenching and photobleaching. Q-dots are generally and after administration of the nanoparticle to a subject, renal made from heavy metalions such as Pb" or Cd" and, there clearance of the nanoparticle ranges from about 80% ID fore, are toxic. Burns et al. “Fluorescent core-shell silica (initial dose) to about 100% ID in about 24 hours, or from nanoparticles: towards “Lab on a Particle' architectures for about 90% ID to about 100% ID in about 24 hours. nanobiotechnology”, Chem. Soc. Rev., 2006, 35, 1028-1042. 0011. The present invention provides a fluorescent silica 0006 Fluorescent nanoparticles having an electrically based nanoparticle comprising a silica-based core having a conducting shell and a silica core are known and have utility fluorescent compound positioned within the silica-based in modulated delivery of a therapeutic agent. U.S. Pat. Nos. core; a silica shell Surrounding at least a portion of the core; an 6,344.272, and 6,428,811. A shortcoming of existing fluores organic polymer attached to the nanoparticle; from about 1 to US 2014/024821.0 A1 Sep. 4, 2014

about 30 ligands, or from about 1 to about 20 ligands attached from about 2 to about 30, from about 3 to about 20, or from to the nanoparticle; and a contrast agent or a chelate attached about 4 to about 15. Renal clearance of the nanoparticle after to the nanoparticle. administration of the nanoparticle to a subject may range 0012. The diameter of the nanoparticle ranges from about from about 10% ID (initial dose) to about 100% ID in about 1 nm to about 25 nm, or from about 1 nm to about 8 nm. The 24 hours, from about 30% ID to about 80% ID in about 24 organic polymers that may be attached to the nanoparticle hours, or from about 40% ID to about 70% ID in about 24 include poly(ethylene glycol) (PEG), polylactate, polylactic hours. In one embodiment, after the nanoparticle is adminis acids, Sugars, lipids, polyglutamic acid (PGA), polyglycolic tered to a subject, blood residence half-time of the nanopar acid, poly(lactic-co-glycolic acid) (PLGA), Polyvinyl acetate ticle ranges from about 2 hours to about 25 hours, tumor (PVA), or the combinations thereof. residence half-time of the nanoparticle ranges from about 5 0013 The ligand may be capable of binding to at least one hours to about 5 days, and renal clearance of the nanoparticle cellular component, Such as a tumor marker. The number of ranges from about 30% ID to about 80% ID in about 24 hours. ligands attached to the nanoparticle may also range from 0018 When the nanoparticles in the amount of about 100 about 1 to about 30, from about 1 to about 25, or from about times of the human dose equivalent are administered to a 1 to about 10. Examples of the ligand include peptide, protein, Subject, Substantially no anemia, weight loss, agitation, biopolymer, synthetic polymer, antigen, antibody, microor increased respiration, GI disturbance, abnormal behavior, ganism, virus, receptor, hapten, enzyme, hormone, chemical neurological dysfunction, abnormalities in hematology, compound, pathogen, toxin, Surface modifier, or combina abnormalities in clinical chemistries, drug-related lesions in tions thereof. Peptides such as tripeptide RGD, cyclic peptide organ pathology, mortality, or combinations thereof, is cRGD, cyclic peptide cRGDYC, octreotate, EPPT1 and pep observed in the subject in about 10 to about 14 days. tide analogs of alpha-MSH are encompassed by the present 0019. The present invention also provides a fluorescent invention. Any linear, cyclic or branched peptide containing silica-based nanoparticle comprising a silica-based core com the RGD or alpha-MSH sequence is within the scope of the prising a fluorescent compound positioned within the silica present invention. based core; a silica shell Surrounding at least a portion of the 0014. A contrast agent, Such as a radionuclide including core; an organic polymer attached to the nanoparticle; and a Zr, Cu, Ga, Y, 'I and '77Lu, may be attached to the ligand attached to the nanoparticle, wherein the nanoparticle nanoparticle. The nanoparticle may be attached to a chelate, has a diameter between about 1 nm and about 15 nm. After for example, DFO, DOTA, TETA and DTPA, that is adapted administration of the nanoparticle to a subject, blood resi to bind a radionuclide. dence half-time of the nanoparticle may range from about 2 0015 The nanoparticle of the present invention may be hours to about 25 hours, or from about 2 hours to about 15 detected by positron emission tomography (PET), single pho hours; tumor residence half-time of the nanoparticle may ton emission computed tomography (SPECT), computerized range from about 5 hours to about 2 days; and renal clearance tomography (CT), magnetic resonance imaging (MRI), opti of the nanoparticle may range from about 30% ID to about cal imaging (such as fluorescence imaging including near 80% ID in about 24 hours. The number of ligands attached to infrared fluorescence (NIRF) imaging), bioluminescence the nanoparticle may range from about 1 to about 20, or from imaging, or combinations thereof. about 1 to about 10. The diameter of the nanoparticle may be 0016. A therapeutic agent may be attached to the nanopar between about 1 nm and about 8 nm. A contrast agent, such as ticle. The therapeutic agents include antibiotics, antimicrobi a radionuclide, may be attached to the nanoparticle. Alterna als, antiproliferatives, antineoplastics, antioxidants, endothe tively, a chelate may be attached to the nanoparticle. The lial cell growth factors, thrombin inhibitors, nanoparticle may be detected by PET, SPECT, CT, MRI, immunosuppressants, anti-platelet aggregation agents, col optical imaging, bioluminescence imaging, or combinations lagen synthesis inhibitors, therapeuticantibodies, nitric oxide thereof. A therapeutic agent may be attached to the nanopar donors, antisense oligonucleotides, wound healing agents, ticle. After administration of the nanoparticle to a Subject, therapeutic gene transfer constructs, extracellular matrix blood residence half-time of the nanoparticle may also range components, vasodialators, thrombolytics, antimetabolites, from about 3 hours to about 15 hours, or from about 4 hours growth factoragonists, antimitotics, statin, Steroids, steroidal to about 10 hours. Tumor residence half-time of the nanopar and non-steroidal anti-inflammatory agents, angiotensin con ticle after administration of the nanoparticle to a Subject may Verting enzyme (ACE) inhibitors, free radical scavengers, also range from about 10 hours to about 4 days, or from about PPAR-gamma agonists, small interfering RNA (siRNA), 15 hours to about 3.5 days. The ratio of tumor residence microRNA, and anti-cancer chemotherapeutic agents. The half-time to blood residence half-time of the nanoparticle therapeutic agents encompassed by the present invention also after administration of the nanoparticle to a subject may range include radionuclides, for example, 'Y. 'I and '77Lu. The from about 2 to about 30, from about 3 to about 20, or from therapeutic agent may be radiolabeled, such as labeled by about 4 to about 15. Renal clearance of the nanoparticle may binding to radiofluorine F. also range from about 45% ID to about 90% ID in about 24 0017. After administration of the nanoparticle to a subject, hours after administration of the nanoparticle to a subject. blood residence half-time of the nanoparticle may range from 0020. Also provided in the present invention is a fluores about 2 hours to about 25 hours, from about 3 hours to about cent silica-based nanoparticle comprising a silica-based core 15 hours, or from about 4 hours to about 10 hours. Tumor comprising a fluorescent compound positioned within the residence half-time of the nanoparticle after administration of silica-based core; a silica shell Surrounding at least a portion the nanoparticle to a subject may range from about 5 hours to of the core; an organic polymer attached to the nanoparticle: about 5 days, from about 10 hours to about 4 days, or from and a ligand attached to the nanoparticle, wherein the nano about 15 hours to about 3.5 days. The ratio of tumor residence particle has a diameter between about 1 nm and about 8 nm. half-time to blood residence halftime of the nanoparticle after After administration of the nanoparticle to a Subject, the ratio administration of the nanoparticle to a Subject may range of tumor residence half-time to blood residence half-time of US 2014/024821.0 A1 Sep. 4, 2014 the nanoparticle ranges from about 2 to about 30, and renal 0031 FIG. 2d shows photobleaching data for Cy5 dye clearance of the nanoparticle ranges from about 30% ID to (light gray), 3.3 nm diameter (dark gray), and 6.0 nm diameter about 80% ID in about 24 hours. (black) PEG-coated nanoparticles under ~3.5 mW laser exci 0021. The present invention further provides a method for tation. detecting a component of a cell comprising the steps of: (a) 0032 FIG.3a shows percent of initial particle dose (%ID) contacting the cell with a fluorescent silica-based nanopar retained by blood (black) and tissues: liver (light gray), lung ticle comprising a silica-based core comprising a fluorescent (mid-low gray), spleen (midgray), and kidney (mid-high compound positioned within the silica-based core; a silica gray) for 6.0 nm diameter nanoparticles at various time points shell Surrounding at least a portion of the core; an organic from 10 minto 48 h post-injection (n=3 mice, meanistandard polymer attached to the nanoparticle: from about 1 to about deviation). 30 ligands attached to the nanoparticle; and a contrastagent or 0033 FIG. 3b shows plot of retained particle concentra a chelate attached to the nanoparticle; and (b) monitoring the tion for 3.3 nm (light gray) and 6.0 nm (black) diameter binding of the nanoparticle to the cellor a cellular component nanoparticles and the associated logarithmic decay fits and by at least one imaging technique. half-lives. 0022. The present invention further provides a method for targeting a tumor cell comprising administering to a cancer 0034 FIG. 3c shows plot of estimated particle excretion patient an effective amount of a fluorescent silica based nano for 3.3 nm (light gray) and 6.0 nm (black) diameter nanopar particle comprising a silica-based core comprising a fluores ticles and the associated logarithmic fits and half-lives cent compound positioned within the silica-based core; a (meant standard deviation, n=9 (three mice per time point)). silica shell Surrounding at least a portion of the core; an 0035 FIG. 4 shows in vivo biodistribution of the nanopar organic polymer attached to the nanoparticle; a ligand ticles in non-tumor-bearing and tumor-bearing mice with attached to the nanoparticle and capable of binding a tumor subcutaneous C6 xenografts. (A) Bare silica particles; (B) marker; and at least one therapeutic agent. The nanoparticle PEGylated RGD particles. may be radiolabeled. The nanoparticle may be administered 0036 FIG. 5 shows total specific binding data for cRGD to the patient by, but not restricted to, the following routes: and PEG-ylated dots (i.e., nanoparticles) using flow cytom oral, intravenous, nasal, Subcutaneous, local, intramuscular etry in the Cy5 channel as a function of time (a) and particle or transdermal. concentration (b). 0037 FIG. 6 shows multimodal C dot design for CfB BRIEF DESCRIPTION OF THE DRAWINGS integrin targeting and characterization. 0023 The patent or application file contains at least one 0038 FIG. 6a. Schematic representation of the 124 drawing executed in color. Copies of this patent or patent cRGDY-PEG-ylated core-shell silica nanoparticle with sur application publication with color drawings will be provided face-bearing radiolabels and peptides and core-containing by the Office upon request and payment of the necessary fee. reactive dye molecules (insets). 0024 FIG.1a shows a dynamic light scattering (DLS) plot 0039 FIG. 6b. FCS results and single exponential fits for (number average) of particle size for bare silica (gray) and measurements of Cy5 dyes in solution (black), PEGcoated PEG-coated (black) Cy5-containing silica nanoparticles. (PEG-dot, red), and PEG-coated, cRGDY-labeled dots (blue, 0025 FIG. 1b shows in vivo imaging of spectrally underneath red data set) showing diffusion time differences demixed Cy5 particle fluorescence (pseudocolor) overlaid on as a result of varying hydrodynamic sizes. visible light imaging of nude mice 45 min post-injection with 0040 FIG. 6c. Hydrodynamic sizes (meants.d., n=15), bare silica nanoparticles. and relative brightness comparisons of the free dye with PEG 0026 FIG. 1c shows in vivo imaging of spectrally coated dots and cRGDY-PEG dots derived from the FCS demixed Cy5 particle fluorescence (pseudocolor) overlaid on curves, along with the corresponding dye and particle con visible light imaging of nude mice 45 min post-injection with PEG-ylated Cy5 nanoparticles. centrations. 0041 FIG. 7 shows purification and quality control of 0027 FIG. 1d shows in vivo biodistribution study using 'I-RGDY-PEG-dots using size exclusion column chroma co-registered PET-CT. Upper row is serial co-registered PET tography. Radioactivity (right column) of ''I-RGDdots and CT image 24-hr after injection of ''I-labeled PEG coated '''I-PEG-dots detected by Y-counting and corresponding nanoparticle, flanked by the independently acquired microCT fluorescence signal intensity (Cy5, left column) of ''I- and microPET scans. Lower row is serial microPET imaging. RGDY-PEG-dots and 'I-PEG-dots in each eluted fraction. 0028 FIG. 2a shows fluorescence correlation spectros copy (FCS) data and single exponential fits for Cy5 dye (light 0042 FIG. 8 shows competitive integrin receptor binding gray), 3.3-0.06 mm diameter (dark gray, meant standard studies with 'I-cRGDY-PEG-dots, cKGDY peptide, and deviation, n=9) and 6.0+0.1 nm diameter (black, anti-CfB antibody using two cell types. meanistandard deviation, n=6) Cy5-containing PEG-coated 0043 FIG.8a. High affinity and specific binding of ''I- nanoparticles showing the differences in diffusion time cRGDY-PEG-dots to M21 cells by Y-counting. Inset shows resulting from the different hydrodynamic sizes of the differ Scatchard analysis of binding data plotting the ratio of the ent species. concentration receptor-bound (B) to unbound (or free, F) 0029 FIG. 2b shows absorption and emission spectra of radioligand, or bound-to-free ratio, B/F, versus the receptor Cy5 dye (light gray), 3.3 nm diameter (dark gray) and 6.0 nm bound receptor concentration, B; the slope corresponds to the diameter (black) PEG-coated nanoparticles. dissociation constant, Kd. 0030 FIG. 2C shows relative brightness comparison of 0044 FIG. 8b. O?3-integrin receptor blocking of M21 free dye (light gray) with 3.3 nm (dark gray) and 6.0 nm cells using flow cytometry and excess unradiolabeled cFGD diameter (black) nanoparticles, measured as count rate per or anti-CfB antibody prior to incubation with cRGDY-PEG molecule/particle as determined from the FCS curves. dots. US 2014/024821.0 A1 Sep. 4, 2014

004.5 FIG. 8c. Specific binding of cRGDY-PEG-dots to 0064 FIG. 13b shows small field-of-view PET image 5 M21 as against M21L cells lacking Surface integrin expres minutes after Subdermal injection of multimodal particles sion using flow cytometry. ('''I-RGD-PEG-dots) about the tumor site. 0046 FIG. 8d. Specific binding of cRGDY-PEG-dots to 0065 FIG. 14a shows whole-body dynamic F-fluorode HUVEC cells by flow cytometry. Each bar represents oxyglucose (F-FDG) PET scan demonstrating sagittal, meants.d. of three replicates. coronal, and axial images through the site of nodal disease in 0047 FIG. 9 shows pharmacokinetics and excretion pro the neck. files of the targeted and non-targeted particle probes. 0066 FIG. 14 b shows fused F-FDG PET-CT scans 0048 FIG. 9a. Biodistribution of '''I-cRGDY-PEG-dots demonstrating Sagittal, coronal, and axial images through the in M21 tumor-bearing mice at various times from 4 to 168 h site of nodal disease in the neck. p.i. The inset shows a representative plot of these data for 0067 FIG. 14c shows the whole body miniswine image. 0068 FIG. 15 shows the same image sets as in FIG. 14, but blood to determine the residence half-time (T). at the level of the primary melanoma lesion, adjacent to the 0049 FIG.9b. Biodistribution of I-PEG-dots from 4 to spine on the upper back. 96 h postinjection. 0069 FIG. 16a shows high resolution dynamic PET 0050 FIG. 9c. Clearance profile of urine samples col images following subdermal, 4-quadrant injection of ''I- lected up to 168 hr p. i. of unradiolabeled cRGDY-PEG-dots RGD-PEG-dots about the tumor site over a 1 hour time (n 3 mice, meants.d.). period. 0051 FIG. 9d. Corresponding cumulative % ID/g for (0070 FIG. 16b shows fused PET-CT images following feces at intervals up to 168 hrp.i. (n=4 mice). For biodistri subdermal, 4-quadrant injection of ''I-RGD-PEG-dots bution studies, bars represent the meants.d. about the tumor site over a 1 hour time period. 0052 FIG. 10 shows acute toxicity testing results. 0071 FIG. 16c shows Cy5 imaging (top image), the 0053 FIG.10a. Representative H&E stained liver at 400x resected node (second to top image), and H&E staining (upper frames) and stained kidneys at 200x (lower frames). (lower two images). Mice were treated with a single dose of either non-radiola 0072 FIG. 17 shows a scheme for a nanoparticle with a beled '''I-RGDY-PEG-dots or '''I-PEG-coated dots (control fluorescent dye within the core and a PEG surface-coating. vehicle) via intravenous injection and organs collected 14 The nanoparticle is decorated with triple bonds for subse days later. quent "click chemistry” with both DFO and Tyr3-octreotate 0054 FIG. 10b. Average daily weights for each treatment functionalized with azide groups. group of the toxicity study. Scale bar in FIG.10a corresponds 0073 FIG. 18 shows structures of PEG derivative. Stan to 100 um. dard chemical reactions are used for the production of the 0055 FIG. 11 shows serial in vivo PET imaging of tumor functionalized PEG with triple bonds, which will then be selective targeting. covalently attached to the nanoparticle via the silane group. 0056 FIG. 11a. Representative whole-body coronal 0074 FIG. 19 shows structures of DFO derivatives. microPET images at 4 hrs p.i. demonstrating M21 (left, (0075 FIG.20a shows structures of Tyr3-octreotate. arrow) and M21L (middle, arrow) tumor uptakes of 3.6 and 0076 FIG. 20b shows synthesis of the azide-containing 0.7% ID/g, respectively, and enhanced M21 tumor contrast at acid for incorporation into Tyr3-Octreotate. 24 hrs (right). 0077 FIG. 21a shows a scheme of the production of func 0057 FIG.11b. In vivo uptake of ''I-cRGDY-PEG-dots tionalized nanoparticle with an NIR fluorescent dye within its in O?3 integrin-overexpressing M21 (black, n=7 mice) and core, a PEG surface-coating, DFO chelates and Tyr3-octreo non-expressing M21L (light gray, n=5 mice) tumors and 'I- tate. (0078 FIG. 21b shows a scheme of the production of a PEG-dots in M21 tumors (dark gray, n=5). multimodality Zr-labeled nanoparticle (PET and fluores 0.058 FIG. 11c. M21 tumor-to-muscle ratios for 'I- cence) decorated with Tyr3-octreotate. cRGDY-PEG-dots (black) and 'I-PEG-dots (gray). 007.9 FIG. 21c shows the tetrazine-norbornene ligation. 0059 FIG. 11d. Correlation of in vivo and ex-vivo M21 0080 FIG. 21d shows a scheme of the strategy for the tumor uptakes ofcRGDY labeled and unlabeled probes. Each creation of radiolabeled core-shell nanoparticles using the bar represents the meants.d. tetrazine-norbornene ligation. 0060 FIG. 12 shows nodal mapping using multi-scale I0081 FIG. 21e shows a scheme of the strategy for the near-infrared optical fluorescence imaging. creation of peptide-targeted radiolabeled core-shell nanopar 0061 FIG.12a. Whole body fluorescence imaging of the ticles using the tetrazine-norbornene ligation. Both one-step tumor site (T) and draining inguinal (ILN) and axillary (in which the pre-metallated chelator-norbornene complex is (ALN) nodes and communicating lymphatics channels (bar, reacted with the particle) and two-step (in which the chelator LC) 1-hr p. i. in a Surgically-exposed living animal. is metallated after conjugation) pathways are shown. 0062 FIG. 12b. Corresponding co-registered white-light I0082 FIG. 22 shows microscopic images demonstrating and high-resolution fluorescence images (upper row) and co-localization between cRGF-PEG-nanoparticles and fluorescence images only (lower row) revealing nodal infra lysotracker red in the endocytotic pathway. structure of local and distant nodes, including high endothe I0083 FIGS. 23a-23i Image-guided SLN (sentinel lymph lial venules (HEV). The larger scale bar in (b) corresponds to node) Mapping: Pre-operative PET imaging. (a,b) Axial CT 500 um. images reveal a left pelvic soft tissue mass (a, arrow) and left 0063 FIG. 13a shows the experimental setup of using flank SLN (b. arrow). (c,d) Axial 'F-FDG PET images show spontaneous miniswine melanoma model for mapping lymph localized activity within the tumor (c. arrow) and left flank node basins and regional lymphatics draining the site of a SLN (d, arrow) following i.v. tracer injection. (e) Axial and (f) known primary melanoma tumor. coronal 'I-cRGDY-PEG-C dot co-registered PET-CT US 2014/024821.0 A1 Sep. 4, 2014

images show site of local injection about the pelvic lesion (e. lymphatic channels (curved arrows) within the neck follow arrow). (g) Corresponding axial and (h) coronal co-registered ing local particle tracer injection. PET-CT images localize activity to the SLN (g, arrow). (i) I0087 FIGS. 27a-27o Assessment of treatment response Radioactivity levels of the primary tumor, SLN (in vivo, ex after radiofrequency ablation (RFA) using '''I-cRGDY vivo), and a site remote from the primary tumor (i.e., back PEG-C dots. (a-c) Single-dose particle radiotracer localiza ground), using a handheld gamma probe. tion of the SLN. (a) Baseline coronal CT (white arrowhead), 0084 FIGS. 24a-24q Image-guided SLN mapping: Real (b) PET (black arrowhead), and (c) fused PET-CT images time intraoperative optical imaging with correlative histol (white arrowhead) following a peritumoral injection. (b-d) ogy. Intraoperative SLN mapping was performed on the ani Tumor particle tracer activity. (b) PET-avid exophytic left mal shown in FIGS. 23a-23i. (a-i) Two-channel NIR optical pelvic mass (black arrow). (c., d) Combined PET-CT images imaging of the exposed nodal basin. Local injection of Cy5. showing a hypermetabolic lesion (white arrow) and particle 5-incorporated particles displayed in dual-channel model (a) tracer flow within a draining lymphatic channel (asterisk) RGB color and (b) NIR fluorescent channels (white). (c-f) towards the SLN (curved arrow). (e.f) Pre-ablation axial CT Draining lymphatics distal to the site of injection. Fluores images locate the SLN (e, white arrowhead) prior to RFA cence signal within the main draining proximal (c,d), mid (e), electrode placement (f, arrow) into the node (below and distal (f) lymphatic channels (arrows) extending toward crosshairs). (g) Pre-ablation fused PET-CT reveals increased the SLN (N). Smaller caliber channels are also shown (ar SLN activity (posterior to cross-hairs). (h) Post-ablation PET rowheads). Images of the SLN displayed in the (g) color and CT scan shows mildly reduced activity at the SLN site, ante (h) NIR channels. (i) Image of the exposed SLN. (-m) Images rior to the needle tip. (i) Corresponding pre-ablation H&E of SLN in the color and NIR channels during (k) and fol staining of core biopsy tissue from the SLN confirms pig lowing (1.m) excision, respectively. (n) Low power view of mented tumor infiltration (bar=200 um). () High magnifica H&E stained SLN shows cluster of pigmented cells (black tion of boxed area in (i) reveals large, rounded pigmented box) (bar=1 mm). (o) Higher magnification of (n) reveals clusters of melanoma cells (bar-50 um). (k) Post-ablation rounded pigmented melanoma cells and melanophages H&E staining shows necrotic changes within a partially (bar 50 um). (p) Low power view of HMB45-stained SLN tumor-infiltrated node (box) and multifocal hemorrhages confirms presence of metastases (blackbox, bar=500pum). (q) (bar 500 um). (1) High magnification of (k) reveals signifi Higher magnification in (p) reveals clusters of HMB45+ cant tissue necrosis (arrowheads) within the metastatic node, expressing melanoma cells (bar 100 um). in addition to lymphoid tissue (bar-50 um). (m) TUNEL 0085 FIGS. 25a-25k Discrimination of inflammation staining of metastatic SLN before ablation (bar-20 um). (n) from metastatic disease: Comparison of 'F-FDG and I Post-ablation TUNEL staining demonstrating focal areas of cRGDY-PEG C dot tracers. (a-d) Imaging of inflammatory necrosis with adjacent scattered tumor foci and normal nodal changes using 'F-FDG-PET with tissue correlation. (a) tissue (NT) (bar 500 um). (o) High magnification of boxed Axial CT scan of the 'F-FDG PET study shows calcification area in (n) shows positive TUNEL staining, consistent with within the left posterior neck (arrows). (b) Fused axial F necrosis (bar-20 um). FDG PET-CT reveals hypermetabolic activity at this same I0088 FIGS. 28A-28C. Core-shell hybrid silica nanopar site (arrows). Increased PET signal is also seen in metaboli ticle platform ('I-cRGDY-PEG-C dots) and overview of cally active osseous structures (asterisks). (c) Low- and (d) study design. (A) Schematic of the hybrid (PET-optical) inor high-power views of H&E-stained calcified tissue demon ganic imaging probe (right) showing the core-containing strate extensive infiltration of inflammatory cells. (e-k) Meta deep-red dye and Surface-attached polyethylene glycol static disease detection following injection of ''I-cRGDY (PEG) chains that bear cRGDY peptide ligands and radiola PEG C dots about the tumor site. (e) Preinjection axial CT bels for detecting human CfB integrin-expressing tumors scan of ''I-cRGDY-PEG-C dots shows calcified soft tissues (left). (B) Absorption-matched spectra (left: red, black within the posterior neck (arrows). (f) Co-registered PET-CT curves) and emission spectra (right) for free (blue curve) and shows no evident activity corresponding to calcified areas encapsulated (green curve) dyes revealing increased fluores (arrow), but demonstrates a hypermetabolic node on the right cence of encapsulated fluorophores. (C) Timeline of clinical (arrowhead). (g) Axial CT at a more superior level shows trial events. Biol specs, biological specimens (blood, urine). nodes (arrowheads) bilaterally and a calcified focus (arrow). I0089 FIGS. 28D-28E. Whole body distribution and phar (h) Fused PET-CT demonstrates PET-avid nodes (N) and macokinetics of ''I-cRGDY-PEG-C dots. (D) Maximum lymphatic drainage (curved arrow). Calcification shows no intensity projection (MIP) PET images at 2-(left), 24 activity (arrow). (i) Low- and () high-power views confirm (middle) and 72-(right) hours p. i. of ''I-cRGDY-PEG-C dots the presence of nodal metastases. (k) Single frame from a reveal activity in bladder (), heart (yellow arrow), and bowel three-dimensional (3D) PET image reconstruction shows (white arrowhead). (E) Decay-corrected percent injected multiple bilateral metastatic nodes (arrowheads) and lym dose per gram (% ID/g) of urine and plasma collected at phatic channels (arrow). Bladder activity is seen with no approximately 30 min, 4h, 24h and 72 h following injection significant tracer accumulation in the liver. Scale bars: 500 of the particles was determined by gamma-counting; indi um (c,d); 100 um (i,j). vidual plots were generated for each patient. ROIs were I0086 FIGS. 26a-26c show 3D Integrated F-FDG and drawn on major organs for each patients PET scans for each '*'I-cRGDY-PEG-C dot PET-CT. (a-c) 3D Volume rendered patient to derive standardized uptake values and % ID/g. images were generated from CT and PET imaging data shown (0090 FIG. 29 Metabolic analyses of biological speci in FIGS. 7a-7k. (a) PET-CT fusion image (coronal view) mens. (A, B) Time-dependent activity concentrations (% shows no evident nodal metastases (asterisks). Increased ID/gx100) in plasma and urine, respectively, decay-corrected activity within bony structures is identified. (b,c) High-reso to the time of injection. (C-H) RadioTLC (4:1 acetic acid: lution PET-CT fusion images showing coronal (b) and supe methanol as mobile phase) of plasma and urine specimens rior views (c) of bilateral metastatic nodes (open arrows) and (decay-corrected counts per minute, cpm). (C-E) Chromato US 2014/024821.0 A1 Sep. 4, 2014

grams of plasma show a single peak at 0.5—(C), 3-(D) and of temperature (4°C., 25°C., 37°C.) and concentration (25 24—(E) hours p. i. (F-H). Chromatograms of urine specimens nM, 100 nM) using anti-CfB integrin receptor antibody and reveal two peaks at 0.5—(F), 3–(G), 24 (H) hours p.i. flow cytometry. Anti-CfB integrin receptor antibody concen Insets (G,H) show respective data scaled to a maximum of 50 trations were 250 times (i.e., 250x) the particle concentration. cpm. (I-K) Chromatograms of standards: injectate (I), radio B. Uptake of crGDY-PEG-C dots in M21 cells, as against iodinated (''I) peptide (J) and free ''I (K). Vertical lines M21L cells lacking normal Surface integrin expression by discriminate peaks corresponding to the particle tracer (long flow cytometry. C. Selective particle uptake in HUVECs dashes; R, 0.04), ''I-cRGDY (short dashes: R-0.2) and using anti-CfB integrin receptor antibody and flow cytom ''I (dotted; R-07). etry. D. cFGDY-PEG-C dot (1 uM, red) colocalization assay 0091 FIG. 30 Whole-body PET-CT imaging of particle with endocytic (transferrin-Alexa-488, FITC-dextran, green) biodistribution and preferential tumor uptake following sys and lysosomal markers (LysoTracker Red) after 4h particle temic injection of ''I-cRGDY-PEG-C dots (A) Reformatted incubation using M21 cells. Colocalized vesicles (yellow), coronal CT demonstrates a well-defined, hypodense left Hoechst counterstain (blue). Scale bar-15 lum. Each data hepatic lobe metastasis (arrowhead). (B) Coronal PET image point (A-C) represents the meaniSD of 3 replicates. at 4 hours p.i. demonstrates increased activity along the 0101 FIG. 40. Expression levels of phosphorylated FAK, peripheral aspect of the tumor (arrowhead), in addition to the Src, MEK, Erk1/2, and Akt in M21 cells. A. FAK/Src com bladder, gastrointestinal tract (stomach, intestines), gallblad plex transduce signals from integrin cell Surface receptors via der, and heart. (C) Co-registered PET-CT localizes activity to activation of downstream signaling pathways (PI3K-Akt, the tumor margin. Ras-MAPK) to elicit a range of biological responses (boxes 0092 FIG. 31 Multimodal imaging of particle uptake in a indicate assayed protein intermediates). B. Western blots of pituitary lesion. (A-B) Multiplanar contrast-enhanced MR phosphorylated and total protein expression levels of key axial (A) and Sagittal (B) images at 72 hours p.i.demonstrate pathway intermediates after exposure (2 h, 37°C.) of Go/G a Subcentimeter cystic focus (arrows) within the right aspect phase-synchronized M21 cells to 100 nMcRGDY-PEG-C- of the anterior pituitary gland. (C-D) Co-registered axial (C) dots relative to cells in serum-deprived (0.2% FBS) media and sagittal (D) MRI-PET images reveal increased focal (i.e., control). After trypsinization of cells and re-suspension activity (red) localized to the lesion site. (E-F) Axial (E) and of the pellet in lysis buffer, proteins were resolved by 4-12% sagittal (F) PET-CT images localize activity to the right gradient SDS-PAGE and analyzed by anti-FAK 397, pFAK aspect of the sella. (G-I) Axial PET images at 3 hours (G), 24 576/577, p-Src, pMEK, pFrk1/2, and pAkt antibodies. Anti hours (H) and 72 hours (I) p.i.demonstrate progressive accu bodies against FAK, Src, MEK, Erk, and Akt were also used mulation of activity (SUVmax) within the sellar region along to detect the amount of total protein. C. Graphical summary of with a corresponding decline in background activity about the percent signal intensity changes in phosphorylated to total lesion. (J) Tumor-to-brain (T/B) and tumor-to-liver (T/L) protein (Adobe Photoshop CS2; see Methods) for particle activity ratios increasing as a function of post-injection times. exposed versus serum-deprived cells. GF, growth factors: EC, 0093 FIG. 32. Structure of N-Ac-Cys-(Ahx)-D-Lys endothelial cell; ECM, extracellular matrix. ReCCMSH (or alpha MSH) peptide used for nanoparticle 0102 FIG. 41. Signaling induction and inhibition studies conjugation. in M21 cells using PF-573228 (PF-228), a FAK inhibitor. A. 0094 FIG.33. Structure of the original ReCCMSH target Western blots of phosphorylated and total protein expression ing molecule. levels using the foregoing process of FIG. 2 with the addition 0095 FIGS. 34A and 34.B. Competitive binding studies of 250 nM or 500 nM PF-228 (0.5 h, 37° C.) to cells prior to using a melanocortin-1 receptor agonist ('I-NDP). N-Ac particle exposure. B. Summary of percent intensity changes Cys-(Ahx)-D-Lys-ReCCMSH (or alpha-MSH) conjugated of phosphorylated to total protein expression levels in par particles (FIG.34A) had stronger affinity for cultured B16/F1 ticle-exposed versus control cells with and without PF-228. melanoma cells than a scrambled sequence version of the (0103 FIG. 42. Effect of cRGDY-PEG-C dots on M21 molecule (FIG. 34B). cellular migration using time lapse imaging. A. Time-depen 0096 FIGS. 35A and 35B. Dose-response data was dent changes in cell migration using ORISTM collagen coated obtained as a function of targeted particle concentrations plates for a range of particle concentrations (0-400 nM; 37° (FIG.35A) and incubation times (FIG.35B) for both B16F10 C.) in RPMI 1640 media supplemented with 0.2% FBS, as and M21 melanoma cell lines. against Supplemented media alone (controls). Images were 0097 FIG. 36. Human M21 cell survival studies, per captured at time t O (pre-migration) and at Subsequent 24h formed over a range of particle concentrations for a fixed intervals following stopper removal by a Zeiss Axiovert incubation time of 48 hr demonstrated no significant loss of 200M inverted microscope (5x/0.25NA objective) and a scan cell viability. slide module (Metamorph R. Microscopy Automation & 0.098 FIGS. 37A and 37B. I-radiolabeled alpha-MSH Image Analysis Software) for a total of 96 hrs. B. Graphical conjugated nanoparticles demonstrated bulk renal excretion plot of changes in the mean area of closure (%) as a function over a 24hr period in both B16F10 and M21 murine xenograft of concentration using Image.J Software. Mean area of closure models. represents the difference in the areas demarcated by the bor 0099 FIGS. 38A and 38B. Neither B16F10 or M21 der of advancing cells (pixels) at arbitrary time points and Xenograft models showed significant accumulation of the after stopper removal (t=0), divided by the latter area. Qua targeted particle probe in the reticuloendothelial system (i.e., druplicate samples were statistically tested for each group not an RES agent), nor in the kidney. using a one-tailed t-test: *, p=0.011: **, p=0.049; ***, p=0. 0100 FIG. 39. Competitive integrin receptor binding and 036. Scale bars=100 um and 33 Lum (magnified images of X temperature-dependent uptake using cFGDY-PEG-C dots and XV). and anti-CfB antibody for 2 cell types. A. Specific binding 0104 FIG. 43. Effect of cRGDY-PEG-C dots on the and uptake of cRGDY-PEG-C dots in M21 cells as a function migration of HUVEC cells. A. Serial HUVEC migration was US 2014/024821.0 A1 Sep. 4, 2014

assayed using the same process in FIG. 4 over a 24-hr time and HUVEC cells following incubation with excess cFGDY interval. The displayed images and area of closure values peptide using gamma counting. Monolayers of M21 and indicated are representative of a single experiment. B. Mean HUVEC cells were incubated (4 h, 25° C.) with 25 nM of areas of closure (%) were determined over this time interval 'I-cRGDY-PEG-dots in the presence and absence of for a range of particle concentrations (0-400 nM) using cRGDY peptide (85, 170 nM). Binding is expressed as a Image.J Software. Triplicate assays were performed for each percentage of the control (i.e., radioiodinated cRGDY-PEG concentration and time point. One-tailed t-test *, p<0.05. dots). B. Tumor-directed binding of non-radiolabeled Scale bar-76 um. cRGDY-PEG-C dots. M21 and HUVEC cells were incubated 0105 FIG. 44. Inhibition of HUVEC cell migration using for 4 hat 25°C. with two targeted particle concentrations (25 PF-228. A. Same process as in FIG. 5, except cells were nM, 100 nM) or particle controls (PEG-C dots) and assayed exposed to 250 nM and 500 nM PF-228 (0.5h, 37° C.) prior by flow cytometry. to particle exposure or incubation in 0.2% FCS supplemented 0110 FIG. 49. Viability and proliferation of M21 and media. B. Mean area closure (%) forcells incubated under the HUVEC cells as a function of particle concentration and foregoing conditions. C. Tabulated p values for each exposure incubation time. A. B. Absorbance (W. 440 nm) as a measure condition using a one-tailed t-test. Quadruplicate samples of viability in subconfluent, Go/G-phase-synchronized M21 were run for each inhibitor concentration. Scale bar 50 um. (A) and HUVEC (B) cells over a 24 hour period using media 0106 FIG. 45. Modulation of M21 cell spreading and supplemented with 10% or 2% FBS alone, respectively, or adhesion using cFGDY-PEG-C dots. A. Time-lapse imaging with addition of particles (25-200 nM). C. D. Cellular prolif showing changes in cellular attachment and spreading. Cells erative activity in M21 (C) and HUVEC (D) cells over a 93 h were pre-incubated in 0.2% FBS-supplemented RPMI (0.5h, time interval using either media alone or particle-containing 25°C.), without and with particles (400 nM), followed by media, as specified in A, B. seeding in (5 ug/ml) fibronectin-coated 96-well plates. 0111 FIG. 50. Cell signaling changes as a function of Images were captured at t0, 0.5h, 1 h, and 2 h using a Zeiss particle incubation time in M21 cells. Western blots of Axiovert 200M inverted microscope (20x/0.4NA objective) selected phosphorylated and total protein intermediates over and a scan slide module in Metamorph R. B. Graphical plot an 8 h time period. Normalized intensity ratios (i.e., differ showing the mean number of rounded and elongated cells ence of phospho-protein and total protein divided by the within two groups as a function of time: non-particle exposed latter) are graphically illustrated. (elongated, graph #1) and particle exposed (rounded, graph 0112 FIG. 51. Cell signaling modulation as a function of #2). Cells in each of three wells of a 96-well plate were particle concentration in M21 cells. Western blots of selected manually counted in a minimum of three high power fields phosphorylated and total protein intermediates over a range (x200 magnification) and averaged. C. Absorbance (v-650 of particle concentrations (i.e., 0-400 nM). nm: SpectroMax M5 microplate reader) values for 4% 0113 FIG. 52. Cell signaling inhibition studies in M21 paraformaldehyde fixed cells, exposed to media or 400 nM cells. A. Western blots of selected phosphorylated and total cRGDY-PEG C-dots, and treated with methylene blue protein intermediates (serum-deprived media, 100 nM par reagent (1 ml. 1 h, 37°C.), as a measure of cellular attach ticles alone, or 100 nM particles after addition of 250 nM or ment. Scale bar-30 um. Quadruplicate samples were run for 500 nM inhibitor). B. Relative intensities of phospho-protein each group. and B-actin blots in A relative to control cells (0.2% FBS). 01.07 FIG. 46. Influence of cKGDY-PEG-C dots on cell cycle. A. Percentage (%) of viable cells in the G. S., and G DETAILED DESCRIPTION OF THE INVENTION phases of the cell cycle as a function of particle concentration 0114. The present invention provides a fluorescent silica (0, 100, 300 nM) added to Go/G-phase-synchronized M21 based nanoparticle that allows for precise detection, charac cells incubated for a total of 96 hours. Italicized numbers terization, monitoring and treatment of a disease such as above each bar represent the percentage of cells (particle cancer. The invention also provides for a method for detecting incubated, control) in S, G and G phase, determined by flow tumor cells. The method may contain the following steps: (a) cytometry. B. Representative cell cycle histograms are shown administering to a patient a plurality of fluorescent silica for cells under control (i.e., no particle) conditions and after based nanoparticles in a dose ranging from about 0.01 nano incubation with 100 and 300 nM particles. One-way ANOVA: mole/kg body weight to about 1 nanomole/kg body weight, *, p<0.05: **, p<0.005 relative to S-phase control. Insets: from about 0.05 nanomole/kg body weight to about 0.9 nano Group mean values (n=3)+SD for each cell cycle phase. mole/kg body weight, from about 0.1 nanomole/kg body Experiments were performed in triplicate. weight to about 0.9 nanomole/kg body weight, from about 0.2 0108 FIG. 47. Dose-response effects and saturation bind nanomole/kg body weight to about 0.8 nanomole/kg body ing kinetics using cFGDY-PEG-C dots and CfB integrin weight, from about 0.3 nanomole/kg body weight to about 0.7 expressing cells. A. B. Cellular binding/uptake as a function nanomole/kg body weight, from about 0.4 nanomole/kg body of particle concentration (A) and incubation time (B) by flow weight to about 0.6 nanomole/kg body weight, or from about cytometry. Monolayers of M21 and HUVEC cells were incu 0.2 nanomole/kg body weight to about 0.5 nanomole/kg body bated (4h, 25°C.) with increasing concentrations of particles weight, and (b) detecting the nanoparticles. In one embodi (5-600 nM), as well as over a range of incubation times (0.5-4 ment, the nanoparticle comprises a silica-based core having a h; 100 nM) and assayed by fluorescence-activated cell sorting fluorescent compound positioned within the silica-based (FACS) analysis. The percentage of the total events detected core; a silica shell Surrounding at least a portion of the core; an is displayed. Each data point represents meaniSD of 3 repli organic polymer attached to the nanoparticle; a ligand Cates. attached to the nanoparticle and capable of binding a tumor 0109 FIG. 48. Competitive integrin receptor binding with marker; and at least one therapeutic agent. '*'I-cRGDY-PEG-C dots and cFGDY peptide in two cell 0115 The nanoparticle has a range of diameters including types. A. Specific binding of ''I-cRGDY-PEG-dots to M21 between about 0.1 nm and about 100 nm, between about 0.5 US 2014/024821.0 A1 Sep. 4, 2014

nm and about 50 nm, between about 1 nm and about 25 nm, present particles may be advantageous over passively tar between about 1 nm and about 15 nm, or between about 1 nm. geted nanocarriers, as the latter penetrate and non-specifi and about 8 nm. The nanoparticle has a fluorescent compound cally accumulate within the tumor interstitium by enhanced positioned within the nanoparticle, and has greater brightness permeability and retention (EPR) effects. and fluorescent quantum yield than the free fluorescent com 0123 Particle-based imaging systems for cancer diagnos pound. The nanoparticle also exhibits high biostability and tics should be non-toxic, and selectively detect sites of pri biocompatibility. To facilitate efficient urinary excretion of mary and metastatic disease while exhibiting relatively rapid the nanoparticle, it may be coated with an organic polymer, renal clearance. Under these conditions, the likelihood of such as poly(ethylene glycol) (PEG). The small size of the potential toxicity will be reduced given the smaller area under nanoparticle, the silica base and the organic polymer coating the plasma concentration-time curve. In one embodiment, minimizes the toxicity of the nanoparticle when administered these renal clearance properties may be achieved by ultras in vivo. In order to target a specific cell type, the nanoparticle mall particle-based platforms or macromolecular systems may further be conjugated to a ligand, which is capable of that meet effective renal glomerular filtration size cutoffs of binding to a cellular component (e.g., the cell membrane or 10 nm or less. Absence of single-dose acute toxicity and the other intracellular component) associated with the specific minimization of such risks will also be important. A platform cell type, such as a tumor marker or a signaling pathway design should maximize safety through rapid whole-body intermediate. In one embodiment, a therapeutic agent may be clearance and the selection of biokinetic profiles that mini attached to the nanoparticle. To permit the nanoparticle to be mize non-specific uptake in the reticuloendothelial system detectable by not only optical imaging (such as fluorescence (RES), thus reducing potential adverse exposures. imaging), but also other imaging techniques, such as positron 0.124. In one embodiment, the present particles are safe emission tomography (PET), single photon emission com and stable in vivo. The particles exhibit distinctly unique and puted tomography (SPECT), computerized tomography reproducible PK signatures defined by renal excretion. (CT), and magnetic resonance imaging (MRI), the nanopar Coupled with preferential uptake and localization of the ticle may also be conjugated to a contrast agent, Such as a probe at sites of disease, these particles can be used in cancer radionuclide. diagnostics. 0116. The properties of the nanoparticles lead to bulk 0.125. The nanoparticle may have both a ligand and a con excretion through the kidneys, increased potency relative to a trast agent. The ligand allows for the nanoparticle to target a native peptide ligand, enhanced uptake, and preferential specific cell type through the specific binding between the accumulation in tumors compared with normal tissues. This, ligand and the cellular component. This targeting, combined along with the lack of in vivo toxicity, has resulted in a unique with multimodal imaging, has multiple uses. For example, the product resulting in its translation to the clinic. nanoparticles can be used to map metastatic disease. Such as 0117 The present particles can be used to preferentially mapping sentinel lymph nodes (SLN), as well as identifying detect and localize tumors. For example, nanomolar particle tumor margins or neural structures, enabling the Surgeon to tracerdoses administered in a microdosing regime accumu resect malignant lesions under direct visualization and to late and preferentially localize at sites of disease, although not obviate complications during the Surgical procedure. The optimized for targeted detection. ligand may also facilitate entry of the nanoparticle into the 0118. The present nanoparticles may exhibit distinct and cell or barrier transport, for example, for assaying the intra reproducible human pharmacokinetic signatures in which cellular environment. renal clearance predominates (e.g., renal clearance of the 0.126 The nanoparticle can be coupled with a ligandanda nanoparticle is greater than about 90% ID in about 24 hours therapeutic agent with or without a radiolabel. The radiolabel after administration of the nanoparticle to a subject) without can additionally serve as a therapeutic agent for creating a significant RES uptake (e.g., less than about 10%). theranostic platform. This coupling allows the therapeutic 0119 The present nanoparticles are excellent diagnostic particle to be delivered to the specific cell type through the probes, exhibiting optimal physicochemical properties in specific binding between the ligand and the cellular compo humans that enable them to “target and clear the body over nent. This specific binding of the therapeutic agent ensures relatively short time intervals (e.g., hours, days, etc.). selective treatment of the disease site with minimum side 0120. The present particles bearing multiple actively tar effects. geted ligands (e.g., cRGDY and alpha-MSH) demonstrate enhanced binding affinity to the cellular targets compared to Nanoparticle Structure the affinity of a ligand alone. In some embodiments, the I0127. The fluorescent nanoparticle of the present inven present particles binds to a cellular target from about 2 to tion includes a silica-based core comprising a fluorescent about 20 fold greater, from about 3 to about 15 fold greater, compound positioned within the core, and a silica shell on the from about 5 to about 10 fold greater than a ligand alone. core. The silica shell may surround at least a portion of the 0121. In certain embodiments, dual-modality, targeted core. Alternatively, the nanoparticle may have only the core particles specifically assess tumor burden and can discrimi and no shell. The core of the nanoparticle may contain the nate metastatic tumor from chronic inflammatory disease in reaction product of a reactive fluorescent compound and a large animal models of metastatic melanoma. co-reactive organo-silane compound. In another embodi 0122 Targeted particles may enhance receptor binding ment, the core of the nanoparticle may contain the reaction affinity and avidity, increase plasma residence times, bio product of a reactive fluorescent compound and a co-reactive availability and tumor retention, and/or promote intracellular organo-silane compound, and silica. The diameter of the core delivery via internalization. The utilization of such targeted may be from about 0.05 nm to about 100 nm, from about 0.1 probes within Surgical (or medical) oncology settings may nm to about 50 nm, from about 0.5 nm to about 25 nm, from enable highly selective treatment of cancer-bearing tissues, about 0.8 nm to about 15 nm, or from about 1 nm to about 8 potentially reducing attendant complication rates. The nm. The shell of the nanoparticle can be the reaction product US 2014/024821.0 A1 Sep. 4, 2014 of a silica forming compound. The shell of the nanoparticle the dye-rich compact core is completed, tetraethylorthosili may have a range of layers. For example, the silica shell may cate (TEOS) is subsequently added to grow the silica shell be from about 1 to about 20 layers, from about 1 to about 15 that surrounded the core. layers, from about 1 to about 10 layers, or from about 1 to 0.132. The synthesis of the expanded core-shell nanopar about 5 layers. The thickness of the shell may range from ticle is accomplished by co-condensing TEOS with the dye about 0.01 nm to about 90 nm, from about 0.02 nm to about 40 precursor and allowing the mixture to react overnight. After nm, from about 0.05 nm to about 20 nm, from about 0.05 nm. the synthesis of the expanded core is completed, additional to about 10 nm, or from about 0.05 nm to about 5 nm. TEOS is added to grow the silica shell that surrounded the 0128. The silica shell of the nanoparticle may cover only a COC. portion of nanoparticle or the entire particle. For example, the I0133. The synthesis of the homogenous nanoparticles is silica shell may cover about 1 to about 100 percent, from accomplished by co-condensing all the reagents, the dye pre about 10 to about 80 percent, from about 20 to about 60 cursor and TEOS and allowing the mixture to react overnight. percent, or from about 30 to about 50 percent of the nanopar ticle. The silica shell can be either solid, i.e., substantially Fluorescent Compound non-porous, meso-porous, such as semi-porous, or porous. I0134. The nanoparticles may incorporate any known fluo rescent compound, Such as fluorescent organic compound, Synthesis of Nanoparticle dyes, pigments, or combinations thereof. A wide variety of Suitable chemically reactive fluorescent dyes are known, see 0129. The present fluorescent nanoparticle may be synthe for example MOLECULAR PROBES HANDBOOK OF sized by the steps of covalently conjugating a fluorescent FLUORESCENT PROBES AND RESEARCH CHEMI compound, Such as a reactive fluorescent dye, with the reac CALS, 6th ed., R. P. Haugland, ed. (1996). A typical fluoro tive moeties including, but not limited to, maleimide, iodoac phore is, for example, a fluorescent aromatic or heteroaro etamide, thiosulfate, amine, N-Hydroxysuccimide ester, matic compound such as is a pyrene, an anthracene, a 4-sulfo-2,3,5,6-tetrafluorophenyl (STP) ester, sulfosuccinim naphthalene, an acridine, a stilbene, an indole or benzindole, idyl ester, sulfodichlorophenol esters, sulfonyl chloride, an oxazole or benzoxazole, a thiazole or benzothiazole, a hydroxyl, isothiocyanate, carboxyl, to an organo-silane com 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine, a pound, such as a co-reactive organo-silane compound, to carbocyanine, a carbostyryl, a porphyrin, a Salicylate, an form a fluorescent silica precursor, and reacting the fluores anthranilate, an azulene, a perylene, a pyridine, a quinoline, a cent silica precursor to form a fluorescent core; covalently coumarin (including hydroxycoumarins and aminocou conjugating a fluorescent compound, Such as a reactive fluo marins and fluorinated derivatives thereof), and like com rescent dye, to an organo-silane compound, Such as a co pounds, see for example U.S. Pat. Nos. 5,830,912, 4,774,339, reactive organo-silane compound, to form a fluorescent silica 5,187,288, 5,248,782, 5,274,113, 5,433,896, 4,810,636 and precursor, and reacting the fluorescent silica precursor with a 4,812,409. In one embodiment, Cy5, a near infrared fluores silica forming compound, Such as tetraalkoxysilane, to form cent (NIRF) dye, is positioned within the silica core of the a fluorescent core; and reacting the resulting core with a silica present nanoparticle. Near infrared-emitting probes exhibit forming compound, Such as a tetraalkoxysilane, to form a decreased tissue attenuation and autofluorescence. Burns et silica shell on the core, to provide the fluorescent nanopar al. “Fluorescent silica nanoparticles with efficient urinary ticle. excretion for nanomedicine', Nano Letters, 2009, 9 (1), 442 448. 0130. The synthesis of the fluorescent monodisperse core 0.135 Non-limiting fluorescent compound that may be shell nanoparticles is based on a two-step process. First, the used in the present invention include, Cy5, Cy5.5 (also known near-infrared organic dye molecules (e.g., tetramethyl as Cy5++), Cy2, fluorescein isothiocyanate (FITC), tetram rhodamine isothiocynate (TRITC)) are covalently conjugated ethylrhodamine isothiocyanate (TRITC), phycoerythrin, to a silica precursor and condensed to form a dye-rich core. Cy7, fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++). Second, the silica gel monomers are added to form a denser Texas Red, LightCycler-Red 640, LightCycler Red 705, tet silica network around the fluorescent core material, providing ramethylrhodamine (TMR), rhodamine, rhodamine deriva shielding from Solvent interactions that can be detrimental to tive (ROX), hexachlorofluorescein (HEX), rhodamine 6G photostability. The versatility of the preparative route allows (R6G), the rhodamine derivative JA 133, Alexa Fluorescent for the incorporation of different fluorescent compounds, Dyes (such as Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor Such as fluorescent organic compounds or dyes, depending on 633, Alexa Fluor 555, and Alexa Fluor 647), 4,6-diamidino the intended nanoparticle application. The fluorescent com 2-phenylindole (DAPI), Propidium iodide, AMCA, Spectrum pounds that may be incorporated in the dye-rich core can Green, Spectrum Orange, Spectrum Aqua, Lissamine, and cover the entire UV-Vis to near-IR absorption and emission fluorescent transition metal complexes, such as europium. spectrum. U.S. patent application Ser. Nos. 10/306,614, Fluorescent compound that can be used also include fluores 10/536,569 and 11/119,969. Wiesner et al., PEG-coated cent proteins, such as GFP (green fluorescent protein), Core-shell Silica Nanoparticles and Mathods of Manufactire enhanced GFP (EGFP), blue fluorescent protein and deriva and Use, PCT/US2008/74894. tives (BFP, EBFP, EBFP2, AZurite, mKalamal), cyan fluo 0131 For the synthesis of the compact core-shell nanopar rescent protein and derivatives (CFP, ECFP, Cerulean, CyPet) ticle, the dye precursor is added to a reaction vessel that and yellow fluorescent protein and derivatives (YFP, Citrine, contains appropriate amounts of ammonia, water and solvent Venus, YPet). WO2008142571, WO2009056282, and allowed to react overnight. The dye precursor is synthe WO9922O26. sized by addition reaction between a specific near-infrared 0.136 The silica shell surface of the nanoparticles can be dye of interest and 3-aminopropyltriethoxysilane in molar modified by using known cross-linking agents to introduce ratio of 1:50, in exclusion of moisture. After the synthesis of Surface functional groups. Crosslinking agents include, but US 2014/024821.0 A1 Sep. 4, 2014

are not limited to, divinyl benzene, ethylene glycol antibodies, such as monoclonal or polyclonal antibodies. In dimethacrylate, trimethylol propane trimethacrylate, N,N'- another embodiment, the ligands are receptor ligands. In still methylene-bis-acrylamide, alkyl ethers, Sugars, peptides, another embodiment, the ligand is poly-L-lysine (pLysine). DNA fragments, or other known functionally equivalent 0140. An antigen may be attached to the nanoparticle. The agents. The ligand may be conjugated to the nanoparticle of antigen-attached nanoparticle may be used for vaccination. the present invention by, for example, through coupling reac 0.141. The terms “component of a cell' or “cellular com tions using carbodiimide, carboxylates, esters, alcohols, car ponent” refer to, for example, a receptor, an antibody, a hap bamides, aldehydes, amines, Sulfur oxides, nitrogen oxides, ten, an enzyme, a hormone, a biopolymer, an antigen, a halides, or any other Suitable compound known in the art. nucleic acid (DNA or RNA), a microorganism, a virus, a U.S. Pat. No. 6,268,222. pathogen, a toxin, combinations thereof, and like compo nents. The component of a cell may be positioned on the cell Organic Polymer (e.g., a transmembrane receptor) or inside the cell. In one embodiment, the component of a cell is a tumor marker. As 0.137 An organic polymer may be attached to the present used herein, the term “tumor marker” refers to a molecule, nanoparticle, e.g., attached to the Surface of the nanoparticle. entity or Substance that is expressed or overexpressed in a An organic polymer may be attached to the silica shell of the cancer cell but not normal cell. For example, the overexpres present nanoparticle. The organic polymer that may be used sion of certain receptors is associated with many types of in the present invention include PEG, polylactate, polylactic cancer. Aligand capable of binding to a tumor marker may be acids, Sugars, lipids, polyglutamic acid (PGA), polyglycolic conjugated to the Surface of the present nanoparticle, so that acid, poly(lactic-co-glycolic acid) (PLGA), polyvinyl acetate the nanoparticle can specifically target the tumor cell. (PVA), and the combinations thereof. The attachment of the organic polymer to the nanoparticle may be accomplished by 0142. Aligand may be attached to the present nanoparticle a covalent bond or non-covalent bond. Such as by ionic bond, directly or through a linker. The attachment of the ligand to hydrogen bond, hydrophobic bond, coordination, adhesive, the nanoparticle may be accomplished by a covalent bond or and physical absorption. In one embodiment, the nanoparticle non-covalent bond. Such as by ionic bond, hydrogen bond, is covalently conjugated with PEG, which prevents adsorp hydrophobic bond, coordination, adhesive, and physical absorption. The ligand may be coated onto the surface of the tion of serum proteins, facilitates efficient urinary excretion nanoparticle. The ligand may be imbibed into the surface of and decreases aggregation of the nanoparticle. Burns et al. the nanoparticle. The ligand may be attached to the Surface of “Fluorescent silica nanoparticles with efficient urinary excre the fluorescent nanoparticle, or may be attached to the core tion for nanomedicine', Nano Letters, 2009, 9 (1), 442-448. when the shell is porous or is covering a portion of the core. 0.138. The surface of the nanoparticle may be modified to When the ligand is attached to the nanoparticle through a incorporate at least one functional group. The organic poly linker, the linker can be any Suitable molecules. Such as a mer (e.g., PEG) attached to the nanoparticle may be modified functionalized PEG. The PEGs can have multiple functional to incorporate at least one functional group. For example, the groups for attachment to the nanoparticle and ligands. The functional group can be a maleimide or N-Hydroxysuccinim particle can have different types of functionalized PEGs bear ide (NHS) ester. The incorporation of the functional group ing different functional groups that can be attached to mul makes it possible to attach various ligands, contrast agents tiple ligands. This can enhance multivalency effects and/or and/or therapeutic agents to the nanoparticle. contrast at the target site, which allows the design and opti mization of a complex multimodal platform with improved Ligand targeted detection, treatment, and sensing in vivo. 0.139. A ligand may be attached to the present nanopar 0.143 A variety of different ligands may be attached to the ticle. The ligand is capable of binding to at least one cellular nanoparticle. For example, tripeptide Arg-Gly-Asp (RGD) component. The cellular component may be associated with may be attached to the nanoparticle. Alternatively, cyclic specific cell types or have elevated levels in specific cell peptide cFGD (which may contain other amino acid(s), e.g., types, such as cancer cells or cells specific to particular tissues cRGDY) may be attached to the nanoparticle. Any linear, and organs. Accordingly, the nanoparticle can target a specific cyclic or branched peptide containing the RGD sequence is cell type, and/or provides a targeted delivery for the treatment within the scope of the present invention. RGD binds to CfB and diagnosis of a disease. As used herein, the term “ligand integrin, which is overexpressed at the Surface of activated refers to a molecule or entity that can be used to identify, endothelial cells during angiogenesis and in various types of detect, target, monitor, or modify a physical state or condi tumor cells. Expression levels of O?3 integrin have been tion, Such as a disease state or condition. For example, a shown to correlate well with the aggressiveness of tumors. ligand may be used to detect the presence or absence of a Ruoslahti et al. New perspectives in cell adhesion: RGD and particular receptor, expression level of a particular receptor, integrins. Science 1987: 238:491. Gladson et al. Glioblas or metabolic levels of a particular receptor. The ligand can be, toma expression of vitronectin and alpha V beta 3 integrin. for example, a peptide, a protein, a protein fragment, a peptide Adhesion mechanism for transformed glial cells. J. Clin. hormone, a Sugar (i.e., lectins), a biopolymer, a synthetic Invest. 1991; 88:1924-1932. Seftor et al. Role of the alpha v polymer, an antigen, an antibody, an antibody fragment (e.g., beta 3 integrin in human melanoma cell invasion. Proc. Natl. Fab, nanobodies), an aptamer, a virus or viral component, a Acad. Sci. 1992: 89:1557-1561. receptor, a hapten, an enzyme, a hormone, a chemical com 0144 Synthetic peptide EPPT1 may be the ligandattached pound, a pathogen, a microorganism or a component thereof, to the nanoparticle. EPPT1, derived from the monoclonal a toxin, a Surface modifier, Such as a Surfactant to alter the antibody (ASM2) binding site, targets underglycosylated Surface properties or histocompatability of the nanoparticle MUC1 (uMUC1). MUC1, a transmembrane receptor, is or of an analyte when a nanoparticle associates therewith, and heavily glycosylated in normal tissues; however, it is overex combinations thereof. In one embodiment, the ligands are pressed and aberrantly underglycosylated in almost all US 2014/024821.0 A1 Sep. 4, 2014 human epithelial cell adenocarcinomas, and is implicated in F(ab')2 fragment of J591 (e.g., HuJ591-F(ab')2 fragments, or tumor pathogenesis. Moore et al. In vivo targeting of under humanized J591-F(ab')2 fragments) are used in diagnosing glycosylated MUC-1 tumor antigen using a multimodal prostate cancer or endometrial cancer (e.g., endometrial imaging probe. Cancer Res. 2004; 64:1821-7. Patel et al. endometrioid adenocarcinoma). In another embodiment, MUC1 plays a role in tumor maintenance in aggressive thy nanoparticles bearing the F(ab')2 fragment of J591 can be roid carcinomas. Surgery. 2005: 138:994-1001. Specific anti used to target brain tumor neovasculature for treatment, dis bodies including monoclonal antibodies against uMUC1 may ease progression monitoring. Brain tumor neovasculature has alternatively be conjugated to the nanoparticle in order to been found to overexpress PSMA, as shown from prior target uMUC1. immunohistochemistry evaluations of excised high grade 0145. In one embodiment, peptide analogues of C.-melan glioma specimens. otropin stimulating hormone (C-MSH) are the ligands 0148 Cyclic peptides containing the sequence HWGF are attached to the nanoparticle. Peptide analogues of C-MSH are potent and selective inhibitors of MMP-2 and MMP-9 but not capable of binding to melanocortin-1 receptors (MC1R), a of several other MMP family members. Peptide CTTHWG family of G-protein-coupled receptors overexpressed in FTLC inhibits the migration of human endothelial cells and melanoma cells. Loir et al. Cell Mol. Biol. (Noisy-le-grand) tumor cells. Moreover, it prevents tumor growth and invasion 1999, 45:1083-1092. in animal models and improves Survival of mice bearing 0146 In another embodiment, octreotate, a peptide analog human tumors. CTTHWGFTLC-displaying phage specifi of 14-amino acid somatostatin, is the ligand attached to the cally targets angiogenic blood vessels in vivo. This peptide nanoparticle. Octreotide, which has a longer half-life than and its extension GRENYGHCTTHWGFTLC or Somatostatin, is capable of binding to Somatostatin receptor GRENYGHCTTHWGFTLS can be used as ligands to be (SSTR). SSTR, a member of the G-protein coupled receptor attached to the present nanoparticle. The peptides can also be family, is overexpressed on the Surface of several human radiolabelled, e.g., radioiodinated. Koivunen et al., Tumor tumors. Reubietal. Distribution of Somatostatin Receptors in targeting with a selective gelatinase inhibitor, Nature Bio Normal and Tumor-Tissue. Metab. Clin. Exp. 1990:39:78 technology 17, 768-774 (1999). Penate Medina et al., Lipo 81. Reubi et al. Somatostatin receptors and their subtypes in Somal tumor targeting in drug delivery utilizing MMP-2- and human tumors and in peritumoral vessels. Metab. Clin. Exp. MMP-9-binding ligands, J. Drug Delivery, Volume 2011 1996: 45:39-41. Other somatostatin analogs may alterna (2011), Article ID 160515. Anticancer Research 21:4101 tively be conjugated to the nanoparticle to target SSTR, such 4106 (2005). In one embodiment, 'I labeled matrix metal as Tyr3-octreotide (Y3-OC), octreotate (TATE), Tyr3-octreo loproteinase peptide inhibitor (MMPI)-attached nanopar tate (Y3-TATE), and '''In-DTPA-OC. These somatostatin ticles are used for SLN mapping to stage endometrioid analogues may be utilized for both PET diagnostic imaging CaCC. and targeted radiotherapy of cancer. de Jong et al. Internal 014.9 The number of ligands attached to the nanoparticle ization of radiolabelled DTPA'octreotide and DOTA, may range from about 1 to about 30, from about 1 to about 20, Tyroctreotide: peptides for somatostatin receptor targeted from about 2 to about 15, from about 3 to about 10, from about Scintigraphy and radionuclide therapy. Nucl. Med. Commun. 1 to about 10, or from about 1 to about 6. The small number 1998; 19:283-8. de Jong et al. Comparison of '''In-Labeled of the ligands attached to the nanoparticle helps maintain the Somatostatin Analogues for Tumor Scintigraphy and Radio hydrodynamic diameter of the present nanoparticle which nuclide Therapy. Cancer Res. 1998: 58:437-41. Lewis et al. meets the renal clearance cutoff size range. Hilderbrand et al., Comparison of four Cu-labeled somatostatin analogs in Near-infrared fluorescence: application to in vivo molecular vitro and in a tumor-bearing rat model: evaluation of new imaging, Curr. Opin. Chem. Biol., 14:71-9, 2010. The num derivatives for PET imaging and targeted radiotherapy.J Med ber of ligands measured may be an average number of ligands Chem 1999: 42:1341-7. Krenning et al. Somatostatin Recep attached to more than one nanoparticle. Alternatively, one tor Scintigraphy with Indium-111-DTPA-D-Phe-1-Oct.- nanoparticle may be measured to determine the number of reotide in Man: Metabolism, Dosimetry and Comparison ligands attached. The number of ligands attached to the nano with Iodine-123-Tyr-3-Octreotide. J Nucl. Med 1992: particle can be measured by any suitable methods, which may 33:652-8. or may not be related to the properties of the ligands. For 0147 Various ligands may be used to map sentinel lymph example, the number of cRGD peptides bound to the particle nodes (SLNs). SLN mapping may be used in diagnosing, may be estimated using FCS-based measurements of absolute staging and treating cancer. J591 is an anti-prostate-specific particle concentrations and the starting concentration of the membrane antigen (i.e., anti-PSMA) monoclonal antibody. reagents for cRGD peptide. Average number of RGD pep J591 has been previously used to detect and stage prostate tides per nanoparticle and coupling efficiency of RGD to cancer. Tagawa et al., Anti-prostate-specific membrane anti functionalized PEG groups can be assessed colorimetrically gen-based radioimmunotherapy for prostate cancer, Cancer, under alkaline conditions and Biuret spectrophotometric 2010, 116(4 Suppl): 1075-83. Bander et al., Targeting Meta methods. The number of ligands attached to the nanoparticle static Prostate Cancer with Radiolabeled Monoclonal Anti may also be measured by other suitable methods. body J591 to the Extracellular Domain of Prostate Specific Membrane Antigen, The Journal of Urology 170(5), 1717 Contrast Agent 1721 (2003). Wernicke et al., (2011) Prostate-Specific Mem 0150. A contrast agent may be attached to the present brane Antigen as a Potential Novel Vascular Target for Treat nanoparticle for medical or biological imaging. As used ment of Glioblastoma Multiforme, Arch Pathol Lab Med. herein, the term "contrast agent” refers to a Substance, mol 2011; 135:1486-1489. The F(ab')2 fragment of J591 may be ecule or compound used to enhance the visibility of structures used as a ligand attached to the present nanoparticle. The or fluids in medical or biological imaging. The term “contrast nanoparticle may also be radiolabeled to create a dual-mo agent also refers to a contrast-producing molecule. The dality probe. In one embodiment, nanoparticles bearing the imaging techniques encompassed by the present invention US 2014/024821.0 A1 Sep. 4, 2014 include positron emission tomography (PET), single photon bonding interaction. The therapeutic agent may be associated emission computed tomography (SPECT), computerized with the ligand that is attached to the fluorescent nanoparticle. tomography (CT), magnetic resonance imaging (MRI), opti The therapeutic agent may also be associated with the organic cal bioluminescence imaging, optical fluorescence imaging, polymer or the contrast agent. For example, the therapeutic and combinations thereof. The contrast agent encompassed agent may be attached to the nanoparticle through PEG. The by the present invention may be any molecule, Substance or PEGs can have multiple functional groups for attachment to compound known in the art for PET, SPECT, CT, MRI, and the nanoparticle and therapeutic agent. The particle can have optical imaging. The contrast agent may be radionuclides, different types of functionalized PEGs bearing different func radiometals, positron emitters, beta emitters, gamma emit tional groups that can be attached to multiple therapeutic ters, alpha emitters, paramagnetic metal ions, and Suprapara agents. The therapeutic agent may be attached to the nano magnetic metal ions. The contrast agents include, but are not particle covalently or non-covalently. limited to, iodine, fluorine, copper, Zirconium, lutetium, asta 0.155. As used herein, the term “therapeutic agent” refers tine, yttrium, gallium, indium, technetium, gadolinium, dys to a Substance that may be used in the diagnosis, cure, miti prosium, iron, manganese, barium and barium sulfate. The gation, treatment, or prevention of disease in a human or radionuclides that may be used as the contrast agent attached another animal. Such therapeutic agents include Substances to the nanoparticle of the present invention include, but are recognized in the official United States Pharmacopeia, offi not limited to Zr, Cu, Ga, Y, 'I and '77Lu. cial Homeopathic Pharmacopeia of the United States, official 0151. The contrast agent may be directly conjugated to the National Formulary, or any supplement thereof. nanoparticle. Alternatively, the contrast agent may be indi 0156 Therapeutic agents that can be incorporated with the rectly conjugated to the nanoparticle, by attaching to linkers fluorescent nanoparticles or the ligated-fluorescent nanopar or chelates. The chelate may be adapted to bind a radionu ticles of the invention include nucleosides, nucleoside ana clide. The chelates that can be attached to the present nano logs, small interfering RNA (siRNA), microRNA, oligopep particle may include, but are not limited to, 1,4,7,10-tetraaza tides, polypeptides, antibodies, COX-2 inhibitors, apoptosis cyclododecane-1,4,7,10-tetraacetic acid (DOTA), promoters, urinary tract agents, vaginal agents, vasodilators diethylenetriaminepentaacetic (DTPA), desferrioxamine neurodegenerative agents (e.g., Parkinson's disease), obesity (DFO) and triethylenetetramine (TETA). agents, ophthalmic agents, osteoporosis agents, para-Sym 0152 Suitable means for imaging, detecting, recording or patholytics, para-sympathometics, antianesthetics, prostag measuring the present nanoparticles may also include, for landins, psychotherapeutic agents, respiratory agents, seda example, a flow cytometer, a laser scanning cytometer, a tives, hypnotics, skin and mucous membrane agents, anti fluorescence micro-plate reader, a fluorescence microscope, a bacterials, anti-fungals, antineoplastics, cardioprotective confocal microscope, a bright-field microscope, a high con agents, cardiovascular agents, anti-thrombotics, central ner tent scanning system, and like devices. More than one imag Vous system stimulants, cholinesterase inhibitors, contracep ing techniques may be used at the same time or consecutively tives, dopamine receptor agonists, erectile dysfunction to detect the present nanoparticles. In one embodiment, opti agents, fertility agents, gastrointestinal agents, gout agents, cal imaging is used as a sensitive, high-throughput Screening hormones, immunomodulators, Suitably functionalized anal tool to acquire multiple time points in the same Subject, gesics or general or local anesthetics, anti-convulsants, anti permitting semi-quantitative evaluations of tumor marker diabetic agents, anti-fibrotic agents, anti-infectives, motion levels. This offsets the relatively decreased temporal resolu sickness agents, muscle relaxants, immuno-Suppressive tion obtained with PET, although PET is needed to achieve agents, migraine agents, non-steroidal anti-inflammatory adequate depth penetration for acquiring Volumetric data, and drugs (NSAIDs), Smoking cessation agents, or sympatholyt to detect, quantitate, and monitor changes in receptor and/or ics (see Physicians’ Desk Reference, 55th ed., 2001, Medical other cellular marker levels as a means of assessing disease Economics Company, Inc., Montvale, N.J., pages 201-202). progression or improvement, as well as stratifying patients to 0157. Therapeutic agents that may be attached to the Suitable treatment protocols. present nanoparticle include, but are not limited to, DNA alkylating agents, topoisomerase inhibitors, endoplasmic Therapeutic Agent reticulum stress inducing agents, a platinum compound, an 0153. A therapeutic agent may be attached to the fluores antimetabolite, Vincalkaloids, taxanes, epothilones, enzyme cent nanoparticle, for example, for targeted treatment of a inhibitors, receptor antagonists, therapeutic antibodies, disease. The therapeutic agent may be delivered to a diseased tyrosine kinase inhibitors, boron radiosensitizers (i.e. vel site in a highly specific or localized manner with release of the cade), and chemotherapeutic combination therapies. therapeutic agent in the disease site. Alternatively, the thera 0158. Non-limiting examples of DNA alkylating agents peutic agent may not be released. The fluorescent nanopar are nitrogen mustards, such as Mechlorethamine, Cyclophos ticle conjugated with the ligand can be used for targeted phamide (Ifosfamide, Trofosfamide), Chlorambucil (Mel delivery of a therapeutic agent to a desired location in a phalan, Prednimustine), Bendamustine, Uramustine and variety of systems, such as on, or within, a cell or cell com Estramustine; nitrosoureas, such as Carmustine (BCNU), ponent, within the body of an organism, Such as a human, or Lomustine (Semustine), Fotemustine, Nimustine, Ranimus across the blood-brain barrier. tine and Streptozocin, alkyl Sulfonates, such as BuSulfan 0154 The therapeutic agent may be attached to the nano (Mannosulfan, Treosulfan); AZiridines, such as Carboquone, particle directly or indirectly. The therapeutic agent can be ThioTEPA, Triaziquone, Triethylenemelamine: Hydrazines absorbed into the interstices or pores of the silica shell, or (Procarbazine); Triazenes such as Dacarbazine and Temozo coated onto the silica shell of the fluorescent nanoparticle. In lomide: Altretamine and Mitobronitol. other embodiments where the silica shell is not covering the 0159. Non-limiting examples of Topoisomerase I inhibi entire Surface, the therapeutic agent can be associated with tors include Campothecin derivatives including CPT-11 the fluorescent core, Such as by physical absorption or by (irinotecan), SN-38, APC, NPC, campothecin, topotecan, US 2014/024821.0 A1 Sep. 4, 2014 exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptoth modulators, a 5-chloro-2,4dihydroxypyridine and potassium ecin, lurtotecan, rubitecan, silatecan, gimatecan, diflomote oXonate), ralititrexed (tomudex), nolatrexed (Thymitaq, can, extatecan, BN-80927, DX-8951f, and MAG-CPT as AG337), LY231514 and ZD9331, as described for example in decribed in Pommier Y. (2006) Nat. Rev. Cancer 6(10):789 Papamicheal (1999) The Oncologist 4:478-487. 802 and U.S. Patent Publication No. 200510250854; Proto 0163 Examples of vincalkaloids, include, but are not lim berberine alkaloids and derivatives thereof including berber ited to Vinblastine, Vincristine, Vinflunine, Vindesine and rubine and coralyne as described in Li et al. (2000) Vinorelbine. Biochemistry 39(24):7107-7116 and Gatto et al. (1996) Can 0164. Examples of taxanes include, but are not limited to cer Res. 15(12):2795-2800; Phenanthroline derivatives docetaxel, Larotaxel, Ortataxel, Paclitaxel and Tesetaxel. An including BenZoiphenanthridine, Nitidine, and fagaronine example of an epothilone is iabepilone. as described in Makhey et al. (2003) Bioorg. Med. Chem. 11 0.165 Examples of enzyme inhibitors include, but are not (8): 1809-1820; Terbenzimidazole and derivatives thereof as limited to farnesyltransferase inhibitors (Tipifamib); CDK described in Xu (1998) Biochemistry 37(10):3558-3566; and inhibitor (Alvocidib, Seliciclib); proteasome inhibitor (Bort Anthracycline derivatives including Doxorubicin, Daunoru eZomib); phosphodiesterase inhibitor (Anagrelide; rollip bicin, and Mitoxantrone as described in Foglesong et al. ram); IMP dehydrogenase inhibitor (Tiazofurine); and (1992) Cancer Chemother: Pharmacol. 30(2):123-25, Crow lipoxygenase inhibitor (Masoprocol). Examples of receptor et al. (1994).J. Med. Chem. 37(19):31913194, and Crespi et antagonists include, but are not limited to ERA (Atrasentan); al. (1986) Biochem. Biophys. Res. Commun. 136(2):521-8. retinoid X receptor (Bexarotene); and a sex steroid (Testolac Topoisomerase II inhibitors include, but are not limited to tone). Etoposide and Teniposide. Dual topoisomerase I and II 0166 Examples of therapeutic antibodies include, but are inhibitors include, but are not limited to, Saintopin and other not limited to anti-HER1/EGFR (Cetuximab, Panitumumab); Naphthecenediones, DACA and other Acridine-4-Carboxa Anti-HER2/neu (erbB2) receptor (Trastuzumab); Anti-Ep mindes, Intoplicine and other Benzopyridoindoles, TAS-I03 CAM (Catumaxomab, Edrecolomab) Anti-VEGF-A (Beva and other 7H-indeno.2.1-cQuinoline-7-ones, Pyrazoloacri cizumab); Anti-CD20 (Rituximab, Tositumomab, Ibritumo dine, XR 11576 and other BenZophenazines, XR 5944 and mab); Anti-CD52 (Alemtuzumab); and Anti-CD33 other Dimeric compounds, 7-oxo-7H-dibenzfi Isoquino (Gemtuzumab). U.S. Pat. Nos. 5,776,427 and 7,601,355. lines and 7-oxo-7H-benzoe Perimidines, and Anthracenyl 0.167 Examples of tyrosine kinase inhibitors include, but amino Acid Conjugates as described in Denny and Baguley are not limited to inhibitors to ErbB: HER1/EGFR (Erlotinib, (2003) Curr: Top. Med. Chem. 3(3):339-353. Some agents Gefitinib, Lapatinib, Vandetanib, Sunitinib, Neratinib); inhibit Topoisomerase II and have DNA intercalation activity HER2/neu (Lapatinib, Neratinib); RTK class III: C-kit (Axi Such as, but not limited to, Anthracyclines (Aclarubicin, tinib, Sunitinib, Sorafenib), FLT3 (Lestaurtinib), PDGFR Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Amrubi (Axitinib, Sunitinib, Sorafenib); and VEGFR (Vandetanib, cin, Pirarubicin, Valrubicin, Zorubicin) and Antracenediones Semaxanib, Cediranib, Axitinib, Sorafenib); bcr-abl (Ima (Mitoxantrone and Pixantrone). tinib, Nilotinib, Dasatinib); Src (Bosutinib) and Janus kinase 0160 Examples of endoplasmic reticulum stress inducing 2 (Lestaurtinib). agents include, but are not limited to, dimethyl-celecoxib 0168 Chemotherapeutic agents that can be attached to the (DMC), nelfinavir, celecoxib, and boron radiosensitizers (i.e. present nanoparticle may also include amsacrine, Trabect velcade (Bortezomib)). edin, retinoids (Alitretinoin, Tretinoin), Arsenic trioxide, 0161 Non-limiting examples of platinum based com asparagine depleter Asparaginase/Pegaspargase), Celecoxib, pound include Carboplatin, Cisplatin, Nedaplatin, Oxalipl Demecolcine, Elesclomol, Elsamitrucin, Etoglucid, atin, Triplatin tetranitrate, Satraplatin, Aroplatin, Lobaplatin, Lonidamine, Lucanthone, MitoguaZone, Mitotane, and JM-216. (see McKeage et al. (1997) J. Clin. Oncol. Oblimersen, Temsirolimus, and Vorinostat. 201:1232-1237 and in general, CHEMOTHERAPY FOR 0169. Examples of specific therapeutic agents that can be GYNECOLOGICAL NEOPLASM, CURRENT THERAPY linked, ligated, or associated with the fluorescent nanopar AND NOVEL APPROACHES, in the Series Basic and Clini ticles of the invention are flomoxeffortimicin(s); gentamicin cal Oncology, Angioli et al. Eds., 2004). (s); glucosulfone Solasulfone; gramicidin S. gramicidin(s): 0162 Non-limiting examples of antimetabolite agents grepafloxacin, guamecycline; hetacillin, isepamicin; josamy include Folic acid based, i.e. dihydrofolate reductase inhibi cin; kanamycin(s); flomoxef fortimicin(s); gentamicin(s): tors, such as Aminopterin, Methotrexate and Pemetrexed: glucosulfone Solasulfone; gramicidin S.; gramicidin(s); gre thymidylate synthase inhibitors, such as Raltitrexed, Pemetr pafloxacin; guamecycline; hetacillin, isepamicin; josamycin; exed: Purine based, i.e. an adenosine deaminase inhibitor, kanamycin(s); bacitracin; bambermycin(s); biapenem; brodi Such as Pentostatin, a thiopurine, such as Thioguanine and moprim; butirosin; capreomycin; carbenicillin, carbomycin; Mercaptopurine, a halogenated/ribonucleotide reductase carumonam, cefadroxil, cefamandole, cefatrizine; cefbu inhibitor, such as Cladribine, Clofarabine, Fludarabine, or a peraZone; cefclidin; cefdinir, cefditoren; cefepime; cefe guanine/guanosine: thiopurine, such as Thioguanine; or Pyri tamet, cefixime; cefinenoxime; cefininoX; cladribine; apalcil midine based, i.e. cytosine/cytidine: hypomethylating agent, lin; apicycline: apramycin; arbekacin; aspoxicillin; such as Azacitidine and Decitabine, a DNA polymerase aZidamfenicol; aztreonam, cefodizime; cefonicid; cefopera inhibitor, such as Cytarabine, a ribonucleotide reductase Zone; ceforamide; cefotaxime; cefotetan, cefotiam, cefoZop inhibitor, such as Gemcitabine, or a thymine/thymidine: ran; ce?pimizole; ce?piramide; ce?pirome, cefprozil; cefroxa thymidylate synthase inhibitor, such as a Fluorouracil (5-FU). dine, cefteram, ceftibuten, cefuZonam; cephalexin; Equivalents to 5-FU include prodrugs, analogs and derivative cephaloglycin; cephalosporin C. cephradine; chlorampheni thereof such as 5'-deoxy-5-fluorouridine (doxifluroidine), col; chlortetracycline; clinafloxacin; clindamycin; clomocy 1-tetrahydrofuranyl-5-fluorouracil (florafur), Capecitabine cline; colistin; cyclacillin; dapsone; demeclocycline; diathy (Xeloda), S-I (MBMS-247616, consisting oftegafur and two mosulfone; dibekacin; dihydrostreptomycin; US 2014/024821.0 A1 Sep. 4, 2014

6-mercaptopurine; thioguanine; capecitabine; docetaxel; eto fendosal; fepradinol; flufenamic acid; Tomudex(R) (N-5- poside; gemcitabine; topotecan; vinorelbine; Vincristine; Vin (1,4-Dihydro-2-methyl-4-oxo-6-quin-azolinyl)methylme blastine; teniposide; melphalan; methotrexate, 2-p-Sulfa thylamino-2-thienylcarbonyl-L-glutamic acid), trimetrex nilyanilinoethanol: 4,4'-sulfinyldianiline; ate, tubercidin, ubenimex, vindesine, Zorubicin; argatroban: 4-sulfanilamidosalicylic acid; butorphanol; nalbuphine. coumetarol or dicoumarol. streptozocin, doxorubicin; daunorubicin; plicamycin, idaru 0170 Lists of additional therapeutic agents can be found, bicin; mitomycin C, pentostatin: mitoxantrone; cytarabine; for example, in: Physicians’ Desk Reference, 55th ed., 2001, fludarabine phosphate; butorphanol; nalbuphine. StreptoZo Medical Economics Company, Inc., Montvale, N.J.; USPN cin, doxorubicin; daunorubicin; plicamycin; idarubicin; Dictionary of USAN and International Drug Names, 2000, mitomycin C; pentostatin; mitoxantrone; cytarabine; fludara The United States Pharmacopeial Convention, Inc., Rock bine phosphate; acediasulfone; acetosulfone; amikacin; ville, Md., and The Merck Index, 12th ed., 1996, Merck & amphotericin B; amplicillin; atorvastatin; enalapril; raniti Co., Inc., Whitehouse Station, N.J. dine; ciprofloxacin; pravastatin: clarithromycin; cyclosporin; 0171 The therapeutic agent may also include radionu famotidine; leuprolide; acyclovir, paclitaxel; azithromycin; clides when the present nanoparticle is used in targeted radio lamivudine; budesonide; albuterol; indinavir, metformin; therapy. In one embodiment, low energy beta-emitting radio alendronate; nizatidine; Zidovudine; carboplatin; metoprolol; nuclides, such as 'Lu-chelated constructs, is associated amoxicillin; diclofenac; lisinopril; ceftriaxone; captopril; Sal with the nanoparticle and used to treat relatively small tumor meterol: Xinafoate; imipenem; cilastatin; benazepril; cefa burdens or micrometastatic disease. In another embodiment, clor, ceftazidime; morphine; dopamine; bialamicol; fluvasta higher energy beta emitters, such as yttrium-90 ('Y), may be tin, phenamidine; podophyllinic acid 2-ethylhydrazine; used to treat larger tumor burdens. Iodine-131 (I) may also acriflavine; chloroaZodin; arsphenamine; amicarbilide; ami be used for radiotherapy. noquinuride; quinapril; oxymorphone; buprenorphine; floXu 0172. The surface of the nanoparticle may be modified to ridine; dirithromycin; doxycycline; enoxacin; enviomycin; incorporate at least one functional group. The organic poly epicillin, erythromycin; leucomycin(s); lincomycin; lom mer (e.g., PEG) attached to the nanoparticle may be modified efloxacin; lucensomycin; lymecycline; meclocycline; mero to incorporate at least one functional group. For example, the penem; methacycline; micronomicin; midecamycin(s): functional group can be a maleimide or N-Hydroxysuccinim minocycline; moxalactam; mupirocin, nadifloxacin; natamy ide (NHS) ester. The incorporation of the functional group cin; neomycin; netilmicin; norfloxacin, oleandomycin; makes it possible to attach various ligands, contrast agents oxytetracycline; p-sulfanilylbenzylamine; panipenem; paro and/or therapeutic agents to the nanoparticle. momycin; paZufloxacin; penicillinN, pipacycline; pipemidic 0173. In one embodiment, a therapeutic agent is attached acid; polymyxin; primycin; quinacillin: ribostamycin; rifa to the nanoparticle (Surface or the organic polymer coating) mide: rifampin: rifamycin SV: rifapentine; rifaximin: ristoce via an NHS ester functional group. For example, tyrosine tin, ritipenem; rokitamycin; rollitetracycline; rosaramycin; kinase inhibitor such as dasatinib (BMS) or chemotherapeu roXithromycin; Salazosulfadimidine; sancycline; sisomicin; tic agent (e.g., taxol), can be coupled via an ester bond to the sparfloxacin; spectinomycin; spiramycin; Streptomycin; suc nanoparticle. This ester bond can then be cleaved in an acidic cisulfone, Sulfachrysoidine; Sulfaloxic acid; Sulfamidoch environment or enzymatically in vivo. This approach may be rysoidine, Sulfanilic acid, Sulfoxone; teicoplanin; temafloxa used to deliver a prodrug to a Subject where the drug is cin; temocillin, tetroxoprim; thiamphenicol; thiazolsulfone; released from the particle in vivo. thiostrepton, ticarcillin; tigemonam, tobramycin; to Sufloxa 0.174 We have tested the prodrug approach by coupling cin; trimethoprim; trospectomycin; trovafloxacin; tuberacti small molecule inhibitor dasatinib with the PEG molecules of nomycin; Vancomycin; aZaserine; candicidin(s); chlorphen the nanoparticle. Based on biodistribution results and the esin; dermostatin(s); filipin; fungichromin; mepartricin; human drug dosing calculations, the nanoparticle has been nystatin; oligomycin (S); perimycin A; tubercidin; 6-azauri found to have unique biological properties, including rela dine; 6-diazo-5-oxo-L-norleucine; aclacinomycin(s); ancit tively rapid clearance from the blood compared to tumors and abine; anthramycin; azacitadine; azaserine; bleomycin(s): Subsequent tumor tissue accumulation of the therapeutic ethyl biscoumacetate; ethylidene dicoumarol; iloprost, lami agent, which Suggests that a prodrug approach is feasible. The fiban; taprostene; tioclomarol; tirofiban; amiprilose; bucil functionalized nanoparticle permits drugs to be dosed mul lamine; gusperimus; gentisic acid; glucamethacin, glycol tiple times, ensuring that the drug concentration in the tumor salicylate; meclofenamic acid, mefenamic acid; mesalamine; is greater than that specified by the IC-50 in tumor tissue, yet niflumic acid, olsalazine; oxaceprol; S-enosylmethionine; will not be dose-limiting to other organ tissues, such as the salicylic acid; salsalate: Sulfasalazine; tolfenamic acid; caru heart, liver or kidney. The therapeutic agent and nanoparticle bicin; carzinophillin A; chlorozotocin, chromomycin(s): can be radiolabeled or optically labelled separately, allowing denopterin: doxifluridine; ediatrexate; eflornithine; ellip independent monitoring of the therapeutic agent and the tinium; enocitabine; epirubicin; mannomustine; menogaril; nanoparticle. In one embodiment, radiofluorinated (i.e., 'F) mitobronitol; mitolactol; mopidamol, mycophenolic acid; dasatinib is coupled with PEG-3400 moieties attached to the nogalamycin; olivomycin(s); peplomycin; pirarubicin; piritr nanoparticle via NHS ester linkages. Radiofluorine is crucial exim; prednimustine; procarbazine; pteropterin; puromycin; for being able to independently monitor time-dependent ranimustine; streptonigrin; thiamiprine, mycophenolic acid; changes in the distribution and release of the drug from the procodazole; romurtide; sirolimus (rapamycin); tacrolimus; radioiodinated ('''I) fluorescent (Cy5) nanoparticle. In this butethamine; femalcomine; hydroxytetracaine; naepaine; way, we can separately monitor the prodrug (dasatinib) and orthocaine;piridocaine; salicylalcohol, 3-amino-4-hydroxy nanoparticle. This permits optimization of the prodrug design butyric acid; aceclofenac; alminoprofen; amfenac, bro compared with methods in the prior art where no dual-label mfenac; bromosaligenin; bumadizon; carprofen; diclofenac; ing approach is used. In another embodiment, radiotherapeu diflunisal; ditazol; enfenamic acid; etodolac, etofenamate; tic iodine molecules (i.e., I-131), or other therapeutic gamma US 2014/024821.0 A1 Sep. 4, 2014

or alpha emitters, are conjugated with PEG via a maleimide novel 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid functional group, where the therapeutic agent may not disso (DOTA) derivatives for chemoselective attachment to unpro ciate from the PEG in vivo. tected polyfunctionalized compounds. Chemistry—a Euro 0.175. A generalizable approach referred herein as "click pean Journal 13, 6082–6090. In a second embodiment, a chemistry’ is described below. In order for the present nano maleimide functional group and a thiol group may be intro particle to readily accommodate large ranges of ligands, con duced onto the nanoparticle and the desired ligand (or con trast agents orchelates, the Surface of the nanoparticle may be trast agent, chelate), with the nanoparticle having the male modified to incorporate a functional group. The nanoparticle imidefunctional group, the ligand (or contrast agent, chelate) may also be modified with organic polymers (e.g., PEGs) or having the thiol group, or vice versa. The double bond of chelates that can incorporate a functional group. In the mean maleimide readily reacts with the thiol group to form a stable time, the ligand, contrast agent, or therapeutic agent is modi carbon-sulfur bond. In a third embodiment, an activated ester fied to incorporate a functional group that is able to react with functional group, e.g., a Succinimidyl ester group, and an the functional group on the nanoparticle, or on the PEGs or amine group may be introduced onto the nanoparticle and the chelating agents attached to the nanoparticle under Suitable conditions. Accordingly, any ligand, contrast agent or thera desired ligand, contrast agent or chelate. The activated ester peutic agent that has the reactive functional group is able to be group readily reacts with the amine group to form a stable readily conjugated to the nanoparticle. This generalizable carbon-nitrogen amide bond. In a fourth embodiment, the approach is referred herein as "click chemistry’, which would click chemistry involves the inverse electron demand Diels allow for a great deal of versatility to explore multimodality Alder reaction between a tetrazine moiety and a strained applications. In the chemical reactions of "click chemistry. alkene (FIG. 21c). Devaraj et al., (2009) Fast and Sensitive two molecular components may be joined via a selective, Pretargeted Labeling of Cancer Cells through a Tetrazine/ rapid, clean, bioorthogonal, and/or biocompatible reaction. trans-Cyclooctene Cycloaddition. Angewandte Chemie-In Kolb et al., (2001) Click Chemistry: Diverse Chemical Func ternational Edition 48,7013-7016. Devarajet al., (2008) Tet tion from a Few Good Reactions. Angewandte Chemie Inter razine-Based Cycloadditions: Application to Pretargeted national Edition 40, 2004-2021. Lim et al., (2010) Bioor Live Cell Imaging. Bioconjugate Chemistry 19, 2297-2299. thogonal chemistry: recent progress and future directions. Blackman et al., (2008) Tetrazine ligation: fast bioconjuga Chemical Communications 46, 1589-1600. Sletten et al., tion based on inverse electron demand Diels-Alder reactivity. (2009) Bioorthogonal chemistry: fishing for selectivity in a Journal of the American Chemical Society 130, 13518 sea of functionality. Angewandte Chemie International Edi 13519. The ligation can be selective, fast, biocompatible, tion 48,6973-6998. and/or bioorthogonal. Unlike many Diels-Alder reactions, 0176 Any suitable reaction mechanism may be adapted in the coupling is irreversible, forming a stable pyridazine prod the present invention for "click chemistry', so long as facile ucts after the retro-Diels-Alder release of dinitrogen from the and controlled attachment of the ligand, contrast agent or reaction intermediate. A tetrazine moiety and a strained alk chelate to the nanoparticle can be achieved. In one embodi ene may be introduced onto the nanoparticle and the desired ment, a free triple bond is introduced onto PEG, which is ligand (or contrast agent, chelate), with the nanoparticle hav already covalently conjugated with the shell of the nanopar ing the tetrazine moiety, the ligand (or contrastagent, chelate) ticle. In the meantime, an azide bond is introduced onto the having the strained alkene, or vice versa. A number of differ desired ligand (or contrast agent, chelate). When the PEGy ent tetraZinc strained alkene pairs can be used for the reaction, lated nanoparticle and the ligand (or contrast agent, chelate) including, e.g., the combination of 3-(4-benzylamino)-1.2.4. are mixed in the presence of a copper catalyst, cycloaddition 5-tetrazine (TZ) and either norbornene- or trans-cyclooctene of azide to the triple bond will occur, resulting in the conju dervatives. Schochet al., (2010) Post-Synthetic Modification gation of the ligand with the nanoparticle. One example of of DNA by Inverse-Electron-Demand Diels-Alder Reaction. click chemistry is the Cu(I)-catalyzed 3+2] Huisgen Journal of the American Chemical Society 132, 8846-8847. cycloaddition between an azide and alkyne. Moses et al., Devaraj et al., (2010) Bioorthogonal Turn-On Probes for (2007) The growing applications of click chemistry. Chemi Imaging Small Molecules Inside Living Cells. Angewandte cal Society Reviews 36, 1249-1262. Glaser et al., (2009) Chemie International Edition 49. Haun et al., (2010) Bioor Click labelling in PET radiochemistry. Journal of Labelled thogonal chemistry amplifies nanoparticle binding and Compounds & Radiopharmaceuticals 52, 407-414. Mindt et enhances the sensitivity of cell detection. Nature Nanotech al., (2009) A “Click Chemistry” Approach to the Efficient nology 5, 660-665. Rossin et al., (2010) In vivo chemistry for Synthesis of Multiple Imaging Probes Derived from a Single pretargeted tumor imaging in live mice. Angewandte Chemie Precursor. Bioconjugate Chemistry 20, 1940-1949. New et International Edition 49, 3375-3378. Li et al., (2010) Tetra al., (2009) Growing applications of "click chemistry’ for Zine-trans-cyclooctene ligation for the rapid construction of bioconjugation in contemporary biomedical research. Cancer 18-F labeled probes. Chemical Communications 46, 8043 Biotherapy and Radiopharmaceuticals 24, 289-301. Wang et 8045. Reiner et al., (2011) Synthesis and in vivo imaging of a al., (2010) Application of “Click Chemistry’ in Synthesis of 18F-labeled PARP1 inhibitor using a chemically orthogonal Radiopharmaceuticals. Progress in Chemistry 22, 1591 Scavenger-assisted high-performance method. Angewandte 1602. Schultz et al., (2010) Synthesis of a DOTA-Biotin Chemie International Edition 50, 1922-1925. For example, Conjugate for Radionuclide Chelation via Cu-Free Click the surface of the nanoparticles may be decorated with tetra Chemistry. Organic Letters 12, 2398-2401. Martin et al., Zine moieties, which can Subsequently be conjugated to nor (2010) A DOTA-peptide conjugate by copper-free click bornene-modified ligand (or contrast agent, or chelate) chemistry. Bioorganic & Medicinal Chemistry Letters 20, (FIGS. 21d and 21e). In one aspect, the nanoparticles are 4805-4807. Lebedev et al., (2009) Clickable bifunctional coated with tetrazine, which can then be modified with the radiometal chelates for peptide labeling. Chemical Commu DOTA chelates using the tetrazine-norbornene ligation, and nications 46, 1706-1708. Knor et al., (2007) Synthesis of radiolabeled with Cu. US 2014/024821.0 A1 Sep. 4, 2014

Residence/Clearance Time In Vivo may range from about 1 to about 30, or from about 1 to about 10. The diameter of the nanoparticle may be between about 1 0177. After administration of the present nanoparticle to a nm and about 8 nm. A contrast agent, Such as a radionuclide, subject, the blood residence half-time of the nanoparticles may be attached to the nanoparticle. Alternatively, a chelate may range from about 2 hours to about 25 hours, from about may be attached to the nanoparticle. The nanoparticle may be 3 hours to about 20 hours, from about 3 hours to about 15 detected by PET, SPECT, CT, MRI, optical imaging, biolu hours, from about 4 hours to about 10 hours, or from about 5 minescence imaging, or combinations thereof. A therapeutic hours to about 6 hours. Longer blood residence half-time agent may be attached to the nanoparticle. After administra means longer circulation, which allows more nanoparticles to tion of the radiolabeled targeted nanoparticle to a Subject, accumulate at the target site in vivo. Blood residence half blood residence half-time of the nanoparticle may also range time may be evaluated as follows. The nanoparticles are first from about 3 hours to about 15 hours, or from about 4 hours administered to a subject (e.g., a mouse, a miniswine or a to about 10 hours. Tumor residence half-time of the nanopar human). At various time points post administration, blood ticle after administration of the nanoparticle to a Subject may samples are taken to measure nanoparticle concentrations also range from about 10 hours to about 4 days, or from about through Suitable methods. 15 hours to about 3.5 days. The ratio of tumor residence 0178. In one embodiment, after administration of the half-time to blood residence half-time of the targeted nano PEGylated (or control) nanoparticle to a subject, blood resi particle after administration of the nanoparticle to a subject dence half-time of the nanoparticle may range from about 2 may range from about 2 to about 30, from about 3 to about 20, hours to about 15 hours, or from about 4 hours to about 10 or from about 4 to about 15. Renal clearance of the nanopar hours. Tumor residence half-time of the nanoparticle after ticle may also range from about 45% ID to about 90% ID in administration of the nanoparticle to a Subject may range about 24 hours after administration of the nanoparticle to a from about 5 hours to about 2 days, from about 10 hours to Subject. about 4 days, or from about 15 hours to about 3.5 days. The ratio of tumor residence half-time to blood residence halftime 0182. In one embodiment, to estimate residence (or clear of the nanoparticle after administration of the nanoparticle to ance) half-time values of the radiolabeled nanoparticles (T, a subject may range from about 2 to about 30, from about 3 to 2) in blood, tumor, and other major organs/tissues, the per about 20, or from about 4 to about 15. Renal clearance of the centage of the injected dose per gram (% ID/g) values are nanoparticle after administration of the nanoparticle to a Sub measured by sacrificing groups of mice at specified times ject may range from about 10% ID (initial dose) to about following administration of the nanoparticles. Blood, tumor, 100% ID in about 24 hours, from about 30% ID to about 80% and organs are harvested, weighed, and counted in a scintil ID in about 24 hours, or from about 40% ID to about 70% ID lation Y-counter. The % ID/g values are corrected for radio in about 24 hours. In one embodiment, after the nanoparticle active decay to the time of injection. The resulting time is administered to a subject, blood residence half-time of the activity concentration data for each tissue are fit to a nanoparticle ranges from about 2 hours to about 25 hours, decreasing monoexponential function to estimate tissue/or tumor residence half-time of the nanoparticle ranges from gan T12 values. about 5 hours to about 5 days, and renal clearance of the 0183. After administration of the present nanoparticle to a nanoparticle ranges from about 30% ID to about 80% ID in Subject, the renal clearance of the present nanoparticles may about 24 hours. range from about 45% ID (initial dose) to greater than 90% ID 0179. After administration of the present nanoparticle to a in about 24 hours, from about 20% ID to about 90% ID in Subject, the tumor residence half-time of the present nanopar about 24 hours, from about 30% ID to about 80% ID in about ticles may range from about 5 hours to about 5 days, from 24 hours, from about 40% ID to about 70% ID in about 24 about 10 hours to about 4 days, from about 15 hours to about hours, from about 40% ID to about 60% ID in about 24 hours, 3.5 days, from about 20 hours to about 3 days, from about 2.5 from about 40% ID to about 50% ID in about 24 hours, about days to about 3.1 days, from about 1 day to 3 days, or about 43% ID in about 24 hours, from about 10% ID to about 100% 73.5 hours. ID in about 24 hours, from about 40% ID to about 100% ID in 0180. The ratio of the tumor residence half-time to the about 24 hours, from about 80%ID to about 100% ID in about blood residence half-time of the nanoparticle may range from 24 hours, from about 90% ID to about 95% ID in about 24 about 2 to about 30, from about 3 to about 20, from about 4 to hours, from about 90% ID to about 100% ID in about 24 about 15, from about 4 to about 10, from about 10 to about 15, hours, or from about 80% ID to about 95% ID in about 24 or about 13. hours. Renal clearance may be evaluated as follows. The 0181. In one embodiment, the present invention provides a nanoparticles are first administered to a subject (e.g., a fluorescent silica-based nanoparticle comprising a silica mouse, a miniswine or a human). At various time points post based core comprising a fluorescent compound positioned administration, urine samples are taken to measure nanopar within the silica-based core; a silica shell Surrounding at least ticle concentrations through suitable methods. a portion of the core; an organic polymer attached to the 0184. In one embodiment, renal clearance (e.g., the frac nanoparticle; and a ligand attached to the nanoparticle, tion of nanoparticles excreted in the urine over time) is wherein the nanoparticle has a diameter between about 1 nm assayed as follows. A subject is administered with the present and about 15 nm. After administration of the PEGylated nanoparticles, and urine samples collected over a certain time (control) nanoparticle to a Subject, blood residence half-time period (e.g., 168 hours). Particle concentrations at each time of the nanoparticle may range from about 2 hours to about 25 point are determined using fluorometric analyses and a serial hours, or from about 2 hours to about 15 hours; tumor resi dilution calibration curve generated from background-cor dence half-time of the nanoparticle may range from about 5 rected fluorescence signal measurements of urine samples hours to about 2 days; and renal clearance of the nanoparticle mixed with known particle concentrations (% ID). Concen may range from about 30% ID to about 80% ID in about 24 tration values, along with estimates of average daily mouse hours. The number of ligands attached to the nanoparticle urine Volumes, are used to compute cumulative% ID/gurine US 2014/024821.0 A1 Sep. 4, 2014

excreted. In another embodiment, renal clearance of radiola tumor tissue contrast relative to normal muscle, compared beled nanoparticles is assayed by measuring urine specimen with decreased tumor tissue contrast relative to normal activities (counts per minute) over similar time intervals muscle for non-targeted nanoparticles (i.e., corresponding using, for example, Y-counting, and after nanoparticle admin nanoparticles not attached with ligands), Suggesting that the istration to compute cumulative urine excretion. targeted nanoparticles are tumor-selective. 0185. In a third embodiment, to assess cumulative fecal 0190. In another embodiment, the targeted and non-tar excretion, feces are collected in metabolic cages over similar geted nanoparticles both show efficient renal excretion over time intervals after administration of the nanoparticles and the same time period. Nearly half of the injected dose is specimen activities determined using a Y-counter. excreted over the first 24 hrs post-injection and about 72% by 0186. When the nanoparticles in the amount of about 100 96 hrs, Suggesting that the bulk of excretion occurred in the times of the human dose equivalent are administered to a first day post-injection. By contrast, fecal excretion profiles Subject, Substantially no anemia, weight loss, agitation, of the targeted nanoparticles indicate that, on average, 7% and increased respiration, GI disturbance, abnormal behavior, 15% of the injected dose is eliminated over 24 and 96 hrs, neurological dysfunction, abnormalities in hematology, respectively. abnormalities in clinical chemistries, drug-related lesions in 0191 The physicochemical and biological parameters of organ pathology, mortality, or combination thereof are the non-toxic nanoparticles, along with its multimodal imag observed in about 10 to about 14 days. ing capabilities (e.g., PET and optical imaging), expand the 0187. When the present nanoparticle contains at least one range of their potential biomedical applications. The applica attached ligand, the multivalency enhancement of the nano tions include (a) long-term monitoring: the extended blood particle (e.g., compared to the ligand alone) may range from circulation time and corresponding bioavailability of the about 1.5 fold to about 10 fold, from about 2 fold to about 8 nanoparticles highlight their versatility for both early and fold, from about 2 fold to about 6 fold, from about 2 fold to long-term monitoring of various stages of disease manage about 4 fold, or about 2 fold. ment (Such as diagnostic screening, pre-treatment evaluation, 0188 The nanoparticles of the present invention show therapeutic intervention, and post-treatment monitoring) unexpected in vitro and in vivo physicochemical and biologi without restrictions imposed by toxicity considerations; (b) cal parameters in view of the prior art. For example, the blood improved tumor penetration: the clearance properties of the residence half-time estimated for the ligand-attached nano targeted nanoparticles (e.g., their renal clearance is slower particles (e.g., about 5.5 hrs for cRGD-attached nanopar that of the molecular probes in the prior art) will be useful for ticles) is substantially longer than that of the corresponding various types of biological applications. For example, the ligand (e.g., about 13 minutes for cRGD). Montet et al. Mul nanoparticles would be particularly useful in cases of poorly tivalent effects of RGD peptides obtained by nanoparticle vascularized and relatively inaccessible solid tumors in which display. J Med Chem. 49, 6087-6093 (2006). Extended blood localization of agents is typically slow after systemic admin residence half-times may enhance probe bioavailability, istration; (c) multimodal imaging capabilities: these modali facilitate tumor targeting, and yield higher tumor uptake over ties can be combined at multiple scales (i.e., whole body to longer time periods. In one embodiment, the tumor residence cellular levels) for acquiring complementary, depth-sensitive half-time for the targeted nanoparticles (i.e., ligand-attached biological information. For example, in SLN mapping, deep nanoparticles) is about 13 times greater than blood residence nodes can be mapped by PET interms of their distribution and half-time, whereas the tumor residence half-time for the non number, while more precise and detailed localization of targeted nanoparticles (i.e., corresponding nanoparticles not Superficial nodes can be obtained by fluorescence imaging; attached with ligands) is only about 5 times greater than blood and (d) targeted therapeutics: longer clearance of the targeted residence half-time. This difference Suggests Substantially nanoparticles from tumor compared to that from blood may greater tumor tissue accumulation of the targeted nanopar be exploited for combined diagnostic/therapeutic applica ticles compared with the non-targeted nanoparticles. In cer tions, in which the nanoparticles can serve as a radiothera tain embodiments, given the number of ligands attached to the peutic or drug delivery vehicle. nanoparticle, the present nanoparticles show unexpected high-affinity binding (e.g., K. 0.51 nM and ICso 1.2 nM for Pharmaceutical Compositions cRGD-attached nanoparticle), multivalency enhancement 0.192 The present invention further provides a pharma (e.g., more than 2 fold enhancement for cRGD-attached ceutical composition comprising the present nanoparticle. nanoparticles compared to cRGD peptide alone), significant The pharmaceutical compositions of the invention may be differential tumor uptake (e.g., cRGD-attached PEG-nano administered orally in the form of a suitable pharmaceutical particles show about 3 to 4 fold increase in differential tumor unit dosage form. The pharmaceutical compositions of the uptake relative to the PEG-coated nanoparticles over 72 hrs invention may be prepared in many forms that include tablets, post-administration), and significant tumor contrast relative hard or soft gelatin capsules, aqueous solutions, Suspensions, to normal muscle (e.g., about 3 to 5 fold over 72 hrs post and liposomes and other slow-release formulations, such as administration) based on tumor-to-muscle uptake ratios. shaped polymeric gels. 0189 In one embodiment, three-fold activity-concentra 0193 Suitable modes of administration for the present tion increases were found for ligand-attached nanoparticles in nanoparticle or composition include, but are not limited to, integrin-expressing tumors over controls (e.g., ligand-at oral, intravenous, rectal, Sublingual, mucosal, nasal, oph tached nanoparticles in non-integrin expressing tumors, or thalmic, Subcutaneous, intramuscular, transdermal, intrader corresponding nanoparticles not attached with ligands in inte mal. Subdermal, peritumoral, spinal, intrathecal, intra-articu grin-expressing tumors) at the time of maximum tumor lar, intra-arterial, Sub-arachnoid, bronchial, and lymphatic uptake (about 4 hrs post-injection of the nanoparticles). In administration, and other dosage forms for systemic delivery addition, tumor-to-muscle uptake ratios for targeted nanopar of active ingredients. The present pharmaceutical composi ticles (i.e., ligand-attached nanoparticles) reveal enhanced tion may be administered by any method known in the art, US 2014/024821.0 A1 Sep. 4, 2014

including, without limitation, transdermal (passive via patch, 0200 Pharmaceutical compositions suitable for rectal gel, cream, ointment or iontophoretic); intravenous (bolus, administration wherein the carrier is a Solid are most prefer infusion); Subcutaneous (infusion, depot); transmucosal ably presented as unit dose Suppositories. Suitable carriers (buccal and Sublingual, e.g., orodispersible tablets, wafers, include cocoa butter and other materials commonly used in film, and effervescent formulations; conjunctival (eyedrops); the art, and the Suppositories may be conveniently formed by rectal (Suppository, enema)); or intradermal (bolus, infusion, admixture of the pharmaceutical composition with the Soft depot). The composition may be delivered topically. ened or melted carrier(s) followed by chilling and shaping in 0194 Oral liquid pharmaceutical compositions may be in molds. the form of, for example, aqueous or oily Suspensions, solu 0201 Pharmaceutical compositions suitable for vaginal tions, emulsions, syrups or elixirs, or may be presented as a administration may be presented as pessaries, tampons, dry product for constitution with water or other suitable creams, gels, pastes, foams or sprays containing, in addition vehicle before use. Such liquid pharmaceutical compositions to the nanoparticles and the therapeutic agent, Such carriers may contain conventional additives Such as Suspending are well known in the art. agents, emulsifying agents, non-aqueous vehicles (which 0202 For administration by inhalation, the pharmaceuti may include edible oils), or preservatives. cal compositions according to the invention are conveniently 0.195 The nanoparticle pharmaceutical compositions of delivered from an insufflator, nebulizer or a pressurized pack the invention may also be formulated for parenteral adminis or other convenient means of delivering an aerosol spray. tration (e.g., by injection, for example, bolus injection or Pressurized packs may comprise a suitable propellant Such as continuous infusion) and may be presented in unit dosage dichlorodifluoromethane, trichlorofluoromethane, dichlo form in ampules, pre-filled Syringes, Small Volume infusion rotetrafluoroethane, carbon dioxide or other suitable gas. In containers or multi-dose containers with an added preserva the case of a pressurized aerosol, the dosage unit may be tive. The pharmaceutical compositions may take Such forms determined by providing a valve to deliver a metered amount. as Suspensions, solutions, or emulsions in oily or aqueous 0203 Alternatively, for administration by inhalation or vehicles, and may contain formulating agents such as Sus insufflation, the pharmaceutical compositions of the inven pending, stabilizing and/or dispersing agents. Alternatively, tion may take the form of a dry powder composition, for the pharmaceutical compositions of the invention may be in example, a powder mix of the pharmaceutical composition powder form, obtained by aseptic isolation of sterile solid or and a suitable powder base Such as lactose or starch. The by lyophilization from solution, for constitution with a suit powder composition may be presented in unit dosage form in, able vehicle, e.g., sterile, pyrogen-free water, before use. for example, capsules or cartridges or, e.g., gelatin or blister 0196. For topical administration to the epidermis, the packs from which the powder may be administered with the pharmaceutical compositions may be formulated as oint aid of an inhalator or insufflator. ments, creams or lotions, or as the active ingredient of a 0204 For intra-nasal administration, the pharmaceutical transdermal patch. Suitable transdermal delivery systems are compositions of the invention may be administered via a disclosed, for example, in A. Fisher et al. (U.S. Pat. No. liquid spray, Such as via a plastic bottle atomizer. Typical of 4,788,603), or R. Bawa et al. (U.S. Pat. Nos. 4.931,279; these are the MistometerR) (isoproterenol inhaler-Wintrop) 4,668,506; and 4,713.224). Ointments and creams may, for and the MedihalerR) (isoproterenol inhaler Riker). example, beformulated with an aqueous or oily base with the 0205 Pharmaceutical compositions of the invention may addition of Suitable thickening and/or gelling agents. Lotions also contain other adjuvants such as flavorings, colorings, may be formulated with an aqueous or oily base and will in anti-microbial agents, or preservatives. general also contain one or more emulsifying agents, stabi 0206. It will be further appreciated that the amount of the lizing agents, dispersing agents, Suspending agents, thicken pharmaceutical compositions required for use in treatment ing agents, or coloring agents. The pharmaceutical composi will vary not only with the therapeutic agent selected but also tions can also be delivered via ionophoresis, e.g., as disclosed with the route of administration, the nature of the condition in U.S. Pat. Nos. 4,140,122; 4,383,529; or 4,051,842. being treated and the age and condition of the patient and will 0197) Pharmaceutical compositions suitable for topical be ultimately at the discretion of the attendant physician or administration in the mouth include unit dosage forms such as clinician. For evaluations of these factors, see J. F. Brien et al., lozenges comprising a pharmaceutical composition of the Europ. J. Clin. Pharmacol., 14, 133 (1978); and Physicians invention in a flavored base, usually Sucrose and acadia or Desk Reference, Charles E. Baker, Jr., Pub., Medical Eco tragacanth; pastilles comprising the pharmaceutical compo nomics Co., Oradell, N.J. (41st ed., 1987). Generally, the sition in an inert base such as gelatin and glycerin or Sucrose dosages of the therapeutic agent when used in combination and acacia; mucoadherent gels, and mouthwashes compris with the fluorescent nanoparticles of the present invention can ing the pharmaceutical composition in a suitable liquid car be lower than when the therapeutic agent is administered 1. alone or in conventional pharmaceutical dosage forms. The 0198 For topical administration to the eye, the pharma high specificity of the fluorescent nanoparticle for a target ceutical compositions can be administered as drops, gels (S. site, such as a receptor situated on a cells Surface, can provide Chrai et al., U.S. Pat. No. 4.255.415), gums (S. L. Lin et al., a relatively highly localized concentration of a therapeutic U.S. Pat. No. 4,136,177) or via a prolonged-release ocular agent, or alternatively, a Sustained release of a therapeutic insert (A. S. Michaels, U.S. Pat. No. 3,867,519 and H. M. agent over an extended time period. Haddad et al., U.S. Pat. No. 3,870,791). 0207. The present nanoparticles or compositions can be 0199 When desired, the above-described pharmaceutical administered to a subject. The Subject can be a mammal, compositions can be adapted to give Sustained release of a preferably a human. Mammals include, but are not limited to, therapeutic compound employed, e.g., by combination with murines, rats, rabbits, simians, bovines, Ovine, Swine, certain hydrophilic polymer matrices, e.g., comprising natu canines, feline, farm animals, sport animals, pets, equine, and ral gels, synthetic polymer gels or mixtures thereof. primates. US 2014/024821.0 A1 Sep. 4, 2014

Uses of Nanoparticles dictate a therapeutic or Surgical intervention, e.g., by deter 0208. The present invention further provides a method for mining whether a lesion is cancerous and should be removed detecting a component of a cell comprising the steps of: (a) or non-cancerous and left alone, or in Surgically staging a contacting the cell with a fluorescent silica-based nanopar disease, e.g., intraoperative lymph node staging, sentinel ticle comprising a silica-based core comprising a fluorescent lymph node (SLN) mapping, e.g., nerve-sparing procedures compound positioned within the silica-based core; a silica for preserving vital neural structures (intraparotid nerves). shell Surrounding at least a portion of the core; an organic 0215. The methods and compositions of the invention may polymer attached to the nanoparticle: from about 1 to about be used in metastatic disease detection, treatment response 25 ligands (from about 1 to about 20 ligands, or from about 1 monitoring, SLN mapping/targeting, nerve sparing proce to about 10 ligands, or other ranges; see discussions here dures, residual disease detection, targeted delivery of thera within) attached to the nanoparticle; and a contrast agent or a peutics (combined diagnostic/therapeutic platform), local chelate attached to the nanoparticle; and (b) monitoring the delivery of non-targeted, drug-bearing nanoparticles (cath binding of the nanoparticle to the cellor a cellular component eter delivery), blood-brain barrier therapeutics, treatment of (and/or its potential intracellular uptake) by at least one imag inflammatory/ischemic diseases (i.e., brain, heart, urinary ing technique. The imaging technique may be PET, SPECT, tract, bladder), combined treatment and sensing of disease CT, MRI, optical bioluminescence or fluorescence imaging, (e.g., Rationetric pH sensing, oxygen sensing), etc. and combinations thereof. 0216. The methods and compositions of the invention can 0209. The location of the cellular component can be also be used in the detection, characterization and/or deter detected and determined inside a metabolically active whole mination of the localization of a disease, especially early cell, in a whole cell lysate, in a permeabilized cell, in a fixed disease, the severity of a disease or a disease-associated con cell, or with a partially purified cell component in a cell-free dition, the staging of a disease, and/or monitoring a disease. environment. The amount and the duration of the contacting The presence, absence, or level of an emitted signal can be can depend, for example, on the diagnostic or therapeutic indicative of a disease state. The methods and compositions objectives of the treatment method, such as fluorescent or of the invention can also be used to monitor and/or guide antibody-mediated detection of upregulated signaling path various therapeutic interventions, such as Surgical and cath way intermediates (i.e., Akt, NF-KB), disease states or con eter-based procedures, and monitoring drug therapy, includ ditions, the delivery of a therapeutic agent, or both. The ing cell based therapies. The methods of the invention can amount and the duration of the contacting can also depend on also be used in prognosis of a disease or disease condition. the relative concentration of the fluorescent nanoparticle to Cellular subpopulations residing within or marginating the the target analyte, particle incubation time, and the state of the disease site, such as stem-like cells ("cancer stem cells) cell for treatment. and/or inflammatory/phagocytic cells may be identified and 0210. The present invention further provides a method for characterized using the methods and compositions of the targeting a tumor cell comprising administering to a cancer invention. With respect to each of the foregoing, examples of patient an effective amount of a fluorescent silica-based nano Such disease or disease conditions that can be detected or particle comprising a silica-based core comprising a fluores monitored (before, during or after therapy) include cancer cent compound positioned within the silica-based core; a (for example, melanoma, thyroid, colorectal, ovarian, lung, silica shell Surrounding at least a portion of the core; an breast, prostate, cervical, skin, brain, gastrointestinal, mouth, organic polymer attached to the nanoparticle; a ligand kidney, esophageal, bone cancer), that can be used to identify attached to the nanoparticle and capable of binding a tumor Subjects that have an increased Susceptibility for developing marker; and at least one therapeutic agent. cancer and/or malignancies, i.e., they are predisposed to 0211. The nanoparticle may be radiolabeled. The nanopar develop cancer and/or malignancies, inflammation (for ticle may be radiolabelled using any Suitable techniques. The example, inflammatory conditions induced by the presence of radiolabelling may be automated. In one embodiment, the cancerous lesions), cardiovascular disease (for example, ath nanoparticle is radiolabelled using the FASTlab radiochem erosclerosis and inflammatory conditions of blood vessels, istry synthesis platform (GE Healthcare). The synthesis ischemia, stroke, thrombosis), dermatologic disease (for parameters may be fine-tuned to achieve high reproducibility, example, Kaposi's Sarcoma, psoriasis), ophthalmic disease radiochemical yield/purity, labeling efficiency, and relatively (for example, macular degeneration, diabetic retinopathy), short synthesis times. New particle tracers may be accommo infectious disease (for example, bacterial, viral, fungal and dated on the same FASTlab module. parasitic infections, including Acquired Immunodeficiency 0212. The nanoparticle may be administered to the patient Syndrome), immunologic disease (for example, an autoim by, but not restricted to, the following routes: oral, intrave mune disorder, lymphoma, multiple Sclerosis, rheumatoid nous, nasal, Subcutaneous, local, intramuscular or transder arthritis, diabetes mellitus), central nervous system disease mal. (for example, a neurodegenerative disease, such as Parkin 0213. In certain embodiments, it may be desirable to use a son's disease or Alzheimer's disease), inherited diseases, mixture of two or more types of fluorescent nanoparticles metabolic diseases, environmental diseases (for example, having different properties to evaluate different tissue types. lead, mercury and radioactive poisoning, skin cancer), bone 0214. The methods and compositions of the invention can related disease (for example, osteoporosis, primary and meta be used to help a physician or Surgeon to identify and char static bone tumors, osteoarthritis) and a neurodegenerative acterize areas of disease, such as cancers and inflammatory/ disease. infectious processes, including, but not restricted to, cancers 0217. The methods and compositions of the invention, of the skin (melanoma), head & neck, prostate, brain, and therefore, can be used, for example, to determine the presence bowels, to distinguish diseased and normal tissue. Such as and/or localization of tumor and/or co-resident stem-like detecting tumor margins that are difficult to detect using an cells (“cancer stem cells'), the presence and/or localization of ordinary operating microscope, e.g., in brain Surgery, to help inflammatory cells, including the presence of activated mac US 2014/024821.0 A1 Sep. 4, 2014 20 rophages, for instance in peritumoral regions, the presence -0.5 kDa), per protocols in PCT/US2008/074894, to prevent and in localization of vascular disease including areas at risk opSonization and further enhance particle clearance while for acute occlusion (i.e., Vulnerable plaques) in coronary and maintaining a small hydrodynamic size. This treatment peripheral arteries, regions of expanding aneurysms, unstable decreased liver retention and resulted in increased renal fil plaque in carotid arteries, and ischemic areas. The methods tration into the bladder at 45 min post-injection by NIR fluo and compositions of the invention can also be used in identi rescence imaging (FIG.1c), with bladder fluorescence visible fication and evaluation of cell death, injury, apoptosis, necro out to 24 h. The probes were well tolerated, with no adverse sis, hypoxia and angiogenesis. PCT/US2006/049222. effects or animal deaths observed over the course of the study. 0218. The following examples are presented for the pur Serial co-registered PET-CT imaging 24-hr after injection of poses of illustration only and are not limiting the invention. 'I-labeled PEG coated nanoparticles (FIG. 1 d, upper row) Example 1 demonstrated a small amount of residual bladder activity, as well as activity overlying the liver/gastrointestinal tract (cen Preparation and Characterization of PEG-Coated ter), flanked by independently acquired microCT and micro Nanoparticles PET scans. Serial microPET images confirmed findings on 0219. Nanoparticles containing an NIR-emitting dye (Cy NIR fluorescence imaging. The half-time of blood residence 5) were synthesized and functionalized by PEGylation of the '''I-labeled PEGylated nanoparticles based on time according to well-established protocols as disclosed in PCT/ dependent changes in blood activity over a 96-hour period US2008/074894 and Stober et al. Controlled growth of was found to be 7.3 hours. For the '''I-labeled, RGD-bound monodispersed silica spheres in the micron size range. Col nanoparticles, the half-time of blood residence was found to loid Interface Sci. 1968; 26:62-69. Ohnishi et al. J. Mol. be 5.6 hours. Imaging 2005, 4:172-181. Cy5 malemide was reacted with a 0221 Based on these in vivo data, a more detailed biodis co-reactive organo silane compound, (3-Mercaptopropyl) tribution and clearance study of coated nanoparticles was tromethoxysilane to form a fluorescent silica precursor. This undertaken on two sets of PEGylated Cy5-containing par fluorescent silica precursor was co-condensed with tetraethy lorthosilicate to form a fluorescent silica based core. A PEG ticles to assess the effects of probe size on biodistribution. silane compound, with methoxy-terminated poly(ethylene Nanoparticles with hydrodynamic diameters of 3.3+0.06 and glycol) chains (PEG, ~0.5 kDa) Methoxy(Polyethyleneoxy) 6.0-0.1 nm, as measured by fluorescence correlation spec Propyl-Trimethoxysilane, was added to the fluorescent silica troscopy (FCS), were generated (FIG. 2a). Prior to in vivo based core to form a PEG coating on the core. PEG-coated studies, particle photophysical properties were investigated nanoparticles were dialyzed to physiological saline (0.15M to establish their performance levels versus free dye. Despite NaCl in H2O) through 3500 MWCO Snakeskin Dialysis the extremely small particle size, silica-encapsulated dye Membranes and sterile-filtered. All samples were optical den molecules exhibited photophysical enhancements over free sity-matched at their peak absorption wavelength (640 nm) dye that scaled with particle size, including significant prior to injection. Hydrodynamic size measurements were increases in brightness, as determined by absorption and achieved by Dynamic Light Scattering (DLS) and Fluores emission spectroscopy (FIG. 2b) and FCS (FIG. 2c). Com cence Correlation Spectroscopy (FCS). Briefly, particles dia pared to the free dye, the 3.3 and 6.0 nm diameter nanopar lyzed to water were measured on a Brookhaven Instruments ticles exhibited 2- and 3-fold increases in photobleaching Company 200SM static/DLS system using a HeNe laser half-life, respectively, when irradiated with a high power 635 (w-632.8 nm). Due to overlap of the dye absorption with the nm laser (FIG. 2d). Thus, these nanoparticle probes were excitation Source, 15-min integration times were used to found to be both brighter and more photostable than their free achieve acceptable signal-to-noise ratios. For FCS, particles dye counterparts. were dialyzed to water, diluted into 0.15M NaCl, and mea 0222. In addition to semiquantitative evaluation of in vivo sured on a Zeiss LSM 510 Confocor 2 FCS (HeNe 633 nm nanoparticle behavior from whole-body imaging, ex-vivo excitation). The instrument was calibrated for size prior to all analysis of tissue homogenates and fluids was performed measurements. Comparison of the relative brightness of using a fluorescence plate reader, which allowed calibrated PEGylated nanoparticles with free dye was determined from quantitation of variations observed in NIR fluorescence imag FCS curves, measured as count rate per molecule/particle. ing. Samples were grouped as "retained' (liver, kidney, lung, spleen homogenates, and blood) and "excreted' (urine) Example 2 Sources of particle fluorescence, were background-corrected and were converted to percent of the initial dose (% ID) per Renal Clearance of PEG Coated Nanoparticles animal based on calibration curves. Tissue analysis showed 0220 Fluorescent core-shell silica nanoparticles, having a minimal particle retention in major organs, with most of the hydrodynamic radius of about 3 nm, were synthesized. These fluorescence attributed to circulating blood (FIG. 3a). Net nanoparticles were found to be in the 6-10 nm diameterrange, particle retention, calculated as the sum of the “retained as shown by dynamic light scattering (DLS) results (FIG.1a). components, was fit with an exponential decay curve to deter In vivo whole-body NIR fluorescence imaging of bare (no mine the kinetics of excretion (FIG. 3b). Larger particles PEG coat) silica nanoparticles, on the order of 6-nm and exhibited a longer tissue half-life (t(3.3 nm)=190 min, 3.3-mm, in nude mice showed considerable renal clearance 45 t(6.0 nm)=350 min) and greater initial organ retention. min post-injection with a significant accumulation remaining After 48 h, the 6-mm particle exhibited minimal retention in in the liver (FIG. 1b). Eventual excretion into the enterohe the body (R(6.0 nm)=2.4+0.6% ID). Urine samples col patic circulation occurred during the ensuing 24 h. On the lected at the time of sacrifice, in conjunction with serial basis of these results, particles were covalently coated with dilution calibration data, was used to estimate the total renal methoxy-terminated poly(ethylene glycol) chains (PEG, clearance based on a conservative estimate of the average US 2014/024821.0 A1 Sep. 4, 2014

urine volume excreted per unit time. By this method, the % ID Materials and Methods excreted over time for both particle sizes (FIG. 3c) was esti mated. Synthesis of cRGDY-PEG-Nanoparticles and PEG-Nanoparticles Example 3 0227 Particles were prepared by a modified Stober-type Fluorescent Silica Nanoparticles Conjugated with silica condensation as described previously. Wiesner et al., CfB Integrin-Targeting Peptide (Melanoma Model) PEG-coated Core-shell Silica Nanoparticles and Mathods of Manufactire and Use, PCT/US2008/74894. Larson, et al., 0223) To synthesize a multimodal (optical-PET) nanopar Silica nanoparticle architecture determines radiative proper ticle with high affinity for tumor marker C. B. integrin, cyclic ties of encapsulated chromophores. Chem. Mater. 20, 2677 RGD pentapeptide (RGDYC) was conjugated to the nanopar 2684 (2008). Bogush, et al., Preparation of Monodisperse ticle via a Cys-maleimide linkage. The tyrosine linker, Y, was Silica Particles: Control of Size and Mass Fraction. J. Non used to Subsequently attach a radiolabel. Male athymic nude Cryst. Solids, 104,95-106 (1988). Sadasivan, et al., Alcoholic mice were injected subcutaneously into their flanks with C6 Solvent Effect on Silica Synthesis NMR and DLS Investi rat glioma cells. At -0.5 cm in diameter, mice were IV gation. J. Sol-Gel Sci. Technol. 12, 5-14 (1998). Herz, et al., injected with either bare silica nanoparticles (FIG. 4A) or Large Stokes-Shift Fluorescent Silica Nanoparticles with PEG-ylated RGD nanoparticles (FIG.4B, -500 nm/kg). FIG. Enhanced Emission over Free Dye for Single Excitation Mul 4 shows the in vivo biodistribution in non-tumor-bearing and tiplexing. Macromol Rapid Commun. 30, 1907-1910 (2009). tumor-bearing mice using whole body optical imaging. Tyrosine residues were conjugated to PEG chains for attach 0224. In vitro binding of targeted (RGD-bound) and non ment of radioiodine or stable iodine moieties. Hermanson, targeted (PEG-coated) nanoparticles to Clf-integrin-posi Bioconjugate Techniques, (Academic Press, London, ed. 2, tive human melanoma cell lines (M21) was investigated as 2008). All samples were optical density-matched at their peak part of a dose response study using flow cytometry (FIGS. absorption wavelength (640 nm) prior to radiolabeling. 5A, 5B). Particle binding/uptake was evaluated as a function cRGD peptides were attached to functionalized PEG chains of time (FIG. 5A) and concentration (FIG. 5B). via a cysteine-maleimide linkage, and the number of cFGD peptides bound to the particle was estimated using FCS-based Example 4 measurements of absolute particle concentrations and the starting concentration of the reagents for cRGD peptide. Fluorescent Silica Nanoparticles Conjugated with CfB Integrin-Targeting Peptide and Nodal Mapping Hydrodynamic Size and Relative Brightness Comparison (Melanoma Model) Measurements by Fluorescence Correlation Spectroscopy 0225. We utilized a biocompatible material, silica, which (FCS) has an architecture that could be precisely tuned to particle 0228 Particles dialyzed to water were diluted into physi sizes optimized for renal clearance. We attached Small target ological saline (0.15 M. NaCl in HO) and measured on a ing peptides and a radioactive label to the particle Surface for Zeiss LSM 510 Confocor 2 FCS using HeNe 633-nm excita serial PET imaging measurements in a well-characterized in tion. The instrument was calibrated for size prior to all mea Vivo human melanoma model, and mapped draining lymph surements. Diffusion time differences were used to evaluate nodes and lymphatic channels using an encapsulated near variations in the hydrodynamic sizes of the dye and particle infrared (NIR) dye and multi-scale optical fluorescence imag species. Relative brightness comparisons of the free dye and ing methods. Ballou et al., Sentinel lymph node imaging the PEG- and the RGDY-PEG nanoparticles were performed using quantum dots in mouse tumor models. Bioconjugate using count rates per molecule/particle. Chem. 18, 389-396 (2007). Kim et al., Near-infrared fluores cent type II quantum dots for sentinel lymph node mapping. Radiolabeling of C Dot Conjugates Nat. Biotechnol. 22, 93-97 (2003). Tanaka et al. Image guided oncologic Surgery using invisible light: completed 0229 Radiolabeling of the cRGDY-PEG and PEG-nano pre-clinical development for sentinel lymph node mapping. J particles was performed using the IODOGEN method Surg Oncol. 13, 1671-1681 (2006). Toxicity testing was also (Pierce, Rockford, Ill.). Piatyszek, et al., Iodo-gen mediated performed and human normal-organ radiation doses derived. radioiodination of nucleic acids. J. Anal. Biochem. 172,356 Specifically, we synthesized about 7 nm diameter, near-infra 359 (1988). Activities were measured by gamma (Y)-counting red (NIR) dye-encapsulating core-shell silica nanoparticles, and fluorescence measured using a Varian fluorometer (exci coated with PEG chains and surface-functionalized with a tation 650 nmfemission 680). Small number (about 6-7) of targeting peptides and radiola bels. Cells and Cell Culture 0226 We demonstrate that these probes simultaneously are non-toxic, exhibit high-affinity/avidity binding, efficient 0230 Human melanoma M21 and M21 variant (M21-L, excretion, and significant differential uptake and contrast C. negative) cell lines were maintained in RPMI 1640 media/ between tumor and normal tissues using multimodal molecu 10% fetal BSA, 2 mM L-glutamine penicillin, and strepto lar imaging approaches. The sensitive detection, localization, mycin (Core Media Preparation Facility, Memorial Sloan and interrogation of lymph nodes and lymphatic channels, Kettering Cancer Center, New York). Human umbilical enabled by the NIR dye fluorescence, highlights the distinct venous cord endothelial cells (HUVECs) were cultured in potential advantage of this multimodal platform for detecting M199 media/10% fetal bovine serum, 20 ug/ml endothelial and staging metastatic disease in the clinical setting, while cell growth factor, 50 ug/ml heparin, penicillin and strepto extending the lower range of nodal sizes that can be detected. mycin. US 2014/024821.0 A1 Sep. 4, 2014 22

In Vitro Cell-Binding and Molecular Specificity of animal welfare. Male athymic nu/nu mice (6-8 weeks old, '*'I-cRGD-PEG-Nanoparticles Taconic Farms Inc, Hudson, N.Y.) were provided with water containing potassium iodide solution to block uptake by the 0231. To assay particle binding and specificity for M21 thyroid gland of any free radioiodine in vivo, and maintained cells, 24-well plates were coated with 10 ug/ml collagen type on a Harlan Teklad Global Diet 2016, ad libitum, as detailed I (BD Biosciences, Bedford, Mass.) in phosphate buffered elsewhere 10. To generate M21 or M21L xenografts, equal saline (PBS) and incubated (37° C., 30 min). M21 cells (3.0- volumes of cells (-5x10/100 ul) and matrigel were co-in 4.0x105 cells/well) were grown to confluency and washed jected Subcutaneously into the hindleg in different mice. with RPMI 1640 media/0.5% bovine serum albumin (BSA). '''I-cRGD-PEG-nanoparticles (0-40 ng/ml) were added to Tumor sizes were regularly measured with calipers, yielding wells and cells incubated (25° C., 4 hours), washed with average tumor volumes of 200 mm. RPMI 1640 media/0.5% BSA, and dissolved in 0.2 MNaOH. Radioactivity was assayed using a 1480 Automatic Gamma In Vivo Pharmacokinetic and Residence Half-Time (T) Counter (Perkin Elmer) calibrated for iodine-124. Nonspe Measurements cific binding was determined in the presence of a 1000-fold 0234 Time-dependent activity concentrations (% ID/g), excess of crGD (Peptides International, Louisville, Ky.). corrected for radioactive decay to the time of injection, were Scatchard plots of the binding data were generated and ana measured by sacrificing groups of mice at specified times lyzed using linear regression analyses (Microsoft Excel following i.v. injection of ''I-cRGDY-PEG-nanoparticles or 2007) to derive receptor-binding parameters (Kd, Bmax, 'I-PEG-nanoparticles (~20 uCi/mouse) and harvesting, IC50). weighing, and counting blood, tumor, and organs in a scintil lation y-counter calibrated for '''I. The resulting time-activ In Vitro Cell-Binding Studies Using Optical Detection ity concentration data for each tissue were fit to a decreasing Methods monoexponential function to determine the values of T and 0232. Maximum differential binding of cRGDY-PEG A, the tissue/organ residence halftime and Zero-time inter nanoparticles and PEG-nanoparticles to M21 cells was deter cept, respectively, of the function. mined for a range of incubation times and particle concentra 0235. The fraction of particles excreted in the urine over tions using flow cytometry, with optimum values used in time was estimated using previously described methods. competitive binding and specificity studies. Cells (3.0x10 Burns, et al., Fluorescent Silica Nanoparticles with Efficient cells/well) were washed with RPMI 1640 media/0.5% BSA, Urinary Excretion for Nanomedicine, Nano Letters 9,442-8 detached using 0.25% trypsin/EDTA, and pelleted in a micro (2009). Briefly, mice were injected i.v. with either 200 ul centrifuge tube (5 min at 1400 rpm, 25°C.). Pellets were unlabeled cRGDY-PEG-nanoparticles or PEG-nanoparticles, resuspended in BD FACSFlow solution (BD Biosciences, and urine samples collected over a 168-hr period (n=3 mice San Jose, Calif.) and analyzed in the Cy5 channel to deter per time point). Particle concentrations at each time point mine the percentage of particle-bound probe (FACSCalibur, were determined using fluorometric analyses and a serial Becton Dickinson, Mountain View, Calif.). Competitive dilution calibration curve generated from background-cor binding studies were additionally performed following incu rected fluorescence signal measurements of urine samples bation of cFGDY-PEG-nanoparticles (2.5 ng/ml) with M21, mixed with known particle concentrations (% ID). Concen M21L, and HUVEC cells in the presence of excess cKGD tration values, along with estimates of average daily mouse and/or mouse monoclonal anti-human integrin CfB fluores urine volumes, were then used to compute the cumulative% cein-conjugated antibody (Millipore, Temecula, Calif.) and ID/g urine excreted over time. To assess cumulative fecal analyzed by flow cytometry. To assess potency of the RGDY excretion, metabolic cages were used to collect feces over a PEG nanoparticles relative to the cKGD peptide, anti-adhe similar time interval after i.v. injection of 200ul 'I-cRGDY sion assays were performed. Ninety-six-well microtiter PEG-nanoparticles (n=4 mice per time point). Specimen plates were coated with vitronectin in PBS (5 g/ml), fol activities were measured usingay-countercalibrated for '''I. lowed by 200 ul of RPMI/0.5% BSA (1 h, 37° C). Cells (3x10/100ul/well) were incubated in quadruplicate (30 min, Dosimetry 25°C.) with various concentrations of crGDY-PEG-nano 0236 Time-activity functions derived for each tissue were particles or cRGD peptide in RPMI/0.1% BSA, and added to analytically integrated (with inclusion of the effect of radio vitronectin-coated plates (30 min, 37°C.). Wells were gently active decay) to yield the corresponding cumulative activity rinsed with RPMI/0.1% BSA to remove non-adherent cells; (i.e. the total number of radioactive decays). ''Imouse organ adherent cells were fixed with 4% PFA (20 min, 25°C.) and absorbed doses were then calculated by multiplying the stained with methylene blue (1 h, 37°C.) for determination of cumulative activity by the '"I equilibrium dose constant for optical densities, measured using a Tecan Safire plate reader non-penetrating radiations (positrons), assuming complete (Wex=650 nm, Wem=680 nm, 12 nm bandwidth). The multi local absorption of such radiations and ignoring the contribu Valent enhancement factor was computed as the ratio of the tion of penetrating radiations (i.e., Y-rays). Eckerman, et al., cRGD peptide to cRGDY-PEG-dot IC50 values. Montet, et Radionuclide Data and Decay Schemes, 2nd ed. Reston, Va.: al., Multivalent effects of RGD peptides obtained by nano Society of Nuclear Medicine; 1989. The mouse normal-organ particle display. J Med Chem. 49, 6087-6093 (2006). cumulated activities were converted to human normal-organ Animal Models and Tumor Innoculation cumulated activities by adjustment for the differences in total-body and organ masses between mice and humans (70 0233 All animal experiments were done in accordance kg Standard Man). Cristy, et al., Specific absorbed fractions with protocols approved by the Institutional Animal Care and of energy at various ages from internal photon Sources Use Committee of Memorial Sloan-Kettering Cancer Center (I-VII). Oak Ridge National Laboratory Report ORNL/TM and followed National Institutes of Health guidelines for 8381/V1-7. Springfield, Va.; National Technical Information US 2014/024821.0 A1 Sep. 4, 2014

Service, Dept of Commerce: 1987. The human normal-organ with 650+20 nm NIR excitation and 710-nm long-pass emis cumulated activities calculated were entered into the sion filters. Whole-body optical images (Cambridge OLINDA dosimetry computer program to calculate, using the Research Instruments Maestro imager) were additionally formalism of the Medical Internal Dosimetry (MIRD) Com acquired and spectrally deconvolved as reported previously. mittee of the Society of Nuclear Medicine, the Standard-Man Burns, et al., Fluorescent Silica Nanoparticles with Efficient organ absorbed doses. Loevinger, et al., MIRD Primer for Urinary Excretion for Nanomedicine, Nano Letters 9,442-8 Absorbed Dose Calculations (Society of Nuclear Medicine, (2009). New York, 1991). Stabin, et al., OLINDA/EXM: the second generation personal computer Software for internal dose Statistical Analysis assessment in nuclear medicine. J Nucl Med. 46, 1023-1027 0240 Statistical analyses comparing groups of tumor (2005). mice receiving targeted/non-targeted probes or bearing M21/ Acute Toxicity Studies and Histopathology M21L tumors, were performed using a one-tail Mann-Whit ney U test, with P-0.05 considered statistically significant. 0237 Acute toxicity testing was performed in six groups For biodistribution studies, the tissue-specific mean % ID/g of male and female B6D2F1 mice (7 wks old, Jackson Labo values of ''I-cRGDY-PEG-(n=7 mice) and 'I-PEG-nano ratories, Bar Harbor, Me...). The treatment group (n=6 males, particles (control, n=5 mice) were compared at each time n=6 females) received unlabeled targeted probe (I-RGDY point, with statistically significant differences intracer activi PEG-nanoparticles) and the control group (n=6 males, n=6 ties observed in blood, tumor, and major organs at 4 and 96 hrs females) unlabeled iodinated PEG-nanoparticles (vehicle, p.i., as well as at 24hrs p.i. for tumor and other tissues (Table '*'I-RGDY-PEG-nanoparticles) in a single i.v. injection (200 1). For tumor targeting studies, differences in mean '% ID/g ul). Untreated controls (n=2 males, n=2 females) were addi values between M21 (n=7) and M21L tumor mice (n=5), as tionally tested. Mice were observed daily over 14 days p.i. for well as mice receiving control probes (n=5), were found to be signs of morbidity/mortality and weight changes, and gross maximal at 4 hrs p.i. (p=0.0015 for both controls), remaining necropsy, histopathology, and blood sampling for hematol significantly elevated at 24 hrs (p=0.0015 and p=0.004, ogy and serum chemistry evaluation was performed at 7- and respectively), 48 hrs (p=0.001 and p=0.003, respectively), 72 14-days p.i (FIG. 10 and Table 3). hrs (p=0.015 and 0.005, respectively), and 96 hrs (p=0.005 for M21-M21L). Tumor-to-muscle ratios for 'I-cRGDY Serial PET Imaging of Tumor-Specific Targeting PEG-nanoparticles (n=7) versus '''I-PEG-nanoparticles 0238 Imaging was performed using a dedicated Small (n=5) were found to be statistically significant at 24 hrs p.i. animal PET scanner (Focus 120 microPET: Concorde Micro (p=0.001) and 72 hrs p. i. (p=0.006), but not at 4 hrs p.i. systems, Nashville,Tenn.). Mice bearing M21 or M21L hind (p=0.35). Goodness of fit values (R2), along with their asso leg tumors were maintained under 2% isoflurane anesthesia ciated p values, were determined for the urine calibration in oxygen at 2L/min during the entire scanning period. One curve (R2=0.973, p=0.01), as well as for the urine (R2>0.95, hour list-mode acquisitions were initiated at the time of i.v. p=0.047) and fecal (R2>0.995, p<0.002) cumulative % ID injection of 200 uCi of ''I-cRGDY-PEG-nanoparticles or excretion curves using non-linear regression analyses (Sig '*'I-PEG-nanoparticles in all mice, followed by serial 30 min maPlot, Systat, v. 11.0). static images over a 96-hour interval. Image data were cor rected for non-uniformity of the Scanner response, dead time Results count losses, random counts, and physical decay to the time of injection. Voxel count rates in the reconstructed images were Nanoparticle Design and Characterization converted to activity concentrations (% ID/g) by use of a measured system calibration factor. Three-dimensional 0241 Cy5 dye encapsulating core-shell silica nanopar region-of-interest (ROI) analysis of the reconstructed images ticles (emission maxima >650 nm), coated with methoxy was performed by use of ASIProsoftware (Concorde Micro terminated polyethylene glycol (PEG) chains (PEG -0.5 systems, Nashville, Tenn.) to determine the mean, maximum, kDa), were prepared according to previously published pro and SD of probe uptake in the tumors. Tumor-to-muscle tocols. Burns, et al., Fluorescent Silica Nanoparticles with activity concentration ratios were derived by dividing the Efficient Urinary Excretion for Nanomedicine, Nano Letters, image-derived tumor % ID/g values by the y-counter muscle 9, 442-8 (2009). Ow, et al., Bright and stable core-shell fluo rescent silica nanoparticles. Nano Lett. 5, 113-117 (2005). % ID/g values. The neutral PEG coating prevented non-specific uptake by the reticuloendothelial system (opsonization). The use of Nodal Mapping Using Combined NIR Fluorescence Imaging bifunctional PEGs enabled attachment of small numbers and Microscopy (-6-7 per particle) of O?3 integrin-targeting cyclic arginine 0239 Nude mice bearing hindleg tumors were injected by glycine-aspartic acid (cRGDY) peptide ligands to maintain a 4-quadrant, peritumoral administration using equal Volumes Small hydrodynamic size facilitating efficient renal clearance. of a 50-ul cRGDY-PEG-dot sample and allowed to perambu Peptide ligands were additionally labeled with 'I through late freely. Following a 30 min to 1-hr interval, mice were the use of a tyrosine linker to provide a signal which can be anesthetized with a 2% isofluorine/98% oxygen mixture, and quantitatively imaged in three dimensions by PET ('I- a Superficial paramidline incision was made vertically along cRGDY-PEG-dots, FIG. 6A); an important practical advan the Ventral aspect of the mouse to Surgically expose the region tage of relatively long-lived 'I (physical half-life: 4.2 d) is from the hindlimb to the axilla ipsilateral to the tumor. In situ that sufficient signal persists long enough to allow radiode optical imaging of locoregional nodes (i.e., inguinal, axillary) tection up to at least several days postadministration, when and draining lymphatics (including axillary region) was per background activity has largely cleared and tumor-to-back formed using a macroscopic fluorescence microscope fitted ground contrast is maximized. Purity of the radiolabeled tar US 2014/024821.0 A1 Sep. 4, 2014 24 geted nanoparticle was >95% by radio thin layer chromatog shown). Montet, et al., Multivalent effects of RGD peptides raphy. Stability of the non-radiolabeled targeted nanoparticle obtained by nanoparticle display. J Med Chem. 49, 6087 is about 1 year by FCS measurements. Particle is excreted 6093 (2006). Li, et al., "Cu-labeled tetrameric and octomeric intact in the urine by FCS analyses. As used herein, “dot' and RGD peptides for small-animal PET of tumor O?3 integrin “nanoparticle' are used interchangeably. A PEG-coated par expression. J. Nucl Med. 48, 1162-1171 (2007). Similar to ticle containing a tyrosine residue for '''I labeling served as M21 cells, excess antibody effectively blockedcRGDY-PEG the control probe ('''I-PEG-dots). Purification of the radio dot receptor binding to HUVEC cells by flow cytometry (FIG. labeled samples by size exclusion chromatography (FIG. 7) resulted in radiochemical yields of >95%. Hydrodynamic 8D). diameters of ~7 nm i.d. were measured for non-radioactive cRGDY-PEG-dots and PEG-dots by fluorescence correlation Biodistribution and Clearance Studies spectroscopy (FCS) (FIGS. 6B and 6C). The relative bright ness of the cRGDY-PEG-dots was determined, on average, to 0243 The time-dependent biodistribution, as well as renal be 200% greater than that of the free dye (FIG. 6C), consistent and hepatobiliary clearance were evaluated by intravenously with earlier results. Burns, et al., Fluorescent Silica Nanopar administering tracerdoses (-0.2 nanomoles) of ''I-cRGDY ticles with Efficient Urinary Excretion for Nanomedicine, PEGdots and 'I-PEG-dots to M21 tumor xenograft mouse Nano Letters, 9,442-8 (2009). Larson, et al., Silica nanopar models (FIG. 9). Although tissue activity-concentrations ticle architecture determines radiative properties of encapsu (percent of the injected dose per gram (% ID/g)) for the lated chromophores. Chem. Mater 20, 2677-2684 (2008). targeted probe were measured over a 196-hr post-injection Based on these physicochemical properties, we anticipated (p.i.) time interval, comparison of the '''I-cRGDY-PEGdot achieving a favorable balance between selective tumor uptake (FIG.9A) and 'I-PEG-dot tracers (FIG. 9B) was restricted and retention versus renal clearance of the targeted particle, to a 96-hr window, as data for the latter was not acquired at 1 thus maximizing target-tissue localization while minimizing week. Statistically significant (p<0.05) differences in tracer normal-tissue toxicity and radiation doses. activities were observed for blood, tumor, and major organs at 4 and 96 hrs p. i., as well as at 24 hrs p.i. for the tumor and In Vitro Receptor Binding Studies several other tissues (Table 1). The targeted probe was almost 0242 To examine in vitro binding affinity and specificity entirely eliminated from the carcass at 1 week p.i (~3% ID). of ''I-cRGDY-PEG-dots and 'I-PEGdots to tumor and The residence halftimes (T2) for blood, tumor, and major vascular endothelial surfaces, C.B. integrin-overexpressing organs for these tracers are shown in Table 2 (columns 2 and (M21) and nonexpressing (M21L) melanoma and human 5). A representative data set (blood residence) is shown in the umbilical vein endothelial (HUVECs) cell lines were used. inset of FIG.9A. A relatively long blood T value of 7.3+1.2 Highly specific linear and saturable binding of the cFGDY hrs was determined for the '''I-PEG-dot. Upon attachment of PEG-dots was observed over a range of particle concentra the crGDY peptide to synthesize the '''I-cRGDY-PEG-dot, tions (0 to 8 ng/ml) and incubation times (up to 5-hrs), with the T value decreased slightly to 5.6+0.15 hrs, but was maximum differential binding at 4-hr and ~2.0 ng/ml particle accompanied by greater probe bioavailability (Table 2, col concentration (data not shown) using flow cytometry. Recep tor-binding specificity of ''I-cRGDY-PEG dots was tested umn 3). The tumor T value for the 'I-cRGDY-PEG-dot using Y-counting methods after initially incubating M21 cells was found to be about 13 times greater than that for blood, with excess non-radiolabeled cRGD and then adding various versus only a 5-fold difference for the '''I-PEG-dot (Table 2, concentrations of the radiolabeled targeted probe (FIG. 8A). columns 2 and 5). Scatchard analysis of the binding data yielded a dissociation equilibrium constant, Kd, of 0.51 nM (FIG. 8A, inset) and TABLE 1 receptor concentration, Bmax, of 2.5 pM. Based on the Bmax value, the CfB integrin receptor density was estimated to be Biodistribution study p-values comparing 10x10 per M21 cell, in reasonable agreement with the pre 2I-cRGDY-PEG- and 2 I-PEG-dotsf viously published estimate of 5.6x10" for this cell line. Cress man, et al., Binding and uptake of RGD-containing ligands to Post-injection times (hours) cellular O?3 integrins. Int J Pept Res Ther: 15, 49-59 (2009). Incremental increases in integrin-specific M21 cellular Tissue 4 24 96 uptake were also observed over a temperature range of 4 to 37° C. Suggesting that receptor-mediated cellular internal Blood O.OO1 O. 113 O.O10 ization contributed to overall uptake (data not shown). Addi Tumor O.O45 O.O12 O.OO1 tional competitive binding studies using the targeted probe Heart O.O19 O.231 O.OO1 showed complete blocking of receptor-mediated binding Lungs O.O39 O.OO6 with anti-CfB integrin antibody (FIG.8B) by flow cytometry. Liver O.OO1 O.O33 O.O28 No significant reduction was seen in the magnitude of recep tor binding (-10% of M21) with M21L cells (FIG. 8C) using Spleen O.OO1 O.208 O.OO1 either excess cRGDY or anti-CB integrin antibody. These Small Intestine O.OO1 O.046 O.OO2 results were confirmed by additional y-counting studies, and Large Intestine O.OO1 0.137 O.OO3 a 50% binding inhibition concentration, IC50, of 1.2 nM was Kidneys O.356 O.OO1 determined for the '''I-cRGDY-PEG-dot. An associated mul Muscle O.OO1 O.007 O.OO1 tivalent enhancement factor of greater than 2.0 was found for Brain O.OO1 O.074 O.OO1 the cRGDY-PEG-dot relative to the monomeric cFGD pep tide using an anti-adhesion assay and M21 cells (data not US 2014/024821.0 A1 Sep. 4, 2014

TABLE 2

Mouse Humanif 12'I-RGDYPEG 124I-PEG 124IRGDYPEG 124I-PEG T1/2 A. Absorbed Dose T. A. Absorbed Dose Absorbed Dose Target Organ (h) (% ID/g) (radimCi) (h) (% ID/g) (radimCi) (radimCi) Blood 5.9 18.8 626 7.3 4.7 189 (see red marrow below) Heart 6.8 7.0 266 34.1 O.8 120 0.307 (Wall) O.087 Lungs 8.5 5.7 267 37.7 3.0 498 O.298 O.263 Liver 65.9 3.9 935 52.5 1.4 294 O.486 O-234 Spleen 423 45.6 1071 27.4 45.7 410 3.20 O.254 195. 286 Small Intestine 30.3 1.8 251 13.2 O.9 61 O.304 O.115 Large Intestine 23.9 2.0 228 49.2 O.S 99 0.427 (U) O.209 0.724 (L) O416 Kidneys 66.O 3.0 712 33.0 2.0 388 2.50 O.32O Muscle 27.7 O.8 105 47.1 O.2 38 O.227 O.O60 13.9 0.4 29 8.5 O.2 8 O.187 O.149 73.5 1.5 380 37.0 O.9 146 na na (see osteogenic cells) O.400 O.083 Breasts O.141 O.042 Gallbladder Wall O.289 O.097 Stomach Wall O.26S O.06S Ovaries O.303 O.124 Pancreas O.389 O.O81 Red Marrow 1.07 O.O84 Osteogenic Cells O.2O3 O.127 Skin O.158 O.O38 Testes O.186 O.O73 Thymus O.173 O.OS2 Thyroid O.188 O.043 Urinary Bladder Wall 2.01 1.65 Uterus O.333 O.171 Total Body O.O34 0.075 Effective Dose Equivalent (remimCi) O.863 O.256 Effective Dose (remimCi) O.S99 O.232 70-kg Standard Man, U (upper), L (lower), mouse melanoma model, bone activity much lower than other tissues (not reported)

0244. By appropriate mass-adjusted translation of the foregoing biodistribution data to man, human normal-organ radiation doses were derived and found to be comparable to those of other commonly used diagnostic radiotracers (Table 2, columns 8, 9). Along with the finding that the targeted probe was non-toxic and resulted in no tissue-specific patho logic effects (i.e., no acute toxicity) (FIG. 10 and Table 3). first-in-man targeted and nontargeted molecular imaging applications with these agents are planned. TABLE 3 Organ Histopathology for 'I-RGDY-PEG-DOTS vs - I-PEG-DOTS Treatment

UNTREATED 27I-PEG-DOTS 27I-RGDY-PEG-DOTS Sex

M F F F F

Heart N N Thymus N N Trachea N N Lungs N N Kidneys N N Liver N N Random cellular clusters Gallbladder NP N NP Pancreas N N N Chronic lymph N US 2014/024821.0 A1 Sep. 4, 2014 26

TABLE 3-continued Organ Histopathology for T-RGDY-PEG-DOTS vs T-PEG-DOTS Treatment UNTREATED 127I-PEG-DOTS 127I-RGDYPEG-DOTS Sex

M F M. M. F F M M F F Spleen N N N N N N N Salivary gland N N N N N N N Esophagus N N N N N N N Stomach N N N N N N N Small intestine N N N N N N N Follic lymph. hyperplasia Large intestine Mesenteric lymph node Submandibular lymph S. Adrenals Thyroid Testes Epididymides Seminal vesicles Coagulating glands Prostate Ovary Uterus Cervix Mammary gland Urinary bladder Bones joint Bone marrow Spinal cord Brain Pituitary P Skin Subcut. inflammation Skeletal muscle Peripheral nerves N N: normal, U; unavailable, NP: not present, 1: minimal. 2: mild F: focal, MF; multifocal 0245. In another study to confirm that '''I-RGD-PEG dots 1x10 moles/mouse, a dose equivalent to an excess of 100 are non-toxic after intravenous administration in mice, formal times the PEGylated RGD silica nanoparticles dose required single dose toxicity testing was performed over the course of for Phase 0 imaging studies, is safe and nontoxic in B6D2F1 2 weeks using '7I-RGD-PEG dots at about 100 times of the mice. human dose equivalent. ''I-PEG dots served as the control 0246. Efficient renal excretion was found for the ~7-mm particle. In Summary, the procedure was as follows. Twenty diameter targeted and non-targeted probes over a 168-hr time eight, 8 week old B6D2F1 mice were used in the acute tox period by fluorometric analyses of urine samples. Fluores icity study and were divided into a treatment and control cence signals were background-corrected and converted to group. The treatment group (n-6 males+6 females) received particle concentrations (% ID/ul) based on a serial dilution one dose '''I-PEGylated RGD silica nanoparticles at a dose calibration scheme (FIG. 9C, inset; Table 4, column 2). of 1x10 moles/mouse intravenously, and the control group Burns, et al., Fluorescent Silica Nanoparticles with Efficient (n-6 males+6 females) received the same amount of vehicle. Urinary Excretion for Nanomedicine, Nano Letters, 9,442-8 Two mice/group (one male and one female/group) were sac (2009). Concentration values, along with age-dependent con rificed on day 7 post dose and clinical chemistry, hematology servative estimates of the average urine excretion rate, per and tissue specific histopathology were done at autopsy. All mitted the cumulative% ID excreted to be computed (Table 4. remaining animals (n=5 males+5 females/group) were column 4). Drickamer, Rates of urine excretion by house observed for 14 days following treatment. Four untreated mouse (mus domesticus): differences by age, sex, Social sta mice (two males and two females) were used as reference. tus, and reproductive condition. J. Chem. Ecol. 21, 1481 The conclusion of the studies was that no adverse events were 1493 (1995). Nearly half of the injected dose (about 43% ID) observed during dosing or the following 14-days observation was observed to be excreted over the first 24hrs p.i.and ~72% period. No mortality or morbidity was observed. Clinical ID by 96 hrs, FIG. 9C), suggesting that the bulk of excretion observations included the absence of the following: anemia, has occurred in the first day p.i. No significant particle fluo weight loss, agitation, increased respiration, GI disturbance, rescence in urine could be detected 168 hrs p.i. Fecal excre abnormal behavior, neurological dysfunction, abnormalities tion profiles of the '''I-cRGDY-PEG-dot indicated that, on in hematology, abnormalities in clinical chemistries, or drug average, 7% ID and 15% ID of the injected dose was elimi related lesions in terms of organ pathology. Thus, a single nated over 24 and 96 hrs, respectively (FIG.9D). FCS analy injection of ''I-PEGylated RGD silica nanoparticles at sis of urine samples obtained at multiple time points after US 2014/024821.0 A1 Sep. 4, 2014 27 injection of the targeted probe revealed that the particle was revealed drainage into and persistent visualization of adjacent excreted intact and without release of the encapsulated dye inguinal and popliteal nodes over this interval, with Smaller (data not shown). and/or more distant nodes and lymphatics more difficult to visualize. By contrast, the non-targeted probeyielded shorter TABLE 4 term (~1 hr) visualization of local nodes with progressively weaker fluorescence signal observed (data not shown). Upon Urine Concentration and Cumulative Excretion Data Surgical exposure, this observation was found to be the result Computed of more rapid particle diffusion from the tumor site, as com Time Concentration Avg. Urine Cumulative % ID pared with the extended retention observed with the targeted (hr) (% IDful) Volume (ul) Excreted probe. 7.0 mm O O.O O.O O 0250 We next performed representative lymph node map RGDYPEG 1 O.292 41.6 6.07 ping over multiple spatial scales using live-animal whole dot 4 O.O26 166.7 26.1 body optical imaging (FIG. 12A) and NIR fluorescence 24 O.O16 1OOO. 43.4 microscopy techniques (FIG. 12B) to visualize lymphatic 96 O.OO)4 3974. 72.2 drainage from the peritumoral region to the inguinal and axillary nodes in Surgically exposed living animals. In addi tion, higher-resolution fluorescence images (FIG.12B, lower Serial Whole Body PET Studies row) permitted more detailed intranodal architecture to be visualized, including high endothelial Venules, which facili 0247 PET imaging of integrin expression in M21 and tate passage of circulating naive lymphocytes into the node, M21L Subcutaneous hindleg Xenograft mouse models was and which may have important implications for nodal staging performed at multiple time points p.i. following i.v. injection and the ability to detect micrometastases at earlier stages of of ''I-cRGDY-PEG-dots or I-PEG-dots (control). Repre disease. Smaller, less intense lymphatic branches were also sentative whole-body coronal microPET images at 4hrs (left: visualized by fluorescence microscopy in the axillary region M21 tumor; middle. M21L tumor) and 24 hrs (right: M21 (data not shown). Thus, the small size of the targeted probe tumor) p.i.are shown in FIG. 11A. The specific targeting of not only permits the first draining (or sentinel node), proximal the CfB integrin-overexpressing M21 tumoris clearly visible to the tumor to be visualized, but also enables visualization of from these images. Average tumor '% ID/g and standard more distant nodes and of the pattern of lymphatic drainage to deviations are shown for groups of M21 (n=7) and M21L (control) tumors (n=5) receiving the targeted 'I-cRGDY be visualized. PEG-dots, as well as for M21 tumor mice (n=5) receiving Discussion non-targeted 'I-PEG-dot tracer (FIG. 11B). At the time of maximum tumor uptake (~4 hrs p. i.), three-fold activity-con 0251 We report on non-toxic, high-affinity, and efficiently centration increases (in 96 ID/g) were seen in the M21 tumors cleared silica nanoparticles for tumor-selective targeting and over the controls. Differences were statistically significant at nodal mapping, having successfully addressed a number of all time points p. i. (p<0.05) except at 1 hr (p=0.27). the current challenges associated with other particle tech 0248 Image-derived tumor-to-muscle uptake (% ID/g) nologies. This is the first targeted nanoparticle that, on the ratios for the '''I-cRGDY-PEG-dots revealed enhanced basis of its favorable properties, can be said to be clinically tumor contrast at later times (~24-72 hrs p. i.), while that for translatable as a combined optical-PET probe. The comple '''I-PEG-dots declined (FIG. 11C). This finding suggested mentary nature of this multimodal probe, coupled with its that 'I-cRGDY-PEG-dots were, in fact, tumor-selective, Small size (~7-nm diameter), may facilitate clinical assess which became more apparent as the blood activity was ment by enabling the seamless integration of imaging data cleared during the initial 24-hr period (compare FIG. 11C acquired at different spatial, temporal, and sensitivity Scales, with inset of FIG. 9A). A statistically significant correlation potentially providing new insights into fundamental molecu was found between PET-derived tumor tissue % ID/g values lar processes governing tumor biology. for both '''I-cRGDY-PEG-dots and 'I-PEGdots, and the 0252) Our in vitro results show receptor-binding specific corresponding ex-Vivo y-counted tumor 96 ID/g values (cor ity of the ~7-mm targeted particle probe to M21 and HUVEC relation coefficient r=0.94, P-0.0016: FIG. 11D), confirming cells. Similar findings have been reported with receptor-bind the accuracy of PET for non-invasively deriving quantitative ing assays using the same cell types, but with the monovalent biodistribution data. form of the peptide. Cressman, et al., Binding and uptake of RGD-containing ligands to cellular CfB integrins. Int J Pept In Vivo NIR Fluorescence Imaging and Microscopy Res Ther. 15, 49-59 (2009). Importantly, the multivalency enhancement of the cFGDY-bound particle probe, along with 0249 We performed in vivo fluorescence imaging studies the extended blood and tumor residence time T values, are using our Small, targeted nanoparticles for mapping local/ key properties associated with the particle platform that are regional nodes and lymphatic channels, thus overcoming the not found with the monovalent form of the peptide. foregoing limitation. Importantly, the multimodal nature and (0253) The relatively long blood T value of 7.3+1.2 hrs Small size of our targeted particle probe can be exploited to estimated for the 'I-PEG-dot tracer may be related to the visualize a range of nodal sizes and lymphatic branches in our chemically neutral PEG-coated surface, rendering the probe melanoma model following 4-quadrant, peritumoral admin biologically inert and significantly less Susceptible to phago istration, simulating intraoperative human sentinel lymph cytosis by the reticuloendothelial system. That a reduction in node mapping procedures. Initially, serial NIR fluorescence the T value to 5.6+0.15 hrs was found for the '''I-cRGDY microscopy was performed in intact mice over a 4-hr time PEG-dot tracer is most likely the result of recognition by period using either the targeted or non-targeted particle target integrins and/or more active macrophage activity. probes. Peritumoral administration of the targeted probe However, it is substantially longer than published blood T. US 2014/024821.0 A1 Sep. 4, 2014 28 values of existing crGDY peptide tracers (~13 minutes), and ability of this nanoprobe highlights its use as a versatile tool results in greater probe bioavailability, facilitating tumor tar for both early and long-term monitoring of the various stages geting and yielding higher tumor uptakes over longer periods of disease management (diagnostic screening, pre-treatment of time. Montet, et al., Multivalent effects of RGD peptides evaluation, therapeutic intervention, and post-treatment obtained by nanoparticle display. J Med Chem. 49, 6087 monitoring) without restrictions imposed by toxicity consid 6093 (2006). In addition, the tumor T value for the I erations. An additional important advantage is that while cRGDY-PEG-dot was about 13 times greater than that for rapidly cleared probes may be useful for certain applications blood, versus only a fivefold difference for the '''I-PEG-dot, where target tissue localization is itself rapid, localization of Suggesting Substantially greater target-tissue localization of many agents in often poorly vascularized and otherwise rela the former than the latter. Such mechanistic interpretations of tively inaccessible solid tumors will likely be slow following the in vivo data can be exploited to refine clinical diagnostic, systemic administration. Thus, the current nanoparticle plat treatment planning, and treatment monitoring protocols. form expands the range of applications of such agents, as the 0254 The results of this study underscore the clear-cut kinetics of target tissue localization are no longer limiting. advantages offered by PET, a powerful, quantitative, and Furthermore, deep nodes can be mapped by PET in terms of highly sensitive imaging tool for non-invasively extracting their distribution and number while more precise and detailed molecular information related to receptor expression levels, localization of Superficial nodes can be obtained by NIR binding affinity, and specificity. The greater accumulation in fluorescence imaging. Finally, the relatively prolonged resi and slower clearance from M21 tumors, relative to surround dence of the targeted probe from tumor relative to that from ing normal structures, allows discrimination of specific tumor blood, in addition to its multivalency enhancement, may be uptake mechanisms from non-specific mechanisms (i.e., tis exploited for future theranostic applications as a radiothera Sue perfusion, leakage) in normal tissues. A small component peutic or drug delivery vehicle. of the M21 tumor uptake, however, presumably can be attrib uted to vascular permeability alterations (i.e., enhanced per Example 5 meability and retention effects). Seymour, Passive tumor tar geting of soluble macromolecules and drug conjugates. Crit. Fluorescent Silica Nanoparticles Conjugated with Rev. Ther. Drug Carrier Syst. 9, 135-187 (1992). This non CfB Integrin-Targeting Peptide and/or specific mode of uptake reflects a relatively small portion of uMUC1-Targeting Peptide (Thyroid Cancer and the overall tumor uptake at earlierp.i. time points based on the Squamous Cell Carcinoma (SCC) Models) observed 9% ID/g increases in mice receiving the control (0257 AcRGD peptide (Peptides International), having a tracer ('''I-PEG-dots, FIG. 11B). At 1-hr p.i., no significant cysteine end functionality, will be attached to the PEG-ylated % ID/g increases were seen in the M21 tumors over the nanoparticle viaathiol-maleimide linkage. The nanoparticles controls. This observation may reflect the effects of differen can optionally further be functionalized by a synthetic pep tial perfusion in the first hour, with tumor accumulation and tide ligand, EPPT1. The nanoparticles will be characterized retention primarily seen at later p.i. times (i.e., 24 hrs). Fur on the basis of particle size, size distribution, and pho ther, in comparison with the clinically approved peptide tobleaching. tracer, F-galacto RGD, nearly two-fold greater uptake in M21 tumors was found for the '''I-cRGDY-PEG-dots34, Characterization of Nanoparticle-Peptide Conjugates while additionally offering advantages of multivalent bind ing, extended blood circulation times, and greater renal clear 0258 For assessing photophysical properties on a per aCC. particle basis, spectrophotometry, spectrofluorometry, and 0255 One advantage of a combined optical-PET probe is multiphoton fluorescence correlation spectroscopy (FCS) the ability to assess anatomic structures having sizes at or well will be used to determine the particle size, brightness, and size below the resolution limit of the PET scanner (i.e., the so distribution. Size data will be corroborated by scanning elec called partial-volume effect), which may undermine detec tron microscopy and dynamic light scattering (DLS) mea tion and quantitation of activity in lesions. For instance, in surements. Ow et al. Bright and stable core-shell fluorescent Small-animal models, assessment of metastatic disease in silica nanoparticles. Nano Letters 2005; 5, 113. Average num Small local/regional nodes, important clinically for mela ber of RGD peptides per nanoparticle and coupling efficiency noma staging and treatment, may not be adequately resolved of RGD to functionalized PEG groups will be assessed colo by PET imaging, given that the size of the nodes observed are rimetrically under alkaline conditions and Biuret spectropho typically on the order of system spatial resolution (1-2 mm). tometric methods (a 450 nm, maximum absorbance). By utilizing a second complementary and sensitive imaging 0259. The nanoparticle conjugates will be iodinated via modality, near-infrared (NIR) fluorescence imaging, func tyrosine linkers to create a radiolabeled ('''I)(T2-4 d) and tional maps revealing nodal disease and lymphatic drainage stable (‘’I) form by using Iodogen (Pierce, Rockford, Ill.). patterns can be obtained. Ballou, et al., Sentinel lymph node The end product will be purified by using size exclusion imaging using quantum dots in mouse tumor models. Biocon chromatography. jugate Chem. 18, 389-396 (2007). While further studies investigating the distribution of intranodal cFGDY-PEG-dot Evaluation of In Vitro Targeting Specificity and fluorescence in relation to metastatic foci are needed to deter Biodistribution Patterns of the RGD- and mine whether sensitive localization of such foci can be RGD-EPPT-Nanoparticles. achieved, these results clearly demonstrate the advantages of 0260 CfB integrin and uMUC1 expression patterns in working with such a combined optical-PET probe. thyroid and squamous cell carcinoma (SCC) cell lines will be 0256 In the clinic, the benefits of such a combined plat evaluated against known CfB integrin-negative and O?3. form for tumor staging and treatment cannot be overstated. integrin-positive (M21-L and M21 human melanoma cell The extended blood circulation time and resulting bioavail lines, respectively) and uMUC1-negative and uMUC1-posi US 2014/024821.0 A1 Sep. 4, 2014 29 tive (U87, H-29 cell lines, respectively) controls using anti Fluorescence Imaging System (CRI, Woburn, Mass.) at 0. integrin and anti-uMUC 1 antibodies. Cell lines highly 0.5, 1, 2, 4, 6, 12, and 24 hrs. At 24-h, mice are euthanized, and expressing Clf-integrin and/or MUC1 will be selected for major tissues/organs dissected, weighed, and placed in 6-well differential binding studies with RGD- and RGD-EPPT plates for ex-Vivo imaging. Fluorescence emission will be nanoparticles, as well as for in vivo imaging. analyzed using regions-of-interest (ROIs) over tumor, 0261 Quantitative cell binding assays will assess the selected tissues, and reference injectates, employing spectral labeling efficiency of tumor cells, and biodistribution studies unmixing algorithms to eliminate autofluorescence. Dividing assaying uptake in tumor, organs, and fluids will be per average fluorescence intensities of tissues by injectate values formed using radioiodinated nanoparticle conjugates ('''I- will permit comparisons to be made among the various tis RGD-nanoparticles, 'I-RGD-EPPT-nanoparticles). To Sues/organs for each injected nanoparticle conjugate. compare PET uptake data of nanoparticle conjugates with 0269. Dynamic MicroPET Imaging Acquisition and that observed initially using optical NIRF imaging, each Analysis. Two groups of tumor-bearing mice (n=5/tumor) nanoparticle conjugate will also be iodinated to create a radio will be injected with radiolabeled 'I-nanoparticle conju labeled ('''I) and stable ('''I) form. gates (radiotracers), and dynamic PET imaging performed 0262 Fluorescence Microscopy with RGD- and RGD for 1-hr using a Focus 120 microPETTM (Concorde Micro EPPT-C-dots. Differential binding of RGD-nanoparticles and systems, TN). One-hour list-mode acquisitions are initiated at RGD-EPPT-nanoparticles to thyroid carcinoma/SCC cell the time of IV injection of -25.9 MBq (700 uCi) radiotracers. lines highly expressing Clf-integrin and/or MUC1, versus Resulting list-mode data are reconstructed in a 128x128x96 control lines will be visualized by fluorescence microscopy. matrix by filtered back-projection. ROI analysis of recon 0263 Animal models. All animal experiments will be structed images is performed using ASIProTM software (Con done in accordance with protocols approved by the Institu corde Microsystems, TN) to determine the mean and SD of tional Animal Care and Use Committee and following NIH radiotracer uptake (% ID/g) in tumors, other organs/tissues, guidelines for animal welfare. and left ventricle (LV). Additional data will be obtained from 0264. In vivo Biodistribution: Male athymic nude mice static images at 24-, 48-, and 72-hr post-injection time points. (6-8 week old, n=5 per tumor) will be subcutaneously (s.c.) A three-compartment, four-parameter kinetic model will be injected in both flanks with integrin-negative/-positive or used to characterize tracer behavior in vivo. For this analysis, uMUC1-negative/-positive tumors of different tissue origins arterial input is measured using an ROI placed over the LV. (n=3/each tumor). At 0.5 cm in diameter (i.d.), mice will be injected intravenously (IV) with I-labeled nanoparticle Example 6 conjugates (~500 nm/kg). Animals are sacrificed at 0.5.1, and 24-hrs later, with removal of tumors, organs, and fluids for Nodal Mapping in Miniswine weighing and counting (gamma counter). Biodistribution 0270. Real-time intraoperative scanning of the nodal basin results will be expressed as the percentage of injected dose cannot be practically achieved at the present time, as these per gram of tissue. systems are generally too cumbersome and expensive for use 0265 Quantitative Cell Binding Assay. Labeling effi in the operating Suite or may be unable to provide the neces ciency will be assessed by incubating fixed numbers of car sary field-of-view or tissue contrast. Further, there are no cinoma cells highly expressing Clf-integrin and/or MUC1, clinically promising, biostable fluorophore-containing with pre-selected concentrations of ''I-labeled nanoparticle agents, offering improved photophysical features and longer conjugates for 1-hr in a humidified CO, atmosphere at 37°C. circulation lifetimes over parent dyes, available to enhance Cells are extensively washed, lysed with 0.1% Triton X, with tissue contrast for extended nodal mapping/resection proce cell lysates counted in a gamma counter. dures. With this animal study, we will show that advances in both multimodal particle probes and real-time molecular Assess of Relative Differences in Tumor-Specific Targeting imaging device technologies can be readily translated to a Using. In Vivo Multimodality (PET-NIRF) Imaging. variety of future human clinical trials. Such transformative 0266. As a high-throughput diagnostic screening tool, technologies can significantly impact standard intraoperative optical NIRF imaging can be used to evaluate relative differ cancer care by providing state-of-the-art targeted visualiza ences in the biodistribution of progressively functionalized tion tools for facilitating metastatic SLN detection and nanoparticle conjugates in vivo with increased sensitivity and enabling accurate delineation of node(s) from adjoining temporal resolution. Semi-quantitative data on tumor-spe anatomy to minimize risk of injury to crucial structures. Ben cific targeting can also be derived. These preliminary studies efits include extended real-time in vivo intraoperative map facilitate the selection of cell lines strongly expressing mark ping of nodal disease spread and tumor extent in the head and ers of interest for further detailed quantitation of biodistribu neck. Deep nodes can be mapped by PET, while precise and tion and tumor-specific targeting using PET. detailed localization of superficial nodes can be obtained by 0267 Whole-body microPETTM and NIRF optical imag NIR fluorescence imaging. The small size of the particle ing will be performed over a 1-week period to assess differ probe may also extend the lower limit of nodal sizes that can ential uptake in flank tumors. The results of these studies will be sensitively detected. The net effect of the proposed non be validated with fluorescence microscopy of tumors ex-vivo. toxic, multimodal platform, along with the application of 0268 Serial InVivo NIRF Imaging. Mice will be injected combined diagnostic/treatment procedures, has important bilaterally with CfB integrin-negative and CfB integrin implications for disease staging, prognosis, and clinical out positive cells or with uMUC1-negative and uMUC1-positive come for this highly lethal disease. cells (n=5/tumor). After tumors reach -0.5 cm i.d., stable iodinated and non-iodinated nanoparticle conjugates (RGD, Disease Target. 27I-RGD, RDG-EPPT, 27I-RGD-EPPT) will be injected IV. 0271. In addition to melanoma, a number of other tumors Serial imaging will be performed using the MaestroTM InVivo (i.e., breast, lung, and brain) overexpress CVB3 integrin US 2014/024821.0 A1 Sep. 4, 2014 30 receptors and could serve as disease targets. Metastatic mela mapping study are roughly comparable to the average diam noma has a very poor prognosis, with a median Survival of eter of an albumin molecule and 2-3 times Smaller than the less than 1 year. Successful management relies on early iden average diameter of a typical antibody. Relative to peptide tification with adequate Surgical excision of the cancer. Sur tracers, the targeted particle probe is less prone to extravasa gical removal of the primary disease, screening, and treat tion and is associated with extended circulation halftimes that ment for regional lymph node spread is standard-of-care in enhance tumor targeting. Importantly, 124I-cRGDY-PEG the US to accurately stage disease and tailor treatment. The dots demonstrate key in vitro and in vivo properties in M21 recently revised staging guidelines recognize the presence of tumors necessary for clinical translation. microscopic nodal metastases as a hallmark of advanced stage disease leading to dramatically reduced Survival. Materials and Methods. Knowledge of pathologic nodal status is critical for early risk stratification, improved outcome predictions, and selection of 0274 Spontaneous melanoma Sinclair miniature Swine patient Subgroups likely to benefit from adjuvant treatment (10-12 kg, Sinclair Research Center, MO) were injected intra (therapeutic nodal dissection, chemotherapy) or clinical tri venously with 5 mCi'F-fluoro-deoxyglucose (F-FDG) for als. whole-body screening of nodal and/or organ metastases. Miniswine underwent 1-hr dynamic F-FDG PET whole Sentinel Lymph Node (SLN) Mapping. body PET scan using a clinical PET scanner 40 minutes after 0272 SLN mapping techniques, routinely used in staging injection to screen for metastatic disease, followed by CT melanoma, identify the specific node(s) that are at highest risk scan acquisition for anatomic localization. Then miniswine of tumor metastases. This procedure identifies patients har were Subdermally injected in a 4-quadrant pattern about the boring metastatic disease for further treatment. Standard-of tumor site (head and neck sites preferentially) with multimo care techniques rely on injection of radioactive technetium dal '''I-RGD-PEG-dots 48 hrs after F-FDG PET, and a ("Tc) sulfur colloid dye around the primary tumor for SLN second dynamic PET-CT scan performed to assess for addi localization, followed by the intraoperative use of a gamma tional nodal metastases. probe to measure radioactivity in lymphatic structures within 0275 Miniswine were taken to the operating room for an exposed nodal basin. Blue dye injected about the primary identification of nodes. Optical fluorescence imaging was tumor can help delineate small SLN(s) from adjacent tissue, performed using large field-of-view near infrared fluores but the technique is unreliable and liable to complications. cence camera system, Smaller field-of-view modified endo Current SLN mapping and biopsy techniques have limita scope, and a modified stereomacroscope for obtaining higher tions, and account for higher rates of non-localization of resolution fluorescence images within the exposed Surgical SLN(s) in the head and neck compared to other anatomic bed. sites. The head and neck region is notorious for its unpredict 0276 Validation of the fluorescent signal was performed able patterns of metastatic disease. The close proximity of the intraoperatively by gamma counting with a clinically-ap primary disease to nodal metastases in this region makes proved hand-held PET device within the operative bed to intraoperative use of the gamma probe difficult due to inter localize targeted dots transdermally, acquired intraopera ference from the injection site. Importantly, current technol ogy does not allow the Surgeon to visualize the sentinel node tively from skin and the nodes within and nodal basin. and reliably differentiate it from adjoining fat or other tissues, 0277. The primary melanoma skin lesion was excised, and placing vital structures (i.e., nerves) at risk for injury during an incision made to allow access to the sentinel node(s). dissection to identify and harvest this node. The small size of Nodal identity was confirmed using handheld PET and multi nodes and wide variation in drainage patterns provides addi scale optical imaging systems, and the nodes in question tional challenges, resulting in a non-localization rate of excised. Specimens were sent for histological assessment for around 10%. metastases and optical confocal microscopy to confirm the presence of both malignancy and nanoparticle fluorescence. Nanoparticles. 0278. Following harvest of the sentinel nodes, the entire 0273. The majority of preclinical studies have used RGD lymph node basin was excised and further evaluated using peptide or peptide-conjugate radiotracers as targeting ligands histological methods (with immunohistochemical markers for imaging CEVfB3-integrin expression. F-galacto-RGD and for melanoma as needed), fluorescence microscopy, and the "Tc-NC100692 are peptide tracers that have been used hand-held PET probe for correlative purposes. This step Successfully in patients to diagnose disease. Peptide tracers helped identify any other malignant nodes within the nodal clear rapidly, which may result in reduced receptor binding basin and the number of ''I-RGD-PEG-dots present in adja and increased background signal from non-specific tissue cent nodes by their appearance on imaging. dispersal. These properties limit the potential of peptide trac (0279 '''I-RGD-PEG-dots was administered subcutane ers for longer-term monitoring. By contrast, nanoparticle ously into the limbs of the animal sequentially. Transit of the probes (~10-100 nm), which have also been used for imaging 'I-RGD-PEG-dots to the inguinal/axillary nodes was fol integrin expression along tumor neovasculature, have lowed using the optical imaging system and hand held PET extended circulation half times for performing longer-term probes to confirm the duration of transit along the lymphatic monitoring (i.e., days). Nanoparticles are typically larger pathways. The draining nodal basins was exposed Surgically than antibodies and radiopharmaceuticals (<10 kDa), and are and the pattern of lymph node drainage observed. The senti associated with slower transmembrane transport, increased nel lymph node was harvested from each site to confirm the reticuloendothelial system (RES) uptake, and enhanced non lymphatic nature of the tissue. Animals were euthanized, and specific uptake due to altered tumor vascular permeability. any further lesions noted on imaging were excised in the The 7 nm diameter targeted nanoparticles used for this SLN necropsy room of the animal facility. US 2014/024821.0 A1 Sep. 4, 2014

Discussion node by H&E staining (lower two images), and we expect 0280 A whole-body 'F-fluorodeoxyglucose (F-FDG) melanoma specificity to be further confirmed using special PET-CT scan revealed a primary melanomatous lesion adja stains (Melan A, HMB45, PNL2, and “melanoma associated cent to the spine on the upperback, as well as a single node in antigen' biogenex clone NKI/C3). We additionally expect the neck, posteriorly on the right side of the animal, which colocalization of the particle with these metastatic clusters of were both FDG-avid, and suspicious for metastatic disease. cells on confocal fluorescence microscopy and high resolu This finding was confirmed after Subdermal, 4-quadrant tion digital autoradiography, confirming metastatic disease injection of ''I-RGD-PEG-dots about the tumor site, which detection. additionally identified two more hypermetabolic nodes, as Example 7 well as the draining lymphatics. Final scan interpretation pointed to 3 potential metastatic nodes. Surgical excision of Fluorescent Silica Nanoparticles Conjugated with the primary lesion, hypermetabolic nodes, and tissue from MC1R-Targeting Peptide (Melanoma Model) other nodal basins in the neck bilaterally was performed after hand-held PET probes identified and confirmed elevated (0285 For the multimodality (PET-NIRF) diagnostic count rates at the location of sentinel node(s). Patchy fluores imaging experiments, the targeting peptide and the radiolabel cence signal measured in the excised right posterior sentinel on the nanoparticle Surface will be exchanged to determine node tissue correlated with sites of melanoma metastases by target specificity, binding affinity/avidity, and detection sen histologic analysis. All hypermetabolic nodal specimens sitivity. Subsequent therapeutic particles will also be synthe were black-pigmented, and found to correlate with the pres sized using therapeutic radiolabels (lutetium-177, 77Lu, ence of distinct clusters of melanoma cells. Thus, the results t'=6.65 d) for targeted killing of MC1R-expressing mela of surgically resected tissue submitted to pathology for H&E noma cells. Combined quantitative PET and optical imaging and staining for other known melanoma markers confirmed findings will be correlated with tumor tissue autoradiography multimodal imaging findings. and optical imaging across spatial scales. For cellular micros 0281 FIG. 13a shows the experimental setup of using copy, an in vivo confocal fluorescence Scanner for combined spontaneous miniswine melanoma model for mapping lymph reflectance and fluorescence imaging will be used. node basins and regional lymphatics draining the site of a known primary melanoma tumor. This intermediate size Example 8 miniswine model is needed to simulate the application of sentinellymph node (SLN) biopsy procedures inhumans, and Fluorescent Nanoparticles for Targeted Radiotherapy more accurately recapitulates human disease. FIG.13b shows 0286 Dose escalation studies with ''I-RGD nanopar small field-of-view PET image 5 minutes after subdermal ticles will be performed and treatment response will be moni injection of multimodal particles ('''I-RGD-PEG-dots) tored weekly, over the course of six weeks, using 'F-FDG about the tumor site. The tumor region, lymph nodes, and the PET. Time-dependent tumor uptake and dosimetry of the lymphatics draining the tumor site are seen as areas of nanoparticle platform will be performed using planar gamma increased activity (black). camera imaging. In vivo imaging data will be correlated with 0282 FIG. 14 shows whole-body dynamic F-fluorode gamma counting of excised tumor specimens. oxyglucose (F-FDG) PET scan (FIG. 14a) and fused F (0287. Male nude mice (6-8wks, Charles River Labs, MA) FDG PET-CT scans (FIG. 14b) demonstrating sagittal, coro will be used for generating hind leg Xenograft models after nal, and axial images through the site of nodal disease in the injection of M21 human melanoma cells (5x10 in PBS). neck. The 'F-FDG PET scan was performed to map sites of Tumors will be allowed to grow 10-14 days until 0.5-0.9 cm metastatic disease after intravenous administration and prior in size. to administration of the radiolabeled nanoparticle probe. A 0288 ''I-based targeted radiotherapy studies. The thera single hypermetabolic node is seen in the neck posteriorly on peutic radionuclide ''I will be used as a radiolabel for tar the right side of the animal (arrows, axial images, upper/lower geted radiotherapy. In estimating the highest possible ''I panels), also identified on the whole body miniswine image dose resulting in no animal deaths and less than 20% weight (FIG. 14c). loss (MTD), a dose escalation study will be carried out in 0283 FIG. 15 shows the same image sets as in FIG. 14, but tumor-bearing nude mice. For a 200 rad dose to blood54, an at the level of the primary melanoma lesion, adjacent to the administered activity of 10 MBq is required, which would spine on the upper back. The PET-avid lesion is identified deliver a dose of 270 rad to tumor. 4 doses of 10 MBq each (arrows, axial images, upper/lower panels), as well as on the will be administered to achieve a tumor dose greater than whole body miniswine image (FIG. 15c). 1000 rad with dose fractionation designed to allow repair and 0284 FIG. 16 shows high resolution dynamic PET (FIG. sparing of bone marrow.' 'I allows for planargamma camera 16a) and fused PET-CT images (FIG. 16b) following subder imaging using a pinhole collimator to measure the time mal, 4-quadrant injection of ''I-RGD-PEG-dots about the dependent tumor uptake and dosimetry of the nanoparticles. tumor site, simulating clinical protocol, over a 1 hour time F-FDG PET allows for quantitative monitoring of tumor period. Three hypermetabolic lymph nodes (arrows) were response, thus providing complementary information. found in the neck, Suggesting metastatic disease. The excised 0289 Based on this data, and in vivo data on the effect of right posterior SLN was excised and whole body near infrared nanoparticles loaded with paclitaxel, a therapy study with the (NIR) fluorescence imaging was performed. Cy5 fluores ''I-RGD-nanoparticle conjugate will be conducted. Two cence signal was detectable within the resected node (FIG. groups of tumor-bearing mice (n=10 per group) will receive 16c, top, Cy5 imaging) on whole-body optical imaging. either four, 10.4-MBq activities once per week for 4 weeks, of Pathological analysis of this black-pigmented node (arrow, i.v.-administered 'I-RGD-nanoparticle conjugates or saline SLN) demonstrated clusters of invading melanoma cells on vehicle (control, n=10), and will be monitored over a 6-week low-(arrows) and high-power cross-sectional views of the period. Treatment response/progression will be quantified on US 2014/024821.0 A1 Sep. 4, 2014 32 the basis of tumor Volume (via caliper measurements). All 0296 Characterization of the functionalized nanoparticle mice from the treatment groups will also be imaged once per preparations will be performed as follows: week (~1 hr sessions) by SPECT imaging (Gamma Medica) 0297 (A) Average number of DOTA chelates per nanopar over a 6 week period. ticle will be determined by standard isotopic dilution assays 0290 F-FDG PET Imaging Acquisition and Analysis. with Cu. Briefly, 'Cu will be added to solutions containing Two groups of tumor-bearing mice (n=10/group) will a known amount of ReCCMSH(Arg11)-nanoparticles. Incu undergo initial PET scanning prior to and then, on a weekly bated Solutions will be spotted on silica gel-coated glass basis after treatment over a 6 week interval. Mice will be plates, developed in 1:1 10% ammonium acetate-to-methanol injected intravenously (i.v.) with 500 uCi F-FDG and static (with EDTA), and analyzed by radio-TLC. While Cu-la 10-minute PET images will be acquired using a Focus 120 beled ReCCMSH(Arg11)-Nanoparticles will remain at the microPETTM (Concorde Microsystems, TN) before and after origin, Cubound to EDTA will migrate. The percent label treatment. Acquired data will be reconstructed in a 128x128x ing efficiency will be plotted against total nanomoles of Cu 96 matrix by filtered back-projection. Region-of-interest added to the reaction mixture. The number of chelates (ROI) analyses of reconstructed images will be performed attached per nanoparticle can be determined from the inflec using ASIProTM software (Concorde Microsystems, TN) to tion point of this curve. determine the mean and SD of radiotracer uptake (% ID/g) in 0298 (B) Average number of ReCCMSH(Arg11) pep tumors. Animals will be sacrificed at the termination of the tides per nanoparticle and coupling efficiency of the ReCC study and tumors excised for gamma counting. MSH(Arg11) to the functionalized PEG groups will be assessed using spectrophotometric methods (w-435 nm, Example 9 maximum absorbance) and the known extinction coefficient of ReCCMSH(Arg11). The incorporation of rhenium offers Fluorescent Nanoparticles Conjugated with the advantage that highly sensitive absorbance measurements Radionuclide Chelate and MC1R-Targeting Peptide of rhenium concentrations can be made on a small sample of product. 0291 PEG-ylated nanoparticles will be conjugated with targeting peptides and macrocyclic chelates binding high In Vitro and InVivo Optical-PET Imaging of Multifunctional specific activity radiolabels. Nanoparticle Nanoparticles in Melanoma Models to Assess 0292 High purity two-arm activated commercially avail Tumor-Specific Targeting and Treatment Response. able PEGs, derivatized with NHS esters or maleimide, will be attached to the silica shell of the nanoparticle using standard 0299 Cu-DOTA-ReCCMSH(Arg11)-nanoparticles procedures. Either of the two functionalized PEG groups will be compared with the native Cu-DOTA-ReCCMSH (NHS esters or maleimide) will be available for further con (Arg11) construct to test targeting capabilities of the nano jugation with either the peptide-chelate construct, cyclic pep particles. tide Re-Cys34,10.D-Phe7c-MSH3-13 (ReCCMSH 0300 Competitive binding assays. The MC1R receptor (Arg11)), or 1,4,7,10-tetraazacyclododecane-N,N',N',N'- positive B 16/F1 murine melanoma lines will be used. The tetraacetic acid (DOTA) linker chelators. The covalent ICso values of ReCCMSH(Arg11) peptide, the concentration attachment of derivatized PEGs to the nanoparticle surface of peptide required to inhibit 50% of radioligand binding, will will be performed in such a manner as to expose different be determined using I-(Tyr2)-NDP7, a radioiodinated functional groups for linking DOTA and peptide-chelate con C-MSH analog with picomolar affinity for the MC1R. Single wells will be incubated at 25°C. for 3 h with approximately structs, as discussed below. 50,000 cpm of 'I-(Tyr2)-NDP in 0.5 ml binding medium Synthesis and Physicochemical Characterization of with 25 mmol/L N-(2-hydroxyethyl)-piperazine-N-(2- Functionalized Nanoparticles. ethanesulfonic acid), 0.2% BSA and 0.3 mmol/L 1.10 phenanthroline, with concentrations of (Arg11)CCMSH 0293 Functionalized nanoparticles will be synthesized by ranging from 10-13 to 10-5 mol/L. Radioactivity in cells and establishing covalent linkages of the following moieties with media will be separately collected and measured, and the data the derivatized PEG groups: processed to compute the ICs value of the Re(Arg11)CC 0294 (A) DOTA chelates for subsequent high-specific MSH peptide with the Kell software package (Biosoft, MO). activity radiolabeling with positron-emitting radiometals 0301 Receptor Quantitation Assay. Aliquots of 5x105 (i.e., "Cu) to permit diagnostic detection with PET imaging. B16/F1 cells will be added to wells, cultured in 200 uL RPMI DOTA will be conjugated to the functionalized PEGs using media, and incubated at 37° C. for 1.5 h in the presence of standard Fmoc chemistry, and purification of the chelated increasing concentrations of 'I-(Tyr2)-NDP (from 2.5 to nanoparticles will be performed by chromatography. Cu 100 nCi) in 0.5 mL of binding media (MEM with 25 mM and '77Lu will be attached to DOTA by incubation of the HEPES, pH 7.4). Cells will be washed with 0.5 mL of ice reaction mixture at 60°C. for 30 min followed by gel filtration cold, pH 7.4, 0.2% BSA/0.01 M PBS twice, and the level of or high pressure liquid chromatography purification. Alterna activity associated with the cellular fraction measured in a tively, PET nuclides, such as 'I.Y. Ga and Zr, may be Y-counter. Nonspecific binding will be determined by incu conjugated to the nanoparticle, either via the DOTA-function bating cells and 'I-(Tyr2)-NDP with non-radioactive NDP alized PEG (radiometals) or tyrosine-functionalized PEG at a final concentration of 10 M. Scatchard plots will be (I). The single photon emitter, ''Lu, obtained in the form obtained by plotting the ratio of specific binding to free I of '77LuCls will be complexed to DOTA for radiotherapy. (Tyr2)-NDP vs. concentration of specific binding (fmol/mil 0295 (B) CMSH melanoma targeting peptide analogue lion cells); Bmax, the maximum number of binding sites, is (ReCCMSH(Arg11)) is cyclized by rhenium. It is necessary the X intercept of the linear regression line. to confirm the ratio of DOTA chelates to ReCCMSH(Arg11) (0302 B 16/F1 murine melanomalines (5x10 in PBS) will moieties on the PEG-ylated nanoparticle surface. be injected subcutaneously into the hind legs of Male nude US 2014/024821.0 A1 Sep. 4, 2014

mice (6-8 week old). The tumors will be allowed to grow be utilized for imaging tumors in live, intact animals at 72-h 10-14 days until 0.5-0.9 cm in size. post-injection. Mice will be maintained under continuous 0303 Biodistribution: A small amount of the Cu isofluorane anesthesia, thus enabling detection and localiza DOTA-ReCCMSH(Arg11)-nanoparticle conjugate (~10 uCi. tion of fluorescence signal from the whole animal/organ level 0.20 ug) will be injected intravenously into each of the mice to the cellular level over a range of magnifications. Whole bearing palpable B 16/F1 tumors. The animals will be sacri animal/macroscopic imaging will be performed with fluores ficed at selected time points after injection (2, 4, 24, 48, 72 cencestereomicroscope (Visen; Nikon SMZ1500) fitted with hours; n=4-5/time point) and desired tissues removed, Cy5 fluorescence filter sets and CCD cameras. Fluorescence weighed, and counted for accumulated radioactivity. Addi confocal laser Scanning microscopy capabilities will be tional mice (n=5) injected with the native radiolabeled con developed. Mice will subsequently be euthanized for autora struct, Cu-DOTA-ReCCMSH(Arg11) (-10 Ci, 0.20 ug) diography in order to map tracer biodistributions at high will serve as the control group, and evaluated 1 h post-injec resolution throughout the tumor volume. Tumors will be tion. To examine in vivo uptake specificity, an additional excised, flashfrozen, serially sectioned (100LL sections) and group of mice (2-h time point) will be pre-injected with 20 ug slide-mounted, with alternating slices placed in contact with of NDP to act as a receptor block immediately prior to the a phosphor plate in a light-tight cassette (up to 1 wk). H&E injection of the Cu-DOTA-ReCCMSH(Arg11) nanopar staining will be performed on remaining consecutive sec ticle conjugate. Major organs and tissues will be weighed and tions. Autoradiographic findings will be correlated with PET gamma-counted, and the percentage-injected dose per gram imaging data and histological results. (% ID/g) determined. 0307. The therapeutic radionuclides 77Lu or 'Y may 0304 Serial In Vivo NIRF Imaging. In parallel with the alternatively be used for targeted radiotherapy. In estimating PET studies below, NIR (fluorescence tomographic imaging, the highest possible 7Lu dose resulting in no animal deaths FMT 225, Visen, Woburn, Mass.) will be performed using a and less than 20% weight loss (MTD), a dose escalation study tunable 680 nm scanning NIR laser beam and CCD before will be carried out in tumor-bearing nude mice. Doses of and after i.v. injection of tumor-bearing animals (n=10). Mice radiopharmaceutical suspected to be at (or near) the MTD will be kept under continuous isoflurane anesthesia, and based on literature values for '77Lu will be evaluated. placed in a portable multimodal-imaging cassette (compat ible with both our FMT 2500 and Focus 120 microPET) for Example 10 FMT scanning before and after injection (1, 2, 4, 6, 12, 24, 48 and 72 hours). The NIR fluorescence image, measured over a Fluorescent Nanoparticles Functionalized to 1-10 minute period, will be reconstructed using the Visen Conjugate with Ligand and Contrast Agent Via proprietary Software and Superimposed onto a normal photo “Click Chemistry” graph of the mouse. The imaging data is quantitative, as the measured intensity is directly related to the NIR fluorophore Synthesis of Nanoparticles Containing Versatile Functional concentration, enabling parametric maps of absolute fluoro Groups for Subsequent Conjugation of Ligand e.g., Peptides) phore concentrations to be generated for co-registration with and Contrast Agent (e.g., Radionuclides). the acquired PET imaging data. 0308. In order to synthesize an array of nanoparticle-pep 0305 Dynamic PET Imaging Acquisition and Analysis. tide-chelate constructs suitable for high-specific activity Two groups of tumor-bearing mice (n=5/group) will be radiolabeling, a "click-chemistry’ approach may be used to placed in the imaging cassette for co-registering sequential functionalize the nanoparticle surface (FIG. 17). This method PET-optical studies. Mice will be injected intravenously is based on the copper catalyzed cycloaddition of azide to a (i.v.); one with radiolabeled 'Cu-DOTA-ReCCMSH(Arg11) triple bond. Such an approach would allow for a great deal of nanoparticle conjugates and the second with native “Cu Versatility to explore multimodality applications. DOTA-ReCCMSH(Arg11) constructs. Following injection, 0309 Nanoparticle synthesis and characterization. The dynamic 1-hr PET images will be acquired using a Focus 120 PEG groups that will be covalently attached will be produced microPETTM (Concorde Microsystems, TN). One-hour list following the scheme in FIG. 14. PEG will be covalently mode acquisitions are initiated at the time of IV injection of attached to the nanoparticle via the silane group. Standard radiolabeled probe (~ 1 mCi). Resulting list-mode data will be chemical pathways will be used for the production of the reconstructed in a 128x128x96 matrix by filtered back-pro functionalized PEG with triple bonds. jection. Region-of-interest (ROI) analyses of reconstructed 0310 Functionalization of nanoparticles with triple images are performed using ASIProTM software (Concorde bonds. To synthesize the bifunctionalized PEGs, the first step Microsystems, TN) to determine the mean and SD of will employ the well studied reaction of activated carboxylic radiotracer uptake (% ID/g) in tumors, other organs/tissues, ester with aliphatic amine (FIG. 18). Alternatively, another and left ventricle (LV). Tracer kinetic modeling of the data Suitable triple-bond bearing amine, for example, p-ami will permit estimation of pharmacokinetic parameters, nophenylacetylene, can be used. The second step of the Syn including delivery, clearance, and Volume of distribution. As thesis also relies on a well-known conjugation reaction. noted, an arterial blood input is measured using an ROI placed Synthesis and Physicochemical Characterization of Func over the LV (as a measure of blood activity). Additional data tionalized Nanoparticles Conjugated with Model Peptides will be obtained from static images at 24 hr, 48 hr., 72 hr and Chelates. post-injection time points. 0311. The functionalized nanoparticle contains both (A) 0306 Fluorescence microscopy and autoradiography of desferrioxamine B (DFO) for subsequent high-specific activ tissues. A combination of optical imaging technologies ity radiolabeling with the positron-emitter zirconium-89 exhibiting progressively smaller spatial scales (i.e., whole (Zr) and (B) the SSTR-targeting peptide, octreotate. body fluorescence imaging, fluorescence macroscopy, and in 0312 Synthesis of DFO with an azide bond. DFO with an Vivo fluorescence confocal laser scanning microscopy) will azide group will be produced by reaction of DFO-B with US 2014/024821.0 A1 Sep. 4, 2014 34 pazido benzoic acid) (FIG. 19) and purified. The "click chem Elman's reagent can be used to quantitate thiols in proteins by istry reaction is a 1,3-dipolar cycloaddition at room tempera absorption measurements. It readily forms a mixed disulfide ture and the conditions are often referred to as “Huigsen with thiols, liberating the chromophore 5-merapto-2-ni Conditions'. Although the reactions can generally be com trobenzoic acid (absorption maximum 410 nm). Only protein pleted at room temperature in ethanol, it may be appropriate thiols that are accessible to this water-soluble reagent are to heat the reaction. The catalyst is often Cu(I)Br, but alter modified. Alternatively, the Measure-iTTM Thiol Assay Kit natives include Cu(I)I or Cu(II)SO4 (with a reductant). Knor from Invitrogen can be used. et al. Synthesis of novel 1,4,7,10-tetraazacyclodecane-1,4,7, 10-tetraacetic acid (DOTA) derivatives for chemoselective In Vivo Testing in Suitable Tumor Models. attachment to unprotected polyfunctionalized compounds. 0317 Subcutaneous xenograft models using AR42J Chemistry, 2007: 13:6082-90. Click reactions may also be tumor-bearing female SCID mice will be generated. Briefly, run in the absence of any catalyst. Alternatively, the NH" AR42J cells (1x10'), will be injected subcutaneously into the group in DFO-B may be converted directly into an azide flanks of female SCID mice. The tumors will be allowed to group. grow 10-12 days until 0.5-0.9 cm in size. 0313 Synthesis of Tyr3-octreotate with an azide. Solid 0318 Radiolabeling of the DFO-nanoparticle by Zr is phase peptide synthesis (SPPS) of Tyr3-octreotate (FIG. expected to proceed in <15 min. at room temperature. Non 20A) will be performed on a peptide synthesizer. Briefly, the specifically bound Zr will be removed by addition of EDTA synthesis will involve the Fmoc (9-fluorenylmethoxycarbo followed by a gel filtration step. nyl) method as previous described for this peptide. Briefly, 0319 Receptor binding assays. The receptor binding the instrument protocol requires 25 umol of Subsequent assays will be performed using Zr-DFO-nanoparticles on Fmoc-protected amino acids activated by a combination of membranes obtained from AR42J tumors. The competing 1-hydroxybenzotriazole (HOBt) and 2-(1Hbenzotriazol-1- ligands, natZr-DFO-Nanoparticles and natZr-DFO-octreo yl)-1,1,3,3-tetramethyluronium hexafluorophosphate tate will be prepared by the reaction of high purity natural (HBTU). The Fmoc-protected amino acids will be purchased zirconium oxalate with DFO-octreotate and DFO-Nanopar commercially unless otherwise stated; the pre-packed amino ticles, respectively. Purity of the final products will be con acids will be obtained from Perkin-Elmer (Norwalk, Conn.), firmed by HPLC. IC50 values will be determined according while those unavailable in pre-packed form, such as the to previously published methods, using the Millipore Multi Damino acids and Fmoc-Cys(Acm) will be supplied by Screen assay system (Bedford, Mass.). Data analysis will be BACHEMBioscience, Inc. (King of Prussia, Pa.) or Nova performed using the programs GraFit (Erithacus Software, biochem (San Diego, Calif.). The azide group (for the "click” U.K.), LIGAND (NIH, Bethesda, Md.), and GraphPad chemistry) will be introduced into the peptide backbone via PRISMTM (San Diego, Calif.). coupling of an azide-containing acid to the N-terminus of the 0320 In vitro assays. The AR42J cells will be harvested peptide, while the peptide is still protected and attached to the from monolayers with Cell Dissociation Solution (Sigma resin (FIG. 20B). Chemical Co., St. Louis, Mo.) and resuspended in fresh 0314. Synthesis of functionalized nanoparticles. The next DMEM media at a concentration of 2x106 cells/mL. An step will be to conjugate both the DFO having an azide bond aliquot of about 0.3 pmol of Zr-DFO-nanoparticles will be and Tyr3-octreotate having an azide bond (FIGS. 21A and B) added to 10 mL of cells, incubated at 37°C. with continuous to the nanoparticle. “Click chemistry’ is highly selective, agitation. At 1, 5, 15, 30, 45, 60 and 120 min triplicate 200-uI. quantitative and can be performed very fast and using mild aliquots will be removed and placed in ice. The cells will conditions. The number of combined azide groups from DFO immediately be isolated by centrifugation, and the % uptake and Tyr3-octreotate will be controlled to never exceed the of the compound into the cells will be calculated. number of available triple-bonds; the triple bonds will always 0321 Biodistribution. A small amount of the Zr-DFO be in <5% excess. nanoparticles (~10 uCi 0.20 ug) will be injected intrave 0315 Functionalized nanoparticle characterization. Aver nously into each of the mice bearing palpable AR42J-positive age number of DFO chelates peptide per nanoparticle will be tumors. The animals will be sacrificed at selected time points determined by performing a standard isotopic dilution assay after injection (1, 4, 24, 48, 72 hours; n=4-5) and desired with Zr (or Ga). Zr will be produced on cyclotron and tissues will be removed, weighed, and counted for radioac purified. Briefly, 10 concentrations of 897r-oxalate will be tivity accumulation. Two additional control groups will be added to solutions containing a known amount of DFO-de studied at 1 h post-injections: (A) mice injected with the rived nanoparticles. Following a 30 min. room temperature native radiolabeled peptide Zr-DFO-octreotate (~10 uCi, incubation, the Solutions will be spotted on silica gel coated 0.20 ug), and (B) mice pre-injected with a blockade of Tyr3 glass plates, developed in 1:1 10% ammonium acetate-to octreotate (150 g) to demonstrate receptor-mediated accu methanol (with EDTA) and analyzed by radio-TLC. Whereas mulation of the Zr-DFO-nanoparticles. Tissues including the Zr-DFO-derived nanoparticles will remain at the origin, blood, lung, liver, spleen, kidney, adrenals (STTR positive) nonspecifically bound Zr bound to EDTA will migrate. The muscle, skin, fat, heart, brain, bone, pancreas (STTR posi percent labeling efficiency will be plotted as a function of tive), small intestine, large intestine, and AR42J tumor will be total nanomoles of Zr added to the reaction mixture. The counted. The percentage injected dose per gram (%ID/g) and number of chelates attached to the nanoparticle can then be percentage injected dose per organ (% ID/organ) will be determined from the inflection point of this curve. calculated by comparison to a weighed, counted Standard 0316 Average number of Tyr3-octreotate peptide per Solution. nanoparticle will be determined by assaying the disulfide 0322. In vivo NIRF imaging. Serial imaging will be per bridge of Tyr3-octreoate. Briefly, the disulfide bonds of the formed using the MaestroTM In Vivo Fluorescence Imaging Tyr3-octreotate can be cleaved quantitatively by excess System (CRI, Woburn, Mass.) at 0, 0.5, 1, 2, 4, 6, 12, 24, 48 sodium sulfite at pH 9.5 and room temperature. DTNB or and 72 hrs. At 72-hr, mice will be euthanized, and major US 2014/024821.0 A1 Sep. 4, 2014 tissues/organs dissected, weighed, and placed in 6-well plates iodine for radiotherapy, attached to tyrosine kinase inhibitors, for ex-Vivo imaging. Fluorescence emission will be analyzed or attached to chemotherapeutic drugs such as Taxol.) using regions-of-interest (ROIs) over tumor, selected tissues, and reference injectates, employing spectral unmixing algo Materials and Methods rithms to eliminate autofluorescence. Fluorescence intensi ties and standard deviations (SD) will be averaged for groups 0327 Internalization/uptake studies. Internalization of 5 animals. Dividing average fluorescence intensities of assays and colocalization studies were performed for identi tissues by injectate values will permit comparisons to be fying specific uptake pathways. made among the various tissues/organs for each injected nanoparticle conjugate. 0328 Melanoma cells, including human M21 and mouse 0323. In vivo small animal PET imaging. Small animal B16 cells (-2x10 cells/well), were plated in 8-well chamber PET imaging will be performed on a microPETR-FOCUSTM slides (1.7 cm/well) slides or 24 well plates (1.9 cm/well) system (Concorde Microsystems Inc., Knoxville Tenn.). Mice with a 12 mm rounded coverglass and incubated at 37° C. bearing the AR42J tumors (n=5 per group) will be anesthe overnight. To monitor targeted nanoparticle internalization, tized with 1-2% isoflurane, placed in a Supine position, and cells were incubated with cRGD-PEG dots (0.075 mg/ml) for immobilized in a custom prepared cradle. The mice will 3 hrs at 37°C. To remove unbound particles in the medium, receive 200 uCi of the Zr-DFO-octreotate-nanoparticle cells were rinsed twice with PBS. Confocal microscopy was complex via the tail vein and will be imaged side by side. performed on a Leica inverted confocal microscope (Leica Animals will initially be imaged by acquiring multiple, suc TCS SP2 AOBS) equipped with a HCX PLAPO: 63x1.2NA cessive 10-minute scans continuously from the time of injec Water DICD objective to assess co-localization of cRGD tion over a 1-hr time frame, followed by 10-min static data PEG-dots with organelle-specific stains or antibodies. acquisitions at 2, 4, 24, 48 and 72-hrs post-injection. Standard Images were analyzed using Image.J Software version 1.37 uptake values (SUVs) will be generated from regions of inter (NIH Image: http://rsbweb.nih.gov/i/). est (ROIs) drawn over the tumor and other organs of interest. 0329. Co-localization Assays/Dye-bound markers. In Co-registration of the PET images will beachieved in com order to identify endocytic vesicles involved in C dot inter bination with a microCAT-II camera (Imtek Inc., Knoxville, nalization, colocalization assays in living cells were per Tenn.), which provides high-resolution X-ray CT anatomical formed using dye-bound markers. Cells were coincubated images. The image registration between microCT and PET with nanoparticles and different dyes. The dyes include: 100 images will be accomplished by using a landmark registration nM. Lysotracker red for 30 min to label acidic organelles technique and AMIRA image display software (AMIRA, along endosomal pathway; 2 ug/mL transferrin Alexa 488 TGS Inc, San Diego, Calif.). The registration method pro conjugate to label recycling and Sorting endoSomes (clathrin ceeds by rigid transformation of the microCT images from dependent pathway); 1 mg/mL70 kDa dextran-FITC conju landmarks provided by fiducials directly attached to the ani gate at 37°C. for 30 minto label macropinosomes. mal bed. 0330 Co-localization/Organelle-specific antibodies. 0324 Pharmacokinetic measurements. The biodistribu Immunocytochemistry will be performed with known mark tion and dynamic PET data will provide the temporal concen ers for Golgi and lysosomes. For Golgi, Giantin (Abcam, tration of Zr-DFO-octreotate-nanoparticle in tissue which rabbit polyclonal. 1:2000) will be used for human cells; will allow for characterization of pharmacokinetic param GM-130 (BD Pharmingen, 1 lug/ml) will be used for mouse eters of the agent. cells. For Lysosomes, LC3B (Cell Signaling, rabbit poly 0325 Fluorescence microscopy and autoradiography of clonal, 0.5ug/ml) will be used. tissues ex vivo. Localization of nanoparticle conjugates in 0331. For Giantin or LC3B staining, cells will be blocked tissues will be performed on frozen sections. Imaging by for 30 minutes in 10% normal goat serum/0.2% BSA in PBS. microPET will allow us to evaluate fully the global distribu Primary antibody incubation (rabbit polyclonal anti-Giantin tion in tumors and other non-target tissues. Following the antibody (Abcam catalog it ab24586, 1:2000 dilution) or acute stage of the imaging trial, autoradiography will also be LC3B (Cell Signaling, C#2775, 0.5ug/ml) will be done for 3 performed on the tumors, and this data will be correlated to hours, followed by 60 minutes incubation with biotinylated both the PET imaging and histological results. Consecutive goat anti-rabbit IgG (Vector labs, cathi:PK6101) in 1:200 slices (~10 um) will be taken, alternating slices for autorad dilution. Detection will be performed with Secondary Anti iography and for histological analysis. These sections will body Blocker, Blocker D, Streptavidin-HRP D (Ventana also be analyzed by multichannel fluorescence microscopy in Medical Systems) and DAB Detection Kit (Ventana Medical the NIR channel. Systems) according to manufacturer instructions. Example 11 0332 For GM-130 staining, cells will be blocked for 30 min in Mouse IgG Blocking reagent (Vector Labs, Cath: Particle Internalization Studies MKB-2213) in PBS. The primary antibody incubation (monoclonal anti-GM130, from BD Pharmingen; 0326. The goal of this study is to evaluate the binding and Cathol O822, concentration 1 ug/mL) will be done for 3 internalization of the present nanoparticles to assess their hours, followed by 60 minutes incubation of biotinylated localization in Subcellular organelles and exocytosis. This mouse secondary antibody (Vector Labs, MOM Kit BMK will help study the fate of functionalized particles with dif 2202), in 1:200 dilution. Detection will be performed with ferent targeting moieties and attached therapies. For example, Secondary Antibody Blocker, Blocker D. Streptavidin-HRP both diagnostic nanoparticles (e.g., non-targeted PEG-coated D (Ventana Medical Systems) and DAB Detection Kit (Ven versus cRGD-PEG-coated nanoparticles) and therapeutic tana Medical Systems) according to manufacturer instruc nanoparticles (e.g., cFGD-PEG-nanoparticles attached to tions. US 2014/024821.0 A1 Sep. 4, 2014 36

0333 For temperature-dependent studies, nanoparticles tumor and SLN sites using the portable gamma probe (FIG. will be incubated with cRGD-PEG-nanoparticles at 4°C.,25° 23i), showed a 20-fold increase in activity within the SLN C., and 37° C. to assess fraction of surface bound versus relative to background signal. internalized particles. 0337 For real-time optical imaging of the lymphatic sys 0334 For exocytosis studies, nanoparticles (0.075 mg/ml) tem, a second subdermal injection of ''I-cRGDY-PEG-C will be incubated for 4 hours and chamber slides washed with dots was administered about the tumor site with the skin PBS, followed by addition of fresh media. At time intervals of intact, and the signal viewed in the color (FIG.24a) and Cy5.5 0.5, 1.0, 1.5, 2.5, 4.5, 8.0hrs, cells will be washed, typsinized, fluorescent channels (FIG. 24b). The adjacent nodal basin and fluorescence signal of cells and media measured by fluo was exposed, and fluorescent signal was seen in the NIR rimetry. In dose-response studies, cells will be incubated over channel flowing from the injection site (FIG. 24c) into the a range of concentrations and incubation times, and assayed main proximal (FIGS. 24c, 24d), mid (FIG. 24e), and distal using flow cytometry. In viability studies, cell viability will be (FIG. 24f) lymphatic branches, which drained towards the measured using a trypan blue exclusion assay before and after SLN (FIG. 24f). Smaller caliber lymphatic channels were incubation to assess for toxicity. In time-lapse studies, also visualized (FIGS. 24.d. 24e). The black-pigmented SLN, mechanism of nanoparticle internalization in living cells will viewed in dual-channel mode (FIGS. 24g, 24 h), was further be investigated after incubating cells with nanoparticle con exposed (FIG. 24i) prior to successive nodal excision (FIGS. jugates at different temperatures of incubation (4°C., 25°C., 24i-24 m). Fluorescence signal within the in situ (FIG. 24k) and 37° C.) using an inverted confocal microscope over a and ex vivo (FIG. 24m) nodal specimen was confirmed by 12-hr period at 20 min intervals. gamma emissions using the gamma probe (FIG. 24i), and seen to correspond to scattered clusters of tumor cells on Discussion low-power (box, FIG. 24n) and high-power (FIG. 24o) views from H&E-stained tissue sections. Positive expression of 0335 cRGD-PEG-dots and PEG-dots were found to co localize with Lysotracker Red in M21 and B16 cells suggest HMB45 was identified on low-power (FIG. 24p) and high ing uptake in the endosomal pathway (FIG.22). Data showed power (FIG. 24q) views, consistent with metastatic mela that these particles strongly colocalize with transferrin and Oa. dextran. Regardless of surface functionality and total charge, 0338. Surprisingly, and by contrast to the observed F nanoparticles (6-7 nm in hydrodynamic diameter) studied FDG findings, 'I-RGD-PEG-C dots were found to specifi cally discriminate between metastatic tumor infiltration and appeared to follow the same route. Time lapse imaging in both inflammatory processes in these miniswine. Mechanistic dif cell types demonstrated internalization of functionalized ferences in the behavior of these agents at the cellular and nanoparticles within a small fraction of the plated cells. Par Subcellular levels, as well as the presence of an integrin ticles were eventually delivered to vesicular structures in the targeting moiety on the particle Surface, may account for the perinuclear region. Colocalization assays with Giantin (or observed imaging findings. In multiple miniswine harboring GM-130) is not expected to show nanoparticle fluorescent pathologically-proven inflammatory changes due to granulo signal in the Golgi. matous disease (n=3), 'F-FDG failed to detect metastatic disease, while identifying inflammatory and other metaboli Example 12 cally active sites. These discrepant findings highlighted the Dual-Modality Silica Nanoparticles for ability of the particle tracer to selectively target, localize, and Image-Guided Intraoperative SLN Mapping and stage metastatic disease, while 'F-FDG failed in many cases Interventions to accurately stage cancer spread, instead identifying sites of inflammation. Dual-Modality Silica Nanoparticles for Image-Guided 0339. In a representative miniswine study illustrating Intraoperative SLN Mapping these findings, initial axial 'F-FDG PET-CT scans showed calcification within the left posterior neck on CT (FIG.25a). 0336. These studies were expanded to include optical corresponding to an area of intense activity on the 'F-FDG imaging using the portable ArteNISTM fluorescence camera PET (FIG. 25b). Low-power (FIG. 25c) and high-power system, along with radiodetection using the gamma probe, for (FIG. 25d) views of H&E stained tissue sections revealed performing real-time assessments of the draining tumor lym diffuse inflammatory changes, consistent with granuloma phatics and nodal metastases, as well as assessment of tumor tous disease. Intense 'F-FDG PET activity was additionally burden. In a representative miniswine (FIGS. 23a-23i), initial seen within the metabolically active bone marrow compart preoperative PET-CT scanning was performed using F ment of these young miniswine (FIGS. 25a, 25b). By con FDG and 'I-cRGDY-PEG-C dots using the foregoing imag trast, the particle tracer imaging study identified bilateral ing procedure. Axial CT images revealed a primary pelvic metastatic neck nodes. A right neck node on axial CT imaging tumor (FIG. 23a) and draining SLN (FIG. 23b), which were (FIG.25e) was seen to be PET-avid on co-registered PET-CT seen as areas of increased activity on the corresponding F (FIG. 25f); additional bilateral nodes on a more superior CT FDG PET scan (FIGS. 23c, 23d). These findings were con image (FIG.25g) were also hypermetabolic onfused PET-CT firmed 2 days later by dynamic PET-CT imaging about 5 (FIG. 25h). Moreover, left neck calcifications (FIGS. 25e, minutes after Subdermal, 4-quadrant injection of the particle 25g) showed no PET activity on co-registered scans (FIG. tracer about the tumor site; coregistered axial (FIGS. 23e. 25f 25h). Corresponding H&E-stained SLN tissue sections 23g) and coronal (arrows, 23f 23h) views demonstrate these revealed dark melanomatous clusters on low-power (box, findings. Following pre-operative Scanning, the skin overly FIG. 25i) and high-power views (FIG. 25i), seen to be com ing the SLN site was marked for intraoperative localization, prised of melanoma cells and melanophages. A single frame and the miniswine was transported to the intraoperative Suite. (FIG. 25k) selected from 3D PET reconstructed images again Baseline activity measurements, made over the primary illustrated multiple, bilateral PET-avid neck nodes and asso US 2014/024821.0 A1 Sep. 4, 2014 37 ciated draining lymphatic channels. Importantly, bulk activ needed are reliable endpoints for ablation Success and ity was seen in the bladder 1 hr post-injection without signifi unequivocal detection of residual disease in the postablation cant tracer accumulation over the liver region. period. 0340. The above findings were seen to better advantage on 0344 As a forerunner to performing future ablations of PET-CT fusion MIP images generated from dynamic imag metastatic liver lesions, a proof-of-concept radiofrequency ing data sets acquired over a 1 hour period after 'F-FDG ablation (RFA) study of a larger (i.e., 1-2 cm) SLN was (FIG. 26a) or local particle tracer administration (FIGS. 26b, performed in a miniswine with metastatic melanoma to evalu 26c). For F-FDG, a clear absence of nodal metastases is ate early treatment response in the presence of the particle noted, with diffusely increased activity seen within metaboli tracer. PET-CT imaging findings prior to and after RFA were cally-active bony structures. In contrast to these findings, correlated histologically. Following subdermal injection of '*'I-cRGDY-PEG-C dots detected bilateral metastatic neck 'I-cRGDY-PEG-C dots (-0.6 mCi) about the primary left nodes, along with draining lymphatic channels. pelvic tumor, an initial baseline coronal CT showed a 2.2x1.6 cm SLN (FIG. 27a) superior to the tumor site, which was Dual-Modality Silica Nanoparticles for Image-Guided PET-avid (FIGS. 27b, 27c). The hypermetabolic left pelvic Interventions: Treatment Response. tumor is also shown (FIG. 27b), noting additional particle tracer flow within a draining lymphatic channel on fused 0341 The ability of the particle tracer to discriminate PET-CT images (FIG. 27c, 27d). Additional serial CT scans metastatic disease from tissue inflammatory changes could were acquired to localize the node (FIG. 27e) prior to the potentially be exploited in a variety of therapeutic settings— ablation procedure and guide RFA probe insertion (FIG. 27f) either Surgically-based or interventionally-driven—as treat into the node (below level of crosshairs). On the correspond ment response assessments are often confounded by the pres ing pre-ablation co-registered PET-CT scan, the PET-avid ence of inflammatory changes, making interpretation SLN was seen just posterior to crosshairs (FIG.27g). A partial difficult. Image-guided interventions, such as therapeutic node ablation was performed for 12 minutes using a 2 cm tumor ablations, may specifically benefit from the innovative active tip RFA probe (Cool-tip ablation system, Covidien plc, coupling of new particle platform and imaging device tech Dublin, Ireland). Post-ablation PET-CT showed mildly nologies to (1) enable earlier post-procedural evaluation of reduced tracer activity in the ablation Zone, anterior to the response; (2) verify complete ablation or detect residual electrode tip (FIG. 27h). Pre- and post-ablation imaging find tumor representing treatment failure, and (3) improve tumor ings were confirmed histologically. H&E staining of pre Surveillance strategies. Locally ablative therapies, including ablated core biopsy tissue from the SLN confirmed diffuse microwave ablation, cryoablation, radiofrequency ablation metastatic tumor infiltration on low-power (FIG. 27i) and (RFA), and laser interstitial therapy, induce local thermal high-power (FIG. 27i) views. Post ablation, the extent of injury via an energy applicator insertion into tumors. These metastatic infiltration decreased on H&E stained nodal tissue, methods are typically employed as alternative options in seen on corresponding low- (FIG. 27k) and high-power views patients deemed ineligible for Surgical excision. Further, (FIG. 27). Coagulative necrosis and lymphoid tissue were patients undergoing ablative therapies are often poor Surgical also identified, along with multifocal hemorrhages (FIGS. candidates due to co-morbidities. Widely used in clinical 27k, 271, respectively). TUNEL stained high-power views practice, they offer a distinct advantage, as they can be per prior to ablation reveal scattered neoplastic cells (FIG.27m). formed percutaneously as outpatient procedures with signifi On post-ablation TUNEL staining, focal areas of necrosis cantly less morbidity, and may improve quality of life and (red) were seen on both low- (FIG.27m) and high-power (FIG. Survival in selected patient cohorts. 27o) views. 0342. Accurate post-therapy imaging, typically acquired 1-3 months after an ablation procedure, traditionally utilized CONCLUSIONS contrast enhanced Volumetric imaging, such as CT or MRI. These techniques suffer from a number of drawbacks. First, 0345 Lymph node metastases are a powerful predictor of they are limited to identifying the presence of abnormal outcome for melanoma. Early detection of micrometastases enhancement or growth in the size of the tumor area, consid in regional lymph nodes using SLN mapping may permit the ered primary indicators of residual tumor or recurrent disease. timely stratification of patients to appropriate treatment arms, Diffuse rim enhancement about the ablation Zone on post and can potentially improve patient outcomes. Although the procedural evaluations may be related to inflammation and current standard-of-care SLN mapping and biopsy tech hyperemia in the ablation Zone, and often does not necessarily niques rely on the use of radioactivity-based identification of represent residual tumor. Increasing enhancement, notably SLNs, a number of limitations of this technology exist. These irregular or nodular, is considered suspicious fortumor. How include low spatial resolution, reduced staging accuracy, ever, these interpretations are controversial, as an ablation absence of target specificity, slow tracer clearance that may Zone can look larger than expected for several months post obscure the Surgical field, and the lack of accurate intraop procedure, and enhancement might also reflect granulation or erative visualization to prevent injury to vital structures lying scar tissue formation. in close proximity to SLNs. 0343 Functional methods, such as 'F-FDG PET, have 0346. The recent introduction of newer generation, bio also been used to assess the efficacy and effects of locally compatible particle platforms that can be actively tailored and ablative procedures, but may suffer from an inability to accu refined to overcome these drawbacks according to key design rately discriminate tumor from inflammatory changes. Thus, criteria, while enabling selective probing of critical cancer interpretation of imaging changes (i.e., inflammation, tumor) targets, can offer important insights into cellular and molecu at the tissue level in response to ablative procedures using lar processes governing metastatic disease spread. The addi current morphologic or functional assessments, particularly tional adaptation of Such platforms for multimodality imag at early time intervals, is a significant challenge. What is ing could be used to advantage by the operating Surgeon or US 2014/024821.0 A1 Sep. 4, 2014 interventionalist to explore these processes in a variety of Duncan, R. The dawning era of polymertherapeutics, Nat Rev image-guided procedural settings. Drug Discov 2,347-360 (2003). Wagner, et al., The emerging 0347 One such dual-modality platform, a clinically-trans nanomedicine landscape, Nat Biotechnol 24, 1211-1217 lated integrin-targeting silica nanoparticle developed for both (2006). Scheinberg, et al., Conscripts of the infinite armada: optical and PET imaging, meets a number of key design systemic cancer therapy using nanomaterials, Nat Rev Clin criteria—Small size, Superior brightness, enhanced tumor tis Oncol 7, 266-276 (2010). Miyata et al., Polymeric micelles Sue retention, and low background signal—that make it an for nano-scale drug delivery, React. Func. Polym. 71, 227 ideal agent for SLN localization and staging during SLN 234 (2011). Lee et al., Multifunctional mesoporous silica biopsy procedures when coupled with portable, real-time nanocomposite nanoparticles for theranostic applications, optical camera systems. The ability to discriminate metastatic Acc Chem Res 44, 893-902 (2011). Schroeder et al., Treating disease from tissue inflammatory changes in melanoma mod metastatic cancer with nanotechnology, Nat Rev Cancer 12, els, which are often co-existing processes, may provide a 39-50 (2012). Rosenholm et al., Nanoparticles in targeted more accurate and reliable marker for the assessment of treat cancer therapy: mesoporous silica nanoparticles entering pre ment response in the future. Further investigation in a broader clinical development stage, Nanomedicine (Lond) 7, 111-120 set of cancer types and treatments is warranted using either (2012). Ashley et al. The targeted delivery of multicomponent Surgically-based or interventionally-driven therapies. cargos to cancer cells by nanoporous particle-supported lipid bilayers, Nat Mater 10,389-397 (2011). Vivero-Escoto et al., Example 13 Silica-based nanoprobes for biomedical imaging and thera nostic applications, Chem Soc Rev 41, 2673-2685 (2012). SLN Mapping of Prostate Cancer Tada et al. In vivo real-time tracking of single quantum dots conjugated with monoclonal anti-HER2 antibody in tumors 0348 Multimodal nanoparticle bearing HuJ591-F(ab')2 of mice, Cancer Res 67, 1138-1144 (2007). Yet, despite fragments are novel diagnostic probes for binding prostate extensive particle developments to date, no inorganic fluores specific membrane antigen (PSMA). By synthesizing par cent particle imaging probe has successively made the tran ticles bearing multiple F(ab')2 fragments, we can (1) enhance sition to the clinic as a targeted multifunctional platform binding affinity/potency due to multivalency effects and (2) technology. Altinoglu et al., Near-infrared emitting fluoro alter in vivo distributions, as clearance and uptake will be phore-doped calcium phosphate nanoparticles for in vivo dominated by particle kinetic behavior, rather than the anti imaging of human breast cancer, ACS Nano 2, 2075-2084 body itself. By maintaining particle size below or just at the (2008). Hilderbrand et al., Near-infrared fluorescence: appli renal cut-off of 10-nm diameter, renal clearance is promoted. cation to in Vivo molecularimaging, Curr Opin Chen Biol 14, Further, target-to-background ratios will increase on the basis 71-79 (2010). Choi et al., Design considerations for tumour of improvements in (1) and (2), potentially improving diag targeted nanoparticles, Nat Nanotechnol 5, 42-47 (2010). He nostic specificity and disease staging. et al., Near-infrared fluorescent nanoprobes for cancer Synthesis?Characterization of ''I-J591 F(Ab')2-Bound molecular imaging: status and challenges, Trends Mol Med Particles. 16,574-583 (2010). 0351. Here we describe that a first, ~7-nm fluorescence (0349. To generate PEGylated F(ab')2 constructs, 100ug of nanoparticle, modified as a hybrid (optical/PET) targeted F(ab')2 was added to an eppendorf tube in 100 ul PBS, fol platform by the attachment of radiolabels and cyclic arginine lowed by incubation with 4 ul of 2 mg/ml Traut’s reagent for glycine-aspartic acid-tyrosine (cRGDY) peptide, is not only 1 hat room temperature. Maleimide-PEG (6 mg) dissolved in well-tolerated in metastatic melanoma patients, but may pref buffer solution was then added. The solution was incubated erentially detect and localize presumed integrin-expressing overnight and purified with a PD-10 column prior to particle tumors in a microdosing regime with high signal-to-noise attachment. F(ab')2 fragments are being radiolabeled with ratios. No significant serum protein binding is observed, and iodine-124 ('''I) to create a dual-modality particle platform, the integrity of the particle and its Surface components are and the specific activity, purity, and radiochemical yield will maintained in vivo. Results on safety, pharmacokinetics, be derived. Particle size and concentration will additionally dosimetry and targeting Suggest general utility of this renally be assessed by FCS. excreted particle in cancer diagnosis and for potentially guid ing treatment planning. This dual modality probe constitutes Example 14 a platform that can be tailored to specific tumor types which may improve optical and/or PET-based lesion detection and In-Human Dual-Modality Silica Nanoparticles for cancer staging in humans, as well as drug delivery, potentially Integrin-Targeting in Melanoma leading to better and more personalized cancer care. 0350 Nanomaterials, in particular nanoparticle probes, 0352 We used an ultra-small (~7-nm diameter), dual-mo possess unique physicochemical and biological properties, as dality (optical/PET) inorganic nanoparticle probe for targeted well as surface versatility. By exploiting their surface versa molecular imaging of C. B. integrin-expressing cancers. This tility, biocompatible, multifunctional particles can be selec nanoparticle probe is the first FDA-investigational new drug tively modified with molecular markers that recognize and of its class and properties approved for first-in-human studies localize key cancer targets. Of increasing importance in (FIG. 28a). The fluorescent silica nanoparticle (Cornell, or C operative settings is the need for more robust optically-driven dots) covalently sequesters dye molecules in a core encapsu multifunctional particle probes which can improve real-time lated by a silica shell to prevent dye leaching and to enhance targeted detection of local/regional disease spread about the brightness and photostability. Ow, et al. Bright and stable primary tumor site, thus facilitating treatment. Real-time core-shell fluorescent silica nanoparticles, Nano Lett 5, 113 delineation of disease from critical neural and/or vascular 117 (2005). Burns, et al. Fluorescent silica nanoparticles with structures in intraoperative settings will also be paramount. efficient urinary excretion for nanomedicine, Nano Lett, 9. US 2014/024821.0 A1 Sep. 4, 2014 39

442-448 (2009). Absorption-matched spectra demonstrate primarily excreted by the kidneys, with both kidney and blad this per dye brightness enhancement as differences in inten der wall (after thyroid and tumor, see below), demonstrating sity between the emissions of the encapsulated and free dyes one of the highest 96 ID/g values by 72 hrs p. i. (FIG. 28d); as (FIG. 28b). A neutral layer of surface poly(ethylene glycol) is often the case for renally excreted radiopharmaceuticals, (PEG) chains enables attachment of a small number of cyclic the bladder wall received a higher average absorbed dose than arginine-glycine-aspartic acid-tyrosine (cRGDY) peptides other major organs and tissues. The detailed clinical protocol for potent and selective integrin receptor binding. Benezra, et (FIG. 28C) and the rationale for its design, is further al. Multimodal silica nanoparticles are effective cancer-tar described in Materials and Methods. geted probes in a model of human melanoma, J Clin Invest, 0355 These findings highlight the fact that renal, rather 121, 2768-2780 (2011). Activated integrins induce many than hepatobiliary, excretion is the predominant route of structural and signaling changes within the cell, in addition to clearance from the body. Efficient renal clearance will be regulating differentiation pathways, mediating adhesion contingent upon the design of ultra-small particle-based plat properties, and promoting cell migration, Survival, and cell forms or macromolecular systems that are on the order of the cycle progression. Hood et al., Role of integrins in cell inva effective renal glomerular filtration size cutoffs of about 10 sion and migration, Nat Rev Cancer, 2, 91-100 (2002). The nm or less. Choi, et al., Targeting kidney mesangium by attachment of nuclear imaging labels, such as '''I, via nanoparticles of defined size, Proc Natl AcadSci USA, 108, tyrosine residues amplifies signal sensitivity for serial PET 6656-6661 (2011). A small fraction of the administered activ imaging. The final product, a highly biocompatible and bio ity (less than 5%) was seen as uptake in the stomach and stable tumor-selective particle tracer ('''I-cRGDY-PEG-C- salivary glands of this patient, consistent with free iodine, dots), has previously provided a read-out of integrin receptor which was progressively cleared over the imaging period. status by PET imaging in human melanoma xenografts, thus The particle does not exhibit properties typical of reticuloen defining a distinct class of renally-cleared theranostic plat dothelial agents (i.e., technetium-99m sulfur colloid), whose forms for nanomedicine. Jokerst et al., Molecular imaging uptake reflects macrophage function, principally in liver. with theranostic nanoparticles, Acc Chem Res, 44, 1050-1060 Based on prior data acquired for cRGD radiotracers, no unex (2011). pected foci of activity were observed. Haubner, R., et al. 0353 A first-in-human clinical trial was initiated, employ Synthesis and biological evaluation of a (99m)Tc-labelled ing PET to quantitatively assess the time-dependent tumor cyclic RGD peptide for imaging the alphavbeta3 expression, uptake and biodistribution, radiation dosimetry, and safety of Nukdearmedizin, 43, 26-32 (2004). this agent in a cohort of five patients with metastatic mela 0356. Importantly, these properties point to the rather noma. Implicit in the rationale of using PET imaging is the unique pharmacokinetic behavior exhibited by this inorganic preclinical evidence that the particle radiotracer has no phar dual-modality imaging particle as a renally cleared agent. macologic, radiogenic, or other demonstrable biologic effect. Metabolic analyses of blood (FIG. 29a) and urine (FIG. 29b) Collins, J. M. Phase 0 clinical studies in oncology, Clin Phar specimens by gamma counting revealed at least an order of macol Ther: 85, 204-207 (2009). Kummar et al., Phase 0 magnitude drop in tracer activity over a 72-hour period, with clinical trials: recommendations from the Task Force on essentially no activity remaining at the end of this interval. Methodology for the Development of Innovative Cancer Particle activity was largely confined to the blood plasma Therapies, Eur J Cancer, 45.741-746 (2009). Following a fraction without evidence of significant serum protein bind single dose intravenous (i.v.) injection of approximately 185 ing (data not shown). RadioTLC analyses of plasma samples megabequerels (MBq) (~3.4-6.7 nanomoles, nmol) of the revealed a single peak through 24 h p. i. (FIGS. 29c–29e). '*'I-cRGDY-PEG-particle tracer (specific activity range corresponding to the intact radiolabeled nanoparticle. In 27.8-57.4 GBq/umol) into human subjects (FIG. 28a), three urine specimens, two peaks, one corresponding to the intact whole-body PET-CT scans were acquired over a 72-hour nanoparticle and the other to a more mobile species (identi period to assess pharmacokinetics, in addition to analyzing fied as free iodine), were seen over a 24-hour period (FIGS. metabolites in blood and urine specimens over a two week 29f29h). RadioTLCanalyses of the particle tracer (FIG.29i), interval by gamma counting and radio thin layer chromatog radioiodinated peptide (''I-cRGDY, FIG. 29.j), and free raphy (radioTLC). radioiodine ("I, FIG. 29k) confirmed that the first and sec 0354 Five patients had no adverse events and the agent ond peaks in the radiochromatograms corresponded to the was well tolerated over the study period. Pharmacokinetic intact nanoparticle and free iodine, respectively. Thus, these behavior, expressed as the percentage of the injected dose per findings suggested that no measurable loss of particle integ gram of tissue (% ID/g), Versus time post-injection and the rity occurred over the course of the study, even after excretion corresponding mean organ absorbed doses (FIG. 28d), were through the kidneys. comparable to those found for other commonly used diagnos 0357 Although tumor detection was not the goal of this tic radiotracers. Serial PET imaging of this representative PET microdosing study, Surprisingly, despite the low injected patient (FIG. 28d) showed progressive loss of presumed dose, there was evidence of tumor localization of the particle blood pool activity from major organs and tissues, with no tracer. In the first case, a whole-body PET-CT scan was appreciable activity seen by 72-hour post-injection (p. i.). acquired four hours after i.v. administration of ''I-cRGDY Whole-body clearance half-times in these patients were esti PEG-C-dots in a patient with anorectal mucosal melanoma. mated to range from 13-21 hours. Interestingly, there was no On coronal CT images (FIG. 30a), a large rounded area of notable localization in the liver, spleen, or bone marrow, in decreased density (arrowhead) was seen in the inferior left contrast to many hydrophobic molecules, proteins, and larger lobe of the liver, the site of a known metastatic lesion by prior particle platforms (>10 nm). Although patients were pre fluorodeoxyglucose (F-FDG) PET/CT imaging (data not treated with potassium iodide (KI) to block thyroid tissue shown). On the coronal PET (FIG. 30b) and co-registered uptake, a higher average absorbed thyroid dose was obtained PET and CT scans (FIG.30c), a rim of higher uptake circum in this patient relative to other tissues. Particles were also scribed this lesion (FIG. 30b arrowhead: FIG. 30c) and sub US 2014/024821.0 A1 Sep. 4, 2014 40 sequently cleared by the time of the 24-hour PET scan, sug 0360. The results suggest that the systemically injected gesting some preferential localization in this presumed particle tracer was well-tolerated and safe. Safety measures integrin-expressing metastasis. Significant activity was seen included monitoring of uptake in normal organs, as well as within the bladder, gastrointestinal tract (stomach, intes laboratory toxicity indicators. Secondary objectives were to tines), heart, and the gallbladder. In a second subject, a well estimate radiation doses and assess plasma and urine meta defined cystic lesion was seen in the right anterior lobe of the bolic activity. Safety assessments were based on dosimetry, pituitary gland by axial (FIG. 31a) and sagittal (FIG. 31b) lack of clinical symptoms, and the absence of any laboratory magnetic resonance imaging (MRI). indications of particle (drug) toxicity. 0361. The results obtained from this first-in-human clini 0358. This lesion, a stable finding on prior MRI scans, was cal trial point to a systemically injected particle tracer that presumed to be a pituitary microadenoma, an intracranial exhibited favorable and reproducible PK signatures defined neoplasm known to exhibit malignant properties, such as by renal excretion. In contrast to reticuloendothelial agents neoangiogenesis and progression into peritumoral tissues. (i.e., technetium-99m sulfur colloid) and macromolecules, Precise co-registration of this tracer-avid focus with multi Such as antibodies, there was no appreciable particle tracer planar MRI (FIGS. 31c,31d) and CT (FIG.31e, 31f) images accumulation within the liver, spleen, or bone marrow. Our confirmed its location within the anterior pituitary gland. data clearly indicate that a large proportion of the adminis Initially seen as a focus of intense activity, it progressively tered activity was eliminated via the urinary system (FIGS. increased in intensity over a 72-hour interval (arrow, FIGS. 28E,29B); activity concentrations in the urinary bladder were 31g-31i), accompanied by a corresponding decrease in Sur up to an order of magnitude higher than those in the liver, for rounding background marrow signal, thus yielding higher example. Based on the conservative assumption that all tumor-to-background ratios (i.e., tumor-to-brain (T/B) -6) hepatic activity is ultimately excreted via the hepatobiliary and tumor-to-liver (T/L)-2)) (FIG.31j). PET imaging results route, these data are consistent with ~90% of the administered may be explained on the basis of findings in a prior study activity being excreted via the urinary system and only ~10% showing increased CfB integrin-expression in the paren via the hepatobiliary route. In remaining organs/tissues, nota chyma of a Subset of adenomas, as well as enhanced integrin bly at early time points (2-4 hrs), residual activity largely expression levels in adenomatous stromal cells in relation to reflects that of the blood pool. normal connective tissue cells. Farnoud, et al., Adenomatous 0362 Targeted detection did not serve as a study endpoint, transformation of the human anterior pituitary is associated and dose escalation procedures to achieve maximum uptake with alterations in integrin expression, IntJ Cancer, 67.45-53 at sites of disease were therefore not incorporated into the trial (1996). design (i.e., no attempt was made to adjust particle doses to optimize tumor targeting). However, despite the low nano 0359 Our initial data support the notion that human PET molar amounts used, preferential localization and accumula studies with this targeted imaging vehicle are a rationale tion of the particle tracer occurred in several tumors. In one of approach towards detection and localization of presumed the patients with a presumed pituitary adenoma, PET imaging integrin-expressing tumors. We are planning dose escalation results showed progressive net accumulation of particle tracer methods to determine an optimal balance among safety, activity at the site of the lesion. The results of this study whole-body clearance, and tumor targeting efficiency in more Suggest that our systemically injected particles are well tol advanced clinical trials. Such PET-driven studies may also erated and exhibit a distinctly unique macromolecular PK permit accurate quantification of integrin receptor expression signature, where bulk renal clearance predominates without levels for achieving maximum targeting efficiency, as well as significant RES uptake. This is in contrast to many hydropho detection of alterations in these levels. Further, the use of bic molecules, proteins, and larger-particle platforms (>10 these quantitative molecularimaging tools can yield informa nm). This feature is highly atypical for nanoparticles (which tion on time-dependent changes in particle uptake and accu generally exhibit diameters greater than estimated renal cut mulation within tumors. Kelloff, G.J., et al., The progress and off values, and therefore little renal excretion and slower promise of molecular imaging probes in oncologic drug clearance) and warrants clinical evaluation of our ultraSmall development, Clin Cancer Res, 11, 7967-7985 (2005). For nanoparticle platform. the case of the pituitary adenoma, we were able to compute 0363 These results, along with essential data on safety, the cumulative uptake of particles within this lesion. Specifi pharmacokinetics, and dosimetry of the dual-modality C dot cally, we computed the fraction of the total injected activity imaging platform, Suggest the general utility of this human and the number of particles that accumulated at this site over microdosing technique in terms of yielding key tumor-spe a 72-hour imaging period. Using the measured maximum cific read-outs for cancer diagnostics. Such estimated tumor standardized uptake value (SUV, see Methods) of the accumulated particle tracer loads (or concentrations), along lesion (i.e., 46.5) at 72 hours post-injection (nearly a factor of with knowledge of cellular inhibitory response (i.e., ICso or ten higher than that in normal pituitary tissue), corrected for 50% inhibitory concentration), can potentially be used to partial Volume effects, as well as the approximate mass of the predict therapeutic dosing requirements for a given drug. lesion (i.e., product of the lesion Volume and an assumed density of 1 g/cm), and the patient's body mass, we found that, relative to an injected particle load of 2x10", roughly Methods 1.78x10' particles (0.01% of the injected dose, % ID) or 1 0364 Synthesis and Characterization of cFGDY-PEG-C- part per 10,000 accumulated at the lesion site. The standard dots. uptake values (SUV) is defined as the activity per gram of 0365 For details regarding the synthesis and characteriza tissue divided by the administered activity per gram of body tion of PEGylated and crGDY (Peptides International, Lou mass. Time-dependent changes in the '% ID/g and dosimetry isville Ky.) surface-functionalized fluorescent core-shell of this lesion, in relation to major organs and tissues, are silica nanoparticles (cRGDY-PEG-C-dots) encapsulating the shown in FIGS. 28C and 28d. organic dye, Cy5 (emission maxima ~650 nm, 2 dye equiva US 2014/024821.0 A1 Sep. 4, 2014

lents within the particle core), see an earlier publication of transmission data collected over the same region as for emis this group and references therein. Benezra, M., et al., Multi sion imaging, and registration of the serial data set was per modal silica nanoparticles are effective cancer-targeted formed using CT data sets. probes in a model of human melanoma, J Clin Invest, 121, 2768-2780 (2011). In brief, particles were prepared by a Imaging and Metabolic Analyses. modified Stöber-type silica condensation. Bogush, et al., 0369 For pharmacokinetic and dosimetric analyses, Preparation of monodisperse silica particles: Control of size regions-of-interest (ROIs) were drawn on PET imaging data and mass fraction, J Non-Cryst Solids, 104, 95-106 (1988). (AW Workstation, GE Healthcare, Ridgewood, N.J.) to Herz, et al., Large Stokes-shift fluorescent silica nanoparticles extract mean and maximum standard uptake values (SUVs) with enhanced emission over free dye for single excitation for all major normal organs and tissues, including brain, lung, multiplexing, Macromol Rapid Commun, 30, 1907-1910 left ventricle, liver, spleen, intestine, kidneys, bladder, (2009). Sadasivan, et al., Alcoholic solvent effect on silica muscle, breast, and tumor(s). For pharmacokinetic evalua synthesis—NMR and DLS investigation, J Sol-Gel SciTech tion, SUVs were converted to % ID/g values (i.e., SUV=% mol, 12, 5-14 (1998). Bifunctional PEGs were derivatized ID/g tissuexpatient body mass/100). Organ/tissue uptake data with silanes for attachment to the silica Surface and for pep was supplemented by time-activity data from the blood and tide coupling. cFGDY peptides containing the sequence urine. Venous blood and urine specimens were collected at cyclo-(Arg-Gly-Asp-Tyr) and bearing cysteine residues approximately 30-min, 3-hr, 24-hr, 72-hr, and up to 2 weeks (Peptide International) were attached to functionalized PEG p.i. of ''I-cRGDYPEG-C-dots. Following centrifugation of chains via a cysteine-maleimide linkage. Hydrodynamic whole blood specimens (4000 rpm, 10 min), plasma super radius, brightness, and concentrations of cFGDY-PEG natant, along with urine specimens, were assayed in a scin Cdots, as against free Cy5 dye, were analyzed on a Zeiss LSM tillation well counter (1480 Automatic Gamma Counter, Per 510 Confocor 2 FCS using HeNe 633-nm excitation. kin Elmer. Shelton Conn.) calibrated for '''I. RadioTLC Radiolabeling of cRGDY-PEG-C-dots. analyses were additionally performed on biological speci mens. RadioTLC analyses of the particle tracer, native pep 0366 Tyrosine residues were conjugated to PEG chains tide (crGDY) labeled with ''I, and free iodine (''I) served for attachment of radioiodine moieties (i.e., 'I. 'I). Her as controls to facilitate interpretation. Activities (counts per manson, G. Bioconjugate Techniques, (Academic Press, Lon minute, cpm) were converted to microcuries, decay-cor don, UK, 2008). Yoon, T. J., et al. Specific targeting, cell rected, and adjusted for Volumes aliquoted. Final values were sorting, and bioimaging with Smart magnetic silica core-shell expressed as % ID/g. Retention factor (R) values for the nanomaterials. Small, 2, 209-215 (2006). Radiolabeling of tracer were obtained and used for identification of the parent cRGDY-PEG-C-dots was performed using the IODOGEN compound and possible metabolites. method (Pierce). Piatyszek, et al., Iodo-Gen-mediated radio iodination of nucleic acids, Anal Biochem, 172, 356-359 Radiation Dosimetry. (1988). The radiolabeled product was eluted from PD-10 0370. The standard radiation dosimetry method is an columns and assayed using a dose calibrator (Capintec, Ram adaptation of that promulgated by the MIRD (Medical Inter sey N.J.) and radioTLC: specific activities of the '''I-bound nal Radionuclide Dosimetry) Committee, accounting for the particle fractions were on the order of 1450 millicuries (mCi)/ physical properties of the administered radionuclides ('''I) as umole and radiochemical purity was greater than 95%. well as the biological properties (pharmacokinetics and bio distribution) of the radiopharmaceutical in individual Patient Selection. patients. The '"I emissions and their respective frequencies and energies are obtained from the MIRD Radionuclide Data 0367 Metastatic melanoma subjects with histological and Decay Scheme publications. Serial whole-body PET confirmation of disease and harboring newly diagnosed or scans enabled derivation of normal-organ absorbed dose (rad recurrent tumor were eligible for the trial. Individuals who and rad/mCi) estimates using ROI-derived time-activity data. had medical illness unrelated to melanoma, which would PET scans were acquired with all parameters identical, preclude administration of the particle tracer, were excluded including the scan time. Using the patients total-body mass from the study. Potassium iodide solution (SSKI, 130 mg per (in kg) and the 70-kg Standard Man organ masses, the total day) was administered 2 days prior to and up to 2 weeks after body and organ ROI data (i.e., mean standard uptake values intravenous injection of the radio-iodinated particle tracer (SUVs) were converted to activities (i.e., fraction of the (I-cRGDY-PEG-C-dots, 185 MBq/5 ml) to block thyroid injected dose). The foregoing image-derived time-activity function. This protocol was approved by the Institutional data were fit to exponential functions using a least-squares Review Board of the Memorial Sloan Kettering Cancer Cen fitting algorithm and the resulting time-activity functions ter. Patients were tested for hemotologic, renal, and liver analytically integrated, incorporating the effect of physical function before and after PET and provided signed informed decay of '''I to yield the cumulated activities (or residence COnSent. times) in uCi-hr/uCi in the organs and total body. Cumulated activities were used to calculate '''I-labeled particle mean Image Acquisition and Processing. absorbed doses to the organs (rad/mCi) and effective dose (rem/mCi) for the 70-kg Standard Man anatomic model by 0368 Low-dose spiral CT scans were obtained per stan employing the OLINDA EXMMIRD program. Loevinger et dard procedure, followed by the acquisition of three whole al., MIRD Primer for Absorbed Dose Calculations, Society of body PET scans on a dedicated GE Discovery STE PET/CT Nuclear Medicine, New York, N.Y., 1991. scanner 4, 24, and 72 hours post-injection of the particle tracer. Positron emission data was reconstructed using the Serum Protein Binding Assays. ordered subsets expectation maximization (OSEM) algo 0371 Whole-blood specimens were collected in serum rithm. Images were corrected for attenuation using the CT separator tubes from a metastatic melanoma miniswine US 2014/024821.0 A1 Sep. 4, 2014 42 model (Sinclair Research Center, MO), followed by centrifu 0377 Human M21 cell survival studies, performed over a gation (4000 rpm, 10 min) to isolate the plasma fraction. An range of particle concentrations for a fixed incubation time of aliquot of serum was set aside for gamma counting; the 48 hr demonstrated no significant loss of cell viability (FIG. remaining fraction was treated with ethanol (200 proof, 36). Decon Labs, King of Prussia, Pa.), Vortexed until cloudy, and 0378) I-radiolabeled alpha-MSH conjugated nanopar placed on dry ice (5 min) to promote precipitation of serum ticles demonstrated bulk renal excretion over a 24hr period in proteins. Following centrifugation, Supernatant was collected both B16F10 and M21 murine xenograft models (FIGS.37A, for gamma counting and the pellet was washed repeatedly 37B); no appreciable particle tracer was measured at later with phosphate buffered saline and centrifuged (4000 rpm, 10 time points (i.e. >24 hrs). In addition, neither B16F10 nor min) to collect 100 uL aliquots of Supernatant for gamma M21 Xenograft models showed significant accumulation of counting (1480 Wizard 3"). the targeted particle probe in the reticuloendothelial system (i.e., not an RES agent), nor in the kidney (FIGS. 38A, 38B), Example 15 the latter organ typically a site that accumulates alpha-MSH non-specifically given its net positive charge. Thus, the Alpha-MSH-PEG-Cy5-Particles (MSH attachment of alpha-MSH to the particle probe significantly Peptide-Bound Particles) improved its renal clearance properties and eliminated accu 0372. The N-Ac-Cys-(Ahx)-D-Lys-ReCCMSH (or alpha mulation within the kidneys. MSH) peptide used for nanoparticle conjugation has the Example 16 structure shown in FIG. 32. It is attached to the nanoparticle via the N-terminal cysteine thiol. A spacer of two amino Integrin-Mediated Signaling and Modulation of hexanoic acid units separates the nanoparticle attachment Tumor Biology point from the D-Lys-ReCCMSH targeting molecule. The original ReCCMSH targeting molecule is shown in FIG. 33. 0379 Integrin signaling regulates diverse functions in It was designed to target radionuclide to melanoma tumors for tumour cells, including adhesion/spreading, migration, inva imaging and therapy. The MSH peptide analog could be Sion, proliferation and Survival. In several tumor types, directly radiolabeled with "Tc or Re (at the site of the including melanoma, the expression of particular integrins Re) or via a metal chelator appended to the amino terminus. correlates with increased disease progression and decreased The alpha MSH peptide analog shown in FIG. 32 is quite patient Survival. Integrin-mediated adhesive interactions different from the original MSH analog shown in FIG. 33. have identified novel integrin functions in cell survival The original MSH molecule could not be attached to a nano mechanisms and in the activation of divergent signaling path particle. The nanoparticle MSH peptide contains a free amino ways. It is well known that binding of peptides (or clusters of group containing side chain for radiolabeling. This is cur peptides), containing the RGD sequence, to integrin receptors rently a D-Lysine but could be an amino group terminated leads to cross-linking or clustering of integrins which, in turn, modulates the above processes. It is not clear, however, side chain of 1 to many carbons in length. whether nanoparticles bearing multiple RGD peptide ligands 0373 The conjugation of the N-Ac-Cys-(Ahx)-D-Lys can additionally trigger Such integrin signaling events, as this ReCCMSH (or alpha-MSH) to the nanoparticle allows the will reflect a complex dependency on multiple particle-based nanoparticle to target and bind melanoma cells and tumors. factors (size, charge, composition, Surface chemistry, ligand The resulting particles, or alpha-MSH-PEG-Cy5-C dots were number/type), tumor (or endothelial) cell type, cell receptor about 6-7 nmi.d. using FCS and contained, on average, about density, and particle dosing. Our dose-response Studies dem 2.6 dyes per particle. The number of alpha-MSH ligands per onstrate modest activation of divergent signaling pathways particle was estimated at <10. upon cell exposure to particle concentrations greater than 100 0374 Competitive binding studies using N-Ac-Cys-(Ahx) nM which, in turn, promote M21 and HUVEC cell migration, -D-Lys-ReCCMSH (or alpha-MSH) conjugated particles: In proliferative activity, and alter adhesion properties. a competitive binding assay with a melanocortin-1 receptor Binding of cRGDY-PEG-C-dots to M21 and HUVEC Cells agonist (I-NDP), the alpha MSH conjugated nanoparticles The CfB integrin receptor plays a key role invascular remod had an ICs for cultured B16/F1 melanoma cells of 6.6x10' eling and angiogenesis, demonstrating increased expression M (FIG. 34A), while a scrambled sequence version of the on endothelial and tumor cell surfaces for a variety of tumor molecule had an ICs of 2.3x107M (FIG.34B). In addition, cell lines. This leads to the use of this receptor as a target for there was a 3 order of magnitude difference in binding. For diagnostic and therapeutic purposes. In our case, the cFGDY reference in the same type of competitive binding assay, the PEG-C dots were tested for their ability to bind to melanoma original DOTA-ReCCMSH had an ICs of 2.1 x 10 M. cells (M21) or to human umbilical vascular endothelial cells 0375. The N-Ac-Cys-(Ahx)-D-Lys-ReCCMSH (or (HUVEC) as a function of concentration and time. Initial alpha-MSH) peptide-bound nanoparticle conjugate had bet dose-response studies showed a progressive increase in the ter affinity for the B16/F1 cells than the original DOTA binding ofcRGDY-PEG-C dots to M21 and HUVEC cells as ReCCMSH and much larger affinity than the scramble pep a function of concentration by flow cytometry (FIG. 39, FIG. tide nanoparticle. 47A). Particle binding demonstrated saturation at about 100 0376 Dose-response data was additionally obtained as a nM for both cell lines, with mean % gated values of about function of targeted particle concentrations (FIG. 35A) and 80% for M21 cells and 96% for HUVEC cells. Dose-response incubation times (FIG.35B) for both B16F10 and M21 mela behavior was additionally investigated as a function of par noma cell lines. Saturation concentrations for these lines, ticle incubation times for both cell types after incubating with based on flow cytometry studies, were found to be on the 100 nM cRGDY-PEG-C dots. Maximum binding was order of ~100 nM for these two cell types, and at least 2 hr observed at 2 hours post-incubation, remaining relatively incubation times were needed to maximize binding. constant thereafter (FIG. 47B). US 2014/024821.0 A1 Sep. 4, 2014

Endocytosis and Intracellular Trafficking ofcRGDY-PEG-C- survival (FIGS. 49A, 49B). Further, no time-dependent dots decreases in absorbance were found following multi-dose To elucidate the nature of the pathway/s utilized by cFGDY (n=5) addition of 100 nMcRGDY-PEG-C dots to M21 and PEG-C-dots following their incubation in M21 cells— HUVEC cells, suggesting no alteration in the proliferative whether this is an O?3 integrin receptor-mediated and/or properties of cells (FIGS. 49C, 49D). non-specific uptake process, we examined the temperature Activation of the FAK Pathway by crGDY-PEG-C-dots dependent uptake of these particles for a 4 hour incubation The binding of a ligand to the C.B. integrin receptor is known time at three temperatures: 4, 25°C. and 37°C. The results, to initiate signal transduction pathways. Upon the binding, summarized in FIG. 39A, C indicate an increase in cell integrin clustering occurs which leads to autophosphoryla associated cRGDY-PEG-C-dots at 37° C. as compared to 25° tion of the non-receptor kinase FAKattyrosine 397, a binding C. or to 4°C. in both cells line tested. Moreover, internaliza site for the Src family kinases. Recruitment of Src kinase tion ofcRGDY-PEG-C-dots was partially blocked in the pres results in the phosphorylation of FAK at tyrosine 576/577 and ence of excess (x250) antibody to O?3 receptor in M21 and FAK at tyrosine 925 in the carboxyl-terminal region. The HUVEC cells (FIGS. 39A, 39C), suggesting a component of phosphorylation of FAK at tyrosine 397 is also known to receptor-mediated binding. Additionally, uptake in C. B activate numerous signaling cascades and downstream path negative M21L cells was roughly a factor of 4- to 8-fold lower way intermediates, such as the Ras-Mitogen Activated Pro than that seen with O?3-expressing cells at both 37° C. and tein (MAPK) signaling, which induces activation of Raf7 25°C. (FIG. 39B), respectively. MEK/Erk and phosphatidylinositol 3-kinase (PI3K)/AKT 0380. To further characterize the cellular compartments pathways (AKT, mTOR, S6K) (FIG. 40A). involved in cFGDY-PEG-C dot internalization, we per 0381 To determine whether CfB integrin-binding formed colocalization assays in M21 cells with cRGDY cRGDY-PEG-C-dots modulates the activity of these path PEG-C dots and biomarkers of different endocytotic vesicles. ways, we treated serum-deprived (0.2% FCS) M21 cells with Internalization of the targeted particle (~1 uM, red, 4-hr incu 100 nM of cKGDY-PEG-C-dots for 2 hrs at 37° C.; serum bation) was sensitively detected by an inverted confocal deprived cells treated with 0.2% FCS alone served as con microscope (Leica TCSSP2AOBS) equipped with a HCXPL trols. Western blot analyses of lysates from particle-exposed APO objective (63x1.2NA Water DICD) (FIG. 39D). Using cells revealed enhancement of the phosphorylation levels of endocytotic markers LysoTracker Red (100 nM, green) (FIG. multiple protein intermediates: pFAK-397 and pFAK-576/ 39D) and transferrin-Alexa488, uptake into acidic endocytic 577, Src, pMEK, pErk, and AkT (FIG. 40B), which suggested structures was confirmed, suggesting clathrin-dependent activation of downstream signaling pathways. These find pathway activity and gradual acidification of vesicles. FIG. ings, depicted graphically as normalized intensity ratios, have 39D shows co-localization data between the particle and been expressed relative to their respective total protein levels, acidic endocytic 30 vesicles (yellow puncta). Uptake into and normalized to corresponding values measured under con macropinocytes was also observed with 70-kDa dextran trol conditions (FIG. 40C). Incubation of cells with PEG-C FITC which co-localized with cRGDY-PEG-C dots; this find dots did not augment phosphorylation levels of the proteins ing Suggested a second pathway of internalization. Nuclear tested (data not shown). counterstaining (blue) was done with Hoechst. No particles We evaluated whether the observed activation of downstream entered the nucleus. Time lapse imaging confirms particle signaling pathways was dependent on the phosphorylation of uptake into M21 cells and co-localization with the lysosomal FAK at tyrosine 397 by blocking this pathway with a small marker, Lamp1. molecule inhibitor, PF-573228. This inhibitor interacts with Competitive Of Integrin Receptor Binding and Molecular FAK in the ATP-binding pocket and effectively blocks both Specificity the catalytic activity of FAK protein and subsequent FAK Competitive binding assays showed blocking of receptor phosphorylation on Tyr. Following the addition of two mediated particle binding in M21 and HUVEC cells (FIG. concentrations of PF-573228 to serum-deprived M21 cells 48A) by 80%-85% in the former and 30-40% in the latter previously exposed to 100 nMcRGDY-PEG-C dots, Western using excess (x50-x100) cRGDY peptide and gamma count blot analyses revealed inhibition of the phosphorylation of ing of the radiolabeled particle tracer (124I-PEG-cRGDY-C FAK on Tyr and Src (FIG. 41A), graphically displayed in dots). By contrast to cRGDY-PEG-C dots, significant reduc FIG. 41B Inhibition of MEK or Erk phosphorylation was tions were seen in the magnitude of receptor binding in M21 observed only at the higher inhibitor concentration (FIGS. (-30%-43%) and HUVEC (-13%-27%) cells after incuba 41A, 41B). tion with non-targeted (i.e., PEG-C dots) particle probes by Effect of cRGDY-PEG-C dots on Cellular Migration flow cytometry (FIG. 48B). The O?3 integrin receptor, which is highly expressed on Influence of cFGDY-PEG-C dots on Cell Viability and Pro many types of tumor and angiogenic cells, is known to modu liferation late a number of downstream biological processes, including To demonstrate that cRGDY-PEG-C dots did not adversely cell migration, adhesion, proliferation, invasion, and angio affect cell survival and proliferation, G0/G1 phase-synchro genesis. In the following experiments, we sought to deter nized M21 and HUVEC cells were exposed to a range of mine whether cRGDY-PEG-C dots alter the migration of particle concentrations (25-200 nMcRGDY-PEG-C dots: 15 M21 and HUVEC cells. An initial set of experiments exam min or 24 hr) and incubation times (0-93 hrs) in serum ined time-dependent changes in M21 cell migration, as supplemented media (2%, 10% FBS) at 37° C., and time reflected in mean areas of closure, using time-lapse imaging dependent changes in absorbance were measured using an at three Successively higher particle concentrations (0 optical plate reader (a 440 nm). Relative to controls (i.e., nM-400 nM; 37°C.) during a 96-hour time period (FIG. 42A, serum-Supplemented media), absorbance measurements i-XX). Statistically significant increases in mean area closure were seen to be relatively constant over the range of particle were observed over a 96-hr period, as a percentage of the concentrations tested, Suggesting no significant loss of cell baseline values (t=0), which were relatively constant for the US 2014/024821.0 A1 Sep. 4, 2014 44 particle concentration range used (i.e., ~87%-92%), as com mented media. Over this range, the percentage of cells in the pared to control samples (73%, p<0.05,) (FIG. 42A, B). No S phase rose by 11%, with statistical significance achieved at significant changes were seen at earlier time intervals (FIG. 100 nM (p<0.05) and 300 nM (p<0.005) in relation to con 42B). trols (FIGS. 46A, 46B). Corresponding declines of 6% and Incubation of HUVEC cells (37° C., 20 hours) with a range of 5% were seen in the G1 and G2 phases of the cell cycle, cRGDY-PEG-C dot concentrations of (100-400 nM) in the respectively. Taken together, these data indicate an enhance presence of 0.2% FCS showed a statistically significant ment in S phase in the presence of cRGDY-PEG-C dots. increase in the mean area closure for a concentration of 400 nM (i.e., 34%) (FIGS. 43A, 43B), as compared to 19% (p<0. Materials and Methods 05) for non-particle exposed cells. No appreciable change in migration was observed for the lower particle concentrations Reagents, Antibodies and Chemicals. used (FIGS. 43A, 43B). Particle-exposed cells were seen to (0382 RPMI 1640, fetal bovine serum (FBS), penicillin, exhibit higher migration rates as compared to control cells. streptomycin and HBSS (without calcium and magnesium We further showed that increases in cell migration rates were containing 0.25% trypsin and 0.05% EDTA) were obtained attenuated by the addition of FAK inhibitor, PF-573228. Ini from the Core Media Preparation Facility, Memorial Sloan tial phase contrast images were acquired after incubating Kettering Cancer Center (New York, N.Y.). Anti-polyclonal HUVEC cells with 400 nM particles over a 24 hour time rabbit: phospho-Erk1/2 (p-Erk1/2'' ''), phospho interval (FIG. 44A, i-viii), and mean area closure was deter AKT (p-Akt')phospho-Src (p-Src')phospho-p70S6 mined and graphically displayed relative to serum alone (p-p70S6 '''), phospho-MEK1/2 (p-MEK1/2 °7'), (FIG. 44B). Percentage change in mean areas of closure rela phosho-FAK-'7, phospho-FAK-'777 phosho-FAK tive to controls, and before and after addition of an inhibitor, '', Erk, AKT, Src, p70S6, MEK1/2 (47E6) and FAK were was seen to decrease from +34% to +3%. Statistically signifi obtained from Cell Signaling Technology (Danvers, Mass.). cant differences were found between values for particle-ex Goat anti-rabbit IgG, Goat anti-mouse IgG horseradish per posed cells without inhibitor (p<0.03) and particles treated oxidase (HRP) conjugates were acquired from Santa Cruz with different inhibitor concentrations (250 nM, 500 nM; Biotechnology (Santa Cruz, Calif.). Propidium iodide, p-0.001) relative to serum-deprived controls; differences RNase A, Transferrin-Alexa Fluor 488 conjugate, FITC-Dex between the first two groups were also statistically significant tran, Fluorescein, LysoTracker Red DND-99, LysoTracker (FIG. 44C). Green DND-26, pHrodo Red Dextran and Hoechst were pro Effect of cRGDY-PEG-C dots on Cellular Adhesion and cured from Invitrogen-Life Technologies (Carlsbad, Calif.). Spreading PF-573228 was obtained from TOCRIS bioscience (Ellis The RGD tripeptide is known to be a component of extracel ville, Mo.). Cyclo (Arg-Gly-Asp-D-Tyr-Cys) was from Pep lular matrix constituents, fibronectin and vitronectin. A com tides International (Louisville, Ky.). petitive binding assay was performed using M21 cells (10 Synthesis of cRGDY-PEG-C Dots and PEG-dots. cells/well) pre-incubated (25°C., 30 minutes) with or without 0383 Fluorescent particles, containing the organic dye 400 nMcRGDY-PEG-C dots and fibronectin, after transfer Cy5, were prepared by a modified Stöber-type silica conden ring cells to fibronectin-coated wells. About ~60% of the cells sation, as previously described. Tyrosine residues were con that were not pre-incubated with cRGDY-PEG-C-dots jugated to PEG chains for attachment of radioiodine.cRGDY attached and spread on fibronectin coated plates in the first 30 peptides containing the sequence cyclo-(Arg-Gly-Asp-Tyr), minutes. By contrast, a very low percentage (15%) of M21 and bearing cysteine residues (Peptide International), were cells pre-incubated with 400 nM cKGDY-PEG-C-dots attached to functionalized PEG chains via a cysteine-male attached and spread on the fibronectin coated well, even 120 imide linkage. The number of cRGD ligands per nanoparticle minutes after seeding (FIGS. 45A, 45B). Average cell counts was empirically calculated. of elongated (spreading cells) versus rounded cells over a 2 hour period revealed that in the absence of particles (i.e., Mechanism of PEG Attachment to the C Dot Surface. control condition), cell spreading (elongated' cells) was observed in the majority of cells, while a predominantly 0384 Bifunctional PEGs, MAL-dPEG(R12-NHS ester rounded appearance was seen in the case of particle-ex (Quanta Biodesigns, Ltd) were derivatized with silanes, spe posed cells (FIG. 45A). These results are graphically depicted cifically 3-aminopropyl triethoxysilane (Gelest), for attach in FIGS. 45B and 45C, respectively. Quantification of the ment to the silica Surface and for peptide coupling via reac optical densities of cells after addition of methylene blue tions between the Sulfhydryl groups and maleimide moieties (absorbance) revealed that cells which attached and spread on of the derivatized PEGs. In addition, methoxy-capped PEG fibronectin (i.e., no particle pre-incubation) took up two times chains were added to the particle Surface using functional more methylene blue than particle-exposed cells (FIG. 45D). organosilicon compounds (Si compounds), specifically We observed the same phenomena if vitronectin-coated wells (MeO)3Si-PEG (Gelest), according to modified protocols. were used (data not shown). Altogether, these data demon Briefly, (MeO)3Si-PEG was added, at approximately three strate that cRGDY-PEG-C-dots enhanced the migration and molar excess, to particles in a waterfalcohol basic mixture spreading of cells through the binding to the integrin CfB (~ 1:5 V/v), and the mixture stirred overnight at room tempera receptor found on M21 and HUVEC cells. ture. Influence of cFGDY-PEG-C dots on Cell Cycle in M21 Cells Hydrodynamic Size and Relative Brightness Comparison Since integrin receptors are involved in survival and prolif Measurements by Fluorescence Correlation Spectroscopy eration of the cells, we analyzed the effect of cRGDY-PEG C-dots on cell cycle. Go/G-phase-synchronized M21 cells (FCS). were incubated for 48 hours using two concentrations of 0385. The hydrodynamic radius, brightness, and concen cRGDY-PEG-C-dots (100 nM,300 nM) in 0.2% FCS supple trations of cRGDY-PEG- and PEG-dots, as against free Cy5 US 2014/024821.0 A1 Sep. 4, 2014 dye, were initially determined by dialyzing these particle NaOH. Radioactivity was assayed using a 1480 Automatic samples to water, diluting into physiological saline (0.15 M Gamma Counter (PerkinElmer) calibrated for iodine-124. NaCl in H2O), and analyzing the resulting specimens on a Zeiss LSM 510 Confocor 2 FCS using HeNe 633-nm excita Western Blot (WB): tion. The instrument was calibrated with respect to particle 0390 M21 cells (1x10° cells/six wells plate) were grown size prior to all measurements. Average hydrodynamic sizes in six wells coated with collagen (10 mg/ml), and made of the dye and particle species were estimated based on dif quiescent by growing under serum-deprived conditions. The fusion time differences, while relative differences in bright medium was then changed to 0.2% FCS, and different con ness were assessed using count rates per molecule/particle. centrations (25-400 nM) of cRGDY-PEG-C-dots were added (37°C., 0.5-8 hours). Cells were rinsed twice in ice cold PBS, Cells and Cell Culture: collected by trypsinization, and the pellet re-suspended in 0386 Human melanoma cell line M21 and M21 variant lysis buffer (10 mM Tris, pH-8.5, 150 mM. NaCl, 1 mM M21L (C. negative) were obtained from D. A. Cheresh (Uni EDTA, 1% Triton X-100: ACROS organics, NJ), 1% Na versity of California, San Diego, Calif.). Cells were main deoxycholate, 0.1% SDS, CompleteTM protease inhibitors tained in RPMI 1640 supplemented with 10% fetal bovine (Roche, Indianapolis, Ind.), and phosphatase inhibitor cock serum, 2 mM L-glutamine and Penicillin Streptomycin. tail Tablet—PhosSTOP (Roche, Indianapolis, Ind.). Lysates Human Umbilical Vein Endothelial Cells (HUVECs) were were centrifuged (10 min, 4°C.). Protein concentrations were obtained from LONZA (Walkersville, Md.) cultured in determined by the bicinchoninic acid assay (BCA, Thermo EGM-2 medium containing 2% FBS, and growth factors Scientific, Rockford, Ill.). A 50-ug protein aliquot of each LONZA. fraction was separated by 4-12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and In Vitro Cell Binding Studies Using Optical Detection transferred to a PVDF membrane (Invitrogen, Carlsbad, Methods: Calif.). Membranes were blocked with 5% non-fat dry milk (0387 To assay particle binding for M21, M21-L or (Bio-Rad, Hercules, Calif.) in Tris Buffered Saline (TBS)- HUVEC cells, 24-well plates were coated with 10 ug/ml Tween 0.1%, and signal visualized by ECL chemilumines collagen type I (BDBiosciences) in PBS, incubated at 37°C. cence (Thermo Scientific, Rockford, Ill.) or Immobilon West for 30 minutes, and washed once with PBS. Cells (3.0x10 ern, (Millipore Billerica, Mass.) after applying primary cells/well to 4.0x10 cells/well) were grown to confluency. (1:1000) and secondary (1:2000-1:5000) antibodies. Differential binding of cRGDY-PEG-C-dots to M21 or Cell Cycle Analysis: HUVEC cells was evaluated over a range of incubation times (up to 4 hours) and particle concentrations (10-600 nM) using 0391 Go/G-phase-synchronized M21 cells were incu flow cytometry. After incubation, cells were washed with bated for 48 hours using two concentrations (100 and 300 RPMI 1640 media/0.5% BSA, detached using 0.25% trypsin/ nM) of cRGDY-PEG-C-dots after changing the medium to EDTA, pelleted in a microcentrifuge tube (5 minutes at 153 g, 0.2% FCS. Following trypsinization, cells were centrifuged 25° C.), re-suspended in BD FACSFlow solution (BD Bio (1200 rpm, 5 min), and the pellet suspended in PBS, followed Sciences), and analyzed in the Cy-5 channel to determine the by fixation with 70% ethanol (4°C., 0.5-1 hour). Cells were percentage of particle-bound probe (FACSCalibur, Becton successively re-suspended in 1 ml PBS containing 1% FCS Dickinson, Mountain View, Calif.). and 0.1% Triton X-100, 200 ul PBS containing 25 g/ml propidium iodide, and 100 mg/ml RNaseA (4°C., 60 min Internalization Study: utes). Cell cycle analysis was performed by flow cytometry, 0388. The experiment was done as above but incubated for FACSCalibur, (Mountain View, Calif.) and Phoenix Flow 4 hours at three different temperatures: 4°C., 25° C. and 37° MultiCycle software (Phoenix Flow Systems, Inc., San C. Co-incubation of excess (x250) cRGDY anti-human inte Diego, Calif.). grin O?3 fluorescein-conjugated antibody (Millipore, Bil lerica, Mass.) with cRGDY-PEG-C-dots was used for recep Cell Proliferation: tor blocking to assess specificity. Assays were performed with 0392 Cells were split (1x10 cells/well) in a 96-well plate fixed and live cells. For fixed M21 cells, inverted confocal coated with collagen as described in the in-vitro cell binding microscopy (Leica TCS SP2 AOBS) equipped with a HCX studies. Different concentrations of cRGDY-PEG-C-dots PLAPO objective (63x1.2NA Water DIC D) was used and were added (25-200 nM) for 24-48 hours at 37° C. Then, 20 time-lapse imaging was used to track live M21 cells. Imaging ml of the proliferation reagent WST-1 (Roche, Indianapolis, was acquired following co-incubation of targeted (or control) Ind.) was added to the plate (37° C. 1 hour). For determina particles at several concentrations with the following dye tion of optical densities, we used a SpectraMax5 micro plate markers) for 4 hours: 70-kDa dextran—FITC conjugate (1 reader (Molecular Devices, Sunnyvale, Calif.). Absorbance mg/mL, 37°C. for 30 min: Invitrogen) to label macropino was measured at 440 nm. somes, Lysotracker Red (100 nM; Invitrogen) to label the endocytotic pathway (i.e., acidic organelles), and transferrin Migration Assay: Alexa488 (2 ug/mL. M21 Cells: Competitive Binding Studies: 0393 M21 cells were seeded (6x10" cells/well) using a 0389. To assay specific binding M21 cells were incubated migration kit (OrisTM Collagen I coated plate, PLATYPUS (25°C., 4 hours) with '''I-cRGDY-PEG-C-dots (25 nM) and TEC). Twenty-four hours after seeding the cells, stoppers in excess cKGDY peptide. Cells were then washed with RPMI the plate were removed. Fresh culture media (100 ul) supple 1640 media/0.5% BSA, and dissolved in 0.5 ml of 0.2 M mented with 0.2% FBS was introduced and cRGDY-PEG-C- US 2014/024821.0 A1 Sep. 4, 2014 46 dots were added at several concentrations: 25, 100 and 400 Zero (following particle addition and media replacement) as nM. Every 24 hours thereafter, media was replaced, along follows: difference in area at a given time point (24, 48, 72 or with new particles, over a 72 hr time interval. Prior to incu 96 hr) and at time Zero divided by the same area at time zero bating the plate at 37° C. overnight, time Zero images were multiplied by 100. The resulting values were averaged and a captured by the Axiovert 200M microscope (Carl Zeiss) using standard error computed for each group. a 5x (0.15NA) objective and using a scan slide module in the 0397 Statistics: Metamorph software (molecular devices, PA). Serial micros 0398 All graphical values are plotted as meant-SE, except copy was then performed and images captured every 24 hrs where noted. One-tailed Student's t-test was used to test the for a total of 96 hours post-incubation. The data were ana statistical significance of differences in cellular migration lyzed by using Image.J software. HUVEC cells: HUVEC cells between HUVECs or M21 cells incubated with serum alone were additionally seeded (5x10" cells/well) and, 24 hours or cRGDY-PEG-C dots. One-way analysis of variance later, incubated with several particle concentrations (100, (ANOVA) was used to perform statistical pair-wise compari 200, and 400 nM) after replacement of the media. A similar sons between the percentage of M21 cells in S phase that were microscopy procedure was performed as that for M21 cells, incubated with serum alone, 100 nM or 300 nM cRGDY with serial imaging acquired 20 hours later. PEG-C dots. We assigned statistical significance for all tests at P3O.O5. Adhesion and Spreading Assay: Example 17 0394. The effect ofcRGDY-PEG-C-dots on the binding of M21 cells to fibronectin coated plates was evaluated by ini Sphingomyelin Liposomes for Enhanced Tumor tially coating 96-well micro titer plates with fibronectin in Delivery of Silica Diagnostic/Therapeutic Particles PBS (5 g/ml), followed by 200ul RPMI/0.5% BSA (37°C., 1 hour). Cells (1-3x10" cells/100 ul/well) were incubated 0399. The therapeutic potential of sub-10 nm particles (25° C., 30 minutes), with or without 400 nM of cRGDY (e.g., C dots) as drug delivery vehicles is under investigation. PEG-C-dots in RPMI/0.1% BSA, and added to fibronectin Drug-bound particles have been developed by attaching coated wells (37°C., 30-120 minutes). For quantification of drugs to particle-bound bifunctionalized PEG chains. Model the number of attached cells, wells were rinsed with RPMI/ drugs utilized for this proof-of-concept work are receptor 0.1% BSA to remove non-adherent cells. Adherent cells were tyrosine kinase (RTK) inhibitors. Delivery and accumulation fixed with 4% PFA (25° C., 20 minutes) and stained with of particles therapeutic payloads at the target site can be methylene blue (37° C. 1 hour). The methylene blue was maximized by employing dose escalation Strategies to assess extracted from cells by the addition of 200 ml of 0.1M HCl for enhanced particle uptake and distribution within tumors (37° C. 1 hour). For determination of optical densities we by optical and/or PET imaging. An alternative approach to used a SpectraMax5 micro plate reader and absorbance was enhance particle load delivery utilizes liposomal formula measured at 650 nm. For spreading assay: Time lapse was tions to encapsulate therapeutic particle probes. Liposomes performed (37° C., 2 hours) and images were captured by passively target tumors via the enhanced permeability and Axiovert 200M microscope (Carl Zeiss) using a 20x retention (EPR) effect while protecting payloads from degra (0.15NA) objective and using a scan slide module in the dation in the circulation. Prior to particle encapsulation, Metamorph Software (Molecular Devices). either cRGDY or linker-drug conjugates will be attached to the particle Surface as an active targeting mechanism to maxi Quantitative Analyses: mize delivery to the target site. 0400. Once at the target site, release of particles from 0395. In order to quantify the differences in the size and liposomal formulations into the tumor interstitium can occur intensity between Western blot bands, we performed densi as a result of upregulated enzymatic systems present in tometry of phosphorylated and total protein intermediates tumors. Extracellular release of acid sphingomyelinase (AS using Photoshop CS2 (Adobe, San Jose, Calif.). Bands were Mase, acid SMase or SMase) from tumor cells in response to scanned at 300 dpi (Scanjet 7650, Hewlett Packard, Palo Alto, cellular stress. Such as X-ray irradiation and toxins, leads to Calif.), and converted to grayscale. The lasso tool was then rapid ceramide-mediated cell injury and, Subsequently, apo used to draw a region of interest (ROI) within the boundaries ptosis. Acid SMase hydrolyzes sphingomyelin, present of each band in order to derive the following: area (number of within the liposomal formulations, to ceramide. Ceramide pixels), mean grayscale value within the selected area (0-255) has a role in biological systems as a secondary messenger in and the associated standard deviation. The product of the first the apoptotic process. Ceramide has significantly different two values for each band was computed, and divided by the membrane properties than the parent molecule sphingomy product for the initial band in each set (control band), yielding elin. When sphingomyelin-containing liposomes are cleaved an intensity value for each sample relative to the control. with SMase, there is a dramatic change in the membrane Finally the ratio of phosphorylated protein to total protein and rigidity. Sphingomyelin is a suitable lipid for liposome for the corresponding propagated error (SD) were computed for mulation; this is not true for ceramide, as its incorporation as each sample using the relative intensities. a liposome building block leads to liposome leakage. This 0396 Phase contrast images captured for migration stud leakage will lead to release of liposomal contents. This opens ies were analyzed using Image.J 1.45s (National Institutes of the possibility for the intracellular targeting of a large payload Health, rsbweb.nih.gov/i/) in order to quantify the extent of of silica nanoparticles, functionalized with drugs and/or cell cell migration (i.e., area closure) for M21 cells and HUVECs. internalizing markers (i.e., peptide KLAKLAC and Small At high power views, an enclosed area was drawn adjacent to molecule inhibitors) to monitor and/or treat disease. the rim of attached cells seen in each image after stopper 04.01 To determine whether liposome encapsulation sig removal. The enclosed area for each image was measured nificantly increases particles delivery to the tumor site relative (pixels) and used to calculate percent closure relative to time to non-encapsulated particle probes, we will utilize optical US 2014/024821.0 A1 Sep. 4, 2014 47 and PET imaging approaches to monitor uptake of both sph a ligand attached to the nanoparticle and capable of ingomyelinase liposome encapsulated and non-encapsulated binding a tumor marker, and particle batches in EGFRmt+ expressing brain tumor at least one therapeutic agent; and xenograft models (i.e., H1650, A431) both liposomes and (b) detecting the nanoparticles. particle probes will contain optical markers for visualization. 2. The method of claim 1, wherein the nanoparticle is 0402 Ref: Sochanik A1, Mitrus I, Smolarczyk R. Cichoh administered Subdermally, peritumorally, orally, intrave T. Snietura M. Czaja M. Szala S. Experimental anticancer nously, nasally, Subcutaneously, intramuscularly or transder therapy with vascular-disruptive peptide and liposome-en mally. trapped chemotherapeutic agent. Arch Immunol Ther Exp 3. The method of claim 1, wherein the nanoparticle has a (Warsz). 2010 June; 58(3):235-45. diameter between about 1 nm and about 25 nm. 0403. The foregoing methods for detecting and targeting 4. The method of claim 3, wherein the nanoparticle has a stressed cells involve a formulation comprised of bilayer diameter between about 1 nm and about 8 nm. liposomes each having an initial outer layer of liposome 5. The method of claim 1, wherein the organic polymer is forming lipids, sphingomyelin, or a mixture thereof a second selected from the group consisting of poly(ethylene glycol) inner layer of liposome-forming lipids, sphingomyelin or a (PEG), polylactate, polylactic acids, Sugars, lipids, poly mixture thereof; the first and second layers forming a bilayer glutamic acid (PGA), polyglycolic acid, poly(lactic-co-gly liposome and defining an interior space therein; wherein the colic acid) (PLGA), polyvinyl acetate (PVA), and the combi interior space contains a silica nanoparticle. The silica nano nations thereof. particle and/or liposome will be compromised of optical 6. The method of claim 1, wherein the number of ligands markers and/or PET labels (dye, fluorophore, radiolabel, con attached to the nanoparticle ranges from about 1 to about 25. trast agent), an enzyme substrate, and/or therapeutic agents, 7. The method of claim 6, wherein the number of ligands including cytotoxic drugs, DNA segments, or a radiotracer attached to the nanoparticle ranges from about 1 to about 10. indicator label. The marker- and/or drug-labeled silica par 8. The method of claim 1, wherein the ligand is selected ticles, contained within Sphingomyelin liposomes, contact a from the group consisting of peptide, protein, biopolymer, cell sample under conditions wherein sphingomyelinase is synthetic polymer, antigen, antibody, microorganism, virus, present in or released by the cell. This enzymatic contact will, receptor, hapten, enzyme, hormone, chemical compound, in turn, hydrolyze the sphingomyelin in the liposome to pathogen, toxin, Surface modifier, and combinations thereof. release the silica nanoparticle and/or marker label and/or drug 9. The method of claim 8, wherein the peptide is selected from the hydrophilic (interior) or hydrophobic (bilayer) com from the group consisting of tripeptide RGD, cyclic peptide partments of the liposome. cRGD, octreotate, EPPT1 and peptide analogs of alpha 04.04 The scope of the present invention is not limited by MSH. what has been specifically shown and described hereinabove. 10. The method of claim 1, wherein a contrast agent or a Those skilled in the art will recognize that there are suitable chelate is attached to the nanoparticle. alternatives to the depicted examples of materials, configura tions, constructions and dimensions. Numerous references, 11. The method of claim 10, wherein the contrast agent is including patents and various publications, are cited and dis a radionuclide. cussed in the description of this invention. The citation and 12. The method of claim 11, wherein the radionuclide is discussion of such references is provided merely to clarify the selected from the group consisting of Zr, Cu, Ga, Y. description of the present invention and is not an admission 2I, and 77Lu. that any reference is prior art to the invention described 13. The method of claim 10, wherein the chelate is adapted herein. All references cited and discussed in this specification to bind a radionuclide. are incorporated herein by reference in their entirety. Varia 14. The method of claim 10, wherein the chelate is selected tions, modifications and other implementations of what is from the group consisting of DFO, DOTA, TETA and DTPA. described herein will occur to those of ordinary skill in the art 15. The method of claim 1, wherein the nanoparticle is without departing from the spirit and scope of the invention. detectable by PET, SPECT, CT, MRI, optical imaging, biolu While certain embodiments of the present invention have minescence imaging, or combinations thereof. been shown and described, it will be obvious to those skilled 16. The method of claim 15, wherein the optical imaging is in the art that changes and modifications may be made with fluorescence imaging. out departing from the spirit and scope of the invention. The 17. The method of claim 1, wherein a therapeutic agent is matter set forth in the foregoing description and accompany attached to the nanoparticle. ing drawings is offered by way of illustration only and not as 18. The method of claim 17, wherein the therapeutic agent a limitation. is selected from the group consisting of antibiotics, antimi What is claimed is: crobials, antiproliferatives, antineoplastics, antioxidants, 1. A method for detecting tumor cells comprising the steps endothelial cell growth factors, thrombin inhibitors, immu of: nosuppressants, anti-platelet aggregation agents, collagen synthesis inhibitors, therapeutic antibodies, nitric oxide (a) administering to a patient a plurality of fluorescent donors, antisense oligonucleotides, wound healing agents, silica-based nanoparticles in a dose ranging from about therapeutic gene transfer constructs, extracellular matrix 0.01 nanomole/kg body weight to about 1 nanomole/kg components, vasodialators, thrombolytics, anti-metabolites, body weight, the nanoparticle comprising: growth factoragonists, antimitotics, statin, Steroids, steroidal A silica-based core comprising a fluorescent compound and non-steroidal anti-inflammatory agents, angiotensin con positioned within the silica-based core: Verting enzyme (ACE) inhibitors, free radical scavengers, a silica shell Surrounding at least a portion of the core; PPAR-gamma agonists, small interfering RNA (siRNA), an organic polymer attached to the nanoparticle: microRNA, and anti-cancer chemotherapeutic agents. US 2014/024821.0 A1 Sep. 4, 2014 48

19. The method of claim 17, wherein the therapeutic agent 32. The nanoparticle of claim 31, wherein the peptide is is radiolabeled. selected from the group consisting of tripeptide RGD, cyclic 20. The method of claim 19, wherein the therapeutic agent peptide cFGD, octreotate, EPPT 1 and peptide analogs of is bound to radiofluorine F. alpha-MSH. 21. The method of claim 17, wherein the therapeutic agent 33. The nanoparticle of claim 23, wherein a contrast agent is a radionuclide. is attached to the nanoparticle. 22. The method of claim 21, wherein the radionuclide is 34. The nanoparticle of claim 33, wherein the contrast selected from the group consisting of 'Y. ''I and ''Lu. agent is a radionuclide. 23. A fluorescent silica-based nanoparticle comprising: 35. The nanoparticle of claim 34, wherein the radionuclide a silica-based core comprising a fluorescent compound is selected from the group consisting of Zr, Cu, Ga.Y. positioned within the silica-based core: I and 77Lu. a silica shell Surrounding at least a portion of the core; 36. The nanoparticle of claim 23, wherein a chelate is an organic polymer attached to the nanoparticle; and attached to the nanoparticle. a ligand attached to the nanoparticle, 37. The nanoparticle of claim 36, wherein the chelate is wherein the nanoparticle has a diameter between about 1 adapted to bind a radionuclide. nm and about 15 nm, 38. The nanoparticle of claim 36, wherein the chelate is and after administration of the nanoparticle to a subject, selected from the group consisting of DFO, DOTA, TETA and renal clearance of the nanoparticle ranges from about DTPA. 80% ID (initial dose) to about 100% ID in about 24 39. The nanoparticle of claim 23, wherein the nanoparticle hours. is detectable by PET, SPECT, CT, MRI, optical imaging, 24. The nanoparticle of claim 23, wherein renal clearance bioluminescence imaging, or combinations thereof. of the nanoparticle ranges from about 90% ID to about 100% 40. The nanoparticle of claim39, wherein the optical imag ID in about 24 hours after administration of the nanoparticle ing is fluorescence imaging. to a Subject. 41. The nanoparticle of claim 23, wherein a therapeutic 25. The nanoparticle of claim 23, wherein the number of agent is attached to the nanoparticle. ligands attached to the nanoparticle ranges from about 1 to 42. The nanoparticle of claim 41, wherein the therapeutic about 25. agent is selected from the group consisting of antibiotics, 26. The nanoparticle of claim 25, wherein the number of antimicrobials, antiproliferatives, antineoplastics, antioxi ligands attached to the nanoparticle ranges from about 1 to dants, endothelial cell growth factors, thrombin inhibitors, about 10. immunosuppressants, anti-platelet aggregation agents, col 27. The nanoparticle of claim 23, wherein the nanoparticle lagen synthesis inhibitors, therapeutic antibodies, nitric oxide has a diameter between about 1 nm and about 8 nm. donors, antisense oligonucleotides, wound healing agents, 28. The nanoparticle of claim 23, wherein the organic therapeutic gene transfer constructs, extracellular matrix polymer is selected from the group consisting of poly(ethyl components, vasodialators, thrombolytics, anti-metabolites, ene glycol) (PEG), polylactate, polylactic acids, Sugars, lip growth factoragonists, antimitotics, statin, Steroids, steroidal ids, polyglutamic acid (PGA), polyglycolic acid, poly(lactic and non-steroidal anti-inflammatory agents, angiotensin con co-glycolic acid) (PLGA), Polyvinyl acetate (PVA), and the Verting enzyme (ACE) inhibitors, free radical scavengers, combinations thereof. PPAR-gamma agonists, small interfering RNA (siRNA), 29. The nanoparticle of claim 23, wherein the ligand is microRNA, and anti-cancer chemotherapeutic agents. capable of binding to at least one cellular component. 43. The nanoparticle of claim 41, wherein the therapeutic 30. The nanoparticle of claim 29, wherein the cellular agent is radiolabeled. component is a tumor marker. 44. The nanoparticle of claim 41, wherein the therapeutic 31. The nanoparticle of claim 23, wherein the ligand is agent is bound to radiofluorine F. selected from the group consisting of peptide, protein, 45. The nanoparticle of claim 41, wherein the therapeutic biopolymer, synthetic polymer, antigen, antibody, microor agent is a radionuclide. ganism, virus, receptor, hapten, enzyme, hormone, chemical 46. The nanoparticle of claim 45, wherein the radionuclide compound, pathogen, toxin, Surface modifier, and combina is selected from the group consisting of 'Y. ''I and ''Lu. tions thereof. k k k k k