ES2929315T3 - Silica-based multimodal nanoparticles - Google Patents

Abstract

The present invention provides a silica-based fluorescent nanoparticle that enables accurate detection, characterization, monitoring, and treatment of a disease such as cancer. The nanoparticle has a range of diameters, has a fluorescent compound positioned within the nanoparticle and has higher brightness and fluorescent quantum yield than the free fluorescent compound, exhibits high biostability and biocompatibility, can be coated with an organic polymer, such as polyethylene glycol ) (PEG). The small size of the nanoparticle, the silica base, and the organic polymer coating minimize the toxicity of the nanoparticle when administered in vivo. The nanoparticle can further be conjugated to a ligand. A therapeutic agent can be attached to the nanoparticle. In addition, magnetic resonance imaging (MRI), radionuclides/radiometals, or paramagnetic ions can be conjugated to the nanoparticle. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Silica-based multimodal nanoparticles

field of invention

[0001] The present invention relates to silica-based fluorescent nanoparticles and silica-based fluorescent nanoparticles for use in tumor cell detection methods.

Background of the invention

[0002] Early tumor detection and treatment selection are critical to achieving therapeutic success and long-term survival rates. In their initial stage, many cancers are localized and can be treated surgically. However, well-defined tumor margins are often difficult to visualize with current imaging techniques. This has led to a disproportionate number of invasive biopsies. Probes targeting highly specific molecules are needed for the early detection of molecular differences between normal and tumor cells, such as cancer-specific alterations in receptor expression levels. When combined with high-resolution imaging techniques, probes targeted to specific molecules will greatly improve detection sensitivity, facilitating cancer characterization, monitoring, and treatment.

[0003] Current fluorescence imaging probes typically consist of a single conventional fluorophore ( eg, organic dyes, fluorescent proteins), fluorescent proteins ( eg, GFP), and semiconductor quantum dots (Q-dots). . Individual fluorophores are generally not stable and have limited brightness for imaging. Like dyes, fluorescent proteins tend to exhibit excited-state interactions that can cause stochastic blinking, quenching, and photobleaching. Q spots are usually made of heavy metal ions like Pb2+ or Cd2+ and are therefore toxic. Burns et al. "Fluorescent core-shell silica nanoparticles: towards "Lab on a Particle" architectures for nanobiotechnology", Chem. Soc. Rev, 2006, 35, 1028-1042.

[0004] Fluorescent nanoparticles having an electrically conductive shell and a silica core are known and have utility in the modulated delivery of a therapeutic agent. US Patent Numbers 6,344,272 and 6,428,811. A shortcoming of existing fluorescent nanoparticles is their limited brightness and low detectability as fluorescent probes in scattered systems.

[0005] Document US2013/039848 A1 discloses silica-based fluorescent nanoparticles for the detection, characterization, monitoring and treatment of a disease such as cancer.

[0006] The present silica-based multifunctional fluorescent nanoparticles offer many advantages over other fluorescent probes. Nanoparticles are non-toxic and have excellent photophysical properties (including fluorescent efficiency and photostability), high biocompatibility, and unique pharmacokinetics for molecular diagnostics and therapy. The nanoparticles are relatively small in size and have a PEG surface coating that offers excellent renal clearance. The fluorescent nanoparticles of the present invention contain a fluorescent core and a silica shell. The core-shell architectures, large surface area, and diverse surface chemistry of the nanoparticle allow for multiple functionalities to be delivered simultaneously to a target cell. For example, the nanoparticle can be functionalized with targeting moieties, medical imaging contrast agents, therapeutic agents, or other agents. Targeting moieties on the nanoparticle surface may be tumor ligands which, when combined with nanoparticle-conjugated therapeutics, make the nanoparticle an ideal vehicle for targeting and potentially treating cancer. Webster et al. Optical calcium sensors: development of a generic method for its introduction into the cell by means of conjugated cell-penetrating peptides. Analyst, 2005; 130:163-70. The silica-based nanoparticle can be labeled with contrast agents for PET, SPECT, CT, MRI, and optical imaging.

Resume

[0007] The invention is defined in the claims. The invention provides a fluorescent silica-based nanoparticle comprising:

a silica-based core comprising a fluorescent compound positioned within the silica-based core;

a silica shell that surrounds at least a part of the core;

an organic polymer attached to the nanoparticle; Y

a ligand having the structure N-Ac-Cys-(Ahx) ² -D-Lys-ReCCMSH attached to the nanoparticle, where the nanoparticle is between about 1 nm and about 15 nm in diameter, and after administration of the nanoparticle to a subject, the renal clearance of the nanoparticle ranges from about 80% ID (initial dose) to about 100% ID in about 24 hours.

[0008] The invention also provides a plurality of silica-based fluorescent nanoparticles for use in a method for detecting tumor cells comprising the steps of:

(a) providing a plurality of silica-based fluorescent nanoparticles, said nanoparticles having been administered to a human subject at a dose ranging from about 0.01 nanomol/kg of body weight to about 1 nanomol/kg of body weight, each comprising one of the nanoparticles:

a silica-based core;

a fluorescent compound within the nucleus; Y

a silica shell that surrounds at least a part of the core;

where the nanoparticle is coated with an organic polymer, where 1 to 20 ligands are bound to the polymer-coated nanoparticle to bind to a tumor marker, where the ligands have the structure N-AcCys-(Ahx) ² -D-Lys- ReCCMSH, and

where each of the nanoparticles has a diameter within a range of 1 nm to 15 nm; Y

(b) detecting the nanoparticles, where the nanoparticles are excreted by renal clearance, by the human subject, within a range of 90% of the initial dose (ID) to 100% ID within 24 hours after administration.

[0009] The nanoparticle can be administered by subdermal, peritumoral, oral, intravenous, nasal, subcutaneous, intramuscular or transdermal route.

[0010] The present invention also provides a fluorescent silica-based nanoparticle as described in the claims wherein, after administration of the nanoparticle to a subject, renal clearance of the nanoparticle ranges from about 90% ID to about 100% DI in about 24 hours.

[0011] The present invention provides a fluorescent silica-based nanoparticle as described in the claims further comprising a contrast agent or chelate attached to the nanoparticle.

[0012] In some embodiments, the diameter of the nanoparticle ranges from about 1 nm to about 8 nm.

[0013] Organic polymers that can be attached to the nanoparticle include polyethylene glycol (PEG), polylactate, polylactic acids, sugars, lipids, polyglutamic acid (PGA), polyglycolic acid, poly(lactic-co-glycolic) acid ( PLGA), Polyvinyl Acetate (PVA), or combinations thereof.

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

[0015] The nanoparticle of the present invention can be detected by positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), formation optical imaging (such as fluorescence imaging including near-infrared fluorescence (NIRF) imaging, bioluminescence imaging, or combinations thereof. A therapeutic agent may be attached to the nanoparticle. Therapeutic agents include antibiotics, antimicrobials, antiproliferatives, antineoplastics, antioxidants, endothelial cell growth factors, thrombin inhibitors, immunosuppressants, antiplatelet agents, collagen synthesis inhibitors, therapeutic antibodies, nitric oxide donors, antisense oligonucleotides, healing agents, therapeutic gene transfer constructs, former matrix components tracellular, vasodilators, thrombolytics, antimetabolites, growth factor agonists, antimitotics, statins, steroids, steroidal and non-steroidal anti-inflammatory agents, angiotensin converting enzyme (ACE) inhibitors, free radical scavengers, PPARgamma agonists, RNA interference small (siRNA), microRNA and cancer chemotherapeutic agents. Therapeutic agents encompassed by the present invention also include radionuclides, for example, 90Y, 131I, and 177Lu. The therapeutic agent may be radiolabeled, such as labeled by binding to radiofluorine 0178F.

[0017] Following administration of the nanoparticle to a subject, the mean residence time in the blood of the nanoparticle can range from about 2 hours to about 25 hours, from about 3 hours to about 15 hours, or from about 4 hours to about 10 hours. The median tumor residence time of the nanoparticle after administration of the nanoparticle to a subject can range from about 5 hours to about 5 days, from about 10 hours to about 4 days, or from about 15 hours to about 3.5 days. The ratio of the mean residence time in the tumor to the mean residence time in the blood of the nanoparticle after administration of the nanoparticle to a subject can range from about 2 to about 30, from about 3 to about 20, or Between about 4 and about 15 disclosed herein (but not claimed) are silica-based fluorescent nanoparticles in which renal clearance of the nanoparticle after administration of the nanoparticle to a subject can range from approximately 10% ID (initial dose) to approximately 100% ID in approximately 24 hours, from approximately 30% ID to approximately 80% ID in approximately 24 hours, or from approximately 40% ID to approximately 70% ID in about 24 hours. In one embodiment, after administration of the nanoparticle to a subject, the mean residence time of the nanoparticle in the blood ranges from about 2 hours to about 25 hours, the mean residence time of the nanoparticle in the tumor ranges from about 5 hours to about 5 days, and the renal clearance of the nanoparticle is about 80% ID in about 24 hours.

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

[0019] The present invention also provides a fluorescent silica-based nanoparticle according to the claims, wherein, after administration of the nanoparticle to a subject, the mean residence time in the blood of the nanoparticle can range from about 2 hours to about 25 hours, or between about 2 hours and about 15 hours; The median tumor residence time of the nanoparticle can range from about 5 hours to about 2 days, and the renal clearance of the nanoparticle is about 80% ID in about 24 hours. The number of ligands attached to the nanoparticle can range from 1 to 20, or from 1 to 10. The diameter of the nanoparticle can be between 1 nm and 8 nm. A contrast agent, such as a radionuclide, can be attached to the nanoparticle. Alternatively, a chelate can be attached to the nanoparticle. The nanoparticle can be detected by PET, SPECT, CT, MRI, optical imaging, bioluminescence imaging, or combinations thereof. A therapeutic agent can be attached to the nanoparticle. Following administration of the nanoparticle to a subject, the mean residence time in the blood of the nanoparticle can also range from about 3 hours to about 15 hours, or from about 4 hours to about 10 hours. The median tumor residence time of the nanoparticle after administration of the nanoparticle to a subject can also range from about 10 hours to about 4 days, or from about 15 hours to about 3.5 days. The ratio of the mean residence time in the tumor to the mean residence time in the blood of the nanoparticle after administration of the nanoparticle to a subject can range from about 2 to about 30, from about 3 to about 20, or between about 4 and about 15. Renal clearance of the nanoparticle can also range from about 80% ID to about 90% ID in about 24 hours after administration of the nanoparticle to a subject.

[0020] In the present invention there is also provided a fluorescent silica-based nanoparticle according to the claims, wherein, after administration of the nanoparticle to a subject, the ratio between the mean residence time in the tumor and the mean residence time in the tumor The blood residence time of the nanoparticle ranges from about 2 to about 30, and the renal elimination of the nanoparticle is about 80% ID in about 24 hours.

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

[0022] The silica-based fluorescent nanoparticles described herein can be used in a method of targeting a tumor cell comprising administering to a cancer patient an effective amount of a silica-based fluorescent nanoparticle comprising a core silica-based comprising a fluorescent compound positioned within the silica-based core; a silica shell that surrounds at least a part of the core; an organic polymer attached to the nanoparticle; a ligand attached to the nanoparticle and capable of binding a tumor marker; and at least one therapeutic agent. The nanoparticle may be radiolabeled.

[0023] The nanoparticle can be administered to the patient by, but not limited to, the following routes: oral, intravenous, nasal, subcutaneous, local, intramuscular, or transdermal.

Brief description of the drawings

[0024] The patent file or application contains at least one drawing executed in color. The Office will provide copies of this patent publication or patent application with drawings in color upon request and payment of the necessary fee.

Fig. 1a shows a dynamic light scattering (DLS) plot (numerical average) of particle size for uncoated (grey) and PEG-coated (black) CyS-containing silica nanoparticles.

Fig. 1b shows in vivo images of spectrally separated Cy5 particle fluorescence (pseudocolor) overlaid on visible-light images of nude mice 45 min after injection with nude silica nanoparticles.

Fig. 1c shows in vivo images of spectrally separated Cy5 particle fluorescence (pseudocolor) overlaid on visible light images of nude mice 45 min after injection with PEG-ylated Cy5 nanoparticles.

Fig. 1d shows the in vivo biodistribution study using co-registered PET-CT. The top row is a serial co-registered PETCT image 24 hours after injection of 124I-labeled PEG-coated nanoparticles, flanked by the independently acquired microCT and microTEP scans. The bottom row is a serial microTEP image.

Fig. 2a shows fluorescence correlation spectroscopy (ECF) data and simple exponential fits for dye Cy5 (light grey), 3.3 -4 -0.06 nm in diameter (dark grey, mean standard deviation i, n = 9) and 6.0 0.1 nm in diameter (black, mean standard deviation, n=6) Cy5-containing PEG-coated nanoparticles showing differences in diffusion time resulting from different hydrodynamic sizes of different species .

Fig. 2b shows the absorption and emission spectra of Cy5 dye (light grey), 3.3 nm diameter (dark grey) and 6.0 nm diameter (black) PEG-coated nanoparticles.

Fig. 2c shows the comparison of the relative brightness of the free dye (light grey) with nanoparticles of 3.3 nm (dark grey) and 6.0 nm in diameter (black), measured as count rate per molecule/particle determined at from the ECF curves.

Fig. 2d shows the photobleaching data for Cy5 dye (light gray), 3.3 nm diameter (dark gray) and 6.0 nm diameter (black) PEG-coated nanoparticles under -3.5 laser excitation. mW.

Fig. 3a shows the percentage of the initial panicle dose (%ID) retained by blood (black) and tissues: liver (light grey), lung (low-medium grey), spleen (medium grey) and kidney. (medium-high grey) for 6.0 nm diameter nanoparticles at various time points from 10 min to 48 h post-injection (w = 3 mice, mean ± S.D.).

Fig. 3b shows the plot of the concentration of retained particles for nanoparticles of 3.3 nm (light gray) and 6.0 nm (black) in diameter and the associated log decay fits and half-lives.

Fig. 3c shows the plot of the estimated particle excretion for nanoparticles of 3.3 nm (light gray) and 6.0 nm (black) in diameter and the associated logarithmic fits and half-lives (mean standard deviation, it = 9 (three mice per time point)).

Fig. 4 shows the in vivo biodistribution of the nanoparticles in mice with and without tumors with subcutaneous C6 xenografts. (A) Bare silica particles; (B) PEGylated RGD particles.

Fig. 5 shows the total specific binding data for cRGD and PEG-ylated dots (ie, nanoparticles) using flow cytometry in the Cy5 channel as a function of time (a) and particle concentration (b).

Fig. 6 shows the multimodal C-point design for the identification and characterization of avp3 integrin.

Fig. 6a. Schematic representation of 124I-cRGDY-PEG-ylated core-shell silica nanoparticle with radiolabels and peptides on the surface and core-containing reactive dye molecules (insets).

Fig. 6b. ECF results and simple exponential fits for measurements of Cy5 dyes in solution (black), PEG-coated (PEG spot, red), and cRGDY-labeled PEG-coated spots (blue, below red dataset) showing differences in diffusion time as a result of different hydrodynamic sizes.

Fig. 6c. Hydrodynamic sizes (mean i s.d., n = 15) and relative brightness comparisons of free dye with PEG-coated dots and cRGDY-PEG dots derived from ECF curves, along with corresponding particle and dye concentrations.

Fig. 7 shows purification and quality control of 124I-RGDY-PEG spots using size exclusion column chromatography. Radioactivity (right column) of 124I-RGD dots and 124I-PHG dots detected by y-counting and the corresponding fluorescence signal intensity (Cy5, left column) of 124I-RGDY-PEG dots and 124I-PEG dots in each eluted fraction.

Fig. 8 shows competitive integrin receptor binding studies with 124I-cRGDY-PEG dots, cRGDY peptide and anti-avp3 antibody using two cell types.

Fig. 8a. High affinity and specific binding of 124I-cRGDY-PEG dots to M21 cells by y-counting. Inset shows Scatchard analysis of binding data plotting the ratio of the concentration of bound to receptor (B) to unbound (or free, F) radioligand, or bound to free ratio, B/ F, versus concentration of receptor bound to receptor, B; the slope corresponds to the dissociation constant, Kd. Fig. 8b. Blockade of the avp3 integrin receptor of M21 cells by flow cytometry and excess non-radiolabeled cRGD or anti-α3 antibody prior to incubation with cRGDY-PEG dots.

Fig. 8c. Specific binding of cRGDY-PEG dots to M21 vs. M2iL cells lacking surface integrin expression using flow cytometry.

Fig. 8d. Specific binding of cRGDY-PEG dots to HUVEC cells by flow cytometry. Each bar represents the mean s.d. of three repetitions.

Fig. 9 shows the pharmacokinetics and excretion profiles of the targeted and non-targeted particle probes. Fig. 9a. Biodistribution of 124I-cRGDY-PEG dots in mice bearing M21 tumors at various time points from 4 to 168 h pi. Inset shows a representative plot of these data for blood to determine median residence time (T ^1/2 ).

Fig. 9b. Biodistribution of 124I-PEG dots from 4 to 96 h after injection.

Fig. 9c. Elimination profile of urine samples collected up to 168 h p.i. of non-radiolabeled cRGDY-PEG spots (n=3 mice, mean s.d.).

Fig. 9d. Corresponding cumulative %DI!g for feces at intervals up to 168 h p.i. (n=4 mice). For biodistribution studies, bars represent mean s.d.

Fig. 10 shows the results of acute toxicity tests

Fig. 10a. Representative H&E stained liver at 400x (upper frames) and kidneys stained at 200x (lower frames). Mice were treated with a single dose of non-radiolabeled 127I-RGDY-PEG-dots or 127I-PEG-dots (vehicle control) by intravenous injection and organs harvested 14 days later.

Fig. 10b. Mean daily weights for each treatment group of the toxicity study. The scale bar in Fig. 10a corresponds to 100pm.

Fig. 11 shows tumor selective targeting serial in vivo PET images.

Fig. 11a. Representative whole-length coronal microTEP images at 4 hours p.i. demonstrating M21 (left, arrow) and M2IL (middle, arrow) tumor uptakes of 3.6 and 0.7% ID/g, respectively, and increased M21 tumor contrast at 24 hours (right)

Fig. 11b. In mo uptake of 124I-cRGDY-PEG dots in M21 tumors that overexpress avp3 integrin (black, n = 7 mice) and M21L non-expressing (tight grey, n = 5 mice) and 124I-PEG dots in M2J tumors (dark grey, n=5)

Fig. 11c. Tumor to M21 muscle ratios for 124I-cRGDY-PEG dots (black) and 124I-PEG dots (grey) Fig. 11d. Correlation of in vivo and ex vivo M21 tumor uptake of cRGDY-labeled and unlabeled probes. Each bar represents the mean ± s.d.

Fig. 12 shows node mapping using multi-scale near-infrared optical fluorescence imaging.

Fig. 12a. Whole-body fluorescence images of tumor site (T) and drainage of inguinal (ILN) and axillary (ALN) nodes and communicating lymphatic channels (bar, LC) 1 h pi in a surgically exposed live animal

Fig. 12b. Corresponding co-registered white light and high-resolution fluorescence images (upper row) and fluorescence-only images (lower row) revealing the nodal infrastructure of local and distant nodes, including high endothelial venules (HEVs). The larger scale bar in (b) corresponds to 500 pm.

Fig. 13a shows the experimental setup of using a porcine miniature spontaneous melanoma model to map the lymph node basins and regional lymphatic vessels draining the site of a known primary melanoma tumor.

Fig. 13b shows a small field of view PET image 5 minutes after subdermal injection of multimodal particles (124IRGD-PEG-dots) over the tumor site.

Fig. 14a shows a whole body dynamic 18F-fluorodeoxyglucose (18F-FDG) PET scan showing sagittal, coronal and axial images through the site of nodal disease in the neck. Fig. 14b shows fused 18F-FDG PET-CT scans showing sagittal, coronal and axial images through the site of nodal disease in the neck.

Fig. 14c shows the image of a full-length minipig.

Fig. 15 shows the same image sets as Fig. 14, but at the level of the primary melanoma lesion, adjacent to the spine in the upper back.

Fig. 16a shows high resolution dynamic PET images after subdermal injection of 4 quadrants of 124I-RGD-PEG dots around the tumor site over a 1 hour time period. Fig. 16b shows fused PET-CT images after 4-quadrant subdermal injection of '241-RGD-PF-, G-spots around the tumor site over a 1 hour time period.

Fig. 16c shows images of Cy5 (upper image), the resected nodule (upper second image) and H&E staining (lower two images).

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

Fig. 18 shows structures of the PEG derivative. Standard chemical reactions are used for the production of the triple bond functionalized PEG, which will then be covalently attached to the nanoparticle via the silane group.

Fig. 19 shows structures of DFO derivatives.

Fig. 20a shows Tyr3-octreotate structures.

Fig. 20b shows the synthesis of the azide-containing acid for incorporation into Tyr3-octreotate.

Fig. 21a shows a schematic of the production of nanoparticles functionalized with a NIR fluorescent dye within their core, a PEG surface coating, DFO chelates and Tyr3 octreotate.

Fig. 21b shows a schematic of the production of a multimodal (TEP and fluorescence) 19Zr-labeled nanoparticle decorated with Tyr3-octreotate.

Fig. 21c shows tetrazine-norbornene ligation.

Fig. 21d shows a schematic of the strategy for the creation of radiolabeled core-shell nanoparticles using tetrazine-norbornene ligation.

Fig. 21e shows a schematic of the strategy for the creation of peptide-targeted radiolabeled core-shell nanoparticles using tetrazine-noroornene ligation. One-step (where the premetallized norbornene-chelator complex reacts with the particle) and two-step (where the chelator is metallized after conjugation) pathways are shown.

Fig. 22 shows microscopic images demonstrating colocalization between cRGF-PEG nanoparticles and lys-red in the endocytic pathway.

The Figs. 23a - 23i Image-Guided SLN (Sentinel Lymph Node) Mapping: PET Imaging preoperative (a,b) Axial CT images reveal a left pelvic soft tissue mass (a, arrow) and SLN on the left flank (b, arrow). (c, d) Axial 18F-FDG PET images show localized activity within the tumor (c, arrow) and SLN in the left flank (d, arrow) after injection of the iv tracer, (e) 124I-cRGDY -Axial PEG and (f) Coronal PET-CT images co-registered with point C show the local injection site over the pelvic lesion (e, arrow), (g) Axial and PET-CT co-registered images (h) Coronal corresponding localize activity in the SLN (g, arrow), (i) Radioactivity levels of the primary tumor, SLN (in vivo, ex vivo) and a remote site of the primary tumor (ie, background), using a portable gamma probe.

The Figs. 24a - 24q Image-guided SLN mapping: real-time intraoperative optical imaging with correlative histology. Intraoperative SLN mapping was performed in the animal shown in Figures 23a-23i. (a-i) NIR optical images of two channels of the exposed nodal basin. Local injection of Cy5.5 incorporated particles shown in dual channel model (a) RGB color and (b) NIR fluorescent channels (white), (c-f) Drainage 1).-lymphatics distal to the injection site. Fluorescence signal within the main proximal (c, d), middle (e), and distal (f) draining lymphatic channels (arrows) extending into the SLN ('N'). Smaller caliber channels (arrowheads) are also shown. Images of the SLN shown in the (g) color and (h) NIR channels. (i) Image of the exposed SLN. (j-m) Images of SLNs in the color and NIR channels during (j,k) and after cleavage (l,m), respectively. (n) Low power view of H&E-stained SLNs shows cluster of pigmented cells (black box) (bar = 1 mm), (o) Higher magnification of (n) reveals round pigmented melanoma cells and melanophages (bar = 50 gm). (p) Low-power view of HMB45-stained SLN confirms the presence of metastasis (black box, bar: 500 gm). (q) Higher magnification in (p) reveals clusters of melanoma cells expressing HMB45+ (\)ar:::100 gm).

The Figs. 25a - 25k Discrimination of inflammation from metastatic disease: Comparison of 18F-FDG and 124I-cRGDY-PEG C dot tracers. (a-d) Imaging of inflammatory changes using 18F-FDG-PET with tissue correlation. (a) Axial CT scan of the "F-FDG PET" study showing calcification within the left posterior neck (arrows). (b) Fused 18F-FDG PET-CT scan axial showing hypermetabolic activity at this same site (arrows). Signal PET magnification is also seen in metabolically active bone structures (asterisks).(c) Low and (d) high magnification views of H&E-stained calcified tissue demonstrating extensive inflammatory cell infiltration.(e-k) Detection of metastatic disease after injection of 124I-cRGDY-PEG-C dots around the tumor site.(e) Pre-injection axial computed tomography of 124I-cRGDY-PEG-C dots shows calcified soft tissues within the posterior neck (arrows). , (f) Core-registered PET-CT shows no obvious activity corresponding to calcified areas (arrow), but demonstrates a hypermetabolic node on the right (arrowhead), (g) Axial CT at a higher level shows nodes (arrowheads) bilaterally and a calcified focus (arrow), (h) Fused PET- CT demonstrates PETavid nodules (N) and lymphatic drainage (curved arrow). without activity (arrow), (i) low power and (j) high power views confirm the presence of nodal metastases. (k) Single image of a three-dimensional (3D) PET image reconstruction showing multiple bilateral metastatic nodes (arrowheads) and lymphatic channels (arrow). Bladder activity is observed without significant tracer accumulation in the liver. Scale bars: 500 urn (c, d); 100 gm (ij).

The Figs. 26a-26c show 18F-FDG and 124I-cRGDY-PEG-C PET-CT spots integrated in 3D. (a-c) 3D volume rendered images were generated from CT and PET imaging data shown in Figures 7a-7k. (a) PET-CT fusion image (coronal view) showing no obvious nodal metastases (asterisks). Increased activity is identified within bony structures. (b, c) High-resolution PET-CT fusion images showing coronal (b) and superior (c) views of bilateral metastatic nodes (open arrows) and lymphatic channels (curved arrows) within the neck after local injection of the particle tracer.

The Figs. 27th - 27th Evaluation of response to treatment after radiofrequency ablation (RFA) using 124I-cRGDY-PEG-C dots. (ac) Localization of the SN with single-dose particle radiotracer. (a) Baseline coronal CT (white arrowhead), (b) PET (black arrowhead) and (c) fused PET-CT images (white arrowhead) after peritumoral injection, (bd) Activity of the tumor particle tracer. (b) PET-avid exophytic left pelvic mass (black arrow), (c, d) Combined PET-CT images showing a hypermetabolic lesion (white arrow) and particle tracer flow within a draining lymphatic channel (asterisk ) towards the SLN (curved arrow). (e,f) Axial pre-ablation CT images locate the SLN (e, white arrowhead) prior to placement of the RFA electrode (f, arrow) in the nodule (below the cross), (g) Fused PET-CT prior to ablation reveals increased SLN (post-reticulum) activity. (h) Post-ablation PET-CT scan shows slightly reduced activity at the SLN site, anterior to the needle tip. (i) Corresponding pre-ablation H&E staining of SLN core biopsy tissue confirms pigmented tumor infiltration (bar = 200 gm). (j) High magnification of the boxed area in (i) reveals large round pigmented clumps of melanoma cells (bar--50 gm). (k) Post-ablation H&E staining shows necrotic changes within a partially tumor-infiltrated node (inset) and multifocal hemorrhages (bar = 500 gm). (I) High magnification of (k) reveals significant tissue necrosis (arrowheads) within the metastatic node, in addition to lymphoid tissue (bar = 50 gm). (m) TUNEL staining of metastatic SLN before ablation (bar = 20 gm). (n) Staining Post-ablation TUNEL demonstrating focal areas of necrosis with adjacent scattered tumor foci and normal nodal tissue (NT) (bar = 500 pm). (o) High magnification of the boxed area in (n) shows positive TUNEL staining, consistent with necrosis (bar = 20 pm).

The Figs. 28A-28C. Core-shell hybrid silica nanoparticle platform (124I-cRGDY-PEG-C dots) and study design overview. (A) Schematic of hybrid inorganic imaging (PET-optical) probe (right) showing deep red dye containing core and surface-bound polyethylene glycol (PEG) chains carrying cRGDY peptide ligands and radiolabels to detect human avp3 integrin-expressing tumors (left). (B) Matching absorption spectra (left: red and black curves) and emission spectra (right) for free (blue curve) and encapsulated (green curve) dyes revealing increased fluorescence from encapsulated fluorophores. (C) Timeline of clinical trial events. Biological specifications, biological specimens (blood, urine).

The Figs. 28D-28E. Whole-body distribution and point pharmacokinetics of 124I-cRGDY-PEG-C. (D) PET images of maximum intensity projection (MIP) at 2 (left), 24 (middle) and 72 (right) hours p.i. of 124I-cRGDY-PEG-C dots revealing activity in the bladder (*), heart (yellow arrow), and intestine (white arrowhead). (E) The decay-corrected percent injected dose per gram (%ID/g) of urine and plasma collected approximately 30 min, 4 h, 24 h, and 72 h after particle injection was determined by gamma counting; individual plots were generated for each patient. ROls were drawn on major organs for each patient's PET scans to obtain standardized uptake values and %ID/g.

Fig. 29 Metabolic analysis of biological specimens. (A, B) Time-dependent activity concentrations (% lD/gx 100) in plasma and urine, respectively, decay corrected for injection. (CH) RadioTLC (4:1 acetic acid:methanol as mobile phase) of plasma and urine samples (corrected decay counts per minute, cpm). (CE) Plasma chromatograms show a single peak at 0.5-(C), 3-(D) and 24-(E) hours p.i. (FH). Chromatograms of urine samples reveal two peaks at 0.5-(F), 3-(G), 24-(H) hours p.i. Boxes (G, H) show the respective data scaled to a maximum of 50 cpm. (IK) Chromatograms of standards: injected (I), radioiodinated peptide (131I) (J) and free 131I (K). The vertical lines discriminate the peaks corresponding to the particle tracer (long dashes; Rt = 0.04), 131I-cRGDY (short dashes; Rf-02) and 131I (dotted; Rf = 0.7).

Fig. 30 Whole-body PET-CT images of particle biodistribution and preferential tumor uptake after systemic dot injection of 124I-cRGDY-PEG-C (A) Reformatted coronal CT demonstrates well-dense hypodense metastasis defined in the left hepatic lobe (arrowhead). (B) Coronal PET image at 4 hours p.i. demonstrates increased activity along the peripheral aspect of the tumor (arrowhead), as well as the bladder, gastrointestinal tract (stomach, intestines), gallbladder, and heart. (C) Core-registered PET-CT localizes activity at the tumor margin.

Fig. 31 Multimodal images of particle uptake in a pituitary lesion. (A-B) Axial (A) and sagittal (B) MR images with multiplanar contrast at 72 hours p.i. showing a subcentimeter cystic focus (arrows) on the right aspect of the anterior pituitary. (C-D) Co-registered axial (C) and sagittal (D) MRI-PET images reveal localized increased focal activity (red) at the lesion site. (EF) Axial (E) and sagittal (F) PET-CT images localize activity on the right side of the sella turcica. (G-I) Axial PET images at 3 hours (G), 24 hours (H) and 72 hours (I) p.i. demonstrate a progressive accumulation of activity (SUVmax) within the sellar region along with a corresponding decrease in background activity around the lesion. (J) Ratios of tumor to brain (T/B) and tumor to liver (T/L) activity that increase as a function of post-injection times

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

Fig. 33. Structure of the original ReCCMSH targeting molecule.

The Figs. 34A and 34B. Competitive binding studies using a melanocortin-1 receptor agonist (125I-NDP). N-AcCys-(Ahx) ² -D-Lys-ReCCMSH (or alpha-MSH) conjugated particles (Fig. 34A) had a higher affinity for cultured B 16/FI melanoma cells than a coding sequence version of the molecule (Fig. 34B).

The Figs. 35A and 35B. Dose-response data were obtained as a function of target particle concentrations (Fig. 35A) and incubation times (Fig. 35B) for the B16F10 and M21 melanoma cell lines.

Fig. 36. Survival studies of human M21 cells, performed at a range of particle concentrations during a fixed incubation time of 48 h, did not demonstrate significant loss of cell viability. Figures 37A and 37B. 125I-radiolabeled alpha-MSH-conjugated nanoparticles demonstrated massive renal excretion over a 24-hour period in the BI 6F 10 and 10 murine xenograft models. M21.

The Figs. 38A and 38B. Neither the B16F10 nor the M21 xenograft models showed significant accumulation of the target particle probe in the reticuloendothelial system (i.e., not a RES agent), nor in the kidney.

Fig. 39. Integrin receptor competitive binding and temperature-dependent uptake using cRGDY-PEG-C dots and anti-avp ³ antibody for 2 cell types. A. Specific binding and uptake of cRGDY-PEG-C spots in M21 cells as a function of temperature (4 °C, 25 °C, 37 °C) and concentration (25 iiM, 100 nM) using anti-receptor antibody for avfJj integrin and flow cytometry. Antibody concentrations against avp3 integrin receptor were 250 times (ie, 250x) the particle concentration. B. Uptake of cRGDY-PEG-C spots in M21 cells, compared to M2 JL cells lacking normal surface integrin expression by flow C. Selective uptake of particles in HUVEC using anti-avp.A integrin receptor antibody and D. cRGDY-PEG-C dot (1 gM, red) colocalization assay with flow cytometry with endocytic (transferrin-Alexa-488, FITC-dextran, green) and lysosomal markers (LysoTracker Red) after 4 h incubation of particles using M21 cells. Vesicles attached (yellow), Hoechst counterstain (blue). Scale bar = 15 pm. Each data point (AC) represents the mean ± SD of 3 replicates.

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

Fig. 41. Signaling induction and inhibition studies in M2J cells using PF-573228 (PF-228), an FAK inhibitor. A. Western blots of phosphorylated and total protein expression levels using the above procedure in Fig. 2 with the addition of either 250 nM or 500 nM PF-228 (0.5 h, 37 °C) to the cells prior to exposure to particles. B. Summary of percent intensity changes from phosphorylated to total protein expression levels in particle-exposed cells versus control cells with and without PF-228.

Fig. 42. Effect of cRGDY-PEG-C dots on M21 cell migration using time-lapse imaging. A. Time-dependent changes in cell migration using ORlS™ collagen-coated plates for a range of particle concentrations (0 - 400 nM; 37 °C) in RPMI 1640 media supplemented with 0.2% FBS, compared to supplemented means alone (controls). Images were captured at time t=0 (before migration) and at subsequent 24-h intervals after cap removal with a Zeiss Axiovert 200M inverted microscope (5x/.25NA objective) and scanning slide module ( Metamorph® microscopy automation and image analysis software) for a total of 96 h. B. Graphical representation of changes in mean area of closure (%) as a function of concentration using ImageJ software. The mean closure area represents the difference in the areas bounded by the border of the advancing cells (pixels) at arbitrary time points and after removing the plug (t:::O), divided by the latter area. Quadruplicate samples were statistically analyzed for each group using a one-tailed t-test: *, p=.01 1; **, p= 049; ***, p=.036. Scale bars = 100 pm and 33 pm (enlarged images of x and xv).

Fig. 43. Effect of cRGDY-PEG-C dots on HUVEC cell migration. A. Serial HUVEC migration was analyzed using the same procedure in Fig. 4 over a 24 hour time interval. Images shown and closure area values indicated are representative of a single experiment. B. Mean areas of closure (%) during this time interval for a range of particle concentrations (0-400 nM) were determined using ImageJ software. Assays were performed in triplicate for each concentration and time point. One-tailed t-test *, p<0.05. Scale bar = 76 gm.

Fig. 44. Inhibition of HUVEC cell migration using PF-228. A. Same procedure as Fig. 5, except cells were exposed to 250 nM and 500 nM PF-228 (0.5 h, 37 °C) prior to particle exposure or incubation in supplemented medium with 0.2% ECF. B. Mean area of closure (%) for cells incubated under the above conditions. C. Tabulated p-values for each exposure condition using a one-tailed t-test. Samples were analyzed in quadruplicate for each concentration of inhibitor. Scale bar = 50 gm.

Fig. 45. Modulation of M21 cell expansion and adhesion using cRGDY-PEG-C dots. A. Time-lapse images showing changes in cell attachment and spread. Cells were preincubated in RPMI supplemented with 0.2% FBS (0.5 h, 25 °C), without and with particles (400 nM), followed by seeding in fibronectin-coated 96-well plates (5 pg/ml ). Images were captured at t = 0, 0.5h, 1h and 2h using a Zeiss Axiovert 200M inverted microscope (20x/.4NA objective) and a Metamorph® scanning slide module. B. Graph showing the mean number of rounded and elongated cells within two groups as a function of time: not exposed to particles (elongated, graph #1) and exposed to particles (rounded, graph #2). Cells in each of three wells of a 96-well plate were manually counted in a minimum of three high power fields (x200 magnification) and averaged. C. Absorbance values (A=650 nm; SpectroMax M5 microplate reader) for cells fixed with 4% paraformaldehyde, exposed to 400 nM cRGDY-PEG-C media or spots, and treated with methylene blue reagent (1 mL; lh, 37 °C), as a measure of cell attachment. Scale bar == 30 pm. Samples were run in quadruplicate for each group.

Fig. 46. Influence of cRGDY-PEG-C spots on the cell cycle. A. Percentage (%) of viable cells in the Gi, S, and G ² phases of the cell cycle as a function of the concentration of particles (0, 100, 300 nM) added to Go/GI phase synchronized M21 cells incubated for a total of of 96 hours. The numbers in italics above each bar represent the percentage of cells (incubated particles, control) in S, G ¹ and G ² phases, determined by flow cytometry. B. Representative cell cycle histograms are shown for cells under control conditions (ie, no particles) and after incubation with 100 and 300 nM particles. One-way ANOVA: *, p<.05; **, p<.005 relative to S phase control. Insets: Group mean values (n=3): t: SD for each cell cycle phase. The experiments were performed in triplicate.

Fig. 47. Dose-response effects and saturation binding kinetics using cRGDY-PEG-C dots and cells expressing cu-Ps integrin. A, B. Cell binding/uptake as a function of particle concentration (A) and incubation time (B) by flow cytometry. Monolayers of M21 cells and HUVEC were incubated (4 h, 25 °C) with increasing concentrations of particles (5 - 600 nM), as well as for a range of incubation times (0.5 - 4 h, 100 nM) and were analyzed using fluorescence activated cell sorting (FACS) analysis. The percentage of total events detected is displayed. Each data point represents the mean SD of 3 replicates

Fig. 48. Competitive binding of integrin receptor with 124I-cRGOY-PEG-C dots and cRGDY peptide in two cell types. A. Specific binding of 124I-cRGDY-PEG dots to M21 and HUVEC cells after incubation with excess cRGDY peptide using gamma counting. Monolayers of M21 and HUVEC cells were incubated (4 h, 25 °C) with 25 nM 124 I-cRGDY-PEG dots in the presence and absence of cRGDY peptide (85, 170 nM). Binding is expressed as a percentage of control (ie, radioiodinated cRGDY-PEG spots).

B. Tumor-directed binding of non-radiolabeled cRGDY-PEG-C dots. M21 and HUVEC cells were incubated for 4 h at 25 °C with two concentrations of specific particles (25 nM, 100 nM) or particle controls (PEG-C spots) and analyzed by flow cytometry.

Fig. 49. Viability and proliferation of M21 and HUVEC cells as a function of particle concentration and incubation time. A, B. Absorbance (Aex = 440 nm) as a measure of viability in subconfluent Go/Gi phase-gated M21 cells (A) and HUVEC (B) over a 24-h period using media supplemented with 10% or 2% FBS alone. , respectively, or with addition of particles (25-200 nM). C,D.

Cell proliferative activity in M21 (C) and HUVEC (D) cells over a 93-h time interval using media alone or media containing particles, as specified in A, B.

Fig. 50. Changes in cell signaling as a function of particle incubation time in M21 cells. Western blots of selected total and phosphorylated intermediate proteins over a time period of 8 hours. The normalized intensity ratios (i.e., the difference of phosphoprotein and total protein divided by the latter) are illustrated graphically.

Fig. 51. Modulation of cell signaling as a function of particle concentration in M21 cells. Western blots of selected total and phosphorylated intermediate proteins over a range of particle concentrations (ie, 0...400 nM).

Fig. 52. Cell signaling inhibition studies in M21 cells. A. Western blot of selected total and phosphorylated intermediate proteins (serum-starved media, 100 nM particles alone, or 100 nM particles after addition of 250 nM or 500 nM inhibitor). B. Relative intensities of phosphoprotein and p-actin transfers in A relative to control cells (0.2% FBS).

Detailed description of the invention

[0025] The present invention provides a fluorescent silica-based nanoparticle according to the claims that enables accurate detection, characterization, monitoring and treatment of a disease such as cancer. The invention it also provides a plurality of silica-based fluorescent nanoparticles according to the claims for use in a method for detecting tumor cells. The method may contain the following steps: (a) administering to a patient a plurality of silica-based fluorescent nanoparticles in a dose ranging from about 0.01 nanomol/kg body weight to about 1 nanomol/kg body weight body weight, from about 0.05 nanomole/kg body weight to about 0.9 nanomole/kg body weight, from about 0.1 nanomole/kg body weight to about 0.9 nanomole/kg body weight, from about 0.2 nanomole/kg body weight to about 0.8 nanomole/kg body weight, from about 0.3 nanomole/kg body weight kg body weight to about 0.7 nanomole/kg body weight, from about 0.04 nanomole/kg body weight to about 0.6 nanomole/kg body weight, from about 0.2 nanomole/kg body weight to about 0.5 nanomole/kg body weight, and (b) detecting nanoparticles. In one embodiment, the nanoparticle further comprises at least one therapeutic agent.

[0026] The nanoparticle has a range of diameters including about 1 nm to about 15 nm, or about 1 nm to about 8 nm. The nanoparticle has a fluorescent compound positioned within the nanoparticle and has higher brightness and fluorescent quantum yield than the free fluorescent compound. The nanoparticle also exhibits high biostability and biocompatibility. To facilitate efficient urinary excretion of the nanoparticle, it can be coated with an organic polymer, such as polyethylene glycol (PEG). The small size of the nanoparticle, the silica base, and the organic polymer coating minimize the toxicity of the nanoparticle when administered in vivo. To target a specific cell type, the nanoparticle is conjugated to a ligand, which is capable of binding to a cellular component ( eg, the cell membrane or other intracellular component) associated with the specific cell type, such as a tumor marker or an intermediate of the signaling pathway. In one embodiment, a therapeutic agent can be attached to the nanoparticle. Allow the nanoparticle to be detectable not only by optical imaging (such as fluorescence imaging), but also by other imaging techniques, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography ( CT) and nuclear magnetic resonance (NMR), the nanoparticle can also be conjugated with a contrast agent, such as a radionuclide.

[0027] The properties of nanoparticles lead to massive excretion via the kidneys, higher potency relative to a native peptide ligand, enhanced uptake, and preferential accumulation in tumors compared to normal tissues. This, along with the lack of toxicity in vivo, has resulted in a unique product that translates into the clinic.

[0028] The present particles can be used to preferentially detect and localize tumors. For example, doses of nanomolar particle tracers administered in a microdosing regimen accumulate and preferentially localize to disease sites, although they are not optimized for targeted detection.

[0029] The present nanoparticles may exhibit distinct and reproducible human pharmacokinetic signatures in which renal clearance predominates ( eg, renal clearance of the nanoparticle is greater than about 90% ID at about 24 hours after administration of the nanoparticle to a subject) without significant RES uptake (eg, less than about 10%).

[0030] The present nanoparticles are excellent diagnostic probes, exhibiting optimal physicochemical properties in humans that allow them to "target and clear" the body in relatively short time intervals (eg, hours, days, etc.).

[0031] The present particles carrying multiple actively targeted ligands ( eg, cRGDY and alpha-MSH) demonstrate higher binding affinity to cellular targets compared to the affinity of a single ligand. In some embodiments, the present particles bind to about 2 to about 20 times greater, about 3 to about 15 times greater, about 5 to about 10 times greater cellular target than a ligand alone.

[0032] In certain embodiments, dual-modality targeting particles specifically assess tumor burden and can discriminate metastatic tumor from chronic inflammatory disease in large animal models of metastatic melanoma.

[0033] Targeting particles can enhance receptor binding affinity and avidity, increase plasma residence times, bioavailability and tumor retention, and/or promote intracellular delivery through internalization. The use of such targeted probes within surgical (or medical) oncology settings may allow highly selective treatment of cancerous tissues, potentially reducing rates of concomitant complications. The present particles may be advantageous over passively targeted nanocarriers, as the latter non-specifically penetrate and accumulate within the tumor interstitium through enhanced permeability and retention (EPR) effects.034*

[0034] Particle-based imaging systems for cancer diagnosis must be non-toxic and selectively detect sites of primary and metastatic disease while exhibiting relatively rapid renal clearance. Under these conditions, the probability of potential toxicity will be reduced given the smaller area under the plasma concentration-time curve. In one embodiment, these renal clearance properties can be achieved by platforms based on ultrasmall particles or macromolecular systems that meet effective renal glomerular filtration size limits of 10 nm or less. The absence of acute single-dose toxicity and the minimization of such risks will also be important. The design of a platform should maximize safety through rapid whole-body clearance and selection of biokinetic profiles that minimize non-specific uptake into the reticuloendothelial system (RES), thereby reducing potential adverse exposures.

[0035] In one embodiment, the present particles are safe and stable in vivo. The particles exhibit distinctively unique and reproducible pharmacokinetic signatures defined by renal excretion. Together with the preferential uptake and localization of the probe at disease sites, these particles can be used in the diagnosis of cancer.

[0036] The nanoparticle can have both a ligand and a contrast agent. The ligand allows the nanoparticle to target a specific cell type through specific binding between the ligand and the cellular component. This targeting, combined with multimodal imaging, has multiple uses. For example, nanoparticles can be used to map metastatic disease, such as sentinel lymph node (SLN) mapping, as well as to identify tumor margins or neural structures, allowing the surgeon to resect malignant lesions under direct visualization and avoid complications during surgery. surgery procedure. The ligand can also facilitate entry of the nanoparticle into the cell or barrier transport, for example, to analyze the intracellular environment.

[0037] The nanoparticle can be coupled with a ligand and a therapeutic agent with or without a radiolabel. The radiolabel can further serve as a therapeutic agent to create a theranostic platform. This coupling allows the therapeutic agent to be delivered to the specific cell type through specific binding between the ligand and the cellular component. This specific targeting of the therapeutic agent ensures selective treatment of the disease site with minimal side effects.

Nanoparticle structure

[0038] The fluorescent nanoparticle of the present invention includes a silica-based core comprising a fluorescent compound positioned within the core and a silica shell on the core. The silica shell surrounds at least a part of the core. The core of the nanoparticle may contain the reaction product of a reactive fluorescent compound and a coreactive organosilane compound. In another embodiment, the core of the nanoparticle may contain the reaction product of a reactive fluorescent compound and a co-reactive organosilane compound, and silica. The nanoparticle shell may be the reaction product of a silica-forming compound. The nanoparticle shell can have a variety of layers. For example, the silica shell may be from about 1 to about 20 layers, from about 1 to about 15 layers, from about 1 to about 10 layers, or from about 1 to about 5 layers.

[0039] The silica shell of the nanoparticle can cover only a part of the nanoparticle or the entire particle. For example, the silica shell can cover 1 to 100 percent, 10 to 80 percent, 20 to 60 percent, or 30 to 50 percent of the nanoparticle. The silica shell can be solid, ie, substantially non-porous, mesoporous, such as semi-porous, or porous.

Nanoparticle synthesis

[0040] The present fluorescent nanoparticle can be synthesized by the steps of: covalently conjugating a fluorescent compound, such as a reactive fluorescent dye, with reactive moieties including, but not limited to, maleimide, iodoacetamide, thiosulfate, amine, N-hydroxysuccimide ester, 4-sulfo-2,3,5,6-tetrafluorophenyl ester (STP), sulfosuccinimidyl ester, sulfodichlorophenol esters, sulfonyl chloride, hydroxyl, isothiocyanate, carboxyl, to an organosilane compound, such as a co-reactive organosilane compound, to form a fluorescent silica precursor, and reacting the fluorescent silica precursor to form a fluorescent core; covalently conjugating a fluorescent compound, such as a reactive fluorescent dye, with an organosilane compound, such as a co-reactive organosilane compound, to form a fluorescent silica precursor, and reacting the fluorescent silica precursor with a silica-forming compound, as tetraalkoxysilane, to form a fluorescent core; and reacting the resulting core with a silica-forming compound, such as a tetraalkoxysilane, to form a silica layer on the core, to provide the fluorescent nanoparticle.041*

[0041] The synthesis of the fluorescent monodisperse core-shell nanoparticles is based on a two-step process. First, organic near-infrared dye molecules ( eg, tetramethylrhodamine isothiocyanate (TRITC)) are covalently conjugated to a silica precursor and condense to form a dye-rich core. Second, silica gel monomers aggregate to form a denser silica lattice around the fluorescent core material, providing protection against solvent interactions that can be detrimental to photostability. The versatility of the preparative route allows for the incorporation of different fluorescent compounds, such as fluorescent organic compounds or dyes, depending on the intended application of the nanoparticles. Fluorescent compounds that can be incorporated into the dye-rich core can cover the entire absorption and emission spectrum from UV-Vis to near-IR. US Patent Application Nos. 10/306,614, 10/536,569 & 11/119,969. Wiesner et al., PEG-coated core-shell silica nanoparticles and methods of manufacture and use, PCT/US2008/74894.

[0042] For synthesis of the compact core-shell nanoparticle, the dye precursor is added to a reaction vessel containing appropriate amounts of ammonia, water, and solvent and allowed to react overnight. The dye precursor is synthesized by addition reaction between a specific near-infrared dye of interest and 3-aminopropyltriethoxysilane in a 1:50 molar ratio, without moisture. Once the synthesis of the dye-rich compact core is complete, tetraethyl orthosilicate (TEOS) is added to grow the silica shell surrounding the core.

[0043] Synthesis of the expanded core-shell nanoparticle is achieved by co-condensing TEOS with the dye precursor and allowing the mixture to react overnight. After synthesis of the expanded core is complete, additional TEOS is added to grow the silica shell surrounding the core.

[0044] The synthesis of the homogeneous nanoparticles is achieved by condensing together all the reagents, the dye precursor and TEOS and allowing the mixture to react overnight.

fluorescent compound

[0045] Nanoparticles can incorporate any known fluorescent compound, such as an organic fluorescent compound, dyes, pigments, or combinations thereof. A wide variety of suitable chemically reactive fluorescent dyes are known, see, for example, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, 6th ed., RP Haugland, ed. (nineteen ninety six). A typical fluorophore is, for example, a fluorescent aromatic or heteroaromatic compound, such as a pyrene, an anthracene, a naphthalene, an acridine, a stilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-amino -7-nitrobenz-2-oxa-l,3-diazole (NBD), a cyanine, a carbocyanine, a carbostyril, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, a coumarin (including hydroxycoumarins and aminocoumarins and fluorinated derivatives thereof), and similar compounds, see, for example, Ee Pat. US numbers 5,830,912, 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,433,896, 4,810,636, and 4,812,409. In one embodiment, Cy5, a near infrared fluorescent (NIRF) dye, is placed within the silica core of the present nanoparticle. Near-infrared emitting probes show decreased tissue attenuation and autofluorescence. Burns et al. "Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine", Nano Letters, 2009, 9 (1), 442-448.

[0046] Non-limiting fluorescent compounds that can be used in the present invention include Cy5, Cy5.5 (also known as Cy5++), Cy2, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRlTC), phycoerythrin, Cy7, fluorescein ( FAM), Cy3, Cy3.5 (also known as Cy3++), Texas Red, LightCycler-Red 640, LightCycler Red 705, tetramethylrhodamine (TMR), rhodamine, rhodamine derivative (ROX), hexachlorofluorescein (HEX), rhodamine 6G (R6G ), rhodamine derivative JA133, Alexa Fluorescent Dyes (as Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 555, and Alexa Fluor 647), 4',6-diamidino-2-phenylindole (DAPI), Propidium, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lisamine, and fluorescent transition metal complexes, such as Europium. Fluorescent compounds that can be used also include fluorescent proteins, such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein and derivatives (CFP , ECFP, Cerulean, CyPet) and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet). WO2008142571, WO2009056282, WO9922026.

[0047] The surface of the silica shell of nanoparticles can be modified using known crosslinking agents to introduce functional groups onto the surface. Crosslinking agents include, but are not limited to, divinylbenzene, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, N,N'-methylene-bis-acrylamide, alkyl ethers, sugars, peptides, DNA fragments, or other known functionally equivalent agents. The ligand can be conjugated with the nanoparticle of the present invention, for example, by coupling reactions using carbodiimide, carboxylates, esters, alcohols, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, halides, or any other suitable compound known in the art. the art. US Patent No. 6,268,222.

organic polymer

[0048] An organic polymer is attached to the present nanoparticle, eg, attached to the surface of the nanoparticle. An organic polymer can be attached to the silica shell of the present nanoparticle. The organic polymer that can be used in the present invention includes PEG, polylactate, polylactic acids, sugars, lipids, polyglutamic acid (PGA), polyglycolic acid, poly(lactic-co-glycolic) acid (PLGA), polyvinyl acetate (PVA) , and combinations thereof. The attachment of the organic polymer to the nanoparticle can be achieved by covalent bonding or non-covalent bonding, such as ionic bonding, hydrogen bonding, hydrophobic bonding, coordination, adhesive, and physical absorption. In one embodiment, the nanoparticle is covalently conjugated to PEG, which prevents serum protein adsorption, facilitates efficient urinary excretion, and reduces aggregation of the nanoparticle. Burns et al. "Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine", Nano Letters. 2009, 9(1), 442-448049

[0049] The surface of the nanoparticle can be modified to incorporate at least one functional group. the polymer organic ( eg, PEG) attached to the nanoparticle can be modified to incorporate at least one functional group. For example, the functional group may be a maleimide ester or n-hydroxysuccinimide (NHS). The incorporation of the functional group allows various ligands, contrast agents and/or therapeutic agents to be attached to the nanoparticle.

ligand

[0050] A ligand according to the claims is bound to the present nanoparticle. The ligand is capable of binding to at least one cellular component. The cellular component to which the ligands attached to the nanoparticles can bind can be associated with specific cell types or have elevated levels in specific cell types, such as cancer cells or specific cells of particular tissues and organs. Accordingly, the nanoparticle can be targeted to a specific cell type and/or provide targeted delivery for treatment and diagnosis of disease. As used herein, the term "ligand" refers to a molecule or entity that can be used to identify, detect, target, monitor, or modify a physical state or condition, such as a disease state or condition. For example, a ligand can be used to detect the presence or absence of a particular receptor, the expression level of a particular receptor, or the metabolic levels of a particular receptor. A ligand can be, for example, a peptide, a protein, a protein fragment, a peptide hormone, a sugar (i.e., lectins), a biopolymer, a synthetic polymer, an antigen, an antibody, an antibody fragment ( e.g., Fab, nanobodies), an aptamer, a virus or viral component, a receptor, a hapten, an enzyme, a hormone, a chemical compound, a pathogen, a microorganism or a component thereof, a toxin, a modifier of surface, such as a surfactant to alter the surface properties or histocompatibility of the nanoparticle or of an analyte when a nanoparticle is associated with it, and combinations thereof. In one aspect disclosed herein but not claimed, the ligands are antibodies such as monoclonal or polyclonal antibodies. In another aspect described herein, the ligands are receptor ligands. In yet another aspect described herein, the ligand is poly-L-lysine (pLysine).

[0051] An antigen can be attached to the nanoparticle. The antigen-bound nanoparticle can be used for vaccination.

[0052] The terms "component of a cell" or "cellular component" refer, for example, to a receptor, an antibody, a hapten, an enzyme, a hormone, a biopolymer, an antigen, a nucleic acid (DNA or RNA), a microorganism, a virus, a pathogen, a toxin, combinations thereof, and similar components. The component of a cell can be placed on the cell ( eg, a transmembrane receptor) or within the cell. In one embodiment, the component of a cell is a tumor marker. As used herein, the term "tumor marker" refers to a molecule, entity, or substance that is expressed or overexpressed in a cancer cell but not in a normal cell. For example, overexpression of certain receptors is associated with many types of cancer. A ligand capable of binding a tumor marker can be conjugated to the surface of the present nanoparticle so that the nanoparticle can be specifically targeted to the tumor cell.

[0053] A ligand can be attached to the present nanoparticle directly or through a linker. Attachment of the ligand to the nanoparticle can be achieved by covalent bonding or non-covalent bonding, such as ionic bonding, hydrogen bonding, hydrophobic bonding, coordination, adhesive, and physical absorption. The ligand can be coated on the surface of the nanoparticle. The ligand can be embedded in the surface of the nanoparticle. The ligand can be attached to the surface of the fluorescent nanoparticle or can be attached to the core when the shell is porous or covers a part of the core. When the ligand is attached to the nanoparticle via a linker, the linker can be any suitable molecule, such as a functionalized PEG. PEGs can have multiple functional groups to bind to the nanoparticle and ligands. The particle can have different types of functionalized PEGs that carry different functional groups that can bind multiple ligands. This can enhance the effects of multivalence and/or contrast at the target site, allowing for the design and optimization of a complex multimodal platform with enhanced targeted screening, treatment, and detection in vivo.

[0054] A variety of different ligands can be attached to the nanopanicle. For example, the tripeptide Arg-Gly-Asp (RGD) can be attached to the nanoparticle described herein but not claimed. Alternatively, the cyclic peptide cRGD (which may contain other amino acid(s), eg, cRGDY) can be attached to the nanoparticle. Any linear, cyclic or branched peptide containing the RGD sequence is within the scope of the present disclosure. RGD binds to the avp3 integrin, which is overexpressed on the surface of activated endothelial cells during angiogenesis and in various types of tumor cells. Expression levels of avp3 integrin have been shown to correlate well with the aggressiveness of tumors. Ruoslahti et al. New perspectives in cell adhesion: RGD and integrins. Science 1987;238,491. Gladson et al. Glioblastoma expression of vitronectin and integrin alpha v beta 3. Adhesion mechanism for transformed glial cells. J.Clin. Invest. 1991; 88:1924-1932. Seftor et al. Role of the alpha v beta 3 integrin in human melanoma cell invasion. Proc. Natl. Acad. Sci 1992; 89:1557-1561.05*

[0055] The synthetic peptide EPPT1 may be the ligand attached to the nanoparticle described herein but not claimed. EPPT1, derived from the monoclonal antibody binding site (ASM2), targets subglycosylated MUC1 (uMUC 1). MUC1, a transmembrane receptor, is heavily glycosylated in normal tissues; however, it is aberrantly overexpressed and underglycosylated in nearly all human epithelial cell adenocarcinomas, and is implicated in tumor pathogenesis. Moore et al. In vivo targeting of underglycosylated MUC-1 tumor antigen using a multimodal imaging probe. Cancer Res. 2004; 64:1821-7. Patel et al. MUC 1 plays a role in tumor maintenance in aggressive thyroid carcinomas. Surgery. 2005; 138:994-1001. Specific antibodies, including monoclonal antibodies against uMUC1, can alternatively be conjugated to the nanoparticle described herein to target uMUC1.

[0056] In one aspect described herein but not claimed, peptide analogs of a-melanotropin-stimulating hormone (aMSH) are the ligands attached to the nanoparticle. α-MSH peptide analogues are capable of binding to melanocortin-1 receptors (MC1R), a family of G protein-coupled receptors overexpressed in melanoma cells. Loire et al. Mol.Biol cell. (Noisy-le-grand) 1999, 45:1083-1092.

[0057] In another aspect described herein but not claimed, octreotate, a 14 amino acid peptide analogue of somatostatin, is the ligand bound to the nanoparticle. Octreotide, which has a longer half-life than somatostatin, is capable of binding to the somatostatin receptor (SSTR). SSTR, a member of the G protein-coupled receptor family, is overexpressed on the surface of various human tumors. Reubi et al. Distribution of Somatostatin Receptors in Normal and Tumor-Tissue. Metab. clin. File 1990; 39:78-81. Reubi et al. Somatostatin receptors and their subtypes in human tumors and in peritumoral vessels. Metab. clin. Exp. 1996:45:39-41. Other somatostatin analogs can alternatively be conjugated to the nanoparticle described herein but not claimed to target SSTR, such as Tyr3-octreotide (Y3-OC), octreotate (TATE), Tyr3-octreotate (Y3-TATE), and 111In-DTPA- OC. These somatostatin analogues can be used for both PET diagnostic imaging and targeted radiation therapy of cancer. de Jong et al. Internalization of radiolabelled [DTPA0]octreotide and [DOTA0, Tyr3]octreotide: peptides for somatostatin receptor targeted scintigraphy and radionuclide therapy. Nucl. Med. Comm. 1998; 19:283-8. de Jong et al. Comparison of 111 In-Labeled Somatostatin Analogues for Tumor Scintigraphy and Radionuclide Therapy. Cancer Res. 1998;58:437-41. Lewis et al. Comparison of four 64Cu-labeled somatostatin analogs in vitro and in a tumor-bearing rat model: evaluation of new derivatives for PET imaging and targeted radiotherapy. J Med Chem 1999;42:1 341-7. Krenning et al. Somatostatin Receptor Scintigraphy with Indium-111-DTPA-D-Phe-1-Octreotide in Man: Metabolism, Dosimetry and Comparison with lodine-123-Tyr-3-Octreotide. J Nucl. Med. 1992;33:652-8.

[0058] Various ligands can be used to map sentinel lymph nodes (SLNs). SLN mapping can be used to diagnose, stage, and treat cancer. J591 is an anti-prostate specific membrane antigen (ie, anti-PSMA) monoclonal antibody. J591 has previously been used to detect and stage prostate cancer. Tagawa et al., nti-prostate-specific membrane antigen-based radioimmunotherapy for prostate cancer, Cancer, 2010, 116(4 Suppl): 1075-83. Bander et al., Targeting Metastatic Prostate Cancer with Radiolabeled Monoclonal Antibody J591 to the Extracellular Domain of Prostate Specific Membrane Antigen, The Journal of Urology 170(5), 1717-1721 (2003). Wernicke et al., (2011) Prostate-Specific Membrane Antigen as a Potential Novel Vascular Target for Treatment of Glioblastoma Multiforme, Arch Pathol Lab Med. 2011; 135:1486-1489. The F(ab ^')2 fragment of J591 can be used as a ligand attached to the nanoparticle described herein but not claimed. The nanoparticle can also be radiolabeled to create a dual-modality probe. In one aspect described herein, nanoparticles bearing the J591 F(ab') ² fragment ( eg, HuJ591-F(ab')2 fragments or humanized J591-F(ab')2 fragments) they are used to diagnose prostate cancer or endometrial cancer ( eg, endometrial endometrioid adenocarcinoma). In another aspect described herein, nanoparticles carrying the F(ab ^')2 fragment of J591 can be used to target the neovasculature of a brain tumor for treatment, monitoring disease progression. Brain tumor neovasculature has been found to overexpress PSMA, as shown in previous immunohistochemical evaluations of excised high-grade glioma specimens.

[0059] Cyclic peptides containing the HWGF sequence are potent and selective inhibitors of MMP-2 and MMP-9 but not several other members of the MMP family. The CTTHWGFTLC peptide inhibits the migration of human endothelial cells and tumor cells. Furthermore, it prevents the growth and invasion of tumors in animal models and improves the survival of mice with human tumors. Phage displaying CTTHWGFTLC specifically target angiogenic blood vessels in vivo. This peptide and its extension GRENYGHCTTHWGFTLC or GRENYGHCTTHWGFTLS can be used as ligands to bind to the nanoparticle described in this document but not claimed. Peptides can also be radiolabeled, eg radioiodinated. Koivunen et al., Tumor targeting with a selective gelatinase inhibitor, Nature Biotechnology 17, 768-774 (1999). Penate Medina et al., Liposomal tumor targeting in drug delivery using MMP-2- and MMP-9-binding ligands, J. Drug Delivery, Volume 2011 (2011), Article ID 160515. Anticancer Research 21:4101-4106 (2005) . In one aspect described herein, 124I-labeled matrix metalloproteinase peptide inhibitor (MMPI)-bound nanoparticles are used for mapping SLNs to classify endometrioid cancer.06*

[0060] The number of ligands bound to the nanoparticle can range from 1 to 20, 2 to 15, 3 to 10, 1 to 10, or 1 to 6. A small amount of ligands bound to the nanoparticle helps to maintain the hydrodynamic diameter of the present nanoparticle that meets the renal clearance cutoff size range. Hilderbrand et al., Nearinfrared fluorescence: application to in vivo molecular imaging, Curr. Opinion. Chem Biol, 14:71-9,2010. The number of ligands measured can be an average number of ligands bound to more than one nanoparticle. Alternatively, a nanoparticle can be measured to determine the number of bound ligands. The number of ligands bound to the nanoparticle can be measured by any suitable method, which may or may not be related to the properties of the ligands. For example, the number of cRGD peptides bound to the particle as described herein but not claimed can be estimated using ECF-based measurements of absolute particle concentrations and the concentration starting point of reagents for cRGD peptide. The average number of RGD peptides per nanoparticle and the coupling efficiency of RGD to functionalized PEG groups can be evaluated colorimetrically under alkaline conditions and Biuret spectrophotometric methods. The number of ligands bound to the nanoparticle can also be measured by other suitable methods.

contrast agent

[0061] A contrast agent may be attached to the present nanoparticle for biological or medical imaging. As used herein, the term "contrast agent" refers to a substance, molecule, or compound used to enhance the visibility of structures or fluids in medical or biological images. The term "contrast agent" also refers to a contrast-producing molecule. Imaging techniques encompassed by the present invention include positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), formation of optical bioluminescence imaging, optical fluorescence imaging, and combinations thereof. The contrast agent encompassed by the present invention can be any molecule, substance, or compound known in the art for PET, SPECT, CT, MRI, and optical imaging. The contrast agent can be radionuclides, radiometals, positron emitters, beta emitters, gamma emitters, alpha emitters, paramagnetic metal ions, and supraparamagnetic metal ions. Contrast agents include, but are not limited to, iodine, fluorine, copper, zirconium, lutetium, astatine, yttrium, gallium, indium, technetium, gadolinium, dysprosium, iron, manganese, barium, and barium sulfate. Radionuclides that can be used as a contrast agent attached to the nanoparticle of the present invention include, among others, 89Zr, 64Cu, 06*8*Ga, 86Y, 124I, and 177Lu.

[0062] The contrast agent can be directly conjugated to the nanoparticle. Alternatively, the contrast agent can be indirectly conjugated to the nanoparticle by binding to linkers or chelates. The chelate can be adapted to bind to a radionuclide. Chelates that can be attached to the present nanoparticle can include, among others, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), deferoxamine (DFO), and triethylenetetramine (TETA).

[0063] Suitable means for imaging, detecting, recording, or measuring the present nanoparticles may also include, for example, a flow cytometer, a laser scanning cytometer, a fluorescence microplate reader, a fluorescence microscope, a microscope confocal, a bright light microscope. Holding microscope, a high-content scanning system, and similar devices. More than one imaging technique can be used at the same time or consecutively to detect the present nanoparticles. In one embodiment, optical imaging is used as a high-throughput, sensitive scanning tool to acquire multiple time points in the same subject, allowing semi-quantitative assessments of tumor marker levels. This compensates for the relatively low temporal resolution obtained with PET, although PET is needed to achieve adequate depth of penetration to acquire volumetric data and to detect, quantify, and monitor changes in receptor levels and/or other cellular markers as a means of assess disease progression. or improvement, as well as stratifying patients in appropriate treatment protocols.

therapeutic agent

[0064] A therapeutic agent can be attached to the fluorescent nanoparticle, eg, for targeted treatment of disease. The therapeutic agent can be administered to a diseased site in a highly specific or localized manner with delivery of the therapeutic agent to the disease site. Alternatively, the therapeutic agent may not be released. The ligand-conjugated fluorescent nanoparticles can be used for targeted delivery of a therapeutic agent to a desired location in a variety of systems, such as on or within a cell or cellular component, within the body of an organism, such as a human. , or through the blood-brain barrier.

[0065] The therapeutic agent can be attached to the nanoparticle directly or indirectly. The therapeutic agent can be absorbed into the interstices or pores of the silica shell or coat the silica shell of the fluorescent nanoparticle. In other embodiments where the silica shell does not cover the entire surface, the therapeutic agent may associate with the fluorescent core, such as by physical absorption or binding interaction. The therapeutic agent can be associated with the ligand that is bound to the fluorescent nanoparticle. The therapeutic agent may also be associated with the organic polymer or contrast agent. For example, the therapeutic agent may be attached to the nanoparticle via PEG. PEGs can have multiple functional groups to be attached to the nanoparticle and therapeutic agent. The particle can have different types of functionalized PEGs that carry different functional groups that can bind multiple therapeutic agents. The therapeutic agent can be attached to the nanoparticle covalently or non-covalently.

[0066] As used herein, the term "therapeutic agent" refers to a substance that can be used in the diagnosis, cure, mitigation, treatment, or prevention of disease in a human or other animal. Such therapeutic agents include substances recognized in the Official United States Pharmacopeia, the Official Homeopathic United States Pharmacopeia, the Official National Formulary, or any supplements thereto.

[0067] Therapeutic agents that can be incorporated with the fluorescent nanoparticles or ligated fluorescent nanoparticles of the invention include nucleosides, nucleoside analogs, small interfering RNA (siRNA), microRNA, oligopeptides, polypeptides, antibodies, COX-2 inhibitors , promoters of apoptosis, urinary tract agents, vaginal agents, vasodilators, neurodegenerative agents ( eg, Parkinson's disease), obesity agents, ophthalmic agents, osteoporosis agents, parasympatholytics, parasympathomitics, anti-anesthetics, prostaglandin agents, psychotherapeutics, respiratory agents, sedatives, hypnotics, cutaneous and mucosal membrane agents, antibacterials, antifungals, antineoplastics, cardioprotective agents, cardiovascular agents, antithrombotics, central nervous system stimulants, cholinesterase inhibitors, contraceptives, dopamine receptor agonists, agents for dis erectile function, fertility agents, gastrointestinal agents, gout agents, hormones, immunomodulators, suitably functionalized analgesics or general or local anesthetics, anticonvulsants, antidiabetic agents, antifibrotic agents, antiinfectives, motion sickness agents, muscle relaxants, immunosuppressive agents, migraine headache agents, nonsteroidal anti-inflammatory drugs (NSAIDs), smoking cessation agents, or sympatholytics (see Physicians' Desk Reference, 55th ed., 2001, Medical Economics Company, Inc., Montvale, NJ, pages 201-202) .

[0068] Therapeutic agents that can be bound to the present nanoparticle include, but are not limited to, DNA alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress-inducing agents, a platinum compound, an antimetabolite, vincalkaloids, taxanes, epothilones, inhibitors enzyme agents, receptor antagonists, therapeutic antibodies, tyrosine kinase inhibitors, boron (ie, velcade) radiosensitizers, and chemotherapeutic combination therapies.

[0069] Non-limiting examples of DNA alkylating agents are nitrogen mustards, such as mechlorethamine, cyclophosphamide (ifosfamide, trofosphamide), chlorambucil (melphalan, prednimustine), bendamustine, uramustine, and estramustine; nitrosoureas, such as carmustine (BCNU), lomustine (Semustine), fotemustine, nimustine, ranimustine, and streptozocin; alkyl sulfonates, such as Busulfan (Mannosulfan, Treosulfan); aziridines, such as carboquone, thioTEPA, triaziquone, triethylenemelamine; hydrazines (procarbazine); triazenes such as dacarbazine and temozolomide; Altretamine and Mitobronitol.

[0070] Non-limiting examples of topoisomerase I inhibitors include camptothecin derivatives including CPT-II (irinotecan), SN-38, APC, NPC, camptothecin, topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin, lurtotecan, rubitecan, silatecan, gimatecan, diflomotecan, extatecan, BN-80927, DX-8951f and MAG-CPT as described in Pommier Y. (2006) Nat Rev. Cancer 6(10):789-802 and patent publication of ee . USA

200510250854; Protoberberine alkaloids and derivatives thereof including berberrubine and coralyne as described in Li et al. (2000) Biochemistry 39(24):7107-7116 and Gatto et al. (1996) Cancer Res. 15(12):2795-2800; Phenanthroline derivatives including Benzo[i]phenanthridine, Nitidine, and fagaronine as described in Makhey et al. (2003) Bioorg, Med. Chem. 11(8): 1809-1820; Terbenzimidazole and derivatives thereof as described in Xu (1998) Biochemistry 37(10):3558-3566; and Anthracycline derivatives including Doxorubicin, Daunorubicin, and Mitoxantrone as described in Foglesong et al. (1992) Cancer Chemother. Pharmacol. 30(2):123-]25, Crow et al. (1994) J. Med. Chem. 37(19):31913194, and Crespi et al. (1986) Biochem. Biophys. Res. Comm. 136(2):521-8. Topoisomerase II inhibitors include, but are not limited to, etoposide and teniposide. Dual topoisomerase I and II inhibitors include, but are not limited to, Saintopin and other naphthecediones, DACA and other acridine-4-carboxamines, Intoplicin and other benzopyridoindoles, TAS-103 and other 7-hindeno[2,1-c]quinoline-7-pyrazoloacridine , XR 11576 and other benzophenazines, XR 5944 and other dimeric compounds, 7-oxo-7H-dibenz[f,ij]lsoquinolines and 7-oxo-7H-benzo[e]Perimidines, and anthracenyl-amino acid conjugates as described in Denny and Baguley (2003) Curr. Top. Med. Chem 3(3):339-353. Some agents inhibit topoisomerase II and have DNA intercalating activity, including but not limited to anthracyclines (aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin) and anthracenediones (mitoxantrone and pixantrone).

[0071] Examples of endoplasmic reticulum stress inducing agents include, but are not limited to dimethylcelecoxib (CDM), nelfinavir, celecoxib, and boron radiosensitizers (ie, velcade (bortezomib)).

[0072] Non-limiting examples of platinum-based compounds include carboplatin, cisplatin, nedaplatin, oxaliplatin, triplatin tetranitrate, satraplatin, aroplatin, lobaplatin, and JM-216. (see McKeage et al. (1997) J. Clin. Oncol. 201:1232-1237 and generally, CHEMOTHERAPY FOR GYNECOLOGICAL NEOPLASM, CURRENT THERAPY AND NOVEL APPROACHa Ch ES, in the Basic and Clinical Oncology series, Angioli et al. Eds ., 2004).073

[0073] Non-limiting examples of antimetabolite agents include folic acid-based inhibitors, ie, dihydrofolate reductase, such as aminopterin, methotrexate and pemetrexed; thymidylate synthase inhibitors, such as Raltitrexed, Pemetrexed; purine-based, i.e., an adenosine deaminase inhibitor, such as pentostatin, a thiopurine, such as thioguanine and mercaptopurine, a halogenated reductase inhibitor/ribonucleotide, such as cladribine, clofarabine, fludarabine, or a guanine/guanosine:thiopurine, such as thioguanine ; or pyrimidine-based, i.e., cytosine/cytidine: hypomethylating agent, such as azacitidine and decitabine, a DNA polymerase inhibitor, such as cytarabine, a ribonucleotide reductase inhibitor, such as gemcitabine, or an inhibitor of thymine/thymidine: thymidylate synthase, such as fluorouracil (5-FU). 5-FU equivalents include prodrugs, analogs, and derivatives thereof, such as 5'-deoxy-5-fluorouridine (doxyfluroidine), 1-tetrahydrofuranyl-5-fluorouracil (ftorafur), capecitabine (Xeloda), S-1 (MBMS -247616, consisting of tegafur and two modulators, a 5-chloro-2,4-dihydroxypyridine and potassium oxonate), ralititrexed (tomudex), nolatrexed (Thymitaq, AG337), LY231514, and ZD9331, as described, for example, in Papamicheal (1999) The Oncologist 4:478-487.

[0074] Examples of vincalkaloids include, but are not limited to, vinblastine, vincristine, vinflunine, vindesine, and vinorelbine.

[0075] Examples of taxanes include, but are not limited to, docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel. An example of an epothilone is iabepilone.

[0076] Examples of enzyme inhibitors include, but are not limited to farnesyl transferase inhibitors (Tipifamib); CDK inhibitor (Alvocidib, Seliciclib); proteasome inhibitor (bortezomib); phosphodiesterase inhibitor (anagrelide; rolipram); IMP dehydrogenase inhibitor (Thiazophyirin); and lipoxygenase inhibitor (Masoprocol). Examples of receptor antagonists include, among others, ERA (Atrasentan); retinoid X receptor (Bexarotene); and a sex steroid (testolactone).

[0077] Examples of therapeutic antibodies include, but are not limited to anti-HER1/EGFR (Cetuximab, Panitumumab); Anti-HER2/neu receptor (erbB2) (Trastuzumab); Anti-EpCAM (Catumaxomab, Edrecolomab) Anti-VEGF-A (Bevacizumab); Anti-CD20 (Rituximab, Tositumomab, Ibritumomab); Anti-CD52 (Alemtuzumab); and Anti-CD33 (Gemtuzumab). US Patent Numbers 5,776,427 and 7,601,355.

[0078] Examples of tyrosine kinase inhibitors include, but are not limited to, ErbB inhibitors: HER1/EGFR (Erlotinib, Gefitinib, Lapatinib, Vandetanib, Sunitinib, Neratinib); HER2/neu (Lapatinib, Neratinib); RTK class III: C-kit (Axitinib, Sunitinib, Sorafenib), FLT3 (Lestaurtinib), PDGFR (Axitinib, Sunitinib, Sorafenib); and VEGFR (vandetanib, semaxanib, cediranib, axitinib, sorafenib), bcr-abl (imatinib, nilotinib, dasatinib); Src (Bosutinib) and Janus kinase 2 (Lestaurtinib).

[0079] Chemotherapeutic agents that can be attached to the present nanoparticle can also include amsacrine, trabectedin, retinoids (alitretinoin, tretinoin), arsenic trioxide, asparagine-reducing asparaginase/pegaspargase), celecoxib, demecolcine, elesclomol, elsamitrucine, etoglucid , lonidamina, lucantona., Mitoguazona, Mitotano, Oblimersen, Temsirolimus and Vorinostat.

[0080] Examples of specific therapeutic agents that can bind, bind or associate with the fluorescent nanoparticles of the invention are flomoxef; fortimicin(s); gentamicin(s); glycosulfone solasulfone; gramicidin S; gramicidin(s); grepafloxacin; guamocycline; hetacillin; isepamycin; josamycin; kanamycin(s); flomoxef; fortimicin(s); gentamicin(s); glycosulfone solasulfone; gramicidin S; gramicidin(s); grepafloxacin; guamecycline; hetacillin; isepamycin; josamycin; kanamycin(s); bacitracin; bambermycin(s); biapenem; brodymoprim; butyrosine; capreomycin; carbenicillin; carbomycin; carumonamon; cefadroxil; cefamandole; cefatrizine; cefbuperazone; cefclidine; cefdinir; cefditoren; cefepime; cefetameth; cefixime; cefinenoxime; cefininox; cladribine; apalcillin; apicycline; apramycin; arbekacin; aspoxicillin; azidamphenicol; aztreonam; cefodizime; cefonicida; cefoperazone; ceforamide; cefotaxime; cefotetan; cefotiam; cefozopran; cefpimizole; cefipyramide; cephyroma; cefprozil; cephroxadine; cefteram; ceftibuten; cefuzonam; cephalexin; cephaloglycin; cephalosporin C; cephradine; chloramphenicol; chlortetracycline; clinafloxacin; clindamycin; clomocycline; colistin; cyclacillin; dapsone; demeclocycline; diathymosulfone; dibekacin; dihydrostreptomycin; 6-mercaptopurine; thioguanine; capecitabine; docetaxel; etoposide; gemcitabine; topotecan; vinorelbine; vincristine; vinblastine; teniposide; melphalan; methotrexate; 2-p-sulfaniliananilinoethanol; 4,4'-sulfinyldianiline; 4-sulfanylamidosalicylic acid; butorphanol; nalbuphine. streptozocin; doxorubicin; daunorubicin; plicamycin; idarubicin; mitomycin C; pentostatin; mitoxantrone; cytarabine; fludarabine phosphate; butorphanol; nalbuphine. streptozocin; doxorubicin; daunorubicin; plicamycin; idarubicin; mitomycin C; pentostatin; mitoxantrone; cytarabine; fludarabine phosphate; acediasulfone; acetosulfone; amikacin; amphotericin B; ampicillin; atorvastatin; enalapril; ranitidine; ciprofloxacin; pravastatin; clarithromycin; cyclosporine; famotidine; leuprolide; acyclovir; paclitaxel; azithromycin; lamivudine; budesonide; albuterol; indinavir; metformin; alendronate; nizatidine; zidovudine; carboplatin; metoprolol; amoxicillin; diclofenac; lisinopril; ceftriaxone; captopril; salmeterol; xinafoate; imipenem; cilastatin; benazepril; cefaclor; ceftazidime; morphine; dopamine; bialamicol; fluvastatin; phenamidine; 2-ethylhydrazine podophyllinic acid; acriflavine; chloroazodine; arsphenamine; amicarbilide; aminoquinuride; quinapril; oxymorphone; buprenorphine; floxuridine; dirithromycin; doxycycline; enoxacin; enviomycin; epicillin; erythromycin; leucomycin(s); lincomycin; lomefloxacin; lucensomycin; lymecycline; meclocycline; meropenem; metacycline; micronomycin; midecamycin(s); minocycline; moxalactam; mupirocin; nadifloxacin; natamycin; neomycin; netilmicin; norfloxacin; oleandomycin; oxytetracycline; p-sulfanylylbenzylamine; panipenem; paromomycin; pazufloxacin; penicillin N; pipacycline; pipemidic acid; polymyxin; primicin; quinacillin; ribostamycin; rifamide; rifampicin; rifamycin SV; rifapentine; rifaximin; ristocetin; ritipenem; rokitamycin; rolitetracycline; rosaramycin; roxithromycin; salazosulfadimidine; sancycline; sisomycin; sparfloxacin; spectinomycin; spiramycin; streptomycin; succisulfone; sulfacrysoidine; sulfaloxic acid; sulfamidochrysoidine; sulfanilic acid; sulfoxone; teicoplanin; temaftoxacin; Temocillin; tetroxoprim; thiamphenicol; thiazolesulfone; thiostrepton; ticarcillin; tigemonam; tobramycin; tosulfloxacin; trimethoprim; trospectomycin; trovafloxacin; tuberactinomycin; vancomycin; azaserin; candicidin(s); chlorphenesin; dermatostatin(s); Filipina; fungichromin; mepartricin; nystatin; oligomycin(s); perimycin A; tubercidin; 6-azauridine; 6-diazo-5-oxo-L-norleucine; aclacinomycin(s); ancitabine; anthramycin; azacitadine; azaserin; bleomycin(s); ethyl biscoumacetate; ethylidene dicoumarol; iloprost; lamifiban; taprostene; thioclomarol; tirofiban; amiprilose; bucillamine; gusperimus; gentisic acid; glucamethacin; glycol salicylate; medofenamic acid; Mefenamic acid; mesalamine; niflumic acid; olsalazine; oxaceprole; S-enosylmethionine; salicylic acid; salsalate; sulfasalazine; tolfenamic acid; carubicin; carzinophyllin A; chlorozotocin; chromomycin(s); denopterin; doxifluridine; edatrexate; eflornithine; elliptinium; enocitabine; epirubicin; mannomustine; menogaril; mitobronitol; mitolactol; mopidamol; mycophenolic acid; nogalamycin; Olivomycin(s); peplomycin; pyrarubicin; pyritrexim; prednimustine; procarbazine; pteropterin; puromycin; ranimustine; strepthongrin; thiamiprine; mycophenolic acid; procodazole; romurtida; sirolimus (rapamycin); tacrolimus; butetamine; phenalcomine; hydroxytetracaine; naepain; orthocaine; pyridocaine; salicylic alcohol; 3-amino-4-hydroxybutyric acid; aceclofenac; Alminoprofen; amfenac; bromfenac; broniosaligenin; bumadison; carprofen; diclofenac; diflunisal; ditazole; enfenamic acid; etodolac; etofenamate; fendosal; fepradinol; flufenamic acid; Tomudex® (N-[[5-ff(1,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]methylamino]-2-thienyl]carbonyl]-L-glutamic acid), trimetrexate, tubecidine , ubenimex, vindesine, zorubicin; argatroban; coumetarol or dicumarol.

[0081] Lists of additional therapeutic agents can be found, for example, in: Physicians' Desk. Reference, 55th ed., 2001, Medical Economics Company, Inc., Montvale, NJ; USPN Dictionary of USAN and International Drug Names, 2000, The United States Pharmacopeial Convention, Inc., Rockville, Md; and The Merck Index, 12th ed., 1996, Merck & Co., Inc., Whitehouse Station, NJ

[0082] The therapeutic agent can also include radionuclides when the present nanoparticle is used in targeted radiotherapy. In one embodiment, low energy beta-emitting radionuclides, such as 177Lu chelated constructs, are associated with the nanoparticle and used to treat relatively small tumor burdens or micrometastatic disease. In another embodiment, higher energy beta emitters, such as yttrium-90 (90Y), can be used to treat larger tumor burdens. Iodine-131 (131I) can also be used for radiation therapy.

[0083] The surface of the nanoparticle can be modified to incorporate at least one functional group. The organic polymer ( eg, PEG) attached to the nanoparticle can be modified to incorporate at least one functional group. For example, the functional group may be a maleimide ester or n-hydroxysuccinimide (NHS). The incorporation of the functional group allows various ligands, contrast agents and/or therapeutic agents to be attached to the nanoparticle.

[0084] In one embodiment, a therapeutic agent is attached to the nanoparticle (organic polymer surface or coating) through an NHS ester functional group. For example, a tyrosine kinase inhibitor such as dasatinib (BMS) or a chemotherapeutic agent ( eg, taxol) can be coupled via an ester bond to the nanoparticle. This ester bond can then be cleaved in an acidic environment or enzymatically in vivo. This approach can be used to administer a prodrug to a subject where the drug is released from the particle in vivo.

[0085] We have tested the prodrug approach by coupling the small molecule inhibitor dasatinib with the PEG molecules of the nanoparticle. Based on biodistribution results and human drug dosage calculations, the nanoparticle has been found to have unique biological properties, including relatively rapid clearance from the blood compared to tumors and subsequent accumulation of the therapeutic agent. in tumor tissue, suggesting that a prodrug approach is feasible. The functionalized nanoparticle allows drugs to be dosed multiple times, ensuring that the drug concentration in the tumor is greater than that specified by the IC-50 in tumor tissue, but not limiting the dose in other organ tissues, like the heart liver or kidney. The therapeutic agent and nanoparticle can be separately radiolabeled or optically labeled, allowing independent control of the therapeutic agent and nanoparticle. In one embodiment, radiofluorinated (ie, 18F) dasatinib is coupled with moieties of PEG-3400 attached to the nanoparticle through NHS ester linkages. Radiofluoride is critical to being able to independently control time-dependent changes in drug distribution and release from the radioiodinated (124I) fluorescent (Cy5) nanoparticle. In this way, we can separately monitor the prodrug (dasatinib) and the nanoparticle. This allows for optimization of prodrug design compared to prior art methods where a dual labeling approach is not used. In another embodiment, radiotherapeutic iodine (ie, I-131) molecules, or other therapeutic alpha or gamma emitters, are conjugated to PEG through a maleimide functional group, where the therapeutic agent cannot dissociate from PEG in vivo.

[0086] A generalizable approach referred to herein as "click chemistry" is described below. In order for the present nanoparticle to be easily adapted to a wide variety of ligands, contrast agents or chelates, the surface of the nanoparticle can be modified to incorporate a functional group. The nanoparticle can also be modified with organic polymers (eg PEGs) or chelates that can incorporate a functional group. Meanwhile, the ligand, contrast agent or therapeutic agent is modified to incorporate a functional group that is capable of reacting with the functional group of the nanoparticle, or of the PEGs or chelating agents attached to the nanoparticle under suitable conditions. Consequently, any ligand, contrast agent, or therapeutic agent having the reactive functional group can be easily conjugated to the nanoparticle. This generalizable approach is referred to here as "click chemistry", which would allow for great versatility in exploring multimodal applications. In "click chemistry" chemical reactions, two molecular components can be brought together by a selective, rapid, clean, bioorthogonal, and/or biocompatible reaction. Kolb et al., (2001) Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angewandte Chemie International Edition 40, 2004-2021. Lim et al., (2010) Bioorthogonal chemistry: recent progress and future directions. Chemical Communications 46, 1589-1600. Sletten et al., (2009) Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angewandte Chemie International Edition 48, 6973-6998.

[0087] Any suitable reaction mechanism can be adapted in the present invention for "click chemistry", as long as easy and controlled binding of the ligand, contrast agent or chelate to the nanoparticle can be achieved. In one embodiment, a free triple bond is introduced into PEG, which is already covalently conjugated to the nanoparticle shell. Meanwhile, an azide bond is introduced into the desired ligand (or contrast agent, chelate). When the PEGylated nanoparticle and ligand (or contrast agent, chelate) are mixed in the presence of a copper catalyst, azide cycloaddition to the triple bond will occur, resulting in conjugation of the ligand to the nanoparticle. An example of click chemistry is the Cu(I)-catalyzed [3+2] Huisgen cycloaddition between an azide and an alkyne. Moses et al., (2007) The growing applications of click chemistry. Chemical Society Reviews 36, 1249-1262. Glaser et al., (2009) 'Click labelling' in PET radiochemistry. Journal of Labeled Compounds & Radiopharmaceuticals 52,407-414. Mindt et al., (2009) A "Click Chemistry" Approach to the Efficient Synthesis of Multiple Imaging Probes Derived from a Single Precursor. Bioconjugate Chemistry 20, 1940-1949. New et al., (2009) Growing applications of "click chemistry" for bioconjugation in contemporary biomedical research. Cancer Biotherapy and Radiopharmaceuticals 24, 289-301. Wang et al., (2010) Application of "Click Chemistry" in Synthesis of Radiopharmaceuticals. Progress in Chemistry 22, 1591-1602. Schultz et al., (2010) Synthesis of a DOTA-Biotin Conjugate for Radionuclide Chelation via Cu-Free Click Chemistry. Organic Letters 12,2398-2401. Martin et al., (2010) A DOTA-peptide conjugate by copper-free click chemistry. Bioorganic & Medicinal Chemistry Letters 20,4805-4807. Lebedev et al., (2009) Clickable bifunctional radiometal chelates for peptide labelling. Chemical Communications 46, 1706-1708. Knor et al., (2007) Synthesis of novel 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) derivatives for chemoselective attachment to unprotected polyfunctionalized compounds. Chemistry-a European Journal 13, 6082-609. In a second embodiment, a maleimide functional group and a thiol group can be introduced into the nanoparticle and the desired ligand (or contrast agent, chelate), the nanoparticle having the maleimide functional group, the ligand (or contrast agent, chelate) having the thiol group, or vice versa. The maleimide double bond readily reacts with the thiol group to form a stable carbon-sulfur bond. In a third embodiment, an activated ester functional group, eg, a succinimidyl ester group and an amine group can be introduced into the nanoparticle and the desired ligand, contrast agent or chelate. The activated ester group readily reacts with the amine group to form a stable carbon-nitrogen amide bond. In a fourth embodiment, click chemistry involves the reverse electron demand Diels-Alder reaction between a tetrazine moiety and a strained alkene (Figure 21c). Devaraj et al., (2009) Fast and Sensitive Pretargeted Labeling of Cancer Cells through a Tetrazine/trans-Cyclooctene Cycloaddition. Angewandte Chemie-International Edition 48, 7013-7016. Devaraj et al., (2008) Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging. Bioconjugate Chemistry 19,2297-2299. Blackman et al., (2008) Tetrazine ligation: fast bioconjugation based on inverse electron demand Diels-Alder reactivity. Journal of the American Chemical Society 130, 13518-13519. Ligation can be selective, rapid, biocompatible, and/or bioorthogonal. Unlike many Diels-Alder reactions, the coupling is irreversible, forming stable pyridazine products after retro-Diels-Alder release of dinitrogen from the reaction intermediate. A tetrazine moiety and a stretched alkene can be introduced into the nanoparticle and the desired ligand (or contrast agent, chelate), the nanoparticle having the tetrazine moiety, the ligand (or contrast agent, chelate) having the stretched alkene, or vice versa. Several different tetrazine-stretched alkene pairs can be used for the reaction, including, for example, the combination of 3-(4-benzylamino)-1,2,4,5-tetrazine (Tz) derivatives and norbornene- or trans -cyclooctene. Schoch et al., (2010) Post-Synthetic Modification of DNA by Inverse-Electron-Demand Diels-Alder Reaction. Journal of the American Chemical Society 132, 8846-8847. Devaraj et al., (2010) Bioorthogonal Turn-On Probes for Imaging Small Molecules Inside Living Cells. Angewandte Chemie International Edition 49. Haun et al., (2010) Bioorthogonal chemistry amplifies nanoparticle binding and enhances the sensitivity of cell detection. Nature Nanotechnology 5, 660-665. Rossin et al., (2010) In vivo chemistry for pretargeted tumor imaging in live mice. Angewandte Chemie International Edition 49,3375-3378. Li et al., (2010) Tetrazine-transcyclooctene ligation for the rapid construction of 18-F labeled probes. Chemical Communications 46, 8043-8045. Reiner et al., (2011) Synthesis and in vivo imaging of a 18F-labeled PARP1 inhibitor using a chemically orthogonal scavenger-assisted high-performance method. Angewandte Chemie International Edition 50, 1922-1925. For example, the surface of the nanoparticles can be decorated with tetrazine moieties, which can subsequently be conjugated to the norbomene-modified ligand (or contrast agent or chelate) (Figures 21d and 21e). In one aspect, the nanoparticles are coated with tetrazine, which can then be modified with DOTA chelates using tetrazinenorbornene ligation, and radiolabeled with 64Cu.

Residence time/clearance in vivo

[0088] After administration of the present nanoparticle to a subject, the mean residence time in blood of the nanoparticles can range from about 2 hours to about 25 hours, from about 3 hours to about 20 hours, from about 3 hours to about 15 hours, about 4 hours to about 10 hours, or about 5 hours to about 6 hours. A longer blood half-residence time means longer circulation, allowing more nanoparticles to accumulate at the target site in vivo. The half residence time in blood can be evaluated as follows. The nanoparticles are first administered to a subject ( eg, a mouse, mini-pig, or human). At various times after administration, blood samples are taken to measure nanoparticle concentrations by appropriate methods.

[0089] In one embodiment, after administration of the PEGylated (or control) nanoparticle to a subject, the mean blood residence time of the nanoparticle can range from about 2 hours to about 15 hours, or from about 4 hours and approximately 10 hours. The median tumor residence time of the nanoparticle after administration of the nanoparticle to a subject may vary. from about 5 hours to about 2 days, from about 10 hours to about 4 days, or from about 15 hours to about 3.5 days. The ratio of the mean residence time in the tumor to the mean residence time in the blood of the nanoparticle after administration of the nanoparticle to a subject can range from about 2 to about 30, from about 3 to about 20, or between about 4 and about 15. Renal clearance of the nanoparticle after administration of the nanoparticle to a subject ranges from about 80% ID (initial dose) to about 100% ID in about 24 hours. Also described herein (but not claimed) are silica-based fluorescent nanoparticles in which renal clearance of the nanoparticle after administration of the nanoparticle to a subject can range from about 10% ID to about 100%. ID in about 24 hours, between 30% ID and about 80% ID in about 24 hours, or from about 40% ID to about 70% ID in about 24 hours. In one embodiment, after administration of the nanoparticle to a subject, the mean residence time of the nanoparticle in the blood ranges from about 2 hours to about 25 hours, the mean residence time of the nanoparticle in the tumor ranges from about 5 hours to about 5 days, and the renal clearance of the nanoparticle is about 80% ID in about 24 hours.

[0090] Following administration of the present nanoparticle to a subject, the mean residence time in the tumor of the present nanoparticles can range from about 5 hours to about 5 days, from about 10 hours to about 4 days, from about 15 hours to about 3.5 days, about 20 hours to about 3 days, about 2.5 days to about 3.1 days, about 1 day to 3 days, or about 73.5 hours.

[0091] The ratio of the mean residence time in the tumor to the mean residence time in the blood of the nanoparticle can range from about 2 to about 30, from about 3 to about 20, from about 4 to about 15, from about 4 and about 10, about 10 to about 15, or about 13.

[0092] In one embodiment, the present invention provides a fluorescent silica-based nanoparticle according to the claims comprising a silica-based core comprising a fluorescent compound positioned within the silica-based core; a silica shell that surrounds at least a part of the core; an organic polymer attached to the nanoparticle; and a ligand attached to the nanoparticle, wherein the nanoparticle has a diameter between about 1 nm and about 15 nm. Following administration of the PEGylated nanoparticle (control) to a subject, the mean blood residence time of the nanoparticle can range from about 2 hours to about 25 hours, or from about 2 hours to about 15 hours; the median tumor residence time of the nanoparticle can range from about 5 hours to about 2 days; and the renal clearance of the nanoparticle is about 80% ID in about 24 hours. The number of ligands attached to the nanoparticle can range from about 1 to about 10. The diameter of the nanoparticle can be between about 1 nm and about 8 nm. A contrast agent, such as a radionuclide, can be attached to the nanoparticle. Alternatively, a chelate can be attached to the nanoparticle. The nanoparticle can be detected by PET, SPECT, CT, MRI, optical imaging, bioluminescence imaging, or combinations thereof. A therapeutic agent can be attached to the nanoparticle. Following administration of the radiolabeled targeted nanoparticle to a subject, the mean residence time in the blood of the nanoparticle can also range from about 3 hours to about 15 hours, or from about 4 hours to about 10 hours. The median tumor residence time of the nanoparticle after administration of the nanoparticle to a subject can also range from about 10 hours to about 4 days, or from about 15 hours to about 3.5 days. The ratio of the mean residence time in the tumor to the mean residence time in the blood of the nanoparticle after administration of the nanoparticle to a subject can range from about 2 to about 30, from about 3 to about 20, or between about 4 and about 15.

[0093] In one embodiment, to estimate the mean residence time (or clearance) values of the radiolabeled nanoparticles (T ¹ / ² ) in blood, tumor and other major organs/tissues, the percentage of the values of the Injected doses per gram (%ID/g) are measured by sacrificing groups of mice at specific times after administration of the nanoparticles. Blood, tumor and organs are collected, weighed and counted in a scintillation counter. %DI/g values are corrected for radioactive decay at the time of injection. The resulting activity-time concentration data for each tissue are fitted to a monoexponential decreasing function to estimate tissue/organ T ^1/2 values.094*

[0094] Also described herein (but not claimed) are silica-based fluorescent nanoparticles in which renal clearance can range from approximately 45% ID (initial dose) to greater than 90% ID in approximately 24 hours, from about 20% ID to about 90% ID in about 24 hours, from about 30% ID to about 80% ID in about 24 hours, from about 40% ID to about 70% ID in about 24 hours, from about 40 % ID to 60% ID in about 24 hours, 40% ID to 50% ID in about 24 hours, or 43% ID in about 24 hours, 10% ID to about 100% ID in about 24 hours, from about 40% ID to about 100% ID in about 24 hours.

[0095] Following administration of a fluorescent silica-based nanoparticle according to the claims, renal clearance ranges from about 80% ID to about 100% ID in about 24 hours, from about 90% ID to about 95% ID DI in about 24 hours, from about 90% DI to about 100% DI in about 24 hours, or from about 80% DI to about 95% DI in about 24 hours. Renal clearance can be assessed as follows. The nanoparticles are first administered to a subject ( eg, a mouse, mini-pig, or human). At various times after administration, urine samples are taken to measure nanoparticle concentrations by appropriate methods.

[0096] In one embodiment, renal clearance ( eg, fraction of nanoparticles excreted in urine over time) is analyzed as follows. A subject is administered the present nanoparticles and urine samples are collected over a certain period of time ( eg, 168 hours). Particle concentrations at each time point are determined using fluorometric assays and a serial dilution calibration curve generated from background-corrected fluorescence signal measurements of pooled urine samples with known particle concentrations (%ID). . The concentration values, together with estimates of the average daily mouse urine volumes, are used to calculate the cumulative %ID/g urine excreted. In another embodiment, renal clearance of radiolabeled nanoparticles is analyzed by measuring urine sample activities (counts per minute) over similar time intervals using, for example, counts after nanoparticle administration to calculate excretion. of accumulated urine.

[0097] In a third embodiment, to assess cumulative fecal excretion, feces are collected in metabolic cages for similar time intervals after administration of the nanoparticles and sample activities determined using a y-counter.

[0098] When nanoparticles in an amount of approximately 100 times the equivalent human dose are administered to a subject, there is substantially no anemia, weight loss, agitation, increased respiration, gastrointestinal disturbances, abnormal behavior, neurological dysfunction, abnormalities in hematology, clinical chemistry abnormalities, drug-related lesions in organ pathology, mortality, or a combination thereof are seen in about 10 to about 14 days.

[0099] When the present nanoparticle contains at least one bound ligand, the multivalency increase of the nanoparticle ( eg, compared to ligand alone) can range from about 1.5-fold to about 10-fold, from about 2-fold and about 8 times, about 2 times to about 6 times, about 2 times to about 4 times, or about 2 times.

[0100] The nanoparticles of the present invention show physicochemical and biological parameters in vitro and in vivo that are unexpected in view of the state of the art. For example, the estimated median residence time in blood for ligand-bound nanoparticles ( eg, about 5.5 hours for the unclaimed cRGD-bound nanoparticles) is substantially longer than that of the corresponding ligand ( p eg, around 13 minutes for cRGD). Montet et al. Multivalent effects of R ^g D peptides obtained by nanoparticle display. J Med Chem. 49, 6087-6093 (2006). Extended blood residence half-times can improve probe bioavailability, facilitate tumor targeting, and produce greater tumor uptake for longer periods of time. In one embodiment, the mean residence time in the tumor for targeted nanoparticles (i.e., ligand-bound nanoparticles) is approximately 13-fold greater than the mean residence time in blood, while the mean residence time in the tumor for untargeted nanoparticles (ie, the corresponding nanoparticles not bound with ligands) is only about 5 times longer than the mean residence time in blood. This difference suggests substantially greater tumor tissue accumulation of targeted nanoparticles compared to untargeted nanoparticles. In certain embodiments, given the number of ligands bound to the nanoparticle, the present nanoparticles show unexpected high-affinity binding ( eg, 0.51 nM Kd and 1.2 nM IC for the ^cRGD -bound nanoparticle not claimed), multivalence enhancement ( eg, plus more than 2-fold increase for unclaimed cRGD-adhered nanoparticles compared to cRGD peptide alone), significant differential tumor uptake (eg, nanoparticles coated over 72 hours post-dose) and significant tumor contrast relative to normal muscle ( eg, about 3 to 5 times during the 72 hours post-dose) based on tumor-to-muscle uptake ratios.

[0101] In one embodiment, three-fold activity concentration increases were found for ligand-bound nanoparticles in integrin-expressing tumors over controls ( eg, ligand-bound nanoparticles in integrin-non-expressing tumors, or corresponding non-ligand-bound nanoparticles in integrin-expressing tumors) at the time of maximum tumor uptake (about 4 hours after injection of the nanoparticles). Furthermore, tumor-to-muscle uptake ratios for targeted nanoparticles (i.e., ligand-bound nanoparticles) reveal increased contrast of tumor tissue relative to normal muscle, compared to decreased contrast of tumor tissue relative to normal muscle. normal muscle for untargeted nanoparticles (ie, corresponding nanoparticles). non-ligand-bound nanoparticles), suggesting that targeted nanoparticles are selective for tumors.012

[0102] In another embodiment, targeted and untargeted nanoparticles show efficient renal excretion during the same time period. Almost half of the injected dose is excreted during the first 24 hours post-injection and about 72% at 96 hours, suggesting that most of the excretion occurred on the first day post-injection. In contrast, the fecal excretion profiles of the target nanoparticles indicate that, on average, 7% and 15% of the injected dose is eliminated in 24 and 96 hours, respectively.

[0103] The physicochemical and biological parameters of non-toxic nanoparticles, along with their multimodal imaging capabilities ( eg, PET and optical imaging), broaden the range of their potential biomedical applications. Applications include (a) long-term monitoring: the long blood circulation time and corresponding bioavailability of nanoparticles highlight their versatility for early and long-term monitoring of various stages of disease treatment (such as diagnostic screening, evaluation pre-treatment, therapeutic intervention and post-treatment monitoring) with no restrictions imposed by toxicity considerations; (b) Enhanced tumor penetration: The clearance properties of targeted nanoparticles ( eg, their renal clearance is slower than that of state-of-the-art molecular probes) will be useful for several types of biologic applications. For example, nanoparticles would be particularly useful in cases of poorly vascularized and relatively inaccessible solid tumors where localization of agents is often slow after systemic administration; (c) multimodal imaging capabilities: these modalities can be combined at multiple scales (ie, from whole body to cellular levels) to acquire complementary depth-sensitive biological information. For example, in SLN mapping, deep nodes can be mapped by PET in terms of their distribution and number, while more precise and detailed localization of superficial nodes can be obtained by fluorescence imaging; and (d) targeted therapies: the longer clearance of targeted nanoparticles from the tumor compared to that from the blood can be exploited for combined diagnostic/therapeutic applications, where the nanoparticles can serve as a radiotherapeutic or drug delivery vehicle. .

pharmaceutical compositions

[0104] The claimed nanoparticles can be provided in a pharmaceutical composition comprising the present nanoparticle. The pharmaceutical compositions of the invention may be administered orally in the form of a suitable pharmaceutical unit dosage form. The pharmaceutical compositions of the invention can be prepared in many forms including tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow release formulations, such as shaped polymer gels.

[0105] Suitable modes of administration for the present nanoparticle or composition include, but are not limited to oral, intravenous, rectal, sublingual, mucosal, nasal, ophthalmic, subcutaneous, intramuscular, transdermal, intradermal, subdermal, peritumoral, spinal, intrathecal , intra-articular, intra-arterial, subarachnoid, bronchial and lymphatic administration, and other dosage forms for systemic administration of active ingredients. The present pharmaceutical composition can be administered by any method known in the art, including, but not limited to, transdermal (passive via patch, gel, cream, ointment, or iontophoretic); intravenous (bolus, infusion); subcutaneous (infusion, depot); transmucosal (buccal and sublingual, eg, orodispersible tablets, wafers, films, and effervescent formulations; conjunctival (eye drops); rectal (suppository, enema)); or intradermal (bolus, infusion, depot). The composition can be administered topically.

[0106] Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle prior to use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.

[0107] The nanoparticle pharmaceutical compositions of the invention may also be formulated for parenteral administration (eg, by injection, eg, bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion containers or multidose containers with an added preservative. Pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the pharmaceutical compositions of the invention may be in powder form, obtained by aseptic isolation of a sterile solid or by lyophilization of solution, for reconstitution with a suitable vehicle, e.g. sterile, pyrogen-free water, prior to use. .018*

[0108] For topical administration to the epidermis, the pharmaceutical compositions can be formulated as ointments, creams or lotions, or as the active ingredient of a transdermal patch. Suitable transdermal delivery systems are described, for example, in A. Fisher et al. (US Patent No. 4,788,603), or R. Bawa et al. (US Patent Nos. 4,931,279; 4,668,506; and 4,713,224). Ointments and creams can, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will generally also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Pharmaceutical compositions can also be administered by ionophoresis, for example, as described in US Patent Numbers 4,140,122; 4,383,529; or 4,051,842.

[0109] Pharmaceutical compositions suitable for topical administration in the mouth include unit dosage forms such as lozenges comprising a pharmaceutical composition of the invention in a flavored base, typically sucrose and acadia or tragacanth; tablets comprising the pharmaceutical composition in an inert base such as gelatin and glycerin or sucrose and acacia; mucoadherent gels and mouthwashes comprising the pharmaceutical composition in a suitable liquid vehicle.

[0110] For topical administration to the eye, the pharmaceutical compositions can be administered in the form of drops, gels (S. Chrai et al., US Patent No. 4,255,415), gums (SL Lin et al. , US Patent) or through an extended release ocular insert (AS Michaels, US Patent No. 3,867,519 and HM Haddad et al., US Patent No. 23,870,791).

[0111] When desired, the pharmaceutical compositions described above can be adapted to give a sustained release of an employed therapeutic compound, for example, by combination with certain hydrophilic polymeric matrices, for example, comprising natural gels, synthetic polymeric gels or mixtures. thereof.

[0112] Pharmaceutical compositions suitable for rectal administration in which the vehicle is a solid are most preferably presented as unit dose suppositories. Suitable vehicles include cocoa butter and other materials commonly used in the art, and suppositories may conveniently be formed by mixing the pharmaceutical composition with the softened or melted vehicle(s) followed by cooling and molding.

[0113] Pharmaceutical compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or aerosols containing, in addition to the nanoparticles and therapeutic agent, such carriers well known in the art.

[0114] For administration by inhalation, the pharmaceutical compositions according to the invention are conveniently administered from an insufflator, nebulizer or a pressurized pack or other convenient means for delivering an aerosol. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.

[0115] Alternatively, for administration by inhalation or insufflation, the pharmaceutical compositions of the invention may take the form of a dry powder composition, for example, a powder mixture of the pharmaceutical composition and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form, for example, in capsules or cartridges or, for example, gelatin or blister packs from which the powder can be administered with the aid of an inhaler or insufflator.

[0116] For intranasal administration, the pharmaceutical compositions of the invention may be administered via a liquid aerosol, such as through a plastic bottle spray. Typical of these are the Mistometer® (isoproterenol inhaler--Wintrop) and the Medihaler® (isoproterenol inhaler--Riker).

[0117] The pharmaceutical compositions of the invention may also contain other adjuvants such as flavorings, colorants, antimicrobial agents or preservatives.

[0118] It will further be appreciated that the amount of pharmaceutical compositions required for use in treatment will vary not only with the therapeutic agent selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient. patient and will be, ultimately, at the discretion of the treating physician or clinician. For evaluations of these factors, see JF Brien et al., Europ. J.Clin. Pharmacol., 14, 133 (1978); and Physicians' Desk Reference, Charles E. Baker, Jr., Pub., Medical Economics Co., Oradell, NJ (41st ed., 1987). Generally, doses of the therapeutic agent when used in combination with the fluorescent nanoparticles of the present invention can be lower than when the therapeutic agent is administered alone or in conventional pharmaceutical dosage forms. The high specificity of the fluorescent nanoparticle for a target site, such as a receptor located on the surface of a cell, can provide a relatively localized concentration of a therapeutic agent or, alternatively, a sustained release of a therapeutic agent over a prolonged period of time. .0192

[0119] The present nanoparticles or compositions can be administered to a subject. The subject can be a mammal, preferably a human. Mammals include, but are not limited to, murine, rat, rabbit, ape, bovine, ovine, porcine, canine, feline, farm animal, sporting animal, pet, equine, and primate.

Use of nanoparticles

[0120] The fluorescent silica-based nanoparticle described herein is useful in a method for detecting a component of a cell comprising the steps of: (a) contacting the cell with a fluorescent silica-based nanoparticle comprising a silica-based core comprising a fluorescent compound positioned within the silica-based core; a silica shell that surrounds at least a part of the core; an organic polymer attached to the nanoparticle; from about 1 to about 25 ligands (from about 1 to about 20 ligands, or from about 1 to about 10 ligands, or other ranges; see discussions herein) bound to the nanoparticle; and a contrast agent or a chelate attached to the nanoparticle; and (b) monitoring the binding of the nanoparticle to the cell or to a cellular component (and/or its potential intracellular uptake) by at least one imaging technique. The imaging technique can be PET, SPECT, CT, MRI, optical bioluminescence or fluorescence imaging, and combinations thereof.

[0121] The location of the cellular component can be detected and determined within a metabolically active whole cell, in a whole cell lysate, in a permeabilized cell, in a fixed cell, or with a partially purified cellular component in a cell-free environment. . The amount and duration of contact may depend, for example, on the diagnostic or therapeutic goals of the treatment method, such as antibody-mediated or fluorescent detection of upregulated signaling pathway intermediaries (i.e., Akt, NF-kB ), disease states or conditions, the administration of a therapeutic agent, or both. The amount and duration of contact may also depend on the relative concentration of the fluorescent nanoparticle with respect to the target analyte, the incubation time of the particle, and the condition of the cell for treatment.

[0122] The silica-based fluorescent nanoparticles described herein can be used in a method of targeting a tumor cell comprising administering to a cancer patient an effective amount of a silica-based fluorescent nanoparticle comprising a core silica-based comprising a fluorescent compound positioned within the silica-based core; a silica shell that surrounds at least a part of the core; an organic polymer attached to the nanoparticle; a ligand bound to the nanoparticle and capable of binding to a tumor marker; and at least one therapeutic agent. The nanoparticle may be radiolabeled. The nanoparticle can be radiolabeled using any suitable technique. Radiolabeling can be automated. In one embodiment, the nanoparticle is radiolabeled using the FASTlab radiochemistry synthesis platform (GE Healthcare). Synthesis parameters can be tuned to achieve high reproducibility, radiochemical yield/purity, labeling efficiency, and relatively short synthesis times. New particle tracers can be accommodated in the same FASTlab module.

[0123] The nanoparticle can be administered to the patient by, but not limited to, the following routes: oral, intravenous, nasal, subcutaneous, local, intramuscular, or transdermal.

[0124] In certain embodiments, it may be desirable to use a mixture of two or more types of fluorescent nanoparticles that have different properties to assess different types of tissue. The invention can be used to help a physician or surgeon identify and characterize areas of disease, such as cancers and inflammatory/infectious processes, including, but not limited to, cancers of the skin (melanoma), head and neck, prostate, brain, and intestines. , to distinguish diseased from normal tissue, such as detecting tumor margins that are difficult to detect using an ordinary surgical microscope, for example, in brain surgery, to help dictate surgical or therapeutic intervention, for example, determining whether a lesion is cancerous and should be removed or not cancerous and left alone, or in surgical staging of disease, eg, intraoperative lymph node staging, sentinel lymph node (SLN) mapping, eg, nerve-sparing procedures to preserve structures vital neural (intraparotid nerves).

[0125] The invention can be used in detection of metastatic disease, monitoring of response to treatment, mapping/targeting of SLNs, nerve-sparing procedures, detection of residual disease, targeted delivery of therapies (combined diagnostic/therapeutic platform ), local delivery of non-targeted drugs -containing nanoparticles (catheter delivery), blood-brain barrier therapies, treatment of inflammatory/ischemic diseases (i.e., brain, heart, urinary tract, bladder), combination therapy, and disease screening ( g., radiometric pH detection, oxygen detection), etc.

[0126] The invention can also be used in the detection, characterization and/or determination of the localization of a disease, especially an early disease, the severity of a disease or a condition associated with the disease, the staging of a disease and/or or monitoring of a disease. The presence, absence or level of an emitted signal may be indicative of a disease state. The invention can also be used to monitor and/or guide various therapeutic interventions, such as catheter-based and surgical procedures, and monitor drug therapy, including cell-based therapies. The invention may also be used in the prognosis of a disease or disease condition. Cellular subpopulations residing within or bordering the disease site, such as stem cell-like cells ("cancer stem cells") and/or inflammatory/phagocytic cells, can be identified and characterized using the invention. With respect to each of the foregoing, examples of such diseases or disease conditions that may be detected or monitored (before, during, or after therapy) include cancer (eg, melanoma, thyroid, colorectal, ovarian, lung, breast , prostate, cervical, skin, brain, gastrointestinal, mouth, kidney, esophagus, bone cancer), which can be used to identify subjects who have an increased susceptibility to developing cancer and/or malignancies, i.e., are predisposed to developing cancer and /or malignancies, inflammation (for example, inflammatory conditions induced by presence of cancerous lesions), cardiovascular disease (for example, atherosclerosis and inflammatory conditions of the blood vessels, ischemia, stroke, thrombosis), dermatological disease (for example, Kaposi's sarcoma, psoriasis), ophthalmic disease (for example, degenerative disease, diabetic retinopathy), infectious disease (for example, bacterial, viral, fungal, and parasitic infections, including acquired immunodeficiency), immunological disease (for example, an autoimmune disorder, lymphoma, multiple sclerosis, rheumatoid arthritis, diabetes mellitus), of the central nervous system (for example, a neurodegenerative disease, such as Parkinson's disease or Alzheimer's disease), hereditary diseases, metabolic diseases, environmental diseases (for example, lead, mercury and radioactive poisoning, skin cancer), related diseases with the bones (for example, osteoporosis, t primary and metastatic bone tumors, osteoarthritis) and a neurodegenerative disease.

[0127] The invention, therefore, can be used, for example, to determine the presence and/or location of co-resident and/or tumor cell-like cells ("cancer stem cells"), the presence and/or localization of inflammatory cells, including the presence of activated macrophages, for example in peritumoral regions, the presence and localization of vascular disease including areas at risk of acute occlusion (i.e., vulnerable plaques) in coronary and peripheral arteries, regions of expanding aneurysms , unstable plaque in carotid arteries and ischemic areas. The invention can also be used in the identification and assessment of cell death, injury, apoptosis, necrosis, hypoxia, and angiogenesis. PCT/US2006/049222.

[0128] The following examples are presented for illustrative purposes only and do not limit the invention. Examples not within the scope of the claims are provided for reference.

Example 1 Preparation and characterization of PEG-coated nanoparticles

[0129] Nanoparticles containing a NIR-emitting dye (Cy-5) were synthesized and functionalized by PEGylation according to well-established protocols as described in PCT/US2008/074894 and Stober et al. Controlled growth of monodispersed silica spheres in the micron size range. Colloid Interface Sci, 1968; 26:62-69. Ohnishi et al. J. Mol. Imaging 2005, 4:172-181. Maleamide Cy5 was reacted with a coreactive organosilane compound, (3-mercaptopropyl)tromethoxysilane to form a fluorescent silica precursor. This fluorescent silica precursor was co-fused with tetraethylortho silicate to form a fluorescent silica-based core. A PEG-silane compound, with methoxy-terminated polyethylene glycol (PEG, -0.5 kDa) methoxy(polyethyleneoxy)propyl]-trimethoxysilane chains, was added to the fluorescent silica-based core to form a PEG coating on the nucleus. PEG-coated nanoparticles were dialyzed to physiological saline (0.15 M NaCl in H2O) through 3500 MWCO snakeskin dialysis membranes and filter-sterilized. All samples were compared for optical density at their wavelength of maximum absorption (640 nm) prior to injection. Hydrodynamic size measurements were achieved by dynamic light scattering (DLS) and fluorescence correlation spectroscopy (ECF). Briefly, dialyzed particles in water were measured on a Brookhaven Instruments Company static/DLS 200SM system using a HeNe laser (A = 632.8 nm). Due to the overlap of dye absorption with the excitation source, integration times of 15 min were used to achieve acceptable signal/noise ratios. For ECF, the particles were dialyzed in water, diluted in 0.15 M NaCl and measured on a Zeiss LSM 510 Confocar 2 ECF (HeNe excitation 633 nm). The instrument was size calibrated prior to all measurements. Comparison of the relative brightness of the PEGylated nanoparticles with free dye was determined from the ECF curves, measured as count rate per molecule/particle.

Example 2 Renal clearance of PEG-coated nanoparticles

[0130] Fluorescent core-shell silica nanoparticles were synthesized, which had a hydrodynamic radius of approximately 3 nm. These nanoparticles were found to be in the range of 6 to 10 nm in diameter, as shown by dynamic light scattering (DLS) results (Figure la), in vivo whole-body NIR fluorescence imaging of naked silica nanoparticles ( without PEG coating), on the order of 6 nm and 3.3 nm, in nude mice showed considerable renal clearance 45 min after injection with significant accumulation remaining in the liver (Figure 1b). Final excretion into the enterohepatic circulation occurred over the next 24 h. Based on these results, the particles were covalently coated with methoxy-terminated poly(ethylene glycol) chains (PEG, -0.5 kDa), according to the protocols in PCT/US2008/074894, to prevent opsonization and further improve the removal of particles while maintaining a small hydrodynamic size. This treatment decreased hepatic retention and resulted in increased renal bladder filtration at 45 min post-injection by NIR fluorescence imaging (Figure 1c), with bladder fluorescence visible up to 24 h. The probes were well tolerated and no adverse effects or animal deaths were observed during the course of the study. Serial co-registered PET-CT images 24 hours after injection of 124I-labeled PEG-coated nanoparticles (Figure 1d, top row) demonstrated a small amount of residual bladder activity as well as activity lining the liver. /GI tract (middle), flanked by independently acquired microCT and microTEP scans. Serial microTEP imaging confirmed the results on NIR fluorescence imaging. The mean residence time in blood of 124I-labeled PEGylated nanoparticles as a function of time-dependent changes in blood activity over a 96-hour period was found to be 7.3 hours. For 124I-labeled RGD-bound nanoparticles, the median residence time in blood was found to be 5.6 hours.

[0131] Based on these in vivo data, a more detailed study of biodistribution and removal of coated nanoparticles was carried out on two sets of PEGylated Cy5-containing particles to assess the effects of probe size on biodistribution. Nanoparticles with hydrodynamic diameters of 3.3 ± 0.06 and 6.0 ± 0.1 nm, measured by fluorescence correlation spectroscopy (ECF) were generated (Figure 2a). Prior to the in vivo studies, the photophysical properties of the particles were investigated to establish their performance levels against the free dye. Despite the extremely small particle size, the silica-encapsulated dye molecules exhibited photophysical improvements over the free dye that scaled with particle size, including significant increases in brightness, as determined by absorption and emission spectroscopy (Figure 2b). ) and ECF (Figure 2c). Compared with the free dye, nanoparticles of 3.3 and 6.0 nm in diameter exhibited 2-fold and 3-fold increases in photobleaching half-life, respectively, when irradiated with a high-power 635-nm laser (Figure 2d). Therefore, these nanoparticle probes were found to be brighter and more photostable than their free-dye counterparts.

[0132] In addition to semi-quantitative assessment of nanoparticle behavior in vivo from whole-body images, ex vivo analysis of tissue and fluid homogenates was performed using a fluorescence plate reader, allowing calibrated quantification of nanoparticles. Variations observed in NIR fluorescence images. Samples were pooled as "retained" (liver, kidney, lung, spleen, and blood homogenates) and "excreted" (urine) particle fluorescence sources, background corrected, and converted to percentage starting dose (%ID). ) per animal based on calibration curves. Tissue analysis showed minimal particle retention in major organs, with most of the fluorescence attributed to circulating blood (Figure 3a). Net particle retention, calculated as the sum of the "retained" components, was fitted to an exponential decay curve to determine excretion kinetics (Figure 3b). Larger particles exhibited a longer tissue half-life (t1/2(3.3 nm)::: 190 min, t1/2(6.0 nm):::: 350 min) and greater initial organ retention . After 48 h, the 6 nm particle exhibited minimal retention in the body (Rtotal (6.0 nm) = 2460.6% ID). Urine samples collected at sacrifice, along with serial dilution calibration data, were used to estimate total renal clearance based on a conservative estimate of the average urine volume excreted per unit time. Using this method, the % ID excreted over time was estimated for both particle sizes (Figure 3c).

Example 3 Integrin O v 0 3 Targeted Peptide-Conjugated Fluorescent Silica Nanoparticles (Melanoma Model)

[0133] To synthesize a multimodal nanoparticle (optical PET) with high affinity for the avp3 integrin tumor marker, cyclic RGD pentapeptide (RGDYC) was conjugated to the nanoparticle via a Cys-maleimide bond. The tyrosine linker, Y, was used to subsequently attach a radiolabel. Male athymic nude mice were injected subcutaneously into their flanks with C6 rat glioma cells. At -0.5 cm in diameter, mice were intravenously injected with bare silica nanoparticles (Fig. 4A) or RGD-pegylated nanoparticles (Fig. 4B, ~500 nm/kg). Fig. 4 shows in vivo biodistribution in tumor-free and tumor-bearing mice using whole-body optical imaging.

[0134] The in vitro binding of targeted (RGD-bound) and untargeted (PEG-coated) nanoparticles to avp integrin-positive human melanoma cell lines (M21) was investigated as part of a dose-response study using cytometry. of flow (Figures 5A, 5B). Particle binding/uptake was assessed as a function of time (Fig. 5A) and concentration (Fig. 5B).

Example 4 Fluorescent Silica Nanoparticles Conjugated with Peptide Targeting 0 /0 3 Integrin and Nodal Mapping (Melanoma Model)

[0135] We used a biocompatible material, silica, which has an architecture that could be fine-tuned to optimized particle sizes for renal clearance. We attached small targeting peptides and a radioactive label to the particle surface for serial PET imaging measurements in a well-characterized in vivo human melanoma model, and mapped lymph nodes and lymphatic drainage channels using a near-infrared dye. (NIR) encapsulation and multiscale optical fluorescence imaging methods. Ballou et al., Sentinel lymph node imaging using quantum dots in mouse tumor models. Bioconjugate Chem. 18, 389-396 (2007). Kim et al., Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat. Biotechnol. 22, 93-97 (2003). Tanaka et al, Image-guided oncologic surgery using invisible light: completed preclinical development for sentinel lymph node mapping. J Surg Oncol. 13, 1671-1681 (2006). Toxicity tests were also performed and human normal organ radiation doses were derived. Specifically, we synthesized near-infrared (NIR) dye encapsulation shell-and-core silica nanoparticles of approximately 7 nm in diameter, coated with PEG chains, and surface-functionalized with a small amount (about 6-7) of peptides. guidance and radio markers.

[0136] We demonstrate that these probes are simultaneously non-toxic, exhibit high affinity/avidity binding, efficient excretion, and significant differential uptake and contrast between tumor and normal tissues using multimodality molecular imaging approaches. Sensitive detection, localization, and interrogation of lymph nodes and lymphatic channels, enabled by NIR dye fluorescence, highlights the clear potential advantage of this multimodal platform to detect and stage metastatic disease in the clinical setting, while expanding the lower range of node sizes that can be detected.

MATERIALS AND METHODS

Synthesis of cRGDY-PEG nanoparticles and PEG nanoparticles

[0137] The particles were prepared by a modified Stober-type silica condensation as described above. Wiesner et al., PEG-coated Core-shell Silica Nanoparticles and Mathods of Manufacture and Use, PCT/US2008/74894. Larson, et al., Silica nanoparticle architecture determines radiative properties of encapsulated chromophores. Chem. Mater. 20,2677-2684 (2008). Bogush, et al., Preparation of Monodisperse Silica Particles: Control of Size and Mass Fraction J. Non-Cryst. Solids, 104, 95-106 (1988). Sadasivan, et al., Alcoholic Solvent Effect on Silica Synthesis-NMR and DLS Investigation. J. Sol-Gel Sci. Technol. 12, 5-14 (1998). Herz, et al., Large Stokes-Shift Fluorescent Silica Nanoparticles with Enhanced Emission over Free Dye for Single Excitation Multiplexing. Macromol Rapid Commun. 30, 1907-1910 (2009). Tyrosine residues were conjugated to PEG chains for attachment of radioactive iodine or stable iodine moieties. Hermanson, Bioconjugate Techniques, (Academic Press, London, ed. 2, 2008). All samples were equalized in optical density at their wavelength of maximum absorption (640 nm) prior to radiolabeling. The cRGD peptides were linked to functionalized PEG chains via a cysteine-maleimide bond, and the amount of cRGD peptides bound to the particle was estimated using ECF-based measurements of absolute particle concentrations and the initial concentration of the reagents for the analysis. cRGD peptide.

Hydrodynamic Size and Relative Brightness Comparison Measurements Using Fluorescence Correlation Spectroscopy (ECF)

[0138] Water dialyzed particles were diluted in physiological saline (0.15 M NaCl in H ² O) and measured on a Zeiss LSM 510 Confocor 2 ECF using 633-nm HeNe excitation. The instrument was size calibrated prior to all measurements. Differences in diffusion time were used to assess variations in the hydrodynamic sizes of the dye species and particles. Comparisons of relative brightness of free dye and PEG and RGDY-PEG nanoparticles were performed using per molecule/particle count rates.

Radioactive labeling of conjugate C-spots

[0139] Radiolabeling of cRGDY-PEG and PEG nanoparticles was performed using the IODOGEN method (Pierce, Rockford, IL). Piatyszek, et al., Iodine-mediated radioiodination of nucleic acids. J.Anal. Biochemistry 172, 356-359 (1988). Activities were measured by gamma (y) counting and fluorescence was measured using a Varian fluorometer (excitation 650 nm/emission 680).

Cells and cell culture

[0140] M21 and M21 variant human melanoma cell lines (M21-L, av negative) were maintained in RPMI 1640/10% fetal BSA media, 2 mM penicillin L-glutamine and streptomycin (Core Media Preparation Facility). ., Memorial Sloan Kettering Cancer Center, New York). Human umbilical venous cord endothelial cells (HUVEC) were cultured in M199 medium/10% fetal bovine serum, 20 mg/ml endothelial cell growth factor, 50 mg/ml heparin, penicillin and streptomycin.

In vitro cell binding and molecular specificity of 124I-cRGD-PEG nanoparticles

[0141] To test particle binding and specificity for M21 cells, 24-well plates were coated with 10 mg/ml type I collagen (BD Biosciences, Bedford, MA) in phosphate buffered saline (PBS) and incubated. (37 °C, 30 min). M21 cells (3.0-4.0 x 10 5 cells/well) were grown to confluence and washed with RPMI 1640/0.5% bovine serum albumin (BSA) medium. 124I-cRGD-PEG nanoparticles (0 - 4.0 ng/ml) were added to the wells and the cells were incubated (25 °C, 4 hours), washed with RPMI 1640 medium/0.5% BSA and they were dissolved in 0.2 M NaOH. Radioactivity was assayed using a 1480 automatic gamma counter (Perkin Elmer) calibrated for iodine-124. Nonspecific binding was determined in the presence of a 1000-fold excess of cRGD (Peptides International, Louisville, KY). Scatchard plots of the binding data were generated and analyzed by linear regression analysis (Microsoft Excel 2007) to obtain receptor binding parameters (Kd, Bmax, IC50).

In vitro cell attachment studies using optical detection methods

[0142] The maximum differential binding of cRGDY-PEG nanoparticles and PEG nanoparticles to M21 cells was determined for a range of incubation times and particle concentrations using flow cytometry, with optimal values used in competitive assays. Binding and specificity studies. Cells (3.0 x 105 cells/well) were washed with RPMI 1640 medium/0.5% BSA, detached with 0.25% trypsin/EDTA, and pelleted in a microfuge tube (5 min at 1400 rpm, 25°C). Pellets were resuspended in BD FACSFlow solution (BD Biosciences, San Jose, CA) and analyzed in the Cy5 channel for percentage of particle-bound probe (FACSCalibur, Becton Dickinson, Mountain View, CA). In addition, competitive binding studies were performed after incubation. of cRGDY-PEG nanoparticles (2.5 ng/ml) with M21, M21L and HUVEC cells in the presence of excess cRGD and/or fluorescein-conjugated mouse monoclonal anti-human avp3 integrin. antibody (Millipore, Temecula, CA) and analyzed by flow cytometry. To assess the potency of the RGDY-PEG nanoparticles relative to the cRGD peptide, anti-adhesive assays were performed. Ninety-six well microtiter plates were coated with vitronectin in PBS (5 pg/ml), followed by 200 µl of 0.5% RPMI/B ^s A (1 hr, 37°C). Cells (3x104/100 µl/well) were incubated in quadruplicate (30 min, 25 °C) with various concentrations of cRGDY-PEG-nanoparticles or cRGD-peptide in RPMI/0.1% BSA, and added to glass-coated plates. vitronectin (30 min, 37°C). Wells were gently rinsed with RPMI/0.1% BSA to remove non-adherent cells; adherent cells were fixed with 4% PFA (20 min, 25 °C) and stained with methylene blue (1 h, 37 °C) for optical densities, measured with a Tecan Safire plate reader (Aex = 650 nm, A em = 680 nm, 12 nm bandwidth). Multivalent potentiation factor was calculated as the ratio between cRGD peptide and cRGDY-PEGdot IC50 values. Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem. 49, 6087-6093 (2006).

Animal models and tumor inoculation

[0143] All animal experiments were performed in accordance with protocols approved by the Memorial Sloan-Kettering Cancer Center's Institutional Animal Care and Use Committee and followed the National Institutes of Health guidelines for animal welfare. Nu/nu athymic male mice (6-8 weeks old, Taconic Farms Inc, Hudson, NY) were given water containing potassium iodide solution to block uptake by the thyroid gland of any free radioiodine in vivo, and maintained on a Harlan Teklad Global Diet 2016, ad libitum, as detailed elsewhere 10 . To generate M21 or M21L xenografts, equal volumes of cells (~5x106/100 pl) and matrigel were co-injected subcutaneously into the hind paw in different mice. Tumor sizes were regularly measured with calipers, resulting in mean tumor volumes of 200 mm3.

Measurements of mean residence time and in vivo pharmacokinetics (T ¹ / ² )

[0144] Time-dependent activity concentrations (%ID/g), corrected for radioactive decay at the time of injection, were measured by sacrificing groups of mice at specific times after iv injection of 124l-cPGDY-PEG natioparticles or 124I-PEG nanoparticles (~20 pCi/mouse) and from the collection, weighing, and counting of blood, tumors, and organs in a 124L calibrated scintillation counter. The resulting activity-time concentration data for each tissue were fitted to a monoexponential decreasing function to determine the T ^1/2 and A values, the tissue/organ half-residence time, and the zero time intercept, respectively, of the function .

[0145] The fraction of particles excreted in the urine over time was estimated using previously described methods. Burns, et al., Fluorescent Silica Nanoparticles with Efficient Urinary Excretion for Nanomedicine. Nano Letters 9, 442-8 (2009). Briefly, mice were injected iv with 200 µl of cRGDY-PEG nanoparticles or unlabeled PEG nanoparticles, and urine samples were collected over a period of 168 h (n = 3 mice per time point). Particle concentrations at each time point were determined using fluorometric assays and a serial dilution calibration curve generated from background-corrected fluorescence signal measurements of mixed urine samples with known particle concentrations (%Dl). . The concentration values, along with estimates of average daily mouse urine volumes, were then used to calculate the cumulative %Dl/g urine excreted over time. To assess cumulative fecal excretion, metabolic cages were used to collect feces for a similar time interval after iv injection of 200 µl of 124µl-cRGDY-PEG nanoparticles (n=4 mice per time point). The activities of the samples were measured using a counter ^and calibrated to 124l.

dosimetry

[0146] The derived time-activity functions for each tissue were analytically integrated (including the effect of radioactive decay) to produce the corresponding cumulative activity (ie, the total number of radioactive decays). Absorbed doses in 124L mouse organs were then calculated by multiplying the cumulative activity by the 124L equilibrium dose constant for non-penetrating (positron) radiations, assuming complete local absorption of such radiations and ignoring the contribution of non-penetrating radiations. penetrating (i.e., rays ^and ). Eckerman, et al., Radionuclide Data and Decay Schemes, 2nd ed. Reston, VA: Society of Nuclear Medicine; 1989. Mouse normal organ cumulative activities were converted to human normal organ cumulative activities by adjusting for differences in total body and organ masses between mice and humans (70 kg standard man). Cristy, et al., Age-Specific Absorbed Energy Fractions of Internal Photon Sources (1-Vll). Oak Ridge National Laboratory Report ORNL/TM-8381/V1-7 Springfield, VA: National Technical Information Service, Department of Commerce; 1987. Calculated human normal organ cumulative activities were entered into the OLINDA dosimetry software to calculate, using the Society of Nuclear Medicine Medical Internal Dosimetry (MIRD) Committee formalism, absorbed doses to Standard-Man organs . Loevinger, et al., MIRD Primer for Absorbed Dose Calculations (Society of Nuclear Medicine, New York, 1991). Stabin, et al., OLINDA/EXM: The Second Generation Personal Computer Software for Internal Dose Assessment in Nuclear Medicine. J Nucl Med. 46, 1023-1027 (2005).

Acute toxicity studies and histopathology

[0147] Acute toxicity tests were performed on six groups of male and female B6D2F1 mice (7 weeks old, Jackson Laboratories, Bar Harbor, ME). The treatment group (n=6 men, n=6 women) received an unlabeled targeted probe (127IRGDY-PEG nanoparticles) and the control group (n=6 men, n=6 women) received iodinated PEG nanoparticles without label (vehicle, 127I-RGDY-PEG-nanoparticles) in a single iv injection (200 mL). Untreated controls (n=2 males, n-2 females) were tested further. Mice were observed daily for 14 days pi for signs of morbidity/mortality and weight changes, and gross necropsies, histopathology, and blood samples for haematological and serum chemistry evaluation were performed at 7 and 14 days pi (Fig. 10 and Table 3).

Tumor-specific targeting serial PET imaging

[0148] Imaging was performed using a dedicated small animal PET scanner (Focus 120 microTEP; Concorde Microsystems, Nashville, TN). Mice bearing M21 or M21L hindlimb tumors were maintained under anesthesia with 2% isoflurane in oxygen at 2 L/min throughout the scan period. One-hour ready-mode acquisitions were initiated at the time of iv injection of 200 pCi of 121I-cRGDY-PEG nanoparticles or 124I-PEG nanoparticles into all mice, followed by 30-min serial static imaging for a 96 hour interval. Imaging data was corrected for inconsistent scanner response, dead-time count misses, random counts, and physical deterioration at the time of injection. Voxel count rates in the reconstructed images were converted to activity concentrations (%ID/g) by using a measured system calibration factor. Three-dimensional region-of-interest (ROI) analysis of the reconstructed images was performed using ASIPro software (Concorde Microsystems, Nashville, TN) to determine the mean, maximum, and standard deviation of probe uptake in the samples. tumors. Tumor to muscle concentration ratios were obtained from activity by dividing the image-derived tumor %ID/g values by the y-counter muscle %ID/g values.

Node mapping using combined NIR fluorescence imaging and microscopy

[0149] Nude mice bearing tumors in the hind paws were injected by 4-quadrant peritumoral administration using equal volumes of a 50 pl cRGDY-PEG dot sample and allowed to roam freely. After a 30 min to 1 h interval, mice were anesthetized with a 2% isofluorine/98% oxygen mixture, and a superficial paramidline incision was made vertically along the ventral aspect of the mouse to surgically expose the region from the hind leg to the armpit. homolateral to the tumor. In situ optical images of locoregional (ie, inguinal, axillary) nodes and draining lymphatics (including axillary region) were performed using a fluorescence macroscopic microscope equipped with 650±20 nm NIR excitation and emission filters. long step of 710 nm. Whole-body optical images (Cambridge Research Instruments Maestro imager) were additionally acquired and spectrally deconvoluted as previously reported. Burns, et al., Fluorescent Silica Nanoparticles with Efficient Urinary Excretion for Nanomedicine, Nano Letters 9, 442-8 (2009).

[0150] Statistical analysis Statistical analyzes comparing groups of tumor mice receiving targeted/non-targeted probes or bearing M21/M21L tumors were performed using a one-tailed Mann-Whitney U test, with P<0.05 considered statistically significant. . For biodistribution studies, the tissue-specific mean %ID/g values of 124I-cRGDY-PEG- (n=7 mice) and 124I-PEG nanopanicles (control, n=5 mice) were compared at each point of study. time, with results statistically significant differences were observed in the tracer activities in blood, tumor, and major organs at 4 and 96 h pi, as well as at 24 h pi for tumor and other tissues (Table 1). For tumor targeting studies, differences in mean %ID/g values were found between M21 (n=7) and M21L (n=5) tumor mice, as well as mice receiving control probes (n=5). =5), were maximum at 4 h pi (p=0.0015 for both controls), remaining significantly elevated at 24 hrs (p=0.0015 and p=0.004, respectively), 48 hrs (p=0.001 and p=0.003, respectively) , 72 hours (p=0.015 and 0.005, respectively) and 96 hours (p=0.005 for M21-M21L). Tumor to muscle ratios for 124IcRGDY-PEG nanoparticles (n = 7) versus 124I-PEG nanoparticles (n = 5) were found to be statistically significant at 24 hours pi (p = 0.001) and 72 hours pi (p = 0.001). = 0.006), but not at 4 h pi (p=35). Goodness-of-fit (R2) values were determined, along with their associated p-values, for the urine calibration curve (R2=0.973, p=0.01), as well as for urine (R2 > 0.95, p=0.047 ) and fecal (R2>0.995, p<0.002) cumulative % ID excretion curves using nonlinear regression analysis (SigmaPlot, Systat, v. 11.0).

RESULTS

Design and characterization of nanoparticles

[0151] Core-shell silica nanoparticles encapsulating Cy5 dye (emission maxima >650 nm), coated with methoxy-terminated (PEG -0.5 kDa) polyethylene glycol (PEG) chains, were prepared according to previously published protocols. Burns, et al., Fluorescent Silica Nanoparticles with Efficient Urinary Excretion for Nanomedicine, Nano Letters, 9, 442-8 (2009). Ow, et al., Bright and stable core-shell fluorescent silica nanoparticles. Nano Lett, 5, 113-117 (2005). The neutral PEG coating prevented non-specific uptake by the reticuloendothelial system (opsonization). The use of bifunctional PEGs allowed the binding of small amounts (~6-7 per particle) of arginine-glycine-cyclic aspartic acid (cRGDY) peptide ligands targeting integrin avp3 to maintain a small hydrodynamic size that facilitates efficient renal clearance. Peptide ligands were further labeled with 124I using a tyrosine linker to provide a signal that can be quantitatively imaged in three dimensions by PET (124I-cRGDY-PEG-dots, Fig. 6A); An important practical advantage of the relatively long-lived 124I (physical half-life: 4.2 days) is that sufficient signal persists long enough to allow detection by radio until at least several days after administration, when background activity it has been greatly lightened and the contrast between the tumor and the background is maximized. The purity of the radiolabeled targeted nanoparticle was >95% by radio thin layer chromatography. The stability of the non-radiolabeled targeted nanoparticle is approximately 1 year based on ECF measurements. The particle is excreted intact in the urine by ECF analysis. As used herein, "dot" and "nanoparticle" are used interchangeably. A PEG-coated bead containing a tyrosine residue for 124I labeling served as a control probe (124I-PEG dots). Purification of the radiolabeled samples by size exclusion chromatography (Fig. 7) resulted in radiochemical yields of >95%. Hydrodynamic diameters of ~7 nm ID for cRGDY-PEG spots and non-radioactive PEG spots were measured by fluorescence correlation spectroscopy (FCS) (Fig. 6B and 6C). The relative brightness of the cRGDY-PEG dots was determined to be, on average, 200% greater than that of the free dye (Fig. 6C), consistent with previous results. Bums, et al., Fluorescent Silica Nanoparticles with Efficient Urinary Excretion for Nanomedicine, Nano Letters. 9, 442-8 (2009). Larson, et al., Silica nanoparticle architecture determines radiative properties of encapsulated chromophores. Chem. Mater. 20, 2677-2684 (2008). Based on these physicochemical properties, we anticipate achieving a favorable balance between selective tumor uptake and retention versus renal clearance of the target particle, thereby maximizing target tissue localization and minimizing normal tissue toxicity and doses of the target particle. radiation.

In vitro receptor binding studies

[0152] To examine the in vitro binding affinity and specificity of 124I-cRGDY-PEG dots and 124I-PEG dots to tumor and vascular endothelial surfaces, avp3 integrin overexpressing (M21) and non-expressing (M21L) cell lines were used. human umbilical vein endothelial and melanoma (HUVEC). Highly specific linear and saturable binding of cRGDY-PEG dots was observed over a range of particle concentrations (0 to 8 ng/mL) and incubation times (up to 5 hours), with peak differential binding at 4 hours and ~2.0 ng/ml particle concentration (data not shown) using flow cytometry. The receptor binding specificity of 124I-cRGDY-PEG dots was tested using y-counting methods after initially incubating M21 cells with excess non-radiolabeled cRGD and then adding various concentrations of the radiolabeled targeting probe (Fig. 8A). ). Scatchard analysis of the binding data produced a dissociation equilibrium constant, Kd, of 0.51 nM (Fig. 8A, inset) and a receptor concentration, Bmax, of 2.5 pM. Based on the Bmax value, the avp ³ integrin receptor density was estimated to be 1.0 x 10 4 per M21 cell, in reasonable agreement with the previously published estimate of 5.6 x 10 4 for this cell line. Cressman, et al., Binding and uptake of RGD-containing ligands to cellular avp3 integrins. Int J Pept Res Ther. 15, 49-59 (2009). Incremental increases in integrin-specific M2I cellular uptake were also observed over a temperature range of 4 to 37 °C, suggesting that receptor-mediated cellular internalization contributed to overall uptake (data not shown). Further studies of competitive binding using the targeted probe showed a complete block of receptor-mediated binding with anti-avp3 integrin antibody (Fig. 8B) by flow cytometry. No significant reduction in the magnitude of receptor binding (~10% of M21) was observed with M21L cells (Fig. 8C) using excess cRGDY or anti-avp3 integrin antibody. These results were confirmed by additional y-count studies and a 50% inhibition of binding concentration, IC50, of 1.2 nM was determined for the 121I-cRGDY-PEG site. An associated multivalent enhancement factor of greater than 2.0 was found for the cRGDY-PEG site relative to the monomeric cRGD peptide using an anti-adhesion assay and M21 cells (data not shown). Montet, et al., Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem. 49, 6087-6093 (2006). Li, et al., 64 Culabeled tetrameric and octomeric RGD peptides for small-animal P ^e T of tumor avp3 integrin expression. J. Nucl Med. 48, 1162-1171 (2007). Similar to M21 cells, excess antibody effectively blocked cRGDY-PEG dot receptor binding to HUVEC cells by flow cytometry (Fig. 8D).

Biodistribution and clearance studies

[0153] Time-dependent biodistribution as well as renal and hepatobiliary clearance were assessed by intravenously administering tracer doses (~0.2 nanomoles) of 124I-cRGDY-PEG dots and 124I-PEG dots to mouse models with M21 tumor xenograft (Figure 9). Although tissue activity concentrations (percent injected dose per gram (% ID/g)) for the target probe were measured over a post-injection time interval (pi) of 196 hours, the 124I-cRGDY point comparison -PEG (Fig. 9A) and 124I-PEG dot tracers (Fig. 9B) were restricted to a 96 hour window, as data for the latter was not acquired within 1 week. Statistically significant differences (p<0.05) were observed in tracer activities for blood, tumor, and major organs at 4 and 96 hours pi, as well as at 24 hours pi for tumor and various other tissues ( Table 1). The targeted probe was almost completely removed from the carcass at week pi (~3% ID). The mean residence times (T ¹ / ² ) for blood, tumor and major organs for these tracers are shown in Table 2 (columns 2 and 5). A representative data set (blood residence) is shown in the inset of Fig. 9A. A relatively long blood T ^1/2 value of 7.3 ± 1.2 hours was determined for the 124I-PEO point. After cRGDY peptide binding to synthesize the 124I-cRGDY-PEG dot, the T ^1/2 value decreased slightly at 5.6 ± 0.15 h, but was accompanied by increased bioavailability of the probe (Table 2, column 3). The tumor T ^1/2 value for the 124IcRGDY-PEG spot was found to be approximately 13-fold greater than that of blood, versus a difference of only 5-fold for the 124I-PEG spot (Table 2, columns 2 and 5). ).

Table 1

p-values from the biodistribution study comparing 124l-cRGDY-PEG and 124l-PEG dots

Table 2

oss e ec va remm , , 70-kg Man. Standard. U ( upper), L ( lower), mouse melanoma model, bone activity much lower than other tissues ( not reported)

[0154] By appropriate mass-adjusted translation of pre-human biodistribution data, human normal organ radiation doses were derived and found to be comparable to other commonly used diagnostic radiotracers (Table 2, columns 8 , 9). Along with the result that the targeted probe was nontoxic and did not produce tissue-specific pathological effects (ie, no acute toxicity) (Fig. 10 and Table 3), the first applications of targeted and nontargeted molecular imaging in humans with these Agents are scheduled.

Table 3

N: normal, U: not available, NP: not present, 1: minimal, 2: soft, F: focal, MF: multifocal

[0155] In another study to confirm that 127I-RGD-PEG dots are non-toxic after intravenous administration in mice, formal single-dose toxicity tests were performed over the course of 2 weeks using 127I-RGD-PEG dots. PEG approximately 100 times the equivalent human dose of 127I-PEG dots served as control particle. In summary, the procedure was as follows. Twenty-eight 8-week-old B6D2F1 mice were used in the acute toxicity study and divided into a treatment and control group. The treatment group (n= 6 males 6 females) received a dose of RGD 127I-PEGylated silica nanoparticles at a dose of 1 x 109 mol/mouse intravenously, and the control group (n= 6 males 6 females) received the same amount of vehicle. Two mice/group (one male and one female/group) were sacrificed on day 7 post-dose and tissue-specific clinical chemistry, hematology, and histopathology studies were performed at autopsy. All remaining animals (n=5 males 5 females/group) were observed for 14 days after treatment. Four untreated mice (two males and two females) were used as reference. The conclusion of the studies was that no adverse events were observed during dosing or the following 14-day observation period. No mortality or morbidity was observed. Clinical observations included the absence of the following: anemia, weight loss, agitation, increased respiration, gastrointestinal disturbances, abnormal behavior, neurologic dysfunction, hematology abnormalities, clinical chemistry abnormalities, or drug-related lesions in terms of pathology organic. Thus, a single injection of RGD 127I-PEGylated silica nanoparticles at 1x10-9 mol/mouse, a dose equivalent to a 100-fold excess of the dose of RGD PEGylated silica nanoparticles required for Phase 0 imaging studies, it is safe and non-toxic in B6D2F1 mice.

[0156] Efficient renal excretion was found for the ~7 nm diameter targeting and non-targeting probes over a time period of 168 hours by fluorometric analysis of urine samples. Fluorescence signals were background corrected and converted to particle concentrations (% ID/pl) according to a serial dilution calibration scheme (Fig. 9C, inset; Table 4, column 2). Burns, et al., Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine, Nano Letters. 9,442-8 (2009). The concentration values, together with conservative age-dependent estimates of the average urinary excretion rate, allowed calculation of the cumulative %ID excreted (Table 4, column 4). Drickarner, Rates of urine excretion by house mouse (mus domesticus): differences by age, sex, social status and reproductive condition. J. Chem. Ecol. 21, 1481-1493 (1995). Almost half of the injected dose (about 43% ID) was observed to be excreted during the first 24 h pi and ~72% ID at 96 h, Fig. 9C), suggesting that most of the excretion occurred on the first day pi No significant particle fluorescence could be detected in the urine 168 h pi The fecal excretion profiles of the 124I-cRGDY-PEG-dot indicated that, on average, 7% ID and 15% ID of the injected dose was eliminated for 24 and 96 hours, respectively (Figure 9D). ECF analysis of urine samples obtained at multiple times after injection of the targeting probe revealed that the particle was excreted intact and without release of the encapsulated dye (data not shown).

Table 4

Urinary concentration data and cumulative excretion

Whole-body serial PET studies

[0157] PET imaging of integrin expression in M21 and M21L hind paw subcutaneous xenograft mouse models was performed at multiple time points pi after iv injection of 124I-cRGDY-PEG- dots or 124I dots -PEG (control). Representative whole-body coronal microTEP images at 4 hours (left: M21 tumor; center: M21L tumor) and 24 hours (right: M21 tumor) are shown in Fig. 11A pi Specific targeting of integrin-overexpressing M21 tumor avp3 is clearly visible in these images. Mean %ID/g tumor and standard deviations are shown for groups of M21 (n=7) and M21L (control) (n=5) tumors receiving the targeted 124I-cRGDY-PEG dots, as well as for M21 tumor mice (n=5) receiving an untargeted 124I-PEG dot tracer (Fig. 11B). At the time of maximum tumor uptake (~4 hours pi), three-fold increases in activity concentration (in %ID/g) were observed in M21 tumors over controls. The differences were statistically significant at all pi time points (p<0.05) except 1 hour (p=0.27).

[0158] Image-derived tumor-to-muscle uptake ratios (%ID/g) for 124I-cRGDY-PEG dots revealed enhanced tumor contrast at later times (~24-72 hr pi), whereas for 124I -peg -dots declined (Fig. 11C). This result suggested that the 124I-cRGDY-PEG spots were, in fact, tumor-selective, which became more apparent as blood activity abated during the initial 24-hour period (compare Fig. 11C with Fig. 11C). inset of Fig. 9A). A statistically significant correlation was found between the PET-derived %ID/g tumor tissue values for 124I-cRGDY-PEG dots and 124I-PEG dots, and the corresponding ex-counted y-tumor %ID/g values. -alive (correlation coefficient r = 0.94, P <0.0016; Fig. 11D), confirming the accuracy of PET for obtaining quantitative biodistribution data non-invasively.

In vivo NIR fluorescence imaging and microscopy

[0159] We performed in vivo fluorescence imaging studies using our small targeted nanoparticles to map local/regional nodules and lymphatic channels, thereby overcoming the above limitation. Importantly, the multimodal nature and small size of our targeted particle probe can be exploited to visualize a variety of lymph node and branch sizes in our melanoma model after 4-quadrant peritumoral delivery, simulating cell mapping procedures. Intraoperative human sentinel lymph nodes. Initially, serial NIR fluorescence microscopy was performed on intact mice over a 4-h time period using either the targeted or untargeted particle probes. Peritumoral administration of the directed probe revealed drainage and persistent visualization of adjacent inguinal and popliteal nodes during this interval, with lymph nodes smaller and/or more distant and more difficult to visualize. In contrast, the untargeted probe produced short-term (~1 hour) visualization of local nodules with a progressively weaker fluorescence signal observed (data not shown). Following surgical exposure, this observation was found to be the result of more rapid particle diffusion from the tumor site, compared to the prolonged retention observed with the targeted probe.

[0160] Next, we performed representative mapping of lymph nodes at multiple spatial scales using whole-body optical imaging of live animals (Fig. 12A) and NIR fluorescence microscopy techniques (Fig. 12B) to visualize lymphatic drainage from the peritumoral region to the inguinal and axillary lymph nodes. in live surgically exposed animals. In addition, higher-resolution fluorescence images (Fig. 12B, bottom row) allowed visualization of more detailed intranodal architecture, including tall endothelial venules, which facilitate passage of circulating naïve lymphocytes into the node, and which may have important implications for lymph node staging and the ability to detect micrometastases in early stages of the disease. Smaller and less intense lymphatic branches were also visualized by fluorescence microscopy in the axillary region (data not shown). Therefore, the small size of the targeted probe not only allows visualization of the first drainage (or sentinel node), close to the tumor, but also allows visualization of more distant nodes and visualization of the lymphatic drainage pattern.

DISCUSSION

[0161] We report non-toxic, high-affinity, and efficiently cleared silica nanoparticles for nodule mapping and selective tumor targeting, having successfully addressed a number of current challenges associated with other particle technologies. This is the first targeted nanoparticle that, on the basis of its favorable properties, can be said to be clinically translatable as a combined optical PET probe. The complementary nature of this multimodal probe, along with its small size (~7 nm in diameter), may facilitate clinical evaluation by allowing seamless integration of imaging data acquired at different spatial, temporal, and sensitivity scales, which could provide new insights into the fundamentals. molecular processes that govern tumor biology.

[0162] Our in vitro results show the receptor binding specificity of the ~7 nm targeting particle probe to M21 and HUVEC cells. Similar results have been reported with receptor binding assays using the same cell types, but with the monovalent form of the peptide. Cressman, et al., Binding and uptake of RGD-containing ligands to cell (avp3 integrins. Int J Pept Res Ther. 15, 49-59 (2009). Importantly, multivalent enhancement of the cRGDY-bound particle probe , together with the prolonged blood and tumor T ¹ / ² residence time values, are key properties associated with the particle platform that are not found with the monovalent form of the peptide.

[0163] The relatively long blood T ^1/2 value of 7.3 ± 1.2 hours estimated for the 124I-PEG dot tracer may be related to the chemically neutral PEG coated surface, making the probe is biologically inert and significantly less susceptible to phagocytosis by the reticuloendothelial system. That a reduction in the T ^1/2 value was found at 5.6 ± 0.15 h for the 124I-cRGDY-PEG dot tracer is probably the result of recognition by the target integrins and/or an activity of more active macrophages (~13 minutes) and results in greater bioavailability of the probe, which facilitates tumor targeting and results in greater tumor uptake for longer periods of time. Montet, et al., Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem. 49, 6087-6093 (2006). In addition, the T ^1/2 value of the tumor for the 124I-cRGDY-PEG spot was approximately 13-fold that of the blood, compared with only a five-fold difference for the 124I-PEG spot, suggesting a substantially greater localization in the target tissue of the former than the latter. These mechanistic interpretations of in vivo data can be harnessed to refine protocols for clinical diagnosis, treatment planning, and treatment monitoring.

[0164] The results of this study underline the clear advantages offered by PET, a powerful, quantitative and highly sensitive imaging tool for non-invasively extracting molecular information related to receptor expression levels, binding affinity and the specificity. The greater accumulation and slower clearance of M21 tumors, relative to surrounding normal structures, allows one to differentiate specific mechanisms of tumor uptake from non-specific mechanisms (ie, tissue perfusion, leakage) in normal tissues. However, a small component of M21 tumor uptake can presumably be attributed to alterations in vascular permeability (ie, enhanced permeability and retention effects). Seymour, Passive tumor targeting of soluble macromolecules and drug conjugates. Crit, Rev. Ther. Drug Carrier Syst. 9, 135-187 (1992). This non-specific uptake mode reflects a relatively small portion of the total tumor uptake at earlier pi time points based on the %ID/g increases observed in mice receiving the control marker (124I-PEG points, Fig. 11B). At hour pi, no significant increases in %ID/g were observed in M21 tumors over controls. This observation may reflect the effects of differential perfusion in the first hour, with tumor accumulation and retention seen primarily at later pi times (ie, 24 hours). In addition, compared to the clinically approved peptide tracer, 18F-galacto RGD, almost two-fold increased uptake was found in M21 tumors for 124I-cRGDY-PEG-34 sites, while additionally offering advantages of multivalent binding, prolonged blood circulation, and increased renal clearance.

[0165] An advantage of a combined PET-optical probe is the ability to assess anatomical structures that have sizes at or well below the resolution limit of the PET scanner (i.e., the so-called partial volume effect), which can undermine the detection and quantification of activity in lesions. For example, in small animal models, the evaluation of metastatic disease in small local/regional nodes, clinically important for the staging and treatment of melanoma, may not be adequately resolved by PET imaging, since the size of the observed nodes is usually of the order of spatial resolution of the system (1-2 mm). By using a second complementary and sensitive imaging modality, near-infrared (NIR) fluorescence imaging, functional maps can be obtained that reveal nodal disease and lymphatic drainage patterns. Ballou, et al., Sentinel Lymph Node Imaging Using Quantum Dots in Mouse Tumor Models. Bioconjugate Chem. 18, 389-396 (2007). Although further studies investigating the distribution of cRGDY-PEG point intranodal fluorescence in relation to metastatic foci are needed to determine whether sensitive localization of such foci can be achieved, these results clearly demonstrate the advantages of working with an optical probe. Combined PET of this type.

[0166] In the clinic, the benefits of such a combined platform for tumor staging and treatment cannot be underestimated. The prolonged blood circulation time and resulting bioavailability of this nanoprobe highlight its use as a versatile tool for early and long-term monitoring of various stages of disease management (diagnostic screening, pretreatment assessment, therapeutic intervention, and post-monitoring). treatment) without restrictions imposed by toxicity considerations. An additional important advantage is that while rapidly clearing probes may be useful for certain applications where target tissue localization is itself rapid, localization of many agents in often poorly vascularized and relatively inaccessible solid tumors it will probably be slow after systemic administration. Therefore, the current nanoparticle platform expands the range of applications of such agents, since the localization kinetics of the target tissue is no longer limiting. Furthermore, deep nodes can be mapped by PET in terms of their distribution and number, whereas more precise and detailed localization of superficial nodes can be obtained by NIR fluorescence imaging. Finally, the relatively long residence of the targeted probe in the tumor relative to that in the blood, in addition to its multivalent potentiation, can be exploited for future theranostic applications as a radiotherapeutic or drug delivery vehicle.

Example 5 Fluorescent Silica Nanoparticles Conjugated with Ov03 Integrin Targeting Peptide and/or uMUC1 Targeting Peptide (Thyroid Cancer and Squamous Cell Carcinoma (SCC) Models )

[01687] A cRGD peptide (Peptides International), having a cysteine end functionality will be attached to the PEG-ylated nanoparticle via a thiol-maleimide bond. The nanoparticles can optionally be further functionalized by a synthetic peptide ligand. EPPT1. Nanoparticles will be characterized based on particle size, size distribution, and photobleaching.

Characterization of nanoparticle-peptide conjugates

[0168] To assess the photophysical properties per particle, spectrophotometry, spectrofluorometry, and multiphoton fluorescence (ECF) correlation spectroscopy will be used to determine particle size, brightness, and size distribution. The size data will be corroborated by scanning electron microscopy and dynamic light scattering (DLS) measurements. Ow et al. Bright and stable core-shell fluorescent silica nanoparticles. NanoLetters 2005; 5, 113; 5, 113. The average number of RGD peptides per nanoparticle and the coupling efficiency of RGD to functionalized PEG groups will be evaluated colorimetrically under alkaline conditions and Biuret spectrophotometric methods (A = 450 nm, maximum absorbance).

[0169] Nanoparticle conjugates will be iodinated through tyrosine linkers to create a form radiolabeled (124I) (T ^1/2 -4 d) and stable (127I) using lodogen (Pierce, Rockford, IL). The final product will be purified by size exclusion chromatography.

Evaluation of the in vitro targeting specificity and biodistribution patterns of RGD and RGD-EPPT nanoparticles.

[0170] The expression patterns of avp3 and uMUC1 integrin in thyroid and squamous cell carcinoma (SCC) cell lines will be assessed against avp3 integrin negative and avp3 integrin positive (M21-L and M21 human melanoma cell lines, respectively). and uMUC1-negative and uMUC1-positive (U8728, H-29 cell lines, respectively) controls using anti-integrin and anti-uMUC1 antibodies. Cell lines that highly express the integrin avp3 and/or MUC1 will be selected for differential binding studies with RGD and RGD-EPPT nanoparticles, as well as for in vivo imaging.

[0171] Quantitative cell binding assays will assess tumor cell labeling efficiency and biodistribution studies. Uptake assays will be performed in tumors, organs and fluids using radioiodinated nanoparticle conjugates (124I-RGD nanoparticles, 124I-RGD-EPPT nanoparticles). To compare the PET uptake data of nanoparticle conjugates with those initially observed using optical NIRF imaging, each nanoparticle conjugate will also be iodinated to create a radiolabeled (124I) and stable (127I) form.

[0172] Fluorescence microscopy with RGD- and RGD-EPPT-C spots. Differential binding of RGD nanoparticles and RGD-EPPT nanoparticles to thyroid carcinoma/SCC cell lines that highly express integrin avp3 and/or MUC1, versus control lines, will be visualized by fluorescence microscopy.

[0173] Animal models. All animal experiments will be conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee and following NIH guidelines for animal welfare.

[0174] In Vivo Biodistribution: Male athymic nude mice (6-8 weeks old, n=5 per tumor) with integrin-negative/positive or uMUC1-negative/positive tumors will be injected subcutaneously (sc) on both flanks. tissues of different origin (n=3/each tumor). With a diameter of 0.5 cm (ID), mice will receive an intravenous (IV) injection of 124I-labeled nanoparticle conjugates (~500 nm/kg). Animals are sacrificed at 0.5, 1, and 24 brs later, with removal of tumors, organs, and fluids for weighing and counting (gamma counter). Biodistribution results will be expressed as the percentage of injected dose per gram of tissue.

[0175] Quantitative cell binding assay. Labeling efficacy will be assessed by incubating fixed numbers of carcinoma cells expressing a high amount of avp3 integrin and/or MUC1, with preselected concentrations of 124I-labeled nanoparticle conjugates for 1 hour in a humidified CO ² atmosphere at 37°C. Cells are washed extensively, lysed with 0.1% Triton X, and cell lysates are counted in a gamma counter.

Assessment of disturbing differences in tumor-specific targeting using multimodality in vivo imaging (TEP-NIRP).

[0176] As a high-throughput diagnostic screening tool, NIRF optical imaging can be used to assess relative differences in the biodistribution of progressively functionalized nanoparticle conjugates in vivo with increased sensitivity and temporal resolution. Semi-quantitative data on the specific targeting of the tumor can also be derived. These preliminary studies facilitate the selection of cell lines that strongly express markers of interest for more detailed quantification of biodistribution and tumor-specific targeting by PET.

[0177] Whole body microTEP™ and NIRF optical imaging will be performed over a one week period to assess differential uptake in flank tumors. The results of these studies will be validated with ex-vivo tumor fluorescence microscopy.

[0178] Serial in vivo NIRF imaging. Mice will be injected bilaterally with avp3 integrin negative and avp3 integrin positive cells or with uMUC1 negative and uMUC1 positive cells (n=5/tumor). After the tumors reach ~0.5 cm in inner diameter, stable iodinated and non-iodinated nanoparticle conjugates (RGD, 127I-RGD, RDG-EPPT, 127I-RGD-EPPT) will be injected intravenously. Serial images will be performed using the Maestro™ In Vivo Fluorescence Imaging System (CRI, Woburn, MA) at 0, 0.5, 1,2,4, 6,12, and 24 hours. At 24 h, mice are sacrificed and major tissues/organs are dissected, weighed, and plated in 6-well plates for ex vivo imaging. Fluorescence emission will be analyzed using regions of interest (ROI) on reference tumors, selected and injected tissues, employing spectral demixing algorithms to remove autofluorescence. Dividing the tissue average fluorescence intensities by the injected values will allow comparisons between the various tissues/organs for each injected nanoparticle conjugate.

[0179] Acquisition and analysis of dynamic MicroTEP images. Two groups of tumor-bearing mice (n=5/tumor) will be injected with radiolabeled 124I nanoparticle conjugates (radiotracers) and dynamic PET images will be performed for 1 hour with a Focus 120 microTEP™ (Concorde Microsystems, TN). One hour list mode acquisitions are initiated at the time of IV injection of ~25.9 MBq (700 mCi) of radiotracers. The resulting list-mode data is reconstructed into a 1283128396 matrix using filtered backprojection. ROI analysis of the reconstructed images is performed using ASIPro™ software (Concorde Microsystems, TN) to determine the mean and mean of radiotracer uptake (%ID/g) in tumors, other organs/tissues, and left ventricle ( SAW). Additional still image data will be obtained at 24, 48, and 72 hours post-injection. A four-parameter, three-compartment kinetic model will be used to characterize the behavior of the marker in vivo. For this analysis, arterial input is measured using an ROI placed over the LV.

Example 6 - Lymph node mapping in mini pig

[0180] Real-time intraoperative scanning of the nodal basin cannot be achieved in practice at present, as these systems are generally too cumbersome and expensive to use in the operating room or may be unable to provide the necessary field of view or tissue contrast. Furthermore, there are no biostable and clinically promising fluorophor-containing agents, offering improved photophysical characteristics and longer circulation lifetimes than the parent dyes, available to enhance tissue contrast for extended node mapping/resection procedures. With this animal study, we will show that advances in both multimodal particle probes and real-time molecular imaging device technologies can be readily translated to a variety of future human clinical trials. These transformative technologies can have a significant impact on standard intraoperative cancer care by providing targeted, state-of-the-art visualization tools to facilitate detection of metastatic SLN and enable precise delineation of nodes from neighboring anatomy to minimize risk of injury to structures. crucial. Benefits include extended real-time in vivo intraoperative mapping of the spread of nodal disease and the extent of tumor in the head and neck. Deep nodes can be mapped by PET, while precise and detailed localization of superficial nodes can be obtained by NIR fluorescence imaging. The small size of the particle probe can also extend the lower limit of the nodal sizes that can be sensitively detected. The net effect of the proposed non-toxic multimodal platform, together with the application of combined diagnostic/treatment procedures, has important implications for disease staging, prognosis, and clinical outcome of this highly lethal disease.

[0181] Disease target . In addition to melanoma, a number of other tumors (ie, breast, lung, and brain) overexpress avp3 integrin receptors and could serve as disease targets. Metastatic melanoma has a very poor prognosis, with a median survival of less than 1 year. Successful management relies on early identification with adequate surgical removal of the cancer. Surgical removal of the primary disease, detection, and treatment of regional lymph node spread is the standard of care in the US to accurately classify the disease and tailor treatment. Recently revised staging guidelines recognize the presence of microscopic nodal metastases as a hallmark of late-stage disease leading to dramatically reduced survival. Knowledge of the pathological status of the nodes is critical for early risk stratification, better outcome predictions, and selection of subgroups of patients likely to benefit from adjuvant therapy (therapeutic nodal dissection, chemotherapy) or clinical trials.

[0182] Sentinel lymph node ( SLN) mapping. SLN mapping techniques, which are routinely used in melanoma staging, identify the specific nodes that have the highest risk of tumor metastasis. This procedure identifies patients harboring metastatic disease for further treatment. Standard care techniques rely on injection of radioactive technetium (99mTc) sulfur colloid dye around the primary tumor for localization of the SLN, followed by intraoperative use of a gamma probe to measure radioactivity in lymphatic structures within a exposed nodal basin. Blue dye injected into the primary tumor can help delineate small SLNs from adjacent tissue, but the technique is unreliable and prone to complications. Current SLN mapping and biopsy techniques have limitations and report higher rates of non-localization of SLN(s) in the head and neck compared to other anatomic sites. The head and neck region is notorious for its unpredictable patterns of metastatic disease. The proximity of the primary disease to nodal metastases in this region makes intraoperative use of the gamma probe difficult due to injection site interference. It is important to note that current technology does not allow the surgeon to visualize the sentinel node and reliably differentiate it from fat or other adjacent tissues, putting vital structures (i.e., nerves) at risk of injury during surgery. dissection to identify and collect this node. The small size of the nodes and the wide variation in drainage patterns present additional challenges, resulting in a non-localization rate of around 10%. Nanoparticles . Most preclinical studies have used RGD peptides or peptide-conjugated radiotracers as targeting ligands to image avp3 integrin expression. 18F-galacto-RGD and 99mTc-NC100692 are peptide tracers that have been used successfully in patients to diagnose disease. Peptide tracers are rapidly cleared, which can result in reduced receptor binding and increased background signal from non-specific tissue scattering. These properties limit the potential of peptide tracers for longer-term follow-up. In contrast, nanoparticle probes (~10-100 nm), which have also been used to image integrin expression throughout tumor neovasculature, have extended half the circulation times for monitoring. longer term (ie days). Nanoparticles are typically larger than antibodies and radiopharmaceuticals (<10 kDa) and are associated with slower transmembrane transport, increased reticuloendothelial system (RES) uptake, and increased non-specific uptake due to impaired vascular permeability. of the tumor. The 7 nm diameter targeted nanoparticles used for this SLN mapping study are roughly comparable to the average diameter of an albumin molecule and 2-3 times smaller than the average diameter of a typical antibody. Relative to peptide tracers, the targeted particle probe is less prone to extravasation and is associated with prolonged circulation times that improve tumor targeting. Importantly, 124I-cRGDY-PEG dots demonstrate key properties in vitro and in vivo in M21 tumors required for clinical translation.

Materials and methods.

[0183] Sinclair miniature pigs with spontaneous melanoma (10-12 kg, Sinclair Research Center, MO) were injected intravenously with 5 mCi of 18F-fluoro-deoxyglucose (18F-FDG) for detection of nodal metastases and/or or organs throughout the body. The mini pig underwent a 1-h dynamic whole-body 18F-FDG PET scan using a clinical PET scanner 40 min post-injection to detect metastatic disease, followed by CT acquisition for anatomic localization. Mini-pigs were then injected subdermally in a 4-quadrant pattern around the tumor site (preferably head and neck site) with multimodal 124I-RGD-PEG dots 48 hours after 13F-FDG PET , and a second dynamic PET-CT scan was performed to assess for additional nodal metastases.

[0184] Mini pigs were brought into the operating room for identification of nodes. Optical fluorescence imaging was performed using a large field of view near-infrared fluorescence camera system, a smaller field of view modified endoscope, and a modified stereomacroscope to obtain higher resolution fluorescence images within the exposed surgical bed.

[0185] Validation of the fluorescent signal was performed intraoperatively by gamma counting with a clinically approved portable PET device within the surgical bed to transdermally localize specific, intraoperatively acquired points of the skin and nodes within the nodal basin.

[0186] The primary skin lesion of the melanoma was excised and an incision was made to allow access to the sentinel node(s). Node identity was confirmed by portable PET and multiscale optical imaging systems, and the nodes in question were excised. Samples were sent for histological evaluation of metastases and optical confocal microscopy to confirm the presence of malignancy and nanoparticle fluorescence.

[0187] After collection of the sentinel nodes, the entire lymph node basin was excised and further evaluated using histological methods (with immunohistochemical markers for melanoma as necessary), fluorescence microscopy, and the portable PET probe for correlative purposes. This step helped to identify any other malignant nodules within the nodule socket and the number of 124I-RGD-PEG dots present in adjacent nodules by their appearance on images.

[0188] 124I-RGD-PEG dots were administered subcutaneously to the limbs of the animal sequentially. The transit of the 124I-RGD-PEG dots to the inguinal/axillary nodes was followed using the optical imaging system and portable PET probes to confirm the duration of transit along the lymphatic pathways. The drainage node basins were surgically exposed and the drainage pattern of the lymph nodes was noted. Sentinel lymph nodes were removed from each site to confirm the lymphatic nature of the tissue. The animals were euthanized, and any other lesions seen on the images were excised in the necropsy room of the animal facility.

Discussion

[0189] A whole-body 18F-fluorodeoxyglucose (18F-FDG) PET-CT revealed a primary melanomatous lesion adjacent to the spine in the upper back, as well as a single nodule in the neck, posteriorly on the left side. right side of the animal, which were both FDG avid and suspicious of metastatic disease. This result was confirmed after 4-quadrant subdermal injection of 124I-RGD-PEG points around the tumor site, which further identified two more hypermetabolic nodes as well as draining lymphatics. Final scan interpretation pointed to 3 potential metastatic nodules. Surgical removal of the primary lesion, hypermetabolic nodes, and tissue from other nodal basins in the neck was performed bilaterally after portable PET probes identified and confirmed elevated count rates at the sentinel node locations. The irregular fluorescence signal measured in the excised right posterior sentinel node tissue was correlated with melanoma metastasis sites by histological analysis. All hypermetabolic node samples had black pigmentation and were found to correlate with the presence of distinct groups of melanoma cells. Therefore, the results of surgically resected tissue subjected to pathology for H&E and staining for other known melanoma markers confirmed the multimodality imaging results.

[0190] Fig. 13a shows the experimental setup of using a spontaneous porcine minimelanoma model to map the lymph node basins and regional lymphatic vessels draining the site of a known primary melanoma tumor. This intermediate-sized mini-pig model is needed to simulate the application of sentinel lymph node (SLN) biopsy procedures in humans and more accurately recapitulate human disease. Fig. 13b shows a small field of view PET image 5 min after subdermal injection of multimodal particles (124I-RGD-PEG-dots) over the tumor site. The tumor region, lymph nodes, and lymphatic vessels draining the tumor site are seen as areas of increased activity (black).

[0191] Fig. 14 shows whole body dynamic 18F-fluorodeoxyglucose (18F-FDG) PET scan (Figure 14a) and fused 18F-FDG PET-CT scans (Figure 14b) showing sagittal, coronal and axial images through the site of nodal disease in the neck. 18F-FDG PET scanning was performed to map sites of metastatic disease after intravenous administration and before administration of the radiolabeled nanoparticle probe. A single hypermetabolic nodule is seen at the back of the neck on the right side of the animal (arrows, axial images, upper/lower panels), also identified in the whole-body mini-pig image (Figure 14C). Fig. 15 shows the same image sets as Fig. 14, but at the level of the primary melanoma lesion, adjacent to the spine in the upper back. The PET avid lesion is identified (arrows, axial images, upper/lower panels), as well as in the whole-body mini-pig image (Figure 15c).

[0193] Fig. 16 shows high-resolution dynamic PET (Figure 16a) and fused PET-CT (Figure 16b) images after 4-quadrant subdermal injection of 124I-RGD-PEG dots over the tumor site, simulating a clinical protocol, during a period of time of 1 hour. Three hypermetabolic lymph nodes (arrows) were found in the neck, suggesting metastatic disease. The excised right posterior SLN was excised and whole-body near-infrared fluorescence (NIR) imaging was performed. The Cy5 fluorescence signal was detectable within the resected nodule (Figure 16c, top, Cy5 images) on whole-body optical images. Pathologic analysis of this black-pigmented nodule (arrow, SLN) demonstrated clusters of invasive melanoma cells in low-magnification (arrows) and high-magnification cross-sectional views of the nodule using H&E staining (bottom two images), and we expect melanoma-specificity for be further confirmed using special stains (Melan A, HMB45, PNL2, and "melanoma-associated antigen" biogenex clone NKI/C3). Furthermore, we expect colocalization of the particle with these metastatic clusters of cells on confocal fluorescence microscopy and high-resolution digital autoradiography, confirming the detection of metastatic disease.

Example 7 MC1R Targeted Peptide-Conjugated Fluorescent Silica Nanoparticles (Melanoma Model)

[0194] For multimodality imaging (TEP-NIRF) experiments, the targeting peptide and radiolabel, the nanoparticle surface will be swapped to determine target specificity, binding affinity/avidity, and detection sensitivity . Subsequent therapeutic particles will also be synthesized using therapeutic radiotracers (lutetium-177, 177Lu, t1/2 = 6.65 d) for the targeted killing of MCIR-expressing melanoma cells. Combined quantitative PET and optical imaging results will be correlated with tumor tissue autoradiography and optical imaging across spatial scales. For cell microscopy, an in vivo confocal fluorescence scanner will be used to obtain combined reflectance and fluorescence images.

Example 8 Fluorescent Nanoparticles for Targeted Radiotherapy

[0195] Dose escalation studies will be performed with 131I-RGD nanoparticles and treatment response will be monitored weekly, over the course of six weeks, using 18F-FDg PET. Time-dependent tumor uptake and dosimetry of the nanoparticle platform will be performed using planar gamma camera imaging. In vivo imaging data will be correlated with the gamma count of excised tumor samples.

[0196] Male nude mice (6-8 weeks old, Charles River Labs, MA) will be used to generate hindlimb xenograft models after injection of M21 human melanoma cells (5x105 in PBS). Tumors will be allowed to grow for 10 to 14 days until they are 0.5 to 0.9 cm3 in size.

[0197] 131I-based targeted radiotherapy studies. Therapeutic radionuclide 131I will be used as a radiolabel for targeted radiotherapy. To estimate the highest possible dose of 131I that produces no animal deaths and less than 20% weight loss (MTD), a dose escalation study will be performed in tumor-bearing nude mice. For a dose of 200 rad to the blood54, an administered activity of 10 MBq is required, which would provide a dose of 270 rad to the tumor. Four doses of 10 MBq each will be administered to achieve a tumor dose greater than 1000 rad with dose fractionation designed to allow bone marrow repair and preservation. 131I allows imaging of planar gamma cameras using a pinhole collimator to measure time-dependent tumor uptake and nanoparticle dosimetry. 18F-FDG PET allows quantitative monitoring of tumor response, thus providing complementary information.

[0198] Based on these data, and in vivo data on the effect of paclitaxel-loaded nanoparticles, it is will carry out a therapy study with the 131I-RGD- nanoparticle conjugate. Two groups of tumor-bearing mice (n = 10 per group) will receive four doses of 10.4 MBq once a week for 4 weeks, of 131I-RGD nanoparticle conjugates administered intravenously or saline vehicle (control, n = 10), and will be monitored for a period of 6 weeks. Response/progression to treatment will be quantified on the basis of tumor volume (via caliper measurements). All mice in treatment groups will also be imaged once a week (~1h sessions) using SPECT imaging (Gamma Medica) for a period of 6 weeks.

[0199] Acquisition and analysis of PET images with 18F-FDG. Two groups of tumor-bearing mice (n=10/group) will undergo an initial PET scan before and then weekly after treatment for a 6-week interval. Mice will be injected intravenously (iv) with 500 pCi of 18F-FDG and 10 min still PET images will be acquired using a Focus 120 microTEP™ (Concorde Microsystems, TN) before and after treatment. The acquired data will be reconstructed in a 128x1 28x96 matrix by filtered rear projection. Region of interest (ROI) analyzes of the reconstructed images will be performed using ASIPro™ software (Concorde Microsystems, TN) to determine the mean and OD of radiotracer uptake (%ID/g) in the tumors. Animals will be sacrificed at the end of the study and tumors will be excised for gamma counting.

Example 9 Fluorescent Nanoparticles Conjugated to Radionuclide Chelate and MCIR Target Peptide

[0200] PEG-ylated nanoparticles will be conjugated to target peptides and macrocyclic chelates that bind to high specific activity radiolabels.

[0201] High purity two-armed activated commercially available PEGs, derivatized with NHS or maleimide esters, will be attached to the silica shell of the nanoparticle using standard procedures. Either of the two functionalized PEG groups (NHS or maleimide esters) will be available for further conjugation with the peptide chelate construct, the cyclic peptide Re-[Cys3,4,10,D-Phe7]a-MSH3-13 (ReCCMSH (Arg11)), or 1,4,7,10-tetraazacyclododecane-W,W',W'W-tetraacetic acid (DOTA) chelating linker. Covalent attachment of derivatized PEGs to the nanoparticle surface will be performed in such a way as to expose different functional groups for binding DOTA and peptide chelate constructs, as discussed below.

Synthesis and physicochemical characterization of functionalized nanoparticles.

[0202] The functionalized nanoparticles will be synthesized by establishing covalent bonds of the following residues with the derivatized PEG groups:

(A) DOTA chelates for subsequent high-specific activity radiolabeling with positron-emitting radiometals (ie, 64Cu) to allow diagnostic detection with PET imaging DOTA will be conjugated to the functionalized PEGs using standard Fmoc chemistry, and purification of the chelated nanoparticles will be carried out by chromatography. 64Cu and 177Lu will bind to DOTA by incubating the reaction mixture at 60 °C for 30 min, followed by gel filtration or high pressure liquid chromatography, purification. Alternatively, TEP nuclides, such as 124I, 86Y. 68Ga and 89Zr, can be conjugated to the nanoparticle, either through DOTA-functionalized PEG (radiometals) or tyrosine-functionalized PEG (124I). The single photon emitter, 177Lu, obtained as 177LuCl ³ , will be complexed with DOTA for radiotherapy.

(B) The aMSH melanoma-targeted peptide analogue (ReCCMSH(Arg11)) is cyclized with rhenium. It is necessary to confirm the ratio of DOTA chelates to ReCCMSH residues (Arg11) on the surface of pegylated nanoparticles.

[0203] The characterization of the functionalized nanoparticle preparations will be carried out as follows:

(A) The mean number of DOTA chelates per nanoparticle will be determined by standard isotope dilution assays with 64Cu. Briefly, 64Cu will be added to solutions containing a known amount of ReCCMSH (Arg11) nanoparticles. The incubated solutions will be plated on silica gel-coated glass plates, developed in 10% ammonium acetate-tomethanol 1:1 (with EDTA) and analyzed by radio-TLC. While the 64Cu-labeled ReCCMSH(Arg11) nanoparticles will remain at the origin, the EDTA-bound 64Cu will migrate. Percent labeling efficiency will be plotted against the total nanomoles of 64Cu added to the reaction mixture. The number of attached chelates per nanoparticle can be determined from the inflection point of this curve.

(B) The average number of ReCCMSH (Arg11) peptides per nanoparticle and the coupling efficiency of ReCCMSH (Arg11) to the functionalized PEG groups will be evaluated by spectrophotometric methods (A = 435 nm, maximum absorbance) and the known extinction coefficient of ReCCMSH (Arg 11). The incorporation of rhenium offers the advantage that highly sensitive absorbance measurements of rhenium concentrations can be made in a small sample of product.

In vitro and in vivo optical-PET imaging of multifunctional nanoparticles in melanoma models to assess tumor-specific targeting and response to treatment.

[0204] The 64Cu-DOTA-ReCCMSH(Arg11) nanoparticles will be compared to the native 64Cu-DOTA-ReCCMSH(Arg11) construct to test the targeting capabilities of the nanoparticles.

[0205] Competitive binding assays. The B16/F1 murine melanoma lines with positive MC1R receptor will be used. ReCCMSH(Arg11) peptide IC ⁵⁰ values, the concentration of peptide required to inhibit 50% of radioligand binding, will be determined using 125I-(Tyr2)-NDP7, a radioiodinated α-MSH analog with picomolar affinity for the peptide. MCIR. Individual wells will be incubated at 25 °C for 3 h with approximately 50,000 cpm 125I -(Tyr2)-NDP in 0.5 mL binding medium with 25 mmol/L N-(2-hydroxyethyl)-piperazine-N -(2-ethanesulfonic acid), 0.2% BSA and 0.3 mmol/l 1,10-phenanthroline], with concentrations of (Arg11)CCMSH ranging from 10-13 to 10-5 mol/l. Radioactivity in cells and media will be collected and measured separately, and the data will be processed to calculate the IC ⁵⁰ value of the Re(Arg11)CCMSH peptide with the Kell software package (Biosoft, MO).

[0206] Receptor quantification assay. Aliquots of 5x105 B16/F1 cells will be added to the wells, grown in 200 µL RPMI medium, and incubated at 37 °C for 1.5 h in the presence of increasing concentrations of 125 I -(Tyr2)-NDP (from 2, 5 to 100 nCi) in 0.5 ml of binding medium (M EM with 25 mM HEPES, pH 7.4). Cells will be washed twice with 0.5 mL of ice-cold 0.2% BSA/0.01 M PBS, pH 7.4, and the level of activity associated with the cell fraction will be measured in a y-counter. Non-specific binding will be determined by incubating cells and 125 I-(Tyr2)-NDP with non-radioactive NDP at a final concentration of 10 pM. Scatchard plots will be obtained by plotting the ratio of specific binding to free 125I-(Tyr2)-NDP against the concentration of specific binding (fmol/million cells); fimax, the maximum number of binding sites, is the X intercept of the linear regression line.

[0207] B16/F1 murine melanoma lines (5x105 in PBS) will be injected subcutaneously into the hind paws of male nude mice (6-8 weeks old). Tumors will be allowed to grow for 10 to 14 days until they are 0.5 to 0.9 cm3 in size.

[0208] Biodistribution: A small amount of the 64Cu-DOTA-ReCCMSH(Arg11) nanoparticle conjugate (~10 pCi, 0.20 pg) will be injected intravenously into each of the mice bearing palpable B16/F1 tumors. Animals will be sacrificed at selected time points post-injection (2, 4, 24, 48, 72 hours; n = 4-5/time point) and the desired tissues will be removed, weighed and counted for accumulated radioactivity. . Additional mice (n=5) injected with the native radiolabeled construct, 64CuDOTA-ReCCMSH(Arg11) (~10 pCi, 0.20 pg) will serve as a control group and will be assessed 1 hour post-injection. To examine the in vivo uptake specificity, an additional group of mice will be pre-injected (2 h time point) with 20 pg NDP to act as a receptor block immediately prior to 64Cu-DOTA-ReCCMSH injection ( Arg11). nanoparticle conjugate. Major organs and tissues will be weighed and gamma counts performed, and the percent injected dose per gram (%ID/g) will be determined.

[0209] Serial in vivo NIRF imaging. In parallel with the PET studies below, NIR (fluorescence tomographic imaging, FMT 225, Visen, Woburn, MA) will be performed using a 680 nm scanning tunable NIR laser beam and CCD before and after iv injection of PET-bearing animals. tumors (n= 10). Mice will be maintained under continuous isoflurane anesthesia and placed in a portable multimodality imaging cassette (compatible with our FMT 2500 and Focus 120 microTEP) for pre- and post-injection FMT scanning (1,2,4,6,12, 24, 48 and 72 hours). The NIR fluorescence image, measured over a period of 1 to 10 minutes, will be reconstructed using the proprietary Visen software and overlaid on a normal photograph of the mouse. The imaging data is quantitative, as the measured intensity is directly related to the NIR fluorophore concentration, allowing parametric maps of absolute fluorophore concentrations to be generated for co-registration with the acquired PET imaging data.

[0210] Acquisition and analysis of dynamic PET images. Two groups of tumor-bearing mice (n=5/group) will be placed in the imaging cassette for co-registration of sequential PET optical studies. Mice will be injected intravenously (iv); one with radiolabeled 64Cu-DOTA-ReCCMSH(Arg11) nanoparticle conjugates and the second with native 64Cu-DOTA-ReCCMSH(Arg11) constructs. After injection, 1-hour dynamic PET images will be acquired using a Focus 120 microTEPTM (Concorde Microsystems, TN). One hour list mode acquisitions are started at the time of IV injection of the radiolabeled probe (~1 mCi). The resulting list mode data will be reconstructed into a 1283128396 matrix using filtered backprojection. Region of interest (ROI) analyzes of the reconstructed images are performed with ASIPro™ software (Concorde Microsystems, TN) to determine the mean and standard deviation of radiotracer uptake (%ID/g) in tumors, other organs/tissues and left ventricle. (LV). Tracer kinetic modeling of the data will allow estimation of pharmacokinetic parameters, including delivery, clearance, and volume of distribution. As indicated, arterial blood inflow is measured using an ROI placed over the LV (as a measure of blood activity). Additional data will be obtained from still images at 24h, 48h, 72h post injection.

[0211] Fluorescence microscopy and autoradiography of tissues. A combination of optical imaging technologies that exhibit progressively smaller spatial scales (ie, whole-body fluorescence imaging, fluorescence macroscopy, and in vivo fluorescence confocal laser scanning microscopy) will be used to image tumors in live intact animals. at 72 h after injection. Mice will be kept under continuous anesthesia with isoflurane, which will allow detection and localization of the fluorescence signal from the whole animal/organ level down to the cellular level at a range of magnifications. Macroscopic/whole animal images will be performed with fluorescence stereomicroscope (Visen; Nikon SMZ1500) equipped with Cy5 fluorescence filter sets and CCD cameras. Fluorescence confocal laser scanning microscopy capabilities will be developed. Subsequently, the mice will be sacrificed for autoradiography in order to map biodistributions of the marker at high resolution throughout the tumor volume. Tumors will be excised, snap frozen, serially sectioned (1D0p sections) and mounted on slides, with alternate sections placed in contact with a phosphor plate in a light-tight cassette (up to 1 week). H&E staining will be performed on the remaining consecutive sections. Autoradiographic results will be correlated with PET imaging data and histological results.

[0212] Therapeutic radionuclides 177Lu or 90Y may alternatively be used for targeted radiotherapy. To estimate the highest possible dose of 177Lu that produces no animal deaths and less than 20% weight loss (MTD), a dose escalation study will be performed in tumor-bearing nude mice. Doses of radiopharmaceuticals suspected to be at (or close to) the MTD will be evaluated based on literature values for 177Lu.

Example 10 Functionalized Fluorescent Nanoparticles for Conjugation with Ligand and Contrast Agent by "Click Chemistry"

Synthesis of nanoparticles containing versatile functional groups for subsequent conjugation of ligand ( eg peptides) and contrast agent ( eg radionuclides).

[0213] In order to synthesize a matrix of nanoparticle peptide chelate constructs suitable for high specific activity radiolabeling, a "click chemistry" approach can be used to functionalize the nanoparticle surface (Figure 17). This method is based on the copper-catalyzed cycloaddition of azide to a triple bond. Such an approach would allow for great versatility in exploring multimodal applications.

[0214] Synthesis and characterization of nanoparticles. The PEG groups that will be covalently attached will be produced following the scheme of Fig. 14. The PEG will be covalently attached to the nanoparticle through the silane group. Standard chemical pathways will be used for the production of the triple bond functionalized PEG.

[0215] Functionalization of nanoparticles with triple bonds. To synthesize the bifunctionalized PEGs, the first step will employ the well-studied reaction of activated carboxylic ester with aliphatic amine (Figure 18). Alternatively, another amine bearing a suitable triple bond may be used, for example p-aminophenylacetylene. The second step of the synthesis is also based on a known conjugation reaction.

Synthesis and physicochemical characterization of functionalized nanoparticles conjugated with model peptides and chelates.

[0216] The functionalized nanoparticle contains (A) desferrioxamine B (DFO) for subsequent radiolabeling of highly specific activity with the positron emitter zirconium-89 (89Zr) and (B) the SSTR-targeting peptide, octreotate.

[0217] Synthesis of DFO with an azide bond. DFO with an azide group will be produced by reacting DFO-B with pazido benzoic acid (Figure 19) and purified. The "click chemistry" reaction is a 1,3-dipolar cycloaddition at room temperature and the conditions are often referred to as "Huigsen conditions". Although the reactions can generally be completed at room temperature in ethanol, it may be appropriate to heat the reaction. The catalyst is usually Cu(I)Br, but alternatives include Cu(I)I or Cu(II)SO4 (with a reductant). Knor et al. Synthesis of new derivatives of 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) for chemoselective binding to deprotected polyfunctionalized compounds. Chemistry, 2007;13:6082-90. Click reactions can also be carried out in the absence of any catalyst. Alternatively, the NH3+ group in DFO-B can be directly converted to an azide group.

[0218] Synthesis of Tyr3-octreotate with an azide. Solid phase peptide synthesis (SPPS) of Tyr3-octreotate (Figure 20A) will be performed on a peptide synthesizer. Briefly, the synthesis will involve the Fmoc (9-fluorenylmethoxycarbonyl) method as described above for this peptide. Briefly, the instrument protocol calls for 25 pmol of subsequent Fmoc-protected amino acids activated by a combination of 1-hydroxybenzotriazole (HOBt) and 2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU ). Fmoc-protected amino acids will be purchased commercially unless otherwise indicated; prepackaged amino acids will be obtained from Perkin-Elmer (Norwalk, TC), while those not available in prepackaged form, such as D-Amino Acids and Fmoc-Cys(Acm), will be supplied by BACHEM Bioscience, Inc. (King of Prussia, PA) or Novabiochem (San Diego, CA). The azide group (for "click" chemistry) will be introduced into the peptide backbone by coupling an azide-containing acid to the N-terminus of the peptide, while the peptide is still protected and bound to the resin (Figure 20B). .

[0219] Synthesis of functionalized nanoparticles. The next step will be to conjugate both the DFO that has an azide bond and the Tyr3-octreotate that has an azide bond (Figures 21A and B) with the nanoparticle. "Click chemistry" is highly selective, quantitative, and can be performed very quickly and under mild conditions. The number of combined azide groups of DFO and Tyr3-octreotate will be controlled to never exceed the number of triple bonds available; the triple bonds will always be in excess of <5%.

[0220] Characterization of functionalized nanoparticles. The mean number of DFO chelated peptides per nanoparticle will be determined by performing a standard isotope dilution assay with 89Zr (or 68Ga). 89Zr will be produced in a cyclotron and purified. Briefly, 10 concentrations of 89Zr-oxalate will be added to solutions containing a known amount of DFO-derived nanoparticles. After 30 min. After incubation at room temperature, solutions will be plated on silica gel-coated glass plates, developed in 10% ammonium acetate to methanol 1:1 (with EDTA) and analyzed by radio-TLC. While the 89Zr-DFO-derived nanoparticles will remain at the source, the 89Zr non-specifically bound to EDTA will migrate. The percentage of labeling efficiency will be plotted as a function of the total nanomoles of 89Zr added to the reaction mixture. The number of chelates bound to the nanoparticle can be determined from the inflection point of this curve.

[0221] The average number of Tyr3-octreotate peptide per nanoparticle will be determined by analyzing the disulfide bridge of Tyr3-octreotate. Briefly, the disulfide bonds of Tyr3 octreotate can be cleaved quantitatively with excess sodium sulfite at pH 9.5 and at room temperature. DTNB or Elman's reagent can be used to quantify thiols in proteins by uptake measurements. It readily forms a mixed disulfide with thiols, releasing the chromophore 5-merapto-2-nitrobenxoic acid (maximum absorption 410 nm). Only protein thiols that are accessible to this water-soluble reagent are modified. Alternatively, Invitrogen's Measure-iT™ Thiol Assay Kit can be used.

In vivo tests in suitable tumor models.

[0222] Subcutaneous xenograft models will be generated using female SCID mice bearing AR42J tumors. Briefly, AR42J (13107) cells will be injected subcutaneously into the flanks of female SCID mice. The tumors will be allowed to grow for 10 to 12 days until they are 0.5 to 0.9 cm3 in size.

[0223] Radiolabeling of the DFO nanoparticle by 89Zr is expected to take place in <15 min. at room temperature. Non-specifically bound 89Zr will be removed by addition of EDTA followed by a gel filtration step.

[0224] Receptor binding assays. Receptor binding assays will be performed using 89Zr-DFO nanoparticles on membranes obtained from AR42J tumors. The competing ligands, natZr-DFO-Nanoparticles and natZr-DFO-octreotate will be prepared by the reaction of high purity natural zirconium oxalate with DFO-octreotate and DFO-Nanoparticles, respectively. The purity of the final products will be confirmed by HPLC. IC ⁵⁰ values will be determined according to previously published methods, using the Millipore MultiScreen Assay System (Bedford, MA). Data analysis will be performed using GraFit (Erithacus Software, UK), LIGAND (NIH, Bethesda, MD) and GraphPad PRISMTM (San Diego, CA) software.

[0225] In vitro assays. AR42J cells will be harvested from cell dissociation solution monolayers (Sigma Chemical Co., St. Louis, MO) and resuspended in fresh DMEM medium at a concentration of 2 x 106 cells/ml. An aliquot of approximately 0.3 pmol of 89Zr-DFO nanoparticles will be added to 10 mL of cells, incubated at 37°C with continuous shaking. At 1, 5, 15, 30, 45, 60 and 120 min, 200 pl aliquots will be withdrawn in triplicate and placed on ice. The cells will be immediately isolated by centrifugation and the % uptake of the compound into the cells will be calculated.

[0226] Biodistribution. A small amount of 89Zr-DFO nanoparticles (~10 pCi, 0.20 pg) will be injected intravenously into each of the mice bearing palpable AR42J positive tumors. Animals will be sacrificed at selected time points after injection (1.4, 24, 48, 72 hours; n = 4-5) and desired tissues will be removed, weighed, and counted for accumulation of radioactivity. Two additional control groups will be studied 1 hour after the injections: (A) mice injected with the native radiolabeled peptide 89Zr-DFO-octreotate (~10 pCi, 0.20 pg), and (B) mice pre-injected with a block of Tyr3-octreotate (150 pg) to demonstrate receptor-mediated accumulation of 89Zr-DFO nanoparticles. Tissues including blood, lung, liver, spleen, kidney, adrenal glands (STTR positive), muscle, skin, fat, heart, brain, bone, pancreas (STTR positive), small intestine, large intestine, and AR42J tumor will be counted. The percentage injected dose per gram (%Dl/g) and the percentage injected dose per organ (%Dl/organ) will be calculated in comparison to a counted and weighed standard solution.

[0227] NIRF imaging in vivo. Serial images will be performed using the MaestroTM In Vivo Fluorescence Imaging System (CRI, Woburn, MA) at 0, 0.5, 1.2, 4, 6, 12, 24, 48, and 72 hours. At 72 h, mice will be sacrificed and major tissues/organs dissected, weighed, and plated in 6-well plates for ex vivo imaging. Fluorescence emission will be analyzed using regions of interest (ROI) on reference tumors, selected and injected tissues, employing spectral demixing algorithms to remove autofluorescence. Fluorescence intensities and standard deviations (SD) will be averaged for groups of 5 animals. Dividing the tissue average fluorescence intensities by the injected values will allow comparisons between the various tissues/organs for each injected nanoparticle conjugate.

[0228] PET imaging of small animals in vivo. PET images of small animals will be performed on a microTEP®-FOCUS™ system (Concorde Microsystems Inc, Knoxville TN). Mice bearing AR42J tumors (n=5 per group) will be anesthetized with 1-2% isoflurane, placed supine, and immobilized in a custom-made cradle. Mice will receive 200 pCi of the 89Zr-DFO-octreotate-nanoparticle complex via the tail vein and will be imaged side by side. Initially, animals will be imaged by acquiring multiple successive 10-minute scans continuously from the time of injection over a 1-hour time period, followed by 10-minute static data acquisitions at 2, 4 , 24, 48 and 72 hours after injection. Standard uptake values (SUVs) will be generated from regions of interest (ROIs) drawn over the tumor and other organs of interest. Co-registration of the PET images will be accomplished in combination with a microCAT-II camera (Imtek Inc., Knoxville, TN), which provides high-resolution anatomical CT X-ray images. Image registration between microCT and PET images will be accomplished using a historical registration technique and AMIRA image viewing software (AMIRA, TGS Inc, San Diego, CA). The registration method proceeds by rigid transformation of microCT images from reference points provided by fiducials directly attached to the animal's bedding.

[0229] Pharmacokinetic measurements. Biodistribution and dynamic PET data will provide the temporal concentration of 89Zr-DFO-octreotate-nanoparticle in the tissue that will allow characterization of the pharmacokinetic parameters of the agent.

[0230] Fluorescence microscopy and autoradiography of ex vivo tissues. Localization of nanoparticle conjugates in tissues will be performed on frozen sections. MicroTEP imaging will allow us to fully assess the global distribution in tumors and other non-target tissues. After the acute stage of the imaging trial, autoradiography will also be performed on the tumors, and these data will be correlated with histological and PET imaging results. Consecutive sections (~10 pm) will be taken, alternating sections for autoradiography and histological analysis. These sections will also be analyzed by multichannel fluorescence microscopy in the NIR channel.

Example 11 Particle internalization studies

[0231] The objective of this study is to evaluate the binding and internalization of the present nanoparticles to assess their localization in subcellular organelles and exocytosis. This will help to study the fate of particles functionalized with different targeting moieties and adjunct therapies. For example, both diagnostic nanoparticles ( eg, untargeted PEG-coated nanoparticles versus cRGD-PEG) and therapeutic nanoparticles ( eg, iodine-linked cRGD-PEG nanoparticles for radiotherapy, linked to tyrosine inhibitors kinase or linked to chemotherapeutic drugs such as Taxol.).

Materials and methods

[0232] Internalization/absorption studies. Internalization assays and colocalization studies were performed to identify specific uptake pathways.

[0233] Melanoma cells, including human M21 and mouse B16 cells (~2x105 cells/well), were seeded in 8-well chamber slide plates (1.7 cm2/well) or 24-well plates ( 1.9 cm2/well) with a diameter of a rounded coverslip of 12 mm. and incubated at 37°C overnight. To monitor internalization of targeted nanoparticles, cells were incubated with cRGD-PEG dots (0.075 mg/ml) for 3 hours at 37°C. To remove unbound particles in the medium, cells were rinsed twice with PBS. Confocal microscopy was performed on a Leica inverted confocal microscope (Leica TCS SP2 AOBS) equipped with an HCX PL APO: 63x 1.2NA Water DICD objective to assess colocalization of cRGD-PEG dots with organelle-specific stains or antibodies. Images were analyzed using ImageJ software version 1.37 (NIH Image; http://rsbweb.nih.gov/ij/).

[0234] Dye-bound markers/colocalization assays. In order to identify endocytic vesicles involved in C-spot internalization, live cell colocalization assays were performed using dye-bound markers. The cells were coincubated with nanoparticles and different dyes. Stains include: 100 nM Lysotracker Red for 30 min to label acidic organelles along the endosomal pathway; 2 pg/ml transferrin Alexa 488 conjugate to label recycling and sorting endosomes (clathrin-dependent pathway); 1 mg/mL70kDa dextran-FITC conjugate at 37 °C for 30 min to label macropinosomes.

[0235] Co-localization/organelle-specific antibodies. Immunocytochemistry with known Golgi markers and lysosomes will be performed. For Golgi, Giantin (Abcam, rabbit polyclonal, I:2000) for human cells will be used; GM-130 (BD Pharmingen, 1 pg/ml) will be used for mouse cells. For Lysosomes, LC3B (Cell Signaling, rabbit polyclonal, 0.5 pg/ml) will be used.

[0236] For Giantin or LC3B staining, cells will be blocked for 30 minutes in 10% normal goat serum/0.2% BSA in PBS. Incubation of the primary antibody (rabbit anti-Giantin polyclonal antibody (Abcam catalog no. ab24586, 1:2000 dilution) or LC3B (cell signaling, C#2775, 0.5 ug/ml) will be performed for 3 hours, followed by 60 min incubation with biotinylated goat anti-rabbit IgG (Vector labs, cat no: PK6101) at a dilution of 1:200 Detection will be performed with the secondary antibody blocker, blocker D, streptavidin-HRP D (Ventana Medical Systems) and DAB detection kit (Ventana Medical Systems) according to the manufacturer's instructions.

[0237] For GM-130 staining, cells will be blocked for 30 min in Mouse IgG Blocking Reagent (Vector Labs, cat#: MKB-2213) in PBS. Incubation of the primary antibody (monoclonal anti-GM130, from BD Pharmingen; Cat#610822, concentration 1ug/ml) will be performed for 3 hours, followed by 60 minutes of incubation of biotinylated mouse secondary antibody (Vector Labs, MOM Kit BMK- 2202), in a dilution of 1:200. Detection will be performed with Secondary Antibody Blocker, D Blocker, Streptavidin-HRP D (Ventana Medical Systems) and the DAB detection kit (Ventana Medical Systems) according to the manufacturer's instructions.

[0238] For temperature-dependent studies, nanoparticles will be incubated with cRGD-PEG nanoparticles at 4°C, 25°C, and 37°C to assess the fraction of surface-bound versus internalized particles.

[0239] For exocytosis studies, nanoparticles (0.075 pg/ml) will be incubated for 4 hours and chamber slides washed with PBS, followed by addition of fresh media. At time intervals of 0.5, 1.0, 1.5, 2.5, 4.5, 8.0 hours, cells will be washed, stained, and the fluorescence signal of cells and media will be measured by fluorimetry. In dose-response studies, cells will be incubated at a range of concentrations and incubation times, and analyzed by flow cytometry. In viability studies, cell viability will be measured by trypan blue exclusion assay before and after incubation to assess toxicity. In time-lapse studies, the mechanism of nanoparticle internalization into living cells will be investigated after incubating cells with nanoparticle conjugates at different incubation temperatures (4 °C, 25 °C and 37 °C) using an inverted confocal microscope on a period of 12 h at intervals of 20 min.

Discussion

[0240] cRGD-PEG dots and PEG dots were found to co-localize with Lysotracker Red in M21 and B16 cells, suggesting uptake in the endosomal pathway (Figure 22). The data showed that these particles strongly colocalize with transferrin and dextran. Regardless of surface functionality and total charge, the nanoparticles (6-7 nm hydrodynamic diameter) studied seemed to follow the same route. Time-lapse imaging in both cell types demonstrated internalization of functionalized nanoparticles within a small fraction of the plated cells. Finally, the particles were delivered to vesicular structures in the perinuclear region. Colocalization assays with Giantin (or GM-130) are not expected to show fluorescent signal from nanoparticles in the Golgi apparatus.

Example 12 Dual-Modality Silica Nanoparticles for Image-Guided Intraoperative SLN Mapping and Interventions

Dual-Modality Silica Nanoparticles for Image-Guided Intraoperative SLN Mapping

[0241] These studies were expanded to include optical imaging using the portable ArteMIS™ fluorescence camera system, along with radiodetection using the gamma probe, to perform real-time assessments of tumor-draining lymphatics and nodal metastases, as well as assessment of the tumor burden. In a representative minipig (Figures 23a-23i), an initial preoperative PET-CT scan was performed using 18F-FDG and 124I-cRGDY-PEG-C dots using the above imaging procedure. Axial CT images revealed a primary pelvic tumor (Figure 23a) and SLN drainage (Figure 23b), which were seen as areas of increased activity on the corresponding 18F-FDG PET scan (Figures 23c, 23d). These results were confirmed 2 days later by dynamic PET-CT images approximately 5 minutes after 4-quadrant subdermal injection of the particle tracer around the tumor site; Coregistered axial (Figures 23e, 23g) and coronal (arrows, 23f, 23h) views demonstrate these results. After the preoperative scan, the skin overlying the SLN site was marked for intraoperative localization and the minipig was transported to the intraoperative room. Baseline activity measurements, performed on the primary tumor and SLN sites using the portable gamma probe (Figure 23i), showed a 20-fold increase in activity within the SLN relative to background signal.

[0242] To obtain real-time optical imaging of the lymphatic system, a second subdermal injection of 124I-cRGDY-PEG-C dots was administered over the tumor site with intact skin, and the signal was viewed in color (Figure 24a ) and Cy5.5 fluorescent channels (Figure 24b). The adjacent nodal basin was exposed and a fluorescent signal was observed in the NIR channel flowing from the injection site (Figure 24c) into the main proximal (Figures 24c, 24d), middle (Figure 24e) and distal (Figure 24f) lymphatic branches. ), which drained towards the SN (Figure 24f). Smaller caliber lymphatic channels were also visualized (Figures 24d, 24e). The black-pigmented SLN, viewed in dual-channel mode (Figures 24g, 24h), was further exposed (Figures 24i) before successive nodal excision (Figures 24j-24m). The fluorescence signal within the nodal specimen in situ (Figure 24k) and ex vivo (Figure 24m) was confirmed by gamma emissions using the gamma probe (Figure 24i), and was found to correspond to scattered clusters of tumor cells at low power ( inset, Figure 24n) and high power views (Figure 24o) of H&E-stained tissue sections. Positive HMB45 expression was identified in low power (Figure 24p) and high power (Figure 24q) views, consistent with metastatic melanoma.

[0243] Surprisingly, and in contrast to the observed 18F-FDG results, 124I_RGD_PEG_C dots were found to specifically discriminate between metastatic tumor infiltration and inflammatory processes in these minipigs. The mechanistic differences in the behavior of these agents at the cellular and subcellular level, as well as the presence of an integrin-targeting moiety on the particle surface, may explain the observed imaging results. In several minipigs harboring pathologically proven inflammatory changes due to granulomatous disease (n=3), 18F-FDG failed to detect metastatic disease, while identifying inflammatory and metabolically active sites. These conflicting results highlighted the ability of the marker particle to selectively target, localize, and stage metastatic disease, while 18F-FDG failed in many cases to accurately stage the spread of cancer, rather than identify sites of inflammation.

[0244] In a representative mini-pig study illustrating these results, initial 18F-FDG PET-CT axial scans showed calcification within the left posterior neck on CT (Figure 25a), corresponding to an area of intense activity in the left posterior neck. PET scan with 18FFDG (Figure 25b). Low power (Figure 25c) and high power (Figure 25d) views of H&E-stained tissue sections revealed diffuse inflammatory changes, consistent with granulomatous disease. In addition, intense 18F-FDG PET activity was observed within the metabolically active compartment of the bone marrow of these young minipigs (Figures 25a, 25b). In contrast, particle tracer imaging identified bilateral metastatic nodes in the neck. A right neck node on the axial CT image (Figure 25e) was observed to be PET-avid on co-registered PET-CT (Figure 25f); the additional bilateral nodes on a more superior CT image (Figure 25g) were also hypermetabolic on fused PET-CT (Figure 25h). In addition, the left neck calcifications (Figures 25e, 25g) did not show PET activity on co-registered scans (Figure 25f, 25h). The corresponding H&Stained SLN tissue sections revealed dark clumps of melanoma in low power (inset, Figure 25i) and high power (Figure 25j) views, which were seen to be composed of melanoma cells and melanophages. A single frame (Figure 25k) selected from reconstructed 3D PET images again illustrated multiple bilateral PET-avid neck nodes and associated draining lymphatic channels. Importantly, bulk activity was observed in the bladder 1 h after injection without significant accumulation of the marker over the liver region.

[0245] The above results were best seen on PET-CT fusion MIP images generated from dynamic imaging data sets acquired over a 1 hour period after administration of 18F-FDG (Figure 26a) or particle tracer local (Figures 26b, 26c). For 18F-FDG, there is a clear absence of nodal metastases, and a diffuse increase in activity is seen within metabolically active bone structures. In contrast to these results, 124I-cRGDY-PEG-C dots detected bilateral metastatic nodes in the neck, along with draining lymphatic channels.

Dual-modality silica nanoparticles for image-guided interventions: response to treatment.

[0246] The ability of the particle marker to discriminate metastatic disease from inflammatory changes in tissues could potentially be exploited in a variety of therapeutic settings, whether surgical or interventional-driven, as assessments of response to treatment often they are confused by the presence of inflammatory changes, making interpretation difficult. Image-guided interventions, such as therapeutic tumor ablations, may specifically benefit from the innovative coupling of new particle platform and imaging device technologies to (1) enable earlier post-procedure response assessment; (2) verify complete ablation or detect residual tumor representing treatment failure, and (3) improve tumor surveillance strategies. Local ablation therapies, including microwave ablation, cryoablation, radiofrequency ablation (RFA), and interstitial laser therapy, induce local thermal lesions through the insertion of an energy applicator into tumors. These methods are generally employed as alternative options in patients deemed ineligible for surgical excision. Additionally, patients undergoing ablative therapies are often poor surgical candidates due to comorbidities. Widely used in clinical practice, they offer a clear advantage in that they can be performed percutaneously as outpatient procedures with significantly lower morbidity and can improve quality of life and survival in selected patient cohorts.

[0247] Accurate post-therapy images, typically acquired 1-3 months after an ablation procedure, traditionally used contrast-enhanced volumetric imaging, such as CT or MRI. These techniques suffer from a number of drawbacks. First, they are limited to identifying the presence of enhancement or abnormal growth in the size of the tumor area, considered primary indicators of residual tumor or recurrent disease. Diffuse border enhancement around the ablation site on postprocedure evaluations may be related to inflammation and hyperemia in the ablation site and often does not necessarily represent residual tumor. Increasing enhancement, markedly irregular or nodular, is considered suspicious for a tumor. However, these interpretations are controversial, as an ablated area may appear larger than expected for several months after the procedure, and enhancement may also reflect granulation or scar tissue formation.

[0248] Functional methods, such as 18F-FDG PET, have also been used to assess the efficacy and effects of ablative procedures, but may suffer from an inability to accurately discriminate tumor from inflammatory changes. Therefore, interpretation of imaging changes (ie, inflammation, tumor) at the tissue level in Response to ablative procedures using current morphological or functional assessments, particularly at early time intervals, is a significant challenge. What is needed are reliable endpoints for ablation success and unequivocal detection of residual disease in the postablation period.

[0249] As a precursor to how to perform future ablations of metastatic liver lesions, a proof-of-concept radiofrequency ablation (RFA) study of a larger SLN (ie, 1-2 cm) was performed in a minipig. with metastatic melanoma to assess response to treatment in the presence of the particle tracer. The results of PET-CT images before and after RFA were histologically correlated. After subdermal injection of 124I-cRGDY-PEG-C dots (~0.6 mCi) over the left primary pelvic tumor, an initial baseline coronal CT showed an SLN of 2.2 x 1.6 cm (Figure 27a). superior to the tumor site, which was avid PET (Figures 27b, 27c). Also shown is the hypermetabolic left pelvic tumor (Figure 27b), which shows additional particle tracer flow into a draining lymphatic channel on fused PET-CT images (Figure 27c, 27d). Additional serial CT scans were acquired to locate the nodule (Figure 27e) prior to the ablation procedure and guide the insertion of the RFA probe (Figure 27f) into the nodule (below the level of the withers). On the corresponding pre-ablation coregistered PET-CT scan, the PET-avid SLN was seen just posterior to the withers (Figure 27g). Partial node ablation was performed for 12 minutes using a 2 cm active-tip RFA probe (Cool-tip ablation system, Covidien plc, Dublin, Ireland). Post-ablation PETCT showed slightly reduced tracer activity in the ablation zone, anterior to the electrode tip (Figure 27h). Imaging results before and after ablation were confirmed histologically. H&E staining of previously ablated core biopsy tissue from the SLN confirmed diffuse metastatic tumor infiltration in low magnification (Figure 27i) and high magnification (Figure 27j) views. After ablation, the extent of metastatic infiltration was decreased in H&E-stained nodal tissue, seen in the corresponding low (Figure 27k) and high (Figure 27l) magnification views. Coagulative necrosis and lymphoid tissue were also identified, along with multifocal hemorrhages (Figures 27k, 27I, respectively). High-resolution images stained with TUNEL before ablation reveal scattered neoplastic cells (Figure 27m). On post-ablation TUNEL staining, focal areas of necrosis (red) were observed in low (Figure 27n) and high magnification (Figure 27o) views.

conclusions

[0250] Lymph node metastases are a strong predictor of melanoma outcome. Early detection of regional lymph node micrometastases using SLN mapping may allow timely stratification of patients into appropriate treatment arms and potentially improve patient outcomes. Although current standard SLN biopsy and mapping techniques are based on the use of radioactivity-based identification of SLNs, there are several limitations of this technology. These include low spatial resolution, reduced staging accuracy, lack of target specificity, slow tracer clearance that can obscure the surgical field, and lack of accurate intraoperative visualization to avoid injury to vital structures in close proximity to the SLNs.

[0251] The recent introduction of next-generation biocompatible particle platforms that can be actively tailored and refined to overcome these drawbacks according to key design criteria, while enabling selective probing of critical cancer targets, may offer insights. important information on the cellular and molecular processes that govern metastasis. spread of the disease. The further adaptation of such platforms for multimodality imaging could be exploited by the surgeon or interventionalist to explore these processes in a variety of image-guided procedural settings.

[0252] One such dual-modality platform, a clinically-developed translated integrin-targeted silica nanoparticle for both optical and PET imaging, meets a number of key design criteria: small size, superior brightness, improved tumor tissue retention and low background signal - making it an ideal agent for SLN localization and staging during SLN biopsy procedures when combined with real-time handheld optical camera systems. The ability to discriminate metastatic disease from inflammatory tissue changes in melanoma models, which are often coexisting processes, may provide a more accurate and reliable marker for future treatment response assessment. Further research is warranted in a broader set of cancer types and treatments using surgical-based or intervention-driven therapies.

Example 13 SLN mapping of prostate cancer

[0253] Multimodal nanoparticles carrying HuJ591-F(ab')2 fragments are novel diagnostic probes for binding to prostate-specific membrane antigen (PSMA). By synthesizing particles containing multiple F(ab') ² fragments, we can (1) improve binding affinity/potency due to multivalent effects and (2) alter in vivo distributions, as clearance and uptake will be dominated by the kinetic behavior of the particle, rather than the antibody itself. By keeping the particle size below or just at the renal limit of 10 nm in diameter, renal clearance is promoted. In addition, target-to-history ratios will increase based on the improvements in (1) and (2), potentially improving diagnostic specificity and disease staging.

Synthesis/characterization of particles bound to 124I-J591 F(ab')?.

[0254] To generate PEGylated F(ab ^')2 constructs, 100 ug of F(ab ^')2 was added to an eppendorf tube in 100 ml PBS, followed by incubation with 4 ml of 2 mg/ml Traut's reagent. for 1 hour at room temperature. Then maleimide-PEG (6 mg) dissolved in buffer was added. The solution was incubated overnight and purified with a PD-10 column prior to particle binding. F(ab ^')2 fragments are radioactively labeled with 124-iodine (124I) to create a dual-modality particle platform and specific activity, purity, and radiochemical yield will be obtained. ECF will also assess the size and concentration of the particles.

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

[0255] Nanomaterials, particularly nanoparticle probes, possess unique physicochemical and biological properties, as well as surface versatility. Exploiting the versatility of their surface, the biocompatible and multifunctional particles can be selectively engineered with molecular markers that recognize and localize key cancer targets. Increasingly in surgical settings is the need for more robust optically driven multifunctional particle probes that can improve real-time targeted detection of local/regional spread of disease around the primary tumor site, thus facilitating treatment . Real-time delineation of disease from critical neural and/or vascular structures in intraoperative settings will also be paramount. Duncan, R, The dawning era of polymer therapeutics, Nat Rev Drug Discov 2, 347-360 (2003). Wagner, et al., The emerging nanomedicine landscape, Nat Biotechnol 24, 1211-1217 (2006). Scheinberg, et al., Conscripts of the infinite armada: systemic cancer therapy using nanomaterials, Nat Rev Clin Oncol 7, 266-276 (2010). Miyata et al., Polymeric micelles for nano-scale drug delivery, React. Func. Polym. 71, 227-234 (2011). Lee et al., Multifunctional mesoporous silica nanocomposite nanoparticles for theranostic applications, Acc Chem Res 44,893-902 (2011). Schroeder et al., Treating metastatic cancer with nanotechnology, Nat Rev Cancer 12, 39-50 (2012). Rosenholm et al., Nanoparticles in targeted cancer therapy: mesoporous silica nanoparticles entering preclinical development stage, Nanomedicine (Lond) 7, 111-120 (2012). Ashley et al. The targeted delivery of multicomponent charges to cancer cells by nanoporous particle-supported lipid bilayers, Nat Mater 10, 389-397 (2011). Vivero-Escoto et al., Silica-based nanoprobes for biomedical imaging and theranostic applications, Chem Soc Rev 41,2673-2685 (2012). Tada et al., In vivo real-time tracking of single quantum dots conjugated with monoclonal anti-HER2 antibody in tumors of mice, Cancer Res 67, 1138-1144 (2007). However, despite extensive particle developments to date, no inorganic fluorescent particle imaging probe has successively transitioned into the clinic as a specific multifunctional platform technology. Altinoglu et al., Near-infrared emitting fluorophore-doped calcium phosphate nanoparticles for in vivo imaging of human breast cancer, ACS Nano 2, 2075-2084 (2008). Hilderbrand et al., Near-infrared fluorescence: application to in vivo molecular imaging, Curr Opin Chem Biol 14, 71-79 (2010). Choi et al., Design considerations for tumor-targeted nanoparticles, Nat Nanotechnol 5,42-47 (2010). He et al., Near-infrared fluorescent nanoprobes for cancer molecular imaging: status and challenges, Trends Mol Med 16, 574-583 (2010).

[0256] Here we describe that a first -7 nm fluorescence nanoparticle, modified as a hybrid targeting platform (optical/TEP) by binding radiolabels and cyclic arginine-glycine-aspartic-tyrosine acid (cRGDY) peptide, is not only well tolerated in patients with metastatic melanoma, but can preferentially detect and localize integrin-expressing tumors in a microdosing regimen with a high signal-to-noise ratio. No significant binding to serum proteins is observed and the integrity of the particle and its surface components are maintained in vivo. The results on safety, pharmacokinetics, dosimetry, and targeting suggest a general utility of this renally excreted particle in the diagnosis of cancer and to potentially guide treatment planning. This dual-modality probe provides a platform that can be tailored to specific tumor types that may improve PET-based and/or optical lesion detection and cancer staging in humans, as well as drug delivery, potentially leading to to better and more personalized cancer care.

[0257] We used an ultra-small (~7 nm diameter) dual-modality inorganic nanoparticle (optical/TEP) probe for targeted molecular imaging of avp3 integrin-expressing cancers. This nanoparticle probe is the FDA's first investigational new drug of its class and properties approved for first-in-human studies (Figure 28a). Fluorescent silica nanoparticle (Cornell or C-dots) covalently sequesters dye molecules in a core encapsulated by a silica shell to prevent dye leaching and improve brightness and photostability. Ow, et al. Bright and stable core-shell fluorescent silica nanoparticles, Nano Lett 5, 113-117 (2005). Burns et al. Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine, Nano Lett, 9, 442-448 (2009). The coincident absorption spectra demonstrate this per-dye brightness enhancement as intensity differences between the emissions from the encapsulated and free dyes (Figure 28b). A neutral layer of poly(ethylene glycol) (PEG) surface chains allows the binding of a small amount of cyclic arginine-glycine-aspartic acid-tyrosine (cRGDY) peptides for potent and selective integrin receptor binding. Benezra et al. Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma, J Clin Invest, 121, 2768-2780 (2011). Activated integrins induce many structural and signaling changes within the cell, in addition to regulating differentiation pathways, mediating adhesion properties, and promoting cell migration, survival, and cell cycle progression. Hood et al., Role of integrins in cell invasion and migration, Nat Rev Cancer, 2, 91-100 (2002). Binding of nuclear imaging tags, such as 124I, through tyrosine residues amplifies signal sensitivity for serial PET imaging. The final product, a highly biocompatible and biostable tumor-selective particle tracer (121I-cRGDY-PEG-C dots), previously provided a readout of the status of the integrin receptor by PET imaging in human melanoma xenografts, thus defining a distinct class of renally cleared theranostic platforms for nanomedicine. Jokerst et al., Molecular imaging with theranostic nanoparticles, Acc Chem Res, 44, 1050-1060 (2011).

[0258] A first human clinical trial was initiated using PET to quantitatively assess time-dependent tumor uptake and biodistribution, radiation dosimetry, and safety of this agent in a cohort of five patients with metastatic melanoma. Implicit in the rationale for the use of PET imaging is preclinical evidence that the particle radiotracer has no demonstrable pharmacological, radiogenic, or other biological effect. Collins, JM Phase 0 clinical studies in oncology, Clin Pharmacol Ther, 85, 204-207 (2009). Kummar et al., Phase 0 clinical trials: recommendations from the Task Force on Methodology for the Development of Innovative Cancer Therapies, Eur J Cancer, 45,741-746 (2009). Following a single dose intravenous (iv) injection of approximately 185 megabecquerels (MBq) (~3.4-6.7 nanomoles, nmol) of the 124I-cRGDY-PEG particle marker (specific activity range 27.8 - 57 0.4 GBq/pmol) in human subjects (Figure 28a), three whole-body PET-CTs were acquired over a 72-hour period to assess pharmacokinetics, in addition to analyzing blood and urine samples for metabolites over a two-hour interval. weeks by gamma counting and radium thin layer chromatography (radioTLC).

[0259] Five patients had no adverse events and the agent was well tolerated during the study period. The pharmacokinetic behaviour, expressed as the percentage of the injected dose per gram of tissue (%ID/g), versus the time post-injection and the corresponding mean doses absorbed by the organs (Figure 28d), was comparable to that found for other commonly used diagnostic radiotracers. Serial PET images of this representative patient (Figure 28d) showed a progressive loss of putative blood pool activity from major organs and tissues, with no appreciable activity observed 72 hours post injection (pi). Whole body elimination half times in these patients were estimated to range from 13 to 21 hours. Interestingly, there was no notable localization to the liver, spleen, or bone marrow, in contrast to many hydrophobic molecules, proteins, and larger (>10 nm) panicle platforms. Although the patients were previously treated with potassium iodide (K1) to block thyroid tissue uptake, a higher mean absorbed thyroid dose relative to other tissues was obtained in this patient. Particles were also mainly excreted by the kidneys, with both the kidney and bladder wall (after thyroid and tumor, see below), demonstrating one of the highest %DI/g values at 72 hours. pi (Figure 28d); as is often the case with renally excreted radiopharmaceuticals, the bladder wall received a higher mean absorbed dose than other major organs and tissues. The detailed clinical protocol (Fig. 28C) and the rationale for its design are described in more detail in Materials and Methods.

[0260] These results highlight the fact that renal, rather than hepatobiliary, excretion is the predominant route of elimination from the body. Efficient renal clearance will depend on the design of platforms based on ultrasmall particles or macromolecular systems that are on the order of effective renal glomerular filtration size limits of approximately 10 nm or less. Choi, et al., Targeting kidney mesangium by nanoparticles of defined size, Proc Natl Acad Sci USA, 108, 6656-6661 (2011). A small fraction of the administered activity (less than 5%) was observed as uptake in the stomach and salivary glands of this patient, consistent with free iodine, which was progressively removed over the imaging period. The particle does not display the typical properties of reticuloendothelial agents (ie technetium-99m sulfur colloid), the uptake of which reflects macrophage function, primarily in the liver. Based on previous data acquired for the cRGD radiotracers, no unexpected foci of activity were observed. Haubner, R. et al. Synthesis and biological evaluation of a (99m)Tc-labelled cyclic RGD peptide for imaging the alphavbeta3 expression, Nuklearmedizin, 43, 26-32 (2004).

[0261] Importantly, these properties point to the rather unique pharmacokinetic behavior exhibited by this dual-modality imaging inorganic particle as a renally cleared agent. Metabolic analyzes of blood (Figure 29a) and urine (Figure 29b) specimens by gamma counting revealed a drop of at least one order of magnitude in tracer activity over a 72-hour period, with virtually no activity remaining at the end of this period. interval. Particle activity was largely limited to the fraction of blood plasma without evidence of significant binding to serum proteins (data not shown). RadioTLC analyzes of the plasma samples revealed a single peak over 24 h pi (Figures 29c-29e), corresponding to the intact radiolabeled nanoparticle. In urine samples, two peaks, one corresponding to the intact nanoparticle and the other to a more mobile species (identified as free iodine), were observed over a period of 24 hours (Figures 29f-29h). RadioTLC analyzes of the particle tracer (Figure 29i), radioiodinated peptide (131I-cRGDY, Figure 29j), and free radioactive iodine (131I, Figure 29k) confirmed that the first and second peaks in the radiochromatograms corresponded to the intact nanoparticle. and free iodine, respectively. Therefore, these results suggested that no measurable loss of particle integrity occurred over the course of the study, even after excretion via the kidneys.

[0262] Although tumor detection was not the goal of this PET microdosing study, surprisingly, despite the low injected dose, there was evidence of tumor localization of the particle tracer. In the first case, a whole-body PET-CT was acquired four hours after iv administration of 124I-cRGDY-PEG-C-dots in a patient with anorectal mucosal melanoma. On coronal CT images (Figure 30a), a large rounded area of decreased density (arrowhead) was seen in the left lower lobe of the liver, the site of a known metastatic lesion from previous fluorodeoxyglucose (18F-FDG) PET/CT imaging (data not shown). On coronal PET (Figure 30b) and co-recorded PET and CT scans (Figure 30c), an edge of increased uptake circumscribed this lesion (Figure 30b arrowhead; Figure 30c) and subsequently cleared at the time of the scan. 24-hour PET scan, suggesting some preferential localization in this putative integrin-expressing metastasis. Significant activity was seen within the bladder, gastrointestinal tract (stomach, intestines), heart, and gallbladder. In a second subject, a well-defined cystic lesion was observed in the right anterior lobe of the pituitary gland by axial (Figure 31 a) and sagittal (Figure 31 b) magnetic resonance imaging (MRI).

[0263] This lesion, a stable finding on previous MRI scans, was presumed to be a pituitary microadenoma, an intracranial neoplasm known to exhibit malignant properties, such as neoangiogenesis and progression to peritumoral tissues. Accurate co-registration of this tracer-avid focus with multiplanar MRI (Figures 31c, 31d) and CT (Figure 31e, 31f) images confirmed its location within the anterior pituitary gland. Initially seen as a focus of intense activity, it progressively increased in intensity over a 72-h interval (arrow, Figures 31g-31i), accompanied by a corresponding decrease in surrounding background medulla signal, resulting in higher ratios from tumor to background (ie, from tumor to brain (T/B) - 6) and from tumor to liver (T/L) - 2)) (Figure 31j). The PET imaging results can be explained on the basis of the results of a previous study that showed increased avp3 integrin expression in the parenchyma of a subset of adenomas, as well as enhanced integrin expression levels in adenomatous stromal cells relative to normal connective tissue cells. Farnoud, et al., Adenomatous transformation of the human anterior pituitary is associated with alterations in integrin expression, Int J Cancer, 67, 45-53 (1996).

[0264] Our initial data support the idea that human PET studies with this targeted imaging vehicle are a rational approach toward detection and localization of suspected integrin-expressing tumors. We are planning dose escalation methods to determine an optimal balance between safety, whole-body clearance, and tumor detection efficiency in more advanced clinical trials. Such PET-directed studies may also allow for the precise quantification of integrin receptor expression levels to achieve maximum targeting efficiency, as well as the detection of alterations in these levels. Furthermore, the use of these quantitative molecular imaging tools can provide insight into time-dependent changes in particle uptake and accumulation within tumors. Kelloff, GJ, et al., The progress and promise of molecular imaging probes in oncologic drug development, Clin Cancer Res, 11, 7967-7985 (2005). For the case of the pituitary adenoma, we were able to calculate the cumulative uptake of particles within this lesion. Specifically, we calculated the fraction of total injected activity and the number of particles that accumulated at this site over a 72-h imaging period. Using the maximum measured standardized uptake value (SUVmax, see Methods) of the lesion (ie, 46.5) at 72 hours post-injection (almost a factor often higher than that of normal pituitary tissue), corrected for effects of partial volume, as well as the approximate mass of the lesion (i.e., the product of the volume of the lesion and an assumed density of 1 g/cm3) and the patient's body mass, we found that, relative to a particle load injection of 2 x 1015, approximately 1.78 x 10” particles (0.01% of the injected dose, %ID) or 1 part per 10,000 accumulated at the lesion site. Standard Absorption Values (SUVs) are defined as the activity per gram of tissue divided by the activity delivered per gram of body mass. Time-dependent changes in %ID/g and dosimetry for this lesion, relative to major organs and tissues, are shown in Figures 28c and 28d.

[0265] The results suggest that the systemically injected particle tracer was well tolerated and safe. Safety measures included control of uptake in normal organs, as well as laboratory indicators of toxicity. Secondary objectives were to estimate radiation doses and to assess the metabolic activity of plasma and urine. Safety assessments were based on dosimetry, lack of clinical symptoms, and absence of laboratory indications of particulate (drug) toxicity.

[0266] The results obtained from this first human clinical trial point to a systemically injected particle tracer that exhibited favorable and reproducible PK signatures defined by renal excretion. Unlike reticuloendothelial agents (ie, technetium-99m sulfur colloid) and macromolecules such as antibodies, there was no appreciable accumulation of particle tracers in the liver, spleen, or bone marrow. Our data clearly indicate that a large proportion of the administered activity was eliminated via the urinary system (Figs. 28E, 29B); activity concentrations in the urinary bladder were up to an order of magnitude higher than those in the liver, for example. Based on the conservative assumption that all hepatic activity is ultimately excreted via the hepatobiliary route, these data are consistent with ~90% of administered activity being excreted via the urinary system and only ~10% via the urinary system. the hepatobiliary route. In the remaining organs/tissues, especially at early time points (2-4 hours), the residual activity largely reflects that of the blood pool.

[0267] Targeted detection did not serve as the study endpoint, and therefore dose escalation procedures to achieve maximum uptake at disease sites were not incorporated into the trial design (i.e., no attempted to adjust particle doses to optimize tumor targeting). However, despite the low nanomolar amounts used, preferential localization and accumulation of the particle tracer occurred in various tumors. In one of the patients with a suspected pituitary adenoma, PET imaging results showed a progressive net accumulation of particle tracer activity at the lesion site. The results of this study suggest that our systemically injected particles are well tolerated and exhibit a signature distinctively unique "maciomolecular" pharmacokinetics, where bulk renal clearance predominates without significant RES uptake. This is in contrast to many hydrophobic molecules, proteins, and larger particle platforms (>10 nm). This feature is highly atypical for nanoparticles (which generally exhibit diameters larger than estimated renal cut-off values and thus poor renal excretion and slower clearance) and warrants clinical evaluation of our ultrasmall nanoparticle platform.

[0268] These results, together with essential data on safety, pharmacokinetics, and dosimetry from the dual-modality C-spot imaging platform, suggest the general utility of this human microdosing technique in terms of producing key tumor-specific readouts. for the diagnosis of cancer. Such estimated burdens (or concentrations) of accumulated particle tracers in the tumor, together with knowledge of the cellular inhibitory response (i.e., IC ⁵⁰ or 50% inhibitory concentration), can potentially be used to predict therapeutic dosage requirements. for a given drug.

methods

[0269] Synthesis and characterization of cRGDY-PEG-C spots. For details on the synthesis and characterization of PEGylated and cRGDY (Peptides International, Louisville KY) surface-functionalized fluorescent core and shell silica nanoparticles (cRGDY-PEG-C spots) encapsulating the organic dye, Cy5 (maximal emission ~ 650 nm, 2 dye equivalents within the particle core), see a previous publication in this group and the references it contains. Benezra, M., et al., Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma, J Clin Invest, 121,2768-2780 (2011). Briefly, the particles were prepared by a modified Stober-type silica condensation. Bogush, et al., Preparation of Monodisperse Silica Particles: Control of Size and Mass Fraction, J Non-Cryst Solids, 104, 95-106 (1988). Herz, et al., Large stokes-shift fluorescent silica nanoparticles with enhanced emission over free dye for single excitation multiplexing, Macromol Rapid Commun, 30, 1907-1910 (2009). Sadasivan, et al., Alcoholic solvent effect on silica synthesis-NMR and DLS investigation, J Sol-Gel Sci Technol, 12, 5-14 (1998). Bifunctional PEGs were derivatized with silanes for binding to the silica surface and for peptide coupling. cRGDY peptides containing the cyclo-(Arg-Gly-AspTyr) sequence and carrying cysteine residues (Peptide International) were linked to functionalized PEG chains via a cysteine-maleimide bond. Hydrodynamic radius, brightness, and concentrations of cRGDY-PEG-Cdots, compared to free Cy5 dye, were analyzed on a Zeiss LSM 510 Confocor 2 ECF using 633 nm HeNe excitation.

[0270] Radiolabeling of cRGDY-PEG-C points. Tyrosine residues were conjugated to PEG chains for attachment of radioactive iodine moieties (ie 124I, 131I). Hermanson, G, Bioconjugate Techniques, (Academic Press, London, UK, 2008). Yoon, TJ, et al. Specific targeting, cell sorting, and bioimaging with smart magnetic silica core-shell nanomaterials. Small, 2, 209-215 (2006). Radiolabeling of cRGDY-PEG-C-dots was performed using the IODOGEN method (Pierce). Piatyszek, et al., Mud-Gen-mediated radioiodination of nucleic acids, Anal Biochem, 172, 356-359 (1988). Radiolabeled product was eluted from PD-10 columns and analyzed using a dose calibrator (Capintec, Ramsey NJ) and radioTLC; the specific activities of the particulate fractions bound to 124l were of the order of 1450 millicuries (mCi)/mmol and the radiochemical purity was greater than 95%.

[0271] Selection of patients. Subjects with metastatic melanoma with histological confirmation of the disease and harboring a newly diagnosed or recurrent tumor were eligible for the trial. People who had a medical condition unrelated to melanoma, which would preclude administration of the particle marker, were excluded from the study. Potassium iodide solution (SSK1, 130 mg per day) was administered 2 days before and up to 2 weeks after intravenous injection of radioiodinated particle tracer (124I-cRGDY-PEG-C-dots, 185 MBq/5 mL) to block the thyroid function. This protocol was approved by the Institutional Review Board of Memorial Sloan Kettering Cancer Center. Patients underwent haematological, renal, and hepatic function tests before and after PET, and signed informed consent.

[0272] Image acquisition and processing. Low-dose spiral CT scans were obtained by standard procedure, followed by the acquisition of three whole-body PET scans on a dedicated GE Discovery STE PET/CT scanner 4, 24, and 72 hours after particle tracer injection. Positron emission data were reconstructed using the ordered subset expectations maximization (OSEM) algorithm. Images were corrected for attenuation using CT transmission data collected in the same region as for emission images, and serial data set registration was performed using CT data sets.

[0273] Imaging and metabolic analysis. For pharmacokinetic and dosimetric analyses, regions of interest (ROI) were drawn on PET imaging data (AW Workstation, GE Healthcare, Ridgewood, NJ) to extract mean and maximum standard uptake (SUV) values for all major organs and normal tissues, including brain, lung, left ventricle, liver, spleen, intestine, kidney, bladder, muscle, breast, and tumor(s). For pharmacokinetic evaluation, SUVs were converted to %ID/g values (ie, SUV = %1 D/g tissue x patient body mass/100). Organ/tissue uptake data were supplemented with blood and urine time-activity data. Urine and venous blood samples were collected at approximately 30 min, 3 h, 24 h, 72 h, and up to 2 weeks pi of 124I-cRGDY-PEG-C data. After centrifugation of the whole blood samples (4000 rpm, 10 min), the plasma supernatant, together with the urine samples, were analyzed in a scintillation well counter. (1480 Automatic Gamma Counter, Perkin Elmer, Shelton TC) calibrated for 124I. RadioTLC analyzes were additionally performed on biological samples. RadioTLC analyzes of the particle tracer, 131I-labeled native peptide (cRGDY), and free iodine (131I) served as controls to facilitate interpretation. Activities (counts per minute, cpm) were converted to microcuries, decay corrected, and adjusted for aliquoted volumes. Final values were expressed as %DI/g. Retention factor (Rf) values for the tracer were obtained and used to identify the parent compound and potential metabolites.

[0274] Radiation dosimetry. The standard radiation dosimetry method is an adaptation of the one promulgated by the MIRD Committee (Medical Internal Radionuclide Dosimetry), which takes into account the physical properties of the radionuclides (124I) administered, as well as the biological properties (pharmacokinetics and biodistribution) of the radiopharmaceutical. in individual patients. The 124I emissions and their respective frequencies and energies are obtained from the MIRD Radionuclide Data and Decay Scheme publications. Serial whole-body PET scans allowed the derivation of estimates of absorbed dose to normal organs (rad and rad/mCi) using ROI-derived time-activity data. PET scans were acquired with all parameters identical, including scan time. Using the patient's total body mass (in kg) and standard 70-kg male organ masses, the whole-body and organ ROI data (i.e., standard mean uptake (SUV) values) were converted into activities (ie, fraction of the injected dose). The time-activity data derived from previous images were fitted to exponential functions using a least-squares fitting algorithm and the resulting time-activity functions analytically integrated, incorporating the effect of the physical decay of 124I to produce the cumulative activities (or residence times) in pCi-hr/pCi in organs and total body. were used to calculate mean absorbed doses to organs (rad/mCi) and effective dose (rem/mCi) from 124I-labeled particles for the 70 kg Standard Man anatomical model using the OLINDA EXM MIRD program. Loevinger et al., MIRD Primer for Absorbed Pose Calculations, Society of Nuclear Medicine, New York, NY, 1991.

[0275] Serum protein binding assays. Whole blood samples were collected in serum separator tubes from a metastatic melanoma minipig model (Sinclair Research Center, MO), followed by centrifugation (4000 rpm, 10 min) to isolate the plasma fraction. An aliquot of serum was reserved for gamma counting; the remaining traction was treated with ethanol (200 test, Decon Labs, King of Prussia, PA), shaken until cloudy, and placed on dry ice (5 min) to promote precipitation of serum proteins. After centrifugation, the supernatant for gamma counting was collected and the pellet was repeatedly washed with phosphate-buffered saline and centrifuged (4000 rpm, 10 min) to collect 100 pL aliquots of supernatant for gamma counting (1480 Wizard 3").

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

[0276] The peptide N-Ac-Cys-(Ahx) ² -D-Lys-ReCCMSH (or alpha MSH) used for nanoparticle conjugation has the structure shown in Fig. 32. It binds to the nanoparticle at through the N-terminal cysteine thiol. A spacer of two aminohexanoic acid units separates the nanoparticle attachment point from the targeting molecule D-Lys-ReCCMSH. The original ReCCMSH targeting molecule is shown in Fig. 33. It was designed to target radionuclides to melanoma tumors for imaging and therapy. The MSH peptide analogue could be radiolabeled directly with 99mTc or 188Re (at the Re site) or through a metal chelator attached to the amino terminus.

[0277] The alpha MSH peptide analog shown in Fig. 32 is quite different from the original MSH analog shown in Fig. 33. The original MSH molecule could not bind to a nanoparticle. The nanoparticle MSH peptide contains a free amino group containing a side chain for radiolabeling. This is actually a D-lysine, but could be a side chain terminated with an amino group from 1 to many carbons in length.

[0278] Conjugation of N-Ac-Cys-(Abx) ² -D-Lys-ReCCMSH (or alpha-MSH) to the nanoparticle allows the nanoparticle to target and bind to melanoma and tumor cells. The resulting particles, or alpha-MSH-PEG-Cy5-C spots, were approximately 6-7 nm in diameter using ECF and contained, on average, approximately 2.6 dyes per panicle. The number of alpha-MSH ligands per particle was estimated to be <10.

[0279] Competitive binding studies using N-Ac-Cys-(Ahx) ² -D-Lys-ReCCMSH (or alpha-MSH) conjugated particles: In a competitive binding assay with a melanocortin-1 receptor agonist ( 125I-NDP), alpha MSH-conjugated nanoparticles had an IC ⁵⁰ for cultured B16/F1 melanoma cells of 6.6x10 -10 M (Fig. 34A), while a coded sequence version of the molecule had an IC ⁵⁰ of 2.3x10-7 M (Fig. 34B). In addition, there was a 3-order-of-magnitude difference in binding. As a reference in the same type of competitive binding assay, the original DOTA-ReCCMSH had an IC ⁵⁰ of 2.1x10 -9 M.

[0280] The N-Ac-Cys-(Ahx) ² -D-Lys-ReCCMSH (or alpha-MSH) peptide-linked nanoparticle conjugate had better affinity for B16/F1 cells than the original DOTA-ReCCMSH and much higher affinity than mixed peptide nanoparticles.

[0281] Dose-response data were additionally obtained as a function of particle concentrations target (Fig. 35A) and incubation times (Fig. 35B) for the B16F10 and M21 melanoma cell lines. Saturation concentrations for these lines, based on flow cytometry studies, were found to be on the order of ~100 nM for these two cell types, and incubation times of at least 2 hours were needed to maximize binding.

[0282] Survival studies of human M21 cells, performed at a range of panicle concentrations during a fixed incubation time of 48 hours, did not demonstrate significant loss of cell viability (Fig. 36).

[0283] 125 I-radiolabeled alpha-MSH-conjugated nanoparticles demonstrated bulk renal excretion over a 24-h period in murine xenograft models B16F10 and M21 (Figs. 37A, 37B); no appreciable particle tracers were measured at later time points (ie, >24 hours). Furthermore, neither the B16F10 nor the M21 xenograft models showed significant accumulation of the target particle probe in the reticuloendothelial system (ie, not a RES agent), nor in the kidney (Figs. 38A, 38B), the last organ typically a site that accumulates alpha-MSH not specifically given its net positive charge. Therefore, the binding of alpha-MSH to the particle probe significantly improved its renal clearance properties and eliminated accumulation within the kidneys.

Example 16 Integrin-mediated signaling and modulation of tumor biology

[0284] Integrin signaling regulates various functions in tumor cells, including adhesion/spreading, migration, invasion, proliferation, and survival. In several types of tumors, including melanoma, the expression of particular integrins is correlated with increased disease progression and decreased patient survival. Integrin-mediated adhesive interactions have identified new roles for integrin in cell survival mechanisms and in the activation of divergent signaling pathways. It is well known that binding of peptides (or groups of peptides), containing the RGD sequence, to integrin receptors leads to integrin cross-linking or clustering which, in turn, modulates the above processes. However, it is unclear whether nanoparticles containing multiple RGD peptide ligands can additionally trigger such integrin signaling events, as this will reflect a complex dependence on multiple particle-based factors (size, charge, composition, surface chemistry, number/ ligand type), tumor (or endothelial) cell type, cell receptor density, and particle dosage. Our dose-response studies demonstrate modest activation of divergent signaling pathways upon cell exposure to particle concentrations greater than 100 nM which, in turn, promote M21 and HUVEC cell migration, proliferative activity, and alter cell properties. accession.

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

[0285] The avp3 integrin receptor plays a key role in vascular remodeling and angiogenesis, demonstrating increased expression on endothelial and tumor cell surfaces for a variety of tumor cell lines. This leads to the use of this receptor as a target for diagnostic and therapeutic purposes. In our case, cRGDY-PEG-C dots were tested for their ability to bind to melanoma cells (M21) or to human umbilical vascular endothelial cells (HUVECs) as a function of concentration and time. Initial dose-response studies showed a progressive increase in cRGDY-PEG-C dot binding to M21 and HUVEC cells as a function of concentration by flow cytometry (Figure 39, Figure 47A). Particle binding demonstrated saturation at around 100 nM for both cell lines, with mean % dependent values of around 80% for M2I cells and 96% for HUVEC cells. Dose response behavior was further investigated as a function of particle incubation times for both cell types after incubation with 100 nM cRGDY-PEG-C dots. Maximum binding was observed 2 hours after incubation, remaining relatively constant thereafter (Figure 47B).

Endocytosis and intracellular trafficking of cRGDY-PEG-C dots

[0286] To elucidate the nature of the pathway(s) used by cRGDY-PEG-C dots after their incubation in M21 cells, whether it is an avp3 integrin receptor-mediated process and/or of non-specific absorption, we examined the temperature-dependent absorption of these particles during an incubation time of 4 hours at three temperatures: 4°, 25°C and 37°C. The results, summarized in Fig. 39A,C, indicate an increase in cell-associated cRGDY-PEG-C spots at 37°C compared to 25°C or 4°C in both cell lines tested. Furthermore, the internalization of cRGDY-PEG-C dots was partially blocked in the presence of excess (x250) of antibodies against the avp3 receptor in M21 and HUVEC cells (Figures 39A, 39C), suggesting a receptor-mediated binding component. In addition, uptake in avp3-negative M21L cells was approximately a factor of 4 to 8 fold lower than that observed with avp3-expressing cells at 37°C and 25°C (Figure 39B), respectively.

[0287] To further characterize the cellular compartments involved in the internalization of cRGDY-PEG-C dots, we performed colocalization assays in M21 cells with cRGDY-PEG-C dots and biomarkers of different endocytic vesicles. Internalization of the target particle (~1 mM, red, 4 hour incubation) was sensitively detected using an inverted confocal microscope (Leica TCS SP2 AOBS) equipped with an HCX PL APO objective (633 1.2NA Water DIC D) (Figure 39D). Using the endocytic markers LysoTracker Red (100 nM, green) (Figure 39D) and transferrin-Alexa488, uptake into acidic endocytic structures was confirmed, suggesting clathrin-dependent pathway activity and gradual acidification of the vesicles. Fig. 39D shows the colocalization data between the particle and the acidic endocytic vesicles (yellow dots). Uptake was also observed in macropinocytes with 70 kDa dextran-FITC that co-localized with cRGDY-PEG-C dots; this result suggested a second pathway of internalization. Nuclear counterstaining (blue) was performed with Hoechst. No particle entered the nucleus. Time-lapse imaging confirms particle uptake in M21 cells and colocalization with the lysosomal marker, Lamp1.

Competitive avP3 integrin receptor binding and molecular specificity

[0288] Competitive binding assays showed blockage of receptor-mediated particle binding in M21 and HUVEC cells (Figure 48A) by 80-85% in the former and 30-40% in the latter using excess (350 3100) of cRGDY peptide and gamma count of radiolabeled particle tracer (spots 1241-PEG-cRGDY-C). In contrast to cRGDY-PEG-C spots, significant reductions in the magnitude of receptor binding were observed in M21 cells (~30-43%) and HUVEC (~13-27%) after incubation with untargeted cells (ie, PEG-C spots) particle probes by flow cytometry (Figure 48B).

Influence of cRGDY-PEG-C dots on cell viability and proliferation

[0289] To demonstrate that cRGDY-PEG-C dots did not adversely affect cell survival and proliferation, G0/G1 phase-gated M21 cells and HUVECs were exposed to a range of particle concentrations (25-200 nM cRGDY dots). -PEG-C; 15 min or 24 h) and incubation times (0 - 93 h) in serum-supplemented media (2%, 10% FBS) at 37 °C, and time-dependent changes in absorbance were measured using a optical plate reader (A = 440 nm). Relative to controls (ie, serum-supplemented media), absorbance measurements were observed to be relatively constant over the range of particle concentrations tested, suggesting that there was no significant loss of cell survival (Figures 49A, 49B). ). Furthermore, no time-dependent decreases in absorbance were found after the addition of multiple doses (n = 5) of 100 nM cRGDY-PEG-C dots to M21 and HUVEC cells, suggesting no alteration in proliferative properties. of the cells (Figures 49C, 49D).

Activation of the FAK pathway by cRGDY-PEG-C points

[0290] Binding of a ligand to the avp3 integrin receptor is known to initiate signal transduction pathways. Upon binding, integrin clustering occurs leading to autophosphorylation of the non-receptor FAK kinase at tyrosine 397, a binding site for Src-family kinases. Recruitment of Src kinase results in phosphorylation of FAK at tyrosine 576/577 and FAK at tyrosine 925 in the carboxyl-terminal region. FAK phosphorylation at tyrosine 397 is also known to activate numerous signaling cascades and downstream pathway intermediates, such as Ras mitogen-activated protein (MAPK) signaling, which induces Raf/MEK/Erk activation and Phosphatidylinositol 3-kinase (PI3K) /AKT pathways (AKT, mTOR, S6K) (Figure 40A).

[0291] To determine whether avp3 integrin-binding cRGDY-PEG-C dots modulate the activity of these pathways, we treated serum-starved M21 cells (0.2% ECF) with 100 nM of cRGDY-PEG-C dots for 2 hours at 37°C; serum-starved cells treated with 0.2% ECF only served as controls. Western blot analysis of particle-exposed cell lysates revealed enhanced phosphorylation levels of multiple intermediate proteins: pFAK-397 and pFAK-576/577, Src, pMEK, pErk, and AkT (Figure 40B), suggesting activation of downstream signaling pathways. These results, plotted as normalized intensity ratios, were expressed relative to their respective total protein levels and normalized to the corresponding values measured under control conditions (Figure 40C). Incubation of cells with PEGC dots did not increase the phosphorylation levels of the proteins tested (data not shown).

[0292] We evaluated whether the observed activation of downstream signaling pathways was dependent on FAK phosphorylation at tyrosine 397 by blocking this pathway with a small molecule inhibitor, PF-573228. This inhibitor interacts with FAK at the ATP-binding pocket and effectively blocks both the catalytic activity of the FAK protein and the subsequent phosphorylation of FAK on Tyr397. After the addition of two concentrations of PF-573228 to serum-starved M21 cells previously exposed to 100 nM cRGDY-PEG-C spots, Western blot analyzes revealed inhibition of FAK phosphorylation at Tyr397 and Src (Figure 41A). , which is shown graphically in Figure 41B. Inhibition of MEK or Erk phosphorylation was observed only at the highest inhibitor concentration (Figures 41A, 41B).

Effect of cRGDY-PEG-C dots on cell migration

[0293] The avp3 integrin receptor, which is highly expressed on many types of tumor and angiogenic cells, is known to modulate a number of downstream biological processes, including cell migration, adhesion, proliferation, invasion, and angiogenesis. In the following experiments, we sought to determine whether cRGDY-PEG-C dots alter the migration of M21 and HUVEC cells. An initial set of experiments examined time-dependent changes in M21 cell migration, as reflected in mean closure areas, using time-lapse imaging at three successively higher particle concentrations (0 nM - 400 nM; 37 °C) for a period of 96 hours. period of time (Figure 42A, i-xx). Statistically significant increases in mid-area closure were observed over a 96-hour period, as a percentage of baseline values (t=0), which were relatively constant for the particle concentration range used (i.e., ~87% - 92%). compared to control samples (73%; p<0.05) (Figure 42A, B). No significant changes were observed at earlier time intervals (Figure 42B).

[0294] Incubation of HUVEC cells (37°C, 20 hours) with a range of cRGDY-PEG-C spot concentrations of (100 - 400 nM) in the presence of 0.2% ECF showed a statistically significant increase in mid-area closure for a concentration of 400 nM (ie, 34%) (Figures 43A, 43B), compared to 19% (p<0.05) for cells not exposed to particles. No appreciable change in migration was observed for the lowest particle concentrations used (Figures 43A, 43B). Cells exposed to particles were observed to exhibit higher migration rates compared to control cells.

[0295] We further show that increases in cell migration rates were attenuated by the addition of the FAK inhibitor, PF-573228. Initial phase contrast images were acquired after incubating HUVEC cells with 400 nM particles for a 24-h time interval (Figure 44A, i-viii), and median area closure was determined and graphically displayed relative to serum alone (Figure 44B). The percentage change in mean areas of closure relative to controls, and before and after inhibitor addition, was observed to decrease from 34% to 3%. Statistically significant differences were found between the values of cells exposed to particles without inhibitor (p<0.03) and particles treated with different concentrations of inhibitor (250 nM, 500 nM; p<0.001) in relation to controls deprived of inhibitor. serum; the differences between the first two groups were also statistically significant (Figure 44C).

Effect of cRGDY-PEG-C dots on cell adhesion and spread

[0296] The RGD tripeptide is known to be a component of the extracellular matrix constituents fibronectin and vitronectin. A competitive binding assay was performed using M21 cells (104 cells/well) preincubated (25°C, 30 minutes) with or without 400 nM cRGDY-PEG-C spots and fibronectin, after transferring the cells to fibronectin-coated wells. Approximately ~60% of cells not previously incubated with cRGDY-PEG-C dots adhered and spread onto fibronectin-coated plates within the first 30 min. In contrast, a very low percentage (15%) of M21 cells preincubated with 400 nM cRGDY-PEG-C dots adhered and spread over the fibronectin-coated well, even 120 min after plating (Figures 45A, 45B). Average cell counts of 'elongated' cells (spreading cells) vs. 'rounded' cells over a 2 hour period revealed that, in the absence of particles (i.e., control condition), cell spread (cells 'spreading') was observed. elongated') in most cells, whereas a predominantly "rounded" appearance was observed in the case of cells exposed to particles (Figure 45A). These results are graphically represented in Figures 45B and 45C, respectively. Quantification of the optical densities of the cells after the addition of methylene blue (absorbance) revealed that cells that attached and were spread on fibronectin (ie, without particle preincubation) absorbed twice as much methylene blue as cells. cells exposed to particles (Figure 45D). We observed the same phenomena if vitronectin-coated wells were used (data not shown). Taken together, these data demonstrate that cRGDY-PEG-C dots enhanced cell migration and spread through binding to the avp3 integrin receptor found on M21 and HUVEC cells.

Influence of cRGDY-PEG-C points on the cell cycle in M21 cells

[0297] Since integrin receptors are involved in cell survival and proliferation, we analyzed the effect of cRGDY-PEG-C dots on the cell cycle. G0/G1 synchronized M21 cells were incubated for 48 hours using two concentrations of cRGDY-PEG-C spots (100 nM, 300 nM) in medium supplemented with 0.2% ECF. Over this range, the percentage of cells in S phase increased by 11%, with statistical significance reached at 100 nM (p<0.05) and 300 nM (p<0.005) relative to controls (Figures 46A, 46B). Corresponding decreases of 6% and 5% were observed in the G1 and G2 phases of the cell cycle, respectively. Taken together, these data indicate an enhancement in S phase in the presence of cRGDY-PEGC dots.

MATERIALS AND METHODS

[0298] Reagents, Antibodies and Chemical Products. RPM1 1640, fetal bovine serum (FBS), penicillin, streptomycin, and HBSS (without calcium and magnesium containing 0.25% trypsin and 0.05% EDTA) were obtained from the Core Media Preparation Facility, Memorial Sloan Kettering Cancer Center ( New York, NY). Anti-polyclonal rabbit: phospho-Erk1/2 (pErk1/2Thr202/TyR204), phospho-AKT (p-AktSeR473), phospho-Src (p-SrcTyR419), phospho-p70 S6 (p-p70S6ThR389), phospho-MEK1 Were obtained from Technology cell signaling (Danvers, MA). Goat anti-rabbit IgG and goat anti-mouse horseradish peroxidase (HRP) conjugates were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Propidium iodide, RNase A, Transferrin-Alexa Fluor 488 conjugate, FITC-Dextran, Fluorescein, LysoTracker Red DND-99, LysoTracker Green DND-26, pHrodo Red Dextran, and Hoechst were obtained from Invitrogen-Life Technologies ( Carlsbad, CA). PF-573228 was obtained from TOCRIS bioscience (Ellisville, MO). Cyclo (Arg-Gly-Asp-D-TyrCys) was from Peptides International (Louisville, KY).

[0299] Synthesis of cRGDY-PEG-C dots and PEG dots . Fluorescent particles, containing the organic dye Cy5, were prepared by a modified Stober-type silica condensation, as described above. Tyrosine residues were conjugated to PEG chains for radioactive iodine binding. cRGDY peptides containing the cyclo-(Arg-Gly-Asp-Tyr) sequence and carrying cysteine residues (Peptide International) were linked to functionalized PEG chains through a cysteine-maleimide bond. The number of cRGD ligands per nanoparticle was calculated empirically.

[0300] Mechanism of binding of PEG to the surface of point C. Bifunctional PEGs, the MAL-dPEG®12-NHS ester (Quanta Biodesigns, Ltd) were derivatized with silanes, specifically 3-aminopropyltriethoxysilane (Gelest), to bind them to the silica surface and for peptide coupling via reactions between the sulfhydryl groups and the maleimide residues of the derivatized PEGs. In addition, methoxy-capped PEG chains were added to the particle surface using functional organosilicon compounds (Si compounds), specifically (MeO)3Si-PEG (Gelest), according to modified protocols. Briefly, (MeO)3SiPEG, in an approximately three molar excess, was added to the particles in a basic water/alcohol mixture (~1:5 v/v), and the mixture was stirred overnight at room temperature.

[0301] Comparison measurements of hydrodynamic size and relative brightness by fluorescence correlation spectroscopy (ECF). The hydrodynamic radius, brightness, and concentrations of cRGDY-PEG and PEG spots, compared to free Cy5 dye, were initially determined by dialysing these particle samples in water, diluting them in physiological saline (0.15 M NaCl in H ² O) and analyzing resulting specimens on a Zeiss LSM 510 Confocor 2 ECF using 633 nm HeNe excitation. The instrument was calibrated with respect to particle size before all measurements. Average hydrodynamic sizes of dye species and particles were estimated based on diffusion time differences, while relative differences in brightness were assessed using per molecule/particle count rates.

[0302] Cells and cell culture: The human melanoma cell line M21 and the M21 variant M21L (av negative) were obtained from DA Cheresh (University of California, San Diego, CA). Cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine and penicillin streptomycin. Human umbilical vein endothelial cells (HU-VEC) were obtained from LONZA (Walkersville, MD) cultured in EGM-2 medium containing 2% FBS and LONZA growth factors.

[0303] In vitro cell binding studies using optical detection methods: To assay particle binding to M21, M21-L or HUVEC cells, 24-well plates were coated with 10 pg/ml type I collagen (BD Biosciences). in PBS, incubated at 37°C for 30 min, and washed once with PBS. Cells (3.0 x 101 cells/well to 4.0 x 105 cells/well) were grown to confluence. Differential binding of eRGDY-PEG-C dots to M21 or HUVEC cells was assessed over a range of incubation times (up to 4 hours) and particle concentrations (10-600 nM) by flow cytometry. After incubation, cells were washed with RPMI 1640 medium/0.5% BSA, detached with 0.25% trypsin/EDTA, pelleted in a microfuge tube (5 min at 153 g, 25°C). ), were resuspended in BD FACSFlow solution (BD Biosciences), and analyzed in the Cy-5 channel for percentage of particle-bound probe (FACSCalibur, Becton Dickinson, Mountain View, CA).

[0304] Internalization study: The experiment was performed as above but incubated for 4 hours at three different temperatures: 4°C, 25°C and 37°C. Co-incubation of an excess (x250) of cRGDY anti-human integrin avp3 fluorescein-conjugated antibody (Millipore, Billerica, MA) with cRGDY-PEG-C dots for receptor blocking was used to assess specificity. The assays were performed with fixed and live cells. For fixed M21 cells, inverted confocal microscopy (Leica TCS SP2 AOBS) equipped with an HCX PL APO objective (63x 1.2NA Water DIC D) was used and time-lapse imaging was used to track live M21 cells. Images were acquired after co-incubation of specific (or control) particles at various concentrations with the following dye markers) for 4 hours: 70 kDa FITC-dextran conjugate (1 mg/mL, 37 °C for 30 min; Invitrogen) to label macropinosomes, Lysotracker Red (100 nM; Invitrogen) to label the endocytic pathway (ie, acidic organelles), and Alexa488 transferrin (2 pg/ml).

[0305] Competitive binding studies: To test specific binding, M21 cells were incubated (25°C, 4 hours) with 124IcRGDY-PEG-C dots (25 nM) and excess cRGDY peptide. Cells were then washed with RPMI 1640 medium/0.5% BSA and dissolved in 0.5 mL 0.2 M NaOH. Radioactivity was assayed using a 1480 Automatic Gamma Counter (Perkin Elmer) calibrated for iodine. 124.

[0306] Western blot (WB): M21 cells (1x10 6 cells/six-well plate) were cultured in six wells coated with collagen (10 mg/ml), and prepared quiescent by growing under serum-starved conditions. The medium was then changed to 0.2% ECF and different concentrations (25-400 nM) of cRGDY-PEG-C spots were added (37°C, 0.5-8 hours). Cells were rinsed twice in ice-cold PBS, collected by trypsinization, and the pellet resuspended in lysis buffer (10 mM Tris pH 8.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 ; ACROS organics, NJ), 1% Na deoxycholate, 0.1% SDS, Complete™ protease inhibitors (Roche, Indianapolis, IN), and Phosphatase inhibitor cocktail Tablet-PhosSTOP (Roche, Indianapolis, IN ). Lysates were centrifuged (10 min, 4°C). Protein concentrations were determined by the bicinchoninic acid assay (BCA, Thermo Scientific, Rockford, IL). A 50 pg protein aliquot was separated from each fraction. by 4 to 12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Invitrogen, Carlsbad, CA). Membranes were blocked with 5% skim milk powder (Bio-Rad, Hercules, CA) in Tris Buffered Saline (TBS)-0.1% Tween, and the signal was visualized by ECL chemiluminescence (Thermo Scientific, Rockford, CA). IL) or Immobilon Western, (Millipore Billerica, MA) after applying primary (1:1000) and secondary (1:2000-1:5000) antibodies.

[0307] Cell cycle analysis: G0/G1 synchronized M21 cells were incubated for 48 hours using two concentrations (100 and 300 nM) of cRGDY-PEG-C spots after changing medium to 0.2% ECF . After trypsinization, the cells were centrifuged (1200 rpm, 5 min) and the pellet was suspended in PBS, followed by 70% ethanol fixation (4 °C, 0.5-1 hour). Cells were successively resuspended in 1 ml PBS containing 1% ECF and 0.1% Triton X-100, 200 ml PBS containing 25 gg/ml propidium iodide and 100 mg/ml RNaseA (4 °C, 60 minutes). Cell cycle analysis was performed using flow cytometry, FACSCalibur, (Mountain View, CA) and Phoenix Flow MultiCycle software (Phoenix Flow Systems, Inc., San Diego, CA).

[0308] Cell proliferation: cells were split (1x104 cells/well) in a collagen-coated 96-well plate as described for in vitro cell attachment studies. Different concentrations of cRGDY-PEG-C spots (25-200 nM) were added for 24-48 hours at 37°C. Then, 20 ml of WST-1 proliferation reagent (Roche, Indianapolis, IN) was added to the plate (37°C, 1 hour). For the determination of the optical densities, we used a SpectraMax5 microplate reader (Molecular Devices, Sunnyvale, CA). The absorbance was measured at 440 nm.

[0309] Migration assay: M21 cells: M21 cells (6x10 4 cells/well) were seeded using a migration kit (Oris™ collagen-1 coated plate, PLATYPUS TEC). Twenty four hours after seeding the cells, the plugs were removed from the plate. Fresh culture media (100 mL) supplemented with 0.2% FBS was introduced and cRGDY-PEG-C spots were added at various concentrations: 25, 100, and 400 nM. Thereafter, every 24 hours, the medium was replaced, along with new particles, for a time interval of 72 hours. Before incubating the plate at 37 °C overnight, time zero images were captured with the Axiovert 200M microscope (Carl Zeiss) using a 5x (.15NA) objective and using a scanning slide module in Metamorph software (devices molecular, PA). Serial microscopy was then performed and images were captured every 24 hours for a total of 96 hours after incubation. Data was analyzed using imaging software. HUVEC cells: HUVEC cells were seeded further (5x10 4 cells/well) and, 24 hours later, incubated with various concentrations of particles (100, 200 and 400 nM) after replacing the medium. A microscopy procedure similar to that of M21 cells was performed, with serial images being acquired 20 hours later.

[0310] Adhesion and Spread Assay: The effect of cRGDY-PEG-C dots on the binding of M21 cells to fibronectin-coated plates was evaluated by initially coating 96-well microtiter plates with fibronectin in PBS (5 gg/ml), followed by 200 ml of RPMI/0.5% BSA (37°C, 1 hour). Cells (1-3 x 10 4 cells/100 ml/well) were incubated (25 °C, 30 min), with or without 400 nM cRGDY-PEG-C spots in RPMI/0.1% BSA, and added to fibronectin coated wells (37°C, 30-120 minutes). For quantification of the number of adhered cells, the wells were rinsed with RPMI/0.1% BSA to remove non-adhered cells. Adherent cells were fixed with 4% PFA (25°C, 20 minutes) and stained with methylene blue (37°C, 1 hour). Methylene blue was extracted from the cells by the addition of 200 ml of 0.1 M HCl (37°C, 1 hour). For the determination of the optical densities, a SpectraMax5 microplate reader was used and the absorbance at 650 nm was measured. For the dispersion assay: a time lapse (37 °C, 2 hours) was performed and images were captured with an Axiovert 200M microscope (Carl Zeiss) using a 20x objective (.15NA) and using a slide module of scan in Metamorph software (Molecular Devices).

[0311] Quantitative analyses: To quantify the differences in size and intensity between Western blot bands, we performed densitometry of total and phosphorylated protein intermediates using Photoshop CS2 (Adobe, San Jose, CA). Bands were scanned at 300 dpi (Scanjet 7650, Hewlett Packard, Palo Alto, CA) and converted to grayscale. The Lasso tool was then used to draw a region of interest (ROI) within the boundaries of each band to obtain the following: area (number of pixels), mean grayscale value within the selected area (0-255) and the associated value. Standard deviation. The product of the first two values for each band was calculated and divided by the product of the initial band in each set (control band), giving an intensity value for each sample relative to the control. Finally, the ratio of phosphorylated protein to total protein and the corresponding propagation error (SD) were calculated for each sample using the relative intensities.

[0312] Phase contrast images captured for migration studies were analyzed using lmageJ 1.45s (National Institutes of Health, rsbweb.nih.gov/ij/) to quantify the extent of cell migration (i.e., closure area) for M21 and HUVEC cells. In high power views, a closed area was drawn adjacent to the edge of the attached cells seen in each image after plug removal. The enclosed area for each image (pixels) was measured and used to calculate percentage closure relative to time zero (after particle addition and medium replacement) as follows: difference in area in a given time point (24, 48, 72, or 96 h) and at time zero divided by the same area at time zero multiplied by 100. The resulting values were averaged and a standard error was calculated for each group.

[0313] Statistics: All graphical values are plotted as mean ± SE, except where indicated. The t-test was used One-tailed Student's test to test the statistical significance of differences in cell migration between HUVEC or M21 cells incubated with serum alone or cRGDY-PEG-C dots. One-way analysis of variance (ANOVA) was used to perform pairwise statistical comparisons between the percentage of M21 cells in S phase that were incubated with serum alone, 100 nM or 300 nM cRGDY-PEG-C spots. We assigned statistical significance for all tests at P < 0.05.

Example 17 Sphingomyelin Liposomes for Enhanced Release of Therapeutic/Diagnostic Silica Particles in Tumors

[0314] The therapeutic potential of particles below 10 nm ( eg, C spots) as drug delivery vehicles is under investigation. Drug-bound particles have been developed by binding drugs to particle-bound bifunctionalized PEG chains. The model drugs used for this proof-of-concept work are receptor tyrosine kinase (RTK) inhibitors. Delivery and accumulation of therapeutic particle payloads at the target site can be maximized by employing dose escalation strategies to assess enhanced particle uptake and distribution within tumors by optical and/or PET imaging. An alternative approach to improve particle charge delivery uses liposomal formulations to encapsulate therapeutic particle probes. Liposomes passively target tumors through enhanced permeability and retention (EPR) effect while protecting payloads from degradation in the circulation. Prior to particle encapsulation, cRGDY or linker-drug conjugates will bind to the particle surface as an active targeting mechanism to maximize delivery to the target site.

[0315] Once at the target site, the release of particles from liposomal formulations into the tumor interstitium may occur as a result of upregulated enzyme systems present in tumors. Extracellular release of acid sphingomyelinase (ASMases, acid SMase, or SMase) from tumor cells in response to cellular stress, such as X-ray irradiation and toxins, leads to rapid ceramide-mediated cell injury and subsequently apoptosis . Acid SMase hydrolyzes sphingomyelin, present in liposomal formulations, to ceramide. Ceramide has a role in biological systems as a secondary messenger in the apoptotic process. Ceramide has significantly different membrane properties than the parent molecule sphingomyelin. When sphingomyelin-containing liposomes are cleaved with SMase, a dramatic change in membrane stiffness occurs. Sphingomyelin is a suitable lipid for liposome formulation; this is not true for ceramide, since its incorporation as a liposome building block leads to liposome leakage. This leakage will lead to the release of the liposomal content. This opens the possibility for intracellular targeting of a large payload of silica nanopanicles, functionalized with drugs and/or cell internalization markers (ie, KLAKLAC peptide and small molecule inhibitors) to monitor and/or treat diseases.

[0316] To determine if liposome encapsulation significantly increases particle delivery to the tumor site relative to non-encapsulated particle probes, we will use optical and PET imaging approaches to monitor the uptake of both encapsulated and particle batches. not encapsulated in sphingomyelinase liposomes in the brain expressing EGFRmt+. tumor xenograft templates (ie H1650, A431) both liposomes and particle probes will contain optical markers for visualization.

[0317] Ref: Sochanik A1, Mitrus I, Smolarczyk R, Cichon T, Snietura M, Czaja M, Szala S. Experimental anticancer therapy with vascular-disruptive peptide and liposome-entrapped chemotherapeutic agent. Arch Immunol Ther Exp (Warsz). June 2010; 58(3):23545.

[0318] Previous methods for detecting and targeting stressed cells involve a formulation comprised of bilayer liposomes, each of which has an initial outer layer of liposome-forming lipids, sphingomyelin, or a mixture thereof; a second inner layer of liposome-forming lipids, sphingomyelin, or a mixture thereof; the first and second layers forming a bilayer liposome and defining an interior space therein; wherein the interior space contains a silica nanoparticle. The silica nanoparticle and/or liposome will be compromised with optical markers and/or PET tags (dye, fluorophore, radiolabel, contrast agent), an enzyme substrate, and/or therapeutic agents, including cytotoxic drugs, DNA segments, or a radiotracer label indicator. Labeler and/or drug-labeled silica particles contained within sphingomyelin liposomes are contacted with a cell sample under conditions where sphingomyelinase is present or released by the cell. This enzymatic contact, in turn, will hydrolyze the sphingomyelin in the liposome to release the silica nanoparticle and/or label marker and/or drug from the hydrophilic (interior) or hydrophobic (bilayer) compartments of the liposome.

[0319] The scope of the present invention is defined by the claims and is not limited by what has been specifically shown and described above. Those skilled in the art will recognize that suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions exist. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. The matter set forth in the above description and accompanying drawings is offered by way of illustration only and not as a limitation.

Claims (20)

1. A plurality of silica-based fluorescent nanoparticles for use in a method for detecting tumor cells comprising the steps of: (a) providing a plurality of silica-based fluorescent nanoparticles, said nanoparticles having been administered to a human subject at a dose range of from about 0.01 nanomoles/kg of body weight to about 1 nanomoles/kg of body weight, comprising each of the nanoparticles: a silica-based core; a fluorescent compound within the nucleus; Y a silica shell that surrounds at least a part of the core; where the nanoparticle is coated with an organic polymer, where 1 to 20 ligands are bound to the polymer-coated nanoparticle to bind to a tumor marker, where the ligands have the structure NAc-Cys-(Ahx) ² -D-Lys- ReCCMSH, and where each of the nanoparticles has a diameter within a range of 1 nm to 15 nm; Y (b) detecting the nanoparticles, where the nanoparticles are excreted by renal clearance, by the human subject, within a range of 90% of the initial dose (ID) to 100% ID in 24 hours after administration. 2. A fluorescent silica-based nanoparticle comprising: a silica-based core comprising a fluorescent compound positioned within the silica-based core; a silica shell that surrounds at least a part of the core; an organic polymer attached to the nanoparticle; Y a ligand having the structure N-AoCys-(Ahx) ² -D-Lys-ReCCMSH attached to the nanoparticle, where the nanoparticle has a diameter between about 1 nm and about 15 nm, and after administration of the nanoparticle to a subject, renal clearance of the nanoparticle ranges from about 80% ID (initial dose) to about 100% ID in approximately 24 hours. The plurality of nanoparticles for use according to claim 1 or the nanoparticle of claim 2, wherein the nanoparticle has a diameter between about 1 nm and about 8 nm. The plurality of nanoparticles for use according to claim 1 or the nanoparticle of claim 2, wherein the organic polymer is selected from the group consisting of poly(ethylene glycol) (PEG), polylactate, polylactic acids, sugars, lipids, acid polyglutamic acid (PGA), polyglycolic acid, poly(lactic-co-glycolic) acid (PLGA), polyvinyl acetate (PVA) and combinations thereof. The plurality of nanoparticles for use according to claim 1 or the nanoparticle of claim 2, wherein the number of ligands bound to the nanoparticle ranges from about 1 to about 10. The plurality of nanoparticles for use according to claim 1 or the nanoparticle of claim 2, wherein a contrast agent or chelate is attached to the nanoparticle. The plurality of nanoparticles for use according to claim 6 or the nanoparticle of claim 6, wherein the contrast agent is a radionuclide. 8. The plurality of nanoparticles for use according to claim 7 or the nanoparticle of claim 7, wherein the radionuclide is selected from the group consisting of 89Zr, 64Cu, 68Ga, 86Y, 124I and 177Lu. The plurality of nanoparticles for use according to claim 6 or the nanoparticle of claim 6, wherein the chelate is adapted to bind to a radionuclide. 10. The plurality of nanoparticles for use according to claim 6 or the nanoparticle of

claim 6, wherein the chelate is selected from the group consisting of DFO, DOTA, TETA, and DTPA. The plurality of nanoparticles for use according to claim 1 or the nanoparticle of claim 2, wherein the nanoparticle is detectable by PET, SPECT, CT, MRI, optical imaging, bioluminescence imaging, or combinations thereof. same. The plurality of nanoparticles for use according to claim 11 or the nanoparticle of claim 11, wherein the optical imaging is fluorescence imaging. 13. A therapeutic agent attached to a silica-based fluorescent nanoparticle, for use in medicine; wherein said nanoparticle comprises: a silica-based core; a fluorescent compound within the nucleus; Y a silica shell that surrounds at least a part of the core; where the nanoparticle is coated with an organic polymer, where 1 to 20 ligands are bound to the polymer-coated nanoparticle to bind to a tumor marker, where the ligands have the structure N-Ac-Cys-(Ahx) ² -D- Lys-ReCCMSH, and where each of the nanoparticles has a diameter within a range of 1 nm to 15 nm; wherein the nanoparticles are excreted by renal clearance within a range of 90% of the initial dose (ID) to 100% ID in 24 hours after administration to a human subject. The therapeutic agent for use according to claim 13, wherein the therapeutic agent is selected from the group consisting of antibiotics, antimicrobials, antiproliferatives, antineoplastics, antioxidants, endothelial cell growth factors, thrombin inhibitors, immunosuppressants, antiplatelet agents, collagen synthesis inhibitors, therapeutic antibodies, nitric oxide donors, antisense oligonucleotides, wound healing agents, therapeutic gene transfer constructs, extracellular matrix components, vasodilators, thrombolytics, antimetabolites, growth factor agonists , antimitotics, statins, steroids, anti-steroidal and non-steroidal -inflammatory agents, angiotensin-converting enzyme (ACE) inhibitors, free radical scavengers, PPAR-gamma agonists, small interfering RNA (siRNA), microRNA, and chemotherapeutic agents against cancer. The therapeutic agent for use according to claim 13, wherein the therapeutic agent is radiolabeled. The therapeutic agent for use according to claim 15, wherein the therapeutic agent is bound to radiofluorine 18F. The therapeutic agent for use according to claim 13, wherein the therapeutic agent is a radionuclide. The therapeutic agent for use according to claim 17, wherein the radionuclide is selected from the group consisting of 90Y, 131I and 177Lu. The plurality of nanoparticles for use according to claim 1 or the nanoparticle of claim 2, wherein the fluorescent compound is Cy5 or Cy5.5. 20. The plurality of nanoparticles for use according to claim 1 or the nanoparticle of claim 2, wherein the mean residence time in blood of the nanoparticle after administration is from 2 hours to 25 hours (for example, from 3 hours to 15 hours, eg, 4 hours to 10 hours) or where the median tumor residence time of the nanoparticle after administration is 5 hours to 5 days ( eg, 10 hours to 4 days, eg, 15 hours to 3.5 days).