Activity based probe and uses thereof for imaging

Abstract

Provided is a compound including at least one carrier moiety associated with a plurality of CT imaging moieties, and with at least one enzyme interacting moiety as well as uses thereof in diagnosis.

Claims

1. A compound comprising at least one carrier moiety bound to a plurality of CT imaging moieties, and with at least one enzyme interacting moiety, wherein the plurality of CT imaging moieties are selected from gold-based moieties, and wherein the at least one carrier is a nanoparticle, is attached to a (a) gold-based moiety comprising a plurality of gold nanoparticles (GNPs); and (b) the at least one enzyme interacting moiety is acyloxymethyl ketone (AOMK); wherein the GNPs and the at least one enzyme interacting moiety are linked to an oligomer or a polymer CM: ##STR00024## wherein K is a point of connectivity to GNP and is S—; B is a point of covalent connectivity to the at least one enzyme interacting moiety and is —C(O)O X is a heteroatom selected from —NH—, O and S; n is 2; and m is an integer from 60 to 120.

2. The compound according to claim 1, wherein the GNP has an average diameter of between 5 nm and 200 nm, or between 10 nm and 100 nm, or of 10 nm, 30 nm and 100 nm.

3. A diagnostic formulation comprising at least one compound according to claim 1.

4. The compound according to claim 1, wherein m is between 65 and 70.

5. The compound according to claim 1, wherein X is —O—.

6. The compound according to claim 1, wherein the GNPs have an average size of between 5 nm and 200 nm.

7. The compound according to claim 1, wherein the GNPs are surface-modified with ligands.

8. The compound according to claim 7, wherein the ligands are antibodies or peptides.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

(2) FIGS. 1A-1F provide pictorial characterization of GNP-based compounds; FIGS. 1A-1C are TEM images of 100 nm (FIG. 1A), 30 nm (FIG. 1B), 10 nm (FIG. 1C) GNP-based compounds; FIG. 1D-1F are UV-Vis spectra of 10 nm (FIG. 1D), 30 nm (FIG. 1E), 100 nm (FIG. 1F) GNP-based compounds.

(3) FIGS. 2A-2G are structural depictions representing various derivatives of GNP-based compounds with GNP size of 10 nm, 30 nm and 100 nm; the curved lines represents ethylene oxide polymer/oligomer of molecular weights 5 kDa or 3 kDa, as indicated, —OMe and COOH denote groups at most-exposed ends of the ethylene oxide polymer/oligomer, COOH denotes points of interaction with the enzyme interacting moiety (“GB111”), the % values indicate % of GB111 substituted ethylene oxide polymer/oligomer relative to the total number of ethylene oxide polymer/oligomers on a GNP, FIG. 2A represents GNP with a 5 kDa polymer/oligomer, with 10% GB111 substitution; FIG. 2B represents GNP with a 5 kDa polymer/oligomer, with 50% substitution; FIG. 2C represent GNP with a 5 kDa polymer/oligomer with 100% substitution; FIG. 2D represents GNP with 5 kDa and 3 kDa polymer/oligomer, with 10% substitution on the 3 kDa moieties; FIG. 2E represents GNP with 5 kDa and 3 kDa polymer/oligomer, with 50% substitution on the 3 kDa moieties; FIG. 2F represents GNP with a 3 kDa polymer/oligomer, with 100% substitution; FIG. 2G represents control GNP with no GB111 substitutions.

(4) FIG. 3 shows SDS gels exhibiting competitive inhibition of recombinant cathepsin B GNP-based compounds; the indicated concentrations of probes were added to samples for 1 h, followed by 30 min labeling of the residual activity with GB123, a fluorescent tag, the samples were separated on SDS gel and scanned for fluorescence.

(5) FIG. 4 shows SDS gels exhibiting inhibition of endogenous cathepsin activity in intact NIH3T3 cells.

(6) FIGS. 5A-5F are bar graphs showing cell viability determined by methylene blue assay after incubating the cells with various concentrations of GNP-based compounds for 24 hours (FIGS. 5A-SC) and 48 hours (FIGS. 5D-5F), data are presented as the mean f SD (n=6).

(7) FIGS. 6A and 6B are plots of concentrations of contrast agents relative to HU detected by CT: FIG. 6A at 35 keV and FIG. 6B at 85 keV.

(8) FIGS. 7A-7L are non-invasive micro-CT imaging of cancer in mice. CT scans of mice 72 h post-injection of 10, 30 and 100 nm GNPs-ABPs (FIGS. 7B, 7D, 7F) or 10, 30 and 100 nm GNPs (as control) (FIGS. 7A, 7C, 7E) (5 mg gold per mouse); FIGS. 7A-7F—representative CT images of volume rendered 3D images represent X-ray absorption of bones and gold, dashed line circle indicate tumor location, FIGS. 7G-7L—2D axial cross section image of the same mice, accumulation of GNP-ABP at the tumor leads to significant signal enhancement (shown in circle).

(9) FIGS. 8A-8F are graphs showing GNP-based compounds uptake in-vivo in different organs, FIGS. 8A-8C show percentage of tumor volume containing gold, extracted from CT images of tumors at 0, 24, 48 and 72 h after IV injection. The higher accumulation in tumors was detected with 10 nm & 30 nm GNP-ABPs (T) compared to GNP (NT) through all time points. Black bars GNP-compounds (T), white bars GNP (NT, without the active group). FIGS. 8D-8F show average distribution of gold in the main organs 72 hours after injection of GNP-ABPs (T) or GNP (NT) (10, 30, 100 nm) detected by ICP MS.

(10) FIGS. 9A and 9B demonstrates comparison for GNP-based compounds M, revealing the strong size dependent uptake in favor of small 10 nm GNP-ABPs (T). FIG. 9A shows GNP size comparison in terms of the percent of tumor volume containing gold as analyzed from CT scans post injection, FIG. 9B shows gold accumulation in major organs detected by ICP-MS for 10, 30 and 100 nm of GNP-based compounds (T).

(11) FIGS. 10A-10F are TEM images of section of tumor tissue taken from a tumor bearing mice injected with 10 nm (FIGS. 10A and 10D), 30 nm (FIGS. 10B and 10E) or 100 nm (FIGS. 10C and 10F) GNP-probe as GNP-ABP (T) (FIGS. 10A-10C) or GNP (NT—no ABP) (FIGS. 10D-10F), Scale bars are: 2 μm to 200 nm.

(12) FIGS. 11A-11F biochemical evaluation of iodine-based compounds: FIGS. 11A-11D show labeling of recombinant cathepsins B (FIGS. 11A and 11D) and recombinant cathepsins L activity (FIGS. 11B and 11E) by IN-ABPs by competitive assay, FIGS. 11C and 11F show cell permeability of IN-ABPs, detection of cathepsin inhibition in NIH-3T3 intact cells by labeling of residual protease activities by competitive assay.

(13) FIGS. 12A-12F show biochemical evaluation of Iodine-based compounds (IN-ABP); FIG. 12A shows labeling of recombinant cathepsins B activity with IN-ABPs by competitive assay, FIG. 12B shows recombinant cathepsins L activity with IN-ABPs by competitive assay, FIG. 12C shows direct labeling of recombinant cathepsin B by Cy5 labelled IN-ABPs, FIG. 12D shows direct labeling of recombinant cathepsin L by Cy5 labeled IN-ABPs, FIG. 12E shows cell permeability of IN-ABPs, detection of cathepsin inhibition in NIH-3T3 intact cells by labeling of residual protease activities by competitive assay, FIG. 12F shows detection of cathepsin labeling in NIH3T3 intact cells by direct labeling of protease activity in comparison to small fluorescent cathepsin ABP GB123.

(14) FIGS. 13A and 13B are bar representations showing viability assay of Iodine tagged probes at 24 h (FIG. 13A) and 48 h (FIG. 13B), no toxicity was found in any of the probes check in the first 24 hours while a slight toxicity was observed at 10 μM of HG 82, 86, and 87 after 48 hours.

(15) FIGS. 14A-14D show signal to background of tumor fluorescence using IN-ABP (FIG. 14A) and control contrast agents (FIG. 14C), tumor bearing mice were injected with Cy5 labeled IN-ABP (in dark diamonds) and control compounds (in light triangles), Non-invasive fluorescence was monitored prior and post injection up to 8 hours using an in vivo Imaging System (IVIS) Kinetic equipped with a 640 nm excitation filter and Cy5 emission filter and the Fluorescent signals were quantified at the tumor area and on the back of the mouse as the background. The tumor to background ratio is presented, IN-ABPs show significantly higher tumor to background signal.

(16) FIGS. 15A and 15B are bar representations showing pharmacokinetics in vivo X-ray computed tomography, FIG. 15A—in vivo pharmacokinetic comparative study of IN-ABP HG92 and HG31, FIG. 15B—in vivo pharmacokinetic comparative study of IN-ABP HG90 and HG99.

(17) FIGS. 16A and 16B show in vivo X-ray computed tomography, FIG. 16A representative picture of a mouse before and 24 h post-injection of IN-ABP (HG92), FIG. 16B show a representative picture of a mouse before and 24 h post-injection of control IN-ABP (HG31).

(18) FIG. 17 is a bar graph showing in vivo X-ray computed tomography pharmacokinetic comparative study of IN-ABP HG82 and HG86.

(19) FIGS. 18A-18D are in vivo X-ray computed tomography, a representative picture of a mouse 5 h post-injection of HG82 that specifically targets cathepsin activity within the tumor, FIG. 18A coronal plane, FIG. 18B transverse plane and FIG. 18C sagittal Plane, FIG. 18D tomographic view, tumor detected was colored gray.

(20) FIG. 19 presents SDS of tumors and organs being lysed and equal protein samples were spread by SDS-PAGE, followed by fluorescent scanning of the gel. Specific labeling of cathepsins by HG92 is detected in kidney, liver, spleen and tumor tissues.

(21) FIG. 20 is an exemplary compound of the invention in some embodiments.

(22) FIG. 21 is a carrier moiety.

(23) FIGS. 22-26 are examples of a 5 kDa PEG-OMe moiety.

(24) FIG. 27 is an example of a 3 kDa PEG moiety.

(25) FIG. 28 is a synthesis of multiple iodine tagged CT-ABP (Acetylated and PEGylated).

(26) FIG. 29 is a synthesis of multiple iodine tagged CT-ABP (Acetylated and PEGylated).

(27) FIG. 30 is a synthesis of multiple iodine tagged CT-ABP (Acetylated, PEGylated and Cy5 labelled).

(28) FIG. 31 is a synthesis of multiple iodine tagged CT-ABP (Acetylated, PEGylated and Cy5 labelled).

DETAILED DESCRIPTION OF EMBODIMENTS

Example 1: Gold Derivatives of Activity Based Probe

(29) Synthesis of GB111-NH.sub.2: Fmoc-Lys(Boc)-AOMK (365.2 mg, 0.594 mmol) was de-protected by 25% trifluoroacetic acid (TFA)/dichloromethane (DCM) (v/v) during 30 minutes to receive the free amine, solvent was removed by co-evaporation with toluene in vacuo. 2-Chlorotrityl chloride resin (371.1 mg) was loaded by shaking of resin with the Fmoc-Lysine(NH.sub.2)-AOMK (1 eq.) dissolved in anhydrous DCM and diisopropylethylamine (DIEA) (3.5 eq, 360μl) for 1.5 hours. Methanol (1 ml/gr resin) was added, and the resin was shaken for 30 minutes followed by washing with DCM and DMF. The Fmoc protecting group was removed by two quick washes with 5% diethylamine (DEA)/DMF (v/v) followed by a 5 min incubation with 5% DEA and then washed with DCM and DMF. De-protection was verified by Kiser test. The peptide was elongated by addition of a solution of N-benzyloxycarbonyl-phenyalanine (Cbz-Phe), (3 eq., 226 mg), Hydroxybenzotriazole (HOBT), (3 eq., 101.3 mg) and benzotriazol-1-yl-oxytripyrrolidino phosphonium hexafluorophosphate (PyBOP), (3 eq., 116 mg) in DMF for 2 hours. The resin was then washed with DCM and DMF and the final peptide product was cleaved from the resin by addition of 7% TFA/DCM (v/v) for 7 to 15 minutes. The cleaved solution was collected and solvent was removed by co-evaporation with toluene. The crude peptide was further dried in vacuo and purified by HPLC. GB111-NH.sub.2 eluted at 44% ACN to yield a white powder, 78.94 mg, 0.138 mmol, M/z+1 574, 56% yield relative to resin loading. Scheme 1 shows GB111NH.sub.2 synthesis.

(30) ##STR00015##

(31) Synthesis of PEGylated gold nanoparticles: GNP stabilized in citrate buffer were purchased from Cytodiagnostic Inc. at three different sizes: 10, 30, 100 nm. A protective layer of PEG was absorbed on the surface of the GNPs at different molar ratios and different lengths consisting of a mixture of thiol-polyethylene-glycol (mPEG-SH) and a heterofunctional thiol-polyethylene-acid (SH-PEG-COOH) (Creative PEGWorks, Winston Salem, N.C.).

(32) In a typical process, to 1.0 ml of GNP solution (OD 1) was added 11 μl of PEG mixture solution at different concentrations to obtain the following final molar ratios: 10, 1, 0.1, 0.01 mM. The reaction mixture was vortexed immediately and then incubated at room temperature for two hours. After PEGylation, each solution was centrifuged at 14000, 9400, 8600 rpm (10, 30, 100 nm respectively) for 20 min and re-dispersed with Milli-Q water by gentle shaking. This process was repeated three times. From the resultant supernatant solution a small part was analyzed by measuring absorption spectra, hydrodynamic size and zeta potential. The mixture was stored in refrigerator at 4° C. PEGylation at 0.01 mM final concentration was found to be the most efficient and stable.

(33) Conjugation of GB111-NH2 to PEGylated gold nanoparticles: PEG-GNP were re-dispersed in 1 ml of DMF:DDW (1:10) several times. EDC (1.13 μmol, 0.21 mg) and NHS (1.13 μmol, 0.13 mg) 1.5 eq relative to the PEG-COOH moiety, were dissolved together and added to GNP-PEG solution for 30 min. at room temperature, providing active sites on GNPs that further undergo amidation reaction with the GB11-NH.sub.2. The amount of peptide added is 0.38 mg (75 nmol) per 2.5 mg of molecular gold (1:1 ratio to activated moiety). The mixture was incubated overnight at room temperature followed by centrifugation and re-dispersion in DMF to remove unbound peptide in the solution.

(34) Preparation of HS-PEG-GB111 and PEGylatien to GNPs: Anhydrous DMF (3 ml) was added to dissolve the ortho-pyridyldisulfide-PEG-succinimidyl ester (OPSS-PEG-NHS, 5 kD) powder (1 eq., 10 mg, 2 μmol), then 1.14 mg (1 eq., 2 μmol) of GB111-NH.sub.2 was added and mixed in, and finally DIEA (3 eq., 1.0 μL, 6 μmol) was added. The solution was stirred at room temperature for 2 hours. The product fraction was lyophilized and purified by HPLC. The OPPS-HS-PEG-GB 111 conjugate, eluted at 65% ACN to yield a yellowish powder, 8.1 mg, 1.45 μmol, 73% yield.

(35) Dithiothreitol (DTT)-3.37 mg (3 eq., 21.8 μmol) and DIEA (6 eq., 43.6 μmol) were dissolved in 1.5 μL MeOH then added to the dissolved OPPS-SH-PEG-GB111 36.3 mg (1 eq., 7.2 μmol) in 674 μL DDW to give 1M concentration. The reaction mixture was stirred for 3 h to reduce the disulfide and obtain a free thiol at the end of the PEG-GB 111 polymer. The resultant powder was harvested through precipitation in dry diethyl ether. PEGylation using HS-PEG-GB111 to GNPs at 0.01 mM molar ratio was carried out in the same way as for HS-PEG-COOH. After centrifugation was performed three times, the final GNP pellets were re-suspended in 0.2 ml Milli-Q water to concentrate the solution.

(36) Chemical evaluation: Dynamic light scattering (DLS) and ζ potential measurements were conducted in DDW using a Zetasizer nano-ZSP (Malvern). Transmission electron microscopy (TEM) analysis was performed using JEOL JEM-1400Plus by applying ˜10 μl of samples resuspended in DDW to a 200 or 400-mesh copper grids covered by carbon-stabilized Formvar film (SPI, West Chester, Pa.). The samples were dried over night before scan performed at different kV. Thermogravimetric analysis (TGA) to establish the mass ratio between organic moiety (PEG) and gold atoms was performed using Mettler Toledo instrument. Temperature Program: heat from 30° C. to 450° C. with rate of 10° C./min in nitrogen atmosphere with a purge rate of 20 mL/min. This protocol led to a density of 0.21 PEG/nm.sup.2, which corresponds to the low PEG density particle group (density lower than 1 PEG/nm.sup.2). This result is consistent with the low concentration of PEG mixture taken for the process.

(37) Recombinant cathepsin labeling: Recombinant human Cathepsin-B, 0.7 μg or Cathepsin-L, 0.6 μg or Cathepsin-S, 0.7 μg in reaction buffer (50 mM acetate, 2 mM DTT and 5 mM MgCl.sub.2 at pH 5.5) were treated in room temperature with inhibitor (GNP-PEG-GB111) 0.01-10 μM probe or vehicle for 1 hour. Indicated concentrations of GB123 were added to samples for 30 minutes at r.t. The reaction was stopped by addition of sample buffer ×4 (40% glycerol, 0.2M Tris/HCl 6.8, 20% b-mercaptoethanol, 12% SDS and 0.4 mg/ml bromophenol blue), samples were boiled, separated on a 12.5% SDS gel and scanned for fluorescence by Typhoon scanner FLA 9500, excitation/emission 650/670 nm.

(38) Evaluation of probes permeability to intact cells, competition assay: NIH-3T3 cells (1×10.sup.5 cells/well) were seeded in a twelve-well plate one day before treatment. Cells were treated with vehicle or 0.1, 1, 10 μM probes that were pre-dissolved in 0.1% DMF, 0.9% DDW. After 4, 6 or 24 hours of probe incubation residual cathepsin activity was labeled with GB123 (1 μM). Cells were washed with PBS and lysed by addition of sample buffer. Lysates were boiled for 10 minutes and centrifuged. Protein determination was performed by butterfly assay and separated by 12.5% SDS gel. Residual labeled proteases in cells were visualized by scanning the gel with a Typhoon scanner FLA 9500, excitation/emission wavelengths of 650/670 nm. Direct labeling was preformed similarly, after probe treatment cells were lysed and equal protein was separated by Gel that was scanned for Cy5 fluorescence.

(39) Cells viability assay: NIH-3T3 cells (5×10.sup.3-24 h, 3×10.sup.3-48 h) and 4T1 cells (6×10.sup.3-24 h, 4×10.sup.3-48 h) were seeded in 96-well plate and incubated for 24 h for cell attachment. GNP-ABPs dispersed in fresh culture media were added at equivalent concentration to each well (200 μl) and incubated for 24 or 48 hours at 37° C. After continuous exposure to various concentrations of GNP-ABPs, the medium was discarded to remove free particles in the solution and cell survival was investigated by standard methylene blue assay.

(40) Animal Care: All animals were maintained on a 12:12 h light/dark cycle under fixed conditions of temperature (23 C) and humidity (50%), with free access to food and water. All experimental procedures were approved by the Animal Care Committee of Hebrew University of Jerusalem and in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

(41) In vivo imaging: 4T1 cells were grown to subconfluency, followed by detachment with trypsin, spin down and resuspention in 0.5% BSA in sterile PBS and 25% matrigel. Cells (1×10.sup.6 per spot in a total volume of 20 μl) were injected subcutaneously at the indicated locations into 3-4 week-old male BALB/c mice under isoflurane anesthesia. Tumors were typically established 9-11 days after cells injections, compounds were injected intravenously via the tail vein as follows: in a total volume of 200 μl from stock solution of 25 mg/ml (5 mg of GNP per mouse). Mice anesthetized with isoflurane were imaged before, at 24, 48 and 72 hours post injection. Micro-CT scanner (Skyscan High Resolution Model 1176) equipped with 64 detectors with a nominal resolution of 35 μm was used (0.2 mm aluminum filter, a tube voltage of 40 kV and 500 mA). Reconstruction was performed using SkyScanNRecon software. Ring artifact reduction, Gaussian smoothing (3%), and beam hardening correction (25%) were applied. Volume rendered three-dimensional (3D) images were generated using SkyScan CT-Volume (“CTVol”) software.

(42) Flame Gold Analysis (ICP-MS): The tissues obtained from the experimental animals were melted with aqua regia acid, a mixture of nitric acid and hydrochloric acid in a volume ratio of 1:3. The samples were then evaporated, filtered and diluted to a final volume of 7 mL. Au wavelength lamp was used in order to determine the gold concentration in the samples. A calibration curve with known gold concentrations was prepared (commonly: 1, 2, 5 and 10 mg/L). Gold concentration in each sample was determined according to its absorbance value with correlation to the calibration curve. Each sample was analyzed in triplicate and averages and standard deviations were taken.

(43) Preparation of tissues for transmission electron microscopy: Tissue was dissected from animal into PBS (Phosphate Buffered Saline pH 7.4) and fixed in 2% paraformaldehyde, 2.5% Glutaraldeyde in 0.1M Cacodylate buffer (pH 7.4) for several hours at room temp. The tissues were then rinsed 4 times, 10 minutes each, in cacodylate buffer and post fixed and stained with 1% osmium tetroxide, 1.5% potassium ferricyanide in 0.1M cacodylate buffer for 1 hour. Tissues were then washed 4 times in cacodylate buffer followed by dehydration in increasing concentrations of ethanol consisting of 30%, 50%, 70%, 80%, 90%, 95%, for 10 minutes each step followed by 100% anhydrous ethanol 3 times, 20 minutes each, and propylene oxide 2 times, 10 minutes each. Following dehydration, the tissues were infiltrated with increasing concentrations of Agar 100 resin in propylene oxide, consisting of 25, 50, 75, and 100% resin for 16 hours each step. The tissues were then embedded in fresh resin and let polymerize in an oven at 60° C. for 48 hours. Embedded tissues in blocks were sectioned with a diamond knife on an LKB 3 microtome and ultrathin sections (80 nm) were collected onto 200 Mesh, thin bar copper grids. The sections on grids were sequentially stained with Uranyl acetate for 5 minutes and Lead citrate for 2 minutes and viewed with Tecnai 12 TEM 100 kV (Phillips, Eindhoven, the Netherlands) equipped with MegaView II CCD camera and Analysis® version 3.0 software (SoftImaging System GmbH, Münstar, Germany).

(44) Results:

(45) Synthesis of single GNP-ABPs was done. Briefly, the peptide was synthesized using Solid-phase peptide synthesis (SPPS), starting with Boc protected Fmoc-Lysine bromomethyl ketone that was reacted with dimethyl benzoic acid in solution to form the AOMK. Next, the Boc protecting group was removed to receive Fmoc-Lysine (NH.sub.2)-AOMK, that was loaded on a chlorotrityl resin via ε-amine of the lysine. The Fmoc protecting group was removed and the Lysine-AOMK was elongated via the free amine of the lysine with protected Cbz-Phenylalanine-OH. The dipeptide-AOMK was cleaved from the resin and purified by HPLC to give GB111-NH.sub.2 with 56% yield (data not shown).

(46) The surface modification conditions of GNP stabilized in citrate buffer were evaluated. A protective layer of PEG was absorbed on the surface of the GNPs at different molar ratios and different lengths consisting of a mixture of thiol-polyethylene-glycol (mPEG-SH) and a heterofunctional thiol-polyethylene-acid (SH-PEG-COOH) (Creative PEGWorks, Winston Salem, N.C.). In a process, to GNP solution (OD 1) a PEG mixture solution was added at different concentrations to obtain the following final molar ratios: 1, 0.5, 0.1, 0.01 mM. The reaction mixture was vortexed immediately and then incubated at room temperature for two hours. After PEGylation, each solution of 10, 30, 100 nm was centrifuged at 14000, 9400, 8600 rpm, respectively, for 20 min and re-dispersed with Milli-Q water by gentle shaking. Average size of PEG-GNP was confirmed by both dynamic light scattering (DLS) and TEM spectroscopy.

(47) The stability of PEG-GNPs was performed by ZETA potential spectroscopy and was evaluated immediately after preparation and 2, 4 weeks following the preparation with no change.

(48) TABLE-US-00002 TABLE 1 Size and ZETA potential of 10 nm, 30 nm and 100 nm GNPs- compounds as described herein GNP-ABPs Size [d, nm] Zeta [mV] 10 nm 34.39 ± 0.4 −21.1 ± 2.1 30 nm 75.75 ± 0.2 −24.0 ± 1.3 100 nm  137.4 ± 0.1 −25.7 ± 0.4

(49) Characterization of GNP-PEG-GB111 at different sizes of 10, 30, 100 nm is shown in FIG. 1A-1F. FIG. 1A-1C demonstrates the core size of the probe and FIG. 1D-1F show that each probe exhibit a unique wavelength representing its size before (dashed line) and after (full line) chemical modifications. As can be seen, 10 nm-521 nm, 30 nm-524 nm and 100 nm-574 nm. For each size, bare GNPs and GNP-ABPs were measured. The absence of shift indicating GNP stability.

(50) In order to proceed with these GNP-ABPs to biochemical evaluation, a quantitative analysis to determine the amount of peptide on each particle was performed. thermogravimetric analysis (TGA) was used to establish the mass ratio between organic moiety (PEG) and gold atoms. Because PEG has a simple linear-chain-bond structure, which consist of —C—O— and —C—C— in the backbone chain with a similar bond energies (82˜83 kcal/mol), thermal dissociation occurs in a narrow temperature range. The main step is seen at the beginning stands for H.sub.2O evaporation (11 minutes) and partially breakage of PEG bonds. At 330° C. the second step is seen, which represents the PEG burning of 18.9 μg (data not shown).

(51) PEG density.fwdarw.0.21 PEG/nm.sup.2 on 30 nm GNP

(52) Based on these calculations, the number of peptide moiety on each particle was estimated for further biochemical evaluation.

(53) FIGS. 2A to 2G show the library of GNP compounds with different GNP size of 10 nm, 30 nm and 100 nm that were generated. Different percentages of PEG-COOH moiety were used to bind GB111NH.sub.2 (“GB111”). The polyethylene oxide was modified with OMe or COOH groups at one end of the ethylene oxide polymer/oligomer. As the COOH groups were used to bind ABP, the % denoted in each one of the Figs. indicate the % of COOH groups being substituted with ABP and as such the % denoted the % of ABP substituted ethylene oxide polymer/oligomer relative to the total number of ethylene oxide polymer/oligomers on a GNP.

(54) Based on the in vitro results shown herein, the GNP with 10% ABP with ABP substituted on the shorter PEG (3 kDa) and OMe on the 5 kDa PEG was selected for further in vivo analysis with different sizes of 10 nm, 30 nm and 100 nm

(55) Biochemical Evaluations

(56) The biochemical evaluation of these probes started with evaluation for their ability to bind recombinant human cathepsins B. Recombinant human cathepsins B was incubated with increasing concentrations of the probes for 1 hour. Residual activity was detected by a fluorescently labeled cathepsin ABP that was added to samples for 30 minutes at r.t. The free probe was separated from the enzyme-probe complex by SDS PAGE, the detection of the probe-enzyme complex was done by a fluorescent scanning of the gel at Cy5 fluorescence.

(57) FIG. 3 shows blots of GNP-ABP derivatives (structures presented in FIG. 2) indicating that 30 nm GNP-ABP derivatives showed competitive inhibition of recombinant cathepsin B (10 and 100 nm) showed the same pattern, shown in bottom panel for DTS-(iv). Derivatives DTS-(ii) and DTS-(v) baring 50% targeting moiety showed no inhibition activity at all.

(58) However, compound DTS-iv, 10% targeting moiety on 3 kDa PEG, for all particle sizes showed competitive inhibition of recombinant cathepsin B. Size dependent inhibition was observed.

(59) Evaluation of the probes cell permeability and capability of labeling cellular cathepsin enzymes was performed using competitive inhibition assay as well. The competitive inhibition assay was carried out by incubation of NIH-3T3 cells with GNP probes for 4, 6 and 24 hours. Next, the residual cathepsin activity was labeled by GB123 1 μM during 2 hours. Cell lysates were separated on SDS-PAGE followed by scanning the gel for fluorescence at: 650/570 nm to visualize labeling of residual cathepsin activity by GB123. FIG. 4 shows results obtained with different GNP-ABPs in NIH3T3 cells (4T1 cells showed a similar pattern—data not shown) which were treated with indicated probes in growth medium for 4 h followed by labeling of residual enzyme activity by GB123 for 2 hours. A control DTS-(vii) was applied. A dose response inhibition of cathepsin activity was detected with the probes. Size dependent pattern was observed, the smaller the particle—the higher the enzyme inhibition.

(60) Prior to using the GNP-ABPs in non-invasive animal model, their biocompatibility was evaluated on NIH-3T3 and 4T1 cells, selected as model for normal and cancer cells respectively. Viability was determined by methylene blue assay after incubating the cells with increasing concentrations of GNP-ABPs for 24 and 48 hours (FIGS. 5A-5F). As can be seen, GNP-ABPs for 24 and 48 hours displays only minor cytotoxicity with 10 nm probes at the highest concentration tested for 48 hours.

(61) The detection level of the various tags using dual energy micro—CT (Skyscan) was evaluated. Each tag was scanned separately in increasing concentrations (0.010 to 10 mg/ml) using a poly-propylene 384 well plate. FIG. 6 shows HU detected by CT as a function of the contrast agent concentration. As can be seen linear correlation were detected with R.sup.2 above 0.98 for all contrast agents in both low energy 35 keV, and high energy 85 keV. The minimal detected concentration in water are presented in Table 2.

(62) TABLE-US-00003 TABLE 2 The minimal detected concentration of gold, gadolinium and iodine by dual beam micro-CT at high and low energies. [mg/ml] HU Beam E GNP 30 nm 1 29 ± 3.5 35 keV GNP 30 nm 1 20 ± 8.8 85 keV GNP 100 nm 0.5 13 ± 3.5 35 keV GNP 100 nm 0.1 16 ± 8.8 85 keV Gadolinium 0.05 17 ± 3.5 35 keV Gadolinium 0.1 38 ± 8.8 85 keV Iodine 0.05 10 ± 3.5 35 keV Iodine 0.5 15 ± 8.8 85 keV
Non-Invasive Imaging of Cancer in Mice Modules

(63) To determine the efficacy of targeting compared to passive mechanism of GNP-ABPs syngeneic mouse model was used. 4T1 cells were injected subcutaneously on the back of 3-4 week-old male BALB/c mice under isoflurane anesthesia. After tumors were established, each mouse received 200 μl of a 25 mg/ml solution of targeted or control GNP-ABPs by tail vain injection. The mice were scanned before, at 24, 48 and 72 hours post injection by a Micro-CT scanner.

(64) The need to perform comparative analysis between different mice injected with different size of GNPs, lead us to seek for different approach than HU quantification that might be misleading in this case. The diversity in size tumor dictates different X-ray absorption values in different mice as well as the same mouse scanned at different time points. Larger tumor, exhibit higher HU values derived from tumor volume but not necessary due to GNP uptake per gram tissue. To quantify the exact amount of GNPs that reached the tumor tissue in each time point the tissue absorbance and GNP absorbance were analyzed for the same ROI apart. The number of voxels for soft tissue absorbance characterized by low energies, hence was defined by us as 25-255 in gray scale, while gold (and bone) 53-255 for higher energies. The minimum threshold for gold detection was derived from pre-scan of the mouse, showing no voxels in the tumor at that grayscale index. After total voxel number was derived from the image for each range, the labeling percentage of voxels per each tumor was calculated (% labeling=num. of vox.sub.53-255/num. of vox.sub.25-255). Significant CT signal enhancement from the tumor could be detected after 24 hours for targeted 10 nm and 30 nm GNP-ABP probe than that of non-targeted nanoparticle (without ABP). This specific signal increased over time and became highly elevated at 72 hours post probe injection excluding the 100 nm GNP-ABP, which exhibited poor active and passive accumulation through all time points as presented in volume rendered 3D and 2D axial cross section images FIG. 7A-7L. The smaller non targeted ABPs accumulated to certain steady state concentration due to EPR effect, slightly enhancing the CT signal. Accumulation of targeted-GNP showed a uniform accumulation in the peripheral region of the tumor, presumably due to solid nature of the tumor. Derived images reflected a pattern of which larger size of GNP-ABPs, lead to lower tumor uptake. The highest signal enhancement was detected with 10 nm targeted ABP.

(65) Bio-Distribution of GNP-ABPs

(66) Probe distribution was monitored to follow pharmacokinetics and signal accumulation in the tumors. After the last time point, the mice were sacrificed and the tumor and other selected organs were taken to inductively coupled plasma mass spectrometry (ICP-MS) tests to determine the gold amount accumulated in each organ. As seen in FIG. 8D-8F, GNP-ABPs showed gold uptake in the spleen, kidney and liver for both pathways: passive and active. As previously published, contrast agents larger than 6 nm avoid renal clearance, hence excreted from the blood pool by phagocytic cells in the reticuloendothelial system, occurring primarily in the liver and spleen leading to accumulation of contrast in those organs. In addition, in our case, targeted GNPs' liver uptake could be explain by high cathepsins' activity. However, despite the targeting of the GNPs, the maximal tumor uptake was lower than it was in the liver and spleen and comparable to kidneys accumulation only for 10 nm GNP-ABPs (FIG. 8A-8F). Previous publications showed that higher PEG concentration on the GNP surface, decreases the spleen accumulation and enhances tumor and liver uptake. Based on the chemical evaluation, it was suspected that the GNP-ABPs developed here are in low density “mushroom” configuration, hence the high spleen uptake, dominantly for 100 nm ABPs. Tumor uptake was prevalently stronger for targeted GNP-ABPs derived from ICP-MS analysis and perfectly consistent with average tumor uptake extracted from CT images (100 nm GNP-ABPs only ICP-MS data). Both 10 nm and 30 nm T-GNP showed significant higher accumulation in tumors compared to NT-GNP.

(67) FIGS. 9A and 9B demonstrates the comparison for targeted ABPs, revealing the strong size dependent uptake in favor of small GNPs. The highest signal was detected with the 10 nm probe. The gold signal in the tumor increased over time and became highly elevated at 72 h post injection. 60 nm probe showed lower accumulation within the tumor, nevertheless both 10 and 30 nm showed significant priority compared to 100 nm probe. Furthermore, the number of voxels containing gold were reflected by ICP-MS analysis. 10 nm GNP-ABPs showed highest liver uptake, raising the necessity of better understanding of long term toxicity. One might suggest that smaller particles can't produce efficient contrast enhancement due to lower local density, however it was suggested their compensation ability by higher accumulation.

(68) Other tumors part were taken to TEM to visualize GNP probes accumulation in biological tissue. Tumor was resected 72 h post IV injection of the probe. FIGS. 10A-10F shows that uptake of targeted CNP-ABPs by the tumor was clearly visualized for 10 nm & 30 nm proposing their accumulation in the lysosomes known in reach cathepsin activity. A small amount of non-targeted GNPs (without ABPs) was also observed due to passive uptake. For 100 nm n: difference was observed between passive and active accumulation (i.e GNP with ABP or without), possibly due to extensive spleen accumulation leading to short blood circulation hence low availability of GNP-ABPs. The images depict localization within the endosome/lysosome within the tumor cells. TEM results support one more time that the CT attenuation increase, derives from GNP-ABPs localization within the tumor cells.

Example 2: Iodine Derivatives of Activity Based Probe

(69) General methods: Unless otherwise noted, all resins and reagents were purchased from commercial suppliers and used without further purification. Iopanoic acid was purchased from TCI, (Japan). All water-sensitive reactions were performed in anhydrous solvents under positive pressure of nitrogen or argon. Reactions were analyzed by a Liquid Chromatography Mass Spectrometer (LC-MS) (Thermo Scientific MSQ-Plus), attached to an Accela UPLC system. Unless otherwise stated, all final compounds were purified by C18 reverse phase HPLC in acetonitrile/water gradient. Proton NMR spectra were recorded on Varian Mercury 500 MHz spectrometer in deuterated solvent. Proton chemical shifts are reported in ppm (δ) relative to tetramethylsilane with the solvent resonance employed as the internal standard (DMSO-d.sup.6, δ 7.26 ppm). Recombinant human cathepsins B and L were prepared as described. The animal ethics committee of the Hebrew University approved all animal procedures. Male BALB/c mice (3-4 week-old) were used in all in vivo assays, 3 mice were used for each compound, repeating each experiment at least three times.

(70) Synthesis of 2,5-dioxopyrrolidin-1-yl 2,3,5-triiodobenzoate (11): To a solution of 2,3,5-triiodobenzoic acid (0.2 mmol, 1 eq.) in anhydrous CH.sub.2Cl.sub.2 (5 mL) under argon were added N-hydroxysuccinimide (0.4 mmol, 2 eq.), DIEA (0.6 mmole, 3 eq.) and EDC-HCl (0.2 mmol, 2 eq.). The reaction mixture was stirred for 3 h at ambient temperature. DCM was added to the crude residue and the resulting organic phase was washed with water, brine and saturated NaHCO.sub.3. The organic phase was dried over MgSO.sub.4 and solvent was removed in vacuo. Crude product was purified by C18 reverse phase HPLC using water-acetonitrile gradient with 0.1% TFA, product was eluted with 70% acetonitrile, to obtain (11) as a white solid (0.16 mmol, 82% yield).

(71) Synthesis of 2-(3-acetamido-2,4,6-triiodobenzyl)butanoic acid (12): To a solution of Iopanoic acid (0.88 mmol, 1 eq.) in tetrahydrofuran (10 mL) were added acetyl chloride (1.76 mmol, 2 eq.) and the solution was stirred at room temperature for 18 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2, washed with water and brine. The organic phase was dried over MgSO.sub.4 and solvent was removed in vacuo. The crude product (0.81 mmol, 92%) obtained was used for step without purification.

(72) LCMS: RT 2.13 (linear gradient 5-95% of ACN/H.sub.2O, 7 min), ESI-MS (m/z): 613.66 [M+H].sup.+, 635.58 [M+Na].sup.+.

(73) Synthesis of 2,5-dioxopyrrolidin-1-yl 2-(3-acetamido-2,4,6-triiodobenzyl) butanoate (13): To a solution of 12 (0.81 mmol, 1 eq.) in anhydrous CH.sub.2Cl.sub.2 (10 mL) under argon were added N-hydroxysuccinimide (1.62 mmol, 2 eq.), DIEA (2.43 mmol, 3 eq.) and EDC-HCl (1.62 mmol, 2 eq.). The reaction mixture was stirred for 3 h at ambient temperature. CH.sub.2Cl.sub.2 was added to the crude residue and the resulting organic phase was washed with water, brine and saturated NaHCO.sub.3. The organic phase was dried over MgSO.sub.4 and solvent was removed in vacuo. The crude product (13) was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, the product was eluted with 60% acetonitrile, to obtain Iopanoic Acid-Succinamide Ester (IPA-SE, 13) as a white solid (0.58 mmol, 72% yield).

(74) LCMS: RT 2.34 (linear gradient 5-95% of ACN/H.sub.2O, 7 min), ESI-MS (m/z): 711.51 [M+H].sup.+, 733.50 [M+Na].sup.+.

(75) .sup.1H-NMR (300 MHz, DMSO-d.sup.6): δ 9.96 (d, J=9.8 Hz, 1H, amide NH), 8.40 (s, 1H, Ar—H), 3.45 (m, 2H, CH.sub.2), 3.10 (m, 1H, CH), 4.32 (m, 2H), 2.81 (s, 4H, 2XCH.sub.2), 2.03 (s, 3H, CH.sub.3), 1.81 (s, 1H, CH), 1.47 (m, 1H, CH), 0.93 (t, 3H, CH.sub.3).

(76) Synthesis of Iodide CT-ABP (14a-b): To a solution of GB111NH.sub.2 (0.009 mmol, 1 eq.) were added iodine tag SE (0.14 mmol, eq. 1.5), DIEA (0.027, 3 eq.) in anhydrous DMSO (10 mL) and the solution was stirred at room temperature for 12 h. The reaction mixture was diluted with CH.sub.2C.sub.2 (5 mL), washed with water, brine and saturated NaHCO.sub.3. The organic phase was dried over MgSO.sub.4 and solvent was removed in vacuo. The crude product was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, the product was eluted with 65% acetonitrile, to obtain (14a-b) as a white solid.

(77) 14a: LCMS: RT 3.51 (linear gradient 5-95% of ACN/H.sub.2O, 7 min), ESI-MS (m/z): 1057.16 [M+2].sup.+, 1079.49 [M+H+Na].sup.+.

(78) 14b: LCMS: RT 3.22 (linear gradient 5-95% of ACN/H.sub.2O, 7 min), ESI-MS (m/z): 1170.63 [M+2].sup.+.

(79) Synthesis of 5-benzyl-8-(2-((2,6-dimethylbenzoyl)oxy)acetyl)-3,6,14-trioxo-1-phenyl-2-oxa-4,7,13-triazaheptadecan-17-oic acid (15): To a solution of GB111-NH.sub.2 (0.087 mmol, 1 eq.) in CH.sub.2Cl.sub.2 (5 mL) were added succinic anhydride (0.096 mmol, 1.1 eq.) and the solution was stirred at room temperature for 3 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2, washed with water and brine. The organic phase was dried over MgSO.sub.4 and solvent was removed in vacuo. The crude product was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, the product was eluted with 57% acetonitrile, to obtain (15) as a white solid (0.074 mmol, 85% yield).

(80) LCMS: RT 2.69 (linear gradient 5-95% of ACN/H.sub.2O, 7 min), ESI-MS (m/z): 674.93 [M+H].sup.+.

(81) Synthesis of GB111NH.sub.2-PAMAM-Go (16): To a solution of PAMAM-Go (0.3 mmol, 1.5 eq.) were added mixture of 15 (0.2 mmol, 1 eq.), PyBOP (0.4 mmol, 2 eq.), HOBt (0.4 mmol, 2 eq.) and DIEA (0.6 mmol, 3 eq.) in anhydrous DMF at 0° C. Then the reaction mixture was stirred for 12 h at ambient temperature. DCM was added to the crude residue and the resulting organic phase was washed with water, brine and saturated NaHCO.sub.3. The organic phase was dried over MgSO.sub.4 and solvent was removed in vacuo. The crude product was purified by C4 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, the product was eluted with 25% acetonitrile, to obtain (16) as a white solid (0.134 mmol, 67% yield).

(82) LCMS: RT 1.53 (linear gradient 5-95% of ACN/H.sub.2O, 7 min), ESI-MS (m/z): 1173.27 [M+H].sup.+.

(83) Synthesis of Iodide CT-ABP (17a-b): To a solution of 16 (0.009 mmol, 1 eq.) were added iodine tag SE (0.14 nmol, 3.0 eq.), DIEA (0.027, 3 eq.) in anhydrous DMSO (2 mL) and the solution was stirred at room temperature for 12 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (5 mL), washed with water, brine and saturated NaHCO.sub.3. The organic phase was dried over MgSO.sub.4 and solvent was removed in vacuo. The crude product was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, the product was eluted with 65% acetonitrile, to obtain (17a-b) as a white solid.

(84) 17a: LCMS: RT 2.80 (linear gradient 5-95% of ACN/H.sub.2O, 7 min), ESI-MS (m/z): 1310.40 [M/2+2].sup.+.

(85) 17b: LCMS: RT 2.53 (linear gradient 5-95% of ACN/H.sub.2O, 7 min), ESI-MS ((m/z): 1479.89 [M/2+H].sup.+.

(86) Synthesis of Iodide CT-ABP (18): To a solution of 16 (0.009 mmol, 1 eq.) were added Iodine tag SE (13, 0.14 mmol, 1.0 eq), DIEA (0.027, 3 eq.) in anhydrous DMSO (2 mL) and the solution was stirred at room temperature for 12 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (5 mL), washed with water, brine and saturated NaHCO.sub.3. The organic phase was dried over MgSO.sub.4 and solvent was removed in vacuo. The crude product was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, product was eluted with 65% acetonitrile, to obtain (18) as a white solid (0.0051 mmol, 57% yield).

(87) LCMS: RT 12.07 (linear gradient 30-95% of ACN/H.sub.2O, 20 min), ESI-MS (m/z): 1181.40 [M+H].sup.+.

(88) Synthesis of 5-benzyl-8-(2-((2,6-dimethylbenzoyl)oxy)acetyl)-3,6,14-trioxo-1-phenyl-2-oxa-4,7,13-triazaheptadecan-17-oic acid (19): To a solution of GB111-NH.sub.2 (0.087 mmol, 1 eq.) in CH.sub.2Cl.sub.2 (5 mL) were added glutaric anhydride (0.096 mmol, 1.1 eq.) and the solution was stirred at room temperature for 3 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2, washed with water and brine. The organic phase was dried over MgSO.sub.4 and solvent was removed in vacuo. The crude product was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, the product was eluted with 57% acetonitrile, to obtain (19) as a white solid (0.074 mmol, 95%).

(89) LCMS: RT 2.65 (linear gradient 5-95% of ACN/H.sub.2O, 7 min), ESI-MS (m/z): 688.35 [M+H].sup.+, 710.38 [M+Na].sup.+.

(90) Synthesis of GB111NH-SE (20): To a solution of 19 (0.81 mmol, 1 eq.) in anhydrous DMF (5 mL) under argon were added N-hydroxysuccinimide (1.62 mmol, 2 eq.), DIEA (2.43 mmol, 3 eq.) and EDC-HCl (1.62 mmol, 2 eq.). The reaction mixture was stirred for 12 at ambient temperature. Ethyl acetate was added to the crude residue and the resulting organic phase was washed with water, brine and saturated NaHCO.sub.3. The organic phase was dried over MgSO.sub.4 and solvent was removed in vacuo. The crude product was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, product was eluted with 65% acetonitrile, to obtain (20) as a white solid (0.58 mmol, 82% yield).

(91) LCMS: RT 2.81 (linear gradient 5-95% of ACN/H.sub.2O, 7 min), ESI-MS (m/z): 785.08 [M+H].sup.+, 817.00 [M+N].sup.+.

(92) .sup.1H-NMR (300 MHz, DMSO-d.sup.6): δ 8.50 (d, J=7.2 Hz, 1H, amide NH), 7.79 (s, 1H, amide NH), 7.64 (d, J=7.9 Hz, 1H, amide NH), 7.27 (m, 10H, Ar—H), 7.20 (m, 1H, Ar—H), 7.10 (d, J=15.4 Hz, 2H, Ar—H), 5.09 (d, J=10.1 Hz, 1H, CH Aomk), 4.95 (s, 2H, CH.sub.2), 4.84 (m, 2H, 2XCH), 4.32 (m, 2H), 3.00 (s, 2H, CH.sub.2), 2.79 (s, 4H, 2XCH.sub.2), 2.64 (s, 2H, CH.sub.2), 2.29 (s, 6H, CH.sub.3), 2.15 (s, 2H, CH.sub.2), 1.80 (s, 2H, CH.sub.2), 1.54 (m, 2H, CH.sub.2), 1.35 (m, 2H, CH.sub.2), 1.28 (m, 2H, CH.sub.2).

(93) Synthesis of six, seven and eight iodine tagged compounds (22, 23, 24): To a solution of PAMAM G1 (0.009 mmol, 1 eq.) were added IPA-SE (13, 0.14 mmol, 4.0 eq), DIEA (0.027, 3 eq.) in anhydrous DMSO (2 mL) and the solution was stirred at room temperature for 12 h. The reaction mixture was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, product was eluted with 45% acetonitrile, to obtain six, seven and eight iodine tagged products (22, 23, 24 respectively) as a white solid (12, 9, 22% yields receptively).

(94) 22: LCMS: RT 2.71 (linear gradient 5-95% of ACN/H.sub.2O, 10 min), ESI-MS (m/z): 1250.91 [M/4+H].sup.+, 1000.89 [M/5+H].sup.+.

(95) 23: LCMS: RT 2.80 (linear gradient 5-95% of ACN/H.sub.2O, 10 min), ESI-MS (m/z): 1399.56 [M/4+H].sup.+.

(96) 24: LCMS: RT 2.90 (linear gradient 5-95% of ACN/H.sub.2O, 7 min), ESI-MS (m/z): 1548.08 [M/4+H].sup.+.

(97) Synthesis of six iodine tagged probe HG81 (25): To a solution of 22 (0.009 mmol, 1 eq.) were added GB111NH-SE (20, 0.14 mmol, 1.0 eq), DIEA (0.027, 3 eq.) in anhydrous DMSO (2 mL) and the solution was stirred at room temperature for 12 h. The reaction mixture was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, the product was eluted with 41% acetonitrile, to obtain HG81 (25) and HG81a (26) as a white solid.

(98) HG81 (25): LCMS: RT 3.27 (linear gradient 5-95% of ACN/H.sub.2O, 10 min), ESI-MS (m/z): 1885.76 [M/3+H].sup.+, 414.71 [M/4+H].sup.+.

(99) HG81a (26): LCMS: RT 3.82 (linear gradient 5-95% of ACN/H.sub.2O, 10 min), ESI-MS (m/z): 1578.45[M/4+H].sup.+.

(100) Synthesis of seven iodine tagged probe, HG78 (27): To a solution of 23 (0.009 mmol, 1 eq.) were added GB111NH-SE (20, 0.14 mmol, 1.0 eq), DIEA (0.027, 3 eq.) in anhydrous DMSO (2 mL) and the solution was stirred at room temperature for 12 h. The reaction mixture was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, the product was eluted with 47% acetonitrile, to obtain HG78 (27) as a white solid (0.0074 mmol, 81% yield).

(101) LCMS: RT 3.53 (linear gradient 5-95% of ACN/H.sub.2O, 10 min), ESI-MS (m/z): 1563.72 [M/3+H].sup.+.

(102) Synthesis of Cy5 labeled I-CT-ABP, HG92 (28): To a solution of above, six iodine tagged activity based probe (25, 0.009 mmol, 1 eq.) were added Cy5-NHS ester (0.14 mmol, 1.0 eq), DIEA (0.227, 3 eq.) in anhydrous DMSO (2 mL) and the solution was stirred at room temperature for 12 h. The reaction mixture was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, the product was eluted with 45% acetonitrile, to obtain HG92 (28) as a blue solid (0.007 mmol, 78% yield).

(103) LCMS: RT 3.14 (linear gradient 5-95% of ACN/H.sub.2O, 10 min), ESI-MS (m/z): 1577.83 [M/4+H].sup.+.

(104) Synthesis of Cy5 labeled control I-CT-ABP, HG31 (29): To a solution of 23 (0.009 mmol, 1 eq.) were added Cy5-NHS ester (0.14 mmol, 1.0 eq), DIEA (0.027, 3 eq.) in anhydrous DMSO (2 mL) and the solution was stirred at room temperature for 12 h. The reaction mixture was purified by C18 reverse phase HPLC using a water-acetonitrile gradient with 0.1% TFA, the product was eluted with 51% acetonitrile, to obtain (29) as a blue solid (0.0077 mmol, 86% yield).

(105) LCMS: RT 2.92 (linear gradient 5-95% of ACN/H.sub.2O, 10 min), ESI-MS (m/z): 1558.83 [M/4+H].sup.+, 1247.25 [M/5+H].sup.+.

(106) Synthesis of PAMAM-G3 conjugated nanometric probes (I-CT-ABP): To a solution of G3 PAMAM dendrimers ((NH.sub.2).sub.32-G3; PAMMA-G3; 100 mg; 0.014 mmol) DIEA (0.46 mmol, 32 eq.) in anhydrous DMSO (3 mL) was added IPA-SE (13, 0.23 mmol, 16.0 eq) and the mixture was stirred at room temperature for 12 h. The resulting mixture was first purified by 8 kD dialysis against Methanol and DI water for 3 runs each and then lyophilized to yield (IPA).sub.16(NH.sub.2).sub.16-G3 (30) as a sticky white solid (211 mg; 88%). To the resulting conjugate, (IPA).sub.16(NH.sub.2).sub.16-G3 (30, 200 mg, 0.0122 mmol, 1.0 eq)), was added GB111NH-SE (20, 0.0122 mmol, 1.0 eq), DIEA (0.39 mmol, 32 eq.) in anhydrous DMSO (2 mL) and the mixture was stirred at room temperature for 2 h. The mixture was then purified by 8 kD dialysis against Methanol and DI water for 3 runs each and then lyophilized to yield (IPA).sub.16(NH.sub.2).sub.15(GB).sub.1-G3 (31) as a sticky white solid (190 mg; 91%). To the protease activated conjugate, (IPA).sub.16(NH.sub.2).sub.15(GB).sub.1-G3 (31, 20 mg, 0.0012 mmol, 1.0 eq), was added acetic anhydride (0.037 mmol, 32 eq), DIEA (0.075 mmol, 64 eq.) in anhydrous DMSO (2 mL) and the mixture was stirred at room temperature for 12 h. The mixture was then purified by 8 kD dialysis against Methanol and DI water for 3 runs each and then lyophilized to yield (IPA).sub.16(GB).sub.1 (Ac).sub.15-G3 (32) as a brown solid (19 mg; 91%). To the protease activated conjugate, (IPA).sub.16(NH.sub.2).sub.15(GB).sub.1-G3 (31, 20 mg, 0.0012 mmol, 1.0 eq), was added PEG(4) and PEG750 NHS ester (0.037 mmol, 32 eq), DIEA (0.075 mmol, 64 eq.) in anhydrous DMSO (2 mL) separately and the mixture was stirred at room temperature for 12 h. The each reaction mixture was then purified by 8 kD dialysis against Methanol and DI water for 3 runs each and then lyophilized to yield (IPA).sub.16(GB).sub.1(PEG-4).sub.15-G3 (33) as a white solid (20 mg; 83.9%) and (IPA).sub.16(GB).sub.1(PEG-750).sub.15-G3 (34) as a white solid (28 mg; 89.9%). A negative controls, G3 dendrimers without GB111 synthesis: To the free amine conjugate, (IPA).sub.16(NH.sub.2).sub.16-G3 (30, 20 mg, 0.0012 mmol, 1.0 eq), was added acetic anhydride (0.039 mmol, 32 eq), DIEA (0.078 mmol, 64 eq.) in anhydrous DMSO (2 mL) and the mixture was stirred at room temperature for 12 h. The mixture was then purified by 8 kD dialysis against Methanol and DI water for 3 runs each and then lyophilized to yield (IPA).sub.16(Ac).sub.16-G3 (35) as a brown solid (18 mg; 86.3%). To the free amine conjugate, (IPA).sub.16(NH.sub.2).sub.16-G3 (30, 20 mg, 0.0012 mmol, 1.0 eq), was added PEG(4) and PEG750 NHS ester (0.039 mmol, 32 eq), DIEA (0.078 mmol, 64 eq.) in anhydrous DMSO (2 mL) separately and the mixture was stirred at room temperature for 12 h. The mixture was then purified by 8 kD dialysis against Methanol and DI water for 3 runs each and then lyophilized to yield (IPA).sub.16(PEG-4).sub.16-G3 (36) as a white solid (21 mg; 86.6%) and (IPA).sub.16(PEG-750).sub.16-G3 (37) as a white solid (29 mg; 89.5%). The number of conjugated IPA molecules per dendrimer was determined to be 16, on average using .sup.1H-NMR spectroscopy. The characteristic peaks at δ (ppm) 7-8 belong to the phenyl proton (16 H's) of the aromatic ring of IPA. The number of conjugated GB111 molecules per dendrimer was determined to be one, on average, using 1H-NMR spectroscopy. The characteristic peaks at δ (ppm) 7-8 belong to the dimethyl proton (6 H's) of the aromatic ring of GB111. The number of conjugated Acetyl, PEG(4) and PEG-750 per dendrimer was determined to be on average, using .sup.1H-NMR spectroscopy.

(107) HG87 (30): .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ 9.89 (bs, 16H, amide NH), 8.31 (s, 16H, Ar—H), δ 7.94-7.70 (brs, 60H, amide protons), 3.23-2.91 (bs, ˜120H), 2.71-2.53 (brs, ˜184H), 2.42-2.29 (brs, ˜58H), 2.23-2.07 (bs, ˜120H), 2.03 (s, 48H, CH.sub.3), 1.61 (m, 16H, CH), 1.30 (m, 16H, CH), 0.71 (t, 48H, CH.sub.3).

(108) HG82 (31): .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ 9.93 (bs, 16H, amide NH), 8.35 (s, 16H, Ar—H), δ 7.96-7.76 (brs, 60H, amide protons), 7.34-7.23 (m, 10H, Ar—H), 7.23-7.16 (m, 3H, Ar—H), 7.13-7.07 (m, 3H, Ar—H), 5.78 (m, 2H, CH.sub.2), 3.53-2.96 (bs, ˜120H), 2.71-2.53 (brs, ˜184H), 2.46-2.33 (m, ˜58H), 2.31 (s, 8H, CH.sub.3), 2.26-2.07 (bs, ˜120H), 2.02 (s, 48H, CH.sub.3), 1.87 (s, 2H, CH.sub.2), 1.68 (m, 16H, CH), 1.54 (m, 2H, CH.sub.2), 1.35 (m, 16H, CH), 1.21 (m, 2H, CH.sub.2), 0.71 (t, 48H, CH.sub.3).

(109) HG86 (32): .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ 9.91 (bs, 16H, amide NH), 8.34 (s, 16H, Ar—H), 8.01-7.71 (m, 60H, amide protons), 7.33-7.24 (m, 13H, Ar—H), 7.21-7.17 (m, 3H, Ar—H), 7.11-7.07 ((m, 3H, Ar—H), 4.96 (m, 2H, CH.sub.2), 3.25-3.15 (m, ˜120H), 3.14-2.99 (brs, ˜154H), 2.76-2.53 (m, ˜120H), 2.46-2.33 (m, ˜58H), 2.30 (s, 9H, CH.sub.3), 2.27-2.10 (brs, ˜120H), 2.01 (s, 48H, COCH.sub.3), 1.79 (s, 42H, COCH.sub.3), 1.90 (s, 4H, CH.sub.2), 1.66 (m, 16H, CH), 1.52 (m, 4H, CH.sub.2), 1.34 (m, 16H, CH), 1.24 (m, 2H, CH.sub.2), 0.74 (t, 48H, CH.sub.3).

(110) HG95 (33): .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ 9.92 (bs, 16H, amide NH), 8.35 (s, 16H, Ar—H), 5 7.95-7.76 (brs, 60H, amide protons), 7.33-7.23 (m, 15H, Ar—H), 7.22-7.18 (m, 4H, Ar—H), 7.12-7.08 (m, 4H, Ar—H), 4.97 (brs, 2H, CH.sub.2), 3.52-3.44 (brs, 20H), 3.25-3.15 (brs, 50H), 3.14-2.99 (bs, 120H), 2.72-2.59 (brs, ˜108H), 2.45-2.34 (brs, ˜58H), 2.33-2.27 (brs, ˜40H), 2.26-2.11 (brs, ˜120H), 2.01 (s, 48H, CH.sub.3), 1.66 (m, 16H, CH), 1.53 (m, 2H, CH.sub.2), 1.33 (m, 16H, CH), 1.23 (m, 2H, CH.sub.2), 0.75 (t, 48H, CH.sub.3).

(111) HG96 (34): .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ 9.93 (bs, 16H, amide NH), 8.35 (s, 16H, Ar—H), δ 7.95-7.81 (brs, 60H, amide protons), 7.34-7.24 (m, 16H, Ar—H), 7.22-7.18 (m, 4H, Ar—H), 7.11-7.08 (m, 4H, Ar—H), 4.97 (bs, 2H, CH.sub.2), 3.57-3.46 (bs, ˜500H), 3.27-3.43 (m, ˜400H), 3.25-3.15 (brs, 120H), 3.14-3.01 (brs, ˜120H), 2.42-2.35 (brs, ˜58H), 2.33-2.27 (brs, ˜40H), 2.34-2.19 (brs, ˜120H), 2.02 (s, 48H, CH.sub.3), 1.66 (m, 16H, CH), 1.54 (m, 2H, CH.sub.2), 1.34 (m, 16H, CH), 1.23 (m, 2H, CH.sub.2), 0.75 (t, 48H, CH.sub.3).

(112) HG33 (35): .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ 9.89 (bs, 16H, amide NH), 8.31 (s, 16H, Ar—H), 8.03-7.72 (m, 60H, amide protons), 7.10-7.06 (m, 3H, Ar—H), 3.23-3.16 (m, ˜120H), 3.13-2.98 (brs, ˜154H), 2.77-2.52 (m, ˜120H), 2.47-2.32 (m, ˜58H), 2.26-2.11 (brs, ˜120H), 2.01 (s, 48H, COCH.sub.3), 1.78 (s, 42H, COCH.sub.3), 1.64 (m, 16H, CH), 1.32 (m, 16H, CH), 0.75 (t, 48H, CH.sub.3).

(113) HG94 (36): .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ 9.95 (brs, 16H, amide NH), 8.36 (s, 16H, Ar—H), δ 7.94-7.70 (brs, 60H, amide protons), 3.52-3.46 (brs, ˜160H), 3.23-2.91 (brs, ˜120H), 2.71-2.53 (brs, ˜184H), 2.42-2.29 (brs, ˜58H), 2.23-2.07 (brs, ˜120H), 2.02 (s, 48H, CH.sub.3), 1.68 (m, 16H, CH), 1.35 (m, 16H, CH), 0.75 (t, 48H, CH.sub.3).

(114) HG97 (37): .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ 9.91 (bs, 16H, amide NH), 8.34 (s, 16H, Ar—H), δ 7.96-7.80 (brs, 60H, amide protons), 3.59-3.45 (bs, ˜500H), 3.42-3.25 (m, ˜400H), 3.23-3.14 (brs, 120H), 3.12-2.98 (brs, ˜120H), 2.41-2.33 (brs, ˜58H), 2.31-2.18 (brs, ˜120H), 2.01 (s, 48H, CH3), 1.67 (m, 16H, CH), 1.33 (m, 16H, CH), 0.73 (t, 48H, CH3).

(115) Synthesis of Cy5 labelled PAMAM-G3 conjugated I-CT-ABP: To the protease activated conjugate, (IPA).sub.16(NH.sub.2).sub.15(GB).sub.1-G3 (31, 30 mg, 0.0017 mmol, 1.0 eq), was added Cy5-NHS ester (0.0017 mmol, 1.0 eq), DIEA (0.112 mmol, 64 eq.) in anhydrous DMSO (2 mL) and the mixture was stirred at room temperature for 2 h, then acetic anhydride (0.056 mmol, 32 eq), was added to the reaction mixture and stirred at room temperature for 12 h. The reaction mixture was then purified by 8 kD dialysis against methanol and DI water for 3 runs each and then lyophilized to yield (IPA).sub.16(GB).sub.1(Cy5).sub.1(Ac).sub.14-G3 (38) as a blue solid (25 mg; 78.1%). To the protease activated conjugate, (IPA).sub.16(NH.sub.2).sub.15(GB).sub.1-G3 (31, 20 mg, 0.0012 mmol, 1.0 eq), was added Cy5-NHS ester (0.0012 mmol, 1.0 eq), DIEA (0.075 mmol, 64 eq.) in anhydrous DMSO (2 mL) and the mixture was stirred at room temperature for 2 h, and then PEG750 NHS ester (0.037 mmol, 32 eq), was added to the reaction mixture and stirred at room temperature for 12 h. The mixture was then purified by 8 kD dialysis against methanol and DI water for 3 runs each and then lyophilized to yield (IPA).sub.16(GB).sub.1(Cy5).sub.1(PEG-750).sub.14-G3 (39) as a blue solid (27 mg; 86.8%). A negative controls, G3 dendrimers without GB111 synthesis: To the free amine conjugate, (IPA).sub.16(NH.sub.2).sub.16-G3 (30, 20 mg, 0.0012 mmol, 1.0 eq), was added Cy5-NHS ester (0.0012 mmol, 1.0 eq), DIEA (0.078 mmol, 64 eq.) in anhydrous DMSO (2 mL) and the mixture was stirred at room temperature for 2 h, then acetic anhydride (0.039 mmol, 32 eq), was added to the reaction mixture and stirred at room temperature for 12 h. The reaction mixture was then purified by 8 kD dialysis against methanol and DI water for 3 runs each and then lyophilized to yield (IPA).sub.16(Cy5).sub.1(Ac).sub.15-G3 (40) as a blue solid 19 mg; 89.3%). To the free amine conjugate, (IPA).sub.16(NH.sub.2).sub.16-G3′ 30, 20 mg, 0.0012 mmol, 1.0 eq), was added Cy5-NHS ester (0.0012 mmol, 1.0 eq), DIEA (0.078 mmol, 64 eq.) in anhydrous DMSO (2 mL) and the mixture was stirred at room temperature for 2 h, and then PEG750 NHS ester (0.039 mmol, 32 eq), was added to the reaction mixture and stirred at room temperature for 12 h. The mixture was then purified by 8 kD dialysis against methanol and DI water for 3 runs each and then lyophilized to yield (IPA).sub.16 (Cy5).sub.1(PEG-750).sub.15-G3 (41) as a blue solid (26 mg; 83.3%). The number of conjugated IPA molecules per dendrimer was determined to be 16, on average using 1H-NMR spectroscopy. The characteristic peaks at δ (ppm) 8.1 belong to the phenyl proton (16 H's) of the aromatic ring of IPA. The number of conjugated GB111 molecules per dendrimer was determined to be one, on average, using .sup.1H-NMR spectroscopy. The characteristic peaks at δ (ppm) 7-8 belong to the dimethyl proton (6 H's) of the aromatic ring of GB111. The number of conjugated Cy5 dyes per dendrimer was determined to be 1, on average, using spectrophotometry. The number of conjugated Acetyl and PEG-750 per dendrimer was determined to be on average, using 1H-NMR spectroscopy.

(116) HG90 (38): .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ 9.93 (bs, 16H, amide NH), 9.86 (m, 6H, cy5H), 9.43 (m, 9H, cy5H), 9.33 (m, 3H, cy5H), 8.35 (s, 16H, Ar—H), 8.24 (m, 4H, cy5H), δ 7.95-7.76 (brs, cOH, amide protons), 7.65 (m, 8H, cy5H), 7.34-7.24 (m, 14H, Ar—H), 7.22-7.18 (m, 3H, Ar—H), 7.11-7.08 (m, 3H, Ar—H), 6.97 (m, 5H, cy5H), 6.79 (m, 9H, cy5H), 6.31 (m, 8H, cy5H), 4.97 (brs, 2H, CH.sub.2), 4.33 (m, 12H, cy5H), 3.26-3.15 (bs, ˜60H), 3.24 (brs, ˜60H), 3.14-3.09 (brs, ˜120H), 2.74-2.56 (brs, ˜120H), 2.46-2.35 (brs, ˜60H), 2.30 (s, 20H), 2.26-2.13 (brs, ˜120H), 2.02 (s, 48H, CH.sub.3), 1.97 (s, 4H), 1.80 (s, 45H, CH.sub.3), 1.69 (m, 16H, CH), 1.54 (m, 2H, CH.sub.2), 1.34 (m, 16H, CH), 1.24 (m, 2H, CH.sub.2), 1.14 (s, ˜20H), 0.75 (t, 48H, CH.sub.3).

(117) HG93 (39): .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ 9.93 (bs, 16H, amide NH), 9.85 (m, 6H, cy5H), 9.46 (m, 9H, cy5H), 9.34 (m, 3H, cy5H), 8.35 (s, 16H, Ar—H), δ 7.95-7.76 (brs, 60H, amide protons), 7.34-7.24 ((m, 14H, Ar—H), 7.22-7.18 (m, 3H, Ar—H), 7.11-7.08 (m, 3H, Ar—H), 6.95 (m, 5H, cy5H), 6.80 (m, 9H, cy5H), 6.33 (m, 8H, cy5H), 4.97 (brs, 2H, CH.sub.2), 3.57-3.46 (bs, ˜400H), 3.24 (brs, ˜60H), 3.20-3.16 (brs, 120H), 3.14-3.01 (brs, ˜120H), 2.64 (brs, ˜50H), 2.33-2.27 (brs, ˜58H), 2.34-2.19 (brs, ˜120H), 2.02 (s, 48H, CH.sub.3), 1.68 (m, 16H, CH.sub., 1.54 (m, 2H, CH.sub.2), 1.34 (m, 16H, CH), 1.24 (m, 2H, CH.sub.2), 1.14 (s, ˜20H), 0.75 (t, 40H, CH.sub.3).

(118) HG9 (40): .sup.1H-NMR (500 MHz, DMSO-d6): δ 9.91 (bs, 16H, amide NH), 9.86 (m, 6H, cy5H), 9.41 (m, 9H, cy5H), 8.34 (s, 16H, Ar—H), 8.21 (m, 4H, cy5H), δ 7.93-7.75 (brs, 60H, amide protons), 6.96 (m, 5H, cy5H), 6.77 (m, 9H, cy5H), 6.30 (m, 8H, cy5H), 4.31 (m, 12H, cy5H), 3.29-3.13 (bs, ˜60H), 3.21 (brs, ˜60H), 3.13-3.07 (brs, ˜120H), 2.73-2.55 (brs, ˜120H), 2.47-2.36 (brs, ˜60H), 2.27-2.11 (brs, ˜120H), 2.03 (s, 48H, CH3), 1.79 (s, 45H, CH3), 1.67 (m, 16H, CH), 1.35 (m, 16H, CH), 1.13 (s, ˜20H), 0.73 (t, 48H, CH3).

(119) HG32 (41): .sup.1H-NMR (500 MHz, DMSO-d6): δ 9.94 (bs, 16H, amide NH), 9.86 (m, 6H, cy5H), 9.43 (m, 9H, cy5H), 9.35 (m, 3H, cy5H), 8.33 (s, 16H, Ar—H), δ 7.96-7.75 (brs, 60H, amide protons), 6.93 (m, 5H, cy5H), 6.83 (m, 9H, cy5H), 6.31 (m, 8H, cy5H), 3.56-3.41 (bs, ˜400H), 3.21 (brs, ˜60H), 3.19-3.16 (brs, 120H), 3.13-3.02 (brs, ˜120H), 2.63 (brs, ˜50H), 2.32-2.26 (brs, ˜58H), 2.33-2.19 (brs, ˜120H), 2.01 (s, 48H, CH.sub.3), 1.67 (m, 16H, CH), 1.35 (m, 16H, CH), 1.13 (s, ˜20H), 0.73 (t, 48H, CH.sub.3).

(120) Recombinant cathepsin labeling, competition assay: Recombinant human Cathepsin L (0.5 μg), Cathepsin B (0.5 μg) were incubated the indicated concentrations of targeted or control iodinated activity based probes (CT-ABP) in reaction buffer (50 mM acetate, 2 mM DTT and 5 mM MgCl.sub.2, pH 5.5) for 120 minutes at 37° C. After probe incubation, residual cathepsin activity was labeled with GB123 for 30 minutes. The reaction was stopped by addition of sample buffer ×4 (40% glycerol, 0.2 M Tris/HCl, pH 6.8, 20% beta-mercaptoethanol, 12% SDS and 0.4 mg/ml bromophenol blue). Samples were then boiled, separated on a 12.5% SDS gel and scanned for fluorescence by a Typhoon scanner FLA 9500 at excitation/emission wavelengths of 645/670 nm.

(121) Recombinant cathepsin direct labeling: Recombinant human Cathepsin L (0.5 μg), Cathepsin B (0.5 μg) were incubated the indicated concentrations of targeted or control Cy5 labelled iodinated activity based probes (CT-ABP) in reaction buffer (50 mM acetate, 2 mM DTT and 5 mM MgCl.sub.2, pH 5.5) for 120 minutes at 37° C. The reaction was stopped by addition of sample buffer ×4 (40% glycerol, 0.2 M Tris/HCl, pH 6.8, 20% beta-mercaptoethanol, 12% SDS and 0.4 mg/mi bromophenol blue). Samples were then boiled, separated on a 12.5% SDS gel and scanned for fluorescence by a Typhoon scanner FLA 9500 at excitation/emission wavelengths of 645/670 nm.

(122) Cell cultures: NIH-3T3 mouse fibroblast cells, Raw 264.7 mouse macrophage cells or 4T1 murine mammary gland epithelial cells were cultured in DMEM (Dulbecco's modified eagle's medium) supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin. Experiments were performed on either day 6 or day 7 after plating in culture. All cells were cultured in a humidified atmosphere of 95% air and 5% CO.sub.2 at 37° C.

(123) Evaluation of probes permeability to intact cells, competition assay: NIH-3T3 cells (100×10.sup.3 cells/well) were seeded in a twelve-well plate one day before treatment. Cells were treated with indicated concentrations of targeted or control iodinated activity based probes (CT-ABP) that were pre-dissolved in 0.1% DMSO in the culture medium. After 24 hours of probe incubation, residual cathepsin activity was labelled with GB123 (1 μM)(final DMSO concentration was maintained at 0.2%). Cells were washed with PBS and lysed by addition of sample buffer. Lysates were boiled for 5 minutes, centrifuged, and separated by 12.5% SDS-PAGE. Residual labelled proteases in cells were visualized by scanning the gel fluorescence by a Typhoon scanner FLA 9500 at excitation/emission wavelengths of 645/670 nm.

(124) Evaluation of probes permeability to intact cells, direct labelling assay: NIH-3T3 mouse fibroblast cells (100×10.sup.3 cells/well) were seeded in a twelve-well plate one day before treatment. Cells were treated with indicated concentrations of targeted or control Cy5 labelled iodinated activity based probes (I-CT-ABP) that were pre-dissolved in 0.1% DMSO in culture medium for 24 hours. Cells were then washed with PBS and lysed by addition of sample buffer. Lysates were boiled for 5 minutes and centrifuged. 100 μg proteins of NIH-3T3 cell lysates or 150 μg of 4T1 cell lysates were then separated by 12.5% SDS-PAGE. Labelled proteases in cells were visualized by scanning the gel fluorescence by a Typhoon scanner FLA 9500 at excitation/emission wavelengths of 645/670 nm.

(125) In vitro Cytotoxicity Assays. Methylene Blue assays were performed in 96-well plates; NIH-3T3 cells (5×10.sup.3 cells/well) were seeded in a 96-well plate one day before treatment. Cells were treated with indicated concentrations of targeted IABNs and non-targeted nanomaterials that were pre-dissolved in 0.1% DMSO in the culture medium. Cells were incubated for 24 h and 48 h and cells were fixed with 2.5% glutaraldehyde incubation for 10 min. Cells were washed with water and borate buffer (0.1M, pH=8.5), Cell number was determined by staining cells with the addition of 100 μl per well of 1% Methylene Blue in borate buffer, followed by incubation at room temperature for at least 1 hour. The stain was aspirated and the plates rinsed by immersion in water, followed by immersion in deionized water. The plates were air-dried and then the stain was released from cells by the addition of 200 μl/well 0.1 N HCl solutions. After 1 hour incubation at 37° C., the absorbance of each 11 at 620 nm was determined on a Cytation 3 microplate reader.

(126) In vivo imaging: 4T1 cells were grown to subconfluency, followed by detachment with trypsin, spin down and resuspension in 0.5% BSA in sterile PBS and 25% matrigel. Cells (1×106 per spot in a total volume of 20 μl) were injected subcutaneously at the indicated locations into 3-4 week-old male BALB/c mice under isoflurane anaesthesia. Tumors were typically established 9-11 days after cells injections, the fur was removed from mice and 100 μI of a 0.5 mg/mice solution of targeted or control Cy5 labelled iodinated activity based probes (CT-ABP) were injected intravenously via the tail vein. Mice anesthetized with isoflurane were then imaged at indicated time points after probe injection using the IVIS kinetic equipped with a 645/670 nm excitation/emission filter.

(127) In vivo imaging of Iodine tagged CT-ABP: in vivo studies were initiated by investigating the probes pharmacokinetics in tumor-bearing mice. This was done to establish the non-invasive imaging capabilities of iodinated activity based probes. 4T1 cells were grown to sub-confluency, followed by detachment with trypsin, and resuspension in 0.5% BSA in sterile PBS and 25% matrigel. Cells (9×105 per spot in a total volume of 20 μl) were injected subcutaneously on the back of 3-4 week-old male BALB/c mice under isoflurane-anesthesia. Tumors were established 9-11 days after cells injections. A solution of targeted I-CT-ABP and control contrast agent were injected intravenously via the tail vein as follows: HG90 or HG99 (exp1), HG92 or HG31 (exp2) dissolved in 10% DMSO in PBS in a total volume of 100 μl of a iodine concentration 0.5 mg/mice. Mice anesthetized with isoflurane were imaged before, at 5 and 24 hours post injection. Micro-CT scanner (Skyscan High Resolution Model 1176) equipped with 64 detectors with a nominal resolution of 35 μm was used. For exp1, the following were used 0.2 mm aluminum filter, a tube voltage of 40 kV and 500 mA, for exp2 the following were used 0.5 mm aluminum filter, 60 kV and 350 mA. Reconstruction was performed using SkyScanNRecon software. Ring artifact reduction, Gaussian smoothing (3%), and beam hardening correction (25%) were applied in both experiments. Volume rendered three-dimensional (3D) images were generated using SkyScan CT-Volume (“CTVol”) software.

(128) Evaluation of the probe's ability to induce apoptosis in vivo after PDT: BALB/c mice bearing two tumor spots of 4T1 cells (see above for details) were injected via the tail vein with Iodine tagged probes (as described above). After 24 hours, post targeted or control Iodine tagged probes injection, mice were anesthetized with isoflurane and sacrificed by cervical dislocation. Tumors, liver, kidneys, and spleen were surgically excised and imaged ex vivo using the same IVIS kinetic as above; fluorescence intensity was calculated by dividing fluorescence signal intensity by area. Tissues were either frozen in liquid nitrogen and lysed using bead beater or bounced in RIPA buffer. Proteins were quantified by Bradford assay; total protein extracts (100 μg) were separated by 12.5% SDS-PAGE and visualized by scanning the gel with a Typhoon scanner FLA 9500 at excitation/emission wavelengths of 645/670 NM, or tumors was incubated for 4 hours with 4% paraformaldehyde, followed by an overnight incubation with 30% sucrose at 4° C. Samples were then embedded in OCT and frozen at −80° C. Tissue was sectioned into 10 μm thick slices using a CM 1900 cryotome (Leica Microsystems, Wetzlar, Germany). The samples were mounted with DAPI Fluoromount-G (Southern Biotech, AL, USA). Fluorescent pictures were taker, with an Olympus FV10i confocal microscope (Olympus, Tokyo, Japan) equipped with Cy5 and DAPI filters.

(129) Results

(130) Iopanoic acid (IPA) and 2,3,5-triiodobenzoic acid (TBA) used as an Iodine tag and linker as an ethylenediamine core PAMAM dendrimer, which may have requisite hydrophilic interaction for good physiological aqueous solubility, which will clear very quickly from bloodstream after intravenous inoculation. Iodide tag were synthesized starting from commercially available 2,3,5-triiodobenzoic acid and Iopanoic acid. The Succinamide Ester (SE) of 2,3,5-triiodobenzoic acid and acetylated Iopanoic acid generated by using N-hydroxysuccinamide and N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) in basic conditions (Scheme 2). Iodine tagged Computed Tomography-Activity Based Probe (I-CT-ABP) (14a-b) were synthesized by reaction of 3 with SE of iodide tag in basic conditions. Two and three tagged Iodide CT-ABP (17a-b) were synthesized starting from 3, which on reaction with succinic anhydride followed by coupling with ethylenediamie core PAMAM-G0 and then reaction with SE of iodide tag in basic conditions (Scheme 3). Further IPA was chosen as an iodine tag for the synthesis of multiple tagged I-CT-ABP because it has good hydrophilic properties and high cell permeability. Six rid seven tagged Iodide CT-ABP (25, 26, 27) were synthesized starting from ethylenediamie core PAMAM-G1 reaction with SE of iodide tag in basic conditions to give six, seven and eight tagged compounds (22, 23, 24), which on reaction with GB111NH-SE in basic conditions to give 25, 26, 27 (Scheme 4).

(131) ##STR00016##

(132) ##STR00017## ##STR00018## ##STR00019##

(133) ##STR00020## ##STR00021## ##STR00022##

(134) TABLE-US-00004 TABLE 3 List of the probes synthesized No. No. of No. No. of Molec- Probe PAMAM Iodine of of free ular Code gen. tag Iodine GB111NH— NH.sub.2 weight 14a — 1 3.00 1.0 — 1055.00 (HG39) 14b — 1 3.00 1.0 — 1168.08 (HG41) 17a G0 3 9.00 1.0 — 2616.82 (HG49) 17b G0 3 9.00 1.0 — 2956.08 (HG51) 18 G0 2 6.00 1.0 1.0 2361.28 (HG77) 24 G1 8 24.00 — — 6187.42 (Control) 25 G1 6 18.00 1.0 1.0 5653.11 (HG81) 26 G1 6 18.00 2.0 — 6308.40 (HG81a) 27 G1 7 21.00 1.0 — 6247.91 (HG78)
Biochemical Evaluations

(135) The biochemical evaluation of the new probes started with evaluation for their ability to bind recombinant human cathepsins B or Cathepsin L. Recombinant human cathepsins B was incubated with increasing concentrations of the probes for 2 hour. Residual activity was detected by a fluorescently labeled cathepsin ABP GB 123 that was added to samples for 30 minutes at r.t. The free probe was separated from the enzyme-probe complex by SDS PAGE, the detection of the probe-enzyme complex was done by a fluorescent scanning of the gel at Cy5 fluorescence, I-CT-ABP 14a, 17a, 14b, 17b, 18, HG78, HG81 and HG81a, were found to bind the cathepsins in a dose respond manner (FIGS. 11A, 11B, 11D and 11E). Evaluation of the probes cell permeability and capability of labeling cellular cathepsin enzymes was performed using competitive inhibition assay as well. The competitive inhibition assay was carried out by incubation of NIH-3T3 cells with all iodine tagged probes for 24 hours. Next, residual cathepsins activity was labeled by GB123 1 μM during 2 hours. Cell lysates were separated on SDS-PAGE followed by scanning the gel for fluorescence to visualize labeling of residual cathepsins activity by GB123 (FIGS. 11C and 11F). Encouragingly, all iodine derivatives showed high cell permeability. For three, six and seven iodine tagged probe showed higher inhibition.

(136) The synthesis of the Cy5 labelled I-CT-ABP were performed using six tagged compounds (25), which on reaction with Cy5 NHS ester in basic conditions to give 28 (HG92). As negative controls, seven tagged compound were on reaction with Cy5 NHS ester to give 29 (HG31) (Scheme 5). The design of the iodine conjugated, generation 3 (G3), ethylenediamine core PAMAM dendrimers used in this study, for the purpose of the designed conjugate was for targeted molecular imaging of cancer and atherosclerosis disease, the conjugate contrast agent is the Iodine, the tumor targeting molecule GB111. In this study, the inventors also evaluated the conjugate contrast agent is the Iopanic acid, the tumor targeting molecule GB111 and the fluorescent molecule Cy5 to achieve the most sensitive detection of targeted cathepsin labelling.

(137) The synthesis, purification, and analysis of the conjugates were performed following standard methods. The NHS ester of acetylated Iopanic acid Iopanoic acid was first conjugated to the ethylenediamine core PAMAM-G3 dendrimer at a mean stoichiometric ratio of 16.0 per dendrimer as determined by 1H NMR. Next, the GB111 was conjugated to the PAMAM-G3, dendrimer at a mean stoichiometric ratio of 1-2 targeting molecule per dendrimer, as determined by 1H NMR. Finally, the remaining surface groups on the dendrimer, initially primary amines, were neutralized by the addition of acetylation, or PEG NHS ester, as previous studies have found that highly cationic PAMAM dendrimers disrupt cellular membranes. As negative controls, conjugates PAMAM-G3 dendrimers without GB111 were synthesized in parallel with HG, with the exemption of the GB111 conjugation steps. Therefore, the only chemical difference between these dendrimers and HG33, HG94, HG97 is the presence of GB111 (Scheme 6). The synthesis, purification, and analysis of the Cy5 labelled conjugates were performed following standard methods. The NHS ester of acetylated Iopanic acid was first conjugated to the PAMAMG3 dendrimer core at a mean stoichiometric ratio of 16.0 per dendrimer as determined by .sup.1H NMR. Next, the GB 111 was conjugated to the PAMAM-G3, dendrimer at a mean stoichiometric ratio of 1-2 targeting molecule per dendrimer and then Cy5 was conjugated to the conjugated dendrimer at a mean stoichiometric ratio of 1-2 fluorophores per dendrimer, as determined by .sup.1H NMR. Finally, the remaining surface groups on the dendrimer, primary amines were neutralized by the addition of acetylation, or PEG NHS ester. As negative controls, conjugates of PAMAM-G3 dendrimers without GB111 were synthesized in parallel with HG99 and HG32, with the exemption of the GB111 conjugation steps. Therefore, the only chemical difference between these dendrimers and HG is the presence of GB 11 (Scheme 7).

(138) ##STR00023##

Scheme 6

Synthesis of multiple iodine tagged CT-ABP (Acetylated and PEGylated). as seen in FIG. 28

Scheme 6 (cont.)

Synthesis of multiple iodine tagged CT-ABP (Acetylated and PEGylated). as seen in FIG. 29

Scheme 7

Synthesis of multiple iodine tagged CT-ABP (Acetylated, PEGylated and Cy5 labelled), as seen in FIG. 30

Scheme 7 (Cont.)

Synthesis of multiple iodine tagged CT-ABP (Acetylated, PEGylated and Cy5 labelled), as seen in FIG. 31

(139) TABLE-US-00005 TABLE 4 List of activity based probes (CT-ABP) synthesized. No. of Iodine No. of No. of Probes PAMAM gen. tag Iodine GB111NH- HG82 G3 16 48.00 ~1.0 HG86 G3 16 48.00 ~1.0 HG95 G3 16 48.00 ~1.0 HG96 G3 16 48.00 ~1.0 HG87 G3 16 48.00 — HG33 G3 16 48.00 — HG94 G3 16 48.00 — HG97 G3 16 48.00 — HG92 G1 6 18.00  1.0 HG90 G3 16 48.00 ~1.0 HG93 G3 16 48.00 ~1.0 HG31 G1 7 21.00 — HG32 G3 16 48.00 — HG99 G3 16 48.00 — No. of No. of No. of PEG-4 PEG-12 Molecular Probes Acetyl (333) (750) No. of Cy5 weight HG82 — — — — ~17098 HG86 ~15.00 — — — ~17743 HG95 — ~15.00 — — ~20368 HG96 — — ~15.00 — ~26623 HG87 — — — — ~16429 HG33 ~16.00 — — — ~17117 HG94 — ~16.00 — — ~19917 HG97 — — ~16.00 — ~26621 HG92 — — — 1.0 6307 HG90 ~14.00 — ~1.0 ~18253 HG93 — — ~14.00 ~1.0 ~26623 HG31 — — — 1.0 6241 HG32 — — ~15.00 ~1.0 ~25989 HG99 ~15.00 — — ~1.0 ~25989
Biochemical Evaluations

(140) Recombinant human cathepsins B was incubated with increasing concentrations of the probes for 2 hour. Residual activity was detected by a fluorescently labeled cathepsin ABP that was added to samples for 30 minutes at r.t. The free probe was separated from the enzyme-probe complex by SDS PAGE, the detection of the probe-enzyme complex was done by a fluorescent scanning of the gel at Cy5 fluorescence, FIG. 12. HG82, HG86, HG95 and HG96 were found to bind the cathepsins in a dose respond manner. To assure that the binding is dependent on protease activity, a control were performed HG87, HG94 and HG97 lacking the reactive moiety. Since no inhibition was detected, it was conclude that the binding of the probes occur in an activity-dependent manner (FIGS. 12A and 12B). Evaluation of I-CT-ABPs in intact cells. The competitive inhibition assay was carried out by incubation of NIH-3T3 cells with all iodine tagged probes for 24 hours. Next, residual cathepsins activity was labeled by GB123 1 μM during 2 hours. Cell lysates were separated on SDS-PAGE followed by scanning the gel for fluorescence at: 645/670 nm to visualize labeling of residual cathepsins activity by GB123 (FIG. 12E). Encouragingly, all iodine derivatives showed high cell permeability. For acetylated and PEGylated probe showed higher inhibition. For control probe, no inhibition was detected, the binding of the probes occur in an activity-dependent manner. Recombinant human cathepsins B was incubated with increasing concentrations of the Cy5 labelled probes for 2 hour. The free probe was separated from the enzyme-probe complex by SDS PAGE, the detection of the probe-enzyme complex was done by a fluorescent scanning of the gel at Cy5 fluorescence. HG92, and HG90, were found to bind the cathepsins in a dose respond manner (FIGS. 12C and 12D). The binding of the all the Cy5 labelled probes occur in an activity-dependent manner. Direct labeling in intact cells. The assay was carried out by incubation of NIH-3T3 cells with Cy5 labelled iodine tagged probes for 24 hours. Cell lysates were separated on SDS-PAGE followed by scanning the gel for fluorescence at: 645/670 nm to visualize labeling of residual cathepsins activity by GB123. Encouragingly, all Cy5 labelled iodine derivatives showed high cell permeability and binding to cellular cathepsins (FIG. 12F). For the PEGylated I-CT-ABP HG93 and HG32 also observed that recombinant cathepsin activity and cell permeability, it's difficult to quantify the selective inhibition due to enhanced permeation and retention (EPR) effect.

(141) Cytotoxicity

(142) Cytotoxicity study of the I-CT-ABPs, the toxic response to the systematically varied physico-chemical properties of successive PAMAM dendrimers. In vitro cytotoxicity of I-CT-ABPs to NIH-3T3 cell lines was investigated. Viability assay was performed for assessment of the cytotoxicity of each I-CT-ABP to the NIH-3T3 cell lines using the methylene blue method. All the I-CT-ABPs shows low toxicity with NIH-3T3 cells in a concentration range from 0.01 to 10 μM after 24 h and 48 h incubation. All the I-CT-ABP does not block the cellular uptake and only shows specific uptake of covalently conjugation to Cathepsin B and L (FIGS. 13A and 173). No toxicity was found in any of the probes check in the first 24 hours (FIG. 13A) while a slight toxicity was observed at 10 μM of HG 82, 86, and 87 after 48 hours (FIG. 13B).

(143) Pharmacokinetic Properties of Cy5 Labelled Iodine Probes In Vivo by Non-Invasive Fluorescence Imaging

(144) The in vivo studies were initiated by investigating the pharmacokinetics of Cy5 labelled I-CT-ABP, HG92, HG90, and respective control in tumor-bearing mice. This was done to establish the non-invasive imaging capabilities of these contrast media. 4T1 cells were grown to sub-confluency, followed by detachment with trypsin, and resuspension in 0.5% BSA in sterile PBS and 25% matrigel. Cells (9×10.sup.5 per spot in a total volume of 20 μl) were injected subcutaneously on the back of 3-4 week-old male BALB/c mice under isoflurane anesthesia. Tumors were established 9-11 days after cells injections. Each mouse received 100 μl probe solution containing 75 nmol compound by tail vain injection. The pharmacokinetics of HG92, and HG90 in tumor-bearing mice relative to control probes lacking the GB111 moiety (HG31, HG99 and HG32 respectively) was evaluated. After I-CT-ABP injection, the fluorescent I-CT-ABP rapidly circulated throughout the animal and high fluorescent signals could be seen in virtually all tissues, including the tumors. A clear signal from the tumor could be detected after 0.5 hours together by the fluorescence of the I-CT-ABP distributed throughout the body. This non-specific fluorescence diminished over time with clearance of the un-bound PAMAM nanomaterics from the body of the mouse, resulting in a tumor-specific signal increases. This specific signal increased over time and reached a maximum at 6-8 hours post I-CT-ABP injection. The HG92 targeted I-CT-ABP first shows an accumulation over 6-8 hours, then a slow decrease, whereas non-targeted contrast agent HG31 shows monoexponetial decay (FIGS. 14A and 14B). For the HG90 targeted I-CT-ABP shows an accumulation over 5-7 hours, then a slow decrease, whereas non-targeted contrast agent HG99 shows monoexponetial decay (FIGS. 14C and 14D). Due to the covalent nature of the targeted I-CT-ABP a significant amount retained in the tumor up to 24 hours post injection. The targeted Cy5 labelled I-CT-ABP circulation in mice was monitored in organ harvested after 24 hour injection, quantified by fluorescence of Cy5 labelled I-CT-ABP. In the targeted I-CT-ABP mice organs, liver, kidney, spleen and tumor shows strong fluorescence intensity, than the non-targeted contrast agent. The fluorescence images indicated strong accumulation of targeted I-CT-ABP in the tumor tissues than non-targeted contrast agent. It then evaluated the probe of HG92 and HG90 demonstrating excellent correlation with the in vivo data. Based on these results, the optimal time for the treatment was determined to be 5-7 hour after administration of the targeted I-CT-ABP (FIG. 14).

(145) CT In Vivo Imaging of Iodine Tagged I-CT-ABP:

(146) The X-ray CT pharmacokinetic behavior of I-CT-ABPs (HG92, HG90 and respective controls) in tumor bearing mice. The single contrast agents' experiment required two CT scan separated by several hours for complete blood clearance of contrast agents in order to quantify and differentiate vascular signal from probe labelled tumor signal. This was done to establish the non-invasive imaging capabilities of targeted I-CT-ABPs. The choosing the ideal targeted contrast agent, the inventors aim to maximize both tumor specificity and total tumor uptake and retention of the I-CT-ABPs. CT images were acquired with subsequent scan obtained at 0 h 5 h and 24 h after injection. The mice were pre scanned before injection by a CT scanner equipped with 64 detectors, 55 kVp & 500 mAs was used for all scans. A CT signal from the tumor could be detected after 5 hours for targeted I-CT-ABP was distributed evenly throughout the body. Images obtained after 24 h injection revealed significant removal of I-CT-ABP from the blood pool and accumulation in the liver, spleen, kidney and tumor observed. The tumor is readily visible in the data for the targeted I-CT-ABP relative to non-targeted. Quantitative analysis of the CT contrast in Hounsfield unit (HU) with tumor size indicated that the tumor labelling of targeted I-CT-ABP (HG9) is more significant than non-targeted nanomaterial (HG99), after 24 h (FIG. 157). While the targeted I-CT-ABP (HG92) shows an insignificant difference to non-targeted nanomaterials (HG31) due to the low number of iodine and low sensitivity X-ray CT (FIG. 15A). After 5 h and 24 h, targeted I-CT-ABP HG92 and HG90 remained visible within the tumor tissues in the CT images, indicating prolonged retention of the I-CT-ABP within tissues (FIGS. 15A and 15B). Here, accumulation of I-CT-ABP in the tumor tissues induces X-ray absorption and increase in density. The kinetics of accumulation of targeted I-CT-ABP HG90 is also different that of the non-targeted one, this specific signal at the time 5 hours and it increased at 24 hours post I-CT-ABP injection. It was noted that this signal in the mouse's body is likely due to a combination of labeling of active cathepsins in organs that have high cathepsin activity and retention of the I-CT-ABP in tissues (FIGS. 16A and 16B). The data shown here are consistent and with increasing efficacy of I-CT-ABP s of tumor that express elevated levels of cathepsin activity, and its extent this theme for a new class of targeted X-ray contrast agent. Similarly to describe above the I-CT-ABP HG82 and HG86 were evaluated as in vivo CT contrast agents. Mice received 100 μl of a 10 mg/ml solution of targeted I-CT-ABPs by tail vain injection. The mice were scanned before, at 5 and 24 hours post injection by a Micro-CT scanner equipped with 64 detectors, 80 kVp & 500 mAs was used for all scans. A CT signal from the tumor could be detected 5 hours post injection of the I-CT-ABP HG82, as seen in FIG. 17. This specific signal was higher at the time 5 h and was reduced at 24 hours post probe injection as seen in FIG. 18D. A clear CT signal was detected within the tumor tissue.

(147) Biodistribution and Cathepsin Labeling of I-CT-ABP and Control:

(148) The tumor tissues was analyzed, as well as tissue from liver, kidney, and spleen. The Cy5 labelled targeted I-CT-ABP (HG92) and non-targeted contrast agent (HG31) treated mouse organ, tissue lysates were prepared and separated by SDS-PAGE, Labeling profiles in all tissues from both Cy5 labelled targeted I-CT-ABP and non-targeted contrast agent treated mice showed the different pattern of activity, cathepsins specific labeling was detected with the higher shift of fluorescent bands between 20-35 kDa, characteristic band for labelled I-CT-ABP-cathepsin enzyme complex. For non-targeted contrast agent, no labeling of cathepsin was detected, the binding of the probes occur in an activity-dependent manner (FIG. 19). The inventors did see I-CT-ABPs cathepsin activity in the tumor, and also saw significant cathepsin activity in other tissue, including spleen, liver, and kidney activity is also presented confirmed by pharmacodynamics therapy. However, the Cy5 labelled targeted I-CT-ABP (HG90, HG93) and non-targeted contrast agent (HG99, HG32) were couldn't separate by SDS-PAGE, due to aggregation of tissue. The microscopy images of tumor tissues show significant difference in fluorescence intensity signal. This additional signal from Cy5 labelled targeted I-CT-ABP HG90 and HG92 is likely to be the pro form of a cysteine cathepsin that can be labeled by the active site I-CT-ABP if it accumulates at sufficient concentrations (data not shown).