Optical fluorescent imaging in histology

Abstract

Compounds and methods are disclosed that are useful for noninvasive imaging in the near-infrared (NIR) spectral range. The NIR is highly sensitive for tumor detection and tracking. The application discloses targeting a tumor-enriched cell surface receptor with a ligand-conjugated fluorescent probe, which specifically allows detection of the tumor relative to the negligible animal autofluorescence.

1 . A method for staining a biological sample for observation, said method comprising:
 a) contacting a biological sample with a compound of the formula:   
       
         
           
           
               
               
           
         
         wherein Cat +  is a cation; 
         r is an integer equal to 1, 2 or 3; 
         X and Y are each independently O, S, Se, or (CH 3 ) 2 C; and 
         R 8  and R 13  are each independently alkyl, L-R 25  or L-B; wherein at least one of R 8  and R 13  is L-B; 
         R 25  is a member selected from the group consisting of optionally substituted alkyl, hydroxyl, thioacetyl and sulfanato; 
         L is a linker; 
         B is a ligand; 
         n is 0, 1, 2 or 3; 
         R 11  and R 12  are each independently F, H, sulfonato, or together with the atoms to which they are bonded form an aromatic ring; and 
         R 16  and R 17  are each independently H, sulfonato, or together with the atoms to which they are bonded form an aromatic ring; and 
         b) illuminating the biological sample to generate an optical response; and 
         c) detecting the optical response. 
       
     
     
         2 . The method of  claim 1 , wherein
 r is 1;   R 11  and R 12  are each independently F, H, or sulfonato;   R 16  and R 17  are each independently F, H, or sulfonato; and   n is 1.   
     
     
         3 . The method of  claim 1 , wherein Cat +  is H +  or a metal ion. 
     
     
         4 . The method of  claim 1 , wherein at least one of X and Y is (CH 3 ) 2 C. 
     
     
         5 . The method of  claim 1 , wherein L is a member selected from the group consisting of a direct link, or a covalent linkage, wherein said covalent linkage is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-60 atoms selected from the group consisting of C, N, P, O, and S, wherein L can have additional hydrogen atoms to fill valences, wherein said linkage contains any combination of ether, thioether, amine, ester, carbamate, urea, thiourea, oxy or amide bonds; or single, double, triple or aromatic carbon-carbon bonds; or phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds; or aromatic or heteroaromatic bonds. 
     
     
         6 . The method of  claim 1 , wherein L is a member selected from the group consisting of a PEG, a block copolymer of PEG-polyurethane and a PEG-polypropylene. 
     
     
         7 . The method of  claim 1 , wherein L is a member selected from the group consisting of a polysaccharide, a polypeptide, an oligosaccharide, a polymer, a co-polymer and an oligonucleotide. 
     
     
         8 . The method of  claim 5 , wherein L is of the formula:
   —X 1 —Y 1 —X 2 —
   wherein:   X 1  is a member selected from the group consisting of a bivalent radical, a direct link, oxygen, an optionally substituted nitrogen and sulfur;   Y 1  is a member selected from the group consisting of a direct link and C 1 -C 10  alkylene optionally interrupted by a heteroatom; and   X 2  is a member selected from the group consisting of a bivalent radical, a direct link, oxygen, an optionally substituted nitrogen and sulfur.   
     
     
         9 . The method of  claim 5 , wherein the bivalent radical of X 1  and X 2  are each independently selected from the group consisting of a direct link, optionally substituted alkylene, optionally substituted alkyleneoxycarbonyl, optionally substituted alkylenecarbamoyl, optionally substituted alkylenesulfonyl, optionally substituted alkylenesulfonylcarbamoyl, optionally substituted arylene, optionally substituted arylenesulfonyl, optionally substituted aryleneoxycarbonyl, optionally substituted arylenecarbamoyl, optionally substituted arylenesulfonylcarbamoyl, optionally substituted carboxyalkyl, optionally substituted carbamoyl, optionally substituted carbonyl, optionally substituted heteroarylene, optionally substituted heteroaryleneoxycarbonyl, optionally substituted heteroarylenecarbamoyl, optionally substituted heteroarylenesulfonylcarbamoyl, optionally substituted sulfonylcarbamoyl, optionally substituted thiocarbonyl, a optionally substituted sulfonyl, and optionally substituted sulfinyl. 
     
     
         10 . The method of  claim 8 , wherein L is —(CH 2 ) r —, wherein r is an integer from 1 to 50. 
     
     
         11 . The method of  claim 1 , wherein both R 8  and R 13  are L-B. 
     
     
         12 . The method of  claim 10 , wherein r is an integer from 1 to 5. 
     
     
         13 . The method of  claim 1 , wherein the compound has the structure. 
       
         
           
           
               
               
           
         
       
     
     
         14 . The method of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         15 . The method of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         16 . The method of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         17 . The method of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         18 . The method of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         19 . The method of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         20 . The method of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         21 . The method of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         22 . The method of  claim 1 , wherein B is a ligand that has affinity for a receptor selected from the group consisting of EGFR, Her-2, PDGFR, IGFR, c-Ryk, c-Kit, CD24, integrins, FGFR, KFGR, VEGFR, TRAIL decoy receptors, retinoid receptor, growth receptor, PPAR, vitamin receptor, glucocordicosteroid receptor, Retinoid-X receptor, RHAMM, high affinity folate receptors, Met receptor, estrogen receptor and Ki67. 
     
     
         23 . The method of  claim 1 , wherein B is selected from the group consisting of somatostatin, endostatin, a carbohydrate, a monosaccharide, a disaccharide, a trisaccharide, an oligosaccharide, aptamer, liposome and PEG. 
     
     
         24 . The method of  claim 1 , wherein B is 2-deoxy-D-glucose, 2-deoxy-D-glucosamine, a glucose derivative, glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, and tagatose. 
     
     
         25 . The method of  claim 1 , wherein B is selected from the group consisting of angiopoietins, angiostatin, angiotensin II, α 2 -antiplasmin, annexin V, β-cyclodextrin tetradecasulfate, endoglin, endosialin, endostatin, epidermal growth factor, fibrin, fibrinopeptide β, fibroblast growth factor, FGF-3, basic fibronectin, fumagillin heparin, hepatocyte growth factor, hyaluronan, insulin-like growth factor, interferon-α,β inhibitors, IL inhibitor, laminin, leukemia inhibitory factor, linomide, metalloproteinases, metalloproteinase inhibitors, antibodies, antibody fragments, monoclonal antibodies, monoclonal antibody fragments, cyclic RGD D  FV, placental growth factor, placental proliferin-related protein, plasminogen, plasminogen activator, plasminogen activator inhibitor-1, platelet activating factor antagonists, platelet-derived growth factor, platelet-derived growth factor receptors, platelet-derived endothelial cell growth factor, pleiotropin, proliferin, proliferin-related protein, selectins, SPARC, snake venoms, substance P, suramin, tissue inhibitor of metalloproteinases, thalidomide, thrombin, thrombin-receptor-activating tetradecapeptide, transforming growth factor-α,β, transforming growth factor receptor, tumor growth factor-α, tumor necrosis factor and vitronectin. 
     
     
         26 . The method of  claim 25 , wherein B is epidermal growth factor. 
     
     
         27 . The method of  claim 1 , wherein B is a compound selected from the group consisting of a tetracycline bone binding agent and a green fluorescein bone binding agent. 
     
     
         28 . The method of  claim 27 , wherein B is calcein. 
     
     
         29 . The method of  claim 1 , wherein the covalent linkage between L and B is selected from the group consisting of a direct bond, an amide bond, an ester bond, an ether bond, an oxime bond, a phosphate ester bond, a sulfonamide bond, a thioether bond, a thiourea bond, and an urea bond. 
     
     
         30 . The method of  claim 25 , wherein the metalloproteinase is matrix metalloproteinase-2. 
     
     
         31 . The method of  claim 25 , wherein the selectin is an E-selectin. 
     
     
         32 . The method of  claim 1 , wherein the biological sample is a member selected from the group consisting of a cell, a fixed cell, a cell extract, a bacteria, a virus, an organelles, and a combination thereof. 
     
     
         33 . The method of  claim 32 , wherein the cell is a tissue, tumor or organ. 
     
     
         34 . The method of  claim 1 , wherein the biological sample is a member selected from the group consisting of a natural or synthetic amino acid, a nucleic acid, a carbohydrate, a polymer, and a lipid membrane. 
     
     
         35 . The method of  claim 1 , wherein the compound is physically inserted into a cell. 
     
     
         36 . The method of  claim 33 , wherein the tumor is imaged, excised, weighed, caliper measurements taken, and tissue sections prepared. 
     
     
         37 . The method of  claim 1 , wherein the compound is at a concentration of about 0.00001 mM to 0.1 mM. 
     
     
         38 . The method of  claim 37 , wherein the compound is at a concentration of about 0.001 mM to about 0.01 mM. 
     
     
         39 . The method of  claim 1 , wherein the optical response is detected by visual inspection. 
     
     
         40 . The method of  claim 1 , wherein the optical response is detected by a device selected from the group consisting of a CCD camera, a video camera, a photographic film, a laser-scanning devices, a fluorometer, a photodiodes, a quantum counter, an epifluorescence microscope, a scanning microscope, a flow cytometer, and a fluorescence microplate reader. 
     
     
         41 . The method of  claim 1 , wherein examination of the sample includes sorting portions of the sample according to its fluorescence response. 
     
     
         42 . The method of  claim 1 , wherein the optical response is detected by an infrared imaging system. 
     
     
         43 . The method of  claim 1 , wherein the compound is illuminated with a member selected from the group consisting of a mercury arc lamp, a xenon lamp, a laser and a laser diode. 
     
     
         44 . The method of  claim 1 , wherein the illumination source is optionally integrated into a laser scanner, a fluorescence microplate reader, a standard or minifluorometer, and a chromatographic detector. 
     
     
         45 . The method of  claim 1 , wherein the compound is excitable at or near the wavelength selected from the group consisting of 633-636 nm, 647 nm, 660 nm, 680 nm, and beyond 700 nm. 
     
     
         46 . A kit for staining a biological sample, said kit comprising:
 a compound according to  claim 1 ;   optionally a buffer; and   instruction for use.