Intramolecularly-quenched near infrared fluorescent probes

Abstract

An intramolecularly-quenched, near infrared fluorescence probe that emits substantial fluorescence only after interaction with a target tissue (i.e., activation) is disclosed. The probe includes a polymeric backbone and a plurality of near infrared fluorochromes covalently linked to the backbone at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites. The probe optionally includes protective chains or fluorochrome spacers, or both. Also disclosed are methods of using the intramolecularly-quenched, near infrared fluorescence probes for in vivo optical imaging.

We claim:  
     
         1 . An intramolecularly-quenched fluorescence probe comprising a polymeric backbone and a plurality of near infrared fluorochromes covalently linked to the backbone at fluorescence-quenching interaction-permissive posit ions separable by enzymatic cleavage at fluorescence activation sites, wherein the backbone is selected from the group consisting of a polysaccharide, a nucleic acid, and a synthetic polymer.  
     
     
         2 . An intramolecularly-quenched fluorescence probe comprising a polymeric backbone and a plurality of near infrared fluorochromes covalently linked to the backbone at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites, wherein the backbone is a D-tyr-D-lys copolymer.  
     
     
         3 . An intramolecularly-quenched fluorescence probe comprising a polymeric backbone and a plurality of near infrared fluorochromes covalently linked to the backbone at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites, wherein each fluorochrome is linked to the backbone by a spacer selected from the group consisting of a polysaccharide, a nucleic acid, and a synthetic cleavable moiety.  
     
     
         4 . An intramolecularly-quenched fluorescence probe comprising a polymeric backbone and a plurality of near infrared fluorochromes covalently linked to the backbone at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites, wherein the fluorochrome is selected from the group consisting of Cy5.5, Cy5, Cy 7, IRD41, IRD700, NIR-1, LaJolla Blue, indocyanine green (ICG), indotricarbocyanine (ITC), and a chelated lanthanide compound.  
     
     
         5 . An intramolecularly-quenched fluorescence probe comprising a polymeric backbone and a plurality of near infrared fluorochromes covalently linked to the backbone at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites, wherein the activation sites are selected from the group consisting of: KK; PIC(Et)FF (SEQ ID NO: 3); HSSKLQ (SEQ ID NO: 4); P(L/Q) G (I/L)AG (SEQ ID NO: 5); and GVVQASCRLA (SEQ ID NO: 6).  
     
     
         6 . An in vivo method of imaging a tumor, the method comprising: 
 (a) administering to a human patient an intramolecularly-quenched fluorescence probe that accumulates preferentially in a the tumor, and comprises a fluorochrome attachment moiety and a plurality of near infrared fluorochromes covalently linked to the fluorochrome attachment moiety at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites;    (b) allowing time for (1) the probe to accumulate preferentially in the tumor, and (2) enzymes in the tumor to activate the probe by enzymatic cleavage at a fluorescence activation site;    (c) illuminating the tumor with near infrared light of a wavelength absorbable by the fluorochromes; and    (d) detecting fluorescence emitted by the fluorochromes,    thereby forming a near infrared image of the tumor.    
     
     
         7 . The method of  claim 6 , wherein the illuminating step and the detecting step are performed endoscopically  
     
     
         8 . An in vivo method of detecting or evaluating an arthritic area in a joint of a human patient, the method comprising: 
 (a) administering to a human patient an intramolecularly-quenched fluorescence probe that accumulates preferentially in an arthritic area, and comprises a fluorochrome attachment moiety and a plurality of near infrared fluorochromes covalently linked to the fluorochrome attachment moiety at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites;    (b) allowing time for (1) the probe to accumulate preferentially in the arthritic area, and (2) enzymes in the arthritic area to activate the probe by enzymatic cleavage at a fluorescence activation site;    (c) illuminating the arthritic area with near infrared light of a wavelength absorbable by the fluorochromes; and    (d) detecting fluorescence emitted by the fluorochromes,    thereby forming an image of the arthritic area.    
     
     
         9 . The method of  claim 8 , wherein the illuminating step and the detecting step are performed endoscopically.  
     
     
         10 . An in vivo method of selectively imaging two different cell or tissue types simultaneously, the method comprising: 
 (a) administering to an animal or human patient two different intramolecularly-quenched fluorescence probes, each of which accumulates preferentially in a target tissue, and comprises a fluorochrome attachment moiety and a plurality of near infrared fluorochromes covalently linked to the fluorochrome attachment moiety at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites, wherein each of the two probes comprises a fluorochrome whose fluorescence wavelength is distinguishable from that of the other flurorochrome, and each of the two probes comprises a different activation site;    (b) allowing time for (1) the probes to accumulate preferentially in the target tissue, and (2) enzymes in the target tissue to activate the probes by enzymatic cleavage at a fluorescence activation site, if the target tissue is present;    (c) illuminating the target tissue with near infrared light of a wavelength absorbable by the fluorochromes; and    (d) separately detecting fluorescence emitted by the two fluorochromes,    thereby forming an image of two different cell or tissue types simultaneously.