Methods and uses for remotely triggered protease activity measurements

Abstract

The present disclosure relates to methods and products associated with in vitro and in vivo protease activity measurements and enzyme profiling. Some aspects of the present disclosure relate to measuring remotely triggered protease activity. In particular, the disclosure relates to methods of in vivo processing of exogenous molecules followed by detection of signature molecules as representative of the presence or absence of active enzymes associated with disease or conditions. The disclosure also relates to products, kits, and databases for use in the methods of the disclosure.

1 - 31 . (canceled) 
     
     
         32 . A method comprising:
 a) administering to a subject a biomarker particle, wherein the biomarker particle comprises a carrier domain linked to a detectable marker via an enzyme substrate, and wherein the biomarker particle comprises a photolabile protecting group positioned at a residue adjacent to an enzyme-target scissile bond in the enzyme substrate and further wherein a liposome encapsulates the carrier domain linked to the detectable marker via the enzyme substrate;   b) exposing the subject to an external force to deactivate the photolabile protecting group;   c) analyzing a biological sample from the subject, wherein the biological sample is not a sample from the site of administration of the biomarker particle; and   d) determining whether the detectable marker is in the biological sample, wherein the presence of the detectable marker in the biological sample is indicative of the enzyme being present in an active form within the subject.   
     
     
         33 . The method of  claim 32 , wherein the liposome comprises a thermosensitive liposome, a pH-responsive liposome, a gold nanoparticle-based liposome, or a reactive oxygen-responsive liposome. 
     
     
         34 . The method of  claim 33 , wherein the thermosensitive liposome is a liposome nanocarrier containing magnetic nanoparticles. 
     
     
         35 . The method of  claim 32 , wherein the photolabile protecting group is a small molecule responsive to different wavelength activations. 
     
     
         36 . The method of  claim 32 , wherein the photolabile protecting group comprises 1-(4,5-dimethoxy-2-nitrophenyl) diazoethane (DMNPE), coumarin or beznoquinolone. 
     
     
         37 . The method of  claim 32 , wherein the photolabile protecting group is a large molecule that provides steric hindrance protection from enzymatic cleavage. 
     
     
         38 . The method of  claim 32 , wherein the biological sample is urine, blood, saliva, or MUCOUS. 
     
     
         39 . The method of  claim 32 , wherein the biomarker particle is a multiplexed library of enzyme susceptible detectable markers. 
     
     
         40 . The method of  claim 32 , wherein the enzyme susceptible detectable markers are mass encoded protease substrates or ligand encoded protease substrates. 
     
     
         41 . The method of  claim 32 , wherein the step of analyzing the biological sample detectable markers comprises identifying mass-encoded protease substrates using mass spectrometry. 
     
     
         42 . The method of  claim 32 , wherein the external force is a magnetic field source. 
     
     
         43 . The method of  claim 42 , wherein the magnetic field source is an alternating magnetic field (AMF). 
     
     
         44 . The method of  claim 32 , wherein the external force is an ultraviolet A (UVA) light source. 
     
     
         45 . The method of  claim 44 , wherein the UVA light is 365 nm. 
     
     
         46 . The method of  claim 44 , wherein the UVA light is administered via photon upconversion or two-photon technology. 
     
     
         47 . The method of  claim 44 , wherein the UVA light is administered via an implantable light source. 
     
     
         48 . The method of  claim 32 , wherein step (c) is performed in vitro. 
     
     
         49 . The method of  claim 32 , wherein step (c) is performed ex vivo. 
     
     
         50 . The method of  claim 32 , wherein step (c) is performed in vivo. 
     
     
         51 . The method of  claim 32 , wherein the enzyme substrate comprises a cancer substrate.