Contrast agents, methods for preparing contrast agents, and methods of imaging

Abstract

Embodiments of the present disclosure provide for contrast agents, methods of making contrast agents, and methods of using contrast agents, and the like.

1 . A contrast agent comprising:
 a) a scaffold protein, and   b) at least one metal ion chelating site, wherein the scaffold protein includes at least one metal ion chelating site that is already present or is integrated into the scaffold protein, wherein the scaffold protein includes a metal ion bound to a metal ion chelating site,   wherein the contrast agent is stable in a physiological environment.   
     
     
         2 . The contrast agent of  claim 1 , wherein the metal is Gd(III). 
     
     
         3 . The contrast agent of  claim 2 , wherein the scaffold protein includes at least one polyethylene glycol (PEG) group attached to the scaffold protein. 
     
     
         4 . The contrast agent of  claim 3 , further comprising a targeting agent attached to the scaffold protein. 
     
     
         5 . The contrast agent of  claim 4 , wherein the targeting agent is selected from the group consisting of: a biomarker probe, a precancerous targeting agent, a cancer targeting agent, a tumor targeting agent, and a probe or agent that targets at least two of a biomarker, a precancerous cell, a cancer cell, and a tumor. 
     
     
         6 . The contrast agent of  claim 3 , wherein one or more metal ion chelating sites are integrated into the scaffold protein, and wherein each of the metal ion chelating sites include one Gd(III) bound to each of the metal ion chelating sites. 
     
     
         7 . The contrast agent of  claim 6 , further comprising a targeting agent attached to the scaffold protein. 
     
     
         8 . The contrast agent of  claim 6 , wherein a Near-IR functional group for Near-IR detection is covalently bound to the scaffold protein. 
     
     
         9 . The contrast agent of  claim 7 , wherein the targeting agent is selected from the group consisting of: a biomarker probe, a precancerous targeting agent, a cancer targeting agent, a tumor targeting agent, and a probe or agent that targets at least two of a biomarker, a precancerous cell, a cancer cell, and a tumor. 
     
     
         10 . The contrast agent of  claim 1 , wherein the scaffold protein includes at least one modification to the protein sequence, wherein the modification of the metal binding site alters the metal selectivity of the binding site, optimizes binding affinity for the metal, or improves serum stability of the contrast agent. 
     
     
         11 . The contrast agent of  claim 1 , wherein the scaffold protein includes at least one polyethylene glycol (PEG) group attached to the scaffold protein. 
     
     
         12 . The contrast agent of  claim 11 , wherein the PEGylation of the contrast agent increased the solubility of protein by more than 100 fold without reducing the metal binding affinity and selectivity relative to a contrast agent that does not include the PEG. 
     
     
         13 . The contrast agent of  claim 11 , wherein the serum stability of the contrast agent is improved relative to a contrast agent that does not include the PEG. 
     
     
         14 . The contrast agent of  claim 11 , wherein PEGylation of the contrast agent increases the in vitro R1 and R2 relaxivities of the contrast agents by at least 2-fold relative to a contrast agent that does not include the PEG. 
     
     
         15 . The contrast agent of  claim 11 , wherein the contrast agent exhibits a significantly-increased blood circulation time relative to a contrast agent not including the PEG. 
     
     
         16 . The contrast agent of  claim 11 , wherein the immunogenicity of the contrast agent is significantly decreased relative to a contrast agent not including the PEG. 
     
     
         17 . The contrast agent of  claim 11 , wherein the PEG has a molecular weight of about 1 to 100 kDa. 
     
     
         18 . The contrast agent of  claim 11 , wherein the PEG has a molecular weight of about 1 to 20 kDa. 
     
     
         19 . The contrast agent of  claim 11 , wherein the PEG is attached to the scaffold protein via an amino acid selected from: lysine, glutamic acid, aspartic acid, cysteine, carboxyl/amino terminals, and combinations thereof. 
     
     
         20 . The contrast agent of  claim 11 , wherein the PEG is attached to the scaffold protein via lysine. 
     
     
         21 . The contrast agent of  claim 1 , wherein at least one metal ion chelating site is embedded within the scaffold protein. 
     
     
         22 . The contrast agent of  claim 1 , wherein at least one metal ion chelating site is substantially embedded within the scaffold protein. 
     
     
         23 . The contrast agent of  claim 22 , wherein the relaxivity of the contrast agent is greater than that of the scaffold protein relative to a contrast agent not including the PEG. 
     
     
         24 . The contrast agent of  claim 1 , wherein the metal is a paramagnetic metal ion selected from the group consisting of: Gd(III), Mn(II), Fe(II), Fe(III), Co(II), Co(III), Ni(III), Mo(V), and V(IV). 
     
     
         25 . The contrast agent of  claim 1 , wherein the metal is an ion of a metal selected from the group consisting of Lanthanide Series metals. 
     
     
         26 . The contrast agent of  claim 1 , wherein the scaffold protein is selected from immunoglobulin proteins. 
     
     
         27 . The contrast agent of  claim 1 , wherein the scaffold protein is selected from proteins derived from a fluorescent protein having a chromophore. 
     
     
         28 . The contrast agent of  claim 1 , further comprising a targeting agent attached to the scaffold protein. 
     
     
         29 . The contrast agent of  claim 28 , wherein the targeting agent is selected from the group consisting of: a biomarker probe, a precancerous targeting agent, a cancer targeting agent, a tumor targeting agent, and a probe or agent that targets of a biomarker, a precancerous cell, a cancer cell, and a tumor. 
     
     
         30 . The contrast agent of  claim 28 , wherein the targeting agent is selected from the group consisting of: a gastrin release peptide (GRP) and a RGD (Arg-Gly-Asp) peptide. 
     
     
         31 . The contrast agent of  claim 1 , wherein the scaffold protein is a natural metal binding site protein, wherein the natural metal binding site protein includes a natural metal binding site, and wherein the metal ion binding site is derived from a natural metal binding site. 
     
     
         32 . The contrast agent of  claim 31 , wherein the natural metal binding protein is selected from a group consisting of: calmodulin, calbindin D9K, troponin C, and parvalbumin. 
     
     
         33 . The contrast agent of  claim 1 , wherein binding the metal ion causes no changes to the protein conformation or to the binding affinity under clinical conditions that would cause premature release of the metal ion, and that the contrast agent will function as a contrast agent. 
     
     
         34 . The contrast agent of  claim 1 , wherein the tailored metal ion binding site is present in an unmodified protein of a scaffold protein. 
     
     
         35 . The contrast agent of  claim 1 , wherein the tailored metal ion binding site is integrated into the scaffold protein. 
     
     
         36 . The contrast agent of  claim 1 , wherein the scaffold protein includes a fragment or domain of a natural metal binding protein. 
     
     
         37 . The contrast agent of  claim 1 , wherein the scaffold protein functions independently or in a complex with another cofactor, ligand or other biomolecule. 
     
     
         38 . A method of imaging a sample comprising:
 administering at least one of the contrast agent of  claims 1 - 37  to the sample;   introducing the sample to an imaging system; and   imaging the sample.   
     
     
         39 . The method as claimed in claimed  38 , wherein the imaging system is selected from the group consisting of: a nuclear magnetic resonance system, a Near-IR system, a PET system, and a combination thereof. 
     
     
         40 . The method as claimed in claimed  38 , wherein the imaging system is an optical imaging system. 
     
     
         41 . A method for preparing a contrast agent comprising the steps of:
 a) selecting a scaffold protein;   b) constructing at least one metal ion chelating site;   c) operatively embedding the metal ion binding site into the protein, wherein the metal ion has contrast agent properties, and   d) attaching at least one polyethylene glycol (PEG) to the scaffold protein.   
     
     
         42 . The method of  claim 41 , wherein attaching includes:
 attaching the PEG to the scaffold protein via an amino acid selected from the group consisting of: lysine, glutamic acid, aspartic acid, cysteine, carboxyl/amino terminals, and a combination thereof.   
     
     
         43 . The method of  claim 41 , wherein attaching includes:
 attaching the PEG to the scaffold protein via lysine.   
     
     
         44 . The method of  claim 41 , wherein the PEGs are attached to amino acid residues so that the PEGs do not substantially interfere with or interfere with the metal ion binding site ability to interact with the metal ion of interest or the conformation of the polymer. 
     
     
         45 . The method of  claim 41 , wherein constructing at least one metal ion chelating site includes constructing at least two metal ion chelating sites. 
     
     
         46 . The method of  claim 41 , further comprising:
 attaching at least one targeting agent to the scaffold protein.