Discrete imaging of hepatic oxidative and nitrosative stress with two-channel nanoparticles for in vivo drug safety screening

• US 9,849,198 B2
• Filed: 07/02/2014
• Issued: 12/26/2017
• Est. Priority Date: 07/02/2013
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Rao, Jianghong, Pu, Kanyi, Shuhendler, Adam
• Primary Examiner(s): Rogers, James
• Application Number:
  US14/321,873
• Publication Number:
  US 20150011878A1
• Time in Patent Office: 1,273 Days
• Field of Search: None
• CPC Class Codes: A61B 5/0071 by measuring fluorescence emission
• CPC Class Codes: A61B 5/14735 comprising an immobilised reagent
• CPC Class Codes: A61B 5/4244 liver
• CPC Class Codes: A61B 5/4848 Monitoring or testing the effects of treatment, e.g. of medication
• CPC Class Codes: A61K 49/0054 Macromolecular compounds, i.e. oligomers, polymers, dendrimers
• CPC Class Codes: A61K 49/0093 Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle

Abstract

Encompassed are embodiments of activatable nanoprobes useful for in vivo longitudinal imaging of drug hepatotoxicity with oxidative and nitrosative stress as the safety biomarkers. Both H₂O₂ and ONOO⁻ are important mediators of radical stress. Two channels of optical detection, intrinsically free from cross-talk, were engineered into superconducting polymer nanoparticles to generate chemiluminescence resonance energy transfer between the conjugated polymer matrix of the nanoparticle and an incorporated chemiluminescent substrate allowing for the luminescent detection of H₂O₂ and fluorescence resonance energy transfer between the polymer matrix and an oxidation-degradable fluorophore for ratiometric detection of ONOO These nanoprobes have been applied for real-time in vivo monitoring of hepatotoxicity resulting from challenges from drugs. In addition to the ability of imaging the dose-dependence of oxidative and nitrosative stress, the positive detection of radical stress that precedes histological changes allow the early and longitudinal detection of drug-induced hepatotoxicity in vivo.

Claims

1. A bifunctional nanoprobe comprising:

(i) a matrix core comprising;

(a) a fluorescent superconducting polymer, wherein said polymer is a 9,9 diarylpolyfluorene polymer or a poly(p-phenylene vinylene) polymer; and

(b) a copolymer comprised of polystyrene and polyethylene glycol monomers and having a plurality of galactose moieties conjugated to the polyethylene glycol and wherein the plurality of galactose moieties are disposed at the surface of the matrix core;

(ii) a chemiluminescent sensor cleavable by hydrogen peroxide and selected from the group consisting of bis-(2,4,5-trichloro-6-(pentyloxycarbonyl)phenyl)oxalate (CPPO), 6-(4-methoxyphenyl)-2-methyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA), and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA); and

(iii) a fluorescent sensor, wherein said fluorescent sensor is 2-[4′

-(β

-carboxyethylthio)-7′

-(1″

,3″

,3″

-trimethylindolenine)-3′

,5′

-trimethyleneheptatrien-1-yl]-1,3,3-trimethylindolenium perchlorate (IR775S) or diaminocyanine.

2. The nanoprobe of claim 1, wherein the 9,9 diarylpolyfluorene polymer is selected from the group consisting of:

poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}], poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole], poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-co-(9,10-anthracene)], poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorenylene}-alt-co-{2,5-bis(N,N′

-diphenylamino)-1,4-phenylene}], poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazol-4,7-diyl)], poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole], poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-diphenylene-vinyl-ene-2-methoxy-5-{2-ethylhexyloxy}-benzene)];

poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-co-(1,4-phenylene)], poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(N,N′

-diphenyl)-N,N′

-di(p-butylphenyl)-1,4-diamino-benzene)], poly[(9,9-dihexylfluorenyl-2,7-diyl)-co-(9,10-anthracene)], poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(N,N′

-bis{4-butyl-phenyl}-benzidineN,N′

-{1,4-diphenylene})], poly[(9,9-dihexylfluorenyl-2,7-diyl)-alt-co-(2-methoxy-5-{2-ethylhexyloxy}-1,4-phenylene)], poly[(9,9-dihexylfluorenyl-2,7-diyl)-alt-co-(9,9′

-spiro-bifluorene-2,7-diyl)], poly[(9,9-dihexylfluorenyl-2,7-diyl)-alt-co-(bithiophene)], poly[(9,9-dihexylfluorenyl-2,7-diyl)-alt-co-(bithiophene)], and poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,4′

-(N-(p-butyl-phenyl))diphenylamine)].

3. The nanoprobe of claim 2, wherein the 9,9 diarylpolyfluorene polymer fluorescent superconducting polymer is poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole].

4. The nanoprobe of claim 1, wherein the poly(p-phenylene vinylene) polymer is selected from the group consisting of:

poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV), poly{[2-[2′

,5′

-bis(2″

-ethylhexyloxy)phenyl]-1,4-phenylenevinylene]-co-[2-methoxy-5-(2′

-ethylhexyloxy)-1,4-phenylenevinylene]}, poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)], and poly[{2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylenephenylene)}-co-{2,5-bis(N,N′

-diphenylamino)-1,4-phenylene}]).

5. A bifunctional nanoprobe comprising:

(i) a matrix core comprising;

(a) a fluorescent superconducting polymer wherein said polymer is poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole] or poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV); and

(b) a copolymer comprised of polystyrene and polyethylene glycol monomers and having a plurality of galactose moieties conjugated to the polyethylene glycol and wherein the plurality of galactose moieties are disposed at the surface of the matrix core;

(ii) a chemiluminescent sensor selected from the group consisting of bis-(2,4,5-trichloro-6-(pentyloxycarbonyl)phenyl)oxalate (CPPO), 6-(4-methoxyphenyl)-2-methyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA), and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA); and

(iii) a fluorescent sensor, wherein said fluorescent sensor is 2-[4′

-(β

-carboxyethylthio)-7′

-(1″

,3″

,3″

-trimethylindolenine)-3′

,5′

-trimethyleneheptatrien-1-yl]-1,3,3-trimethylindolenium perchlorate (IR775S) or diaminocyanine.

6. The bifunctional nanoprobe of claim 5, said nanoprobe comprising:

(i) a matrix core comprising;

(a) a fluorescent superconducting polymer comprising poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole]; and

(b) a copolymer consisting of polystyrene and polyethylene glycol monomers and having a plurality of galactose moieties conjugated to the polyethylene glycol and, wherein the plurality of galactose moieties are disposed at the surface of the matrix core;

(ii) a chemiluminescent sensor, wherein said sensor is bis-(2,4,5-trichloro-6-(pentyloxycarbonyl)phenyl)oxalate (CPPO); and

(iii) a fluorescent sensor, wherein said fluorescent sensor is 2-[4′

-(β

-carboxyethylthio)-7′

-(1″

,3″

,3″

-trimethylindolenine)-3′

,5′

-trimethyleneheptatrien-1-yl]-1,3,3-trimethylindolenium perchlorate (IR775S).