In vivo detection of phospholipase activation

Patent metadata

Field	Value
Publication number	US-11034994-B2
Application number	US-201815967216-A
Country	US
Kind code	B2
Filing date	Apr 30, 2018
Priority date	Mar 4, 2008
Publication date	Jun 15, 2021
Grant date	Jun 15, 2021

Abstract

The present invention is directed to a phospholipid-based NIR molecular beacon, having a phospholipid moiety; with an NIR fluorophore moiety covalently linked to a phospholipid glycerol backbone and a quencher moiety covalently linked to the phospholipid glycerol backbone. Additionally, provided herein is methods of analyzing a sample for the presence of a phospholipase and methods of identifying the activity of a phospholipase in vivo utilizing phospholipid-based NIR molecular beacon.

First claim

What is claimed is:

1. A method of analyzing a sample for the presence of a phospholipase, the method comprising:
contacting a sample suspected of comprising a phospholipase with a phospholipid-based near infrared (NIF) molecular beacon comprising (i) a phospholipid moiety with a glycerol backbone; (ii) a first fluorophore moiety covalently linked to the glycerol backbone either directly or via a linker, wherein said first fluorophore moiety is an NIR fluorophore moiety; and (iii) a fluorescence quencher moiety covalently linked to said phospholipid glycerol backbone either directly or via a linker, wherein the fluorophore moiety, the fluorescence quencher moiety or both are uncharged when covalently linked at either or both sn-1 or sn-2 fatty acyl positions; and
detecting the NIR fluorescence of said NIR fluorophore moiety as a function of time, wherein an increase in the NIR fluorescence as a function of time correlates with the presence of the phospholipase in the sample.

2. The method of claim 1 , wherein said NIR fluorophore is covalently bound to the glycerol backbone at either the sn-1 or sn-2 fatty acyl positions or to the sn-3 head group.

3. The method of claim 2 , wherein said quencher moiety is a second fluorophore covalently bound to the remaining unbound fatty acyl position or the sn-3 head group.

4. The method of claim 3 , wherein said second fluorophore moiety or quencher moiety is covalently linked to the phospholipid moiety either directly or via a linker.

5. The method of claim 1 , wherein said sample is a tumor cell or an extract of a tumor cell.

6. The method of claim 1 , wherein said sample is a cultured cell, a primary cell culture or their combination.

7. A method of monitoring the activity of a phospholipase in vivo, comprising the steps of:
exposing a tissue to a phospholipid-based near infrared (NIR) molecular beacon under conditions effective to permit said phospholipase to cleave said beacon, wherein said beacon comprises (i) a phospholipid moiety with a glycerol backbone; (ii) a first fluorophore moiety covalently linked to the glycerol backbone either directly or via a linker, wherein said first fluorophore moiety is an NIR fluorophore moiety; and (iii) a fluorescence quencher moiety covalently linked to said phospholipid glycerol backbone either directly or via a linker, wherein the fluorophore moiety, the fluorescence quencher moiety or both are uncharged when covalently linked at either or both sn-1 or sn-2 fatty acyl positions; and
detecting the fluorescence of said fluorophore moiety as a function of time, wherein an increase in the quantity or rate of accumulation of fluorescence as compared to a control reaction identifies the activity of the phospholipase.

8. The method of claim 7 , wherein said NIR fluorophore is covalently bound to the glycerol backbone at either the sn-1 or sn-2 fatty acyl positions or to the sn-3 head group.

9. The method of claim 8 , wherein the quencher moiety is a second fluorophore covalently bound to the remaining unbound fatty acyl position or the sn-3 head group.

10. The method of claim 9 , wherein said second fluorophore moiety or quencher moiety is covalently linked to the phospholipid moiety either directly or via a linker.

Assignees

• Univ Pennsylvania

Inventors

• Delikatny Edward J
• Popov Anatoliy V
• Zheng Gang
• Mawn Theresa

Classifications

• G01N33/5758

  involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites · CPC title

• C12Q1/61 Primary

  involving triglycerides · CPC title

• A61K49/0036

  Porphyrins (used in photodynamic therapy A61K41/0071 or A61K41/0076; used as targeting group or modifying agent for targeting a therapeutic compound A61K47/546) · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• A61K41/0071

  PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines · CPC title