Optical imaging probes

• US 9,549,997 B2
• Filed: 04/05/2012
• Issued: 01/24/2017
• Est. Priority Date: 04/08/2011
• Status: Active Grant

• Current Assignee: University of Edinburgh
• Original Assignee: University of Edinburgh
• Inventors: Walton, Tashfeen, Bradley, Mark, Dhaliwal, Kev, Avlonitis, Nikolaos, Haslett, Chris, McDonald, Neil, Debunne, Manuelle
• Primary Examiner(s): Jones, D L
• Application Number:
  US14/110,616
• Publication Number:
  US 20140134110A1
• Time in Patent Office: 1,755 Days
• Field of Search: 424/1.11, 424/1.65, 424/1.69, 424/1.81, 424/1.89, 424/9.1, 424/9.2, 424/9.6
• US Class Current: 1/1
• CPC Class Codes: A61K 49/0041 Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal in vivo G01N
• CPC Class Codes: A61K 49/0043 Fluorescein, used in vivo
• CPC Class Codes: A61K 49/0054 Macromolecular compounds, i.e. oligomers, polymers, dendrimers
• CPC Class Codes: A61K 49/0056 Peptides, proteins, polyamino acids
• CPC Class Codes: A61K 49/006 Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16

Abstract

The present invention relates to methods of visualizing cells especially although not exclusively in vivo using a dye, such as a dendrimer-dye molecule or polybranched-dye molecule which is internalized by the cells and thus permits subsequent visualization by confocal fluorescence endomicroscopy or other optical detectors. There is also provided internally quenched probes for use in visualizing cells especially although not exclusively in vivo by confocal fluorescence endomicroscopy and the use of internally quenched probes in combination with confocal fluorescence endomicroscopy, for visualizing cells by virtue of internalization and dequenching of a probe by the cells. In a particular embodiment the cells are activated neutrophils, such as within the lung of a subject.

Claims

1. A method of visualising a cell or cells within a mixture of cells in vivo using confocal fluorescence endomicroscopy, comprising the step of administering a dye construct to cells of a subject;

and observing a cell or cells which have internalised the dye construct, by observing an increase in fluorescence by a factor of 1.2 or more from said cell or cells using a confocal endomicroscope;

wherein

(a) the dye construct is a poly-branched molecule having any of the following generalized structures;

2. The method according to claim 1 wherein the construct is to be administered locally and fluorescence is to be carried out in the gut;

arteries and veins;

the respiratory system, the brain;

or the reproductive system.

3. The method according to claim 2, wherein the method is carried out in the lung.

4. The method according to claim 3 in order to detect activated neutrophils.

5. The method according to claim 1, wherein the fluorescent moiety *F is independently selected from FAM, rhodamine, a cyanine dye or a BODIPY dye.

6. The method according to claim 1 wherein the dye construct is locally administered to an area of tissue to be imaged.

7. The method according to claim 6 wherein detection of the cells which have internalized the dye construct is carried out within 1-30 min of the dye construct being administered to a subject.

8. The method according to claim 1 wherein the cell or cells to be detected are activated neutrophils.

9. The method according to claim 1 wherein the amount of the construct to be administered to a subject is less than 100 μ

g.

10. The method according to claim 1, wherein the dye construct is administered to the subject by a catheter inserted into the confocal endomicroscope.

11. A method of visualizing a cell or cells within a mixture of cells in vivo using confocal fluorescence endomicroscopy, comprising the step of administering a dye construct to cells of a subject, and observing fluorescence from said cell or cells using a confocal endomicroscope, wherein the dye construct is a poly-branched molecule selected from one of the following structures:

12. The method according to claim 11 wherein detection of the cells which have internalized the dye construct is carried out within 1-30 min of the dye construct being administered to a subject.

13. The method according to claim 11 wherein the cell or cells to be detected are activated neutrophils.