METHODS FOR PRODUCTION AND USE OF SUBSTANCE-LOADED ERYTHROCYTES (S-IEs) FOR OBSERVATION AND TREATMENT OF MICROVASCULAR HEMODYNAMICS

• US 20140308656A1
• Filed: 02/10/2014
• Published: 10/16/2014
• Est. Priority Date: 05/02/2008
• Status: Active Grant

• Current Assignee: Stryker Corporation
• Original Assignee: Novadaq Technologies, Inc. (Stryker Corporation)
• Inventors: Flower, Robert W.
• Granted Patent: US 10,041,042 B2
• Time in Patent Office: Days
• US Class Current: 435/2
• CPC Class Codes: A61K 49/0034 Indocyanine green, i.e. ICG, cardiogreen
• CPC Class Codes: A61K 49/0097 Cells, viruses, ghosts, red blood cells, viral vectors, used for imaging or diagnosis in vivo
• CPC Class Codes: A61K 9/5063 Compounds of unknown constitution, e.g. material from plants or animals oils, fats, waxes, shellac A61K9/5015
• CPC Class Codes: C12N 5/0641 Erythrocytes

Abstract

Disclosed herein are methods, kits, and compositions for medical imaging relating to fluorescent dyes entrapped in erythrocytes. Also disclosed therein are methods, and compositions further comprising erythrocytes entrapping at least one therapeutically active agent, as well as methods for releasing the entrapped therapeutically active agent(s). Disclosed herein also are methods for preparation of the cells entrapping dye and therapeutically active agent(s) in a freeze-dried form that makes them readily available and easy to use in a clinical environment.

Claims

1. A method of making a freeze-dried composition of erythrocyte ghosts comprising:

(a) providing erythrocytes in a blood anti-coagulant solution;

(b) reducing the osmolality of the erythrocyte-containing solution in (a) by dialysis against a dialysis buffer having an osmolality less than 300 mOsm/kg addition of distilled, de-ionized water to form a hypotonic solution to cause pores in the erythrocyte to open and form a dialyzed solution;

(c) combining the dialyzed hypotonic-solution in (b) with indocyanine green and with at least one saccharide;

(d) combining the dye-containing solution in (c) with a resealing solution having an osmolality of at least 1000 mOsm/kg to increase the osmolality, thereby causing the erythrocyte pores to close and entrap the at least one fluorescent dye and at least one saccharide within the erythrocytes;

(e) washing the erythrocytes in an isotonic saline washing solution containing at least one saccharide to remove extracellular dye; and

dye.

(f) freezing or freeze-drying the erythrocytes containing the entrapped dye.

2. The method of claim 1, wherein the dialysis buffer is water.

3. The method of claim 1, wherein the at least one saccharide is selected from the group consisting of mannose, xylose, glucose, trehalose, sucrose, and maltose.

4. The method of claim 1, wherein the provision of erythrocytes comprises obtaining a blood sample from a subject.

5. The method of claim 1, further comprising washing the erythrocytes with an isotonic solution prior to the dialyzing in (b).

6. The method of claim 1, wherein the dialyzed solution in (b) has an osmolality ranging from 80-120 mOsm/kg.

7. The method of claim 1, wherein the concentration of the entrapped dye in (d) ranges from 0.25 to 1.5 mM.

8. The method of claim 1, wherein the concentration of the entrapped dye in (d) ranges from 0.3 to 0.4 mM.

9. The method of claim 1, wherein the concentration of the entrapped dye in (d) is greater than 0.4 mM.

10. The method of claim 1, wherein the indocyanine green dye has a concentration ranging from 0.25 to 3.0 μ

mol/mL of dialyzed solution.

11. The method of claim 1, further comprising the step of adding at least one therapeutically effective agent prior to step (d) to entrap the at least one agent within the erythrocytes.

12. The method of claim 1, wherein the resealing solution has an osmolality of at least 2000 mOsm/kg.

13. The method of claim 1, wherein a source of the erythrocytes in (a) is 0-type blood.

14. The method of claim 1, wherein a source of the erythrocytes in (a) is A-type or B-type blood.

15. The method of claim 1, wherein after step (a), the method further comprises adding a-glucosidase to split off the erythrocyte surface A- and B-agglutinogens.

16. The method of claim 1, wherein the erythrocytes in (d) further entraps a substance that inhibits the destructive formation of ice crystals during cooling.

17. The method of claim 16, wherein the substance is a monosaccharide.

18. The method of claim 16, wherein the monosaccharide is trehalose.