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

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.

1 . A method 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 to cause pores in the erythrocyte to open and form a dialyzed solution;   (c) combining the dialyzed solution in (b) with at least one fluorescent dye;   (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 within the erythrocytes;   e) washing the erythrocytes in an isotonic saline washing solution containing at least one saccharide to remove extracellular dye; and   (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 , further comprising introducing at least one saccharide prior to (d). 
     
     
         4 . The method of  claim 1 , wherein the at least one saccharide is glucose or trehalose. 
     
     
         5 . The method of  claim 1 , wherein the providing in step (a) comprises obtaining a blood sample from a subject. 
     
     
         6 . The method of  claim 1 , further comprising washing the erythrocytes with an isotonic solution prior to the dialyzing in (b). 
     
     
         7 . The method of  claim 1 , wherein the dialyzed solution in (b) has an osmolality ranging from 80-120 mOsm/kg. 
     
     
         8 . The method of  claim 1 , wherein the fluorescent dye is indocyanine green (ICG) dye. 
     
     
         9 . The method of  claim 1 , wherein the concentration of the entrapped dye in (d) ranges from 0.25 to 1.5 mM. 
     
     
         10 . The method of  claim 1 , wherein the concentration of the entrapped dye in (d) ranges from 0.3 to 0.4 mM. 
     
     
         11 . The method of  claim 1 , wherein the concentration of the entrapped dye in (d) is greater than 0.4 mM. 
     
     
         12 . The method of  claim 1 , wherein the at least one fluorescent dye in (c) is indocyanine green dye having a concentration ranging from 0.25 to 3.0 μmoles/mL of dialyzed solution. 
     
     
         13 . 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. 
     
     
         14 . The method of  claim 1 , wherein the resealing solution has an osmolality of at least 2000 mOsm/kg. 
     
     
         15 . The method of  claim 1 , wherein a source of the erythrocytes in (a) is O-type blood. 
     
     
         16 . The method of  claim 1 , wherein a source of the erythrocytes in (a) is A-type or B-type blood. 
     
     
         17 . The method of  claim 1 , wherein after step (a), the method further comprises adding α-β glucosidase to split off the erythrocyte surface A- and B-agglutinogens. 
     
     
         18 . The method of  claim 1 , wherein the erythrocytes in (d) further entraps a substance that inhibits the destructive formation of ice crystals during cooling. 
     
     
         19 . The method of  claim 18 , wherein the substance is a monosaccharide. 
     
     
         20 . The method of  claim 18 , wherein the monosaccharide is trehalose. 
     
     
         21 . The method of  claim 1 , wherein after step (d), the method further comprises adding at least one biocompatible excipient. 
     
     
         22 . The method of  claim 21 , wherein the at least one excipient is polysucrose. 
     
     
         23 . The method of  claim 21 , wherein the at least one excipient concentration ranges from 0.5% to 5%. 
     
     
         24 . The method of  claim 1 , wherein step (d) results in a first population of dye-entrapped erythrocytes, and wherein after step (d), the method further comprises:
 (g) repeating steps (a) and (b) with a second sample of erythrocytes;   (h) repeating step (c) with the product of (g) and at least one fluorescent dye at a concentration higher than that of step (c);   (i) repeating step (d) with the product of (h) to form a second population of dye-entrapped erythrocytes; and   (j) combining the first and second populations.   
     
     
         25 . The method of  claim 24 , further comprising adding at least one therapeutically effective agent at step (g) or (h), wherein the second population further comprises agent-entrapped erythrocytes. 
     
     
         26 . The method of  claim 25 , wherein the at least one dye in the first population has a concentration ranging from 0.3 to 0.4 mM, and the at least one dye in the second population has a concentration greater than 0.4 mM, wherein upon illumination of a field of view at a selected wavelength with laser energy, the erythrocytes of the first population fluoresce. 
     
     
         27 . The method of  claim 26 , wherein the selected wavelength is 805 nm. 
     
     
         28 . The method of  claim 26 , wherein the at least one dye is ICG. 
     
     
         29 . A method for controlled release of a therapeutic agent, comprising:
 (a) administering to a patient, a composition comprising erythrocytes having entrapped therein both the therapeutic agent and a fluorescent dye;   (b) applying radiation to a field of view of a vasculature of interest at a selected wavelength and power to cause the erythrocytes to fluoresce; and   (c) applying an increased power level of the same fluorescence-stimulating radiation to a target vasculature area within the field of view, such that the increased power heats the erythrocytes due to absorption by the entrapped dye, causing them to lyse and release the entrapped therapeutic agent.   
     
     
         30 . The method of  claim 29 , wherein the fluorescent dye is ICG. 
     
     
         31 . The method of  claim 30 , wherein a portion of the erythrocytes contain ICG at a concentration that maximizes fluorescence efficiency, and the remainder contains both the therapeutic agent and ICG at a dye concentration that enhances light absorption, rather than fluorescence. 
     
     
         32 . A freeze-dried composition formed by the method 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 100 mOsm/kg to cause pores in the erythrocyte to open;   (c) combining the dialyzed solution in (b) with at least one fluorescent dye;   (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 within the erythrocytes;   e) washing the erythrocytes in an isotonic saline washing solution containing at least one saccharide to remove extracellular dye; and   (f) freezing or freeze-drying the erythrocytes containing the entrapped dye.   
     
     
         33 . A lyophilized composition comprising at least one fluorescent dye entrapped within erythrocytes. 
     
     
         34 . The composition of  claim 33 , wherein the at least one fluorescent dye is ICG. 
     
     
         35 . The composition of  claim 33 , further comprising at least one therapeutic agent entrapped within the erythrocytes. 
     
     
         36 . The composition of  claim 33 , further comprising at least one saccharide entrapped within the erythrocytes.