DIRECT CELLULAR ENERGY DELIVERY SYSTEM

• US 20110311442A1
• Filed: 06/17/2011
• Published: 12/22/2011
• Est. Priority Date: 05/14/2002
• Status: Active Grant

• Current Assignee: University of Louisville
• Original Assignee: University of Louisville
• Inventors: Ehringer, William D., Chien, Sufan
• Granted Patent: US 9,066,866 B2
• Time in Patent Office: Days
• US Class Current: 424/1.21
• CPC Class Codes: A61K 31/7076 containing purines, e.g. adenosine, adenylic acid
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 23/00 Anaesthetics
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 39/00 General protective or antinoxious agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y10S 977/726 using chemical reaction/biological energy, e.g. ATP
• CPC Class Codes: Y10S 977/907 Liposome

Abstract

A lipid vesicle comprising a phospholipid which is a stable vesicle former and at least one unstable vesicle forming member, wherein the unstable vesicle forming member is selected from the group consisting of a polar lipid which is not a stable vesicle former, a PEG, a raft former and a fusion protein is provided. The vesicle can further comprise a biomolecule, such as for example ATP. Methods of using the vesicle for delivery of biomolecules are also provided.

Claims

1. A vesicle, comprising:

(a) a phospholipid which is a stable vesicle former; and

(b) at least one unstable vesicle forming member, wherein the unstable vesicle forming member is selected from the group consisting of a PEG, a raft former, fusion protein, and a polar lipid having the structure of formula (I)

X-L-(Z)₂ 

 

(I)

wherein X is A or has a structure of formula (II)

2. The vesicle of claim 1, wherein A has a structure selected from the group consisting of formulas (III), (IV), (V), (VI) and (VII)

3. The vesicle of claim 1, wherein the phospholipid which is a stable vesicle former is a phosphatidylcholine.

4. The vesicle of claim 3, wherein the phosphatidylcholine is soy phosphatidylcholine, egg phosphatidylcholine, E. coli extract 5 phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC) or a mixture thereof.

5. The vesicle of claim 1, wherein the unstable vesicle forming member is an unstable vesicle forming polar lipid having a structure selected from the group consisting of formulas (XXIV), (XXV), (XXVI), (XXVII), (XXIX), (XXXI), (XXXII), (XXXIII), and (XXXIV):

6. The vesicle of claim 1, wherein the unstable vesicle forming member is a PEG having a weight of from about 20 to about 8000 repeat units.

7. The vesicle of claim 6, wherein the PEG has a weight of from about 3000 to about 4000 repeat units.

8. The vesicle of claim 7, wherein the PEG has a weight of about 3350 repeat units.

9. The vesicle of claim 1, wherein the unstable vesicle forming member is sphingomyelin.

10. The vesicle of claim 1, wherein the unstable vesicle forming member is a fusion protein selected from the group consisting of fertilin, soluble Nethylmaleimide-sensitive factor attachment protein receptors (SNARES), sec1/munc18 (SM) polypeptides, viral envelope fusion proteins, and annexins.

11. The vesicle of claim 1, wherein the vesicle further comprises a biomolecule.

12. The vesicle of claim 11, wherein the biomolecule is a lipid-soluble biomolecule.

13. The vesicle of claim 12, wherein the lipid-soluble biomolecule is selected from the group consisting of Vitamin E, Vitamin A, Vitamin K, Vitamin D, cholesterol, cholesterol esters, steroids, hopanoids, detergents, fatty acids, bacterial branched fatty acids, isoprenoids, long chain alcohols, lipid-soluble anesthetics, gangliosides, lipopolysaccharides, biotin-labeled phospholipids, membrane ion conductance channels, transport proteins, glucose transporters, adhesion proteins, gap junction proteins, synaptic junction proteins, caspases, adherence proteins, G-proteins, MHC proteins, complement proteins, lipid-soluble viral proteins, cellular receptors, lipid-soluble fluorescent probe molecules, and lipid-soluble radioactive tracer molecules.

14. The vesicle of claim 11, wherein the biomolecule is a water-soluble biomolecule.

15. The vesicle of claim 14, wherein the water-soluble biomolecule is selected from the group consisting of amino acids, polypeptides, proteins, monosaccharides, disaccharides, polysaccharides, nucleotides, polynucleotides, water-soluble vitamins, minerals, high energy phosphates, glycolytic, oxidative intermediates, nicotinadenine dinucleotide (NAD+/NADH), flavin adenine dinucleotide (FAD+/FADH2), water-soluble cellular enzymes, insulin, water-soluble fluorescent probe molecules, watersoluble radioactive tracer molecules, and water-soluble drugs.

16. The vesicle of claim 15, wherein the high energy phosphate is selected from the group consisting of ATP, ADP, AMP, adenosine, CTP, CDP, CMP, cytosine, UTP, UDP, UMP, uracil, GTP, GDP, GMP, guanosine, ITP, TDP, TMP, thymine, ITP, IDP, IMP, inosine, and phosphocreatine.

17. The vesicle of claim 16, wherein the high-energy phosphate biomolecule is ATP.

18. The vesicle of claim 17, wherein the ATP is Mg-ATP.

19. The vesicle of claim 17, wherein the ATP is present at a concentration of about 50 mM or less when the vesicle is in solution.

20. The vesicle of claim 19, wherein the ATP is present at a concentration of from about 0.001 mM to about 50 mM when the vesicle is in solution.

21. The vesicle of claim 20, wherein the ATP is present at a concentration of from about 1 mM to about 50 mM when the vesicle is in solution.

22. The vesicle of claim 1, wherein the vesicle has an absorption rate of at least 1 vesicle absorption per second per cell.

23. The vesicle of claim 22, wherein the vesicle has an absorption rate of at least 10³ vesicle absorptions per second per cell.

24. The vesicle of claim 23, wherein the vesicle has an absorption rate of at least 10⁶ vesicle absorptions per second per cell.

25. The vesicle of claim 1, wherein the vesicle has a mole:

mole ratio of the stable vesicle forming member to the unstable vesicle forming member of 1;

9 to 100,000;

1.

26. The vesicle of claim 25, wherein the vesicle has a mole:

mole ratio of the stable vesicle forming member to the unstable vesicle forming member of 1;

1 to 1,000;

1.

27. The vesicle of claim 1, wherein the vesicle has a hydrodynamic radius of from about 20 nm to about 600 nm.

28. The vesicle of claim 27, wherein the vesicle has a hydrodynamic radius of from about 100 nm to about 300 nm.

39. A method of delivering a biomolecule to a cell, comprising contacting the cell with the vesicle of claim 11.

40. The method of claim 39, wherein the biomolecule is ATP.

41. The method of claim 40, wherein an amount of ATP delivered to the cell is sufficient to meet metabolic demand of the cell.

42. A method for treating a wound, the method comprising contacting the wound with a composition comprising the vesicle of claim 11.

43. The method of claim 42, wherein the composition further comprises becaplermin, fibroblast growth factor, vascular endothelial growth factor, an antibiotic, silver containing compositions, a skin graft composition or combinations thereof.

44. The method of claim 42, the method further comprising contacting the wound with a skin graft composition and the composition comprising the vesicle of claim 12.

45. A method of preserving tissue, comprising contacting tissue with the vesicle of claim 11.

46. A method of improving the productivity of a bioreactor having at least one cell, comprising contacting the cell with the vesicle of claim 11.

51. The vesicle of claim 13, wherein the lipid-soluble biomolecule is selected from the group consisting of alpha-tocopherol, retinol, phyllochinon, and ergocalciferol.

52. The vesicle of claim 5, wherein the phospholipid which is a stable vesicle former is a phosphatidylcholine selected from the group consisting of soy phosphatidylcholine, egg phosphatidylcholine, E. coli extract 5 phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), and mixtures thereof.

53. The vesicle of claim 52, wherein the phospholipid which is a stable vesicle former is selected from the group consisting of soy phosphatidylcholine and DOPC;

and wherein the unstable vesicle forming member has the structure of formula (XXXIII).

29. A vesicle, comprising:

a biomolecule;

soy phosphatidylcholine; and

DOTAP,

wherein the vesicle is unilamellar.

30. The vesicle of claim 29, wherein the biomolecule is ATP.

31. The vesicle of claim 30, wherein the ATP is present at a concentration of from about 0.01 mM to about 50 mM when the vesicle is in solution.

32. The vesicle of claim 29, wherein the vesicle has a mole:

mole ratio of soy phosphatidylcholine to DOTAP of 50;

1.

33. The vesicle of claim 29, wherein the vesicle has a hydrodynamic radius of from about 100 nm to about 300 nm.

47. The vesicle of claim 29, wherein the vesicle has a hydrodynamic radius of from about 10 nm to about 600 nm.

48. The vesicle of claim 30, wherein the ATP is selected from Mg-ATP or Na-ATP.

34. A vesicle, comprising:

a biomolecule;

DOPC; and

DOTAP,

wherein the vesicle is unilamellar.

35. The vesicle of claim 34, wherein the biomolecule is ATP.

36. The vesicle of claim 35, wherein the ATP is present at a concentration of from about 0.01 mM to about 50 mM when the vesicle is in solution.

37. The vesicle of claim 34, wherein the vesicle has a mole:

mole ratio of DOPC to DOTAP of 50;

1.

38. The vesicle of claim 34, wherein the vesicle has a hydrodynamic radius of from about 100 nm to about 300 nm.

49. The vesicle of claim 34, wherein the vesicle has a hydrodynamic radius of from about 10 nm to about 600 nm.

50. The vesicle of claim 35, wherein the ATP is selected from Mg-ATP or Na-ATP.