Direct cellular energy delivery system

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.

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 polar lipid which is not a stable vesicle former, a PEG, a raft former and a fusion protein.    
   
   
       2 . The vesicle of  claim 1 , wherein the phospholipid which is a stable vesicle former or the polar lipid which is not a stable vesicle former has the structure of formula (I):  
       X-L-(Z) 2   (I),  wherein X is H, A, or has a structure of formula (II)                          B is a cation or an alkyl group;    A is H or an alkyl group;    L is an alkyl group further missing two hydrogen atoms; and    each Z is independently H, E, or the structure of formula (XI),                          wherein E is an alkyl or alkenyl, and when one Z is H, the other Z is not H.    
   
   
       3 . The vesicle of  claim 2 , wherein 
 A is H, or has a structure selected from the group consisting of formulas (III), (IV), (V), (VI) and (VII)                          wherein n is an integer from 0 to 4;    L has a structure selected from the group consisting of formulas (VIII), (IX) or (X)                          and E is a bacterial fatty acid selected from the group consisting of iso-branched fatty acids, anteiso-branched fatty acids, 15-methyl fatty acids, trans-unsaturated or cis-unsaturated fatty acids, a-hydroxyl fatty acids, b-hydroxyl fatty acids, a-hydroxyl-b-methyl fatty acids, a,b-dihydroxyl fatty acids, cyclohexyl fatty acids, (Z,Z)-unsaturated fatty acids, a-hydroxyl-(bE)-ene, and 2-hexylcyclopropanedecanoic acid or E has a structure selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), and (XXII)                          
   
   
       4 . The vesicle of  claim 1 , wherein the phospholipid which is a stable vesicle former is a phosphatidylcholine.  
   
   
       5 . The vesicle of  claim 4 , wherein the phosphatidylcholine is soy phosphatidylcholine, egg phosphatidylcholine,  E. coli  extract 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.  
   
   
       6 . 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):  
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
   
   
       7 . 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.  
   
   
       8 . The vesicle of  claim 7 , wherein the PEG has a weight of from about 3000 to about 4000 repeat units.  
   
   
       9 . The vesicle of  claim 8 , wherein the PEG has a weight of about 3350 repeat units.  
   
   
       10 . The vesicle of  claim 1 , wherein the unstable vesicle forming member is a raft former selected from the group consisting of cholesterol and sphingomyelin.  
   
   
       11 . The vesicle of  claim 1 , wherein the unstable vesicle forming member is a fusion protein selected from the group consisting of fertilin, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), sec1/munc18 (SM) polypeptides, viral envelope fusion proteins, and annexins.  
   
   
       12 . The vesicle of  claim 1 , wherein the vesicle further comprises a biomolecule.  
   
   
       13 . The vesicle of  claim 12 , wherein the biomolecule is a lipid-soluble biomolecule.  
   
   
       14 . The vesicle of  claim 13 , wherein the lipid-soluble biomolecule is selected from the group consisting of α-tocopherol (Vitamin E), retinol (Vitamin A), phyllochinon (Vitamin K), ergocalciferol (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.  
   
   
       15 . The vesicle of  claim 12 , wherein the biomolecule is a water-soluble biomolecule.  
   
   
       16 . The vesicle of  claim 15 , 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, water-soluble radioactive tracer molecules, and water-soluble drugs.  
   
   
       17 . The vesicle of  claim 16 , 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, TTP, TDP, TMP, thymine, ITP, IDP, IMP, inosine, and phosphocreatine.  
   
   
       18 . The vesicle of  claim 17 , wherein the high-energy phosphate biomolecule is ATP.  
   
   
       19 . The vesicle of  claim 18 , wherein the ATP is Mg-ATP.  
   
   
       20 . The vesicle of  claim 18 , wherein the ATP is present at a concentration of about 1 M or less.  
   
   
       21 . The vesicle of  claim 20 , wherein the ATP is present at a concentration of from about 0.001 mM to about 200 mM.  
   
   
       22 . The vesicle of  claim 21 , wherein the ATP is present at a concentration of from about 1 mM to about 50 mM.  
   
   
       23 . The vesicle of  claim 1 , wherein the vesicle has an absorption rate of at least 1 vesicle absorption per second per cell.  
   
   
       24 . The vesicle of  claim 23 , wherein the vesicle has an absorption rate of at least 10 3  vesicle absorptions per second per cell.  
   
   
       25 . The vesicle of  claim 24 , wherein the vesicle has an absorption rate of at least 10 6  vesicle absorptions per second per cell.  
   
   
       26 . The vesicle of  claim 1 , wherein the vesicle has a ratio of the stable vesicle forming member to the unstable vesicle forming member of 1:9 to 100,000:1.  
   
   
       27 . The vesicle of  claim 26 , wherein the vesicle has a ratio of the stable vesicle forming member to the unstable vesicle forming member of 1:1 to 1,000:1.  
   
   
       28 . The vesicle of  claim 1 , wherein the vesicle is a unilamellar vesicle.  
   
   
       29 . The vesicle of  claim 1 , wherein the vesicle has a hydrodynamic radius of from about 20 nm to about 600 nm.  
   
   
       30 . The vesicle of  claim 29 , wherein the vesicle has a hydrodynamic radius of from about 100 nm to about 300 nm.  
   
   
       31 . A vesicle, comprising: 
 a biomolecule;    soy phosphatidylcholine; and    DOTAP.    
   
   
       32 . The vesicle of  claim 31 , wherein the biomolecule is ATP.  
   
   
       33 . The vesicle of  claim 32 , wherein the ATP is present at a concentration of from about 0.01 mM to about 200 mM.  
   
   
       34 . The vesicle of  claim 31 , wherein the vesicle has a ratio of soy phosphatidylcholine to DOTAP of 50:1.  
   
   
       35 . The vesicle of  claim 31 , wherein the vesicle has a hydrodynamic radius of from about 100 nm to about 300 nm.  
   
   
       36 . A vesicle, comprising: 
 a biomolecule;    DOPC; and    DOTAP.    
   
   
       37 . The vesicle of  claim 36 , wherein the biomolecule is ATP.  
   
   
       38 . The vesicle of  claim 37 , wherein the ATP is present at a concentration of from about 0.01 mM to about 200 mM.  
   
   
       39 . The vesicle of  claim 36 , wherein the vesicle has a ratio of DOPC to DOTAP of 50:1.  
   
   
       40 . The vesicle of  claim 36 , wherein the vesicle has a hydrodynamic radius of from about 100 nm to about 300 nm.  
   
   
       41 . A method of delivering a biomolecule to a cell, comprising contacting the cell with the vesicle of  claim 12 .  
   
   
       42 . The method of  claim 41 , wherein the biomolecule is ATP.  
   
   
       43 . The method of  claim 42 , wherein an amount of ATP delivered to the cell is sufficient to meet metabolic demand of the cell.  
   
   
       44 . A method for treating a wound, the method comprising contacting the wound with a composition comprising the vesicle of  claim 12 .  
   
   
       45 . The method of  claim 44 , 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.  
   
   
       46 . The method of  claim 44 , the method further comprising contacting the wound with a skin graft composition and the composition comprising the vesicle of  claim 12 .  
   
   
       47 . A method of preserving tissue, comprising contacting tissue with the vesicle of  claim 12 .  
   
   
       48 . A method of improving the productivity of a bioreactor having at least one cell, comprising contacting the cell with the vesicle of  claim 12.