German Rare Donor Program

Transcription

1 German Rare Donor Program A Novel Technology for Preservation of Human Erythrocytes Abbreviated version 1 May 25 Nelly Tsvetkova Center for Biostabilization University of California Davis

2 A common feature of the anhydrobiotic organisms is that they can synthesize large quantities of disaccharides, often sucrose or trehalose. Trehalose Sucrose High glass transition temperature High chemical, acid and thermal stability Low hygroscopicity

3 Trehalose Preserves Dry Biomolecular Assemblages Assemblage Dried without trehalose Dried with trehalose Membranes Fusion, phase No fusion or leakage transitions, leakage Proteins Denatured Structure maintained DNA Fragmentation into No fragmentation short segments Crowe et al. (1984. Science) began establishing the mechanism.

4 Trehalose Sucrose Glucose Tg ( o C) g H 2 O/g Dry Wgt

5 Some real world applications from studies on anhydrobiosis: liposomes for drug delivery. Hydrated liposomes Liposomes dried Liposomes dried with with trehalose trehalose and The dry liposomes rehydrated are shipped in serum bottles. Crowe, J.H. and L.M. Crowe Preservation of liposomes by freeze drying. In: Gregoriadis, G. (ed). Liposome Technology. 2nd Edition. CRC Press. Crowe, J.H. and L.M. Crowe Method for preserving liposomes. U.S. Patent Number 4,857,319.

6 Ongoing work at the Center for Biostabilization: 1. Human Blood Platelets Fresh platelets, obtained from Sacramento Blood Center Freeze-dried without trehalose, rehydrated Freeze-dried with trehalose, rehydrated. They are intact in the rehydrated state. W.Wolkers et al. Cell Pres.Technol.3, 24

7 Fluorescence images showing distribution of lucifer yellow placed outside after: 15 min 1 hr 3.5 hr 5 hr Trehalose enters platelets by an endocytotic pathway regulated by temperature. W.Wolkers et al. BBA 1612, 23

8 Rehydrated platelets respond normally to agonists Stimulation with agonists results in proper clot formation Dose response curves of rehydrated platelets fall within normal physiological range Transmittance (%) Thrombin (1 U/ml) time (minutes) fresh control rehydrated platelets Freeze-dried platelets are stable at room temperature for at least 7 days aggregation (%) control rehydrated aggregation (%) control rehydrated W.Wolkers et al. BBA 1612, Thrombin (U/ml) Ristocetin (mg/ml)

9 Cytoplasmic trehalose (mm) ln rate [mole.m -2.s -1 ] /T [K -1 ] 4 o C 23 o C 37 o C 41 o C Trehalose uptake as a function of time and temperature Duration of incubation (hours) Hemolysis (%) o C 23 o C 37 o C 41 o C Duration of incubation (hours) Satpathy et al. Cryobiology 24

10 ATP (μmol/g Hb) ATP 4 o C, control RBCs 4 o C, + trehalose 37 o C, control RBCs 37 o C, + trehalose Level of ATP and 2,3-DPG in RBCs during loading in 8 mm trehalose/1 mosm ADSOL/K-phosphate (ph 7.2) Duration of incubation (hours) 12 2,3-DPG 1 2,3-DPG (μmol/g Hb) o C, control RBCs physiological concentrations: ATP = μmol/g Hb DPG = μmol/g Hb 2 4 o C, + trehalose 37 o C, control RBCs 37 o C, + trehalose Duration of incubation (hours)

11 Post-rehydration hemolysis and percent methemoglobin in freeze-dried RBCs 12 1 % Hemolysis RBCs in PBS control RBCs, no trehalose Trehaloseloaded RBCs 4 35 % Methemoglobin control RBCs Trehalose-loaded RBCs + MB

12 Percent hemolysis of freeze-dried RBCs as a function of the residual water content % hemolysis Water content (g/g dry weight)

13 Level of ATP and 2,3-DPG in freeze-dried and rehydrated RBCs. The rehydration buffer is supplemented with phosphate, inosine, pyruvate, and adenine (PIPA) physiological concentrations: ATP = DPG = µmol/g Hb 6 4 DPG ATP 2 RBCs in ADSOL Reh. buffer Reh. buffer+pipa

14 Superoxide dismutase (SOD), catalase and acetylcholine esterase (AchE) activity in freshly isolated, trehalose loaded, and freeze-dried and rehydrated RBCs SOD activity (Units/g/Hb) at 525n Fresh Loaded Rehydrated SOD Catalase activity (Units/mg H Fresh Loaded Rehydrated Catalase AChE activity (OD/min/g Hb) at 412n Fresh Loaded Rehydrated AchE

15 A α- helix β-sheet Secondary structure of hemoglobin from fresh RBCs (A), freeze-dried with trehalose (B), and freeze-dried without trehalose (C) RBCs..2. turns Trehalose preserves the secondary structure of hemoglobin during freeze-drying Wavenumber (cm -1 ) B 17 α-helix turns β-sheet Absorbance turns+β-sheet α-helix β-sheet Wavenumber (cm -1 ) Wavenumber (cm -1 ) C

16 Lipid profiles of fresh ( ) and rehydrated ( )RBCs Fresh RBCs, chol:phospholipids, 55:45 Freeze-dried RBCs, chol:phospholipids, 65:35 Chol PE, PS PC, SM

17 % Hemolysis trehalolse loaded RBCs control, no trehalose Long-term stability of freeze-dried RBCs Duration of incubation at 4 C (days) 45 4 trehalose loaded RBCs control, no trehalose 35 % methemoglobin Duration of incubation at 4 C (days)

18 Acknowledgement DARPA Grants and N National Institutes of Health Grants HL5781 and HL6124