Blood substitutes and uses thereof
The invention relates to blood substitute compositions, and methods of use thereof. Described herein are compositions in which hemoglobin is maintained substantially in the reduced form of hemoglobin as opposed to the oxidized methemoglobin form through inclusion of an oxido-reductase enzyme and reducing agent within a vesicle with the hemoglobin. The vesicles additionally can comprise a dismutase, a catalase, and an electron acceptor, each of which contribute to either the maintenance of hemoglobin in the active oxygen carrying state or provide a benefit not achieved with free hemoglobin.
1. A method of encapsulating a blood substitute composition, the method comprising the steps of:
a. mixing a lipid preparation with a composition comprising an oxido-reductase enzyme, a dismutase, a catalase, a reducing agent, and an electron acceptor;
b. extruding the mixture to form lipid vesicles; and
c. isolating lipid vesicles, wherein the lipid preparation comprises at least one lipid selected from the group consisting of: DPPC, DSPC, DOPC, DLPC, DPPG, DSPG, DHPG, DOPG, DSPEG PEG 5000, DSPE-PEG200, and DOPE-PEG5000,
and, wherein electron acceptors include but are not limited to: methylene blue, sodium 2,6-dibromophenol-indophenol, sodium 2,6-dichlorophenol-indophenol, sodium o-cresol indophenols, indigotetrasulfonic acid, indigotrisulfonic acid, indigo carmine, indigomono sulfonic acid, phenosafranin, safranin T, neutral red, and thionine.
2. The method of claim 1 , wherein the oxido-reductase enzyme comprises DT-diaphorase.
3. The method of claim 1 , wherein the dismutase comprises superoxide dismutase.
4. The method of claim 1 , wherein the reducing agent comprises NADH or NADPH.
5. The method of claim 1 , wherein the electron acceptor comprises methylene blue.
6. The method of claim 1 , wherein the lipid preparation comprises:
a. at least one lipid selected from the group consisting of: DPPC, DSPC, DOPC, and DLPC;
b. at least one lipid selected from the group consisting of: DPPG, DSPG, DHPG, and DOPG; and
c. at least one lipid selected from the group consisting of: DSPEG PEG 5000, DSPE-PEG200, and DOPE-PEG5000.
7. The method of claim 1 , wherein the size of the isolated vesicles is comprised between 150 nm to 500 nm inclusive.
8. The method of claim 1 , wherein the size of the isolated vesicles is comprised between of 180 nm to 220 nm inclusive.
9. The method of claim 1 , wherein the size of the isolated vesicles is 200 nm.
10. The method of claim 1 , wherein the lipid preparation comprises: Cholesterol, DPPC, DSPE-PEG5000, and DPPG.
11. The method of claim 10 , wherein the ratio of Cholesterol, DPPC, DSPE-PEG5000, and DPPG is respectively 5:5:0.0333:1.