SELF-ASSEMBLING OXYGEN CARRIER COMPOSITIONS

Abstract

Synthetic blood substitutes and methods for making them. A lipid-amphiphile blood-substitute precursor compound having a hydrophobic fatty acid/acyl moiety, a hydrophilic head moiety including a phosphate group, and a pH responsive moiety. The lipid-amphiphile precursor is configured to self-assemble from a solution mixture of phospholipid and cholesterol in the presence of hemoglobin and an allosteric effector into a hybrid-vesicle resulting from the combined self-assembly of both the amphiphilic lipid-oligomer and the lipids into an advanced vesicular structure containing a hemoglobin/allosteric effector payload.

Claims

1. A lipid-amphiphile precursor compound having the formula: ##STR00006## or a salt or tautomer thereof, wherein: R.sup.1 is a hydrophobic group; R.sup.2 is an amine containing group; and R.sup.3 is chosen from ##STR00007##

2. A compound according to claim 1, wherein R.sup.1 is (CH.sub.2).sub.lCH.sub.3; and l is an integer between 10 and 16, inclusive.

3. A compound according to claim 2, wherein R.sup.1 is (CH.sub.2).sub.lCH.sub.3; and l is an integer between 10 and 16, inclusive.

4. A compound according to claim 3, wherein l is 14.

5. A compound according to claim 1, wherein: R.sup.2 is chosen from (CHR.sup.5).sub.mR.sup.6 and (CHR.sup.5).sub.mNR.sup.7(CH.sub.2).sub.nR.sup.6; each R.sup.5 is independently chosen from H and N(R.sup.7).sub.2; at most one R.sup.5 is not H; R.sup.6 is chosen from N(R.sup.8).sub.2 and N═C(NHR.sup.8).sub.2; each R.sup.7 is independently chosen from H and alkyl; each R.sup.8 is chosen from H and alkyl; m is an integer between 2 and 6, inclusive; and n is an integer between 2 and 6, inclusive.

6. A compound according to claim 5, wherein each R.sup.5 is independently chosen from H and NHCH.sub.3; and at most one R.sup.5 is not H.

7. A compound according to claim 5, wherein R.sup.6 is chosen from N(R.sup.8).sub.2 and N═C(NH.sub.2).sub.2.

8. A compound according to claim 1, wherein R.sup.2 is chosen from ##STR00008##

9. A compound according to claim 1, wherein R.sup.3 is ##STR00009##

10. A compound according to claim 1, wherein R.sup.1 is (CH.sub.2).sub.14CH.sub.3.

11. A composition comprising: (a) a phospholipid; (b) cholesterol (c) an oxygen carrier; (d) a lipid-amphiphile precursor according to claim 1; and (e) an allosteric effector.

12. A composition according to claim 11, wherein the oxygen carrier is hemoglobin (Hb).

13. A composition according to claim 11, wherein the allosteric effector is selected from the group consisting of 2,3-DPG, RSR-13, inositol phosphate, inositol hexaphosphate (IP6), phytic acid, and guanosine triphosphate.

14. A composition according to claim 11, which self assembles into a vesicle having a amphiphilic lipid bi-layer membrane and a payload comprising said oxygen carrier and said allosteric effector.

15. A composition according to claim 11 further comprising a reducing agent.

17. A composition according to claim 15, wherein said reducing agent is selected from leucomethylene blue, n-benzoyl-leucomethylene blue, and methylene blue.

18. A method for making a synthetic oxygen carrier, comprising: preparing a composition comprising (a) a phospholipid; (b) cholesterol; (c) an oxygen carrier; (d) a lipid-amphiphile precursor according to claim 1; and (e) an allosteric effector. allowing said composition to self-assemble into a vesical having a lipid bilayer outer membrane and a payload comprising said oxygen carrier and said allosteric effector; and lyophilizing said self-assembled vesicle.

18. A method according to claim 17, wherein said composition further includes a reducing agent selected from the group consisting of methylene blue, n-benzoyl-leucomethylene blue, and leucomethylene blue.

19. A composition according to claim 11, wherein said composition is lyophilized.

20. A method for preparing a synthetic oxygen carrier, comprising reconstituting a lyophilized composition according to claim 19.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a schematic representation of the compositions and self-assembly according to an embodiment of the invention.

[0024] FIG. 2 is a representation of the synthesis of a precursor designated KC-1003 according to a preferred embodiment of the invention.

[0025] FIG. 3 is a schematic representation of the self-assembly of precursor KC-1003 and hb:allosteric effector into an oxygen carrier particle according to a preferred embodiment of the invention.

[0026] FIG. 4 is a table showing free hemoglobin detected following purification of batches of oxygen carrier particles using different purification methods.

[0027] FIG. 5 is a chart showing the change in oxygen affinity with changes in pH for oxygen carrier particles according to the invention.

[0028] FIG. 6 shows the hydrodynamic diameter of a synthetic blood substitute and TEM image (inset) according to an embodiment of the invention.

[0029] FIG. 7 shows the electrophoretic potential results according to an embodiment of the invention.

[0030] FIG. 8 shows a lyophilized sample of an oxygen carrier particle according to an embodiment of the invention.

[0031] FIG. 9 shows various properties of an oxygen carrier particle according to an embodiment of the invention before lyophilization, four days after reconstitution, and 14 days after reconstitution.

DETAILED DESCRIPTION

[0032] Synthesis of preferred precursor KC-1003 is portrayed in FIG. 2. Lipid-oligomeric amphiphile is preferably synthesized and stored at 4° C. Purity of >90% may be confirmed by qHPLC, 1H NMR, 13C NMR and/or HRMS.

[0033] Precursor Film Preparation [0034] 1a. In the test tube (25×250 mm) precursor [KC 1003], [80.66 mmol] is dissolved in anhydrous chloroform to a concentration of [8 mg/mL]. Chloroform is transferred by glass syringe or measured by glass cylinder. [0035] 1b. [17 mmol] of cholesterol and [2.34 mmol] of [25 mg/mL] [PEG2000-PE] are added to the test tube. Resulting in [100 mM] of surfactant in chloroform. [0036] 1c. The test tube is gently swirled for [1 min] to make homogenous/until solution is clear. [0037] 1d. Precursor solution is passed through a small bed of cotton to ensure no particulate presence. [0038] 1e. Chloroform is evaporated in the test tube under reduced pressure by rotary evaporator. Water bath temp is 50° C. Film is formed on the wall of the test tube and no more liquid chloroform is present. [0039] 1f. The test tube is dried in vacuum oven for [12h], HV at room temperature, 20° C. [0040] 1g. The test tube is weighed again.

[0041] Self-Assembly [0042] 2a. Frozen hemoglobin at is placed at room temperature until melted, 20° C. [0043] 2b. Hemoglobin is transferred to 50 mL centrifuge tube by Eppendorf pipette and the lid is sealed immediately. [0044] 2c. Hemoglobin is transferred to the dried film in the test tube via micropipette to make a [6.339:1] of precursor to payload components Hb. The vessel is immediately sonicated (described in next step). Note: The same volume of stock Hb soln. to chloroform precursor mass is used.

[0045] Particle Size Normalization [0046] 3a. After mixing of payload and membrane components the test tube is immediately probe sonicated for [2 min for <5 mL, 5 min for >5 mL] in ice water. The test tube is swirled by hand on the stationary probe. [0047] 3b. After sonication, rest test tube for at least 15-20 min [actual time here] to achieve equilibrium from Brownian motion. [0048] 3c. Then, the hydrodynamic diameter is checked by DLS (nm and PDI).

[0049] Purification [0050] 4a. After sonication, the contents of the test tube are loaded on the Tangental Flow Filtration System and ran with a 50 nm PS membrane (D02-505U-05-N) at a rate of 130 mL/min with lactated ringers. [0051] 4b. TFF is ran until the flow of outlet was clear by spectrophotometer. [0052] 4c. DLS (hydrodynamic diameter and PDI) are again checked and should be within 10% variance.

[0053] The resulting product is lyophilized for packaging. The lyophilized product is a powder comprising amphiphilic precursor, cholesterol and PEG-PE hemoglobin and allosteric effector, optionally also including cryoprotectants.

[0054] Reconstitution at the original EM production concentration (or concentrated) can be achieved with PBS/water by simple mixing and gentle vortexing/agitation.