Self-assembling oxygen carrier compositions

Abstract

Synthetic blood substitutes and methods for making same. Lipid-amphiphile blood substitute precursor compounds having hydrophobic fatty acid / acyl groups, hydrophilic head groups including phosphate groups, and pH-responsive moieties. The lipid-amphiphile precursors are configured to self-assemble from a solution mixture of phospholipids and cholesterol in the presence of hemoglobin and an allosteric effector into hybrid vesicles that result from the combined self-assembly of both amphiphilic lipid-oligomers and lipids into highly vesicular structures containing the hemoglobin / allosteric effector payload.

Description

[Technical Field]

[0001] The present invention relates to synthetic blood substitute and / or oxygen carrier compositions and methods for making them. Summary of the Invention

[0002] The invention described herein relates to synthetic blood substitutes and methods for making same. More particularly, the invention relates to lipid-amphiphile blood substitute precursor compounds having a hydrophobic fatty acid / acyl moiety, a hydrophilic head moiety containing a phosphate group, and a pH-responsive moiety.

[0003] In accordance with another embodiment of the present invention, there is provided a composition comprising a phospholipid-cholesterol solution, a lipid-amphiphilic precursor compound, hemoglobin, and an allosteric precursor.

[0004] According to a further embodiment of the present invention, lipid-amphiphile precursors are configured to self-assemble from a solution mixture of phospholipids and cholesterol in the presence of hemoglobin and an allosteric effector into hybrid vesicles, which are the result of the combined self-assembly of both amphiphilic lipid-oligomers and lipids into highly vesicular structures containing the hemoglobin / allosteric effector payload. In a preferred embodiment, the structures are hybrid vesicles having diameters of about 80 nm to about 300 nm, with an outer layer consisting of the amphiphilic precursor, cholesterol, and a polyethylene glycol-phosphatidylethanolamine ("PEG-PE") bilayer, and carrying a payload comprising hemoglobin, an allosteric effector, and, optionally, a reducing agent, such as leucomethylene blue, n-benzoylleucomethylene blue, or methylene blue. The result is a novel synthetic blood substitute with a negative net zeta potential, excellent payload retention, and differential gas permeability. Furthermore, unlike the phospholipid bilayer in liposome-based HBOCs, the synthetic blood substitutes of the present invention have tunable membranes that provide greater integrity due to counterionic hemoglobin-precursor interactions and pH responsiveness (different oligomeric amine moieties may be used in the precursors to vary membrane thickness).

[0005] According to a preferred embodiment, the phospholipid is PEG 2000 -PE (20 mol%), the phospholipid to cholesterol ratio is preferably 17.00:2.34, and the allosteric effector can be RSR-13 (efaproxiral) present in a ratio with hemoglobin of 20:1 to 0.1:1, preferably 10:1:1 to 1:1, most preferably 5:1. The lipid-amphiphile-precursor compound can be a compound of Formula I:

[0006] Formula I: [ka]

[0007] According to various alternative embodiments, the pH-responsive moiety is preferably an amine, particularly a tertiary amine, because it allows for fine tuning of the pKa and can exhibit a slightly lower pKa when substituted with a longer hydrophobic chain. Adjustable pKa can be achieved based on hydrophobic modification of the amine, while increasing the hydrophobicity of the alkyl substituent results in a linear decrease in pKa. The abundance of positive charges in the self-assembled vesicle structure creates a unique charge density that drives the pH shuttle associated with hemoglobin's oxygen uptake at high pH and oxygen release at low pH.

[0008] Examples of suitable pH-responsive moieties according to the present invention include chitosan, lysine, ethyleneimine, esteramines, 2-(dimethylamino)ethyl methacrylate, amidoamines, arginine, spermine, spermidine, dimethylethanolamine, urethane, oxylysine, aminoglycolic acid, oxazoline, acrylamide, quaternium-6, piperidine, pyrrolidine, diphenylalanine, 1-adamantyl(5-bromo-2-methoxybenzyl)amine, ornithine, 2-(diisopropylamino)ethyl methacrylate, 4-vinylpyridine, histidine, and β-amino esters.

[0009] According to various other embodiments of the present invention, the acyl group of the lipid amphiphile precursor may constitute a linkage between the hydrophobic fatty acid moiety, the hydrophilic head group containing a phosphate group, and the pH-responsive moiety. According to these embodiments, the pH-responsive moiety may be attached to a phosphoglyceride, including, for example, phatidylcholine, phosphatidylethanolamine, phosphatidyl inositol, phosphatidylserine, or lysophosphatidylcholine. The pH-responsive moiety may be attached to either the sn1 (more preferred) or sn2 position of the phosphoglyceride. The preferred acyl chain length is 16-18, and the preferred ratio of acyl carbon chain to amine carbon chain is 16:4-18:6.

[0010] Thus, the lipid-amphiphile-precursor according to the invention has the following formula: Formulas I and II: [ka] [In the formula, R 1 is a hydrophobic group; R 2 is an amine-containing group; and R 3 teeth, [ka] Select from or a salt or tautomer thereof.

[0011] According to various embodiments, R 1 is alkyl, and optionally one or more R 4 where R 4 are each independently selected from alkyl and halo. According to various preferred embodiments, R 1 is (CH2) l CH3; where l is an integer from 10 to 16, inclusive.

[0012] According to various embodiments, R 2 is (CHR 5 ) m R 6 or (CHR 5 ) m NR 7 (CH2) n R 6 where R 5 are each independently H and N(R 7 )2; at most one R5 is not H; R 6 is N(R 8 )2 and N=C(NHR 8 )2 is selected from; R 7 are each independently selected from H and alkyl; R 8are each selected from H and alkyl; m is an integer from 2 to 6, inclusive; and n is an integer from 2 to 6, inclusive.

[0013] According to various embodiments, R 2 is CHR 5 (CH2) o R 6 or CHR 5 (CHR 5 ) o NR 7 (CH2) p R 6 and;R 5 are each independently H and N(R 7 )2; at most one R5 is not H; R 6 is N(R 8 )2 and N=C(NHR 8 )2 is selected from; R 7 are each independently selected from H and alkyl; R 8 are each selected from H and alkyl; p is an integer from 2 to 6, inclusive; and o is an integer from 1 to 5, inclusive.

[0014] According to various embodiments, R 3 teeth, [ka] is selected from.

[0015] According to various embodiments, R 3 teeth, [ka] is.

[0016] According to a further aspect of the invention, the allosteric effector may be one or more of 2,3-DPG, RSR-13, inositol phosphate, inositol hexaphosphate (IP6), phytic acid, guanosine triphosphate.

[0017] According to yet a further embodiment of the present invention, the phospholipid solution is most preferably a mixture of cholesterol and PEG in a ratio of 17.00:2.34. 2000 According to an alternative embodiment, the phospholipid solution may be a PEG having a molecular weight between 500 Da and 5000 Da if a low or high membrane thickness of the self-assembled particles is desired.

[0018] According to a further embodiment of the present invention, the synthetic blood substitute of the present invention can be prepared by dissolving the lipid-oligomer amphiphile in a solution of phospholipids and then evaporating the phospholipids to form a film. Frozen hemoglobin premixed with an allosteric effector is thawed, transferred to the dry film, and then mixed. After mixing the hemoglobin payload and membrane components, the mixture is sonicated and then allowed to rest to reach equilibrium, after which the hydrodynamic diameter of the self-assembled oxygen carrier particles is measured. The mixture is then filtered until the outlet stream is clear, and the hydrodynamic diameter is rechecked.

[0019] In accordance with a further embodiment of the present invention, the lipid-amphiphile precursor and hemoglobin are self-assembled into the synthetic blood substitute of the present invention, and the resulting product is freeze-dried and packaged. The freeze-dried product is a powder containing the amphiphile precursor, cholesterol, and PEG-PE hemoglobin and an allosteric effector, and optionally a cryoprotectant.

[0020] Reconstitution at the original EM production concentration (or concentration) can be achieved with PBS / water by simple mixing and gentle vortexing / agitation. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram illustrating composition and self-assembly according to an embodiment of the present invention.

[0022] [Figure 2] FIG. 2 is a diagram illustrating the synthesis of a precursor designated KC-1003 according to a preferred embodiment of the present invention.

[0023] [Figure 3] FIG. 3 is a schematic representation of the self-assembly of precursor KC-1003 and hb: an allosteric effector into oxygen carrier particles according to a preferred embodiment of the present invention.

[0024] [Figure 4] FIG. 4 is a table showing the free hemoglobin detected after purification of batches of oxygen carrier particles using various purification methods.

[0025] [Figure 5] FIG. 5 is a graph showing the change in oxygen affinity with the change in pH of oxygen carrier particles according to the present invention.

[0026] [Figure 6] FIG. 6 shows the hydrodynamic diameter and TEM image (inset) of a synthetic blood substitute according to an embodiment of the present invention.

[0027] [Figure 7] FIG. 7 shows the electrophoretic potential results according to an embodiment of the present invention.

[0028] [Figure 8] FIG. 8 shows a freeze-dried sample of oxygen carrier particles according to an embodiment of the present invention.

[0029] [Figure 9] FIG. 9 shows various properties of oxygen carrier particles according to an embodiment of the present invention before lyophilization, after 4 days of reconstitution, and after 14 days of reconstitution.

[0030] Detailed Description The synthesis of the preferred precursor, KC-1003, is shown in Figure 2. The lipid-oligomer amphiphiles are preferably synthesized and stored at 4°C. Purity of >90% can be confirmed by qHPLC, 1H NMR, 13C NMR and / or HRMS.

[0031] Preparation of precursor films 1a. In a test tube (25 x 250 mm), dissolve 80.66 mmol of the precursor [KC 1003] in anhydrous chloroform to a concentration of 8 mg / mL. Transfer the chloroform using a glass syringe or measure it into a glass cylinder. 1b. Add 17 mmol of cholesterol and 2.34 mmol of 25 mg / mL of PEG2000-PE to a test tube, resulting in a 100 mM solution of surfactant in chloroform. 1c. Gently swirl the test tube for 1 minute to homogenize / until the solution becomes clear. 1d. Pass the precursor solution through a small cotton bed to ensure the absence of particulates. 1e. Evaporate the chloroform in the test tube under reduced pressure using a rotary evaporator. The water bath temperature is 50°C. A film forms on the wall of the test tube and no liquid chloroform remains. 1f. Dry the test tube in a vacuum oven at HV, room temperature, 20°C for 12 h. 1g. Reweigh the test tube.

[0032] self-organization 2a. Leave the frozen hemoglobin at room temperature (20°C) until thawed. 2b. Transfer the hemoglobin to a 50 mL centrifuge tube using an Eppendorf pipette and immediately close the lid. 2c. Transfer the hemoglobin to the dry film in the test tube via a micropipette to produce a precursor-to-payload component Hb ratio of 6:339:1. Immediately sonicate the container (as described in the next step). Note: Use a volume of stock Hb soln. equal to the mass of the chloroform precursor.

[0033] Granularity Standardization After mixing of the payload and membrane components, the test tube is immediately sonicated in ice water [<5 mL for 2 minutes, >5 mL for 5 minutes]. The test tube is rotated by hand over a fixed probe. 3b. After sonication, let the test tube rest for at least 15-20 minutes (actual time is shown here) to allow equilibrium to be reached from Brownian motion. 3c. Next, confirm the hydrodynamic diameter by DLS (nm and PDI).

[0034] purification 4a. After sonication, the contents of the test tube are loaded into a Tangental Flow Filtration System using a 50 nm PS membrane (D02-S05U-05-N) and running Ringer's lactate at a rate of 130 mL / min. 4b. Run the TFF until the outlet effluent is clear on the spectrophotometer. 4c. DLS (hydrodynamic diameter and PDI) is checked again and the variation is within 10%.

[0035] The resulting product is lyophilized and packaged as a powder containing the amphiphilic precursor, cholesterol and PEG-PE hemoglobin, and the allosteric effector, and optionally a cryoprotectant.

[0036] Reconstitution of the original EM production concentrate (or concentrates) can be done with PBS / water by simply mixing and gently vortexing / agitating. Furthermore, the present invention includes the following aspects. [Aspect 1] formula: [ka] [In the formula, R 1 is a hydrophobic group; R 2 is an amine-containing group; and R 3 teeth, [ka] Select from or a salt or tautomer thereof. [Aspect 2] R 1 However, (CH 2 ) l CH 3 and l is an integer from 10 to 16, inclusive. [Aspect 3] R 1 However, (CH 2 ) l CH 3 and l is an integer from 10 to 16, inclusive. [Aspect 4] The compound of embodiment 3, wherein 1 is 14. [Aspect 5] R 2 However, (CHR 5 ) m R 6 and (CHR 5 ) m NR 7 (CH 2 ) n R 6 Selected from; R 5 are each independently H and N(R 7 ) 2 Selected from; At most one R 5 but not H; R 6 But N(R 8 ) 2 and N=C(NHR 8 ) 2 Selected from; R 7 are each independently selected from H and alkyl; R 8 are each selected from H and alkyl; m is an integer from 2 to 6, inclusive; and A compound according to any one of aspects 1 to 4, wherein n is an integer from 2 to 6, inclusive. [Aspect 6] R 5 are each independently H and NHCH 3 and at most one R 5 is not H. [Aspect 7] R 6 But N(R 8 ) 2 and N=C(NH 2 ) 2 6. The compound according to embodiment 5, selected from: [Aspect 8] R 2 but,

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Claims

1. formula: 【Chemistry 1】 [In the formula, R 1 is a hydrophobic group; R 2 teeth, 【change】 is selected from; and R 3 teeth, 【Chemistry 2】 Selected from or a salt or tautomer thereof.

2. R 1 However, (CH 2 ) l CH 3 and l is an integer from 10 to 16, or a salt or tautomer thereof.

3. 3. The lipid-amphiphile precursor compound of claim 2, or a salt or tautomer thereof, wherein 1 is 14.

4. R 3 but, 【Transformation 3】 4. The lipid-amphiphile precursor compound of any one of claims 1 to 3, wherein:

5. R 1 However, (CH 2 ) 14 CH 3 5. The lipid-amphiphile precursor compound of any one of claims 1 to 4, wherein:

6. (a) phospholipids; (b) cholesterol; (c) oxygen carrier; (d) a lipid-amphiphile precursor compound according to any one of claims 1 to 5, or a salt or tautomer thereof; and (e) allosteric effectors, A composition comprising:

7. 7. The composition of claim 6, wherein the oxygen carrier is hemoglobin (Hb).

8. 8. The composition of claim 6, 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.

9. 9. The composition of any one of claims 6 to 8, which self-assembles into a vesicle having an amphiphilic lipid bilayer membrane and a payload comprising said oxygen carrier and said allosteric effector.

10. The composition of any one of claims 6 to 9, further comprising a reducing agent.

11. 11. The composition of claim 10, wherein the reducing agent is selected from leucomethylene blue, n-benzoyl-leucomethylene blue, and methylene blue.

12. 1. A method for producing a synthetic oxygen carrier, comprising: (a) phospholipids; (b) cholesterol; (c) oxygen carrier; (d) a lipid-amphiphile precursor compound according to any one of claims 1 to 5, or a salt or tautomer thereof; and (e) allosteric effectors, preparing a composition comprising: allowing the composition to self-assemble into a vesicle having an outer lipid bilayer membrane and a payload comprising the oxygen carrier and the allosteric effector; and freeze-drying the self-assembled vesicles.

13. 13. The method of claim 12, wherein the composition further comprises a reducing agent selected from the group consisting of methylene blue, n-benzoyl-leucomethylene blue, and leucomethylene blue.

14. 12. A method for preparing a synthetic oxygen carrier, comprising lyophilizing a composition according to any one of claims 6 to 11 and reconstituting the lyophilized composition.

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