MICROPARTICLES

Abstract

The invention provides a blood substitute product comprising haemoglobin and a self-assembled microparticle having an acid having two or more acid groups and an organic base in a solvent. The particle is of micron scale. The microparticle may be obtained by contacting a bis-acid and organic base in a hydrophilic solvent, wherein the acid is insoluble or sparingly soluble in the hydrophilic solvent and the organic base is soluble in a hydrophilic solvent.

Claims

1. A blood substitute product comprising mammalian haemoglobin and a self-assembled microparticle in which the microparticle comprises an acid having two or more acid groups and an organic base which is soluble in a hydrophilic solvent.

2. A blood substitute product according to claim 1 in which the blood substitute product comprises a stable polymerized haemoglobin solution, comprising mammalian haemoglobin cross-linked with a self-assembled microparticle.

3. A blood substitute product according to claim 1 in which the microparticle has a particle size of 0.5 to 10 microns.

4. A blood substitute product according to claim 1 in which the molar ratio of acid groups to basic groups in the acid and base is from 0.6 to 1.4:1.

5. A blood substitute product according to claim 1 in which the molar ratio of acid groups to basic groups is from 0.7 to 1.3:1.

6. A blood substitute product according to claim 1 in which the microparticle comprises an acid having two or more acid groups and an organic base obtainable by a process comprising contacting the bis-acid and organic base in a hydrophilic solvent, wherein the acid is insoluble or sparingly soluble in the hydrophilic solvent and the organic base is soluble in a hydrophilic solvent.

7. A blood substitute product according to claim 6 in which the solvent comprises an aqueous solution.

8. A blood substitute product according to claim 6 wherein the solvent comprises a water in oil emulsion within an aqueous phase.

9. A blood substitute product according to claim 1 in which the acid comprises a bis-acid.

10. A blood substitute product according to claim 9 in which the acid comprises a bis-aliphatic acid.

11. A blood substitute product according to claim 1 in which the acid comprises a bis-carboxylic fatty acid in which terminal carboxylic acids are linked by a region which is hydrophobic.

12. A blood substitute product according to claim 1 in which the acid groups are separated by a saturated, or unsaturated aliphatic chain; or substituted saturated, or unsaturated aliphatic chain.

13. A blood substitute product according to claim 1 in which the acid comprises a compound of general formula HOOC(CH.sub.2).sub.nCOON wherein n is sufficiently large that the bis acid is sparingly soluble or insoluble in water.

14. A blood substitute product according to claim 13 in which n is at least 5 and not more than 40.

15. A blood substitute product according to claim 1 in which the acid comprises brassylic acid, sebacic acid and/or azelaic acid.

16. A blood substitute product according to claim 1 in which the organic base comprises an aliphatic amine or an aromatic amine having a basic character or other nitrogen-containing base.

17. A blood substitute product according claim 1 in which the organic base comprises one or more of an alkylated amine and an alkylated polyamine.

18. A blood substitute product according to claim 1 in which the organic base comprises one or more of N-methylmorpholine, N, N-dimethylaminoethanol, 4-dimethylaminopyridine, imidazole, 1-methylamidazole poly(diallyldimethylammonium chloride) (PDAC), didecyldimethylammonium chloride (DDAC), dodecyldipropylenetriamine (DDPT) and poly epsilon lysine.

19. A blood substitute product according to claim 1 in which the microparticle comprises a multi-lamellar structure.

20. A blood substitute product according to claim 1 in which the acid is reacted with haemoglobin to form a cross-linked species.

21.-23. (canceled)

Description

EXAMPLE 1PREPARATION OF SELF-ASSEMBLED MICROPARTICLES

[0048] Brassylic acid (1.54 g, 6.31 mmol) and 4-dimethylaminopyridine (DMAP, 1.54 g, 12.62 mmol) were dissolved in water (10 cm.sup.3) and a sample placed on a microscope. Almost monodispersed spherical entities of 3 m diameter were observed (FIG. 1).

EXAMPLE 2PREPARATION OF SELF-ASSEMBLED MICROPARTICLES

[0049] Brassylic acid (1.54 g, 6.31 mmol) and dimethylaminoethanol (DMAE, 1.12 g, 12.62 mmol) were dissolved in water (10 cm.sup.3) and a sample placed on a microscope. Almost monodispersed spherical entities of 3 m diameter were observed.

EXAMPLE 3PREPARATION OF SELF-ASSEMBLED MICROPARTICLES

[0050] Brassylic acid (1.54 g, 6.31 mmol) and 4-methylmorpholine (NMM, 1.275 g, 12.62 mmol) were dissolved in water (10 cm.sup.3) and a sample placed on a microscope. Almost monodispersed spherical entities of 3 m diameter were observed.

EXAMPLE 4PREPARATION OF SELF-ASSEMBLED MICROPARTICLES

[0051] The above dicarboxylic acid dissolution experiments were also carried out using a range of acids and a range of water soluble organic bases. Some of the combinations tested are listed below. The combinations had an acid group to basic group molar ratio of 0.9 to 1.1:1. All of these combinations formed the spherical entities as described in Example 1. [0052] Pimelic acid plus NMM [0053] Suberic acid plus NMM [0054] Azelaic acid plus NMM [0055] Sebacic acid plus NMM [0056] Sebacic acid plus DMAP [0057] Sebacic acid plus DMAE [0058] Sebacic acid plus imidazole [0059] Dodecanedioic acid plus NMM [0060] Dodecanedioic acid plus DMAP [0061] Dodecanedioic acid plus DMAE [0062] C36 dimer acid plus NMM

EXAMPLE 5POLY(DIALLYLDIMETHYLAMMONIUM CHLORIDE) (PDAC) SPHERISOMES

[0063] PDAC (1.615 g, 10 mmol) was dissolved in water (50 cm.sup.3) and NaOH (0.4 g, 10 mmol) added. Brassylic acid (1.22 g, 5 mmol) was added to this solution and allowed to dissolve overnight. This appeared by visual inspection to be a clear solution but was confirmed to be a suspension of 3 m microparticles when observed under the microscope. A microscope photograph is shown in FIG. 2.

EXAMPLE 6PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES CONTAINING HAEMOGLOBIN

[0064] PDAC (16.167 g of 20% solution, 20 mmol) was dissolved in water (100 cm.sup.3) and NaOH (0.8 g, 20 mmol) added. Brassylic acid (2.44 g, 10 mmol) was added to this solution and allowed to dissolve. Human haemoglobin (2.44 g) was dissolved in water (100 cm.sup.3) and added to the solution of brassylic acid/PDAC SpheriSomes. The mixture was filtered through a 0.5 m cartridge and a sample placed on a microscope. Microspheres of 3 m diameter were still present. A solution of N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCl)(4.6 g, 24 mmol) and 1-hydroxybenzotriazole (HOBt) (0.47 g, 1.2 mmol) were dissolved in water (50 cm.sup.3) and added to the above solution. The cross-linking reaction was left overnight, the resultant particles washed by tangential flow filtration (TFF). It was noted that the supernatant was colourless confirming that the haemoglobin was incorporated into the SpheriSomes. A sample of the SpheriSomes was lyophilised. FIG. 3 shows a scanning electron micrograph of the resultant microspheres.

EXAMPLE 7PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES CONTAINING HAEMOGLOBIN

[0065] PDAC (16.167 g of 20% solution, 20 mmol) was dissolved in water (100 cm.sup.3) and NaOH (0.8 g, 20 mmol) added. Brassylic acid (2.44 g, 10 mmol) was added to this solution and allowed to dissolve. Human haemoglobin (3.66 g) was dissolved in water (100 cm.sup.3) and added to the solution of brassylic acid/PDAC SpheriSomes. The mixture was filtered through a 0.5 m cartridge and a sample placed on a microscope. Microspheres of 3 m diameter were still present. A solution of EDCl (4.6 g, 24 mmol) and HOBt (0.47 g, 1.2 mmol) were dissolved in water (50 cm.sup.3) and added to the above solution. The cross-linking reaction was left overnight, the resultant particles washed by tangential flow filtration (TFF). It was noted that the supernatant was colourless confirming that the haemoglobin was incorporated into the SpheriSomes. FIG. 4 shows a microscope photograph of the resultant microspheres.

EXAMPLE 8PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES CONTAINING HAEMOGLOBIN

[0066] PDAC (16.167 g of 20% solution, 20 mmol) was dissolved in water (100 cm.sup.3) and NaOH (0.8 g, 20 mmol) added. Brassylic acid (2.44 g, 10 mmol) was added to this solution and allowed to dissolve. Human haemoglobin (4.88 g) was dissolved in water (100 cm.sup.3) and added to the solution of brassylic acid/PDAC SpheriSomes. The mixture was filtered through a 0.5 m cartridge and a sample placed on a microscope. Microspheres of 3 m diameter were still present. A solution of EDCl (4.6 g, 24 mmol) and HOBt (0.47 g, 1.2 mmol) were dissolved in water (50 cm.sup.3) and added to the above solution. The cross-linking reaction was left overnight, the resultant particles washed by tangential flow filtration (TFF). It was noted that the supernatant was slightly pink confirming the majority of the haemoglobin was incorporated into the SpheriSomes. FIG. 5 shows a microscope photograph of the resultant microspheres.

EXAMPLE 9PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES CONTAINING HAEMOGLOBIN

[0067] L-Carnitine (1.612 g, 10 mmol) was suspended in water (20 cm.sup.3) and NaOH (0.4 g, 10 mmol) added. Brassylic acid (1.22 g, 5 mmol) was added to this solution and the mixture allowed to dissolve. Human haemoglobin (1.83 g) was dissolved in water (200 cm.sup.3) and added to the solution of Brassylic acid/carnitine SpheriSomes. The mixture was filtered through a 0.5 m cartridge and a sample placed on a microscope. Microspheres of 3 m diameter were present. A solution of EDCl (4.6 g, 24 mmol) and HOBt (0.37 g, 2.4 mmol) were dissolved in water (25 cm.sup.3) and added to the above solution. The cross-linking reaction was left overnight, the resultant particles washed by tangential flow filtration (TFF). It was noted that the supernatant was colourless confirming that the haemoglobin was incorporated into the SpheriSomes.

EXAMPLE 10PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES CONTAINING HAEMOGLOBIN

[0068] Tetraethyl ammonium hydroxide (TEA.OH) (4.2 cm.sup.3 of a 35% solution, 10 mmol) was added to water (20 cm.sup.3) and Brassylic acid (1.22 g, 5 mmol) was added to this solution. The Brassylic acid dissolved immediately to form SpheriSomes. Human haemoglobin (1.83 g) was dissolved in water (200 cm.sup.3) and added to the solution of Brassylic acid/TEA.OH SpheriSomes. The mixture was filtered through a 0.5 m cartridge and a sample placed on a microscope. Microspheres of 3 m diameter were still present. A solution of EDCl (2.3 g, 12 mmol) and HOBt (0.18 g, 1.2 mmol) were dissolved in water (25 cm.sup.3) and added to the above solution. The cross-linking reaction was left overnight, the resultant particles washed by tangential flow filtration (TFF). It was noted that the supernatant was colourless confirming that the haemoglobin was incorporated into the SpheriSomes.