MICROPARTICLES

Abstract

The invention provides a self-assembled microparticle having an acid having two or more acid groups and an organic base in a solvent. The microparticles may form into a macrostructure and provide a scaffold for cell culture. 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. The microparticles have antimicrobial activity and may be used in a wide range of consumer product applications, cell culture and medical products, such as wound dressings.

Claims

1. A self-assembled microparticle comprising an acid having two or more acid groups and an organic base.

2. A microparticle according to claim 1 having a particle size of 0.5 to 10 microns.

3. A microparticle 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.

4. (canceled)

5. A method of making a microparticle according to claim 1 suitable for use as a particulate support comprising contacting a bis-acid and an 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.

6-7. (canceled)

8. A microparticle according to claim 1 wherein the acid comprises a bis-acid.

9-11. (canceled)

12. A microparticle according to claim 1 wherein the acid comprises a compound of general formula HOOC(CH.sub.2).sub.nCOOH wherein n is sufficiently large that the bis acid is sparingly soluble or insoluble in water.

13. A microparticle according to claim 12 wherein n is at least 5 and not more than 40.

14. A microparticle according to claim 1 wherein the acid comprises brassylic acid, sebacic acid and/or azelaic acid.

15. A microparticle according to claim 1 wherein the organic base comprises an aliphatic amine or an aromatic amine having a basic character or other nitrogen-containing base.

16. (canceled)

17. A microparticle according to claim 1 wherein 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.

18. A microparticle according to claim 1 wherein the microparticle comprises a multi-lamellar structure.

19. (canceled)

20. A microparticle according claim 1 where the bis-acid is reacted with the organic base to form a cross-linked species.

21. A microparticle according to claim 1 wherein the organic base is displaced with another reactive base which is then reacted to form a cross-linked species.

22. A macroporous material comprising microparticles according to claim 1, wherein the microparticles are contacted so as to form cross links between the microparticles and thereby form a three dimensional body.

23. A microparticle according to claim 1 and a macroporous material according to claim 22 further comprising a functional material, wherein the functional material absorbed or covalently attached to the self-assembled microparticle and/or macroporous material is selected from a catalyst, an initiator species for peptide synthesis, an initiator species for oligonucleotide synthesis, an initiator species for solid phase organic synthesis, a pharmaceutical active, an agrochemical active, a protein, an enzyme or other biological macromolecule.

24. A medical diagnostic comprising a self-assembled microparticle according to claim 1 or a macroporous material according to claim 22 and further comprising a functional material bound or retained by the support.

25. A medical diagnostic according to claim 24 wherein the functional material comprises an enzyme supported by a polymer.

26-28. (canceled)

29. A wound care product comprising a self-assembled microparticle according to claim 1 or a macroporous material according to claim 22 for use in wound care or treatment of a wound either internal or external to the body.

30. A self-assembled microparticle according to claim 1 or a macroporous material according to claim 22 as a support or three dimensional scaffold for cell culture, regenerative medicine or tissue repair.

31. A method for producing a self-assembled microparticle according to claim 1 or macroporous material according to claim 22 comprising contacting the two-acid having two or more acid groups with an organic base in an aqueous medium.

32. (canceled)

Description

EXAMPLE 1PREPARATION OF SELF-ASSEMBLED MICROPARTICLES

[0078] 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

[0079] 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

[0080] 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

[0081] 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.

Pimelic acid plus NMM
Suberic acid plus NMM
Azelaic acid plus NMM
Sebacic acid plus NMM
Sebacic acid plus DMAP
Sebacic acid plus DMAE
Sebacic acid plus imidazole
Dodecanedioic acid plus NMM
Dodecanedioic acid plus DMAP
Dodecanedioic acid plus DMAE
C36 dimer acid plus NMM

EXAMPLE 5PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES

[0082] 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). Poly-epsilon-lysine (PeK) (2 g, 12.04 mmol of NH.sub.2) was dissolved in water (10 cm.sup.3) and added to the above solution of Brassylic acid/DMAP microspheres. The mixture was filtered through a 0.45 m membrane and a sample placed on a microscope. Microspheres of 3 m diameter were still present. This solution was diluted with water to 100 cm.sup.3. N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCI)(4.6 g, 2.4 mmol) and HONSu (1.38 g, 1.2 mmol) were dissolved in water (10 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) and recovered by lyophilisation (yield 2.35 g). FIG. 2 shows a scanning electron micrograph of the resultant microspheres.

EXAMPLE 6PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES CONTAINING PROTOPORPHYRIN IX, HEME B

[0083] Brassylic acid (0.734 g, 3.3 mmol) and 4-dimethylaminopyridine (DMAP, 0.734 g, 6.6 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). Poly-epsilon-lysine (PeK) (1 g, 6.02 mmol of NH.sub.2) was dissolved in water (10 cm.sup.3) and added to the above solution of Brassylic acid/DMAP microspheres. The mixture was filtered through a 0.45 m membrane and a sample placed on a microscope. Microspheres of 3 m diameter were still present. This solution was diluted with a saturated solution of Heme B (50 cm.sup.3). N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCI)(2.3 g, 1.2 mmol) and HONSu (0.7 g, 0.6 mmol) were dissolved in water (5 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) and recovered by lyophilisation (yield 0.93 g).

EXAMPLE 7PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES

[0084] Sebacic acid (0.619 g, 6.12 mmol) and NMM (0.62 g, 6.12 mmol) were dissolved in water (10 cm.sup.3) and a sample placed on a microscope. Almost monodispersed spherical entities of 2.5 m diameter were observed.

[0085] Poly-epsilon-lysine (PeK) (1 g, 5.83 mmol of NH.sub.2) was dissolved in water (10 cm.sup.3) and added to the above solution of Sebacic acid/NMM microspheres. The mixture was filtered through a 0.45 m membrane and a sample placed on a microscope. Microspheres of 2.5 m diameter were still present. This solution was diluted with water to 50 cm.sup.3. EDCI (2.24 g, 11.7 mmol) and HONSu (2.0 g, 17.4 mmol) were dissolved in water (10 cm.sup.3) and added to the above solution. The cross-linking reaction was left overnight, the resultant particles washed by TFF and recovered by lyophilisation.

EXAMPLE 8PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES

[0086] Sebacic acid (5.06 g, 25 mmol) and imidazole (3.4 g, 50 mmol) were dissolved in water (50 cm.sup.3) and a sample placed on a microscope. Almost monodispersed spherical entities of 2.5 m diameter were observed.

[0087] Poly-epsilon-lysine (PeK) (8.576 g, 50 mmol of NH.sub.2) was dissolved in water (50 cm.sup.3) and added to the above solution of Sebacic acid/imidazole microspheres. The mixture was filtered through a 0.45 m membrane and a sample placed on a microscope. Microspheres of 2.5 m diameter were still present (FIG. 3). This solution was diluted with water to 500 cm.sup.3. EDCI (4.8 g, 25 mmol) was dissolved in water (20 cm.sup.3) and added to the above solution. The cross-linking reaction was left for 1 h then a further 25 mmol of EDCI added before leaving overnight. The resultant particles washed with water by decantation and recovered by lyophilisation (FIG. 4).

EXAMPLE 9PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES

[0088] Sebacic acid (5 g, 24.7 mmol) and (3-Aminopropyl)trimethoxysilane (8.42 g, 46.9 mmol) were dissolved in water (50 cm.sup.3) and a sample placed on a microscope. Almost monodispersed spherical entities of 2.5 m diameter were observed.

[0089] The mixture was left overnight then acidified with concentrated hydrochloric acid. The addition of hydrochloric acid led to the formation of silica within the particles creating a Sebacic acid/silica composite.

EXAMPLE 10PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES

[0090] Sebacic acid (5 g, 24.7 mmol) and N-[3-(Trimethoxysilyl)propyl]ethylenediamine (5.77 g, 51.9 mmol of amine) were dissolved in water (50 cm.sup.3) and a sample placed on a microscope. Almost monodispersed spherical entities of 2.5 m diameter were observed.

[0091] This solution was diluted with water to 500 cm.sup.3. EDCI (20 g, 104 mmol) was dissolved in water (100 cm.sup.3) and added to the above solution. The mixture was left overnight then acidified with concentrated hydrochloric acid. The addition of hydrochloric acid led to the formation of silica within the particles creating a Sebacic acid/silica composite.

EXAMPLE 11PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLES

[0092] Sebacic acid (5 g, 24.7 mmol) and N1-(3-Trimethoxysilylpropyl)diethylenetriamine (4.37 g, 46.9 mmol of amine) were dissolved in water (50 cm.sup.3) and a sample placed on a microscope. Almost monodispersed spherical entities of 2.5 m diameter were observed.

[0093] This solution was diluted with water to 500 cm.sup.3. EDCI (20 g, 104 mmol) was dissolved in water (100 cm.sup.3) and added to the above solution. The mixture was left overnight then acidified with concentrated hydrochloric acid. The addition of hydrochloric acid led to the formation of silica within the particles creating a Sebacic acid/silica composite.

EXAMPLE 12PREPARATION OF A SELF-ASSEMBLED MACROPOROUS CROSS-LINKED SHEET

[0094] Sebacic acid (0.619 g, 6.12 mmol) and NMM (0.62 g, 6.12 mmol) were dissolved in water (10 cm.sup.3) and a sample placed on a microscope. Almost monodispersed spherical entities of 2.5 m diameter were observed.

[0095] Poly-epsilon-lysine (PeK) (1 g, 5.83 mmol of NH.sub.2) was dissolved in water (10 cm.sup.3) and added to the above solution of Sebacic acid/NMM microspheres. The mixture was filtered through a 0.45 m membrane and a sample placed on a microscope. Microspheres of 2.5 m diameter were still present. EDCI (2.24 g, 11.7 mmol) and HONSu (2.0 g, 17.4 mmol) were dissolved in water (10 cm.sup.3) and added to the above solution. The cross-linking reaction was left overnight, the resultant sheets washed with water and dried by lyophilisation. The SEM shown in FIG. 5 clearly demonstrates the fused microsphere structure of the macroporous polymer formed.

EXAMPLE 13PREPARATION OF A SELF-ASSEMBLED MACROPOROUS CROSS-LINKED SHEET

[0096] (12-Phosphonododecyl)phosphonic acid (330 mg, 1 mmol) and NMM (404 mg, 4 mmol were dissolved in water. A sample placed on a microscope confirmed the presence of virtually monodispersed microspheres. PeK (343 mg, 2 mmol NH.sub.2) was dissolved in water (10 cm.sup.3) and added to the bis-phosphonic acid solution prepared above. Microspheres were still present at this stage. EDCI (1.15 g, 6 mmol) dissolved in water (10 cm.sup.3) was added and the mixture immediately poured into a tray. Again microspheres were still present at this stage. A sheet formed after 2 h which was washed thoroughly with water. The final sheet had a rubbery texture.

EXAMPLE 14BONE CELL CULTURE (EXAMPLE OF 3D CELL CULTURE)

[0097] In this Example a self-assembled macroporous sheet as produced in Example 12 was produced. A further product comprising the sheet of Example 12 together with about 10% by weight relative to the scaffold of hydroxyapatite nanoparticle available from Sigma Aldrich under catalogue number 702153 and the cytocompatibility was tested.

[0098] The 9 day growth curve of osteoblasts bone cells demonstrated there was no significant difference between cell viability during the early stages of the culture period when compared to the tissue culture plastic control. There was no significant difference in cell viability between osteoblast cultured on either the carboxyl or hydroxyapatite coated scaffolds. Cellular interaction with the 3D scaffold became more apparent at 40 magnification and actin filament staining highlighted adhesion points where cells had anchored to the 3D scaffold as shown in FIG. 7.

[0099] FIG. 7 shows osteoblasts cultured on a carboxyl functionalized and hydroxyapatite coated 3D scaffolds, a+b) cultured for 48 h at 10 magnification, c+d) cultured for 48 h at 40 magnification, e+f) cultured for 7 days at 10 magnification, g+h) cultured for 7 days at 20 magnification.

[0100] Osteoblasts continued to survive and interact with the scaffold for the rest of the culture period. These results demonstrate that the 3D scaffold with/without the hydroxyapatite coating supports osteoblast growth.

Osteoblast Proliferation

[0101] Osteoblasts were seeded at a density of 110.sup.5 cells on 24 well inserts and cultured under standard tissue culture conditions of 37 C. and 5% CO.sub.2. Dulbecco's Modified Eagle Medium (DMEM) (high glucose+2 mM glutamine) media containing 10% FCS with fungizone and pen/strep supplementation was used. Cellular F-actin was stained with FITC conjugated Phalloidin and then counter-stained with the nuclear stain, Hoechst 33342. Images were taken at time points 24 h, 48 h, and 7 days using a Nikon Eclipse Ti-E phase contrast microscope (Nikon, Tokyo, Japan) (FIG. 6). FIG. 6: Metabolic activity assay (CCK-8) of osteoblasts cultured on a carboxyl and hydroxyapatite coated 3D scaffold over a 9 day culture period.

Cell Proliferation Assay

[0102] A CCK-8 assay kit (Dojindo Laboratories, Kumamoto, Japan) was used to monitor cell proliferation at various time periods during the course of a 9 day cell culture period. A standard curve to determine cell number was carried out following the manufacturer's guidelines. Cells were incubated under standard culture conditions after seeding scaffolds with an initial density of 510.sup.4 cells. At each time-point CCK-8 solution (50 mm.sup.3) was added to the media (500 mm.sup.3) in each well. The cells were then incubated under standard culture conditions for 2 h. An aliquot (3100 mm.sup.3) of solution from each well was pipetted into labelled wells in a 96-well plate. Suitable controls were also used. The absorbance was read at 485 nm using a FLUOstar Optima plate reader (BMG Labtech, Ortenberg, Germany) with a background reading at 600 nm and the results recorded.

EXAMPLE 15BIOCIDE FORMULATIONS

[0103] Biocides for personal care, cosmetics, home care and general disinfection are currently limited by the time they stay in contact with the surface to be treated due to abrasion. For example, surface sprays of the type used for disinfection in hospitals have a limited active lifetime and consequently reduced activity against hospital infections such as MRSA, p. auregenosa and c. difficile. In addition, some surface sprays contain organic solvents such as isopropanol or non-biodegradable components such as silicone oils to reduce abrasive removal of the biocides.

[0104] Cationic and amphoteric biocides such as quaternary ammonium compounds act against pathogens by solubilising the cell membrane, resulting in cell lysis and death. There are many biocides used commercially for disinfection which include chlorhexidine, benzalkonium chloride, climbazole, didecyldimethylammonium chloride, dodecyldipropylenetriamine, which are cationic compounds. Additionally some biocides are polymeric cationic compounds such as Poly(diallyldimethylammonium chloride). These compounds can be readily formulated into spherical microparticles using the technology described herein, which will allow for reduced abrasive removal on surfaces, skin and hair; potentially allowing for controlled release of the biocide. Additionally, biocides containing multiple cationic compounds in the same microparticle are possible and may provide formulations that can be tailored and targeted to specific applications where the source of infection is well defined.

[0105] The samples produced were as follows:

Poly(diallyldimethylammonium chloride) (PDAC) SpheriSomes

[0106] 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 to be a clear solution but was confirmed to be a suspension of 3 m microparticles and a novel formulation of PDAC when observed under the microscope and the results are shown in FIG. 8 shows PDAC-Brassylic acid microparticle formulation.

Didecyldimethylammonium Chloride (DDAC)

[0107] DDAC (9.04 cm.sup.3 of 40% w/v solution, 10 mmol) was diluted with water to 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 to be a hazy solution but was confirmed to be a suspension of 3 m microparticles and a novel formulation of DDAC when observed under the microscope.

Dodecyldipropylenetriamine (DDPT)

[0108] DDPT (9.97 cm.sup.3 of 30% w/v solution, 10 mmol) was diluted with water to 50 cm.sup.3 and Brassylic acid (3.66 g, 15 mmol) was added to this solution and allowed to dissolve overnight. This appeared to be a clear solution but was confirmed to be a suspension of 3 m microparticles and a novel formulation of DDPT when observed under the microscope.

EXAMPLE 16ANTIMICROBIAL WOUND DRESSINGS

[0109] The hydrophilic nature of the porous polymer formed by collision of the biscarboxy fatty acid microparticles is advantageous in for absorbent wound dressings. When the biscarboxy fatty acids are combined with poly--lysine and cross-linked to form such a porous matrix the natural antimicrobial activity of the components of the wound dressing can be retained and enhanced when necessary. In cationic form, where there is an excess of poly--lysine over the fatty acids the materials have been shown to retain the characteristics of the food preservative providing novel antimicrobial wound dressings. The porous nature of the material will allow for improved skin repair as a 3D scaffold combined with a cationic nature capable of destroying microbial biofilms.

[0110] The anti-biofilm capability of a cationic wound dressing was assessed using a mixed species CDC reactor model. The product of Example 13 was employed in these experiments.

[0111] Two mixed species biofilms were prepared as shown below and tested against PBS and a control anionic dressing.

Multi Species Biofilm 1

[0112] Staphylococcus aureus NCTC 8325
Pseudomonas aeruginosa NCIMB 10434
Acinetobacter baumannii ATCC 19606
Staphylococcus epidermidis

Multi Species Biofilm 2

[0113] Staphylococcus aureus NCTC 8325

MRSA

[0114] VRE faecalis NCTC 12201
Candida albicans ATCC MYA-2876 SC5313
Escherichia coli NCTC 12923 Page 3 of 6 DOT 202 (03)

Preparation of Mixed Inoculum 1

[0115] Twenty-four hour cultures of Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus epidermidis were harvested from appropriate agar plate using a sterile swab and suspended in 20 cm.sup.3 of Tryptone Soya Broth (TSB). The mixed species suspension was diluted in TSB to give an overall concentration of 1075106 cfuml-1 and used as the inoculum for the CDC reactor. The CDC reactor was incubated for 72 hours at 37 C. with shaking at 50 rpm in order to encourage biofilm growth.

Preparation of Mixed Inoculum 2

[0116] Twenty-four hour cultures of Staphylococcus aureus, Methicillin-resistant staphylococcus aureus, Vancomycin-resistant Enterococcus, Candida albicans and Escherichia coli were harvested from appropriate agar plate using a sterile swab and suspended in 20 cm.sup.3 of TSB. The mixed species suspension was diluted in TSB to give an overall concentration of 1075106 cfuml-1 and used as the inoculum for the CDC reactor. The CDC reactor was incubated for 72 hours at 37 C. with shaking at 50 rpm in order to encourage biofilm growth.

Biofilm Treatment

[0117] After incubation the test coupons were removed from the CDC reactor and washed 3 times in sterile phosphate buffered saline (PBS) in order to remove planktonic cells. The washed coupons were then treated by sandwiching the coupon between two discs of the wound dressing material. The dressings were activated prior to testing by the addition of 400 mm.sup.3 PBS+1% TSB to each disc. Control coupons were submerged in 1 cm.sup.3 of PBS+1% TSB. All samples were tested in triplicate. Following the 24 hour treatment period, the coupons were placed in 1 cm.sup.3 PBS and sonicated for 15 minutes in order to recover any viable microorganisms attached to the coupons. Recovered microorganisms were quantified using serial dilutions and spread plates.

Mixed Inoculum 1

[0118] Following treatment with the control dressing (A), bacterial recovery was similar to PBS only treatment controls as shown in FIG. 9. No viable organisms were recovered from coupons treated with cationic dressing (B). This represents a greater than 5 log reduction compared to PBS treated controls. The surviving organism post treatment were predominantly Pseudomonas aeruginosa (FIG. 10).

Mixed Inoculum 2

[0119] Treatment with control dressing (A) resulted in a 1.27 log reduction in the number of viable bacteria recovered compared to the PBS treated controls. No viable organisms were recovered from coupons treated with cationic dressing (B). This represents a greater than 7 log reduction compared to the PBS treated controls (FIG. 11). Surviving organisms were mixed species (FIG. 12).