Cross-linked poly-E-lysine non-particulate support

Abstract

The invention provides a non-particulate cross-linked poly--lysine polymer. The poly--lysine and cross linker are linked by amide bonds and may the cross linker has at least two functional groups capable of reacting with an alpha carbon amine of poly--lysine. The polymer is suitably insoluble in water and other solvents and is provided in macro form for example a sheet, article or fiber. The macro form polymer is useful in a wide range of applications including wound treatment, as a medical diagnostic comprising a particulate support and a functional material bound or retained by the support and solid phase synthesis of peptides, oligonucleotides, oligosaccharides, immobilisation of species, cell culturing and in chromatographic separation.

Claims

1. A process for carrying out: (i) solid phase synthesis of peptides, oligonucleotides, or oligosaccharides; (ii) solid phase extraction; (iii) solid phase organic chemistry; (iv) the immobilisation of a species selected from solid phase reagents, chemical catalysts, bio-catalysts, enzymes, proteins, antibodies, whole cells, and polymers; (v) cell culturing; or (vi) preparation of a stationary phase for chromatographic separation; the process comprising employing a non-particulate cross-linked poly--lysine polymer comprising poly--lysine and a cross-linker linked by amide bonds, wherein the cross-linker comprises at least two functional groups capable of reacting with an alpha carbon amine of poly--lysine; and the cross-linker is selected from the group consisting of: (a) a moiety derived from a compound of formula X[CO.sub.2H].sub.n, wherein n is 2 or more and X is an aliphatic chain; (b) a bis-carboxy-polyalkylene glycol; (c) nitrilotriacetic acid; (d) glutaric acid; (e) an amino acid; (f) EDTA; (g) a synthetic peptide containing the tripeptide sequence -Arg-Gly-Asp-; and (h) HOOCCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2NHCO CH.sub.2CH.sub.2COOH.

2. The process of claim 1, wherein the cross-linker comprises a moiety derived from a compound of formula X[CO.sub.2H].sub.n where n is 2 or more and X is a hydrophobic or hydrophilic linking group having a molecular weight of 14 to 250 excluding any functional substituents on the linking group.

3. The process of claim 1, wherein the polymer is insoluble in water.

4. The process of claim 1, wherein the polymer is porous.

5. The process of claim 1, wherein the cross-linked poly--lysine support is used to coat a non-particulate media directly or indirectly.

6. The process of claim 1, wherein the cross-linked poly--lysine support is used to coat and is bound covalently to a non-particulate media.

7. The process of claim 1, wherein the cross-linked poly--lysine is used to coat an organic non-particulate media.

8. The process of claim 1, wherein the cross-linked poly--lysine is used to coat an inorganic non-particulate media.

Description

(1) The invention is illustrated by reference to the accompanying drawings in which:

(2) FIG. 1 shows a diagrammatic representation of poly--lysine.

(3) FIG. 2 shows a diagrammatic representation of poly--lysine cross-linked with a bifunctional carboxylic acid.

(4) FIG. 3 shows a diagrammatic representation of poly--lysine cross-linked with aspartic acid as example.

(5) FIG. 4 shows a diagrammatic representation of poly--lysine cross-linked with cystine.

(6) FIG. 5 shows a diagrammatic representation of poly--lysine cross-linked with nitrilotriacetic acid.

(7) FIG. 6 shows an SEM of the product of Example 1 prior to dissolution of the polyacrylonitrile balloons is shown in FIGS. 7a and 7b show SEM's of the self-assembled supermacroporous sheet of Example 2.

(8) FIGS. 8a, 8b and 8c show sectioned samples of the stained cells showing proliferation throughout the polymer of Example 3.

(9) FIG. 9 shows sectioned samples of the stained cells showing proliferation throughout the polymer in Example 4.

(10) FIG. 10 shows a photograph of the self-assembled supermacroporous tube from Example 6.

(11) FIG. 11 shows an SEM of the resulting cross-linked nanofibre mat from Example 9.

(12) FIG. 12 shows a photograph of the lens of Example 10.

(13) The invention is illustrated by the following non-limiting examples.

EXAMPLE 1

Preparation of Supermacroporous Cross-linked Poly--lysine

(14) Poly--lysine (200 mg, 1 mmol amine content) was dissolved in DMF/water (2.45 cm.sup.3, 1:1 v/v) and NMM (0.137 cm.sup.3, 1.2 mmol) added followed by glutaric anhydride (70 mg, 0.6 mmol of glutaric anhydride i.e. an excess relative to the amine). The reaction was allowed to proceed for 2 hours.

(15) N-Hydroxysuccinimide (143 mg) was added followed by Expancel 920 DEX 80 d30 (80 m polyacrylonitrile balloons)(50 mg, 3 cm.sup.3) and EDCI (224 mg, 1.2 mmol) was added to initiate polymerisation. The mixture was mixed thoroughly for 1 minute cast into a sheet on a polypropylene surface before cutting into discs using a cork borer. The polymerisation was left to go to completion overnight at room temperature.

(16) An SEM of the product prior to dissolution of the polyacrylonitrile balloons is shown in FIG. 6. The cavities shown are approximately 20 to 100 microns in size as shown by comparison with the 300 micron scale bar at the bottom right hand portion of the figure.

(17) The discs prepared above were treated with DMF overnight to dissolve the polyacrylonitrile balloons then washed thoroughly with potassium phosphate buffer (100 mmol/dm.sup.3, pH 7) and water before freeze drying from water.

(18) The supermacroporous cross-linked poly--lysine has been used shown to support three dimensional growth and proliferation of human embryonic stem cells.

EXAMPLE 2

Preparation of a Self-assembled Supermacroporous Cross-linked Poly--lysine Sheet

(19) A solution was prepared containing poly--lysine (4.93 g, 28.7 mmol amine content), dodecanedioic acid (3.47 g, 30 mmol carboxyl content) and sodium hydroxide (1.15 g, 28.7 mmol) in water (100 cm.sup.3).

(20) A solution of EDCI (14.46 g, 75 mmol) and HONSu (1.65 g, 14 mmol) in water (30 cm3) was added to the above solution and the mixture immediately poured into a tray to form a layer 5 mm deep.

(21) After 20-30 minutes the mixture had solidified to form a 5 mm thick supermacroporous sheet. The sheet was washed thoroughly with water then dried by lyophilisation.

(22) SEM's of the self-assembled supermacroporous sheet is shown in FIGS. 7a and 7b.

EXAMPLE 3

3D Culture of Mouse Embryonic Stem Cells (Esc'S) on Self-assembled Supermacroporous Cross-linked Poly--lysine

(23) Supermacroporous discs cut from the above sheet were washed with phosphate buffered saline (3PBS) and UV irradiated for 30 min prior to cell contact.

(24) The discs were seeded with mouse embryonic stem cells and cultured in Advanced high glucose DMEM (Gibco, Invitrogen, UK) supplemented with 1 mM -mercaptoethanol (Gibco, Invitrogen, UK), 2 mM L-glutamine (Gibco, Invitrogen, UK), 1000 U/mL leukaemia inhibitory factor (LIF) (Millipore, UK) and 2% fetal calf-serum (PAA). This medium was changed every second day.

(25) Immuno-staining of ESCs in contact with polymer involved fixing cells in 4% paraformaldehyde (PFA), followed by washing with PBS (3). Cells were incubated with blocking solution (10% fetal calf serum, 0.1% Triton X-100 in PBS) at RT for 40 min. Blocking solution was removed and primary antibody solution was added (Oct4/Nanog), cells were incubated at 4 C. overnight. Cells were washed (3PBS), and secondary antibody solution was added and incubated at RT for 2 h, after which cells were washed (3PBS), and counterstained with nuclei marker, DAPI (1 cm.sup.3 DAPI: 1 L (1/100** working stock+1 mL PBS), incubated at RT in dark for 5 min. Cells were washed three times in PBS and mounted on a slide with a coverslip and fluorescent mountant.

(26) Sectioned samples of the stained cells showing proliferation throughout the polymer are shown in FIGS. 8a, 8b and 8c.

(27) ESCs were seeded onto polymers, to determine ESC attachment and, most importantly the effect the polymer has on ESC self-renewal over time.

(28) In FIG. 8a ESCs were seeded onto super macroporous polymers and allowed to proliferate for 7 days. After 7 days, ESCs and polymers were fixed, gelatine embedded, frozen and sectioned, before co-staining with DAPI (blue) and alkaline phosphatise (red). The polymer supports ESC viability and attachment, and ESCs retain alkaline phosphatase expression. Scale bar represents 100 m and images are representative of entire population.

(29) The experiment was repeated 3 times.

(30) For FIG. 8b ESCs were seeded onto supermacroporous polymers and allowed to proliferate for 7 days. After 7 days, ESCs and polymers were fixed, gelatine embedded, frozen and sectioned, prior to staining with self-renewal marker, nanog (green). The polymer is shown to support ESC attachment; furthermore ESCs remain positive for nanog, thus maintain the capacity to self-renew. Scale bar represents 25 um. Images are representative of entire population.

(31) In FIG. 8c ESCs were seeded onto supermacroporous polymers and allowed to proliferate for 7 days. After 7 days, ESCs and polymers were fixed, gelatine embedded, frozen and sectioned, prior to co-stain with self-renewal marker Oct4 (red) and nuclei marker DAPI (blue). The polymer supports ESC attachment, furthermore ESCs remain positive for Oct4, therefore maintaining the capacity to self-renew. Scale bar represents 50 um. Images are representative of entire population.

(32) The polymer supported ESC attachment and furthermore ESCs retained the expression of alkaline phosphatase for up to 7 days. Similarly, ESCs maintained the expression of self-renewal markers, transcription factors, Nanog and Oct4 after 7 days. Collectively, this suggests that this specific polymer not only supports ESC viability but supports maintenance of ESC pluripotency, crucial in any ESC scale-up culture condition.

EXAMPLE 4

3D Culture of Kidney Cells on Self-assembled Supermacroporous Cross-linked Poly--lysine

(33) Supermacroporous discs cut from the above sheet were washed with phosphate buffered saline (3PBS) and UV irradiated for 30 min prior to cell contact.

(34) The discs were seeded with kidney stem cells (KSC's) and cultured in high glucose DMEM (Gibco, Invitrogen, UK) supplemented with 10% fetal calf serum (PAA), 2 mM L-glutamine (Gibco Invitrogen, UK), 1% NEAA (Gibco, Invitrogen, UK), 1 mM 2--mercaptoethanol (Gibco Invitrogen, UK). This medium was changed every second day.

(35) KSCs (GFP stained) were seeded onto the polymer and attachment/interaction monitored. Initially at day 1, KSCs remained rounded on the surface, however at day 10, KSCs morphology appears typically flattened around the surface of the polymer.

(36) Sectioned samples of the stained cells showing proliferation throughout the polymer are shown in FIG. 9. KSCs GFP were seeded onto supermacroporous polymer and attachment/interaction monitored. Initially at day 1, KSCs remained rounded on the surface, however at day 10, KSCs morphology appears typically flattened around the surface of the polymer.

EXAMPLE 5

Culture of Schwann Nerve Cells on Self-assembled Supermacroporous Cross-linked Poly--lysine

(37) Samples of the supermacroporous polymer were placed in triplicate into wells of a 12 well tissue culture plate and UV sterilized for 1 hour prior to hydrating the samples in Schwann cell growth medium [SCGM (DMEM+10% FBS+GGF+forskolin)]. Two cell densities were seeded (500,000 and 50,000 Schwann cells) onto each of the supermacroporous polymer scaffolds in SCGM. The Alamar blue absorbance assay was used to test cell proliferation.

(38) Alamar blue results show that Schwann cells initially attach and survive in the supermacroporous polymer scaffolds after 24 hours. Overall cell proliferation was allowed to progress over a 5 day period in all samples tested.

(39) In summary, initial Schwann cell attachment and growth was demonstrated by the reduction of Alamar blue at 24 hours on all samples tested. All of the materials tested supported longer-term survival of Schwann cells and are therefore suitable biomaterial for supporting nerve regeneration.

EXAMPLE 6

Preparation of a Self-assembled Supermacroporous Cross-linked Poly--lysine Tube

(40) A solution was prepared containing poly--lysine (4.93 g, 28.7 mmol amine content), dodecanedioic acid (3.47 g, 30 mmol carboxyl content) and sodium hydroxide (1.15 g, 28.7 mmol) in water (100 cm3).

(41) A solution of EDCI (14.46 g, 75 mmol) and HONSu (1.65 g, 14 mmol) in water (30 cm3) was added to the above solution and the mixture immediately poured into a tubular mould.

(42) After 20-30 minutes the mixture had solidified to form a tube of 15 mm external diameter with a wall thickness of 5 mm. The tube was washed thoroughly with water then dried by lyophilisation.

(43) A photograph of the self-assembled supermacroporous tube is shown in FIG. 10.

EXAMPLE 7

Preparation of a Supermacroporous Column Monolith for Chromatographic Separations

(44) A solution was prepared containing poly-s-lysine (0.49 g, 2.9 mmol amine content), dodecanedioic acid (0.35 g, 3.0 mmol carboxyl content) and sodium hydroxide (0.115 g, 2.9 mmol) in water (10 cm.sup.3).

(45) A solution of EDCI (1.45 g, 7.5 mmol) and HONSu (0.165 g, 1.4 mmol) in water (3 cm.sup.3) was added to the above solution and the mixture immediately used to fill an empty HPLC column (4.6 mm diameter x 10 cm).

(46) After 20-30 minutes the mixture had solidified to form a monolith. The monolith was washed thoroughly with water on an HPLC system.

EXAMPLE 8

Immobilisation of Protein A on Cross-linked Poly--lysine Supermacroporous Column Monolith For Antibody Purification Coupling of rProtein A to Cross-linked Poly--lysine

(47) N-hydroxysuccinimide (1 g) was dissolved in cold MES buffer (25 mmol/dm.sup.3, pH 5.0, 2.5 cm.sup.3) and mixed with EDCI (1 g) dissolved in MES buffer (25 mmol/dm.sup.3, pH 5.0, 2.5 cm.sup.3). This solution was passed through the monolith using an HPLC pump. The monolith was washed with MES buffer (25 mmol/dm.sup.3, pH 5.0, 50 cm.sup.3) and immediately, a solution of rProtein A (5 cm.sup.3, 4 mg/cm.sup.3 in 25 mmol/dm.sup.3 MES, pH 5.0) was passed onto the column and allowed to stand overnight. The monolith was washed with Trizma-HCl (30 cm.sup.3 pH 7.4) to block any remaining N-hydroxysuccinimide esters on the polymer. The monolith was washed with water (100 cm.sup.3) and stored in water.

(48) The Protein A based monolith was tested to determine whether it retained Human IgG under standard conditions known to those skilled in the art. The column was shown to retain Human IgG as expected.

EXAMPLE 9

Preparation of an Electrospun Fibre

(49) A solution containing polyacrylonitrile (0.8 g, 150,000 average molecular weight), poly--lysine (0.4 g) and sebacic acid (0.24 g) in DMSO (6 cm.sup.3), was electrospun (24 kV, 0.5 cm.sup.3/hr) on to a roller drum at 40 C. and 30% humidity.

(50) A portion (5 cm2) of the electrospun fibre mat produced was treated with an aqueous solution of EDCI (1 g in 5 cm.sup.3 of water) for 1 h then washed thoroughly with water. The resulting cross-linked fibre mat was washed thoroughly with N,N-dimethylformamide to remove the PAN support, then it was washed with methanol before drying in air.

(51) An SEM of the resulting cross-linked nanofibre mat is shown in FIG. 11.

EXAMPLE 10

Preparation of an Optically Clear Lens

(52) A solution was prepared containing poly--lysine (0.49 g, 2.9 mmol amine content), sebacic acid (0.15 g, 1.45 mmol carboxyl content) and sodium hydroxide (0.06 g, 1.5 mmol) in water (1.5 cm.sup.3).

(53) A solution of EDCI (0.83 g, 4.35 mmol) in water (1 cm.sup.3) was added to the above solution and the mixture immediately used to fill the base of polypropylene test tubes to demonstrate the ability to cast a lens.

(54) After 20-30 minutes the mixture had solidified to form a clear polymer resembling a contact lens. The lens was washed thoroughly with water then left to dry in air.

(55) A photograph of the lens is shown in FIG. 12.