MAY, Jessica Rose (Imperial Innovations Limited, Level 12 Electronic & Electrical Engineering BuildingImperial College,Exhibition Road, London SW7 2AZ, GB)
GENTILINI, Cristina (Imperial Innovations Limited, Level 12 Electronic & Electrical Engineering BuildingImperial College,Exhibition Road, London SW7 2AZ, GB)
STEVENS, Molly Morag (Imperial Innovations Limited, Level 12 Electronic & Electrical Engineering BuildingImperial College,Exhibition Road, London SW7 2AZ, GB)
MAY, Jessica Rose (Imperial Innovations Limited, Level 12 Electronic & Electrical Engineering BuildingImperial College,Exhibition Road, London SW7 2AZ, GB)
GENTILINI, Cristina (Imperial Innovations Limited, Level 12 Electronic & Electrical Engineering BuildingImperial College,Exhibition Road, London SW7 2AZ, GB)
| CLAIMS 1. A process for producing a drawn esterified γ-PGA body comprising: preparing a solution-cast film of esterified γ-PGA, wherein the esterified γ- PGA has a degree of esterification sufficient to allow cold drawing of the solution- cast film; drying the solution-cast film; and cold drawing the solution-cast film to form the drawn esterified γ-PGA body. 2. The process of claim 1, wherein the degree of esterification sufficient to allow cold drawing of the solution-cast film is at least about 50%. 3. The process of claim 1 or 2, wherein the esterified γ-PGA comprises an aliphatic, alicyclic, aromatic, aliphatic-aromatic or aromatic-aliphatic ester of γ-PGA. 4. The process of claim 3, wherein the ester is a C1-4 aliphatic, aromatic or C1-4 aliphatic- aromatic ester (for example a C1-4 aliphatic or benzyl ester). 5. The process of claim 3 or 4, wherein the ester is an ethyl ester, propyl ester or benzyl ester. 6. The process of any preceding claim, wherein the esterified γ-PGA has a degree of esterification of 70- 100%. 7. The process of claim 6, wherein the degree of esterification is 80-100%. 8. The process of claim 7, wherein the degree of esterification is 90-100%. 9. The process of any preceding claim, wherein the solution-cast film is prepared by casting a solution of esterified γ-PGA onto a substrate to form a γ-PGA film. 10. The process of claim 9, wherein the γ-PGA film is dried prior to cold drawing. 11. The process of claim 9 or 10, wherein the solution of esterified γ-PGA comprises 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) as a solvent. 12. The process of any preceding claim, wherein the cold-drawing step is carried out at a temperature of 10-40°C below the crystallisation temperature of the solution- cast esterified γ-PGA film. 13. The process of claim 12, comprising the steps of measuring the crystallisation temperature of the solution-cast esterified γ-PGA film prior to cold-drawing, selecting a temperature of 10-40°C below the crystallisation temperature and carrying out cold drawing at the selected temperature. 14. The process of claim 12 or 13, wherein cold drawing is carried out at a temperature 10-35°C or 15-30°C below the crystallisation temperature. 15. The process of any preceding claim, wherein the process involves the step of esterifying γ-PGA prior to the preparation of a solution-cast film of esterified γ-PGA. 16. The process of any preceding claim, wherein the esterified γ-PGA has a molecular weight of 70-400 kDa. 17. The process of any preceding claim wherein the esterified γ-PGA is drawn to a draw ratio from 2 to 20. 18. An esterified γ-PGA body produced by a process according to any preceding claim. 19. Cold-drawn esterified γ-PGA having a Young's Modulus, E, in the range of 0.2-8 GPa and a tensile strength, σ, in the range of 10-350 MPa. 20. Cold-drawn esterified γ-PGA according to claim 19, having a degree of esterification of 70- 100%. 21. Cold-drawn esterified γ-PGA according to claim 19 or 20, wherein the cold- drawn esterified γ-PGA has been drawn to a draw ratio of 2-20. 22. Cold-drawn esterified γ-PGA according to any one of claims 19 to 21 , having a strain at break, ε, of 4-150%. 23. Cold-drawn esterified γ-PGA according to any one of claims 19 to 22, having one or more of the following features: (i) a Young's Modulus, E, in the range of 0.5-6GPa; (ii) a tensile strength, σ, in the range of 20-250 MPa; and (iii) a degree of esterification of 80-100%. 24. Cold-drawn esterified γ-PGA according to any one of claims 19 to 23, wherein the esterified γ-PGA comprises an aliphatic, alicyclic, aromatic, aliphatic -aromatic or aromatic-aliphatic ester of γ-PGA. 25. Cold-drawn esterified γ-PGA according to claim 24, wherein the ester is a CM aliphatic, aromatic or C1-4 aliphatic-aromatic ester (for example a CM aliphatic or benzyl ester). 26. Cold-drawn esterified γ-PGA according to claim 25, wherein the ester is an ethyl ester, propyl ester or benzyl ester. 27. A device for use in aiding tissue repair, replacement and/or regeneration comprising a drawn esterified γ-PGA body as produced by a process according to any one of claims 1 to 17 or cold-drawn esterified γ-PGA according to any one of claims 19 to 25. 28. The device of claim 19, wherein the device is for use in aiding soft tissue, ligament or tendon repair, replacement and/or regeneration. 29. A tissue scaffold comprising a drawn esterified γ-PGA body as produced by a process according to any one of claims 1 to 17 or cold-drawn esterified γ-PGA according to any one of claims 19 to 25. 30. The tissue scaffold of claim 29, wherein the scaffold is a soft tissue regeneration scaffold, a ligament regeneration scaffold or a tendon regeneration scaffold. 31. A method of repairing, replacing and/or regenerating tissue in a subject in need thereof, comprising implanting or administering a scaffold comprising a drawn esterified γ-PGA body as produced by a process according to any one of claims 1 to 17, cold-drawn esterified γ-PGA according to any one of claims 19 to 25 or the scaffold of claim 30 to an implantation or administration site in the subject in need thereof. 32. A process, esterified γ-PGA body, cold-drawn esterified γ-PGA device, scaffold or method substantially as described herein, with reference to one or more of the examples and/or figures. |
The present invention relates to a process for the preparation of a high tensile strength poly gamma glutamic acid (γ-PGA) ester body by cold drawing of a solution-cast γ- PGA ester film, a body produced by this process and applications of such a body in the production of materials for use in tissue engineering.
Background
One goal in tissue engineering is to repair, renew and restore diseased tissues and organs by means of biomimetic scaffolds. Biomimetic scaffolds play a crucial role in tissue engineering. They aim to provide for the mechanical and structural requirements of the target tissue, while promoting cellular attachment, proliferation, and in-growth, ultimately leading to new tissue formation. Biodegradable polymers of both natural (e.g. collagen) and synthetic (e.g. poly (L-Iactic acid)) origin are widely used in tissue engineering because they are biocompatible and bioresorbable. Materials composed of synthetic polyesters are attractive as they are less immunogenic and structurally more flexible than natural polymers. However, they suffer from poor hydrophilicity, a lack of natural recognition sites and rapid auto catalytic degradation by random hydrolysis to release acidic products that can cause an inflammatory response.
Poly (γ-glutamic acid) (γ-PGA) is a naturally occurring poly (γ-amino acid) of microbial origin produced by several Bacillus species. It is made from D- and L- glutamic acid units and may reach very high molecular weights (up to 1 million), γ- PGA is water-soluble, biodegradable and highly biocompatible. Potential uses of γ- PGA and its derivatives have been explored in recent years for a broad range of industrial fields including food, cosmetics, and water treatment, as well as in biomedical applications. γ-PGA is degraded by a class of extracellular enzymes called γ-glutamyl hydrolases and, as a polyamide, is more resistant than synthetic polyesters to random chain hydrolysis. In biological systems γ-PGA undergoes enzymatic degradation from the surface, rather than bulk hydrolysis. Thus, γ-PGA provides benefits for use as a scaffold material, preventing rapid deterioration in scaffold strength. In addition, due to the presence of the carboxyl group (-COOH) on the side chain, γ-PGA exhibits unique advantages over other materials in terms of scaffold applications. The presence of side chain carboxyl groups offers the possibility of tailoring the properties of the material by chemical modification of the side chains.
Mechanical properties of materials are vital for applications in tissue engineering. For example, tensile strength and Young's modulus, which relates to elasticity of a material (without peraianent deformation) are two crucial features for a material that aims to mimic mechanical properties of tissues, such as ligaments and tendons. Mechanical performance of polymer fibres, tapes and films can be enhanced by tensile drawing techniques aiming to induce high orientation and extension of the constituent polymer chains. This process, also known as 'tensile deformation', has been used in materials science to fabricate ultra-high strength polymeric fibres. For example, high molecular weight polyethylene can be "stretched" to produce ultra- high-strength filaments with a Young's modulus exceeding 150 GPa and a tensile strength of 4 GPa or more (Smith, P. et al, J. Polymer Set Polymer Phys. Edn., 1981(19), 877-888).
Application of a tensile deformation processing method to fibres made of biodegradable and biocompatible polymeric materials opens the way to the fabrication of resorbable scaffolds with an unprecedented range of mechanical properties, for example tailored for the regeneration of injured ligament and tendon tissue.
Summary
The inventors have determined that a cold drawing process can successfully be utilised to produce γ-PGA with high tensile strength if the form of γ-PGA subjected to cold drawing is a solution-cast esterified γ-PGA film.
Accordingly, in a first aspect the present invention provides a process for producing a drawn esterified γ-PGA body, the process comprising:
preparing a solution-cast film of esterified γ-PGA, wherein the esterified γ- PGA has a degree of esterification sufficient to allow cold drawing of the solution- cast film;
drying the solution-cast film; and
cold drawing the solution-cast film to form an esterified γ-PGA body. The degree of esterification sufficient to allow cold drawing of the solution- cast film is preferably at least 50%.
The degree of esterification is preferably at least 70%, more preferably 80-100%, even more preferably 90-100%.
Cold drawing results in extension and alignment of the polymer chains, resulting in highly oriented filaments that exhibit excellent tensile strength and modulus. A schematic diagram of the process at a molecular level is shown in Figure 1. In some embodiments, the esterified γ-PGA comprises an aliphatic, alicyclic, aromatic, aliphatic-aromatic, aromatic-aliphatic or aromatic-alicyclic ester of γ-PGA. The ester may be an aliphatic, alicyclic, aromatic, aliphatic-aromatic or aromatic- aliphatic. Preferably, the ester is an ethyl ester, propyl ester or benzyl ester. More preferably, the ester is a benzyl ester.
The solution cast film is prepared from a solution of esterified γ-PGA in a solvent. In the process of the invention, the solution-cast film is prepared by casting the solution of esterified γ-PGA onto a substrate to form a γ-PGA film. The γ-PGA film is dried to remove solvent prior to cold drawing. Drying may be carried out by allowing evaporation of solvent at room temperature or by heating (for example to a temperature up to about 60°C), optionally under vacuum. The solvent used in the solution casting of esterified γ-PGA is preferably a solvent in which esterified γ-PGA is soluble and which has a low boiling point and high volatility. Preferably, the solvent is an organic solvent, more preferably the solvent is 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). It has been determined that 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) is a particularly useful solvent to allow production of a continuous film because it solubilises esterified γ-PGA and will give complete solvent evaporation at a slow rate, typically over the course of 2-3 days, to give an even film without trapped solvent. An even film is optimal to allow enhanced chain alignment during the cold drawing process. In some embodiments, the cold-drawing step is carried out at a temperature of about 10-40°C below the crystallisation temperature (Tc) of the solution-cast esterified γ- PGA film. Accordingly, the invention encompasses a process comprising the steps of measuring the crystallisation temperature of the solution-cast esterified γ-PGA film prior to cold-drawing, selecting a temperature to carry out cold drawing of 10-40°C below the crystallisation temperature and carrying out cold drawing at the selected temperature. Preferably, cold drawing is carried out at a temperature of 10-35°C below Tc, more preferably 15-30°C below Tc.
The esterified γ-PGA used in the process of the invention preferably has an average molecular weight (Mw) of about 50-500 kDa, more preferably 70-400 kDa.
In some embodiments, the process of the invention involves the step of esterifying γ- PGA prior to the preparation of a solution-cast film of esterified γ-PGA. The esterified γ-PGA body may be an esterified γ-PGA film or fibre. A film is produced by cold drawing of the solution-cast esterified γ-PGA film as-cast. A fibre may be produced by cutting the solution-cast esterified γ-PGA film into strips (for example of width l-2mm) prior to drawing, giving a drawn esterified γ-PGA body in the form of a fibre.
It will be appreciated that the features of the process of the invention as described above can be present in any combination thereof.
In a second aspect, the invention provides an esterified γ-PGA body produced by a process of the first aspect of the invention.
Due to its good mechanical and biological/biocompatible properties an esterified γ- PGA body produced by a process of the first aspect of the invention can be utilised in tissue engineering applications. It has been demonstrated in cell culture that cells will grow on an esterified γ-PGA body produced by a process of the invention. For example, a body as produced by the process of the invention can be used to form tissue repair or suture materials. The materials are of particular use for tissue repair applications where high tensile strength is crucial, for example for tendon and ligament repair. The materials are also of use in soft tissue applications such as wound repair. In a third aspect, the invention provides esterified γ-PGA having a Young's Modulus, E, in the range of 0.2-8 GPa and a tensile strength, σ, in the range of 10-350 MPa. The esterified γ-PGA is esterified γ-PGA that has been drawn by a cold-drawing process. In an embodiment of this aspect of the invention, the cold-drawn esterified γ-PGA has been drawn to a draw ratio of 2-20. In certain embodiments, the draw ratio is 2-18, preferably 8-12.
In some embodiments the degree of esterification in the cold-drawn esterified γ-PGA is at least about 50%, preferably 70-100%, more preferably 80-100%, even more preferably 90-100%. In some embodiments, the degree of esterification is 100%.
In some embodiments of the cold-drawn esterified γ-PGA as described above, one or more of the following features are present:
(i) a strain at break, ε, of 4-150%, preferably 7-40%;
(ii) a Young's Modulus, E, in the range of 0.5-6 GPa, preferably 2-6 GPa;
(iii) a tensile strength, σ, in the range of 20-250 MPa, preferably 50-200 MPa, more preferably 100-200 MPa.
In certain embodiments the esterified γ-PGA of the third aspect of the invention may comprise an aliphatic, alicyclic, aromatic, aliphatic-aromatic or aromatic-aliphatic ester of γ-PGA. Preferably, the ester is a C 1- 4 aliphatic, aromatic or CM aliphatic- aromatic ester (for example a C 1-4 aliphatic or benzyl ester). In certain embodiments, the ester is an ethyl ester, propyl ester or benzyl ester.
It will be appreciated that the features of the esterified γ-PGA of the embodiments of the third aspect of the invention as described above can be present in any combination thereof.
In a fourth aspect, the invention provides a device for use in repairing, replacing and/or promoting regeneration of tissue comprising a drawn esterified γ-PGAbody as produced by process according to the first aspect of the invention. In an embodiment of this aspect of the invention, the device is for use in aiding soft tissue repair, replacement and/or regeneration, for example ligament or tendon repair and regeneration.
In a fifth aspect, the invention provides a tissue scaffold comprising a drawn esterified γ-PGA body as produced by a process of the first aspect of the invention. A tissue scaffold can be used to promote tissue repair and regeneration. In an embodiment, the scaffold is a soft tissue scaffold, for example, a ligament scaffold or a tendon scaffold.
In a sixth aspect, the invention provides a tissue scaffold comprising a drawn esterified γ-PGA body as produced by a process of the first aspect of the invention or esterified γ-PGA of the second aspect of the invention.
In a seventh aspect, the invention provides a method of repairing, replacing and/or promoting regeneration of tissue in a subject in need thereof, comprising implanting or administering to the subject a scaffold comprising a drawn esterified γ-PGA body as produced by a process of the first aspect of the invention or esterified γ-PGA of the second aspect of the invention or a scaffold of the fifth aspect of the invention to an implantation or administration site in need of repair, replacement and/or regeneration of tissue. The implantation or administration site may be a site where there is damage or degeneration of tissue. The tissue may be soft tissue. Exemplary tissues include tendon and ligament tissue. It will be appreciated that the features and embodiments described for the first aspect of the invention apply equally to the second to seventh aspects of the invention mutatis mutandis. Brief Description of the Drawings
The invention may be put into practice in various ways and a number of specific embodiments will be described by way of example to illustrate the invention with reference to the accompanying figures, in which: Figure 1 shows a schematic diagram of the tensile deformation process at a molecular level. The polymer chains are aligned during this process and the overlap length between them determines the strength of the material.
Figure 2 shows a) 1H- and b) 13 C-NMR of γ-PGA-H (in D 2 O) and of γ-PGA esters (in DMSO-d6). y-PGA-Et (A), y-PGA-Pr (B), y-PGA-Bn (C), and γ-PGA-H (D).
Figure 3 shows XRD patterns of Et, Pr and Bn γ-PGA esters.
Figure 4 shows a typical nominal stress/strain curves recorded at room temperature for native y-PGA-H films.
Figure 5 shows nominal stress/strain curves recorded at room temperature for esterified y-PGA films. Figure 6 shows nominal stress/strain curves recorded at room temperature for drawn γ-PGA-Et films drawn at 125°C. Draw ratios are indicated in the figure.
Figure 7 shows nominal stress/strain curves recorded at room temperature for drawn γ-PGA-Pr films draw at 125°C. Draw ratios are indicated in the figure.
Figure 8 shows nominal stress/strain curves recorded at room temperature for drawn y-PGA-Bn films drawn at 115°C. Draw ratios are indicated in the figure. Figure 9 sets out plots showing the implements of tensile deformation on Young's Modulus, E, tensile strength, σ, and strain at break, ε, for ethyl, propyl and benzyl esterified γ-PGA.
Figure 10 shows cell metabolic activity normalized to negative control in the ISO 10993:5 pre-clinical test of esterified γ-PGA films.
Detailed Description
The meanings of terms used in the specification of the present application will be explained below, and the present invention will be described in detail.
The term 'about' is taken to allow a variation of ± 5%, preferably ± 1%, of a defined value.
Native γ-PGA is a polymer comprising recurring units of the formula:
Native γ-PGA is brittle, water soluble and cannot be successfully drawn due to intra/intermolecular hydrogen bonds. Native γ-PGA can be referred to as γ-PGA acid or γ-PGA-H.
Esterified γ-PGA is a polymer as defined above wherein a proportion of the recurring units have been esterified to have the formula:
wherein R is an aliphatic, alicyclic, aromatic, aliphatic-aromatic or aromatic-aliphatic group.
The term "aliphatic" as used herein refers to a straight or branched chain hydrocarbon which is completely saturated or contains one or more units of unsaturation. Thus, aliphatic may be alkyl, alkenyl or alkynyl, preferably having 1 to 30 carbon atoms, 1 to 12 carbon atoms, 1 to 6 carbon atoms or 1 to 4 carbon atoms. It will be appreciated that for alkenyl and alkynyl these ranges are 2 to 30, 2 to 12, 2 to 6 or 2 to 4 carbon atoms. The term "alicyclic" as used herein refers to a saturated or partially unsaturated carbocyclic group having 3 to 8 ring carbon atoms. An alicyclic may be a "cycloalkyl", which as used herein refers to a fully saturated hydrocarbon cyclic group. Preferably, a cycloalkyl group is a C 3 -C 6 cycloalkyl group. An "aromatic group" as referred to herein is a monocyclic or bicyclic aromatic ring having 6 to 10 carbon atoms. Preferably, an aromatic group is phenyl.
The terms "aliphatic-aromatic" and "aromatic-aliphatic" as used herein refers to an aliphatic group as defined above substituted with an aromatic group as defined above and an aromatic group substituted with an aliphatic group, respectively. Preferably, aliphatic- aromatic is benzyl.
An aliphatic, alicyclic, aromatic, aliphatic-aromatic or aromatic-alicyclic group as referred to herein may be unsubstituted or may be substituted by one or more substituents independently selected from the group consisting of halo, aliphatic, alicyclic, alkoxy, alkylamino (monoalkylamino or dialkylamino), acylamino, -N0 2 , - CN, alkylcarbonyloxy, alkoxycarbonyl or alkylcarbonyl.
R is preferably, a C 1-4 aliphatic, aromatic or C 1-4 aliphatic-aromatic group, more preferably C 1 -4 aliphatic or benzyl, even more preferably ethyl, propyl or benzyl.
In some embodiments, the process of the invention involves the step of esterifying γ- PGA prior to the preparation of a solution-cast film of esterified γ-PGA. Esterification can be earned out by mixing an aliphatic, alicyclic, aromatic, aliphatic- aromatic or aromatic-aliphatic halide (of formula R-halide) with γ-PGA in an alkaline solution. Typically, reaction takes place at 60°C for up to 96 hours. Alternatively, esterification can be achieved by activation of the γ-PGA side chain carboxylic group (for example with thionyl chloride) and condensation with an alcohol R-OH, where R can be an aliphatic, alicyclic, aromatic, aliphatic-aromatic or aromatic- aliphatic group. In a further alternative, γ-PGA can be esterified with a diazo compound (for example, diazomethane). Partially esterified γ-PGA can be achieved by reducing reaction time, reaction temperature, halide loading or any combination thereof. Preferred values for R are as described above.
The degree of esterification of the esterified γ-PGA is a measure of the proportion of recurring monomer units within the γ-PGA polymer in which the side chain carboxylic acid group is esterified. The degree of esterification is the number of recurring monomer units in which the side chain carboxylic acid group is esterified /total number of recurring monomer units χ 100. Esterified γ-PGA for use in the process of the invention preferably has at least a 50% degree of esterification, i.e. preferably at least 50% of the recurring units of the polymer are esterified. The esterified γ-PGA having at least a 50% degree of esterification can accordingly be represented as a polymer comprising recurring units (A) and (B):
wherein R is an aliphatic, alicyclic, aromatic, aliphatic-aromatic or aromatic-aliphatic group as defined above and wherein at least 50% of the total recurring units (total number of (A) + (B)) are (B). The degree of esterification can be assessed using Ή NMR. Preferably, the degree of esterification is at least 70%, preferably 80-100%, more preferably 85-100%, even more preferably 90-100%. The degree of esterification in some embodiments is 100%. For esterified γ-PGA with a degree of estenfication of 100%, 100% of the recurring units are (B).
Esterification of γ-PGA allows cold drawing to be successfully performed by reducing intra/inter-molecular hydrogen bonding. In addition, esterification of γ-PGA to a degree of at least 50% beneficially alters solubility characteristics of γ-PGA, reducing its water solubility and instead rendering it soluble in organic solvents. For example, esterification of γ-PGA side groups can reduce the solubility in aqueous solutions while enhancing both the solubility of the polymer in organic solvents and its resistance to hydrolysis. γ-PGA can be produced by Bacillus licheniformis and Bacillus subtilis. The former bacterium was shown to produce amorphous γ-PGA polymer while the latter one produces semi-crystalline or crystalline γ-PGA polymer. A semi-crystalline polymer is a polymer which is partly amorphous and partly crystalline. It is believed that the regular distribution of D and L units in γ-PGA renders the semi-crystalline property. Esterified γ-PGA as used in the process of the present invention is preferably semi- crystalline. γ-PGA starting material used to produce γ-PGA esters may be from any suitable source, including Bacillus licheniformis and Bacillus subtilis. Esterification of γ-PGA introduces a degree of crystallinity and, therefore, the γ-PGA starting material may be amorphous or semi-crystalline. Preferably, the source of γ-PGA is Bacillus subtilis.
In the context of the invention, cold drawing is a process of subjecting a material to tensile deformation by stretching. Cold drawing is carried out on a material in the solid state, i.e. at a temperature below the melting temperature (Tm) of the material. It can be distinguished from hot drawing, which takes place in the melt. In the present invention, cold drawing is preferably carried out at a temperature above the glass transition temperature (Tg) of the esterified γ-PGA polymer and below its crystallisation temperature (Tc) and melting temperature (Tm). Drawing above Tg is beneficial because the amorphous regions of polymer chains are mobile and easy to align in semi-crystalline polymers above Tg. γ-PGA esters have lower Tg than native γ-PGA (as a result of the reduction in H-bonds via esterification), which enables cold drawing to be performed above Tg, but well below the Tm and decomposition temperature. As discussed above, the esterified γ-PGA used in the process of the invention may be semi-crystalline. Cold drawing should preferably be carried out in a single direction, i.e. uniaxially. Preferably, drawing should be carried out to a draw ratio of at least 1.5, preferably at least 2. Drawing can be achieved up to a draw ratio of 16 or more, for example up to 20. Thus, cold drawing is preferably carried out to a draw ratio up to 16. The draw ratio may therefore be 2-20. In certain embodiments, the draw ratio is 2-18, preferably 8-12. Draw ratio can be calculated by marking the esterified γ-PGA at intervals (e.g. 1mm) prior to drawing to determine draw ratio (λ = final/initial length) from displacement of marks.
The cold-drawing step may be carried out at a temperature of 10-40°C preferably 10- 35°C, more preferably 15-30°C, below the crystallisation temperature (Tc) of the solution-cast esterified γ-PGA film. Even more preferably the cold drawing step is carried out 10-20°C below Tc where the esterified γ-PGA is a C 1-4 aliphatic ester or 25-35°C below Tc where the esterified γ-PGA is a benzyl ester. This temperature range has been determined to be optimal for drawing, providing a balance between the increase in crystallinity seen at higher temperatures and the reduction in chain mobility seen at lower temperatures. The crystallisation temperature (Tc) of the solution-cast esterified γ-PGA film can be measured by differential scanning calorimetry (DSC) thermal characterisation and measurement should be carried out after drying of the solution-cast esterified γ-PGA film. A protocol for DSC measurement is described in the following examples. Tc is typically measured as the peak of the crystallisation curve, determined by peak integration.
The esterified γ-PGA on which cold drawing has been performed has improved mechanical properties (e.g. higher tensile strength) than solution-cast esterified γ-PGA film. Moreover, esterified γ-PGA on which cold drawing has been performed has higher tensile strength than γ-PGA fibres produced by alternative processes, such as electrospinning. In the context of this disclosure mechanical properties (i.e. Young's Modulus, tensile strength and strain at break) are measured at room temperature and at a strain rate of 5 mm/min. Mechanical measurements are taken in the direction of drawing. Room temperature can be taken to be about 20-25°C, with an average of 23°C.
In the context of this disclosure Mw is weight-average molecular weight. Mw can be measured, for example, by gel permeation chromatography (GPC)/size exclusion chromatography (SEC) an exemplary protocol of which is described in the following examples. It will be appreciated M w of the esterified γ-PGA has an influence on mechanical properties, with increasing M w tending to an increase in Young's Modulus and tensile strength.
In the process of the invention, cold drawing is carried out on a solution-cast esterified γ-PGA film. The inventors have determined that solution-casting is favourable to other methods of film production because it avoids problems such as degradation which is observed if a melt-casting process is attempted.
It will be appreciated that the various features of the process of the invention as described above can be present in any combination thereof.
Examples
1) Esterification of γ-PGA
6 g of poly (γ-glutamic acid) Na + salt (γ-PGA-Na + produced by Bacillus Subtilis; Natto Science Ltd, Japan) was dissolved in 200 mL of distilled water. The solution was acidified with 6M HCl to a pH of 1.5 and subsequently lyophilized for 48 hours. Next, 0.01 mol of the lyophilized γ-PGA-H was suspended in 150 mL of N-Methyl-2- pyrrolidone (NMP) while stirring for 24 h at 80°C. The mixture was then cooled down to 60°C before adding 0.04 mol of NaHCOs. After 2 hours of stirring, a bromide (EtBr, PrBr or BnBr) was added dropwise to the mixture in a 5 to 9 fold molar excess, after which the mixture was stirred at 60°C for another 36 to 60 hours to produce esterified γ-PGA. Partially esterified γ-PGA can be achieved by reducing the reaction time. For example, 50% esterified γ-PGA was obtained by reducing the reaction time to 24 hours.
The structure and purity of the esterified compounds were confirmed by ! Η- and I3 C- NMR spectroscopy (Figure 2). The XRD pattern indicates that the esterified γ-PGA is semi-crystalline (Figure 3). The glass transition temperatures of ethyl/propyl/benzyl γ- PGA are 66 °C, 40 °C and 40 °C respectively, as determined by DSC. The structure and properties of the produced polymers are shown in Table 1 below.
Table 1 : Structure and properties of polymers. Glass transition temperature, T g; crystallization temperature, T C, and melting temperature, T m , of native powder were determined by differential scanning calorirnetry; average molecular weight, M w , calculated from gel permeation chromatography.
Degradation detected in TGA prior to melting
Double endotherm, second peak at 240 °C DSC measurement protocol - Glass transition temperatures, T g , crystallization temperatures, T c , and melting temperatures, T m , were determined with a Mettler- Toledo DSC822e differential scanning calorimeter (DSC), calibrated with indium and zinc. Samples of 5-7 mg sealed in aluminum crucibles were heated under nitrogen at a rate of 10°C min -1 from 25°C until 20°C above T m .
Gel Permeation Chromatography (GPC) Protocol - GPC analyses of native γ-PGA were carried out in aqueous buffers (0.20 M NaNO 3 and 0.01 M NaH 2 PO 4 at pH 7) using a PL-GPC 120 (Fikrah Technology, Maylasia) with a PL-AS-MT autosampler. Sodium polyacrylate was used as a standard reference. GPC analyses of esterified γ- PGA in organic solvents were performed using the PL-GPC 220 autosampler with NN'-dimethylfoimamide 1 g L -1 LiBr as mobile phase and monodisperse polymethylmethacrylate solutions as a standard reference. GPC can also be referred to as size- exclusion chromatography (SEC).
The protocal used for degree of esterification calculations was: 3 mL of esterified γ- PGA/NMP solution was precipitated in 30 mL cold HCl (pH 1.5), centrifuged at 4000 R.P.M. for 5 min, supernatant removed, and 30 mL HCl added. This was
subsequently repeated twice using diH20, and twice with diethyl ether (Et 2 0). The resulting polymer was dissolved in 600mL DMSO-d6 for 1H-NMR analysis. The degree of esterification was calculated from integration of the NH peak of the esterified γ-PGA against that of -PGA-H (if present) from the 1 H-NMR spectra at 25°C. The resonance frequency was 400 MHz.
2) Production of solution-cast films of esterified γ-PGA
For convenient removal of solution cast films, glass petri dishes were treated with a fresh Piranha solution of 70/30 sulphuric acid/hydrogen peroxide for 1 hour and subsequently washed with excess water. The petri dishes were then placed in a dessicator with a few drops of perfluorotrichlorosilane, evacuated to 20 mbar, left under vacuum to fluorinate overnight, and annealed at 100 °C for one hour in vacuum.
Esterified γ-PGA samples in HFIP (5% wt/wt) were poured into the fluorinated glass petri dishes, covered and the solvent was allowed to evaporate at room temperature (RT) for 48 hrs, followed by 24 hrs under vacuum at 60 °C to remove any remaining solvent. γ-PGA-acid was dissolved in diH 2 0 (5% wt/wt), allowed to evaporate under mild heating (circa 50°C) for 96 hrs, and then placed at 60 °C under vacuum to prevent rehydration.
Sample concentrations were calculated to produce films approximately 100 μm thick. 3) Cytotoxicity and culture stability of esterified γ-PGA
Scaffolds produced for tissue engineering should show biocompatibility and biodegradability. The native γ-PGA polymer backbone is enzymatically degraded. In esterified γ-PGA side groups are coupled through an ester bond. An assessment was carried out to determine whether any side chain hydrolysis or overall dissolution of the modified polymer takes place and whether there are released side groups (alkyl or aryl alcohols) which have an adverse effect on biocompatibility.
Experimental
All reagents for cell culture were used as purchased from Invitrogen (Paisley, UK) unless stated otherwise. Salts were purchased from Sigma-Aldrich (Gillingham, UK), the Triton X-100 from BDH laboratories (Poole, UK). Aluminum Scanning Electron Microscopy (SEM) pin stubs and adhesive carbon discs were purchased from Agar Scientific (Standsted, UK). ISO 10993:5 pre-clinical cytotoxicity test
Esterified γ-PGA films were sterilised for 2 h on each side under UV light and prepared with a material surface area to culture medium ratio of 6 cm^ niL-l according to thin-film requirements outlined in ISO10993:12. Similarly, controls were calculated with an extraction ratio of 3 cm 2 mL -1 [negative, non-toxic PVC
(Med7539 noDop) tubing; positive, organo-tin stabilised PVC sheet, t > 0.5 mm; supplied by Raumedic (Munchberg, Germany)] and sterilised using 70% ethanol for 40 min and allowed to air dry.
The mouse osteoblast cell line MC3T3-E1 (European Collection of Cell Cultures; Salisbury, U.K.) was routinely cultured under standard conditions (37°C, 5% CO2,
100% humidity) in Alpha Minimum Essential Medium (a-MEM) supplemented with 10% (v/v) foetal bovine serum (FBS) and 2 mM L-glutamine (all Invitrogen; Paisley, U.K.).
Sterile samples were soaked in a-MEM for 7 days at 37°C, 5% C0 2 to make the conditioned media. MC3T3-E1 cells were seeded into 96 well plates at a density of 20,000 cells cm-^ in standard culture medium (as above). Cells were imaged on an Olympus 1X51 epifluorescence microscope fitted with a DP-70 camera (London, U.K.). After 24 h, the medium was exchanged with conditioned culture medium (supplemented with 10% v/v FBS and 1% v/v L-glutamine) and diluted by factors of 2, 4, 8, or 16 with standard culture medium for 24 h, upon which the cellular metabolic activity was assessed using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide). For the assay, 20 μL, of a sterile-filtered 5 mg mL -1 solution of MTT in Dulbecco's phosphate buffered saline (DPBS, pH 7.4) was added to the culture media and incubated for 2 h. Upon completion of incubation, media was removed, cells washed with PBS, and 200 DMSO was pipetted in wells to dissolve the formazan product. Finally, 150 μL, of formazan/DMSO was transferred to a fresh 96 well plate and read on a colorimetric plate reader at 592 nm (background subtraction at 620 nm). All results are normalised to negative control conditioned, media-treated MC3T3 -El cell metabolic activity. This test assesses if any toxic dissolution products are released, as they will cause a detectable reduction in cellular metabolic activity level. Polymer films were soaked in cell culture medium at 37°C for 7 days. Murine pre-osteoblastic cells (MC3T3-E1) were then cultured in the conditioned medium overnight and their metabolic activity assessed. MC3T3-E1 cells cultured in conditioned medium soaked with either the negative control or esterified γ-PGA films appeared morphologically normal, while those exposed to positive control conditioned medium displayed a rounded, abnormal morphology. Activity of cells exposed to all experimental film soaked media were similar to that of the negative control as shown in Figure 10. The MTT activity of positive control treated cells was significantly lower than all other tested groups (p < 0.001).
These results indicate that dissolution products released from soaked films did not have a negative effect upon cell metabolic activity. Thus all esterified γ-PGA polymers pass the international standard test for assessing in vilro mammalian cell cytotoxicity and have good potential for use in tissue engineering applications.
LIVE/DEAD® assay
Cell viability on esterified γ-PGA samples was evaluated using a LIVE/DEAD® stain assay (Invitrogen). Primary human foreskin fibroblast cells- 1 (HFF-1; ATCC,
Virginia, USA) were routinely cultured under standard conditions (37°C, 5% C0 2 , 100% humidity) in grown in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 15% v/v FBS and 1% v/v antibiotic-antimycotic (all Invitrogen). Round samples 8 mm in diameter were punched and sterilised by UV light (2 h per side) in polystyrene petri dishes. Following sterilisation, samples were transferred to 12 well plates containing 1 mL DPBS and soaked for 1 h at RT. After pre-wetting, HFF-1 cells (passage two, P2), were seeded at a density of 20,000 cells cm- with 50 μΕ cell suspension per film. Cells were allowed to attach for 1 h before 2 mL fresh media was added. Samples were cultured, changing media every 2 days, for 1 and 7 days. Upon completion of time points, cells were washed with DPBS and 1 mL sterile LIVE/DEAD® solution (0.5 μΜ calcein AM and 0.5 μΜ ethidium homodimer-1 (EthD-1) in DPBS) was added to each well. Plates were incubated at 37 °C for 30 min, after which fluorescence was imaged on an Olympus 1X51 epifluorescence microscope fitted with a DP-70 camera (calcein peak excitation 494 nm, peak emission: 517 nm; EthD-1 peak excitation: 528 nm, peak emission: 617 nm).
Viability of human foreskin fibroblast- 1 (HFF-1) cells (P2) cultured on esterified γ- PGA films was assessed at days 1 and 7. The assay shows live cells with intact cell membranes, which only fluoresce green from esterase cleaved calcein AM in the cytoplasm. Meanwhile, "dead" cells, which have compromised cell membranes, allow EthD-1 to label nucleic acids with red fluorescence.
Fibroblasts on all polymer types remained viable at both time points assessed in this study, as confirmed by the LIVE/DEAD® stain. Visual examination on day 7 shows that cells spread and proliferated over time on all three polymer types. These results, taken together with those from the ISO test indicate that all three γ-PGA
modifications can support human cell attachment and grown and have potential to be candidates for tissue engineering. 4) Tensile deformation of esterified γ-PGA films
The solution cast esterified γ-PGA films were subjected to mechanical tests and tensile deformation to demonstrate improvement of mechanical properties of the polymers. Initial tensile tests at room temperature (RT) were performed to determine the initial mechanical properties of the isotropic, as-cast films.
Initial mechanical testing of as-cast, dried films was performed at room temperature with an Instron Tensile Tester Model 5864 (Norwood, USA) with hydraulic clamps (5 bar) at a cross-head speed of 5 mm min -1 . Dumbbell shaped strips of 1.2 mm width and 4.5 mm gauge length were cut from films. Typical stress-strain curves recorded for as-cast native γ-PGA-H and its esterified forms are shown in Figures 4 and 5, while their corresponding mechanical properties are presented below in Table 2. As shown in the stress-strain plots, esterification of the γ-PGA-H resulted in highly enhanced ductility, as seen in a dramatic increase in strain at break of as much as 50, 150, and 250 times for the benzyl, propyl and ethyl esters, respectively. This enhancement was accompanied by a decrease in values of the stiffness E and of the yield and tensile strength.
Table 2: Mechanical characteristics at RT of native γ-PGA (γ-PGA-H) and differently esterified γ-PGA films
In a second series of experiments, tensile cold drawing of the dried esterified γ-PGA films above their respective T g and below their melting temperature was performed using an Instron Tensile Tester (Model 5864) fitted with an environmental chamber (Model EC43) at a cross-head speed of 2 mm min -1 . Dumbbell shaped strips were cut as with the room temperature (RT) testing and ink marks were printed at 1 mm intervals prior to drawing to determine draw ratios (λ = final/initial length) from their displacement.
As a first step, stress- strain curves were recorded in the temperature range from RT- T m in order to establish the maximum draw ratio (λ max = ε/100 + 1) for subsequent tensile deformation experiments. It was found that all modified films could be drawn to remarkably high draw ratios, generally above λ > 10 at the optimum temperature (125°C for both γ-PGA-Et and γ-PGA-Pr, and 115°C for γ-PGA-Bn). Above these temperatures, λ max rapidly reduced due to excessive macromolecular mobility. It was found that γ-PGA-H film could not be drawn to appreciable draw ratios; this is likely due to a high T g and the limited temperature window between T g and the onset of degradation, caused by hydrogen bonds between the chains.
The different γ-PGA films were drawn at the optimum temperatures to various draw ratios. Ethyl- and propyl- esterified samples were drawn at 125°C while benzyl- esterified samples were drawn at 115°C. At these respective temperatures, maximum draw ratios ( λ max ) were achieved.
Subsequent to drawing (tensile deformation) and determination of draw ratios from the ink mark displacement, samples were subjected to tests at room temperature to determine their mechanical properties. Samples were cut and glued using Al-Fix glue (Novatio; Olen, Belgium) between card frames and allowed to dry for 2h under 200 g weights to ensure a strong bond. The segment length tested, i.e. the separation distance, was 10 mm. Sample width was measured using an optical microscope, while thickness is determined using a micrometer. Mechanical testing of the samples was performed at room temperature using an Instron tensile tester with hydraulic clamps as described above, with a cross-head speed of 5 mm min -1 .
A comparable set of results were generated for polymers with different side chains for polymer films drawn to λ≤10. Nominal stress-strain curves of these drawn films are presented in Figures 6, 7 and 8 for γ-PGA-Et, γ-PGA-Pr, and γ-PGABn, respectively. In all cases, the draw ratio was measured from initial ink mark displacement as indicated in these graphs. Common to all esterified polymer samples, both Young's modulus and tensile strength rapidly increased with increasing draw ratio, while the strain at break decreased.
These characteristic trends are more quantitatively shown in Figure 9, in which the values of E, σ, and ε are plotted as a function of the draw ratio, λ, for all three γ-PGA films. These plots display the positive relationship between λ and E, as well as the negative development of ε with λ. These consistent correlations are indicative of increased polymer chain orientation in the tensile direction.
The mechanical properties were greatly increased over those seen for the as-cast γ- PGA films, yet the strain at break was maintained to similar values of various human tissues. Values of the mechanical characteristics of the drawn films produced in this study, compared to those of the as-cast polymers and native γ-PGA film as cast are presented in Table 3.
Table 3. Mechanical properties of esterified γ-PGA films as-cast and properties achieved by drawing to λ = 10.
The degree of esterification of the polymers produced in the examples set out above was 100% for the ethyl and benzyl esters and, 90% for the propyl ester.
From the presented data it can be concluded that by drawing to λ = 10, the modulus of γ-PGA-Et increased by a factor of about 4.5 and the tensile strength improved by over a factor of 13. Benzyl esterified γ-PGA offered the highest modulus, with a 4.3 fold increase to 5.36 GPa. The maximum tensile strength achieved with γ-PGA-Bn was found to be 149 MPa, a dramatic increase of more than 5 -fold, but still inferior to that of γ-PGA-Et. Drawing γ-PGA-Pr resulted in a nearly double value of its modulus and tripled the tensile strength, with only a 10-fold decrease in strain at break. The above results clearly indicate that the mechanical properties were greatly enhanced for all γ-PGA films. Most importantly, properties achieved approached similar values to those of soft human tissues (see Figure 9), yet their strain at break remained at or above a physiologically relevant 7%.
Although the data presented above relates to polymers drawn to a draw ratio up to 10, higher draw ratios have been achieved for esterified γ-PGA films. For example, the propyl and benzyl γ-PGA as produced in the preceding examples has been drawn above a 15 times draw ratio, with draw ratios up to 20 achievable.
Esterification of γ-PGA polymers shielded H-bonds present in the unmodified form. This allowed tailoring of their mechanical properties over an exceptional range by tensile deformation. Taken together, these results indicate that esterified γ-PGA bodies formed by cold drawing are suitable for use in tissue engineering applications, where mechanically appropriate, biodegradable scaffolds are required. The esterified γ-PGA bodies can be used to form scaffolds for implantation into or administration onto a site where tissue repair, replacement and/or regeneration is required. These scaffolds could be, for example, seeded with cells prior to implantation. Embodiments of the invention have been described by way of example only, ft will be appreciated that variations of the described embodiments may be made which are still within the scope of the invention.
Next Patent: CONTAINER, THE SPACE OF WHICH IS ADJUSTABLE