PL242810B1 - Method of producing a thermoplastic material - Google Patents

Abstract

The subject of the invention is a method of producing a thermoplastic material as a 3D printing filament containing poly(vinyl alcohol), polycaprolactone and chitosan. The method consists in the solution of chitosan saturated with carbon dioxide being compatibilized for processing with thermoplastic raw materials by mixing it with an aqueous solution of hydrolyzed poly(vinyl alcohol), with a degree of hydrolysis of at least 75%, preferably 80%, where the mass fraction of both polymers in relation to to their anhydrous forms is 1:1. Further, the mixture is frozen at a temperature ranging from -80°C to -18°C and freeze-dried at a pressure in the range of 0.9 - 6.0 mbar, with a temperature difference between the lyophilizer condenser and the sample in the range of 70°C to 140°C. The dried composite of chitosan and poly(vinyl alcohol) after grinding is mixed with polycaprolactone and the obtained filament is subjected to extrusion, preferably in two stages.

Description

The subject of the invention is a method of producing a thermoplastic material containing biodegradable components: polycaprolactone and poly(vinyl alcohol) and a biodegradable component of natural origin, having antimicrobial activity - chitosan, applicable as a material for the production of scaffolds for cell cultures, also as a carrier of medicinal preparations or other ingredients bioactive.

From the patent description PL223280 there is known a method of producing a chitosan-protein biopolymer material, applicable to the treatment of tissue defects, especially hard-to-heal wounds, as well as a carrier for medicinal, cosmetic and food preparations. This method consists in the preparation of chitosan in a mixture of acids, which is then subjected to neutralization, separation, washing and saturation of the chitosan slurry with gaseous carbon dioxide and/or dry ice until it dissolves, and then mixed with a dispersion of collagen proteins and/or gelatin.

From the patent description PL222739, a composition based on an aqueous solution of chitosan is known, e.g. in the form of an aerosol and a hydrogel membrane, used as a matrix for plant protection preparations, medical, cosmetic and food products. In the described method, the aerosol consists of a dispersion phase, which is a chitosan hydrogel in the amount of 90% to 98% by weight, a biologically active compound up to 5% by weight and a propellant in the amount of 2% to 10% by weight. The membrane is prepared by preparing a chitosan solution, applying an addition of a biologically active compound and conditioning it at a temperature not lower than 1°C, preferably for 24 hours.

From M. Matet, M-C. Heuzey, E. Pollet, A. AJi, L. Averons, Carbohydr. Polym. 2013, 95, 241-251, a method of obtaining thermoplastic chitosan is known, which consists in adding a solution of a strong acid to the chitosan powder or polyol and a solution of a strong acid and mechanically kneading at 80°C for 15 min at a constant speed of 100 rpm.

From the publication of A. Golchin and M.R. Nourani, Polym. Adv. Technol. 2020, 31, 2443-2452 is known to create nanofibrillary two-layer scaffolds by electrospinning for biomedical applications using polycaprolactone (PCL) as the top layer and chitosan (CS)/polyvinyl alcohol (PVA) as the bottom dressing layer. In this solution, chitosan is dissolved in 2% acetic acid for 6 hours at room temperature. The same amount of dissolution time at 80°C is given to polyvinyl alcohol and combined in a ratio of 3:1 (CS:PVA). This mixture is solution A. In parallel, solution B is prepared, containing polycaprolactone dissolved in a mixture of dimethylformamide and chloroform in a ratio of 1:9. The solutions in alphabetical order are subjected to electrospinning.

In the publication of F. Chogan, T. Mirmajidi, A.H. Rezayan, M. Sharifi, A. Ghahary, J. Nourmohammadi, A. Kamali, M. Rahaie, Acta Biomater. 2020, 113, pp. 144-163, a similar solution was proposed to use the electrospinning technique. The proposed scaffold consists of three layers: two load-bearing layers made of a composite of polycaprolactone (PCL) and chitosan (CS) constituting the outer layers surrounding the middle layer made of poly(vinyl alcohol) (PVA). The presence of PCL in the outer layers is intended to control the release rate of the incorporated drug through the scaffold, while chitosan, a natural polymer, serves as an antibacterial agent capable of preventing infection at the wound site. PVA was used as a carrier of the active substance metformin hydrochloride.

A method of producing a hybrid dressing material is known from A. Shamloo, M. Sarmadi, Z. Aghababaie, M. Vossoughi, Int. J. Pharm. 2018, 537, 278-289. It consists in the production and immobilization of a cell growth factor in an emulsion of a solution of poly(vinyl alcohol) and polycaprolactone dissolved in chloroform and dropping it into a surfactant solution to form microcapsules. Then the chitosan solution in 3% acetic acid is adjusted to pH 7.4 with 0.1M NaOH, combined with 10% PVA solution and 5% gelatin solution in a 2:2:1 volume ratio, mixed with microspheres, frozen and freeze-dried .

From the description of the patent application WO2018031491, there is known a method of producing one or more layered three-dimensional tissue scaffolds based on at least one of the water-insoluble components, which can be: thermoplastic polyurethane (TPU), polycaprolactone (PCL), poly(glycolic acid) (PLGA), poly(lactic acid) (PLA) or high impact polystyrene (HIPS) and at least one of the water-soluble ingredients which may be: poly(vinyl alcohol) (PVA), salt, sugar, sugar glass, polyethylene glycol (PEG) or any non-crosslinked derivative thereof, gelatin and its derivatives, poly(co-glycolic acid), alginate and sodium bicarbonate. These scaffolds can be obtained from the above components or their composites by known methods of rapid prototyping, such as: SLA, DLP, FDM, SLS, SLM, EBM and/or LOM.

From the description of the patent application CN1238063, there is known a method of producing porous scaffolds consisting in preparing a solution of a thermoplastic material, e.g. polycaprolactone in an organic solvent, adding spherical paraffin particles with a particle size of 355-450 μm and polymer, mixing, partial evaporation of the solvent and pressing the resulting paste into a silicone mold and its appropriate pressure. The shaped scaffold is then dried and extracted in n-pentane to remove the paraffinic porogen. The disadvantage of the above method is the use of large amounts of organic solvents.

The description of the patent application WO2017040156 discloses a system for the simultaneous deposition of thermoplastic materials and thermosetting materials, applicable to the construction of tissue scaffolds using the 3D bioprinting method. The thermoplastic material can be polycaprolactone (PCL) and/or polyvinyl alcohol (PVA). As thermosetting materials, derivatives of acrylic compounds can be used: acrylate, diacrylate, methacrylate, as well as epoxides, silicones, polyethylene glycol diacrylate (PEGDA), hyaluronic acid and chitosan. The described system is very complex and requires control of individual stages. The disadvantage of this solution is also the fact that the scaffolds made on the basis of acrylic compounds require proper cleaning. Otherwise, they may have a cytotoxic effect on the deposited cells.

The method of producing a thermoplastic material as a 3D printing filament containing poly(vinyl alcohol), polycaprolactone and chitosan consists in preparing a solution of chitosan in hydroxyacetic or lactic acid or acetic acid, from which the dissolved chitosan is then precipitated with a base solution, the obtained precipitate is subjected to separation and washing, then suspension in an appropriate volume of distilled water so as to obtain a 1% aqueous solution based on the weight of the non-hydrated polymer, successively homogenized and saturated with carbon dioxide while mechanically stirring the suspension until the chitosan is dissolved, is characterized according to the invention in that in the next stage, chitosan is subjected to compatibilization for processing with thermoplastic raw materials by mixing its solution saturated with carbon dioxide with an aqueous solution of hydrolyzed poly(vinyl alcohol), with a degree of hydrolysis of at least 75%, preferably 80%, where the mass fraction of both polymers in relation to their of the anhydrous forms is 1:1. Next, the mixture is frozen at a temperature ranging from -80°C to -18°C and freeze-dried at a pressure in the range of 0.9-6.0 mbar, with a temperature difference between the lyophilizer condenser and the temperature of the shelf on which the drying takes place. in the range from 70°C to 140°C, and then the composite of chitosan and poly(vinyl alcohol) dried in this way, after grinding, is mixed with polycaprolactone, and the obtained filament is subjected to extrusion, preferably in two stages.

The dried composite of chitosan and poly(vinyl alcohol) constitutes 20 to 40% of the mass share, preferably 20%, of the produced material in the form of a filament in a matrix of polycaprolactone, while the concentration of carbon dioxide in the final solution of chitosan used to create the composite with polyvinyl alcohol) has a pH in the range 5.5-6.5 ranges from 10 to 500 ppm.

Preferably, the 3D printing filament is extruded by the method of deposition of fused FDM material at a nozzle temperature of 130-60°C and a nozzle diameter of 0.4-3.0 mm.

During extrusion, poly(vinyl alcohol) acts as a plasticizer/compatibilizer between the chemically different chitosan and polycaprolactone, hence it is important to obtain a homogeneous mixture with chitosan without the use of acid. Obtaining a thermoplastic material by the method described above consists of several uncomplicated stages, the final effect of which is a biodegradable and biocompatible material with antimicrobial activity, which depends on the concentration of chitosan, the degree of deacetylation and the molecular weight of the polymer in the finished material. To obtain the highest possible antimicrobial activity, it is preferable to use chitosan with low molecular weight, high degree of deacetylation and 20% concentration (40% CS-PVA composite) in the filament.

The advantage of this method of obtaining a thermoplastic material as a 3D printing filament is that this material does not have a cytotoxic effect on the L929 fibroblast cell line. In addition, the filament obtained by the method according to the invention, containing from 10% to 20% by weight of chitosan, has antimicrobial activity.

The invention is presented in more detail in embodiment examples.

Example 1

Polycaprolactone (PCL) with a low molecular weight in the range of 50-60 kDa, a melting point of 60°C and a melting index of 8 g/10 min, measured at 100°C, was used to produce a thermoplastic material with antimicrobial activity, poly(vinyl alcohol) ( PVA) with a molecular weight of 9-10 kDa and a degree of hydrolysis of 80%, and chitosan (CS) with a low molecular weight in the range of 50-100 kDa and a degree of deacetylation of at least 75%. In the first stage of the production of a thermoplastic material with antimicrobial activity, a composite of chitosan and poly(vinyl alcohol) was prepared.

g of chitosan was dissolved in 975 g of a 0.3 M aqueous solution of hydroxyacetic acid and mixed with a mechanical stirrer at a rotational speed in the range of 100-200 RPM until a homogeneous, clear solution was obtained. Then, while stirring, a 1 M sodium hydroxide solution was added in portions to the chitosan solution until the pH value of the solution was equal to 8. The chitosan precipitate obtained in this way was separated using a vacuum filtration set. The separated precipitate was alternately washed with distilled water and filtered off until the pH of the washing water reached a value in the range of 6.5-7. The precipitate, washed and separated under reduced pressure, was supplemented with distilled water in such an amount to obtain a 1% aqueous solution based on the weight of the non-hydrated polymer (before precipitation). The chitosan slurry prepared in this way was homogenized for 1 minute at a rotational speed of 10,000 RPM, and then saturated with gaseous carbon dioxide in the amount of 100 mL/min in an open tank with simultaneous mechanical stirring at a rotational speed of 300 RPM until a homogeneous and transparent solution was obtained. , which is tantamount to the dissolution of chitosan at a concentration of carbon dioxide in the range of 10-500 ppm. The pH of the chitosan solution obtained in this way is in the range of 5.5-6.5. The poly(vinyl alcohol) solution was prepared by dissolving its appropriate weight in distilled water at a temperature in the range of 50-60°C, so that the concentration of the polymer solution was 10% (m/m). In the next step, ready solutions of chitosan in carbonic acid and poly(vinyl alcohol) were mixed together in such a way that the mass fraction of both polymers in relation to their anhydrous forms was 1:1.

In the next stage, the composite solutions were frozen at -24°C for 12 hours, and then freeze dried at a pressure of max. 1 mbar and a temperature difference between the temperature of the lyophilizer condenser and the temperature of the shelf on which the drying takes place, equal to at least 70°C, during 72 hours. The freeze-dried CS/PVA composite was subjected to mechanical fragmentation using a mill to form a coarse-grained powder. The comminuted composite was mixed with polycaprolactone granulate so that its share was 40% by weight of the mixture. After thorough mixing of the ingredients, the mixture was extruded at 130°C. The extruded fiber with a diameter of 2-5 mm was cooled with air at a temperature of 20-30°C and granulated in a continuous process. Then, the obtained granulate was subjected to re-extrusion at a temperature of 130°C using a head with a diameter in the range of 1-3 mm.

Example 2

Polycaprolactone (PCL) with an average molecular weight in the range of 70-80 kDa, a melting point of 60°C and a melting index of 11 g/10 min, measured at 100°C, poly(vinyl alcohol) was used to produce a thermoplastic material with antimicrobial activity (PVA) with a molecular weight of 10 kDa and a degree of hydrolysis of 80%, chitosan (CS) with an average molecular weight in the range of 200-250 kDa and a degree of deacetylation of at least 75%. In the first stage of the production of a thermoplastic material with antimicrobial activity, a composite of chitosan and poly(vinyl alcohol) was prepared.

g of chitosan was dissolved in 975 g of a 0.1 M aqueous lactic acid solution and stirred using a mechanical stirrer at a rotational speed of 200 RPM until a homogeneous, clear solution was obtained. Then, while stirring, a 1 M sodium hydroxide solution was added in portions to the chitosan solution until the pH value of the solution was equal to 8. The chitosan precipitate obtained in this way was separated using a vacuum filtration set. The separated precipitate was alternately washed with distilled water and filtered off until the pH of the washing water reached a value in the range of 6.5-7. The precipitate, washed and separated under reduced pressure, was supplemented with distilled water in such an amount to obtain a 1% aqueous solution based on the weight of the non-hydrated polymer (before precipitation). The chitosan slurry prepared in this way was homogenized for 1 minute at a rotational speed of 10,000 RPM, and then saturated with gaseous carbon dioxide in the amount of 75 mL/min in an open tank with simultaneous mechanical stirring at a rotational speed in the range of 200 RPM until a homogeneous and clear solution, which is tantamount to dissolution of chitosan at a concentration of carbon dioxide in the range of 10-500 ppm. The pH of the chitosan solution obtained in this way is in the range of 5.5-6.5. The poly(vinyl alcohol) solution was prepared by dissolving its appropriate weight in distilled water at a temperature in the range of 50-60°C, so that the concentration of the polymer solution was 7.5% (m/m).

In the next step, ready solutions of chitosan in carbonic acid and poly(vinyl alcohol) were mixed together in such a way that the mass fraction of both polymers in relation to their anhydrous forms was 1:1. In the next step, the composite solutions were frozen at -18°C for 18 hours, and then freeze dried at a pressure of max. 1 mbar and the temperature difference between the temperature of the lyophilizer condenser and the temperature of the shelf on which the drying takes place, equal to at least 100°C, during 68 hours. The freeze-dried CS/PVA composite was subjected to mechanical fragmentation using a mill to form a coarse-grained powder. The comminuted composite was mixed with polycaprolactone granulate so that its share was 30% by weight of the mixture. After thorough mixing of the ingredients, the mixture was extruded at 150°C. The extruded fiber with a diameter of 1-3 mm was cooled with air at a temperature of 20-30°C and granulated in a continuous process. Then, the obtained granulate was subjected to re-extrusion at a temperature of 150°C using a head with a diameter in the range of 0.4-3 mm.

Example 3

To produce a thermoplastic material with antimicrobial activity, polycaprolactone (PCL) of high molecular weight in the range of 100-120 kDa, melting point of 60°C and melting index of 5 g/10 min, measured at 160°C, poly(vinyl alcohol) (PVA ) with a molecular weight of 10 kDa and a degree of hydrolysis of 75%, chitosan (CS) with a high molecular weight in the range of 300-400 kDa and a degree of deacetylation of at least 75%. In the first stage of the production of a thermoplastic material with antimicrobial activity, a composite of chitosan and polyvinyl alcohol was prepared.

g of chitosan was dissolved in 990 g of a 0.5 M aqueous solution of hydroxyacetic acid and mixed with a mechanical stirrer at a rotational speed in the range of 100-200 RPM until a homogeneous, clear solution was obtained. Then, while stirring, 0.5 M sodium hydroxide solution was added in portions to the chitosan solution until the pH value of the solution was equal to 8. The chitosan precipitate obtained in this way was separated using a vacuum filtration set. The separated precipitate was alternately washed with distilled water and filtered off until the pH of the washing water reached a value in the range of 6.5-7. The precipitate, washed and separated under reduced pressure, was supplemented with distilled water in such an amount to obtain a 1% aqueous solution based on the weight of the non-hydrated polymer (before precipitation). The suspension of chitosan sediment prepared in this way was homogenized for 2 minutes at a rotational speed of 20,000 RPM, and then saturated with gaseous carbon dioxide in a pressure tank equipped with a safety valve at a working pressure of 2 bar, with simultaneous mechanical stirring at a rotational speed of 300 RPM, until a homogeneous and transparent solution, which is tantamount to dissolving chitosan at a concentration of carbon dioxide in the range of 10-500 ppm. The pH of the chitosan solution obtained in this way is in the range of 5.5-6.5. The poly(vinyl alcohol) solution was prepared by dissolving its appropriate weight in distilled water at a temperature in the range of 50-60°C, so that the concentration of the polymer solution was 5% (m/m).

In the next step, ready solutions of chitosan in carbonic acid and poly(vinyl alcohol) were mixed together in such a way that the mass fraction of both polymers in relation to their anhydrous forms was 1:1. In the next step, the composite solutions were frozen at -80°C for 6 hours, and then freeze dried at a pressure of max. 6 mbar and a temperature difference between the temperature of the lyophilizer condenser and the temperature of the shelf on which the drying takes place, equal to at least 140°C, within 24 hours. The freeze-dried CS/PVA composite was subjected to mechanical fragmentation using a mill to form a coarse-grained powder. The comminuted composite was mixed with polycaprolactone granulate so that its share was 20% by weight.

EN 242810 B1 of the mixtures. After thorough mixing of the ingredients, the mixture was extruded at 160°C. The extruded fiber with a diameter of 2-3 mm was cooled with air at a temperature of 20-30°C and granulated in a continuous process. Then, the obtained granulate was re-extruded at 160°C using a head with a diameter in the range of 0.4-3 m.

Example 4

In order to confirm the receipt of an antimicrobial thermoplastic material that is not degraded due to the presence of acid residues for dissolving chitosan, tests of thermostating the CS-PVA composite (Table 1) and measurements of mechanical properties, activity against Escherichia coli and Staphylococcus aureus bacteria, cytotoxicity and printability by deposition method were carried out. fused fiber (FDM) for the fibers obtained according to examples 1-3.

Table 1. Confirmation of the destructive role of mineral acids normally used to dissolve chitosan in its composites with poly(vinyl alcohol) after a 5-hour temperature treatment at 160°C.

Table 2. Physicochemical, biological and functional properties of the obtained fibers according to examples 1-3.

Example 1 Example 2 Example 3 Tensile strength |MPa| 7.5 8.1 9.2 Tensile elongation [%] 112 110 110 Log Reduction rate of S. aureus 2.38 1.64 0.95 Logarithmic Reduction rate of E.coli 2.05 1.25 0.56 Cytotoxicity against the L929 cell line as cell survival | %| 91 90 88 FDM printability π i., a Hardness [kPa] 6.2 5.5 4.8 Flexibility [-] 0.51 0.54 0.65

The test of thermosetting of composites confirmed that the use of the method of their preparation by combining chitosan hydrogel in carbonic acid and a solution of polyvinyl alcohol, followed by lyophilization of the frozen mixture of these polymers, allows to obtain a thermally stable composite that does not degrade at temperatures up to 160°C. during several hours of heating. This feature is not present in composites of chitosan with poly(vinyl alcohol), in which mineral acid (e.g. acetic, hydrochloric) is present, used in classical methods of dissolving chitosan. Thus, it was proved that the materials produced in this way will not degrade during the extrusion process, which requires elevated temperature, and the poly(vinyl alcohol) chitosan composite can be a component of thermoplastic materials using a polycaprolactone matrix. Antimicrobial activity tests confirmed that both materials exhibit at least bacteriostatic activity, with the material containing 20% chitosan, described in embodiment 1, allowing the reduction of S. aureus and E. coli bacteria by at least 99% at the initial microbial load with cells above of the listed bacteria at the level of 1-5 x 107 CFU/mL culture. In contrast, the material containing 10% chitosan, described in embodiment 3, allows a reduction of S. aureus and E. coli by 50 to 90% with the same initial microbial load with the cells of the above-mentioned bacteria. The MTT cytotoxicity test against the L929 mouse fibroblast line confirmed that cell survival in contact with the produced material is higher than 80%. Such a result means no cytotoxic effect and potential compatibility of the material as a cell culture scaffold.

Claims (4)

1. A method of producing a thermoplastic material as a 3D printing filament containing poly(vinyl alcohol), polycaprolactone and chitosan, where the chitosan solution is prepared in hydroxyacetic acid, lactic acid or acetic acid, from which the dissolved chitosan is then precipitated with a base solution, the resulting precipitate is separated and washing, then suspending in an appropriate volume of distilled water so as to obtain a 1% aqueous solution based on the weight of the non-hydrated polymer, successively homogenized and saturated with carbon dioxide while mechanically stirring the suspension until the chitosan is dissolved, characterized in that chitosan in the next stage subjected to compatibilization for processing with thermoplastic raw materials by mixing its solution saturated with carbon dioxide with an aqueous solution of hydrolyzed poly(vinyl alcohol), with a degree of hydrolysis of at least 75%, preferably 80%, where the mass fraction of both polymers in relation to their anhydrous forms is 1 :1, then the mixture is frozen at a temperature ranging from -80°C to -18°C and freeze-dried under pressure conditions in the range of 0.9-6.0 mbar, with a temperature difference between the lyophilizer condenser and the temperature of the shelf on which drying takes place in the range from 70°C to 140°C, and then the composite of chitosan and poly(vinyl alcohol) dried in this way, after grinding, is mixed with polycaprolactone, and the obtained filament is subjected to extrusion, preferably in two stages. 2. The method of claim The method according to claim 1, characterized in that the dried composite of chitosan and poly(vinyl alcohol) constitutes 20 to 40% by weight, preferably 20%, of the produced material in the form of a filament in a polycaprolactone matrix. 3. The method of claim The method according to claim 1, characterized in that the concentration of carbon dioxide in the final chitosan solution used to create the poly(vinyl alcohol) composite with a pH in the range of 5.5-6.5 ranges from 10 to 500 ppm. 4. The method of claim 1, characterized in that the 3D printing filament is extruded by the method of deposition of fused FDM material at a nozzle temperature of 130-160°C and a nozzle diameter of 0.4-3.0 mm.