Method for production of nanofibres

Technical field

The invention relates to method for production of nanofibres through electrostatic spinning of polymer matrices prepared upon biopolymers of chitosan or collagen.

Next to this, the invention relates to fabric comprising at least one layer of nanofibres produced through electrostatic spinning of biopolymer of chitosan or collagen.

Background art

Biopolymers feature a number of unique properties thanks to which they are suitable for application in medicine, first of all thanks to its biocompatibility and nontoxicity. Important is their application e.g. for production of bandages and plasters, but also for implants and antiadhesive mats, where they expressively decrease the risk of occurrence of postoperative adhesions between the tissues, further in dental medicine, cosmetics and surgery plastic for filling of dermatic or bone defects. Some of the biopolymers are biodegradable, that means decomposable e.g. by action of enzymes. The nanofibrous materials of biopolymers are of a high porosity as well as of a specific surface, are penetrable for oxygen but not penetrable for microbes, at the same time they preserve all above mentioned properties of the used biopolymers. Biopolymer nanofibres are suitable e.g. for healing of burns, when they ensure an optimum humidity at healing and simultaneous removal of exudate from the wound, further for the bandage materials, plasters, etc.

One of the significant biopolymers is chitosan, which is the polycationic polysaccharide composed of β- (1→4) 2-acetamido-2-deoxy-D-glucopyranosic and 2-amino-2-deoxy-D-glucopyranosic units. To its chemical composition it is very similar to cellulose and it is the second most widespread renewable nature source. It is extracted from chitin through alkaline deacetylation, the source of

which are the shellfish, e.g. the shells, mussels, crabs and crayfish, it is also contained in tectrices of insect and also in mushrooms. Chitosan is biodecomposable, biocompatible, thanks to positive charge at physiological pH it is bioadhesive, which is a great advantage in healing of wounds, it has hemostatic effects, thus stops bleeding, it has also antibacterial effects. Presently, thanks to its ability to absorb LDL cholesterol and heavy metals, it is contained in most of the reduction diets. By these properties it is directly designated for application in medicine, e.g. for the already mentioned plasters and bandages, but also for the antiadhesive mats into the body or in dental medicine for filling to stop bleeding. It is also used in biotechnologies for purification of waste waters or liquids, e.g. beer, wine or milk.

Most types of chitosan are insoluble in water, but soluble in organic acids with pH of solution lower than 5. Most frequently are as solvent used the acetic acid, lactic acid, malic acid, oxalic acid, etc. At present great number of scientific laboratories and institutions deal with chitosan. For acquiring nanofibres the method of electrostatic spinning is used. At present the nanofibres from chitosan are produced by means of spinning devices with needle or jet spinning electrode. WO2007093805A1 discloses production of composite fibres from chitosan and alginate, where the maximum content of chitosan reaches 80%, whereas the chitosan fibres coat the surface of alginate fibres. Due to the diameter of fibres, which is 50 μm , these fibres are not nanofibres.

WO2006133118A1 generally deals with biopolymers in a nanofibrous form, where alternate the nanofibrous layers of polymer soluble in water and insoluble in water. Diameters of produced nanofibres are in the range of 1 to 25000 nm, which is not the nano dimension any more. KR100652469B deals with antibacterial nanofibres, which are made of chitosan in mixture with polyethylene terephthalate. As a solvent the trifluorethanol, hexafluoroisopropanol or trifluoroacetic acid is used. Another Korean patent deals with production of nanofibres of chitin or chitosan upon usage of jets as the spinning electrodes. As solvents the N-

methylmorpholineoxid, hexafuoro-2-propanol or hexafluoroacetone hydrate and formic acid are used.

WO2006048829 patent deals with new derivates of chitin in a nanofibrous form for application in medicine, mainly for application to protect skin and as hypodermic fillings. The used biopolymers are oxychitin, chitin of glycolate, chitin of hyaluronate. Also here is for production of nanofibres used the needle spinning electrode.

WO03042251A1 patent discloses production of composites containing chitosan in the form of nanodimensional fibres for the purpose of increasing the activity and solubility mostly for usage in cosmetic. Nevertheless the length of produced nanofibres is considerably limited and it varies in the range of 5 to 200 nm, while their diameter is in the range of 5 to 30 nm, which corresponds rather to the size of nanoparticles than the nanofibres.

Another patent KR1020050048360AA deals with production of nanofibrous nonwoven fabric for tissue engineering, where for production of nanofibres the natural polymer from the group of chitosan, collagen, alginic acid and synthetic polymer is used, e.g. homopolymer of lactic acid, copolymer of lactic acid and of gluconic acid, homopolymer of gluconic acid and their mixtures, while the ratio of natural and synthetic polymer is 4:1 to 1:4. There exists relatively great quantity of published documents, out of which some deal with production of nanofibres of chitosan in mixture with polyethylenoxid. One of them is the article Bin Duan (Journal of Biomaterial Science, Polymer Edition, Vol. 15, 2004, p.797-811), where the maximum rate chitosan : PEO is 2:1 and as solvent the 2% acetic acid is used. For production of nanofibres are applied capillaries and achieved diameter of nanofibres is 80 to 180 nm.

Another document N.Bhattarai (Biomaterials, vol.26, lss.31 , 2005, p.6176-6184) deals with production of nanofibres from the mixture of chitosanPEO in maximum ratio 90:10, where as solvent non-ionic tenside Triton X-100™ is used. As a spinning electrode the syringe is used and the applied voltage is 20 to 25 kV and distance of electrodes is 17 to 20 cm. As a co-solvent dimethylsulfoxide is added.

X.Geng et. al.. deals with spinning of chitosan in concentrated acetic acid (Biomaterials, Vol. 26,2005, p.5427 - 5432). They use 7% chitosan in 90% acetic acid at applied voltage of 4 kV/cm. At higher voltage nanofibres defects occur, when lower concentration of acid is used the problems with too high surface tension of polymer solution arise. As a spinning electrode the nozzle is used and the achieved diameter of nanofibres is 130 nm.

Another possible method for production of nanofibres from chitosan is spinning of chitosan solution with addition of polyvinyl alcohol, like Lei Li and his group (Carbohydrate Polymers, Vol.62, 2006, p.142 - 158) deals with. The original solution is composed of mixture PVA/chitosan in ratio 83/17 (w/w) in 2% acetic acid, while the achieved diameter of nanofibres is 20 to 100 nm. Then PVA is removed from nanofibres through leaching in NaOH.

Another in medicine mostly used biopolymers is collagen which is applicable especially at healing of burns, as implants, for artificial dermal fillings, artificial skin, artificial cartilages, vertebras, etc. Collagen is contained e.g. in skin, vascular walls, cartilages, ligament. There are known 19 types of collagen out of which some are soluble in acetic acid. In medicine are used primarily the types I ₁ II and III. Collagen is formed of three polypeptides creating the α-helix with regularly repeating amino acids Gly-Pro-Hyp. It is insoluble in water and soluble in a few of solvents, out of which the mostly used is hexafluoroisopropanol. Through chemical or thermal degradation the gelatine is obtained.

CN 1944724 is a patent that deals with production of composites of chitosan and collagen. Here as a solvent the hexafluoroisopropanol and trifluoracetic acid or their mixture are used. One of publications which deals with spinning of collagen of type I is article of J.A.Matthews (Biomacromolecules,

Vol.3, 2002,p.232-238), where as a solvent hexafluoroisopropanol is applied.

WO2006068421A1 discloses production of nanofibrres which are formed of polyhydroxyalkanoate, collagen or gelatine. The spinning electrode applied for production of these nanofibres is nozzle or needle, and diameter of nanofibres varies in the range of 50 to 2000 nm.

It is apparent from the mentioned patents or publications, that there is not known any method of continual production of nanofibres of biopolymers, especially of chitosan or collagen. The electrostatic spinning from needles, which form the spinning electrode is interrupted after the drop of polymer solution is consumed. Neither spinning from nozzles or capillaries, into which the polymer solution is delivered, does not represent a continuous process, because the small inner diameters of nozzles or capillaries get obstructed and for the period of cleaning the spinning process must be interrupted. Moreover the nanofibres produced to date through the mentioned methods are not of a sufficient quality and their layer is not uniform.

The goal of this invention is to suggest the method for production of nanofibres of biopolymers through electrostatic spinning, which would remedy the disadvantages of the background art.

Principle of the invention

The goal of the invention has been achieved by production method of nanofibres through electrostatic spinning of polymer matrices prepared upon on biopolymers of chitosan or collagen according to the invention, whose principle consists in that, the biopolymer is before spinning dissolved as clean or in mixture with auxiliary non-toxic polymer in solvent system, which contains an organic or inorganic acid, selected from the group of acetic acid in concentration from 30 % to 90 % of weight, lactic acid, malic acid, thhydrogen-phosphoric acid and their mixtures, and this solution is brought into electrostatic field between the spinning electrode and collecting electrode, while the produced biopolymer nanofibres comprise more than 90 % of weight of biopolymer in dry mass.

At the same time it is advantageous if biopolymer nanofibres comprisen more than 95 % of biopolymer in the dry mass.

Constant quality results at spinning are achieved if the solvent system comprises the acetic acid. For production of nanofibres of chitosan it is advantageous if chitosan of molecular weight lower than 150 kDa is before spinning dissolved in acetic acid

of concentration higher than 50 % wt. together with auxiliary nontoxic polymer PEO.

The fibres of chitosan may be produced also without addition of PEO, as it is shown in the claim 5. For production of nanofibres of collagen it is advantageous if collagen is before spinning dissolved in the solvent system comprising the diluted acetic acid and the auxiliary polymer PEO or PVA soluble in water having concentration of 1 to 3%.

Through the above mentioned method it is possible to perform electrostatic spinning of the mentioned biopolymers constantly at production of nanofibres which correspond as to the quality to electrostatic spinning of other polymers that can be subject to the spinning process.

The constant good spinning results are achieved, if the biopolymer solution in electric electrostatic field for spinning is positioned on surface of the active zone of spinning mean of the spinning electrode.

It is at the same time advantageous, if the biopolymer solution is delivered to electrostatic field for spinning through surface of the spinning electrode.

The spinning electrode is with advantage formed of a rotating spinning electrode of an oblong shape, which by section of its perimeter extends into the biopolymer solution.

In advantageous embodiment such spinning electrode comprises a couple of faces made of electrically non-conductive material, between which are positioned the spinning members created of wire, which are equally distributed around perimeter, parallel with axis of rotation and mutually electrically conductive connected.

It may also be advantageous, if the biopolymer solution in electrostatic field for spinning is situated on surface of the active spinning zone of the spinning means.

Active spinning zone of the cord during spinning has a stable position towards the collecting electrode and biopolymer solution to the active spinning zone of the cord is delivered by applying or by a motion of the cord in direction of its length.

Examples of embodiment

Chitosan is dissolved solitary or in mixture with auxiliary nontoxic polymer, especially the one soluble in water, which is in ideal case biocompatible and biodegradable. The example is polyvinylalcohol, polyethylene oxide or polyvinylpyrrolidone. Chitosan is used in the concentration of 5 - 25 wt. % in dependence on the solvent system, which is formed of organic or inorganic acid, especially the acetic acid, while concentration of acetic acid is higher than 30 % and lower than 90%. Further the lactic acid, malic acid and trihydrogen phosphoric acid or their mixtures may be used. The ratio of chitosan and of the auxiliary polymer is higher than 90:10 to the dry mass of nanofibres. To the solution may be under an increased temperature the reticulate agents added, e.g. dialdehydes, dicarboxylic acids, genipin, trisodium citrate. The process depends on molecular weight of chitosan, grade of deacetylation, concentration, or viscosity, surface tension, temperature and humidity of surroundings and parameters of technology, like rotation and type of electrode, distance between electrodes and applied voltage.

In the particular example of embodiment for production of solution was used 31 ,5g of chitosan in 45Og of 65,7 % acetic acid, which is best if mixed for a period of 24 hours, with possibility of short term warming the solution to 50 ⁰ C to increase the dissolubility of chitosan. After standing the mixture is mixed with 112,5 g of 3% solution of polyethylene oxide in water. The ratio of chitosan and PEO (w/w) is 90,3:9,7 in 100g of dry mass of produced nanofibres. This content of chitosan may be increased nearly to 100% at simultaneous reducing the performance. For electrostatic spinning of the above mentioned solution of biopolymer was used the device for electrostatic spinning of polymer solutions comprising the spinning electrode, which comprises the rotatably mounted spinning means

extending by a portion of its perimeter into the biopolymer solution being present in the reservoir. The rotatable spinning means due to its rotation carries out the biopolymer solution into electrostatic field of a high intensity, which is created by difference of potentials between the spinning electrode and against it arranged collecting electrode, while the section of surface of the rotating spinning means positioned against the collecting electrode represents the active spinning zone of the spinning means. Upon spinning, the biopolymer solution is to be found in electrostatic field on surface of the active spinning zone of the spinning means of the spinning electrode. The rotatable spinning means may be performed for example according to the CZ patent 294274 or according to CZ PV 2006-545 or CZ PV 2007-485.

For production of nanofibres of above described solutions of biopolymers may be used also other types of spinning electrodes, by which the biopolymer solution in electrostatic field for spinning is to be found on surface of active spinning zone of the spinning means of the spinning electrode. Such spinning means may be formed of other types of rotatable spinning electrodes according to the patent 294274 or invention applications mentioned above. Application of rotatable spinning means is not a necessary condition, because spinning of biopolymer solution runs successfully also on the cord spinning electrodes according to the CZ PV 2007-485, at which the active spinning zone of the cord during spinning has a stable position towards the collecting electrode and the biopolymer solution to the active spinning zone of the cord is delivered either by applying or by motion of the cord in direction of its length. In this case the solution of biopolymer in electrostatic field for spinning is to be found on surface of active zone of the spinning means.

Upon the concrete spinning of chitosan solution described above a portion of the solution was poured into a reservoir tub and this was equipped with spinning electrode, especially the cylindric or cord one. The tub with electrode was positioned into a device for production of nanofibres through electrostatic spinning. As a substrate material the polypropylene spunbond of surface weight of 17 g/m ² with antistatic surface finish was used, and as a collecting electrode the non-ionising cylindric electrode according to the CZ PV

2006-477 was used. During the process on the spinning electrode was constantly set the voltage of 60 to 75 kV and the collecting electrode was grounded. The distance between electrodes was 100 to 200 mm. Rotation speed of the spinning electrode was 3 to 10 rot/min and shift of the substrate material was 20 cm/min. Relative humidity of surrounding air was 30% at temperature of 20 ⁰ C.

Using the same method and with a similar result the following solution was subjected to electrostatic spinning.

Collagen of the type I underwent spinning from its solution in diluted acetic acid, so that halogen solvents, which may cause problems in medicine applications, were not used. The residua of acetic acid may be removed by a short term warming of the nanofibrous material. The nanofibres of collagen may be reticulated by the same means as chitosan. Achieved weight ratio of colagenu and auxiliary polymer is higher than 90:10. For production of nanofibres of collagen especially the above mentioned cord spinning electrode is suitable, the applied voltage of 6 to 7 kV/cm.

For production of 12g of 10% collagen solution in 0,5M acetic acid it is mixed 6g of 99% acetic acid and 1 ,2g of 3% PEO in water i.e. 98,5% collagen to 100g of dry mass of nanofibres. If the content of collagen is reduced to the benefit of PEO, performance of the process is increased.

Advantage of the described technology is a high content of biopolymer in nanofibres and a large range of surface density of nanofibres, which is 0,05 to

100 g/m ² . The produced nanofibres of chitosan may have diameter of 10 to 250 nm, the nanofibres of collagen 10 to 200 nm. In all cases a long term continual spinning process has been achieved.

Example 1 Chitosan of a low molecular weight (lower than 15OkDa, with viscosity

0,5% of solution in 0,5% solution of acetic acid between 5 - 30 mpa.s), of the grade of deacetylation at min. 75 % before spinning is dissolved in diluted acetic acid with concentration higher than 50 % and it is left to be mixed for a

period of 12 hours at minimum. After stabilising it is mixed with mixture of auxiliary in water soluble polymer like the PEO of molecular weight 300000- 400000 and concentration 1-3% at temperature to 35 ⁰ C ₁ humidity to 60%, and this solution is delivered into electrostatic field between the spinning electrode and collecting electrode.

Example 2 Before spinning, collagen is dissolved in solvent system comprising the acetic acid 87,5 % of weight, the auxiliary polymer (PEO or PVA) soluble in water (concentration of 1-3%) at temperature to 35°C, humidity to 60% and this solution is brought into electrostatic field between the spinning electrode and collecting electrode.

Industrial applicability

The chitosan as well as collagen nanofibres provide a large possibilities of application, first of all in medicine, and thanks to possibility of nearly any surface density of nanofibres they may be applicable also as substrateless materials, antiadhesive mats, plasters, implants and fillings of undesired bone or dermal defects. Chitosan thanks to hemostatic effects may be used at operation or in dental medicine to stop bleeding along reduction of economic costs and simultaneous speeding of wound healing. Colagen nanofibres will surely find their application solitary as replacement of damaged ligaments, tendons and cartilages or at injury of backbone, e.g. the intervertebral plate, or also for coating of implants, which reduces the negative immunity response of organism after introduction of a foreign element into the body.