Field of the invention

The invention relates to voluminous nanofibrous materials based on hyaluronic acid, its salts or derivatives thereof, a method of preparation thereof, a method of modification thereof by cross-linking, a modified nanofibrous material, nano fibre structure, and their use in medicine or cosmetics.

Background of the invention

The use of hyaluronic acid (HA) in medical devices is attractive because HA is natural biodegradable polymer enhancing cells migration and proliferation and extracellular matrix production (Schmidt, 2010). However the processing of HA into nanofibres is quite difficult. Another drawback is that hyaluronic acid in its natural form is water soluble material which, after its electrostatic spinning into nanofibres, dissolves immediately after coming into contact with water. This is advantageous for some applications only.

Electrostatic spinning of hyaluronic acid (HA), its sodium salt, eventually other derivatives, was described in detail in many patent documents and publications. Its processing by this method is more or less successful but always with low efficiency. The reason is the nature of water solutions of hyaluronic acid. Water solutions of HA reach high viscosity and form gels even at their low molecular weights and concentrations. The solutions of lower concentration are not able to be spun due the insufficient level of twisted polymer chains, and this, together with physical behaviour of the solution, prevents the formation of stable fibres (Kim, 2008). Almost all the undermentioned documents discuss the hyaluronic acid or its derivatives of relatively high molecular weight, usually of Mw 0.4 to 3.5 x 10 ⁶ g/mol) (Kim, 2008; Luo, 2010; Son, 2004), whose processing needs the use of highly diluted solutions because of the high viscosity and surface tension of high molecular HA, and therefore it is non efficient. That is why the research teams solving this problem often use solvents other than water to deal with high viscosity of water solutions of HA, i.e. the solvents as is dimethylformamide (D F), formic acid, trifluoroethane, chloroforme and the like, which are absolutely inappropriate for the use in medical devices and can negatively affect an organism. Furthermore, in the case of production of these materials in larger scales, there would be the need to dispose large amount of toxic waste. From the commercial point of view, it is more advantageous to process the solutions of higher concentrations. During spinning the solutions of high molecular HA of low concentrations, it is necessary to evaporate a large amount of solvent (the solvent can form up to 99.5 % of the solution), therefore the yields of the process, ie. amount of dry nanofibres gained, is extremely low.

The technique known as "electroblowing" or„blowing assisted electrospinning" can be also used for electrostatic spinning of HA (Wang, 2005). This technique is suitable for preparation of water resistant nanofibres from native HA (Mw 3.5 xlO ⁶ g/mol) by spinning of the 2.5 to 2.7% HA dissolved in acidic aqueous solution with the productivity of 1.2 to 3.6 ml/h. The water resistibility of HA nanofibres is reached by exposing the nanofibrous materials to gaseous HC1 However, the use of acids causes undesired degradation of HA. Brenner, 2012, also describes the spinning of HA, 3 % solution of HA (2x10 ⁶ g/mol) was spun from (NaOHDMF) = (4:1) or 1.5 % solution of HA was spun from (K¾OH:DMF) = (2:1) with the maximum rate of 0.9 ml/h. The authors (Liu, 2011) successfully spun the solution of pure HA (Mw 1 xlO ⁶ g/mol) from the rnixture of solvents FA/DMF/water = (50/25/25) of the concentration of (0.8 - 1.2) % with the productivity of 0.3 mL/h. Most of the listed solvents are inappropriate for the use in medical devices. Uppal, 2012, solves the spinning of aqueous solutions, in particular 1 to 4 % solutions, which however did not form fibres alone, therefore the authors had to use surfactants.

Electrostatic spinning of fibres with a certain amount of HA is discussed also in ( R2011116616), where the aqueous solution of alkali salts was used for dissolving HA (molecular weight of 1x10 ^s g/mol - 1. 59x10 ⁹ g/mol), and this solution simultaneously adjusted conductivity of the solution; but the resulting material is used for cell cultivation and cannot be considered voluminous. Other surface nanofibrous materials based on HA are surface nanofibrous materials for cosmetics purposes with high content of vitamins A and E (KR2011110482) that are stabilized by absence of water in a dry nano fibre layer, but it is necessary to add a surfactant to them.

US7662332 discusses electrostatic spinning of HA, its copolymers and mixtures by the method known as electroblowing. However, in the case of low viscosity solutions of low molecular HA, the authors state that polymer chains are not sufficiently twisted due to the low viscosity of the solutions of low molecular HA. The experimental results however relates to the solution of HA of molecular weight of 3.5xl0 ⁶ g/moL with maximum concentration of 3 % w/v, furthermore prepared with the use of an acidic aqueous solution whose evaporation in commercial production process can cause corrosion of the construction parts of spinrung device, degradation of the HA used, and the residual amount of the acid can negatively affect an organism. WO2005033381 describes the preparation of HA fibres by the electroblowing technique including a biomedicine material made of HA with the fibres diameter of 10 to 1000 nm. None of these documents mention voluminous nanofibres materials.

The viscosity of the solution being spun can be reduced with the use of low molecular HA or its derivatives in the combination with fibre forming polymer, for example PEO, PVP or PVA from aqueous solutions; this is described only in JP2009041117 with the assumption of the presence of thermoplastic resin, ie. the spinning supporting agent.

CN1837274 mentions a nanofibre membrane containing hyaluronic acid in the amount of 0 to 100 %. HA, HA/GE, HA/PVA, HA/PEO were spun, the concentrations of the described solutions were in the range of 1.8 to 5 weight % and the productivity was in the range of 0.3 to 18 mL h However, the vo rninous nanofibrous materials are not mentioned.

The production of voluminous nanofibrous materials from HA or its derivatives is even more complicated and it has been successfully performed by electrostatic spinning according to (Kim, 2008) only, but the structure obtained was not stable and it broke down after switching off the voltage source. That is why the researchers started to spill small crystals of NaCl into forming nanofibre layer during the spinning process, and then the crystals were washed out, thereby the nanofibres turned into a gel due their heavy swelling and the loose of nanofibre structure. Then the layer was dried by lyophilisation, which is not only time consuming but also very costly. The nanofibre structure was partly restored, but the fibre diameter was considerably greater despite using the lyophilisation.

The use of conventional nanofibrous materials for scaffolds is very limited. Small dimensions of the interfibrous pores prevent cells migration into nanofibrous material; therefore the cells grow on the surface of material and form 2D structure causing their dedifferentiatioa This problem is usually solved by the use of tubular shapes (W O2010040129) or by interlacing the nanofibre layers and the cells (WO2010042651, US2008112998). In the case of scaffolds, it is in particular important to form a porous structure with large pores for cells colonization; the pores can be prepared by various methods, as is the perforation of nanofibre layers (WO2006106506), cryogenic methods (WO2011004968, US2010248368), freezing and lyophilisation (CN102383267), photolitography, 3D printing and bioprinting, or various foams and spongeous materials.

Regarding to sorption properties of conventional nanofibrous materials made from hydrophilic polymers, they are good ((JP2013049927), but strongly limited by their surface weight. The higher layer thickness, i.e. surface weight, the lower quantity of the liquid absorbed, as the conventional nanofibre layer is compact, more -wrapped (WO2011130110) and it inhibits remarkable swelling related with high sorption.

Thanks to its excellent effects in an organism, HA has been already used in many existing medical devices, and the use of hyaluronic acid and its derivatives, in the form of nanofibre s or hydrogels, is mentioned in many works (e.g. Dawson, 2008; Young, 2006; Bhardwaj, 2010 etc). Several texts focusing directly on developing the HA nanofibres can be found in the literature, for example (Xu, 2009) succeeded in electrostatic spinning of HA (Mw 2x10 ⁶ g/mol), more specifically the solution of the concentration of about 1.5 % of HA in DMF with the peroduction of 3.6 mL/h, and thus they produced ultra-thin fibrous membrane. However Ν,Ν-dimethylfortnamide (DMF) is a toxic substance with respiration toxicity, toxicity in contact with skin, teratogenity, and it can cause a serious damage of eyes, so it is inappropriate for the production of medical devices.

(CN101775704, CN101792955, CN102068339) describe the preparation of nanofibres from hyaluronic acid, that are spun from the mixture of solvents of water - formic acid - N,N- (iimethylformamide; the solution of the concentration of (0.5 - 5) weight % were spun, with the productivity of 0.5 - 1.5 mL/h As it was mentioned above, Ν,Ν-dimefhylformamide (DMF) is a toxic substance with respiration toxicity, toxicity in contact with skin, teratogenity, and it can cause a serious damage of eyes, whilst the formic acid is a corrosive substance. HA fibres with voluminous structure are not mentioned. ( R2009071993) describes spinning of 10% HA in TFE (trifluoroethanol). (CN 101581010) includes nanofibres from the mixture HA/glutin with the content of HA in a dry matter of (60 % - 90 %), with the mixture of trifluoroethanol and water as the solvent. In both the documents lastly mentioned, the solvent used is highly inappropriate for the use in the medicine or tissue engineering. Trifluorethanol is a hazardous substance harmful to the health, both at respiratory and skin contact. Furthermore, voluminous nanofibrous materials based on HA are not mentioned. CN102691176 describes patterned nanofibre membranes from various polymers comprising also HA, but voluminous nanofibrous materials are not mentioned.

Hyaluronic acid in its natural form is water soluble and its dissolving proceeds immediately after corning into contact with moisture; this is undue for various types of materials, especially implants or antiadhesive membranes designed for persisting in an organism for defined period of time. Therefore it is necessary to cross-link the nanofibrous materials. Normally it is realized by immersing the nanofibre layer in a solution containing a crosslinking agent (US7323425AVO2006026104, Wang, 2005). This method however would lead to disruption of the voluminous and fluffy nanofibre structure. The patent document WO2010040129 mentions 3D porous scaffolds from fibres for tissue engineering and regenerative medicine generally consisting of oriented or randomly organized fibres prepared from natural or synthetic materials. Particular layers of these fibres are organized into matrices suitable for cell cultivation. However in this document, the 3D structures are reached by forming the little tubes from surface nanofibre layers. US7704740 describes nanofibre structure where one or more fibres contain one or more bioactive molecules, for example HA; volurninous materials based on HA are not mentioned. Also the patent document CN1958892 does not mention voluminous materials based on HA. The document describes a special surface patterned nanofibrous material that can be prepared f om biological materials by the method named discharge spinning. The pattern is reached by exposing the material to UV radiation through a patterned template, where the radiation induces the crosslinking reaction.

WO2007012050 discusses the forming of nano-fibrilous structures for the cell cultivation, but these structures are on the base of polyamide. O2009002869 describes the nanofibres with reactive groups capable to be activated by photochemical or thermal way and by biologically active substances. WO2005025630 describes the nanofibres for medical devices, systems for controlled drug delivery, materials for tissue engineering, regenerative devices, prothetics, or cosmetics facial masks, among others made from HA. However none of these documents does not state voluminous and fluffy structures from HA nanofibres.

WO2008100534 describes a nano fibrous material combined with continuous secondary phase; the material is a nanofibre scaffold f om oriented or non-oriented nanofibres from biodegradable polymer.

The patent document US20130052712 describes a nanofibre scaffold where the HA is used for coating the substrates or as a bioactive ingredient used on the surface of nanofibres. JP2013049927 discusses the spinning of mixed nanofibres, for example from HA/FVA, together with glutaraldehyde and PVA as the crosslinking agent.

US20110111012 describes also the preparation of nanofibrous materials that can among others contain HA. It describes, among others, a dressing material, that can contain HA, and the method of its preparation. However, the use of various additives for reducing the viscosity of a solution, as is for example HN0 ₃ , NaOH or acetic acid, minimizes the possibilities of the use of this material in medical devices. WO2011086330 discusses the dressing materials that dissolve or change into a gel. The aim of the described nanofibrous material is not exudate drainage from a wound but more likely forming a interlayer moistening the wound by quick dissolving the contact nanofibre layer.

As it was stated above, the preparation of voluminous and fluffy nanofibrous materials and structures is very difficult. So far, such stable materials have been prepared directly by electrostatic spinning, for example by spinning of PLLA from dichloromethane, hexaflouroisopropanol and acetone (WO2007024125), Matrigel® from hexaflouroisopropanol or acetic acid (WO2006138718), PLA and PEO from chloroform or dichloromethane (WO2010132656), and PCL from mixed solution of chloroform-methanol (WO2011130110), PCL in the combination with gelatine and hydroxyapatite from trifluoroethanol (WO2008093341), PLLA from dichloromethane (KR875189), sulfated celullose from H3PO4 (US2010233234), PS nanofibres from THF (WO2008030457).

WO2011130110 discusses in detail the preparation of voluminous nanofibre layers, where the voluminous structure is reached by the use of special collecting electrodes.

Voluminous nanofibrous materials based on HA have been so far described in (Kim, 2008), where the fluffy nanofibre structure was successfully prepared. However after switching off the voltage source, the 3D fluffy structure collapsed (Fischer, 2012) describes the same method of preparation of mixed HA collagen nanofibres. They used DMF as the solvent, which is inappropriate for eventual further use of the material in medicine. They use NaCl for stabilizing the structure, however the fluffy structure disintegrates due to washing out the salt and the fibres stick together, therefore the lyophiusation was included. However the lyophilisation is very expensive, so the suggested method is inappropriate for commercial use.

Crosslinking of hyaluronic acid can be performed by many known methods used for example for forming hydrogels and scaffolds. These reactions often have the drawback of the need of dissolving HA or the course of crosslinking reaction in aqueous environment, as is the case of hydrochloride l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), because the HA fibres dissolve immediately after coming into contact with moisture. Other commonly used crosslinking agents include for example formaldehyde, glutaraldehyde, carbodiimides, and genipine (Zhang, 2011), but most of these substances are inappropriate for the use in medical materials, and, as it has been stated, the cross-linking of voluminous layers by the method needing direct contact with crosslinking solution destroys the desired voluminous structure. Within the scope of (Ji, 2006) the use of thiol derivative of HA, more specifically the HA modified with 3,3'-mf obis(propanomd ydrazide), abbreviated as (HADTPH), was investigated; the HADTPH was synthetized from HA sodium salt (1500 kDa) and spun electrostatically into 3D nanofibre scaffolds. The spinning of 2 % solution of HADTPH was performed from aqueous solution in the mixture with maximum 2 % PEO (900 kDa), with optimal weight ratio of 1:1 to 4:1. Process productivity was 1,2 mL/h. The 3D scaffolds prepared in this way were then cross-linked by poly(ethylenglykol)-diacrylate (PEGDA) and dried for 24 hours, and PEO was washed out by distilled water for 2 days. Nanofibrous material had to be lyophilised to regain the nanofibre structure. During the electrostatical cross-linking described in (Shu, 2002) a spontaneous uncontrolled crosslinking occurred due to air oxidation of thiols forming disulfide bridges.

Another possibility is described in (Foltran, 2010), where the coating from collagen nanofibres and HA was used on dental implants to improve the osteoblasts differentiation. The application of the nanofibre HA layer was preformed from 1 % aqueous solution of HA (Mw lx 10 ⁶ g mol) with the productivity of 0.3 mL/h, and the cross-linking was performed with the use of EDC solution, which is again an inappropriate method.

The paper (Yue, 2011) describes the preparation of hydrophobic films by surface grafting of hyaluronic acid with polydimethylsiloxane (PDMS) and their capability for cochlear implants. The hydrophobization can bring important inhibition of solubility, but also important decreasing of sorption properties. This can be utilized for example for controlled drug release and for decreasing the rate of drug release by inhibition of swelling. At the same time it can bring the necessity of using inappropriate solvents at spinning.

Many studies of the art (Bolgen, 2007; Zong, 2004; Liu, 2012; Hooker, 1999; Chang, 2012; Johns, 2001; Kim, 2004; Pados, 2010; Schnuringer, 2011; Wallwiener, 2006) assess the efficiency of the existing materials used for the prevention of postsurgical adhesions, mainly in abdomen, or for the fillings of various defects, e.g. fistules, in the form of both hquid and solid medical devices, as limited. One of the possibilities of enhancing the properties of anti- adhesion materials based on the principle of physical barriers, that appear to be more effective (Pados, 2010; Schnuringer, 2011), is the use of nanofibrous materials. In this context, nanofibre antiadhesive membranes from various polymers have been already developed, e.g. from polycaprolactone (PCL) spun from chloroform and DMF (B5lgen, 2007; Dinarvand, 2012), PES from DMF, PLLA from DMF and chloroform (Zong, 2004; Dinarvand, 2012), PLGA from DMF from or from the mixture of solvents DMF/THF (Zong, 2004; Lee, 2009; Dinarvand, 2012), PCL spun from THF containing aqueous HA microdispersion (Liu, 2012), nylon 6 spun from formic acid containing silver nanoparticles (Park, 2009), PVDF fromN,N- dimethylacetarnide (DMAc), copolymer PEG and PLLGA from chloroform and DMF (Ma, 2012), copolymer PLA-PEG (Yang, 2009), mixture PLGAPEG-PLA (Zong, 2004) and mixture of alginate and chitosane prepared with the use of glycerol, acetic acid and alcohol (Yeo, 2006). The paper (Chang, 2012) describes mixed nanofibres chitosan/algkiate as anti- adhesion membrane. Almost all the cited solvents systems are inappropriate for the use in medical devices not only in light of application, but also in light of production and toxic waste disposal.

HA based anti-adhesion materials for the prevention of postsurgical adhesion has been already used and some of them have been approved by FDA for human use (Hooker, 19 9). They have the disadvantage of fast degradation and quick assimilation (Chang, 2012). On the other hand, the synthetic materials, as is PCL, with very slow degradation are inappropriate as well because their residues can cause inflammatory reactions leading to more adhesions (Bolgen, 2007; Wallwiener, 2006).

Brief description of the drawings

Fig. 1 - nanofibre pad.

Fig. 2 - nanofibre pad weld formed by induction welding.

Fig. 3 - ultrasonic welding of one layer (NJ_20130618__02).

Fig. 4 - ultrasonic welding of more layers (NJ_20130131_02).

Fig. 5 - ultrasonic welding of more layers (NJ_20130131_02).

Fig. 6 - pressure welding.

Fig. 7 - pressure welding - patterning by matrix printing.

Fig. 8 - change of the nanofibre layer into a gel owing to sorption.

Fig. 9 - dependence of absorbability of HA (3-furfuryl-acroyl-hyaluronane) derivative on surface weight.

Fig. 10 - nanofibres from 10 % solution HA/PEO (90/10), Mw HA 15 x 10 ³ g/mol, Mw PEO 6 x 10 ^s g/moL Collecting electrode - needles, humidity 32.6 % RH, temperature 22 °C.

Fig. 11 - nanofibres from 20% solution HA PEO (80/20), Mw HA 15 x 10 ³ g/mol, Mw PEO 9 x 10 ^s g/moL Collecting electrode - wire, humidity 36,4% RH, temperature 22°C.

Fig. 12 - nanofibres from 10% solution HA/PEO (80/20), Mw HA 39,9 x 10 ³ g/mol, Mw PEO 6 x 10 ⁵ g/moL Collecting electrode - sieve, humidity 16% RH, temperature 22°C.

Fig. 13 - nanofibres from 10% solution HA PEO (80/20), Mw HA 70 x 10 ³ g/moL Mw PEO 6 x 10 ^s g/moL Collecting electrode - sieve, humidity 20% RH, temperature 20°C.

Fig. 14 - nanofibres from 10% solution HA/PEO (80/20), Mw HA 80.4 x 10 ³ g/mol _} Mw PEO 6 x 10 ⁵ g/mol Collecting electrode - sieve, humidity 20% RH, temperature 20°C.

Fig. 15 - nanofibres from 10% solution HAPEO (80/20), Mw HA 80.4 x 10 ³ g/mol, Mw PEO 4 x 10 ⁵ g/mol . Collecting electrode - sieve, humidity 25% RH, temperature 21°C.

Fig. 16 - nanofibres from 10% solution HA/PEO (80/20), Mw HA 80.4 x 10 ⁴ g/mol, Mw PEO 3 x 10 ^s g/moL Collecting electrode - sieve, humidity 25% RH, temperature 21°C.

Fig. 17 - nanofibres from 10% solution HA PEO (80/20), Mw HA 80.4 x 10 ³ g/moL Mw PEO 9 x 10 ⁵ g/moL Collecting electrode- wire, humidity 38.9% RH, temperature 23,8°C. Fig. 18 - nanofibres from 8% solution HA PEO (80/20), Mw HA 13 x 10 ⁴ g/mol, Mw PEO 4 x 10 ^s g/mol. Collecting electrode - wire, humidity 25.3% RH, temperature 22.4°C.

Fig. 19 - nanofibres from 5.33% solution HA/PEO (80/20), Mw HA 25 x 10 ⁴ g/mol, Mw PEO 4 x 10 ⁵ g/mol. Collecting electrode - wire, humidity 25.3% RH, temperature 22.4°C.

Fig. 20 - nanofibres from 4.67% solution HA PEO (80/20), Mw HA 31 x 10 ⁴ g/mol, Mw PEO 4 x 10 ^s g/mol. Collecting electrode - wire, humidity 25.3% RH, temperature 22.4°C.

Fig. 21 - nanofibres from 8% solution HA/PEO (99/1), Mw HA 86.6 x 10 ³ g/mol, Mw PEO 4 x 10 ^s g mol a 4 x 10 ⁶ g/mol mixed in the ratio (1:1). Collecting electrode - sieve, humidity 20% RH, temperature 20°C.

Fig. 22 - nanofibres from 10% solution HA PEO (90/10), Mw HA 92 x 10 ³ g/mol, Mw PEO 6 x 10 ^s g/mol. Collecting electrode - sieve, humidity 23,6% RH, temperature 20.6°C.

Fig. 23 - nanofibres from 6% solution HA/PEO (90/10), Mw HA 92 x 10 ³ g/mol, Mw PEO 6 x 10 ⁵ g/mol. Collecting electrode - sieve, humidity 19.4% RH, temperature 23.2°C.

Fig. 24 - nanofibres from 6% solution HA PEO (30/70), Mw HA 92 x 10 ³ g/mol, Mw PEO 6 x 10 ^s g/moi Collecting electrode - sieve, humidity 23% RH, temperature 22.8°C.

Fig. 25 - nanofibres from 10% solution MHA/PEO (80/20), Mw MHA 1 x 10 ⁵ g/mol, Mw PEO 6 x 10 ⁵ g/moL DS 27%. a) containing dexamethasone, b) corriaining diclofenac. Collecting electrode - needles, humidity 32.8% RH, temperature 22.8°C.

Fig. 26 - nanofibres from 3% solution PHA/PEO (80/20), Mw PHA 25 x 10 ⁴ g/mol, Mw PEO 4 x 10 ⁵ g/mol, DS 48%. a) containig dexamethazone, b) containig diclofenac. Collecting electrode - sieve, humidity 24.6% RH, temperature 22.8°C.

Fig. 27 - nanofibres from 6.25% solution CIHA PEO (80/20), Mw CIHA 116 x 10 ³ g/moL Mw PEO 6 x 10 ⁵ g mol, DS 47%. Collecting electrode - wire, humidity 23,3% RH, temperature 22.8°C.

Fig. 28 - nanofibres from 6% solution HA-TEO/PEO (80/20), Mw HA-TEO 1 x 10 ⁵ g/mol, Mw PEO 6 x 10 ^s g/mol, DS 5%. Collecting electrode - plate, humidity 19.9% RH, temperature 24.2°C.

Fig. 29 - nanofibres from 6% solution HA-TEO/PEO (80/20), Mw HA-TEO 1 x 10 ⁵ g/mol, Mw PEO 6 x 10 ⁵ g/mol, DS 28%. Collecting electrode - plate, humidity 19.9% RH, temperature 24°C. Fig. 30 - nanofibres from 6% solution HA-FU/PEO (80/20), Mw HA-FU 1 x 10 ⁵ g/mol, Mw PEO 6 x 10 ^s g/mol, DS 5%. Collecting electrode - plate, humidity 19.9% RH, temperature 24°C.

Fig. 31 - nanofibres from 6% solution HA-FU/PEO (80/20), Mw HA-FU 1 x 10 ⁵ g/mol, Mw PEO 6 x 10 ⁵ g/mol, DS 20%. Collecting electrode - plate, humidity 18.5% RH, temperature 24°C.

Fig. 32 - nanofibres from 5.56% solution HA-AII/PEO (80/20), Mw HA-AII 96.813 x 10 ³ g/mol, Mw PEO 6 x 10 ^s g/mol, DS 7%. Collecting electrode - sieve, humidity 34.4% RH, temperature 22. PC.

Fig. 33 - nanofibres from 10% solution HA-PY/PEO (80/20), Mw HA-PY 25. lx 10 ³ g/mol, Mw PEO 6 x 10 ^s g/mol, DS 18%. Collecting electrode - plate, humidity 36.4% RH, temperature 22°C.

Fig. 34 - nanofibres from 11 % solution ((HA-CAPA)+(HA-CAPr))/PEO (80/20). Mw HA- CAPA 89.170 x 10 ³ g/moL Mw HA-CAPr 93,910 x 10 ³ g/mol, HA-CAPA and HA-CAPr ratio (1:1), Mw PEO 6 x 10 ^s g/mol, DS 20%. Collecting electrode - plate, humidity 38% RH, temperature 27°C.

Fig. 35 - nanofibres from 6% solution HA PEO (80/20), Mw HA 86.6 x 10 ³ g/mol, Mw PEO 6 x 10 ⁵ g/mol, spun at 15% RH onto needles.

Fig. 36 - nanofibres from 10% solution HA/PEO (80/20), Mw HA 86.6 x 10 ³ g/mol, Mw PEO 6 x 10 ⁵ g/mol, spun at 15% RH onto needles, nanofibre textile section.

Fig. 37 - nanofibres from 10% solution HA/PEO (80/20), Mw HA 86.6 x 10 ³ g/mol, Mw PEO 6 x 10 ⁵ g/mol, spun at 45% RH onto a plate.

Fig. 38 - nanofibres from 6% solution HA PEO (80/20), Mw HA 86.6 x 10 ³ g/mol, Mw PEO 6 x 10 ^s g mol, spun at 15% RH onto a sieve.

Fig. 39 - nanofibres from 6% solution HA/PEO (80/20), Mw HA 86.6 x 10 ³ g/mol ₅ Mw PEO 6 x 10 ^s g/mol, spun at 15% RH onto a plate.

Fig. 40 - nanofibres with iodine generator a) containing NaI0 ₃ , b) containing I. Collecting electrode - sieve, humidity 16% RH, temperature 19.3°C.

Fig. 41 - nanofibres from 8% solution HA/PAA (50/50), Mw HA 15 x 10 ³ g/mol, Mw PAA 45 x 10 ⁴ g/mol. Collecting electrode - wire, humidity 26.5% RH, temperature 22.2°C. Fig. 42 - nanofibres from 8.7% solution HA/PVA (33/67), Mw HA 15 x 10 ³ g/mol, Mw PVA 125 x 10 ³ g/mol. Collecting electrode - wire, humidity 26.5% RH, temperature 22.2°C.

Fig. 43 - influence of Mw HA on solution viscosity.

Fig. 44 - influence of HA content in mixed solution HA PEO on viscosity of the solution. Fig. 45 - encapsulation of additives among nanofibre layers and forming of composite materials with the use of welding.

Subject-matter of the Invention

No research team has succeeded in producing permanently voluminous fluffy nanofibrous materials based on HA by means of electrostatic spinning so far.

The invention is aimed especially at the formation of voluminous nanofibrous structures having a high amount of small interfibre pores providing for excellent sorption properties of the material. The same materials having a higher volumetric weight, i.e. materials which are less fluffy, do not achieve as good sorption properties as the materials of the invention and their sorption decreases with the increasing basis weight of the nanofibrous layer, which does not apply to voluminous materials.

The drawbacks of the prior art are largely removed by the voluminous nanofibrous material according to the invention, the subject-matter of which is that the volumetric weight is within the range of 1 to 100 kg.rn ³ , preferably 1 to 80 kg.rn ³ , more preferably 1 to 50 kg.tn ³ , wherein the absorbability thereof is preferably within the range 0.01 to 100 g of water for 1 g of the dry material, preferably 10 to 100 g or 0.01 to 50 g of the physiological solution for 1 g of the dry material, preferably 10 to 50 g.

Voluminous nanofibrous material according to the invention includes nanofibres which comprise hyaluronic acid or a pharmaceutically acceptable salt thereof or their derivative having at least one functional group selected from the group comprising alkyne, azide, ester, amine, amide, aldehyde, imitie, ether or carboxyl, or a mixture thereof, and further they comprise at least one carrier polymer which is preferably selected from the group comprising polyvinyl alcohol, polyacrylic acid, polyethylene oxide, polyvinyl pyrroHdone.

According to another preferred embodiment of the invention, the voluminous nanofibrous material according to the invention includes nanofibres which comprise hyaluronic acid or a pharmaceutically acceptable salt thereof or their derivative of the general formula I wherein R ¹ are independently OH or an amino group which is -NH- R -alkyne or -NH- R ² -N ₃ or -NH-R ² -heteroaryl, wherein R ² is selected from the group comprising an aliphatic, aromatic, arylaliphatic or heterocyclic group which contains 1-12 carbon atoms,

or an ester group which is -OC(=O)Ci-C ₃₀ alk l or -OC(=0)-C(CH ₃ )=CH ₂ or -OC(=0)CH=CH-R ³ , wherein Ci-Cao alkyl has a linear or branched, saturated or unsaturated chain, where R ³ is an aromatic or heteroaromatic group having at least one or more identical or different heteroatoms selected from the group comprising N, 0, S;

or an aldehydic group which is -CH=0,

provided that at least one R ¹ in the derivative is an amino group, esteric group or aldehydic group; or a combination thereof;

and further, it comprises at least one carrier polymer which is preferably selected from the group comprising polyvinyl alcohol, polyacrylic acid, polyethylene oxide, polyvinyl pyrrolidone.

Preferably, the voluminous nanofibrous material according to the invention comprises an esteric derivative of hyaluronic acid or a pharmaceutically acceptable salt thereof, of the general formula Π

wherein n is an integer within the range of 1 to 5000 dimers,

R ¹ are independently H or -C(=O)Ci-C ₃₀ alkyl or -C(=0)-C(CH ₃ )=CH ₂ or

-C(=0)CH=CH-R ² , wherein C1-C30 alkyl has a linear or branched, saturated or unsaturated chain,

where R ² is an aromatic or heteroaromatic residue having at least or more identical or different heteroatoms selected from the group comprising N, O, S, preferably R ² is selected from the group comprising phenyl, furyl, rurfuryl, thienyL thiophenyl, pyridyl or imidazoyl; provided that at least one R ¹ in the derivative is -C(=O)Ci-C30 alkyl or -C(=0)- -C(CH ₃ )=CH ₂ or -C(=0)CH=CH-R ² ;

R is H* or a pharmaceutically acceptable salt, preferably selected from the group comprising any alkali metal ions, more preferably Na ⁺ , K ⁺ .

The above mentioned esteric derivatives of hyaluronic acid or pharmaceutically acceptable salts thereof, of the general formula II, have the substitution degree (DS) within the range of 1 to 70 %, preferably 1 to 50 %, more preferably 4 to 40 %.

Further, the voluminous nanofibrous material according to the invention may comprise nanofibres containing an amine derivative of hyaluronic acid or its pharmaceutically acceptable salt of the general formula III

wherein n is an integer within the range of 1 to 5000 dimers,

R is H ⁺ or a pharmaceutically acceptable salt preferably selected from the group comprising any alkali metal ions, more preferably Na ⁺ , K ⁺ .

The above mentioned amine derivatives of hyaluronic acid or pharmaceutically acceptable salts thereof of the general formula III have the substitution degree (DS) wili-in the range of 1 to 30 %, preferably 1 to 20 %.

Another variant of the voluminous nanofibrous material according to the invention is the material comprising nanofibres containing an aldehydic derivative of hyaluronic acid or a pharmaceutically acceptable salt thereof, of the general formula IV

where n is an integer within the range of 1 to 5000 dimers;

R is or a pharmaceutically acceptable salt, preferably it is selected from the group including any alkali metal ions, more preferably Na ⁺ , K ^÷ . The above mentioned aldehydic derivatives of hyaluronic acid or pharmaceutically acceptable salts thereof of the general formula IV have the substitution degree (DS) within the range of 1 to 15 %, preferably 1 to 10 %.

A still another variant of the voluminous nanofibrous material according to the invention is the material comprising nanofibres containing a derivative of hyaluronic acid or a

pharmaceutically acceptable salt thereof, of the general formula V, carrying an alkyne group bound via the secondary amino group

and a derivative of hyaluronic acid or a pharmaceutically acceptable salt thereof, of the general formula VI, with an azide group bound via the secondary ammo group:

HA-CAPr wherein n is an integer within the range of 1 to 5000 dimers,

1

R and R are identical or different and include ahphatic, aromatic, arylaliphatic, cycloaliphatic and heterocyclic groups which comprise 1-12 carbons and where R ¹ may represent methyl and R ² may represent 3,6,9-trioxadecane; preferably R ¹ is selected from the group including methyl and phenyl and R ² is selected from the group comprising propyl, phenyl and 3,6,9-tri-oxaundecane

R is H ^1" or a pharmaceutically acceptable salt preferably selected from the group including any alkali metal ions, more preferably Na ⁺ , ⁺ . The above mentioned derivatives of hyaluronic acid or pharmaceutically acceptable salts thereof, of the general formula V and the general formula VI have the substitution degree (DS) within the range of 1 to 15 %, preferably 8 to 15 %.

According to another embodiment the voluminous nanofibrous material according to the invention may comprise at least one adjutant selected from the group carboxymethyl cellulose, gelatin, chitosan, polycaprolactone, polymeric lactic acid, polyamide, polyurethane, poly-(lactid-co-glycolic) acid; a mixture thereof or copolymers thereof.

According to a still another embodiment the voluminous nanofibrous material according to the invention may comprise at least one active substance, preferably selected from the group comprising CaCl ₂ , urea, bee honey, diclophenac, dexamethazone, octenidine, heparine, iodine generator based onNaI0 ₃ and KL

The above mentioned drawbacks of the prior art, i.e. low yields of the electrostatic spinning process due to the processing of HA having high molecular weights which must be diluted to very low concentrations in order to achieve a technologically suitable viscosity, and the necessity of using solvents for the spinning thereof which are unsuitable for use in healthcare, are solved by using aqueous solutions of HA having low molecular weights within the range of 2x10 ³ to 4 xlO ^s g/mol. Such solutions, however, are not able to form fibres by themselves, and here the ability to form fibres is substituted by an addition of a minimal amount of 0.5 to 20 wt.% of a suitable carrier polymer having a high molecular weight, which is at the same time able to decrease the surface tension of the spinning solution.

According to another embodiment of the invention, the content of hyaluronic acid, pharmaceutically acceptable salt thereof or their derivatives is within the range 5 to 99.9 wt.% in the dry matter, preferably 30 to 90 wt.%, more preferably 50 to 90 wt.%, the molecular weight thereof is within the range 2x10 ³ to 4x10 ⁵ g/mol, preferably 15 xlO ³ to 1 xlO ⁵ g/mol. The molecular weight of the carrier polymer is within the range from 2xl0 ³ to 5xl0 ⁶ g/mol. The preferred range of the molecular weight of polyethylene oxide is 3x10 ⁵ to 4x10 ⁶ g mol or the molecular weight range of polyvinyl alcohol is preferably within the range of 6x10 ⁴ to 15xl0 ⁴ g/mol or the molecular weight range of polyvinyl pyrrolidone is preferably within the range of 2x10 ⁴ to 4x10 ⁵ g/mol or the molecular weight range of polyacrylic acid is preferably within the range of 24 xl 0 ⁴ to 50 xl 0 ⁴ g/mol.

Preferably, the diameter of the nano fibres comprised in the voluminous nanofibrous material is within the range of 1 to 1000 nra, preferably 50 to 800 nm, more preferably 80 to 500 nm and it is in the form of a layer. Another embodiment of the invention is the method of production of such voluminous nanofibrous material, as is defined above, the subject-matter of which is that an aqueous spinning solution is prepared, comprising hyaluronic acid, a pharmaceutically acceptable salt thereof or at least one derivative thereof and at least one carrier polymer, which is spun electrostatically in an electrostatic spinning apparatus provided with a spinning electrode and a collecting electrode arranged in the spinning chamber at a relative humidity of 5 to 50 %, preferably 15 to 25 %, wherein the viscosity of the spinning solution is within the range of 0.2 to 25 Pa.s, preferably 0.2 to 10 Pa.s.

This spinning process is carried out preferably at the temperature of 15 to 30 °C, more preferably at 15 to 25 °C.

The collecting electrode has preferably the shape selected from the group comprising a board or a sieve having the thickness of 0.1 to 4 mm, and a wire or a needle having the diameter within the range of 0.01 to 2 mm. The diameter within the range of 0.01 to 2 mm applies to the sieve wire as well. The thickness of the sieve wire can and doesn't have to determine the thickness of the sieve.

The formation of voluminous nanofibrous materials according to the invention is first of ah given by the spinning conditions. The most important of them are the viscosity of the spun solution and the atmospheric conditions. According to the present invention, it is not necessary to use neither any special device, nor any special collecting electrodes. It is possible to either significantly enhance or totally suppress the formation of voluminous nanofibrous materials in the form of layers, by a suitable selection and combination of commonly available collecting electrodes, which allows producing a wide range of products in the same production equipment. Therefore, the advantage is a higher variability of the spinning device, since the same device is able to produce both planar and voluminous materials, depending on the conditions. While narrow collecting electrodes rather enhance the formation of voluminous structures, the wide flat electrodes lead mostly to the formation of compact materials having low volumes. This phenomenon is probably associated with the influence of the so-called electrostatic wind. The method of production according to the invention may be performed within a single operation and in a commonly available spinning device.

Further, it is preferred that the weight ratio of hyaluronic acid, the pharmaceutically acceptable salt thereof or their derivative with respect to the carrier polymer is within the range of 10/90 to 99/1 in the aqueous spinning solution, preferably 80/20 to 99.5/0.5, more preferably 80/20 to 94/6, wherein the molecular weight of hyaluronic acid, the pharmaceutically acceptable salt thereof or their derivative is within the range from 2xl0 ³ to 4x10 ⁵ g/mol, preferably 15x10 ³ to lxl 0 ^s g/mol and the molecular weight of the carrier polymer is within the range from 2x10 ³ to 5x10 ⁶ g/mol. The preferred ranges of the molecular weights of the selected carrier polymers are as defined above.

The concentration of hyaluronic acid, the pharmaceutically acceptable salt thereof or their derivative and the carrier polymer in the aqueous spinning solution is within the range of 0.1 to 60 wt.%, preferably 1 to 50 wt.%, more preferably 5 to 20 wt.%.

The spinning solution may also contain a mixture of water and a water-miscible polar or non-polar solvent selected from the group comprising isopropanol, ethanoL acetone, ethylacetate, dimethyl sulphoxide, acetonitrile, dimethyl formamide, tetrahydrofuran, preferably isopropanol.

The spinning solution may further contain an initiator of crosslinking, preferably (2~hydroxy- 4'-(2-hydroxyethoxy)-2-methylpropio phenone) or l-[4-(2-hydroxyethoxy)phenyl]— 2- hydroxy-2-methyl-l-propan-l-on.

The spinning solution may further contain at least one adjuvant selected from the group comprising carboxymethyl cellulose, gelatin, chitosan, polycaprolactone, polymeric lactic acid, polyamide, polyurethane, poly-(lactid-co-glycolic) acid; and a mixture thereof or copolymers thereof, preferably carboxymethyl cellulose.

The spinning solution may further contain an active substance, preferably selected from the group comprising CaCl ₂ , urea, bee honey, diclophenac, dexamethazone, octenidine, heparine, iodine generator based on NaIC>3 and KI.

Further, according to a preferred embodiment of the method of the invention, it is possible to prepare a voluminous nanofibrous material having a high content of hyaluronic acid, a pharmaceutically acceptable salt thereof or their derivative in nanofibres up to 99.9 wt.% in the dry matter, wherein a mixture of identical or different carrier polymers having various molecular weights is used. Preferably, the spinning is carried out from the spinning solution containing polyethylene oxide having the molecular weight within the range of 3x10 ⁵ to 9x10 ^s g/mol and polyethylene oxide having the molecular weight within the range of lxl 0 ⁶ to 9xl0 ⁶ g/mol. The preferred ratio of both components in the spinning solution is within the range of 9/1 to 1/9, preferably 3/2 to 2/3, the most preferred is 1/1. The high productivity of the present method according to the invention permits an industrial production of voluminous nanofibrous materials and therefore also the preparation of cheaper medical devices designated for coverings of external and internal wounds, having an excellent sorption and for anti-adhesive membranes. If use of voluminous nanofibrous structures in medicine is involved, then it is in a moist environment of the organism, and if immediate dissolution of the material is not desired, it is necessary to modify the materials, e.g. by crosslinking. Crosslinking of the volurninous structures is very important not only for enhancing the stability of the material in a moist environment but also for achieving good sorption abilities.

Crosslinking of biopolymers may be provided by two basic principles. The first is a classical chemical method leading to the formation of a covalent bond. The second variant of crosslinking is based on a physical principle of interacting molecules. The use of energy of light for ensuring the formation of three-dimensional and often randomly arranged structures belongs to the first group and is classified as the so-called photo crosslink, or as photochemical crosslink or photochemical crosslinking. A subgroup of photochemical crosslinking provides several advantages compared to the classical chemical crosslinking method. First of all, it is a time and space control of the reaction proceeding with its high selectivity. It is a method which does not require maceration of the nanofibrous layer in the crosslinking solution, which would be undesirable for the voluminous nanofibrous material according to the invention, when in contact with moisture the pressure of the liquid would compress its voluminous structure.

Another embodiment according to the invention is a method of modification of the voluminous nanofibrous material according to the invention comprising nanofibres containing an acrylic derivative of hyaluronic acid or a pharmaceutically acceptable derivative thereof of the general formula II defined above and/or an amine derivative of hyaluronic acid or a pharmaceutically acceptable derivative thereof of the general formula ΙΠ defined above and/or an aldehydic derivative of hyaluronic acid or a pharmaceutically acceptable derivative thereof of the general formula IV above, or a mixture thereof, wherein the crosslinking is carried out by light radiation within the range of UV-Vis wavelengths. Another preferred embodiment is the method of modification of the voluminous nanofibrous material according to the invention, wherein the nanofibres are crosslinked by light radiation within the range UV-Vis wavelengths of 280 nm to 750 nm, preferably 302 ran. Preferably, the nanofibres contain such HA derivatives carrying photoreactive groups (chromophores), where the crosslinking reaction may be induced after the electrostatic spinning process. By this way, any possible initiation of the crosslinking reaction during the electrostatic spinning and the increase of viscosity associated therewith and the formation of a non-homogenous product is simply avoided. Initiations by heat, UV radiation or microwave radiation are ideal for their simplicity, undemanding and contactless procedure. The resulting modified materials according to the invention exhibit different mechanical properties, different viscosity, different solubility and an enhanced stability in an aqueous environment, compared to the initial polymers. Said properties directly depend on the radiation intensity, energetic dose of radiation, substitution degree (DS) of the respective photoreactive group, mutual intermolecular distance of the photoreactive groups, rigidity of the modified biopolymer, concentration of the respective reagents and the extent of the achieved crosslinking.

It is also possible to crosslink HA acylated by methacrylic acid anhydride photochemically. If a suitable photoinitiator is selected, UVB radiation may be used for initiation of the reaction.

It is also preferred that the modification by crosslinking is performed for 2 minutes to 60 minutes, more preferably 3 minutes to 10 minutes.

Another preferred embodiment is a method of modification of the voluminous nanofibrous material according to the invention, wherein the nanofibres comprising amine derivatives of hyaluronic acid or their pharmaceutically acceptable salts of the general formulae V and VI according to the invention are crosslinked by treating with heat, preferably 40 to 80 °C, more preferably 50 to 70 °C, the most preferred is 60 °C, or by microwave radiation.

In case of modification of the voluminous nanofibrous material according to the invention comprising nanofibres which contain an acryloyl derivative of hyaluronic acid or a pharmaceutically acceptable salt thereof of the general formula II, where at least one R ¹ in the derivative is -C(=0)-C(CH3)=CH ₂ or -C(=0)CH=CH-R ² , as defined above for the acryloyl derivative, the crosslinking brings about the formation of the compound having a cyclobutane ring of the general formula VII

(VII), where

R ² is H or an aromatic or heteroaromatic residue having at least one or more identical or different heteroatoms selected from the group comprising N, 0, S, preferably R ² is selected from the group comprising phenyl, furyl, furfuryl, thienyl, thiophenyl, pyridyl or imidazoyl and

R ^s is the main chain of hyaluronic acid or the pharmaceutically acceptable salt thereof, and the carrier polymer as defined below.

Moreover, in case of modification of the voluminous nanofibrous material according to the invention comprising nanofibres which contain an amine derivative of hyaluronic acid or a pharmaceutically acceptable salt thereof of the general formula III defined above, the crosslinking brings about the formation of a crosslinked 3D amine derivative of the general formula VIII

where R ⁵ is the main chain of hyaluronic acid or the pharmaceutically acceptable salt thereof, the carrier polymer is as defined below.

In case of modification of the voluminous nanofibrous material according to the invention comprising nanofibres which contain an aldehydic derivative of hyaluronic acid or a pharmaceutically acceptable salt thereof of the general formula IV defined above, the crosslinking brings about the formation of a crosslinked aldehydic derivative of the general formula DC

where n is an integer within the range of 1 to 5000 dimers, R is H* or a pharmaceutically acceptable salt selected from the group comprising any alkali metal ions, more preferably Na ⁺ , K ⁺

and the carrier polymer as defined below.

Moreover, in case of modification of the voluminous nanofibrous material according to the invention comprising nanofibres which contain amine derivatives of hyaluronic acid or a pharmaceutically acceptable salt thereof of the general formulae V and VI defined above, which are preferably crosslinked by treatment by heat of microwave radiation, a crosslinked derivative of hyaluronic acid or a pharmaceutically acceptable salt thereof of the general formula X is formed

where R ¹ , R ^z are as defined above for amine derivatives of hyaluronic acid or pharmaceutically acceptable salts thereof of the general formulae V and VT above,

R ⁵ is the main chain of hyaluronic acid or a pharmaceutically acceptable salt thereof, and at least one carrier polymer as defined below.

The modified voluminous nanofibrous material according to the invention may also contain mixtures of the above mentioned crosslinked derivatives of hyaluronic acid or pharmaceutically acceptable salts thereof.

Another preferred embodiment according to the invention is a nanofibrous structure containing voluminous nanofibrous material or modified voluminous nanofibrous material according to the invention, as defined above, wherein it has at least one weld. Reinforcing or bonding of nanofibrous materials by means of a weld has not been described yet as well. It is interesting that the classical forms of materials made of HA, such as common fibres or foils, cannot be bound by welding. Welding may also be used for forming reinforced edges facilitating the manipulation or for areal or local compacting of nanofibrous materials. According to another embodiment, the nanofibrous structure according to the invention comprises at least two layers of the nanofibrous material bound by a weld, preferably one of the layers contains NaI0 ₃ and the other layer contains KI, wherein the layers are located one on the other or are separated by at least one layer of the nanofibrous material according to the invention.

According to yet another embodiment, the nanofibrous structure according to the invention is preferably in the form of a pad having a weld along the circumference of the nanofibrous material. Such a pad having reinforced edges is easy manipulated with, which is very important especially for surgeons. The nanofibrous materials have not only an excellent sorption, but also a good adhesion to moist surfaces. Therefore, they may adhere to moist surfaces including moist surgical gloves. This problem has been solved by forming a weld (Fig. 1, Fig. 2), Le. a compact edge thanks to which the airy material has the desired shape and which facilitates the manipulation significantly. As mentioned above, the voluminous nanofibrous material according to the invention has excellent sorption and retention abilities and moreover, it is fully biodegradable, which allows the application of the material not only on surface, but above all, also inside the body.

Thanks to the excellent sorption, the nanofibrous materials according to the invention can prevent spreading of the moisture by means of common transport mechanisms by transformation to a gel (Fig. 3). That is beneficial e.g. for covering of chronic wounds where it is necessary to provide for draining the exudate away from the wound, at the same time for moistening the wound, but at the same time also for having a dry covering of the wound edges so that the wound does not spread by maceration of said edges and irritation thereof.

Further, it is preferred that the nanofibrous structure according to the invention as described above further contains a layer made of viscose and/or of at least one fusible polymer, preferably selected from the group comprising polyethylene, polypropylene, polyester, polyamide, polylactid acid.

Even more preferably, such materials may be selected from the group comprising polypropylene spunbond, which is a non-woven textile made by spunbond technology, a textile containing 70% of viscose and 30% of polyester (TA 2678), perforated foil made of low-density polyethylene (PE), polyamide textile, textile containing a mixture of polyamide and polyester and a mixture of polyamide, polyester and polylactic acid in the ratio of 70:30 (PA + PES, PA + EL (70; 30)), 100% polyester (PES), textile containing polyamide and polylactic acid in the ratio of 81 : 19 (PA + EL (81 : 19)). Welding may also be used for producing composite materials, into which it is possible to enclose materials or fillings which cannot be spun or for the spinning of which it is necessary to use toxic solvents and the like. Such substances may be locked by means of welding between two voluminous nanofibrous layers according to the invention, e.g. in a composite such as a„tea bag". The filling may be preferably chitin/chitosan - glucane complex or schizophyllan. Thanks to the small size of the interfibre pores, the nanofibrous layer disallows the filling to be dusted off, even though the particle size thereof is small. For a filling according to the invention, also a foil or a textile may be used.

The weld of a nanofibrous structure is formed in a way that a pressure within the range of 0.2 to 0.4 MPa or a temperature within the range of 5 to 80 °C or a combination thereof is applied to the desired location of the nanofibrous material. Preferably, the weld is formed by means of a stamping die or a press.

The possibility of forming a weld is quite surprising because microfibrous materials of the same composition cannot be bound by this way. Moreover, materials based on HA do not belong to fusible materials. Adhesion of a deposited nanofibrous layer on a support textile may be improved by a similar method, i.e. by heat bonding. Heat bonding of nanofibrous materials e.g. based on HA, which cannot normally be bound, has not been disclosed anywhere so far. Another possibility of use is patterning of both areal compact and voluminous nanofibrous materials and layers according to the invention by means of a patterned master which is used for welding. By means of said master, it is possible to obtain locally compacted spots of nanofibrous materials made of hyaluronic acid, salts thereof or derivatives thereof in a desired pattern for use in medical devices, especially in coverings of both internal and external wounds, antiadhesive materials and tampons, but also as carriers of drugs or in cosmetics.

The nanofibrous material or the nanofibrous structure according to the invention may be used in cosmetics or medicine. Preferably, they may be used for the production of sorption materials, especially of wound coverings, tampons, scaffolds or antiadhesive materials or as carriers of drugs or as a material for tissue engineering.

These voluminous nanofibrous materials according to the invention are characterized by a large specific surface and pores which are big enough for enabling a free migration of cells through the material. Therefore, they may be easily used for scaffolds for which it is essential especially to form a porous structure where big pores must be achieved for seeding of cells. Another advantage of the nanofibrous material according to the invention is the excellent sorption properties thereof thanks to the high porosity, whereby they provide a great volume of interfibre pores. This allows massive swelling, and thus sorption and retention of high amount of Hquid, as well as an easy incorporation of adjuvants or active substances and their immediate release. They are also significantly more flexible and they may copy the sha e and the surface of a tissue defect.

The voluminous nanofibrous materials made of HA, salts thereof or their derivatives according to the invention may be used for prevention of post-surgical adhesions especially in the abdominal cavity, such as coverings of internal wounds or for fillings of various defects, such as fistulae. The effectivity of HA in case of antiadhesive materials is due to its ability to lubricate the cells, mamtaining the structural integrity of tissues, regulation of Hquid retention, stimulation of mesothelium regeneration. For this type of application, the modified nanofibrous materials according to the invention having a better stability and slower degradation, optionally mixtures thereof with native HA, may be very suitable.

Such materials fulfill the demanding criteria for the production of medical devices leading to a lower both ecological and economical burden of an eventual industrial production, especially thanks to the use of non-toxic chemicals. The undesired post-surgical adhesions are involved in up to 90% of actions in abdominal cavity and they cause serious comphcations. A higher reactivity of the nanofibrous materials given by the small size of the fibres and the big specific surface, together with the possibility of a very fast release of the incorporated drugs appears to be optimal in the aspect of healing of internal wounds and prevention of adhesions. The sorption properties, a pleasant texture of the nanofibrous material according to the invention and the flexibility are also very beneficial for the prevention of adhesions.

Definitions:

The term„material based on hyaluronic acid" means a material comprising nano fibres containing hyaluronic acid and/or a pharmaceutically acceptable salt thereof, and/or their derivative; the volumetric weight of which is within the range of 1 kg/m ^"3 to 100 kg/ m ^"3 .

The term„derivative of hyaluronic acid" means an ester derivative of HA, amine derivative of HA or an aldehydic derivative of HA, or pharmaceutically acceptable salts thereof.

The term„fUnctional group" means a primary or secondary OH group of hyaluronic acid or of a pharmaceutically acceptable salt thereof, substituted by a group selected from the group comprising alkyne, azide, ester, amine, amide, aldehyde, imine, ether or carboxyl. The term „ester group" means or -OC(=0)CH=CH-R, wherein C1-C30 alkyl has a linear or branched, saturated or unsaturated chain, where R is an aromatic or heteroaromatic group having at least one or more identical or different heteroatoms selected from the group comprising N, 0, S.

The term„ amino group" means - H-R-alkyne or - H-R-N3 or - H-R-heteroaryL, wherein R is selected from the group comprising aliphatic, aromatic, arylaliphatic and heterocyclic groups, as described below.

The term„aldehydic group" means -CH=0.

The term„aliphatic group" means C1-C12 alkane, C2-C12 alkene or C2-C12 aikyne having a linear or branched, saturated or unsaturated chain.

The term„aromatic group or aryl" means a 5 to 12-membered aryl, preferably 5 or 6- membered aryl.

The term„heteroaromatic group or heteroaryl" means a 5 to 12-membered heteroaryl, preferably 5 or 6-membered heteroaryl having at least one or more identical or different heteroatoms selected from the group comprising N, O, S, preferably selected from the group furan or thiophene.

The term„arylaliphatic group" means a group containing a 5 to 12-membered aryl, preferably 5 or 6-membered aryl, as described above, bonded to an aliphatic group described above.

The term„cycloaliphatic group" means a cyclic 3 to 12-membered aliphatic group, as described above.

The term heterocyclic group" means a 5 to 12-membered heterocycle having at least one or more identical or different heteroatoms selected from the group comprising N, 0, S.

The term„iodine generator" means that iodine is generated by a reaction of NaI0 ₃ and I with an addition of acid initiators, said two components are contained in two separate nanofibrous layers and only upon the contact with moisture they are gradually released and iodine is generated.

The term„carrier polymer" means a fibre- fonning polymer having a long chain, the addition of which aUows/facilitates spinning.

The term "spinning electrode" or emitor is an electrode which is in direct contact with the spinning solution. It may be in the form of a nozzle, e.g. a needle-free multinozzle. The term„spinning solution" means a solution, melt or dispersion of the spun polymer.

The term collecting electrode" or collector, is an electrode designated for capturing the forming nanofibrous structures. It may be in the form of a board, sieve, or a wire or a needle.

The term collecting electrode in the form of a sieve" means a collector consisting of a frame and a sieve, the diameter of the sieve wire is within the range of 0.01 to 2 mm.

The term substitution degree" (DS) represents the ratio of the molar amount of the bound substitute with respect to the molar amount of all polysaccharide dimers and it is cited in per cents.

The term„antiadhesive membrane" - biodegradable material which prevents the physical contact of tissues , the adhesion/coales cence of which is undesirable.

The term„layer" mentioned in the text in connection with the voluminous nanofibrous material means a layer of the voluminous nanofibrous material which is formed on the collecting electrode after spinning the polymer.

The term„crosslinking initiator" means a specific chemical compound which initiates the crosslinking reaction of methacryloyl 1^(MHA).

The term„aqueous spinning solution" means a solution comprising hyaluronic acid, a pharmaceutically acceptable salt thereof or at least one of their derivatives and at least one carrier polymer and, as a solvent, water or a mixture of water and a water-miscible polar or non-polar solvent.

The term ₅ fusible polymer" means a polymer, the most preferred is a thermoplast, which is able to convert from the solid state to the liquid state, the most preferably repeatedly, owing to temperature changes within the temperature range of 110 °C to 190 °C.

The term„filling" means any substance which may be enclosed between two nanofibrous layers of the voluminous material according to the invention. The substance must be suitable for cosmetic or medical use and is preferably in solid state, more preferably in the form of a powder, granules, paste, foil, textile.

Preferred embodiments of the invention

Example 1 - Production of voluminous materials from native HA: Volume weight of voluminous fluffy nanofibre samples reaches only of about 1 to 100 kg.m ^"3 , whilst the compact samples have the volume weight of about 200 to 500 kg.m ^'3 . The preparation of voluminous nanofibrous materials lies mainly in proper choice of parameters of the solution being spun and in the welding conditions.

Native HA of various molecular weights according to the Table 1 was spun from spinning solution together with the carrier gas, Le. PEO (polyethylenoxide). The spinning solution was prepared by mixing dry HA and dry PEO in particular ratio (e.g. 8/2 for the HA content of 80% in the dry matter), followed by their dissolution in water to the corresponding concentration. The solution concentration means the content of the polymer rnixture HA PEO in the water solution. The viscosity values in the Table 1 below correspond to the spinning solution viscosity.

Table 1 - Processing of HA of different molecular weights

(Mw HA denotes molecular weight of hyaluronic acid, Mw PEO denotes molecular weight of polyethylenoxide) :

The performance was measured by weighing the produced dry nanofibrous material and expressing the weight during the time, i.e. [g/h]. The fibre diameter was detennmed by image analysis of the images of nanofibre voluminous layers obtained by scarming electron microscopy (Fig. 10 to 20). It is obvious from Table 1 that the highest productivity was reached by using low molecular HA; low molecular HA is preferred also for the formation of voluminous nanofibre layers, because the low viscosity of solutions in combination with the spinning of HA of low Mw promote the forrning of voluminous layers. Besides PEO other carrier polymers can be used, for example PVA or PAA (see Table 42 - Fig. 42).

Table 2 - Other carrier polymers

The spinning was performed by the electrostatic spinning method from the multi nozzle E4 without a needle on the device 4 Spin® from Contipro Biotech Company, onto a collecting electrode, electrical voltage 60 kV, electrodes distance 20 cm, dose speed 80 to 120 μΐ min. Table 3 shows the comparison of the properties of chosen voluminous materials depending on the process conditions and spinning solution properties.

Table 3 - Effect of parameters of a spinning solution and spinning conditions to volume weight of the product

Spinning solutions of different concentrations of polymer rnixture HA PEO were prepared, (Mw HA 86, 6 x 10 ³ g/mol) and PEO (Mw PEO 6 x 10 ⁵ g/mol) mixed in the ratio of 8/2, as stated in the Table 3 above, and then they were spun onto above mentioned collecting electrodes. Thickness of the collecting stainless plate electrode was 1 mm. Thickness of the collecting sieve electrode from stainless wires is 0.1 mm, wire diameter 38 μιη, mesh diameter 78 μπι. The compared voluminous nanofibrous materials are showed in the Fig. 35 to 39 obtained by scanning electron microscopy. Spinning of the 3 ^rd sample was performed at RH 45%. It was revealed that a compact material was formed, with volume weight 4 times higher than it is at voluminous fluffy materials. The volume weight was determined from the thickness of a layered shape, which is formed by layering the individual layers of voluminous material, and from its surface weight. The thickness of layered shape was measured with the use of thick meter 318-221 A Mitutoyo Litematic VL-50A. Fig. 35 shows the section of layered material; unfortunately the voluminous fluffy structure deforms during cutting and its thickness highly reduces. Low viscosity of the spinning solution and low humidity in the spinning chamber affect the formation of voluminous materials in the most important way. The viscosity of starting spinning solution can be influenced for example by mM of the HA used (see Fig. 33) or by the proportion of both the components of the solution, i.e. by HA content in the mixture (see Fig. 34). For the formation of voluminous structures, it is preferred to use a spinning solution with high content of low molecular HA.

The shape of a collecting electrode influences the preparation of voluminous nanofibrous materials in a certain manner, but the volurninous materials can be prepared also on conventional collecting electrodes. The electrodes used are usually thin, as are for example wires, needles etc. In the field with the electrodes so thin, the nanofibres tend to fly from each other in the spmning chamber area.

Example 2 - Preparation of nanofibrous materials with high content of HA

Despite the low molecular HA itself cannot be spun, the spirming is enabled by adding a small amount of carrier spinnable polymer. As the adding of a carrier polymer can be inappropriate for the final application, it is possible to minimize its content up to 1 % weight only.

Nanofibres with high content of HA of 90 to 99 % weight from aqueous solutions (see Table 4) were prepared by the same method as was described in Example 1, on the device 4Spin® from Contipro Biotech Company, from a multi nozzle without a needle E4 onto the collecting mesh electrode, electrical voltage 60 kV, electrodes distance 20 cm, dose speed 80 to 120 μΐ/min. HA 4x 10 ^s g/mol and 4x 10 ⁶ g/mol ratio was 1:1.

Table 4

The productivity was determined by weighing the dry nanofibrous material and expressing the weight during the time, i.e. [g h]. The fibre diameter was determined by image analysis of the images of nanofibre vobminous layers obtained by scanning electron microscopy (Fig. 21 to 23).

Voluminous materials with low content of HA (Table 5, Fig. 24) can be prepared by this method as well.

Table 5

Example 3 - Production of voluminous materials from HA derivatives

Volirmrnous materials containing HA derivatives were prepared by the same method as was described in Example 1 (see Table 6). They were prepared also on the device from Contipro Biotech Company, from a multinozzle without a needle E4 onto the collecting mesh electrode, electrical voltage 60 kV, electrodes distance 20 cm, dose speed 80 to 120 μΐ/min.

Spinning solutions of hydrophobized HA derivatives, for example palmitoyl HA, were prepared with the use of mixed solvent water/isopropylalcohol(IPQ) in the ratio 1:1, i.e. 50 % ofJPA.

The productivity was also determined by weighing the formed dry nanofibrous material and expressing the weight during the time, i.e. [g h]. The fibre diameter was determined by image analysis of the images of nanofibre layers obtained by scanning electron microscopy (Fig. 25 to 34).

Table 6 - Preparation of voluminous nanofibre layers from HA derivatives

Sodium hyaluronane unsaturated aldehyde p-D-GlcA-(l→3)^-D-.d ⁴ ^-6-oxo-GlcNAc-(l was used as HA- AIL

Hyaluronate azidykroine derivative, namely polyfsoo^um- -D-glucuronate-tl-Sl-p-N-acetyl- 6-N-ll-azido-3,6,9-trioxaundecanammy^ was used as HA-CAPA, and sodium hyaluronate propargylamine derivative was used as HA-CAPr, namely poly(sodium-

These derivatives were prepared by HA oxidation mediated by 4-Ac-TEMPO at the presence of sodium hypochlorite and sodium bromide at pH = 9, followed by adding primary amines and reduction by picoline borane at pH = 5 leading to click chemistry substrates. Both the olymers were spun together.

Example 4 - Cross-linking

The chosen voluminous nanofibrous materials described in Example 3 were cross-linked. MHA was spun together with an initiator of cross-hiking reaction; the initiator was (2- hydroxy-4 -(2-hydroxyethoxy)-2-methylpropiophenone) in the amount of 10 weight % of the dry matter. As this compound is not water soluble, in the case of spinning the materials based on methacroyl HA, where the aim was to cross-link the materials, it was necessary to add a necessary amount of isopropylalcohol (IPA) besides the initiator, and the solvent was the mixture water IPA in the ratio of 9/1. The spinning was performed in a standard manner described in previous examples. The cross-linking of voluminous nanofibrous materials prepared from CIHA, HA-TEO, HA-FU, HA-AII, HA-PY and MHA with the content of initiator, as they are described above, was induced by UV radiation. The cross-linking was performed in the UV reactor named UV Crosslmker CL-1000M (302 nm) from Eppendorf Czech&Slovakia Company. This device ensures homogeneous UV radiation in UVB spectrum range (280 nm - 315 nm) with continual power of about 6,75 mW.cm ^"2 , where the maximum value of relative radiation energy is declared at the wave length of 302 nm. The cross-linking was performed for 5 to 60 minutes at 302 nm.

The nanofibres containing the particular derivatives HA-CAPA and HA-CAPr were thermally cross-linked together. They were put in hot air sterilizer Stericell 222 and maintained at 60 °C for 20 hours. The cross-linking can be also performed with the use of micro waves at the power of 1200 W for 30 mm.

Example 5 - Absorbability of materials

Testing of absorption properties of nanofibre voluminous materials described above were performed by the gravimetric method. All the methods are based on deteitaination of the weight of a hquid (absorption medium) which is absorbed by the nanofibres during particular period of time. The materials were measured by the method described in (British Pharmacopoeia, 1995), this method is also known as Free Swell Method. In this method, 0.5 g of chosen voluminous nanofibrous material prepared according to Examples 1 to 3 was removed and immersed into 30 ml of liquid (water or 0.9 % NaCl) where it was kept macerating for 5 minutes. Then it was filtered trough the sintered glass funnel with pores of the size of 100 - 160 μηι. Filtration was performed for 5 minutes. The liquid having passed through the filter was weighted and the weight of the liquid absorbed in fibres was calculated by subtracting it from the starting amount. All the procedure was performed at 23.5 ± 0.5 °C and relative air humidity of 33 ± 2 %.

It is obvious from Fig. 9 that the higher is the surface weight of a nanofibre layer prepared by the standard method, the lower is the absorbability of this compact structures. Therefore it is preferred to prepare the fluffy layers of voluminous materials which are able to absorb large amount of liquid thanks to high number and high volume of inter-fibrous pores and they compensate this decrease (see Table 7). The difference between the values for voluminous and compact samples, with surface weight of about 100 g.m ^"2 , is not so important, as the layering and then the welding of individual compact layers of lower weights are necessary to gain so high weight. However there always remains space between the layers for water retention.

Table 7 - Comparing of various types of materials and their absorbability

Example 6 - Welding/Patterning

Pressure welding, thermal welding or their combination can be used for creating strengthened edges of voluminous nanofibrous materials (Fig. 1 and Fig. 2), they can also serve for creating the fixed joint of two or more nanofibre layers or their combination with other materials. This method can be used also for patterning (Fig. 3 and Fig. 7); the materials with locally compressed structure can be obtained, having different physical properties. The pattern of a material depends on a stamping die and its print. The compression of nanofibre layers structure can be made also surfacialy by conventional pressing. Dissolving and swelling can be importantly suppressed and the rate of release of additives from the nanofibre structure can be decreased by using this technique.

Welding can be performed mainly with the use of presser induced pressure (Fig. 6) and its appropriate combination with heating, induced for example by induction (Induction welding machines - Fig. 1 and Fig. 2), or ultrasound (Ultrasound welding machines - Fig. 4 and Fig. 5).

The welds were created on different matrices. The welds were performed for 5 to 30 s, at the pressure of 0.2 to 0.4 MPa. The patterning is illustrated for example in Fig. 7 and it was performed by printing the patterned matrice at the pressure of 0.2 MPa for 10 s. volummous nanofibrous materials with the content of native FIA/PEO prepared according to Example 1 were used.

A material of more layers consisting of nanofibre layer from native HA/PEO on support polypropylene (PP) non-woven textile made by spun-bond method was prepared. The welding was performed with the use of patterned stamping die, in this case it was warmed at the temperature of 150 °C for 3 s at the pressure of 0.2 to 0.4 MPa. The warmed stamping die was placed on the PP side of the textile to minim alize eventual damage of HA PEO layer caused by heat. Example 7

Voluminous samples containing active substance

Electrostatic spinning is an effective method to prepare additives containing nanofibres, the method consist in adding the additives into the starting spinning solution (see Table 8; Fig 25, 26 and 40).

Table 8

Native HA or its derivatives, PEO, and active substances showed in Table 8 were used for preparing the spinning solution. The spinning was performed according to the description in Example 1.

Example 8 - Preparation of composite materials by welding

Between two nanoSbre layers of volurninous material from native HA PEO prepared according to Example 1, schizophylane in powder form was applied using the conventional methods, see Fig. 45, the created area was smaller than the welding area to prevent inhibition of welding due to presence of a fill between two welded layers. Two outside nanofibre layers are then weld according to the method described in Example 6.