CZ309644B6 - Biocompatible and biodegradable non-woven fibre structure containing submicron fibres based on silicon dioxide, biogenic ions and a functional surface for binding active substances and producing it - Google Patents

Abstract

The solution concerns a biocompatible and biodegradable nonwoven fibre structure with submicron silica-based fibres, biogenic ions and a functional surface for binding active substances. The fibres are created by spinning a sol prepared by the sol-gel method and have directly in their volume during spinning bioactive ions from the group of calcium and/or copper and/or zinc and/or silver ions, and the surface of the fibres is functionalized with aminoalkyl alkoxy silane or epoxy alkyl alkoxy silane.The solution further relates to the method of producing the biocompatible and biodegradable nonwoven fibre structure, in which the sol prepared by the sol-gel method is spun. Prior to spinning, the salt is synthesized from a low-molecular-weight precursor of tetra-alkoxysilane by the sol-gel method in alcohol with the addition of water under acid catalysis, this salt is doped with bioactive ions in the form of salts during its preparation before spinning, and after spinning, the surface of the fibres is functionalized with aminoalkyl alkoxy silane or epoxy alkyl alkoxy silane.

Description

Biocompatible and biodegradable non-woven fiber structure containing submicron fibers based on silicon dioxide, biogenic ions and a functional surface for the binding of active substances and its production method

Field of technology

The invention relates to a stable and at the same time biocompatible and biodegradable non-woven fiber structure containing submicron fibers based on silicon dioxide doped with bioactive ions or their mixture with a surface bearing functional groups modifying the surface properties of the fibers and enabling the possible binding of active substances to this surface.

The invention also relates to a method of producing these non-woven fiber structures.

Current state of the art

Fiber structures containing submicron fibers and nanofibers represent structures with high application potential in medicine and biotechnologies. For the field of medicine, submicron fibrous structures are particularly useful as carriers for tissue engineering of various tissues and also as a wound cover. Here, the similarity of artificially prepared fibrous structures with naturally occurring structures of extracellular matter (ECM) and/or the membrane effect is used, which stimulates the wound environment, promotes so-called moist healing and at the same time enables gas exchange between the wound and the external environment. To achieve this effect, several conditions must be met, the most essential of which is maintaining sufficient porosity in a humid environment.

The healing of damaged or the growth of completely new tissue can also be supported by the presence of signaling and bioactive and biogenic molecules and ions that support cell migration, differentiation or stimulation of the formation of selected ECM components. At the same time, these substances can also be used to decontaminate an infected wound or prevent the occurrence of postoperative infections. Such biogenic substances include, for example, silicon, the presence of which in low concentrations promotes healing and stimulates collagen production. Furthermore, the effect of silver and copper ions as substances with a high antimicrobial effect was described.

In biotechnological applications as well as in medical applications, the conjugation of active molecules to various substrates is used in order to improve their stability, reduce the load of the given reaction environment with free active molecules and extend their effective time when applied in situ. Here it is possible to use a combination of bioactive ions and, for example, conjugated enzymes, whose catalytic activity can be directly dependent on or supported by the presence and gradual release of selected bioactive ions. Such an effect has been described, for example, in the case of calcium ions in relation to the activity of the enzyme trypsin or zinc in relation to the activity of digestive enzymes.

In such a case, the subsequent functionalization of the surface leads to a change in the wettability of the surface with an aqueous solution, which subsequently has two effects. On the one hand, it increases the efficiency of the subsequent binding of the active molecule from aqueous buffers and at the same time leads to an improvement in the kinetics of the release of incorporated ions supporting the activity of the bound molecule. This effect can also be supported by the application of physical methods during the production of the fiber cover, which lead to an improvement in the wettability of the surface before the chemical functionalization (grafting of functional groups) and increase the efficiency of this process and the number of functional groups per unit area.

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From CZ PV 2003-2421 A3, respectively from WO 2005024101 A1, the procedure for the production of nanofibers by electrostatic spinning is known, but the polymer solutions for the preparation of the fibers are not specified in more detail.

CZ PV 2012-549 A3 describes a nanofibrous structure with an immobilized organic agent, which is made up of pure silicon nanofibers with a surface modified with an aminoalkoxysilane and subsequently immobilized organic agent. This nanofibrous structure is produced by electrospinning pure silica nanofibers from the starting sol synthesized by the sol-gel method from tetra alkoxysilane, which are subsequently heat-treated, and then the surface of the nanofibers is modified with an aminoalkoxysilane solution, after which organic agents are immobilized on the thus modified surface of the nanofibers .

CZ 34116 U1, deals with the preparation of pure silica nanofibers with grafted functional groups and an immobilized organic agent. In order to increase the bonding efficiency, plasma surface pretreatment and subsequent chemical modification are combined here.

From CZ PV 2015-406 A3, a method for the production of a hybrid nanofibrous structure with immobilized active substances is known, which consists in the preparation of the starting hybrid sol by the sol-gel method, in such a way that the basic reaction mixture containing tetraalkoxysilane is enriched with a proportion of aminopropyltriethoxysilane, after which the thus prepared hybrid sol is subsequently spun, while the further formed nanofibrous structure is thermally stabilized under specific conditions, namely by applying a temperature of up to 200 °C. Finally, the nanofibrous layer with an active surface created in this way is exposed to a solution for immobilizing the selected active substance, where this active substance is bound to the surface of said nanofibrous layer by means of peptide and/or hydrogen bonds. The technical solution also relates to a hybrid nanofibrous structure, created as a nanofibrous spatial structure based on nanofibers activated by -NH2 groups, where an active substance is bound to the surface of the nanofibers, namely by a peptide bond or a bond by hydrogen bridges, while it is preferably a hybrid nanofibrous layer produced by , as mentioned above, where the active substance is preferably a medicinal substance or medicine. However, in the nanofibrous structures prepared in this way, the availability of functional groups available on the surface of the nanofibers for further binding of active agents is limited.

CZ PV 2012-549 A3, CZ 34116 U1, CZ PV 2015-406 A3 do not contain bioactive ions except for silicon, which is their basic structural component, but whose release from the structure is not declared.

In the document WO 2009018104 A1, methyltrimethoxysilane is used as a starting precursor for the preparation of silicon nanofibers. Without heat treatment of nanofibers according to WO 2009018104 A1 or at low temperatures of heat treatment of nanofibers according to WO 2009018104 A1, due to the presence of a methyl group on their surface, these nanofibers have hydrophobic properties and show a low number of Si-OH groups on the surface needed for possible subsequent modification of the surface with aminoalkylalkoxysilane. For these reasons, the solution according to WO 2009018104 A1 is not suitable for the preparation of initial silicon nanofibers for subsequent surface modification and possible immobilization of organic agents.

Documents JP 20040041335 A, JP 20040161234 A and JP 20040243580 A describe the preparation of an organic-inorganic nanofibrous composite composed of a regular skeleton of polyethylene imide fibers with silicon dioxide layers deposited by the sol-gel method. The resulting composite material is intended to capture or concentrate various substances in the prepared structure, but capture of desired particles occurs only as with a filter, i.e. in the gaps between individual nanofibers, or by simple adsorption of desired particles into the volume of the nanofiber composite.

Wrapping paper according to KR 20090058155 A is made of nanofibers obtained by electrospinning a biodegradable organic polymer with the addition of an oxide salt

- 2 CZ 309644 B6 silicon and silver nitrate. The resulting product has antiseptic and antibacterial properties, but does not have a functional surface and is not suitable for immobilizing organic agents.

KR 20100058372 A describes the preparation of a catalyst from mesoporous silica nanofibers prepared by growth from the void phase and subsequent introduction of the catalyst by means of silane onto the surface and into the pores of the thus formed fibers. The resulting product is declared as a catalyst for various organic reactions, but does not contain biogenic ions and does not serve to immobilize organic agents.

Utility model CZ 34086 U1 describes a wound cover with a nanofibrous layer for the transport of drugs, while the active substance is bound here in carrier nanoparticles - liposomes, for which the nanofibrous structure functions as a support system enabling their application into the wound space. Nanofibers prepared by electrostatic spinning of organosilicate can also be used as supporting nanofibers. These fibers are not otherwise surface functionalized. The disadvantage of this solution is the impossibility of binding other active substances.

From the patent document CZ PV 2012-166 A3 and the utility model CZ 31723 U1, fibrous (nano-fibrous and micro-fibrous) biocompatible structures usable for the treatment of wounds and skin defects are known. In the case of CZ 31723 U1, the structure based on the copolymer of L-lactide and ε-caprolactone does not contain functional groups for binding agents on its surface, nor does it contain bioactive ions. In the case of CZ PV 2012-166 A3, the disadvantage of the material based on carboxymethyl cellulose is its low stability in an aqueous environment, the absence of active substances/ions and limited surface functionality.

Solutions containing ions are also known, or solutions representing the conjugation of enzymes on polymer nanofibers.

Fiber dental implants based on bioglass are known from patent document EP 3042628 A1. However, these do not contain surface-functionalized fibers with a submicron diameter, and their structure is primarily based on the content of phosphate components such as tricalcium phosphate and others. From the patent document WO 2007017756 A2, a solution for the preparation of bioglass-based scaffolds is known. However, these are prepared by layering over a polymer template and do not contain submicron fibers. The situation is the same with other bioglasses, even if these contain bioactive ions, their structure contains other components, it is not surface functional and fibrous.

The lack of the current state of the art is the absence of a comprehensive solution that would simultaneously enable the use of the effect of bioactive ions and the possibility of surface immobilization of active substances in sufficient quantities and achieving their synergistic effect.

The aim of this invention is to eliminate or at least minimize the disadvantages of the prior art.

The essence of the invention

The invention is achieved by a siliceous non-woven fiber structure with a fiber diameter in the submicron scale doped with bioactive ions and at the same time surface functionalized in such a way as to achieve a 2D or 3D structure with increased antibacterial activity or other biological activity, and at the same time enabling the application of the non-woven fiber structure in this form or its further use for the conjugation of molecules on their surface in sufficient quantity thanks to the modification of the surface with aminoalkylalkoxysilane or epoxyalkylalkoxysilane. Such a modification leads to an increase in the wettability of the surface and at the same time enables electrostatic bonding, hydrogen bonding or covalent bonding depending on the use of conjugation chemistry, while the effectiveness of the modification can be increased by adding physical activation of the fiber surface by plasma treatment.

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Silica submicron fibers are used in this invention as a supporting substrate due to their biocompatibility, unique method of biodegradation and also due to the wide possibilities of their surface modifications. Silicon dioxide-based nanostructures have been proven not only biocompatible with different types of tissue, but also bioactive due to the release of orthosilicic acid during their degradation. This takes place through the gradual erosion of the surface, when the porosity of the layer does not decrease due to the swelling of the individual fibers. Another indisputable advantage of silica fibers is the high proportion of silanol Si-OH groups on their surface, which can be used to modify their surface through the covalent bond of a functional alkoxysilane. The frequency of the resulting functional groups introduced to the surface is then modified by the reaction conditions and can also be increased by pre-treating the surface of the fibers before the binding itself.

The essence of the production of a non-woven submicrofibrous structure consists in the preparation of a spinning sol. It is prepared by the sol-gel method from tetra-alkoxysilane through acid catalysis in an alcoholic environment, but without the addition of another auxiliary polymer, and during the preparation process this sol is doped with selected bioactive ions in the form of suitable compounds - most often nitrates. The salt prepared in this way is subsequently processed into a non-woven fabric with fiber diameters on a submicron scale. Depending on the parameters of the spinning sol and the spinning conditions, it is also possible to achieve structures with a majority of nanofibers (i.e. fibers with a diameter of <100 nm) in the structure. The production of a fibrous structure can take place both by the method of electrostatic and centrifugal spinning. The created non-woven fiber structure is subsequently thermally stabilized and possibly treated with a suitable type of plasma. This step increases the effectiveness of the surface functionalization with the alkoxysilane, but is not necessary. Subsequently, the surface is modified with the chosen aminoalkyl alkoxy silane or epoxy alkyl alkoxy silane. This achieves a biocompatible and bioactive nonwoven fiber layer with a functional surface available for the binding of the active substance to its surface through the chosen conjugation chemistry.

During the preparation of spinning sol doped with individual bioactive ions (Ca ²⁺ , Zn ²⁺ , Ag ⁺ , Cu ²⁺ ) or their combination, while the spinning sol is created by the sol-gel method from tetra-alkoxysilane with the involvement of acid catalysis, the following ratios of input raw materials.

In the case of the molar ratio of tetraalkoxysilane to water, this is kept in the range of 0.3 to 0.65, and at the same time, the molar ratio of acid to tetraalkoxysilane is maintained in the range of 0.005 to 1. When doping with bioactive ions, it is then necessary to maintain the molar ratio of salt to tetraalkoxysilane in the range of 0.001 up to 0.25. After the completion of the polycondensation reaction, the concentration of the spinning sol is adjusted to the range of 25 to 45%. By this procedure, a spinning sol is prepared, during the processing of which it is possible to achieve a non-woven fiber structure with a fiber diameter in the submicron scale. The structure of the prepared layer is determined by the selected spinning method, where the use of electrostatic spinning leads to the preparation of mainly 2D structures, while the use of centrifugal spinning enables the creation of 3D structures with a higher proportion and size of inter-fiber pores. The diameter of the fibers in the layer and the thickness of the spun layer is determined by the spinning conditions. Most often, the basis weight ranges from 10 to 50 g/m ² , but these limits are not restrictive. The prepared structures are further heat-treated to achieve higher mechanical stability. Thermal stabilization takes place most often at temperatures up to 350 °C, but further modifications can be carried out without heat treatment. In the same way, the layer can be hydrophilized before the binding of the functional groups by physical treatment with low-pressure plasma or plasma performed at atmospheric pressure. This modification leads to an increase in the number of Si-OH groups on the surface, which subsequently results in a higher functionality of the surface. Plasma treatment can also be used for fibers stabilized at temperatures above 350 °C to re-induce Si-OH lost due to heat treatment carried out in order to achieve higher mechanical resistance of the layer. Silicon nanofibers do not lose their biocompatibility at a higher temperature. This was confirmed for fibers treated up to a temperature of 750 °C.

Subsequently, the surface of the doped silicon fibers is functionalized by the covalent bond of a functional alkoxysilane in the form of an aminoalkylalkyloxysilane or an epoxyalkylalkyloxysilane

- 4 CZ 309644 B6 in a concentration most often of 0.1 to 5% in solution, but it can also be 0.1 to 75%, more preferably 0.1 to 60%, even more preferably 0.1 to 25% and most preferably the above-mentioned 0, 1 to 5%. This surface functionalization does not reduce the biocompatibility of the surface, it modifies its charge and wettability. This modification leads to an improvement in the availability of active ions incorporated into the fiber mass and at the same time can be used for adsorption, electrostatic or covalent binding of other molecules to the surface of the fibers, depending on the specific chosen biotechnological or medical application, so that, if possible, a synergistic effect with the incorporated ions. These two types of functional groups on the surface enable the binding of most active molecules using spontaneous binding or via auxiliary conjugation chemistry. An example of such chemistry can be the use of various types of conjugation linkers, the so-called zero length based on ethyl(dimethylaminopropyl)carbodiimide.

Clarification of drawings

The invention is schematically illustrated in the drawings, which show:

Fig. 1 SEM microscopic morphology of submicron silicon-based fibers containing bioactive zinc ions according to Example 1, Fig. 1a representation of individual classes of fiber diameters in the layer according to Example 1;

Fig. 2 evidence of biocompatibility of submicron silicon-based fibers containing bioactive zinc ions according to Example 1 before and after surface functionalization, evaluated on Hacat cells;

Fig. 3 SEM microscopic morphology of submicron silicon-based fibers containing bioactive copper ions according to example 2;

Fig. 3 and representation of individual classes of fiber diameters in the layer according to example 3;

Fig. 4 evidence of surface functionality and biocompatibility of submicron silicon-based fibers containing bioactive copper ions according to example 2 before and after surface functionalization, evidence of the presence of amino groups on the surface of untreated and treated fibers through methyl orange staining, biocompatibility before and after surface treatment evaluated on Hacat cells;

Fig. 5 SEM microscopic morphology of submicron silicon-based fibers containing bioactive calcium ions according to example 3;

Fig. 5a represents individual classes of fiber diameters in the layer according to example 3;

Fig. 6 SEM microscopic morphology of submicron silicon-based fibers containing a mixture of bioactive silver, copper and zinc ions according to example 4;

Fig. 6a representation of individual classes of fiber diameters in the layer according to example 4;

Fig. 7 evidence of biocompatibility of submicron silicon-based fibers containing a mixture of bioactive silver, copper and zinc ions according to Example 4 before and after surface functionalization, evaluated on Hacat cells; and Fig. 8 evidence of antibacterial activity of submicron silicon-based fibers containing a mixture of bioactive ions against S. gallinarum (left) and E. coli (right) bacteria.

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Examples of implementation of the invention

The invention will be described using examples of the creation of a non-woven fiber structure based on silicon dioxide doped with biogenic ions and with a modified surface, which further enables the binding of active and medicinal substances and other molecules selected according to the specific application. The invention is documented by selected specific examples of implementation, which, however, do not describe all the possibilities of implementing the invention. These options, not described in more detail here, are obvious to the average person familiar with the invention from this text without the need for further inventive activity.

Example 1

Spinning salt was prepared using a modified sol-gel method - by mixing 164 ml of isopropyl alcohol and 200 ml of tetraethoxysilane, water and acid so that the molar ratios correspond to the values of 0.45 molar ratio of tetraethoxysilane to water and at the same time 0.1 molar ratio of acid to tetraethoxysilane. At the same time, zinc nitrate was added as a source of bioactive ions in a ratio of 0.01 moles to tetraethoxysilane. The salt prepared in this way went through the process of hydrolysis, polycondensation and maturation, when it is finally thickened to 33% of the dry matter content of the total weight of the spinning salt. This step is ensured by evaporation of the solvent.

The spinning sol prepared in this way was subsequently processed by the method of electrostatic spinning with direct current (so-called DC electrospinning) from a wire electrode while maintaining the spinning conditions at a spinning distance of 160 mm and a voltage difference between the electrodes of 60 kV. In this way, a non-woven fiber layer was prepared with a mean fiber diameter of 174 nm, its surface weight was determined by the speed of movement of the underlying material at 12 g/m ² . To achieve higher cohesion of the layer, temperature stabilization was performed by exposure to a temperature of 180 °C for 2 hours. This heat treatment did not lead to a significant change in the structure (homogeneity) of the layer or the diameters of the submicron fibers. To achieve surface functionality, the layer was subsequently soaked in a 3% solution of 3-aminopropyltriethoxysilane dissolved in 96% ethanol for 2 hours at room temperature. The stability of functional groups grafted onto the surface after rinsing the excess functionalization agent, carried out by repeated rinsing in ethanol solution, was supported by heat treatment at 110 °C for 30 minutes. In this way, silicon nanofibers with bioactive zinc content and simultaneously with a functional surface containing -NH groups free for further binding in the selected manner were obtained.

The morphology of the thus prepared submicron fibers is shown in Fig. 1 taken by means of electron microscopy, the representation of individual classes of fiber diameters in the layer according to example 1 is shown in Fig. 1a. Biocompatibility with the cells of the skin cover was demonstrated for the material prepared in this way. The test performed in vitro on Hacat human keratinocytes was performed in accordance with the standard ČSN EN ISO 10993-5:2009 so that the eluate of the test sample in different concentrations was prepared by elution for 24 hours. The latter was subsequently exposed to pre-cultured cells for the following 24 hours. The resulting cytocompatibility was evaluated through the metabolic activity of living cells with the tetrazolium salt WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, sodium salt) and subsequent quantification at 450 nm wavelength. The results obtained in the form of proof of biocompatibility of the submicron silicon-based fibers with the content of bioactive zinc ions according to Example 1 before and after surface functionalization, evaluated on Hacat cells, are shown in Fig. 2.

This layer can be applied in this state without further modification, or it can serve to further bind biotechnologically or medically important substances. In this case, for example, as a carrier of the biotechnologically usable laccase enzyme for water treatment.

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Example 2

Spinning sol was prepared using a modified sol-gel method - by mixing 164 ml of isopropyl alcohol and 200 ml of tetraethylorthosilicate (tetraethoxysilane), water and acid so that the molar ratios correspond to the values of 0.4 molar ratio of tetraethoxysilane to water and at the same time 0.11 molar ratio of acid to tetraethoxysilane. At the same time, copper nitrate was added to the mixture as a source of bioactive ions in a ratio of 0.034 moles to tetraethoxysilane. The sol prepared in this way went through the process of hydrolysis, polycondensation and maturation, when it was finally thickened to 35% of the dry matter content of the total weight of the spinning sol. This step was ensured by evaporation of the solvent.

The spinning salt prepared in this way was subsequently processed by the method of electrostatic spinning with direct current (so-called DC electrospinning) from a rod electrode while maintaining the spinning conditions at a spinning distance of 150 mm and a voltage difference of 45 kV between the electrodes. In this way, a non-woven fibrous layer was prepared with an average fiber diameter of 289 nm. To achieve higher cohesion of the layer, temperature stabilization was performed by exposure to a temperature of 180 °C for 2 hours. In order to increase the efficiency of the surface functionalization, the fiber layer was subsequently treated with a corona discharge in an air atmosphere at room temperature, a pressure of ~101 kPa, a power of 800 W and a material feed speed of 1 m/min. The activated layer was then transferred to a functionalization bath consisting of a 3% solution of 3-aminopropyltriethoxysilane dissolved in 96% ethanol, where it was soaked for 1 hour at room temperature. The stability of the functional groups grafted onto the surface was supported by heat treatment at 110 °C for 30 minutes after rinsing the excess functionalization agent, carried out repeatedly in an ethanol solution. In this way, silica nanofibers containing bioactive copper ions and simultaneously with a functional surface containing NH2 groups free for further binding in the selected manner were obtained.

The morphology of the thus prepared submicron fibers is shown in Fig. 3 taken by means of electron microscopy, the representation of individual classes of fiber diameters in the layer according to Example 3 is shown in Fig. 3a. Biocompatibility with the cells of the skin cover was demonstrated for the material prepared in this way. The test performed in vitro on Hacat human keratinocytes was performed in accordance with the standard ČSN EN ISO 10993-5:2009 so that the eluate of the test sample in different concentrations was prepared by elution for 24 hours. The latter was subsequently exposed to pre-cultured cells for the following 24 hours. The resulting cytocompatibility was evaluated through the metabolic activity of living cells with the tetrazolium salt WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, sodium salt) and subsequent quantification at 450 nm wavelength. The viability of the exposed cells exceeded 95% of the cell control viability at all concentrations. At the same time, surface treatment resulted in a slight increase in cell viability compared to the untreated doped layer. Achieved results, in the form of evidence of surface functionality and biocompatibility of submicron silicon-based fibers containing bioactive copper ions according to example 2 before and after surface functionalization, evidence of the presence of amino groups on the surface of untreated and treated surfaces through methyl orange staining, biocompatibility evaluated before and after surface treatment on Hacat cells, are shown in Fig. 4.

This layer can be applied in this state without further modification, or it can serve to further bind biotechnologically or medically important substances. In this case, for example, a combination of an active silicon layer and natural antiseptics, growth factors or other substances supporting angiogenesis and wound healing is suitable. This can be bound by simple adsorption or electrostatic bonding or covalent bonding while maintaining the high porosity of the silicon layer. In the case of protein substances, their presence on the surface can be demonstrated by (non)specific protein staining and their quantification.

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Example 3

Spinning sol is prepared using a modified sol-gel method - by mixing 164 ml of isopropyl alcohol and 200 ml of tetraethylorthosilicate (tetraethoxysilane), water and acid so that the molar ratios correspond to the values of 0.43 molar ratio of tetraethoxysilane to water and at the same time 0.1 molar ratio of acid to tetraethoxysilane. At the same time, calcium nitrate was added to the mixture as a source of bioactive ions in a ratio of 0.0072 moles to tetraethoxysilane. The sol prepared in this way went through the process of hydrolysis, polycondensation and maturation, when it was finally thickened to 29% of the dry matter content of the total weight of the spinning sol. This step was ensured by evaporation of the solvent.

The spinning salt thus prepared was subsequently processed by the centrifugal spinning method while maintaining the spinning conditions of 200 mm and 9000 RPM. In this way, a three-dimensional non-woven fiber layer with a mean value of fiber diameter of 463 nm was prepared. The morphology of the submicron fibers prepared in this way is shown in Fig. 5, the representation of individual classes of fiber diameters in the layer according to example 3 is shown in Fig. 5 a. To achieve higher cohesion of the layer, temperature stabilization was performed by exposure to a temperature of 180 °C for 2 hours. Subsequently, the layer was surface functionalized by soaking for 30 minutes in a 2% mixture of 3-glycidyloxypropyltriethoxysilane and ethanol with a proportion of 15% water. After repeated rinsing in ethanol solution, the layer was dried at room temperature for 48 hours. In this way, silicon nanofibers containing bioactive calcium ions and simultaneously with a functional surface containing epoxy groups free for further binding in the selected manner were obtained.

This layer can be applied in this state without further modification and serve for in situ conjugation (e.g. as a sensor). Or it can be conjugated with biotechnologically or medically important substances to ensure their increased stability during subsequent application. This surface functionalization ensures reactivity with the entire spectrum of substances containing amino groups, sulfanyl groups and others. In this case, for example, a combination of an active silica layer and a conjugated trypsin enzyme used for wound debridement is suitable.

Example 4

Spinning sol is prepared using a modified sol-gel method - by mixing 164 ml of isopropyl alcohol and 200 ml of tetraethylorthosilicate (tetraethoxysilane), water and acid so that the molar ratios correspond to the values of 0.37 molar ratio of tetraethoxysilane to water and at the same time 0.11 molar ratio of acid to tetraethoxysilane. At the same time, silver nitrate, copper nitrate and zinc nitrate were added to the mixture as sources of bioactive ions in a ratio of 0.007/0.004/0.003 moles to tetraethoxysilane. The sol prepared in this way went through the process of hydrolysis, polycondensation and maturation, and was subsequently thickened to 36% of the dry matter content of the total weight of the spinning sol. This step is ensured by evaporation of the solvent.

The spinning salt prepared in this way was subsequently processed by the method of electrostatic spinning with direct current (so-called DC electrospinning) from a wire electrode while maintaining the spinning conditions at a spinning distance of 165 mm and a voltage difference between the electrodes of 65 kV. In this way, a non-woven fiber layer was prepared with a mean fiber diameter of 193 nm. To achieve higher cohesion of the layer, temperature stabilization was performed by exposure to a temperature of 200 °C for 120 minutes. To achieve surface functionality, the layer was then soaked in a 5% solution of 3-aminopropyltriethoxysilane dissolved in 96% ethanol for 30 minutes at room temperature. The stability of the functional groups grafted onto the surface was supported by heat treatment at 110 °C for 30 minutes after rinsing the excess functionalization agent, carried out repeatedly in an ethanol solution. In this way, silicon nanofibers were obtained with a mixture of bioactive silver, copper and zinc ions and at the same time with a functional surface containing -NH2 groups free for further binding.

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The morphology of the thus prepared submicron fibers is shown in Fig. 6 taken through electron microscopy, the representation of individual classes of fiber diameters in the layer according to example 4 is shown in Fig. 6a. The material prepared in this way was proven to be biocompatible with skin cells according to the methodology described in example 1. The results achieved are shown in Fig. 7 in the form of proof of biocompatibility of silicon-based submicron fibers containing a mixture of bioactive silver, copper and zinc ions according to example 4 before the surface functionalization and after it, evaluated on Hacat cells. Furthermore, the antibacterial activity of the layer against two model bacterial strains, Staphylococcus gallinarum and Escherichia coli, was confirmed by the diffusion method. During the test, a bacterial suspension with a concentration of 10 ⁴ bacteria/ml was inoculated onto a dish with culture agar. Subsequently, a triplicate of the tested samples was attached. After incubation under standard conditions (37 °C) for 24 hours, the size of the diffusion zone around the samples was evaluated. An average dimension of 14 mm confirmed the antibacterial activity of the samples against both bacterial strains, as shown in Fig. 8 in the form of evidence of the antibacterial activity of the submicron silicon-based fibers containing a mixture of bioactive ions against S. gallinarum (left) and E. coli (right) bacteria. .

This layer can be applied in this state without further modification and support, for example, the cleaning and healing of contaminated and difficult-to-heal wounds, or it can serve to further bind biotechnologically or medically important substances. In this case, a combination with an antiseptic or antibiotic important for dermatovenerology is suitable. This can be bound by simple adsorption or electrostatic binding while maintaining the high porosity of the silicon layer. In this way, an active layer can be achieved with a synergistic effect combining an antibacterially effective surface-bound substance released within hours to days and bioactive ions ensuring protection against secondary infection after the release of the drug from the surface or infection caused by microorganisms outside the area of effect of the surface-bound active substance.

Claims (7)

1. Biocompatible and biodegradable non-woven fiber structure with submicron fibers based on silicon dioxide, with biogenic ions and with a functional surface for the binding of active substances, characterized by the fact that the fibers are created by spinning a sol prepared by the sol-gel method and are directly in their volume during spinning provided with bioactive ions from the group of calcium and/or copper and/or zinc and/or silver ions, and the surface of the fibers is functionalized with aminoalkylalkoxysilane or epoxyalkylalkoxysilane. 2. The method of producing a biocompatible and biodegradable non-woven fiber structure according to claim 1, in which a sol prepared by the sol-gel method is spun, characterized in that the sol is synthesized before spinning from a low-molecular-weight precursor of a tetra-alkoxysilane by the sol-gel method in alcohol with the addition of water under acidic conditions catalysis, while this salt is doped with bioactive ions in the form of salts already during its preparation before spinning, and after spinning, the surface of the fibers is functionalized with aminoalkyl alkoxy silane or epoxy alkyl alkoxy silane. 3. The method according to claim 2, characterized in that the salt is synthesized in an ethanol or isopropyl alcohol solution prior to spinning, and the molar ratio of tetraalkoxysilane to water is in the range of 0.3 to 0.65, while the pH of the sol during the reaction is adjusted by adding inorganic or organic of acid at a ratio of acid to tetra-alkoxysilane in the range of 0.005 to 1. 4. The method according to claim 2 or 3, characterized in that the sol is thickened before spinning by evaporating excess alcohol to a final concentration of dry matter in the range of 25 and 45% by weight, based on the total weight of the spinning sol. 5. The method according to any one of claims 2 to 4, characterized in that the salt is doped with bioactive ions of calcium and/or copper and/or zinc and/or silver while maintaining the molar ratio of the salt to the tetraalkoxysilane in the range of 0.001 to 0.25. 6. The method according to any one of claims 2 to 5, characterized in that the spinning is carried out by electrostatic and/or centrifugal spinning, after which the fiber structure is heat-treated and possibly also irradiated with plasma to increase the effectiveness of the subsequent surface modification with a selected functional aminoalkylalkoxysilane or epoxyalkylalkoxysilane. 7. The method according to any one of claims 2 to 6, characterized in that the surface of the fibers of the non-woven fiber structure is functionalized after spinning in a solution of functional aminoalkyl alkoxy silane or epoxy alkyl alkoxy silane in water, alcohol or another organic solvent for a period of 0.5 to 12 hours at a concentration of functional of aminoalkyl alkoxy silane or epoxy alkyl alkoxy silane 0.1 to 75%, more preferably 0.1 to 60%, even more preferably 0.1 to 25% and most preferably 0.1 to 5% in the solution.