Filed of Art

The present invention relates to a method of preparation of a polymer composite substrate with immobilized silver nanoparticles and to the substrate obtainable using the method.

Background Art

Nowadays, there are several approaches to tailored immobilization of silver nanoparticles on various substrates (AgNPs) with the aim to provide antimicrobial treatment of the material or to provide antifouling protection (prolongation of lifetime of the filtration/fibrous structures). These approaches, however, do not solve the problem of potential nanoparticle release, potentially release of the silver nanoparticles with their linkers. The Czech patent CZ303502 describes the approach to immobilization of silver nanoparticles (AgNPs) via covalent bonds between the silver and the linker but it does not eliminate possible release of the whole composite, i.e. Ag-linker, from the substrate under mechanical stress or under high pressure of the flow and flow rate of the media - in the case of the filtration materials.

The above-described problem is solved by the present invention, which provides for the immobilization of silver nanoparticles onto the substrate in such a way that effectively prevents possible release of the silver nanoparticles/silver nanoparticles with a polymer linker from the composite substrate. This immobilization process represents rather simplified method as the functionalization of the surfaces can proceed under significantly lower temperatures. The temperature represents a crucial parameter especially from some substrates. Disclosure of the Invention

The present invention introduces a method of preparation of a polymer substrate with immobilized silver nanoparticles, wherein the substrate is prepared from a mixture of water non-soluble polymer and a polymer linker containing Ag-reducing functional groups (i.e. functional groups that are able to reduce Ag ⁺ to Ag°) containing nitrogen atom with a free electron pair. Consequently, the substrate is functionalized with covalently bonded silver nanoparticles, which are generated from an aqueous solution of silver ions. The generated silver nanoparticles are covalently bonded to the substrate via the nitrogen atom and/or sulphur atom of the polymer linker.

Water non- soluble polymer is preferably a synthetic polymer, more preferably selected from a group comprising polyurethanes, polylactide, polyglycolides, poly(lactide-co- glycolide), polyethylene, polypropylene, polyamide, saccharides, as for example cellulose or chitosan.

Ag-reducing functional groups comprise all groups containing nitrogen and at the same time having a free electron pair, i.e. for instance amino-, imino-, imido- functional group.

The polymer linker has preferably molecular weight from 300 g.mol ^"1 to 1,000,000 g.mol ^"1 and has either linear or branched structure. A preferable polymer linker is e.g. poly ethy lenimine . The substrate is nanofibrous and is prepared from a water non-soluble polymer and a polymer linker using electro spinning method. It has been proved that if the substrate is prepared from a mixture of water non- soluble polymer and polymer linker, it is sufficient to prevent the undesired release of the composite of Ag-linker. The release of Ag-linker composite represented the disadvantage in CZ 303502.

Preferably, the polymer linker is involved in the substrate in the concentration from 0.0001 wt. % up to 5 wt.%. The concentration is related to the overall mass of the water non- soluble polymer.

Silver ions might be in a form of a compound containing silver ions, which can be a water soluble silver salt, as e.g. AgN0 ₃ , AgC10 ₄ , AgF, or a water soluble complex compound of silver ions, as e.g. diamminesilver complex. Aqueous solution containing ionic silver reacts with the substrate, preferably in the concentration range of silver ions solution from 0.0001 mol-L ^"1 up to 5 mol-L ^"1 ; preferably at the temperature of from 5 °C to 95 °C, ideally at room temperature, preferably for the period from 1 s up to 60 minutes.

The resulting composite material exhibit the required antimicrobial properties thanks to the covalently bonded silver nanoparticles, which are even firmly anchored through the polymer linker, which itself is anchored into the structure of the substrate. In contrast to the method, when the silver nanoparticles are anchored into the substrate, in the method according to the present invention the overall surface of the silver nanoparticles is available for the antimicrobial action, and the silver nanoparticles do not release from the surface under mechanical stress, which represents a key parameter of such a composite for the use e.g. in the filtration/water treatment technologies or in medicinal applications. The subject of the present invention is further an antimicrobial material containing silver nanoparticles, which structure is based on a substrate formed by a water non-soluble polymer and polymer linker containing a nitrogen atom and/or a sulphur atom onto which silver nanoparticles are covalently bonded. Preferably, the antimicrobial material can be prepared using the method according to the present invention.

Brief Description of Figures

Fig. 1 shows the SEM image of the nanofibers prepared according to the example 1.

Fig. 2 shows the SEM image of the nanofibers prepared according to the example 2.

Fig. 3 shows the SEM image of the nanofibers prepared according to the example 3.

Examples of the Invention Embodiments

Example 1: Polyurethane (PU) fibres with immobilized silver nanoparticles (on all surfaces) Nanofibrous structures of polyurethane (PU), i.e. fibres with a diameter in the range from tens up to hundreds of nanometers, are prepared by electro spinning from the mixture of polyurethane dissolved in dimethylformamide (DMF). The weight percentage of PU dissolved in DMF is 4%. After homogenization of the DMF:PU mixture, the branched polyethylenimine (i.e. hyperbranched polymer linker containing nitrogen atoms with free electron pairs, which can be used for the reduction stabilization of subsequently generated and immobilized silver nanoparticles) was added. The molecular weight of the polyethylenimine was 25,000 g.mol ^"1 (in the concentration of 1 wt.% to the PU mass). The homogenization of the mixture DMF:PU:PEI is followed by electro spinning of this mixture. The fibres are collected on a rotation collector as a compact layer of the mesh structured fibres (i.e. fibres without orientation). This structure contains fibres with immobilized polymer linker in its structure. Thanks to the immobilized linker the fibres can be preferably used for tailored deposition of silver nanoparticles when the fibres are immersed into the precursor of the silver nanoparticles solution, i.e. into silver nitrate with the concentration of 8-10 ^"4 mol.L ^"1 . The mesh structure is taken out of the solution immediately and the structure is washed with water in order to get rid of the residues of the precursor from all the mesh cavities. The whole process is carried out under room temperature. There is a strong coordination-covalent interaction between the heteroatom and silver, and the linker itself is firmly anchored in the substrate structure. Based on the above-mentioned reasons the release of the silver nanoparticles or silver nanoparticles with the linker from the composite material is prevented.

The amount of silver in the prepared sample of PU fibres with immobilized AgNPs was consequently quantified with AAS. Comparable sample was placed into water and onto a shaker and let to be mechanically stressed. After 24 hours, the amount of silver on the composite material was quantified with AAS. The amount of silver on the tested (i.e. first sample) and stressed (i.e. the sample mechanically stressed for 24 hours in water on a shaker) sample was almost identical (note: the difference was 0.1 wt% of silver, which represents a statistical error can correspond with the released Ag ⁺ ions released into the aqueous environment; the antimicrobial effect is probably based on the release of the Ag ⁺ from the silver nanoparticle surface).

The structure of the PU fibres with immobilized molecules of hyperbranched polyethylenimine and consequently immobilized silver nanoparticles on the substrate surface proved to exhibit antimicrobial efficiency against model organisms of strains of Staphylococcus and Escherichia (tested according to the norm JIS1902/IS 020743 for textiles). The tested samples were 5x5 cm in size and the concentration of the inoculum was 10 ⁵ CFU/mL. The samples functionalized with silver nanoparticles proved to supress the growth of bacterial colonies completely in contrast to the blank samples (the PU fibres without functionalization).

Example 2: Fibres of poly(lactide-co-glicolide) (PLGA) with immobilized silver nanoparticles on all surfaces.

Nanofibrous structures of poly(lactide-co-glycolide) (PLGA), i.e. fibres of PLGA with the average diameter of hundreds of nanometers are prepared via electro spinning from the mixture of PLGA in chloroform (CHC1 ₃ , 95%) with the addition of dimethylformamide (DMF; 5%). PLGA with the molecular weight of 50,000 - 75,000 g.mol ^"1 is dissolved in the mixture of chloroform and DMF in the weight ratio of 30%. After homogenization of the mixture of PLGA:DMF:CHCl ₃ on a shaker, hyperbranched polyethylenimine (PEI) with molecular weight 25,000 g.mol ^"1 was added in the concentration of 0.1 wt.% (in the relation to the PLGA mass). (Note: The hyperbranched polymer linker contains numerous nitrogen atoms with free electron pair, which is used for the reduction, stabilization and immobilization of the generated silver particles.) The resulting mixture is again homogenized on a shaker. Subsequently, oriented fibres are prepared via electro spinning and collected on a rotation collector. This way prepared fibres, containing polymer linker in its structure for targeted deposition of silver nanoparticles, are consequently immersed into the solution of silver nanoparticle precursor, i.e. into silver nitrate with the concentration equal to 5- 10 ^" 3 mol.L ^" 1 , for the period of 3 minutes. Then they are taken out and washed with distilled water in order to eliminate the chemical residues, especially traces of silver nitrate as the nanoparticle precursor. There is a strong coordination- covalent interaction between the heteroatoms and silver. The linker itself is firmly anchored in the structure of the fibre, which prevents release of either silver nanoparticles or silver nanoparticles with the polymer linker.

Example 3: Nanofibres of polylactide-co-glycolide (PLGA) with immobilized silver nanoparticles on all fibre surfaces. Nanofibrous structures of polylactide-co-glykolide (PLGA), i.e. fibres with the diameter of tents of nanometers are prepared via electrospinning from the mixture of PLGA dissolved in tetrahydrofuran (THF, 50%) and chloroform (CHC1 ₃ , 50%). PLGA (with the molecular weight of 50,000 - 75,000 g.mol ^"1 ) is dissolved in the mixture of chloroform and THF in the weight ratio of 30%. After homogenization of the mixture of PLGA:THF:CHC1 ₃ on a shaker, hypebranched polymer linker - polyethylenimine is added to the mixture (the molecular weight of polyethylenimine is 750,000 g.mol ^"1 ; note: the hyperbranched polymer linker contains sufficient number of nitrogen atoms with free electron pair, which is used in the process of reduction, stabilization and immobilization of silver nanoparticles)) in the concentration of 0.1 wt.% (in the relation to the PLGA mass). Subsequently, the fibres were prepared via electrospinning. The fibres were collected on a rotation collector without any orientation - in a form called "mesh". This way prepared fibres, involving polymer linker in its structure for targeted deposition of silver nanoparticles, are consequently immersed into the solution of silver nanoparticle precursor, i.e. into silver nitrate with the concentration equal to 1- 10 ^" 2 mol.L ^" 1 , for the period of 3 minutes. Then they are taken out and properly washed with distilled water in order to eliminate the chemical residues, especially the any traces of the silver nitrate as the precursor. There is a strong coordination-covalent interaction between the heteroatoms in polymer linker structure and silver. The linker itself is firmly anchored in the structure of the fibre, which prevents release of either silver nanoparticles or silver nanoparticles with the polymer linker.