WO2012161603A2

WO2012161603A2 - Hybrid material containing silver nanoparticles, method for obtaining the same and use thereof

Info

Publication number: WO2012161603A2
Application number: PCT/PL2012/000035
Authority: WO
Inventors: Maria Nowakowska; Szczepan Zapotoczny; Maria BULWAN; Maciej DŁUGOSZ
Original assignee: Uniwersytet Jagiellonski
Priority date: 2011-05-24
Filing date: 2012-05-24
Publication date: 2012-11-29
Also published as: WO2012161603A3; PL395002A1; PL221411B1

Abstract

The hybrid material is composed of calcium carbonate microparticles with the polyelectrolyte additive and silver nanoparticles (nAg) embedded in its structure. The process of making of the hybrid material comprises of ultrasound-assisted coprecipitation of calcium carbonate in aqueous medium in the presence of polyelectrolyte and in the presence of silver nanoparticles (nAg). The hybrid material can be used as an antibacterial and antifungal agent, especially to protect goose down feathers from negative influence of microorganisms.

Description

Hybrid material containing silver nanoparticles, method for obtaining the same and use thereof

The subject of this invention is a bacteriostatic, antibacterial, fungistatic and antifungal hybrid material consisting silver nanoparticles (nAg) embedded in a calcium carbonate matrix, as well as the method of preparing the hybrid material and its application.

Colloidal silver nanoparticles (nAg) are a well known antibacterial and bacteriostatic material, which is widely used in industry. nAg are bacteriostatically/antibacterially active mainly at the cellular level. Small sizes of nAg allow mechanical disruption of a cell membrane by perforation and enable them to penetrate deep into the cell interior. However the main contribution to the antibacterial effect is associated with silver ion (Ag⁺) release from the surface of nAg. Ag⁺ released inside a cell can readily interact with its negatively charged components, including DNA and RNA, efficiently disrupting microbial life processes. (Feng, Q.L., Wu, J, Chen, G. Q., Cut, F.Z., Kim, T.N., Kim, J. O. "A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus " Journal of Biomedical Materials Research 52 (2000) 662-668; Yamanaka, M., Hara, K., Kudo, J. "Bactericidal Actions of a Silver Ion Solution on Escherichia coli, Studied by Energy-Filtering Transmission Electron Microscopy and Proteomic Analysis " Applied And Environmental Microbiology 71 (2005) 7589-7593). It was proven that nAg of smaller sizes generate more Ag⁺ ions. {Sotiriou, G. A., Pratsinis, S. E. "Antibacterial activity of nanosilver ions and particles ", Environmental Science and Technology, 2010, 44, 5649-5654). Moreover the aggregation of nAg is unfavorable and leads to a decrease in their antimicrobial activity.

The literature is vague about dangers and risks for human organism and environment resulting from extensive nAg usage. Since Ag is a heavy metal it can be anticipated that its reactivity towards mammalian cells will be similar as in the case of fungi/bacteria (reactions of Ag⁺ ions with DNA or thiol moieties in proteins leading to deactivation of these structures). Additionally, nAg, thanks to their small size, can freely penetrate barriers inside the organism and deposit in lungs and other organs eventually leading to inflammation. The problem was noticed and the regulations concerning safety of nAg handling and restrictions regarding their release into the environment are gradually introduced. (Weier, F. W. "Health hazard from occupational exposure to metallic copper and silver dust" American Industrial Hygiene Association Journal 40 (1979) 245-247). Potential toxic activity of nAg may greatly limit their direct biomedical applications (biosensors, bioimaging). Thus, there have been efforts in protecting nAg by coating them with synthetic polymers (e.g., polyvinylpyrrolidone), natural polymers (e.g., polysaccharides), silanes or surfactants to ensure safety of their application (Sotiriou, G.A., Sannomiya, T, Teleki, A., Krumeich, F., Voros, J, Pratsinis, S.E. "Non-Toxic Dry-Coated Nanosilver for Plasmonic Biosensors " Advanced Functional Materials 20 (2008) 4250-4257; Kvitek, L, Panacek, A., Soukupova, J, Kolar, M., Vecerova, R., Prucek, R. et al. "Effect of surfactants and polymers on stability and antibacterial activity of silver nanoparticles (NPs) " Journal of Physical Chemistry C 112 (2008) 5825-5834). Such coatings also prevent the decrease of their antibacterial activity, which is related to nAg aggregation.

nAg are widely used in the industry including textile production. Unfortunately, application and exploitation of such material is associated with release of nAg to the environment and its presence in the postproduction waste which can later influence the wastewater treatment plants processes. Preliminary studies show that in the process of nAg contaminated wastewater treatment (on the stage involving activated sludge) ions resulting from the dissolution of nAg form silver sulfide, which might disrupt the work of the wastewater treatment plant (Kim, B., Park, C.S., Murayama, M., Hochella, M.F. "Discovery and Characterization of Silver Sulfide Nanoparticles in Final Sewage Sludge Products " Environmental Science and Technology 44 (2010) 7509-7514). Therefore it is essential to control the release of nAg from materials containing them.

The paper by Lei, M., Tang, W.H., Cao, L.Z., Li, P.G., Yu, J.G. "Effects of poly (sodium 4-styrene-sulfonate) on morphology of calcium carbonate particles " (Journal of Crystal Growth 294 (2006) 358-366) presents a synthesis of highly porous, microspherical calcium carbonate particles which owe their exceptional properties to the addition of PSS (about 2% of PSS is present in the microparticles according to the described experiment). Calcium carbonate microparticles were previously prepared employing various homopolymers and copolymers, e.g., poly(styrene-b-acrylic acid) PS-b-PAA (Li,X; Hu, Q.; Yue, L; Shen, J;„ Synthesis of Size-Controlled Acid-Resistant Hybrid Calcium Carbonate Microparticles as Templates for Fabricating "Micelles-Enhanced" Poly electrolyte Capsules by the LBL Technique ", Chem.- Eur. J. 12 (2006) 5770-5778), or Copolymers such as carboxymethyl chitosan (Wang, ; Chen.J-S; Zong,J.-Y; Zhao, D.; Li, F; Zhuo, R. X; Cheng, S.-X; "Calcium Carbonate/Carboxymethyl Chitosan Hybrid Microspheres and Nanospheres for Drug Delivery", J. Phys. Chem. C 114 (2010) 18940-18945). The aim of this invention was the development of an antibacterial and antifungal material containing silver nanoparticles capable of prolonged and controlled release of nAg which would limit the contact of nAg with the human organism and the environment and would be easy to store. Particularly important was development of a material suitable for contact with human skin.

Hybrid material of the present invention are silver nanoparticles embedded in a calcium carbonate/polyelectrolyte microparticle matrix.

Preferably, the applied polyelectrolytes are polymers and copolymers, including biopolymers, with ionic moieties such as sulfonic, carboxylic, phosphorous groups.

Preferably, alkali salts of poly(vinylsulfonic acid), poly(methacrylic acid), carboxymethyl cellulose are used as the polyelectrolyte.

Poly(sodium styrenesulfonate), PSS, is the most preferred polyelectrolyte because it is a strong polyelectrolyte and it enables control over spherical microparticles formation at relatively low concentration.

Preferably, the hybrid material is cationically or anionically modified.

Cationic or anionic modifications of the hybrid material by adsorption of at least one layer of ionic polymer are preferred. Synthetic and natural polymers can be employed during the surface modifications. Exemplary cationic polymers are: poly(allylamine), poly(ethyleneimine), poly(vinylpyridine) salts, chitosan, protamine; and exemplary anionic polymers are: poly(methacrylic acid) salts, poly(styrenesulfonic acid) salts, poly(2- acrylamido-2-methylpropane sulfonic acid), carboxymethyl cellulose, sulfonated dextran.

The method of fabrication of the hybrid material according to the invention is characterized by ultrasound assisted co-precipitation of calcium carbonate in an aqueous medium, in the presence of polyelectrolyte, which causes formation of spherical microparticles and in the presence of silver nanoparticles (nAg), which are responsible for bacteriostatic, antibacterial, fungistatic and antifungal properties of the material.

Preferably, the co-precipitation is performed by mixing of water-soluble calcium salt and water-soluble carbonate salt with addition of polyelectrolyte and silver nanoparticles.

The preferred ratio of calcium and carbonate ions is 1 : 1. There is a possibility of applying different ions ratios but a high excess of one salt might negatively influence the process of microparticles co-precipitation.

The concentrations of calcium and carbonate salts are preferably used in the range from 0.001 M to 1 M, most preferably around 10^"2 M. The size and composition of the obtained microparticles can be controlled by careful adjustment of concentrations in the aforementioned range.

The type of the polyelectrolyte employed during co-precipitation determines its required concentration. In the case of PSS the concentration may range from 0.1 to 10 g/dm , most preferably from 1 to 2 g/dm³. Concentration of PSS may be used in wide range but to maintain control over the size and shape of the micropatricles the ratio of the polymer sulfonic moieties to calcium ions not lower than 1 :3 is preferred. The higher is the concentration of the polymer the better is the control over the parameters of the obtained microparticles.

The co-precipitation is done at the temperature from the range of 15-50 °C, preferably at 20-25 °C. The temperature influences the speed of the process as well as composition and size of the obtained microparticles.

During synthesis any calcium salts that are soluble in water may be used. The preferred calcium salts are calcium nitrate and calcium chloride, the most preferred is calcium nitrate. The preferred carbonates are salts of alkali metals such as sodium and potassium, the most preferred is sodium carbonate.

The silver nanoparticles are preferred to be used as an aqueous colloidal dispersion.

Silver nanoparticles obtained during ultrasound assisted reduction of silver ions by trisodium citrate in aqueous medium at the temperature lower than the boiling temperature of water preferably between 70 and 85°C are preferred.

The most preferred method of making the hybrid material is as follows: trisodium citrate solution is added to the aqueous solution of silver nitrate, the mixture is sonicated and heated for 10-60 minutes, preferably in the temperature from 70 to 85°C. Then, to the obtained nAg colloid, the solution of Na₂C0₃ containing PSS and the solution of Ca(N0₃)₂ are added simultaneously. Then ultrasound-assisted co-precipitation is conducted for 1-20min, most preferably for 5 minutes at room temperature. The obtained colloid is then centrifuged to separate the obtained precipitate.

Preferably the hybrid material undergoes subsequent cationic or anionic modification.

Cationic or anionic modification by adsorption of at least one layer of a polyelectrolyte is preferred. Polyelectrolytes might be adsorbed on the surface using layer-by- layer electrostatic self-organisation (alternate adsorptions of polycation and polyanion), {Decker, G.;„ Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites", Science, 277 (1997) 1232).

During surface modifications synthetic as well as natural polymers are preferred. The invention also covers the antibacterial and antifungal application of the hybrid material.

The hybrid material is preferably used to protect aqueous and non-aqueous solutions and dispersions as well as polymeric materials such as lacquers, paints, building materials etc.

The preferred application of the hybrid material is the protection of goose down feathers from adverse influence of microorganisms. Protection of goose down feathers used as a filling in ready products such as blankets, pillows, jackets, suits is preferred.

The hybrid material can be stored in the form of a dry powder as well as in the form of colloidal water suspension.

Thanks to the method of making, described before, calcium carbonate crystalizes in the form of porous, spherical microparticles with nAg firmly embedded inside them. The polyelectrolyte employed during co-precipitation influences the crystallizton of calcium carbonate. In the presence of the polymer calcium carbonate crystallizes in the form of vaterite, which forms a characteristic spherical microparticles. Ultrasound assistance during microparticles fabrication allows to obtain more monodisperse colloidal particles. Ultrasound also causes formation of smaller microparticles which can be more easily attached to the surface of the feathers. Ultrasounds also positively influence the amount of silver nanoparticles embedded in the pores of carbonate microparticles.

Calcium carbonate matrix serves as an nAg carrier that controls their release. It was estimated that silver is firmly embedded in the microparticles that allows its prolonged release even in the acidic environment with pH = 5.4, close to the pH of a healthy skin.

The synthesis of nAg which were used to produce the hybrid material is a modified Lee and Meisel method (Lee P. C, Meisel D. "Adsorption and surface-enhanced Raman of dyes on silver and gold sols" Journal of Physical Chemistry 86 (1982) 3391-3395). Lee and Meisel method is easy and environmentally friendly in caparison with many other methods. The method was modified by introduction of ultrasounds and by decrease of the reaction temperature during the process of silver ions reduction. Ultrasounds causes faster diffusion of ions and nanoparticles leading to more monodisperse colloidal particles. Lower temperature also contributes to the monodispersity of the colloid. So the modification of the synthesis helps to produce more concentrated colloids composed of bigger but monodisperse nAg. Bigger and monodisperse nAg will be easier to embed inside the hybrid matrix (microparticles) and the release process of Ag⁺ will be longer and readily controlled. Carbonate matrix described above serves as a carrier for nAg, which are embedded inside it. It protects nAg from aggregation (nAg retain their antimicrobial activity for a longer time), enables their long-term release (prolonged antibacterial effect) and imparts white color on the material (in contrast to sole nAg which are green and might dye the products covered with them). The . matrix limits direct contact of nAg with skin that makes the material attractive for various biomedical applications. The hybrid material can be stored as a dry powder without the risk of losing its antimicrobial activity (in contrast to sole nAg, which tend to aggregate), and be redispersed if needed in an appropriate amount of water. The material according to the invention serves as an alternative to a material composed of titanium(IV) dioxide which is currently used to protect goose down feathers. Polymer- carbonate microparticles are cheaper in production, non-toxic and easier to apply.

The hybrid material according to the invention was deposited on a high quality down feathers acquired from white Koludzka geese. The adsorption was monitored using optical microscopy and scanning electron microscopy (SEM). In the case of SEM despite high vacuum applied during the measurement the hybrid material could be still observed on the surface of the feathers that certifies its firm attachment.

Microbiological tests were conducted employing bacterial and fungal strains present on a healthy human skin at the physiological conditions. The bacteria were acquired from patients. Microbiological tests were conducted in suspension. Appropriate amounts of materials suspected of bacteriostatic action were added to the bacterial suspensions. The quantity of the bacteria was estimated using densitometric method before and after 24 hours of incubation. The concentration of sole bacteria in the physiological salt did not change (or drops insignificantly up to 5%). The investigation of bacteriostatic action of feathers covered with the hybrid material was conducted in the same manner as that of sole bacteria but the bacterial concentration was measured after separation of the feathers from the mixture to ensure that the substrate did not contribute to the measurement. The hybrid material exhibits bacteriostatic properties despite of very low amount of nAg in the material.

The aim of the surface modification of the hybrid material is to change its surface charge and to enable its deposition on surfaces with different properties. The evaluation of such modifications was performed on flat mica surface. The electrostatic interactions allowed adsorption of cationically and anionically modified materials. Mica, which surface charge is negative, adsorbed microparticles to certain extent dependent on their surface charge. It has been observed that on the 1 cm² surface of mica lxl 0⁵ of unmodified microparticles were adsorbed while favorable electrostatic interactions in the case of cationicaly modified microparticles lead to increase of the number up to 7xl0⁵.

The respective figures represent:

Fig. 1 - AFM picture of silver nanoparticles obtained after 60 min of the synthesis. The nanoparticles were deposited from aqueous colloid on a silica plate, which was previously covered with cationic polymer (PAH, polyallylamine hydrochloride) to enhance electrostatic adsorption.

Fig. 2 - Spectra of nAg dispersions after different duration of the synthesis.

Fig. 3 - SEM images of the hybrid material.

Fig. 4 - UV-Vis spectra of the hybrid material dissolved using EDTA solution.

Fig. 5 - Optical microscopy images of feathers before the deposition of the hybrid material

(A) and after the deposition.

Fig. 6 - SEM images of sole feathers (A) and feathers covered with the hybrid material (B, C).

Fig. 7 - Optical microscopy images of mica surfaces with adsorbed unmodified -CaCOa (A) and μ-CaCOi coated by PAH (B).

Detailed description of the respective steps of the synthesis and characterization of the hybrid material can be found below in examples.

Example 1

nAg synthesis

45 mg of AgN0₃ was dissolved in 250 ml of deionized water and then 5 ml of 1% trisodium citrate was added. The reaction was carried out for 10 to 60 minutes in an ultrasonic bath heated to 75°C. The obtained nAg colloid exhibits a maximum absorption peak at 418 nm. The obtained nanoparticles after 60 minutes of synthesis are spherical and their sizes range from 30 to 50 nm (Fig. 1). The process of the nanoparticles growth was also monitored using UV-Vis method (Fig. 2) - before each measurement the samples were diluted. The absorbance rises along with nAg concentration and the shift of the absorption peak can be attributed to the nAg growth. nAg obtained after 60 min synthesis were used in the following experiments. Example 2

One step synthesis of calcium carbonate matrix with embedded nAg.

The nanoparticles were embedded inside the matrix during this step of the synthesis. 50 ml of 0.03 M Na₂C0₃ with addition of PSS (4.8 g/dm³)and 50 ml 0.03 M Ca(N0₃)₂ were added simultaneously to 20 ml of nAg colloid placed in an ultrasonic bath. Ultrasound-assisted co- precipitation was carried out for 5 minutes at 25°C. The obtained dispersion was centrifuged at 4000 rpm for 5 min. The obtained precipitate was washed three times with deionized water and centrifuged using aforementioned conditions to remove excess of silver. SEM images of the hybrid material ^-CaC0 ) (Fig. 3) certify its high monodispersity (the diameter of the microparticles is about 2 Dm). The microparticles are spherical as it is characteristic for crystals composed of vaterite. They also exhibit high porosity enabling efficient encapsulation of nAg inside them.

Example 3

Quantitative analysis of silver content in the hybrid material

Qualitative analysis was carried out to prove the presence of nAg inside the calcium carbonate matrix. To do this the matrix was dissolved using 0.2 M EDTA and then UV-Vis spectrum of the solution was acquired (Fig. 4). There was a peak in the spectrum with maximum at around 420 nm originating from the surface plasmon resonance of silver nanostructures - the same peak could also be observed in the spectra acquired during the synthesis of nAg (Fig. 2). This directly implies the presence of nAg inside the carbonate matrix. Quantitative analysis of silver embedded inside carbonate matrix was conducted using AAS (Atomic Absorption Spectroscopy) and provided that 1 g of the hybrid material in the form of dry powder contains 0.15 mg nAg. The AAS experiments were done in the following manner. 0.05981 g of dry hybrid material was dissolved in 30 ml of 0.12 M HN0₃ which also provided a baseline for the measurement. The reference samples containing silver in concentrations: 1 ; 2; 3; and 4 mg/dm were prepared by dissolving appropriate amounts of silver in 0.12 M HN0₃. It was found out that 21% of the initial amount of silver was embedded inside the carbonate matrix. According to the microbiological tests (Example 6) even such little amount of nAg embedded inside the hybrid material is enough to ensure its antimicrobial activity. Example 4

Sustained release of silver from the hybrid material.

1200 ml of the obtained hybrid material suspension was centrifuged at 4000 rpm. The material was washed with deionized water and centrifuged using aforementioned conditions three times. The obtained material was divided into two parts. The buffer solutions were prepared by mixing appropriate amounts of 0.2 M Na₂HP0₄ and 0.1 M citric acid, and their pH was measured using pH-meter. Respective buffer solutions were added to each of the two parts of the hybrid material (8 ml) of and the dispersions were left to mix on a magnetic stirrer (Table I). At specified time intervals the samples were centrifuged at 4000 rpm, to separate the solution from the hybrid material. For further analyses the supematants were collected and another 8 ml of the respective buffer solution was added to the separated microparticles so the silver could be release to silver-free medium again.

Table I shows sustained release data acquired using AAS method for different intervals of time. The results indicate long-term release of silver even in acidic and constantly mixed solution.

Table I. Sustained release of silver from the hybrid material to buffer solutions.

Example 5.

Adsorption of the hybrid material on natural down feathers.

The samples of down feather (0.025 g each) were immersed in 10 ml of 0.6% water dispersion of the hybrid material for 30 minutes. Down feathers were then removed from dispersion and dried under nitrogen. The samples were investigated using optical microscopy (Fig. 5) and SEM (Fig. 6). The acquired images confirm attachment of the hybrid material to the surface of the feathers. Example 6

Cationic modification of the hybrid material using cationic polymer and adhesion of such modified microparticles on a surface with specified surface charge.

The obtained hybrid material was immersed in the solution of cationic polymer PAH (1 g/dm in 0.1 M NaCl) for 15 minutes, then the material was separated by centrifugation and washed with deionized water. On the clean surface of mica which has negative surface charge, the modified and unmodified microparticles were adsorbed by immersion of the mica plates in water dispersions of these materials. After the adsorption process the mica plates were washed with deionized water and then dried under nitrogen. The adsorption on mica plates was investigated using optical microscopy (Fig. 7). The obtained images clearly show that the number of cationically modified microparticles adsorbed on negative mica surface is much larger then the number of the unmodified ones.

Example 7

Microbiological tests in suspension.

Microbiological tests were carried out for the hybrid material as well as for the feathers protected with it. All experiments were done in bacterial suspensions (in physiological salt) of a given concentration expressed in McFarland scale. McFarland scale is based on densitometric measurements standardized on barium sulfate colloids. McFarland scale and respective standards concentrations can be found in Table II. The initial rise in the bacterial concentration after addition of the microparticles can be attributed to their contribution to the scattering of the probing light beam. The following changes are connected only with rise or decrease of the bacterial concentration. The bacterial concentration measurements in the case of down feathers were done only after removing the feathers from the suspension.

Decrease in the bacteria concentration was observed for sole nAg colloid (Table III) as well as for the hybrid material of μ-Ο&ΟΟ^ containing nAg (table IV). The hybrid material adsorbed on the surface of down feathers protected it from development of bacteria (table V). On the other hand sole unprotected feathers underwent bacterial contamination. Table II. McFarland scale.

Table III. Antibacterial action of nAg.

Table IV. Antibacterial and antifungal action of the hybrid material of μ-CaCOB containing

Table V. Bacteriostatic action of feathers protected with the hybrid material.

Table VI. Bacterial develo ment on the sole un rotected feathers.

Bacter al suspension without the addition of feathers; measurement done after 24h of incubation.

Bacterial suspension with the addition of unprotected feathers; measurements done after 24h of incubation.

Claims

Claims:

1. Hybrid material containing silver nanoparticles, comprising of calcium carbonate microparticle matrix with addition of a polyelectrolyte and with silver nanoparticles embedded inside.

2. The material according to claim no.l, wherein polymers and/or copolymers containing ionized moieties such as sulfonic, carboxylic, phosphoric, including biopolymers are used as the polyelectrolyte.

3. The material according to claim no.l, wherein alkali metal salts of poly(vinylsufonic acid), poly(methacrylic acid), carboxymethyl cellulose are used as the polyelectrolyte.

4. The material according to claim no.l, wherein sodium polystyrene sulfonate are used as the polyelectrolyte.

5. The material according to claim no.l, wherein it is modified cationically or anionically.

6. The material according to claim no.5, wherein it is modified cationically or anionically by adsorption of at least one layer of ionic polymer.

7. The material according to claim no.5 or 6, wherein it is modified by a synthetic and/or natural polymer.

8. The material according to claim no.5 or 6, wherein it is modified by a polymer selected from a group comprising of polyallylamine, polyethyleneimine, poly(vinylpyridine) salt, chitosan, protamine, poly(methacrylic acid) salt, poly(styrenesulfonic acid) salt, poly(2-acrylamido-2-methylpropane sulfonic acid) salt, carboxymethyl cellulose salt, sulfonated dextran salt.

9. The process of making of the hybrid material containing silver nanoparticles comprising of ultrasound-assisted co-precipitation of calcium carbonate in aqueous medium in the presence of polyelectrolyte and silver nanoparticles.

10. The process according to claim no.9, wherein co-precipitation is done by mixing solutions of water-soluble calcium salt and water-soluble carbonate salt containing addition of polyelectrolyte and silver nanoparticles (nAg).

11. The process according to claim no.10, wherein ratio of calcium to carbonate ions is 1 : 1.

12. The process according to claim no.10, wherein the concentration of calcium salts and carbonate salts used is between 0.001 M andl M.

13. The process according to claim no.12, wherein the concentration of calcium salts and carbonate salts is about 10^" M.

14. The process according to claim no.9, wherein a polymer and/or a copolymer containing ionized moieties such as sulfonic moieties, carboxylic moieties, phosphoric moieties, including biopolymers are used as the polyelectrolyte.

15. The process according to claim no.9 or 14, wherein alkali salts of poly (vinyl sulfonic acid), poly(methacrylic acid), carboxymethyl cellulose are used as the polyelectrolyte.

16. The process according to claim no.9 or 14, wherein polystyrene sulfonate is used as the polyelectrolyte.

17. The process according to claim no.16, wherein polystyrene sulfonate concentration is 0.1-10 g/dm³.

18. The process according to claim no.16 wherein, molar ratio of the polymer sulfonic moieties to the calcium ions is not lower than 1 :3.

19. The process according to claim no.9, wherein the co-precipitation temperature range is 15-50°C.

20. The process according to claim no.10, wherein calcium nitrate or calcium chloride are used as calcium salts and sodium carbonate, potassium carbonate or other alkali salts are used as carbonates.

21. The process of claim 20, wherein calcium nitrate and sodium carbonate are used.

22. The process according to claim no.9, wherein silver nanoparticles are used in the form of colloid suspension.

23. The process according to claim no.9 or 22, wherein silver nanoparticles used are obtained as a result of ultrasound-assisted reduction of silver ions with trisodium citrate in water medium in temperature lower than the water boiling temperature.

24. The process according to claim no.9, wherein to the water solution of silver nitrate solution of trisodium citrate is added, the mixture is sonicated and heated for 10-60 minutes, preferably in 70-85 °C temperature range, then Na₂C0₃ containing addition of PSS and solution of Ca(N0₃)₂ are added to the obtained nAg colloid solution, then ultrasound-assisted co-precipitation takes place for 1-20 minute in room temperature, then the obtain dispersion is centrifuged to obtain the final precipitate.

25. The process according to claim no.9, wherein the hybrid material is modified cationically or anionically.

26. The process according to claim no.25, wherein the cationic or anionic modification is done by adsorption of at least one polymer layer.

27. The process according to claim no.26, wherein the surface is covered using layer-by- layer electrostatic self-assembly technique.

28. The process according to claim no.25, wherein to synthetic or natural polymers are used to cover the surface.

29. Use of the hybrid material consisting of silver nanoparticles embedded in the matrix of calcium carbonate microparticles with addition of the polyelectrolyte as antibacterial and antifungal agent.

30. Use according to claim no.29, wherein the hybrid material is used to protect water solutions and water dispersions or non water solutions and dispersions and polymer materials such as lacquers, paints, building materials.

31. Use according to claim no.29, wherein the hybrid material is used to protect goose down feathers from negative influence of microorganisms.

32. Use according to claim no. 31, wherein the goose down feathers are used as fillings of ready products such as blankets, pillows, jackets, suits.