"METHOD FOR PRODUCING NANOPARTICLES, PROTEIN STABILIZED SILVER NANOPARTICLES, PRODUCTION OF ANTIBACTERIAL TEXTILE PRODUCTS, ANTIBACTERIAL TEXTILE PRODUCTS AND BIOREMEDIATION METHOD"

Field of the Invention

The present invention discloses a synthesis of silver nanoparticles by a fungus extract with reductase activity and the presence of anthraquinone derivatives, for example, Fusarium oxysporum species, associated to proteins as colloidal stabilizer and easy grip as stabilizing material in textile products made of cotton inhibiting microorganisms like Staphylococcus aureus. This invention shows the biological use of this material with the aim of sterilizing textile products that can be used in aprons, other clothing used in the field of healthcare, or sportswear garments. This method includes the treatment of effluents after sterilization of textile products with silver nanoparticles with a new system of bioremediation using the bacteria Chromobacterium violaceum which absorbs the nanoparticles released during the process of washing the impregnated fabrics, avoiding environmental contamination. Background of the Invention The problems with resistant or multi-resistant pathogens are evident today, such as Staphylococcus aureus which is resistant to methicillin and Candida albicans which is

resistant to fluconazole (Schaller e col. Skin Pharmacol. Physiol. 17, 31-36, 2004). The introduction of a new generation of textile products incorporating antimicrobial agents to avoid contamination by way of wounds and infections, has been analyzed (Yin e col. J. Burn Care Rehabil. 20, 195-200, 1999). It is widely known that silver ions and silver-based compounds have strong biocide activity. Hence, silver ions have been used in various kinds of formulations (Sondi and Salopek-Sondi, J. Colloid Interface Sci. 275, 177-182, 2004) and, recently, it has been shown that hybrids of silver nanoparticles with hyperbranched macromolecules exhibit an effective antimicrobial surface (Aymonier et al. Chem. Commun. 24, 3018-3019, 2002). A silver colloid incorporated into the fabric (Contreet-H ®) results in the significant decrease of epithelium-infected pathogens. The silver released onto the keratinocytes infected in the humid healing environment improves the benefit/risk (toxicity) ratio compared with a fabric without silver (Schaller e col. Skin Pharmacol. Physiol. 17, 31-36, 2004). Similar results with E. coli have been obtained with silver nanoparticles (Sondi and Slopek-Sondi, J. Colloid Interface Sci. 275, 177-182, 2004; Baker e col. J. Nanosci. Nanotechnol. 5, 244-249, 2005).

The interest in antibacterial agents in textiles has increased over recent decades due to various environmental contaminations. The publication of various works in this field proves this trend (Yeo e col. J. Materials Sci. 38, 2143-2147,

2003; Yeo and Jeong, Polymer Intern. 52, 1053-1057, 2003). The effectiveness of fabrics treated with nanometric colloidal systems has been demonstrated (Lee e col. J. Materials Sci. 38, 2199- 2204, 2003; Lee and Jeong, Textile Res. J. 74, 442-447, 2004; Jain and Pradeep, Biotechnol. Bioeng. 90, 59-63, 2005).

Various patents related to this method show that nanoparticles of 10 to 40 nm were prepared with a silver oxide surface using the ammoniacal silver nitrate / sodium hydroxide / glucose / nitric acid method. Therefore, the end product is oxidized in the presence of surfactants and hydrogen peroxide (Zhu and Zhu, WO2003101200-A (2003); CN 1473553-A (2004)). Similar nanoparticles of silver and zinc oxide were prepared and impregnated in textile fabrics (Lie e col. CN 1563553-A (2005)). Injections of silver nanoparticles (colloidal) in fabrics are prepared for hospital clothing (Yang, KR 2004823557-A (2004)) and with silver ions sprayed over the fabrics by high-pressure process (Yang and Zhang, CN 1473628- A (2004)). Silver powder nanoparticles (10-100 nm), produced by ultra-sound, silver nitrate/hydrazine, were fixed to the fabric heating the nanoparticle suspension at 60 ⁰ C together with the fabric under agitation for 30 minutes and then cooled (Huang, CN 1557502-A (2004); Huang e col. CN1557150-A (2004)) or metallic silver / nitric acid / polyglycol / hydrazine (Huang CN 1557163-A (2004)). Antibacterial fabrics were also obtained by spraying silver vapor on fabrics in the presence of argonium and

under pressure of 10 mbar which were recoated with a controlled transport layer obtained by polymerization using plasma with 95% oxygen and 5% hexamethyldisiloxane at 0.07 mbar (Wagener e col. WO2005049699-A2 (2005)). As noted above, many procedures for obtaining silver nanoparticles are easy to perform. However, the greatest difficulty lies in preparing particles having controlled size. It is likely that the biological synthesis could be controlled more easily than the other methods and also the nanoparticles would be directly stabilized in the process (proteins). By using the metabolization properties of metals (dissimilation) by fungus, the biosynthesis of inorganic nanomaterials using eukaryotic organisms such as fungus, could be achieved intracellularly (Sastry e col. Current Sci. 85, 162-170, 2003) as well as extracellularly (Ahmad e col. Colloids Surf. B. Biointerfaces 28, 313-318, 2003). In this latest innovation, the fungus Fusarium oxysporium, after growth in a culture under suitable conditions, was separated by centrifugation and the biomass was washed several times with distilled water and then centrifuged. The biomass was placed in sterile conical flasks and then silver nitrate (aqueous solution) was added, and finally the material was incubated at 27 ⁰ C. The growth of the nanoparticles was monitored from time to time showing the production of silver nanoparticles in the range of 5-100 nm (Mukherjee and col. US Patent 6.537.344 (2003)), characterizing a rather large interval and

indicating little control over the size of the particles produced.

Based on the methods of producing silver nanoparticles as described above, there is a difficulty in generating these particles in a narrow diameter range, also by biosynthesis process through fungus (Mukherjee e col. US Patent 6.537.344 (2003)). The other methods, besides the difficulty in obtaining nanoparticles in a narrow range, comprise complex and arduous procedures, apart from posing a serious potential of environmental contamination. Brief Description of the Invention

Faced with the problem found in the state of the art, the present invention relates to a method of preparing silver nanoparticles, impregnation and sterilization of fabrics with nanoparticles without generating effluents. More specifically, the present invention relates to the preparation of silver nanoparticles that can act in methods of impregnating textile products with the aim of sterilizing and allowing successive washing without significant loss of the silver nanoparticles. Still more specifically, the present invention describes the preparation of silver nanoparticles by a biosynthetic method through fungus and fixation thereof onto textile products in a method of recycling the nanoparticles produced without being lost to the environment (zero effluent). It also describes the treatment of the effluent obtained from washing the fabric, so as not to impact the environment with any nanoparticles that may be released during

fabric washing. Said method involves bacterial treatment for bioabsorption of the nanoparticles.

Brief Description of the Drawings

Below is a brief description of the drawings that accompany this specification to enhance the understanding and illustration thereof, and includes the following:

Figure 1 presents the spectrum of absorption in the visible-ultraviolet region recorded based on the reaction time in aqueous solution of nitrate reductase and anthraquinone derivative containing 10-3 M of AgNO3.

Figure 2 shows the result of transmission microscopy of the silver nanoparticles obtained in this patent. These particles are 3 run in diameter.

The energy dispersion spectrum (EDS) of the cotton fabric with silver nanoparticles and the sweeping electronic microscopy (SEM) of the fibers of this fabric can be seen in Figure 3.

The sweeping microscopy of the cotton fiber with the incorporation of the silver nanoparticles, after the antimicrobial activity test against S. aureus, is presented in Figure 4.

Figure 5 presents a sweeping electronic microscopy of cotton fibers without the incorporation of silver nanoparticles (control) after the antimicrobial activity test against S. aureus increased 75 times (A) and 1400 times (B).

Figure 6 presents the visual UV spectra of the wash waters of the fabrics with silver nanoparticles: A) before treatment with C. violaceum, B) after treatment with 10 ² UFC/mL of C. violaceum, C) after treatment with 10 ⁵ UFC/mL of C.

Q violaceum, D) after treatment with 10 UFC/mL of C. violaceum.

Detailed Description of the Invention

Silver nanoparticles are obtained by biosynthetic means and the method of obtaining said nanoparticles comprises the following stages: - cultivation of the fungus in a liquid medium;

- biomass filtration;

- incubation of the filtrate;

- separation of the fungus; and

- addition of AgNO3. The fungus cultivated in the present invention is selected based on its reductase activity and on the presence of an anthraquinone derivative. The nitrate reductase activity and the anthraquinone were evaluated according to the usual methods from literature. As an example of the fungus used in the present embodiment, there are various species of Fusarium oxysporium (5 of different origin) such as: subgentinans, graminearum, alomini, equiseti, solaniglicerum. Additionally, others types and species were used, having nitrate reductase and anthraquinone derivative, such as Desulfovidro sp, Piriformospora, Pisolithus sp,

Streptomyces sp, Sporisorium sp, Penicillium sp, Neurospora sp, Aspergillus, sp, among others.

The liquid phase for growth of the fungus was preferably selected from the means of cultivating mate extract with yeast extract.

The growth of the fungus is obtained during a period of incubation, that is, of cultivation, of around 6 days at a temperature within the range of 25-30 ⁰ C. However, the best growth results of the fungic mass are obtained through incubation at a temperature ranging between 27 and 29 ⁰ C.

After the incubation period, the biomass is submitted to filtration, the fungic mass is re-suspended in sterile distilled water and then undergoes the aqueous phase for a period of around 72 hours preferably at a temperature of 28 ⁰ C. After the period of 72 hours, the biomass is again submitted to filtration, and the fungus is separated from the liquid medium. A volume of around 50 to 10OmL of silver nitrate (AgNOs) was added to the liquid extract generated, that is, to the filtrate, in the concentration in the range of 0.1 to 3OmM, preferably using AgNO ₃ in the range of 0.1 to 30.0 mM, more preferably using AgNO ₃ in the range of 1.5 to 5mM. Thereafter, the substrate was incubated for around 48 hours under heat conditions varying between 25 and 3O ⁰ C, in order to obtain the silver nanoparticles at the end of the incubation period. Additionally, in the method of this present

embodiment, the silver nitrate solution can also be used in proportions between 10-40 mg per gram of biomass, the ratio of water to the fungic mass being 10:1 v/w.

The kinetics of formation of the silver nanoparticles was accompanied by way of visible ultraviolet absorption spectroscopy technique throughout the entire incubation period of the substrate with AgNO ₃ , as shown in

Figure 1.

One particular aspect of the method of obtaining silver nanoparticles that differentiates it from the methods known in the art, as described in document US Patent 6.537.344 (2003), resides in the fact that in the method of this present embodiment, the biomass filtrate was used, that is, the fungal liquid which contained the nitrate reductase enzyme and the anthraquinone derivative.

The silver nanoparticles obtained by means of the method developed in this present embodiment were measured using the transmission electronic microscopy. The results showed that the silver nanoparticles obtained had a diameter varying between 3 and 20nm, most nanoparticles having a diameter of around 3 nm as shown in Figure 2.

The silver nanoparticles now obtained by said method were characterized using the techniques of visible ultraviolet absorption spectroscopy, sweeping and transmission electronic microscopy and by means of the X-Ray fluorescence

technique.

The second objective of the present embodiment consists of the development of a method to produce antibacterial textiles by impregnating the silver nanoparticles in material with a biocide purpose, such as fabrics. Said silver nanoparticles were obtained by way of the method developed and described in the present invention.

For the present embodiment, fabric is understood to be any weave of natural or synthetic fibers, such as cotton, which is used, for example, but not restricted to aprons, plasters, medical materials, and others.

The method of impregnating the silver nanoparticles into materials begins by immersing the fabric in around 10OmL of the silver nanoparticles suspension, causing the fabric to absorb the silver particles. The suspension used in the present embodiment was obtained in the method of obtaining said nanoparticles that are also the subject matter of this present invention.

Immersing the fabric in the suspension of silver nanoparticles was homogenized by way of ultracentrifugation preferably at a time interval of around 10 to 40 hours, at an agitation speed of around 100 to 1000 rpm and preferably at a temperature of 10 to 4O ⁰ C.

After the homogenization period, the substrate (fabric and silver nanoparticle suspension) is filtered and the

supernatant is eliminated from the medium in order to obtain just a concentrate of nanoparticles in the fabric. Said fabric then undergoes drying at a temperature varying between 60 and 8O ⁰ C. Thereafter, at the end of the impregnation process, the fabric is submitted to sterilization.

The supernatant now eliminated contains the silver nanoparticles that were not fixed to the fabric. Said supernatant may be used subsequently in new fabric treatments. Therefore, in order to promote the absorption of said nanoparticles into the fabric, the supernatant is reused, but in a closed circuit, so as not to generate effluent. At the end of the impregnation process, the fabric was submitted to sterilization.

As shown in Figure 3, the effectiveness of the fabric impregnation process was evaluated by means of the presence of the silver nanoparticles in the fibers of the fabric by means of the energy dispersion spectrum in conjunction with sweeping electronic microscopy (SEM-EDS).

An analysis of the X-Ray fluorescence revealed that the result of the impregnation process was a concentration of silver nanoparticles in the fabrics of around 1 to 4% part by weight.

Another particular aspect of the present invention lies in the fact that this present fabric impregnation process of silver nanoparticles differs from the methods known in the art, since the art methods involved impregnation with high

pressure, injection, padding, and the diameter of the particles applied is much greater (1-100 nm) than that obtained for the nanoparticles achieved by way of the method set forth in this present invention. The antibacterial textile products now obtained by the impregnation technique have biological use, since said products can be sterilized and used as aprons or other clothing used in the field of healthcare. Additionally, said textile products can also be used as sportswear garments. The antibacterial activity was evaluated against a gram-positive bacteria and a gram-negative bacteria. The fabrics were tested, introducing a sample of the fabric onto the bacteria culture and a non-ionic agent for around 24 hours.

A sweeping electronic microscopy showed the absence of microorganisms in the fabrics impregnated with silver nanoparticles, as shown in Figure 4, whereas in the control group it was possible to identify the presence of microorganisms in fabric samples not impregnated with silver nanoparticles, as shown in Figure 5. The third objective of the present invention consists of the bioremediation method of the silver nanoparticles. The bioremediation method of the nanoparticles initially comprises a washing stage of the fabrics previously impregnated with silver nanoparticles pursuant to the impregnation process

described herein, which is also the subject matter of this present embodiment.

The fabrics were successively washed by usual washing procedures, such as those carried out in a launderette. Although there is a loss of less than 10% of particles during washing, the effluent generated was submitted to treatment with a suspension of Chromobacterium violaceurn (CCT 3496) in concentrations of 10 ² , 10 ⁵ and 10 ⁸ UFC/mL under agitation at a speed of around 20 to 300 rpm, at a temperature of between 10 and 50 ⁰ C and incubated for a period of 5 to 36 hours associated to proteins such as colloidal stabilizer and easy grip as sterilizing material in textile products made of cotton inhibiting microorganisms like Staphylococcus aureus.

Next, the substrate was filtered and the wash waters were analyzed before and after treatment using the visible ultraviolet absorption spectroscopy technique, demonstrating the complete removal of the silver nanoparticles from the water when the water is treated with C. violaceum in the concentration of 10 ⁸ UFC/mL, as shown in Figure 6. The in vitro results of the antibacterial activity of the silver nanoparticles clearly show that this invention has application in the field of human or animal healthcare, more specifically in the combat of hospital infections and in the protection of wounds and in the process of healing by means of impregnating silver nanoparticles onto textile products.

For an improved understanding of the invention, the invention will now be detailed in the form of examples. However, the examples described below are purely for illustration and are not intended to define limits for the invention. EXAMPLE 1 : DETERMINING THE ANTHRAQUINONE.

The anthraquinone was determined by using the fine-layer chromatography technique in silica gel using chlorophorm-methanol-acetic acid (195:5: 1) obtaining an R _f of 0.65 and, using benzene-nitromethane-acetic acid (75:25:2) obtaining an R _f of 0.85, corresponding to 2-acetyl-3,8-dihydroxy- 6-methoxy anthraquinone or its isomer 2-acetyl-2,8-dihydroxy-6- methoxy anthraquinone. The structure was corroborated by fluorescence data, as already described in literature (Baker and Tatum, J. Ferment. Bioeng. 85, 359-361, 1998). EXAMPLE 2: ANALYSIS OF THE NITRATE REDUCTASE ACTIVITY.

The nitrate reductase activity was determined through the reaction of nitrate with 2,3-diaminophthalene as described in literature (Misko e col. Anal. Biochem. 214, 11-16, 2003).

EXAMPLE 3: PRODUCING SILVER NANOPARTICLES

The fungic growth in liquid phase was carried out in malt extract (2-4%) and yeast extract (0.3-1.0%) preferably at a temperature of around 27 to 29 ⁰ C preferably for a period of 6 days. The biomass was filtered and re-suspended in sterile water,

subsequently undergoing aqueous phase, preferably for a period of 72 hours preferably at a temperature of 28 ⁰ C. After this period, the biomass was filtered generating a liquid extract, to which AgNO ₃ was added. The substrate is preferably incubated for around 48 hours under a temperature ranging between 25 and 30 ⁰ C, in order to obtain the silver nanoparticles at the end of the incubation period. EXAMPLE 4: IMPREGNATION PROCESS.

For example 2, a sample of the fabric was immerged in around 10OmL of the filtrate of silver nanoparticles, which was obtained by way of Example 1 and incubated preferably for a period of 24 hours. Next, it was submitted to ultracentrifugation preferably at a speed of 600 rpm. After this period, the supernatant was eliminated to concentrate the nanoparticles and the fabrics were dried at a temperature of 60-

8O ⁰ C. The concentration of nanoparticles in the fabrics was 2% part by weight.

EXAMPLE 5: EVUATING THE ANTIMICROBIAL ACTION.

The antibacterial activity was evaluated against the species Staphylococcus aureus, a gram-positive bacteria and against the species Klebisiela pneumoniae, a gram-negative bacteria. The fabrics were tested, introducing a sample of the fabric on a culture of S. aureus recently incubated with a concentration of 1.3-1.6 10 ⁵ UFC/mL and 0.5% of non-ionic agent for 24 hours.

EXAMPLE 6: PROCESS OF BIOREMEDIATION OF THE PARTICLES RELEASED IN THE WASH WATERS

The fabrics previously impregnated with the silver nanoparticles were submitted to successive, consecutive washing and the effluent generated was subsequently submitted to treatment. The washing procedure was carried out at least 5 times. The treatment of the effluent generated was carried out with around 4 mL of a suspension containing Chromobacterium violaceum CCT 3496, which was incubated for around 12 hours preferably at a temperature of 30 ⁰ C in a ^" medium of glucose, peptone, yeast extract and tryptophan. Around 100 mL of the wash water was inoculated in said suspension. The suspensions of silver nanoparticles with C. violaceum were kept under agitation preferably at a speed of 120 rpm and at a temperature of around 30 ⁰ C for a period of 24 hours.