WO2011045623A1

WO2011045623A1 - Titanium dioxide nanostructured materials comprising silver and their use as antimicrobials

Info

Publication number: WO2011045623A1
Application number: PCT/IB2009/006900
Authority: WO
Inventors: Tessy María LOPEZ GORNE
Original assignee: Arce Macias, Carlos Francisco; MUÑOZ OCHOA, José Francisco
Priority date: 2009-10-12
Filing date: 2009-10-12
Publication date: 2011-04-21

Abstract

A bactericide or antimycotic agent comprising nanostructured titania, silica or mixed oxides titania-silica with silver and a functional group.

Description

TITANIUM DIOXIDE NANOSTRUCTURED MATERIALS COMPRISING SILVER AND THEIR USE AS ANTIMICROBIALS

DESCRIPTION

This invention is related to the synthesis of nanostructured inorganic silver- titania materials with antibacterial and antimycotic properties.

BACKGROUND FOR THE INVENTION

Since material properties are structure-dependent, new and interesting properties are expected from unusual or complex structures. During the last decade there has been an incredible growing in research and development in the nanoscience and nanotechnology area. The increased focus on the nanoscale has been driven by the significant potential that the application of nanoscience and nanotechnology can provide solutions to basic industrial and societal needs. Materials whose size is reduced to the nanometer scale often exhibit new and unique properties both interesting from an academic and useful from a technological perspective. Technological advances in the field of imaging at the nanoscale have allowed new insight and new mechanistic understanding. Developments within electronic probe microscopes will be highlighted along with a number of examples of current nanotechnology research.

Bactericide is a most important example of nanobiotechnology applications. Silver solutions and silver supported materials have been used as bactericide and fungicide [1-3]. Silver nanoparticles in solution or supported on appropriate substrates are currently used due to their effective action adversely affecting the cellular metabolism and inhibiting cell growth. The chemistry has revealed that an Ag deposit is not toxic to human cells in vivo and is reported to be biocompatible [4-6]. The colloidal and silver salts have several limitations due to the cost of the Ag and their toxicity at high concentrations. Nevertheless, supported silver particles obtained by the sol-gel process 0 to synthesized Ag- nanostructured materials are successful technology when fixed on adequate supports due to their increased bactericide effect. The crystal size and high Ag nanoparticles dispersion are necessary to increase the bactericide effect. The bactericidal activity of silver loaded alumina in deionized water at room temperature was investigated by other research groups. The survival changes of E. coli in the presence of different silver-alumina nanomaterials as a function of time is studied by P. Quintana et al. Under the control condition, the viable cell numbers were counted (see in Fig. 1a in the reference). It was observed that in the presence of Ag/AI₂03 and AgCI/Al₂0₃ the E. coli in the suspension was completely inactivated after only 30 min and 60 min, respectively. Silver supported alumina nanomaterials were so effective that the surviving E. coli cells decreased sharply even in the first 10 min. However, with AI2O3 control, without silver addition, the behavior was similar to that in the control even after 60 min. This means that the adsorption of E. coli on alumina is negligible, and the dramatic decrease in E. coli survival is ascribed to the highly efficient bactericidal activity of Ag/AI₂03 and AgCI/AI₂03. It is well known that Ag+ at high concentrations exhibits bactericidal activity [17-19], and Ag+ eluting from Ag/AI₂0₃ cannot be avoided under the experimental conditions. The quantitative analysis of eluted silver ions by ICP-AES showed that there was 0.897 mg/L Ag+ and 0.081 mg/L Ag+ eluted from Ag/Al₂0₃ and AgCI/AI₂0₃, respectively, when the nanostructured materials were immerged singly in deionized water in the absence of E. coli for 60 min.

The preparation of uniform nanosized particles with specific requirements in terms of size, shape, and physico-chemical properties is of great interest in the formulation of new products with many biotechnology applications 0. Resistance of bacteria to bactericides and antibiotics has increased in recent years due to the progress of resistant strains. Some antimicrobial agents are extremely irritant and toxic and there is interest in finding ways to formulate new types of safe and cost-effective biocide materials. Catalytic oxidation by metallic silver and reaction with dissolved monovalent ion contribute to its bactericidal effect. By the other hand, in low concentrations, silver is nontoxic to human cells. The size-dependent Ag/Ti0₂ interaction of silver nanoparticles with gram-negative bacteria and virus has also been showed 0. The electrostatic attraction between negatively charged bacterial cells and positively charged nanoparticles is fundamental for the activity as bactericide [21-22]. H. M. Coleman et al. [23] report that PVC interacts with titania and can be use as bactericide. Perhaps, the material modification can be made also to improve its behavior against the bacteria, that's why it is important the use of nanoparticles with a high surface area and a better dispersion of the silver particles that increases the contact interface between the bactericidal and the cell [23-24]. As it has been used as photocatalyst for the petrochemical industry, some investigators have also used this idea beside the bacteria taking the silver supported titania as a photocatalytic bactericide [25]. By the other hand, Polyene antimycotics, sometimes referred to as polyene antibiotics, are a class of antimicrobial polyene compounds that target fungi. Amphotericin B, nystatin, and natamycin are examples of polyene antimycotics. Their chemical structures feature a large ring of atoms (essentially a cyclic ester ring) containing multiple conjugated carbon-carbon double bonds (hence polyene) on one side of the ring and multiple hydroxyl groups bonded to the other side of the ring. Their structures also often have a d-mycosamine (a type of amino-glycoside) group bonded to the molecule. The series of conjugated double bonds typically absorbs strongly in the ultraviolet-visible region of the electromagnetic spectrum. These polyene antimycotics are typically obtained from some species of Streptomyces bacteria. The polyenes bind to ergosterol in the fungal cell membrane and promote leakiness which may contribute to fungal cell death.

Other application is the bacterial inactivation of Escherichia coli on Ag-cotton textiles were investigated under different experimental conditions with novel Ag nanoparticles fixed on cotton textiles [26]. ADDITIONAL TITANIA, SILICA OR TIO9-SIO? PATENTS USING SOL-GEL METHOD

US Patent 5492763 - Infection resistant medical devices and process. The resent invention relates generally to medical devices designed to come into contact with body tissue and, more particularly, to medical devices which have been rendered bacteriostatic/bactericidal and a process for making them so. More specifically, it is an object of the present invention to provide a medical device formed from either polymeric, metallic and/or ceramic materials, or a combination of such materials, with a permanent subsurface bacteriostatic/bactericidal stratum to a predetermined depth and introduced therein by injecting bacteriostatic/bactericidal ions of sufficient concentration into the surface of the medical device without adversely affecting the biocompatibility of the device. Albeit the level of concentration of the injected bacteriostatic/bactericidal ions to impart the desired bacteriostatic/bactericidal property to the medical device depends on the material thereof, a minimum sufficient level of bacteriostatic/bactericidal ion concentration is about 1 *10¹⁵ ions/cm³ Since it does not leach, the acquired bacteriostatic/bactericidal property of the medical device remains effective for the useful life of the device. Preferably, the bacteriostatic/bactericidal ions comprise at least one member of the class of Ag, Au, Cu, Pt, Ir, Mg, Pd and its respective compounds and alloys. Preferably, the injection of the bacteriostatic/bactericidal ions is effected in a chamber evacuated to a vacuum of at least about 10^"4 torr. Depending on the substrate, the treated medical device also is thromboresistant and wear resistant.

United States Patent 6254894. The present invention relates to water purification compositions comprising silver and a second material, such as aluminum or zinc metal, to methods of treating or purifying water using this composition. W095/15816, 1995. The bactericidal activity of copper-deposited titanium dioxide thin film (Cu Ti0₂) was investigated under very weak ultraviolet (UV) light illumination. To elucidate the roles of the film photocatalyst and the deposited copper in the bactericidal activity, cells from a copper-resistant Escherichia coli (E. coli) strain were utilized. A decrease in survival rate was not observed with the copper-resistant cells under dark conditions, but when illuminated with a very weak UV intensity of 1 μ\Λ// η², the survival rate decreased, and suggesting photocatalytic bactericidal activity. The decay curve of survival on the Cu/Ti0₂ film under very weak UV light illumination consisted of two steps, similar to the survival change of normal E. coli on T1O₂ films under rather strong UV illumination. The first step is due to the partial decomposition of the outer membrane in the cell envelope by a photocatalytic process, followed by permeation of the copper ions into the cytoplasmic membrane. The second step is due to a disorder of the cytoplasmic membrane caused by the copper ions, which results in a loss of the cell's integrity. These processes explain why the CU/T1O₂ film system shows an effective bactericidal activity even under very weak UV light illumination.

PCT/AU2004/000681. An in-line cleaning and sanitation apparatus for cleaning a liquid, the apparatus including electronic oxidation means to increase the oxidation reduction potential of the liquid, and ionization means to produce ions having an algaecidal or bactericidal effect into the liquid, in that order together with ultrasonic cleaning means to introduce sound waves into the liquid, and wherein the ionization means, the ultrasonic cleaning means and the electronic oxidation means are operated simultaneously for a period to clean and sanitize the liquid in the absence of added salt, chlorine or other chemicals. At least two electrolytic cells, including an electrolytic ionization cell to produce ions having an algaecide or bactericidal effect into the liquid, and an electrolytic oxidization cell to increase the oxidation reduction potential of the liquid. Ultrasonic cleaning means to introduce sound waves into the liquid, wherein the at least two electrolytic cells are provided in the order of electrolytic oxidization cell, and electrolytic ionization cell and are operated simultaneously for a period to clean and sanitize the liquid in the absence of added salt, chlorine or other chemicals.

United States Patent 4906466. An antimicrobial composition for topical use or for incorporation into a coating or structural composition comprises an antimicrobial silver compound, preferably silver chloride, deposited on a physiologically inert oxide synthetic particulate support material in particulate form. A preferred support material is titania containing one or more of the crystalline forms anatase, rutile, and brookite.

US Patent 6444726 - Biocidal compositions. The present invention concerns biocidal compositions. More especially, it concerns compositions comprising a silver compound supported on an oxide support. Many proposals have been made to utilize the antimicrobial action of silver and silver compounds since the Romans discovered the bactericidal or bacteriostatic properties of silver drinking vessels. An antimicrobial composition comprising an antimicrobial silver compound deposited on an inert oxide support. The preferred composition is described as AgCI deposited on a support such as titania.

OBJECTIVES

1. The development of nanostructured materials for use as bactericide or antimycotic agent.

2. Obtain nanostructured materials with silver high dispersed nanoparticles from 0.5 to 15 % wt in concentration. 3. The precursor of silver will be colloidal silver, sulphate, phosphate, chlorine, acetylacetonate or nitrate silver salts. Obtain nanostructured materials with alkoxide:water molar ratio between 1 :4 to 1 :24. Obtain nanostructured materials with alkoxide:solvent molar ratio between 1 :8 to 1 :64. Obtain nanostructured materials with reflux between 45°C to 90°C Obtain nanostructured materials at initial pH from 5 to 12. Using acid and bases both weak and strong to obtain the pH necessary in the initial "sol". The resultant solids are dried from 40 to 100°C during required time. The time of dry is between 24 hours to 144 hours. The heat rate ramp is between 2°C/min to 10°C/min. Optimization of materials to enable control of the following parameters:

• High dispersion of the silver on the support

• Substitution of silver atoms for titanium atoms in the sol-gel silver- titania net in nanostructured material

• Medium Lewis acidity concentration

• Structure: the existence of anatase, brookite or rutile and the mix of 2 or 3 crystalline phases

• Support particle size between 30 to 70 nm

• Metal particle size between 5 to 20 nm Optimization of BET area, pore size distribution, particle size, and degree of functionalization in nanostructured biomaterials. The surface of nanostructure bactericide must have hydroxyl, carboxyl, acid, ammonia, Chloride, ester or ether functional groups. 15. Or the mix of two or more of these functional groups.

16. The concentration of the functional groups varies from 0.001 to 1 mol%

17. Obtain a nanostructured material with high action to eliminate bacteria.

18. Obtain a nanostructured material with high action antimycotic. 19. Obtain a nanostructured material with free radicals.

20. Obtain a nanostructured material capable to release ketaconazole or triclosan or salicylic acid or other chemical molecule to attack in two ways the bacteria and fungus, controlling the liberation time.

21. Obtain a nanostructured material no toxic. 22.Obtain a nanostructured material biocompatible but not ingested.

23. The nanostructured materials will consist of nanostructured titania, silica or mixed oxides titania-silica with silver and additives prepared using sol-gel methods. These materials are functionalized in order to get biocompatible, no toxic and nanostructured particles. Table 1. Textural experimental values for different samples.

Table 2. Zone of inhibition with the Ag-nanoparticles

Bacterial strain Zone of inhibition

(mm)³ AgS0₄ AgAc AgN0₃ AgCI

P. aeruginosa 8.83 9.33 7.66 7.0

K. pneumoniae 7.0 8.33 10.16 7.16

S. marcencens 8.33 8.83 9.00 7.66

P. vulgaris 8.0 8.3 9.00 7.33

E. coli 7.66 8.16 9.66 7.0

S. aureus ATCC 10.12 10.66 10.33 7.0

25923

S. aureus ATCC 8.5 8.0 9.16 7.0

43300

S. typhimurium 8.0 9.0 8.66 8.83

S. dysenteriea 7.66 7.66 8.33 7.66

Disk's diameter was 7 mm

Example 1

Titania supported silver materials were prepared by the sol-gel method using titanium n-butoxide, ethanol and water with a molar ratio equal to 1 :8:4 and the amount of silver precursor to have 2% of silver. The silver precursor used was silver chlorine. Ammonium hydroxide was used to fix the pH 9. The suspension was vigorously stirred until the gel was formed. Finally was dried at 60°C in a muffle with rate heat ramp of 5°C/min. Example 2

Titania supported silver materials were prepared by the sol-gel method using titanium n-butoxide, ethanol and water with a molar ratio equal to 1 :8:4 and the amount of silver precursor to have 2% of silver. The silver precursor used was silver nitrate. Ammonium hydroxide was used to fix the pH 9. The suspension was vigorously stirred until the gel was formed. Finally was dried at 60°C in a muffle with rate heat ramp of 5°C/min.

Example 3

Titania supported silver materials were prepared by the sol-gel method using titanium n-butoxide, ethanol and water with a molar ratio equal to 1 :8:4 and the amount of silver precursor to have 2% of silver. The silver precursor used was silver sulphate. Ammonium hydroxide was used to fix the pH 9. The suspension was vigorously stirred until the gel was formed. Finally was dried at 60°C in a muffle with rate heat ramp of 5°C/min.

Example 4

Titania supported silver materials were prepared by the sol-gel method using titanium n-butoxide, ethanol and water with a molar ratio equal to 1 :8:4 and the amount of silver precursor to have 2% of silver. The silver precursor used was silver acetate. Ammonium hydroxide was used to fix the pH 9. The suspension was vigorously stirred until the gel was formed. Finally was dried at 60°C in a muffle with rate heat ramp of 5°C/min.

Characterization. The Infrared spectroscopy was made in a Perkin Elmer's Spectrophotometer. The XRD were made using Cu Ka radiation, in Siemens D- 500 equipment. The signal intensity was measured by step scanning in the 29 range with a step of 0.03° and a measuring time of 2 s per point. The specific surface areas of the samples were measured on a Quantasorb Sorptometer and calculated from the nitrogen isotherms using the BET method. The mean pore diameter was calculated by the BJH method. The powder samples were analyzed by TEM and SEM microscopy. The particle size was measured by conventional transmission electron microscopy performed on a Zeiss EM910 electron microscope operated at 100 kV, with a 0.4 nm point to point resolution side entry goniometer attached to a CCD Mega Vision III image processor. For the SEM studies, a JEOL 5600 LV scanning electron microscope was used to perform the samples morphology. Bacterial strains. Pseudomona aeruginosa, Klebsiella pneumoniae, Serratia marcencens, Proteus vulgaris, enterophatogenic Escherichia coli (EPEC), Salmonella typhimurium, Shigella dysenteriea, from collection of our laboratory; Sthapylococcus aureus (ATCC 25923), and methicillin-resistant Sthapylococcus aureus (MRSA) (ATCC 43300), were used. Bacterial susceptibility to nanosilver. To examine the susceptibility of bacterial strains to silver nanoparticles, a Kirby-Bauer disk- diffusion method was used, following the recommended criteria of CLSI (12). Disks of Whatman filter paper (7mm) were soaked with a 0.1% solution of AgN0₃, AgS0₄, AgAc, and AgCI-nanoparticles of Ti0₂. Bacterial strains were spread onto Mueller-Hinton agar with cotton swabs from freshly grown bacterial suspension (0.5 McFarland densities). The inoculated agar plates were allowed to dry and then the round disks with Ag-nanoparticles were placed on top of the inoculated agar. The agar plates were incubated at 37°C for 18h. The diameter of the zone of inhibition was measured using caliper. Three experiments were performed to obtain average values for each strain.

Results.

X-ray diffraction of silver acetate and sulfate supported titania nanostructured materials showed an amorfous pattern of incipient anatase mixed with nanocristallytes of Ag-Ti0₂ materials, that can not detected by our diffractometer (Fig. 1). Silver nitrate favored the crystallization of anatase nanoparticles, whereas rutile was formed in the chloride salt sample. According to the microbiological results we can see that the silver nitrate has the best action followed by the acetate, sulfate and finally by the chloride. Anatase cristalline phase to enhance the antibacterial effect and the formation of rutile inhibits this action. The presence of a high density of hydroxyl groups bonded to the titania network in anatase and amorphous phase, explain the high activity of the nitrate, acetate and sulphate precursor silver Ag/Ti02 solids. Menwhile, the silver chloride-titania fovors the growth of rutile at low reaction temperature. The rutile crystalline phase diminishes the catalytic activity of the materials over the culture. The presences of OH groups was characterized by FTIR, Fig. 2a and 2b, and were associated to the bands observed in the high energy region of the spectra at 3390, 3399, 3401 and 3411cm^"1 for all the samples. The bands at 1623, 1627, 1629 are related to the presence of C-H vibrations and 1633 cm^"1 is assigned to the bending frequency O-H bond in water. At 1384 cm^"1 a peak that corresponds to a vibration of the Ti-Ligand bond is observed. In sulfate salt sample, we can be seen a decrease in the intensity of the 1384cm^"1 band and the presence of two bands in 1135 and 1051cm^"1 related to the sulfate anion bonding to the surface. For the acetate salt we can see a band at 1536 related to the C=O group of the acetate. For the sulfate salt we have an important result because the FTIR results talks about a sulfated titania that has a high acidity and a great stability but this decrease the antibiosis in an important way. Instead of this we have a helpful aspect of the sulfated titania, which has been reported to be a solid acid [26-28]. According to the results of the FTIR and the microbiological test we can observe that there is an inverse correlation between the bactericidal action of the bactericide and the OH groups quantity, less OH groups means more bacteria killing.

The band gap values are almost constant in all the samples. These values are closer to that reported for the conventional anatase structures, 3.0-3.1 eV [23]. When the sample is gelled simultaneously with silver salts using different Ag precursors, the initial band gap exhibits the same value as that observed for titania. However, when the hydroxylation is maximum (Eg = 2.77 eV) , anionic vacancies are formed as follows:

2 OH^" + H₂O+V +e- +0₂ ^".

Thus, by dehydroxylation, a great number of vacancies are formed, but the free electrons are "trapped" by the silver. When the pure titania is prepared as reference, the Eg value is higher than that observed for Ag TiO₂ prepared samples. The formation of (V) vacancies must occur by desorption of surface oxygen [29-35]:

0₂ + 0₂(g)+VO₂ V0₂- - V + e^■

Fig. 3 shows the adsorption N₂ isotherms. The Ti0₂-AgN0₃, Ti0₂-Ac and Ti0₂- Cl have a type II isotherm (lUPAC) due to the mesoporous presence. The Ti0₂- S0₄ is the unique sample with a type IV isotherm related to mesoporous solids with a multilayer filling of the surface. All the samples have a hysteresis loop due to ink bottle pore with a big body shape and small and thin neck. Area and mean pore diameter are reported in table 1. The high specific area is an explanation of the good bactericide activity.

Antibacterial properties of Ag/Ti0₂ nanostructured bactericide. The antibacterial properties of silver nanoparticles were evaluated using the disk diffusion test. The filter paper disks with Ag-nanoparticles placed on the bacteria-inoculated agar plates killed all the bacteria under and around them; we observed distinct zones of inhibition (clear areas with no bacterial growth) around the disk depending of bacterial strain and Ag/TiO₂ nanostructured bactericide materials. The zones of inhibition for disks of 0.1 % Ag-nanoparticles solutions are given in Table 1. The main inhibition was presented for AgN0₃, continued for AgAc and AgS0₄. Although, AgCI was a poor inhibition, four bacterial strains were not inhibited with this particle (table 2; Fig. 4). All the bacterial strains were inhibited by AgN0₃-nanoparticles_, principally S. aureus ATCC 43300, K. pneumoniae, E. coli, S. marcencens, P. vulgaris, and S. dysenteriea. However, S. aureus ATCC 25923, P. aeruginosa, and S. typhimurium were inhibited better for AgAc.

The increase of bacterial resistance to antimicrobial agents is a serious problem in the treatment of infectious diseases as well as in epidemiological survey. Progressively more new bacterial strains have emerged with dangerous levels of resistance, including both Gram-positive and Gram-negative bacteria [29]. The bacterial resistance will require precautions that guide to prevention of the emergence and spreading of multi-resistance bacterial strains, and the development of new antimicrobial substance. The results of this study clearly demonstrated that the nanostructured sol-gel Ag/Ti02 have a bactericide effect including highly pathogenic bacteria such as EPEC and MRSA [30-37].

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Claims

The bactericide or antimycotic agent of claim 1 comprising silver high dispersed nanoparticles from 0.5 to 15 % wt in concentration.

The bactericide or antimycotic agent of claim 1 wherein the functional group is selected from hydroxyl, carboxyl, acid, ammonia, chloride, ester, ether, ketaconazole, triclosan or salicylic acid or mixtures thereof.

The bactericide or antimycotic agent of claim 3, wherein the

concentration of the functional groups is from 0.001 to 1 mol%.