WO2008120259A1

WO2008120259A1 - Material of nano-aggregates of tetrasilver tetroxide

Info

Publication number: WO2008120259A1
Application number: PCT/IT2008/000130
Authority: WO
Inventors: Fabio Di Fonzo; Matteo Paolo Bogana; David Dellasega; Anna Facibeni; Carlo Enrico Bottani
Original assignee: Politecnico Di Milano
Priority date: 2007-04-03
Filing date: 2008-02-27
Publication date: 2008-10-09
Also published as: ITMI20070683A1

Abstract

The present invention concerns a new tetrasilver tetroxide-based product having improved antibacterial properties, and a process for the production thereof. In particular, the invention concerns a material of tetrasilver tetroxide nano-aggregates having controlled density and morphology and medical devices comprising the material Of Ag4O4 nano-aggregates according to the invention and a suitable substrate.

Description

Title: "Material of nano-aggregates of tetrasilver tetroxide"

DESCRIPTION

The present invention concerns a material of nano-aggregates of tetrasilver tetroxide having improved antibacterial properties, and a process for its production. In particular, the invention concerns a material of nano-aggregates of tetrasilver tetroxide

(Ag₄O₄) with controlled density and morphology and medical devices comprising said material.

According to the present invention, by "nano-aggregates" it is intended particle groups having sizes from 1 to 500 nm. The antimicrobial properties of metallic silver have been known for centuries, which, once placed in solution, releases ions or free radicals that then damage the outer surface of the bacteria, thus blocking their enzymatic respiratory system and altering their microbic DNA and cell wall. Silver in solution has been used for nearly a century as antimicrobial agent in the treatment of lesions. It is deemed that the extensive use of silver in lesion disinfection, in particular for skin lesions, has occurred mainly due to its non-toxicity for in vivo human cells.

Some products available on the market are nowadays known, which comprise nanocrystalline silver and which provide a continuous release of silver. Among these, for example, there is a product known on the market as Acticoat7® (nanocrystalline silver) sold by Smith & Nephew.

The benefits of the continuous silver release are due to the fact that said continuous release ensures a low but continuous silver dose, which reduces the possibility of damage to the cells and tissues.

Nevertheless, problems are known for such medications related to the over- exposure of the human and animal organism to silver.

US patent No. 5,336,499 of M.S. Antelman discloses a method for inhibiting the growth of pathogen agents in cosmetic and pharmaceutical products. According to such a method, in order to obtain the inhibition the insertion of Ag₄O₄ molecular crystals is provided for in the presence of an oxidising agent comprising persulfate. In particular, said document discloses tetrasilver tetroxide (Ag₄O₄) in single crystals, chemically obtained, i.e. prepared by reaction of silver nitrate with sodium or potassium peroxydisulfate, as schematised below:

4AgNO₃ + 2Na₂S₂O₈ + 8NaOH → Ag₄O₄ + 4Na₂SO₄ + 4NaNO₃ + 4H₂O According to Antelman's thesis, once silver species contact bacteria, a multi- step chain reaction is triggered: first, the formation of a covalent bond with the pathogen agent occurs, then followed by the release of electrical energy (electrocution) through a redox process, and finally there is the release of the highly active singlet oxygen, which causes the death of the pathogen. In particular, it is considered that the redox process involves the following reactions:

Ag(I) - e^" = Ag(II); Ag(III) + e = Ag(II) For Ag₄O₄ molecules:2Ag(I) + 2Ag(UI) = 4Ag(II)

Nevertheless, the tetrasilver tetroxide (Ag₄O₄) obtained according to the teaching of the US patent No. 5,336,499, i.e. chemically produced, shows the drawback that the compound separated by crystallisation contains a high impurity percentage, impurities that must be removed by means of several subsequent washings. It is clear, therefore, that such impurities are definitely undesired, as well as potentially harmful.

A further problem consists of the fact that the precipitate of Ag₄O₄ so obtained has a very low solubility, for raising thereof persulfate is employed (for example Oxone^® of DuPont, as indicated in column 3, lines 14-17 of the US patent No. 5,336,499).

The object of the present invention is therefore to provide a product capable of releasing silver that allows an effective removal of bacteria and prevents the regeneration of the same. Moreover, a further object of the invention is to provide a product that is effective in severe affections and wounds, without using any activating substance.

The above objects have been achieved by a material of nano-aggregates of tetrasilver tetroxide obtainable from the process as indicated in claim 1. Further advantages and preferred embodiments of the invention are indicated in the dependent claims.

The material obtainable from the process according to the invention consists of aggregates of tetrasilver tetroxide of nanometric size, i.e. of nano-aggregates OfAg₄O₄. Further characteristics and advantages of the invention will become evident from the following detailed description with reference to the embodiments of the invention, given as exemplifying and non-limiting, and to the attached figures wherein:

Figure Ia represents a photographic acquisition of the plasma plume generated according to the invention, on which a possible position of the substrate was reported;

Figure Ib represents a photographic acquisition carried out in the preparation of the material according to the invention in Example 1 ;

Figure 2 represents the deposition rate (r) of the tetrasilver tetroxide material of Example 1 as a function of the fluence (F) of the laser;

Figure 3 represents a SEM enlargement of the surface of the tetrasilver tetroxide nano-aggregate material on silicon substrate obtained according to Example 1; Figure 4 represents the Raman spectrum of the tetrasilver tetroxide nano- aggregate material of Example 1;

Figures 5A, 5B, 5C, 5D represent four SEM enlargements of the section of four films of a tetrasilver tetroxide nano-aggregate material on silicon substrate obtained with four Ω values and with different pressures, obtained from Example 1, Example 2, Example 3 and Example 4, respectively;

Figure 6 reports bacteriostatic effectiveness tests of tetrasilver tetroxide material discs of Example 1, carried out on cultures of 1) Escherichia coli, 2) Staphylococcus aureus, 3) Enterococcus faecium, 4) Pseudomonas aeruginosa respectively; Figure 7 represents bacteriostatic effectiveness tests of tetrasilver tetroxide material discs of Example 1, carried out on a culture of Saccharomyces cerevisiae;

Figure 8 represents bacteriostatic effectiveness tests of a comparison material carried out on a culture of Escherichia coli;

Figures 9A and 9B represent bacteriostatic effectiveness tests of tetrasilver tetroxide material discs of Example 1, carried out on liquid medium by using a culture of Escherichia coli; and

Figure 10 represents a graph of the antibacterial activity expressed in CFU/ml over time of the material of Example 1 in comparison with the antibacterial activity of a material of the prior art. In the present invention, with the term:

- "substrate", it is intended any suitable material on whose surface a vapour phase deposit can be carried out;

- "target material", it is intended the silver material which the laser radiation is delivered to;

- "laser", it is intended a laser having wavelength in the spectral region of 100 nm to l500 nm;

- "plasma", it is intended an ionised gas, overall electrically neutral, composed of positive ions and free electrons;

- "plume", it is intended the characteristic three-dimensional form assumed by the plasma generated by the interaction of a laser pulse with the target material, and formed by the species emitted by the surface of the target material itself;

- "x_s", it is intended the deposition distance, i.e. the distance between the point of ablation from the target material, generated by the interaction of a laser pulse, and the median point of the surface of the substrate turned towards the plume;

- "plume axis", it is intended the symmetry axis of the plume that passes through the point of ablation from the target material and is perpendicular to the target material itself;

- "θ", it is intended the angle from the plume axis to the axis on which the deposition distance x_s is measured;

- "plume front", it is intended the external surface of physical discontinuity of the plume;

- "x_p", it is intended the distance between the point of ablation from the target material and the point where the axis identified by x_s intersects the plume front;

- "vacuum chamber", it is intended a chamber wherein it is possible to control the pressure and composition of the atmosphere therein; - "antibacterial agent", in the present invention it is intended both substances having bacteriostatic activity, i.e. capable of inhibiting the proliferation of gram-negative and gram-positive bacteria resistant to antibiotics, and substances having bactericide activity, i.e. capable of killing the same bacteria.

The invention therefore has as object a material of nano-aggregates of tetrasilver tetroxide obtainable by the process according to the present invention comprising the steps of: a) providing" a silver target material in a vacuum chamber, b) providing a substrate in said vacuum chamber, c) delivering a pulsed laser radiation of wavelength of 100 to 1500 nm to the silver target material of step a), such as to generate a plasma plume by ablation of the target material, d) positioning the substrate of step b) at a distance x_s from the point of ablation from the target material of step a), being θ the angle from the plume axis to the axis identified by x_s itself; e) determining the distance x_p from the point of ablation from the target material of step a) to the point wherein the axis identified by the distance x_s intersects the front of the plume generated in step c); f) allowing nano-aggregates of tetrasilver tetroxide to deposit from the plasma plume of step c) on the substrate positioned as in step d), characterised in that, in the vacuum chamber, a gas is present comprising at least 5% oxygen, the remaining being an inert gas, that the angle θ is of 0° to 50° and that the ratio Ω between x_s and x_p is of 0.7 to 1.4.

As far as step a) is concerned, this envisages to provide a silver target material. Preferably, it is 99.9% silver.

In step b), a substrate is provided which is placed in the same vacuum chamber. Any substrate can be employed that is suitable for producing materials for pulsed layer ablation (PLD - Pulsed Laser Deposition), for example silicon. Preferably, said substrate is a material of medical grade. More preferably, it is formed by one or more layers, equal to or different from each other, of a medical grade material selected from the group consisting of cellulose, polymer films, isolated fibres, fabrics and their combinations.

In step c), a pulsed laser radiation of wavelength of 100 to 1500 nm is delivered to the silver target material, being said pulsed laser radiation capable of causing the expulsion by ablation from the target material of chemical species, i.e. positive ions and free electrons, which form a plasma plume. In particular, step c) envisages to focus the laser beam on the surface of the target material by a suitable lens. In the present invention, the angle of incidence of the laser beam on the surface of the target material is preferably of 0° to 90°, more preferably of 30° to 50°, even more preferably is about 40°.

In particular, in the vacuum chamber, the substrate is placed on an appropriate fixed or mobile support, whereas the target material is placed on an appropriate moving system for an advantageously homogenous and uniform ablation of the target material.

The working fluence of laser, F, defined as the ratio between the laser pulse energy and the area of the laser spot, is selected by applying techniques known in the PLD field. Preferably, as will be further described in detail in the following examples, in order to know the working fluence, a calibration curve can be traced by varying the deposition rate of the material of the invention as a function of the fluence, and such a curve can be interpolated with a linear function that will be used for selecting the working fluence itself. Even more preferably, the curve experimentally traced for determining the fluence has a linear region; such a linear region will have a deviation of less than 2% with respect to the interpolation line. In the preferred embodiment, for fluence values in the first part of said linear region, a plasma is generated from the target material, advantageously free from metallic silver drops, whose expulsion, if any, could contaminate the final tetrasilver tetroxide material with metallic silver. More preferably, the working fluence is selected from the first part of the range of values falling within said linear region of the experimental calibration curve having a deviation of less than 2%.

In a preferred and advantageous embodiment, in step c) of the invention, the laser has a fluence of 1 to 4 J/cm² and a pulse energy of 150 to 450 mJ.

More preferably, said embodiment employs an ultraviolet laser, i.e. a laser with wavelength in the range of 100 to 400 nm. Even more preferably, said ultraviolet laser is a KrF excimer laser (λ = 248 nm).

The expansion of the plasma plume, generated by the action of the laser radiation of step c) from the silver target material, slows down over time until stop at an equilibrium condition. Such an equilibrium condition reached by the plume is a function of the energy provided by the pulsed laser radiation and is a function of the pressure in the vacuum chamber. As a matter of fact, it results that the expansion capacity of the plume increases with the increase of energy delivered by the pulsed laser radiation on the target material, whereas it diminishes with the increase of pressure in the vacuum chamber. hi step d), the substrate is positioned at a deposition distance x_s from the target material. The man skilled in the PLD field knows how to orient the substrate inside the vacuum chamber in order to deposit material from the plume on said substrate. As previously defined, x_s is the deposition distance, i.e. the distance between the point of ablation from the target material, generated by the interaction of a laser pulse, and the median point of the surface of the substrate turned towards the plume. According to the present invention, as represented in figure Ia, the axis identified by the distance x_s and the axis of the plume form an angle θ. Such an angle θ, under the merely geometric point of view, can vary from 0° to 90°. It is known to the man skilled in PLD that the deposition rate of the material formed by laser ablation significantly decreases with the increase of θ. Therefore, for the applications of the present invention, the angle θ is of 0° to 50°, since for values of θ greater than 50° the deposition rate of the material according to the invention is disadvantageous^ low.

Preferably, the angle θ is equal to about 0°, being the substrate positioned substantially facing the target material. More preferably, the angle θ is equal to 0°, being the substrate positioned substantially facing the target material and the surface of the substrate itself being substantially parallel to the surface of the target material. According to a preferred embodiment of the invention, in step d) the silver target material of step a) and the substrate of step b) in the vacuum chamber are frontally arranged parallel to each other with θ equal to 0°, at a distance x_s of at least about 2 cm from each other. Even more preferably, such a distance X₅ is from about 3 cm to about 10 cm. Once the working fluence of the laser is selected as described above, in step e) the distance x_p is determined from the point of ablation from the target material to the point wherein the axis identified by the distance x_s intersects the plume front, as represented in figure Ia.

A possible method for determining the distance x_p is photographic acquisition of plume images, for example through a digital camera, at different exposure times. Such an image acquisition of the plume can be made both before and after the positioning of the substrate of step d). Nevertheless, when x_s < x_p, it is advantageous to acquire the plume image before the substrate positioning step d). As illustrated in figure Ia, on the acquired image of the plume, the distance x_p (black coloured segment) is then measured from the point of ablation from the target material to the point wherein the axis identified by the x_s distance (dashed grey coloured segment) intersects the plume front.

Finally, in step f), the tetrasilver tetroxide thus formed then deposits from the plasma on the substrate in nano-aggregate form. Such a deposition takes place in a time preferably of not less than 1 minute. The process according to the invention is characterised in that in the vacuum chamber, a gas is present comprising at least 5% oxygen, being the remaining an inert gas. In particular, such an inert gas is selected from the group consisting of nitrogen and noble gases, i.e. helium, neon, argon, krypton, xenon, radon and mixtures thereof. Preferably, the gas in the vacuum chamber comprises inert gas and at least 10% oxygen; more preferably, the gas in the vacuum chamber is 20% oxygen and 80% nitrogen.

In said vacuum chamber, the pressure is of 1 to 101325 Pa (atmospheric pressure), preferably 20 to 150 Pa, more preferably it is about 60 Pa.

The process according to the invention is moreover characterised in that the ratio between x_p and x_s is defined as Ω; accordingly, Ω is a function of the pressure, of the composition of the gas in the vacuum chamber, of the laser fluence and of the deposition distance x_s.

The inventors of the present invention have surprisingly found that for values of Ω between 0.7 and 1.4, a material of pure tetrasilver tetroxide having the desired morphology is obtained, i.e. Ag₄O₄ in nano-aggregate form. In particular, it should be noted that, for the objects of the present invention, the upper limit of Ω is 1.4 since, as will be clear from the following examples, with the increase of x_s, with respect to x_p, the deposition rate (r) decreases disadvantageously and exponentially, and moreover, with the increase of x_s, the adhesion of the material deposited on the substrate decreases, as well as the structural coherence of the final material itself. On the other hand, for distances x_s < x_p, hence for values of Ω less than 1, an increase of the deposition rate (r) is observed, but also at the same time an increase of the spatial unevenness of the final material. In particular, for Ω less than 0.7, the progressive formation of a central zone is observed clearly different in stoichiometry, crystalline structure and morphology from the rest of the deposited material. The latter is a mixture of metallic silver and under-stoichiometric compounds of silver and oxygen. Hence, the lower limit of Ω is 0.7.

Sets of parameters that give rise to plumes of equal shape and distance x_p, with the same deposition rate, are fully equivalent for the purposes of the process of the invention. Moreover, the physical-chemical processes necessary for producing the material of tetrasilver tetroxide nano-aggregates take place at the plume front.

Hence, according to the present invention, a material of tetrasilver tetroxide nano- aggregates is obtained having controlled density and morphology, Le. having from compact to open porous structure within the range from 0.7 to 1.4.

For the objects of the present invention, values from 0.95 to 1.3 of Ω are further preferred. It is observed, in fact, that such values allow to obtain nano- aggregated Ag₄O₄ materials having controlled density and morphology that are particularly advantageous for the applications of the present invention. More preferably, Ω is about 1, since at such a value a material having advantageous controlled density and morphology can be obtained, while finding the process of the invention the maximum yield and deposition efficiency thereof.

In addition, for the objects of the present invention, values of the angle θ close to 0° are preferred, since, as stated above, the deposition rate of the nano-aggregated

Ag₄O₄ material from the plume increases with the decreasing of the angle θ. More preferably, therefore, θ is equal to 0°, since at this value of θ, the deposition rate is maximum.

In another aspect, the present invention concerns a material of nano-aggregates of tetrasilver tetroxide according to claim 18 obtainable from the above-described process.

In figure 3, an enlargement of the surface of the material of tetrasilver tetroxide nano-aggregates of a first embodiment of the invention on silicon substrate is represented, wherein said nano-aggregates have size of 5 to 80 nm. The confirmation that this is Ag₄O₄ is given by the Raman spectrum reported in figure 4. More preferably, the nano-aggregates of Ag₄O₄ according to the invention have size of 5 to 50 nm, even more preferably of 5 to 10 nm. Without wishing to be bound by any theory, it is deemed that the tetrasilver tetroxide properties depend on size thereof. In particular, by reducing the volume of the crystals, reducing the density and varying the morphology of the nano-aggregate material, the exposed surface area of the crystals themselves is significantly increased, thus increasing the available surface area in order that the chemical reactions occur in a shorter time period.

In a further aspect, the present invention concerns films of tetrasilver tetroxide nano-aggregate material according to claim 22, as shown in figures 5A, 5B, 5C, 5D. According to preferred embodiments, said films have porous structure with controlled density and morphology and are obtainable by the process according to the invention for values of Ω close to or above about 1, advantageously for values of Ω between 0.95 and 1.3. More preferably, said films have porous structure with controlled density and morphology and are obtainable by the process according to the invention in which Ω is equal to about 1. It was observed, as in fact described above, that at this Ω value, the process finds an advantageous compromise between efficiency from the production point of view and morphology of the Ag₄O₄ material with advantageously porous structure, although, as said, a greater overall surface area of the material of the invention is obtained for values of Ω greater than 1, since such a surface area increases with the increase of Ω.

Preferably, the thickness of the Ag₄O₄ nano-aggregate material obtained according to the process of the present invention is between 0.1 and 1 μm.

In another aspect of the present invention, the Ag₄O₄ nano-aggregate material is used as a medicament, as in claim 24. The nature of the nano-aggregates advantageously having a high surface area showed to be decisive in terms of effectiveness of the invention material as a medicament, owing to the high contact offered to the damaged tissues to be treated. In particular, the Ag₄O₄ nano-aggregate material according to the present invention is suitable for the treatment of severe lesions, such as skin lesions, deep ulcers, decubitus ulcer and amputations.

As will be widely demonstrated in the following examples, it was observed that the Ag₄O₄ nano-aggregate material according to the present invention is effective as antibacterial agent in an extremely significant manner against a number of bacteria types, with respect to other known antibacterial materials, whereas against other bacteria types the nano-aggregate material according to the invention resulted effective in a manner comparable with known antibacterial materials. Therefore, the nano- aggregate material according to the present invention is used for the preparation of a medicament for the treatment of severe lesions, as in claim 25. In particular, by "severe lesions" the skin lesions, deep ulcers, decubitus ulcer and amputations are intended to be comprised.

In further another aspect, the present invention concerns a medical device, as in claim 26, comprising the material of tetrasilver tetroxide nano-aggregates or the film of material of tetrasilver tetroxide nano-aggregates and suitable medical grade elements.

In particular, said suitable elements of medical grade are selected from the group consisting of one or more further substrates and optionally one or more suitable excipients. The invention will now be described in detail with reference to production examples, given as illustrative and non- limiting, of material of tetrasilver tetroxide nano- aggregates and effectiveness tests thereof.

EXAMPLES

EXAMPLE 1 - Preparation of a material OfAg₄O₄ nano-aggregates The following materials were placed within a vacuum chamber: a 2 x 3 cm silicon substrate, 10 cellulose discs of 5 mm diameter and a silver target material. The target material was 99.9% silver (commercialised by MaTecK GmbH).

A KrF excimer laser (λ=248 nm) was selected. The focusing of the laser beam on the surface of the target material to generate a plasma plume was carried out through an appropriate lens. In the present example, the angle of incidence of the laser beam on the surface of the target material was 40° and the size of the laser spot was 6.6 mm². Subsequently, synthetic air (20% O₂ and 80% N₂) was introduced in the vacuum chamber at a pressure of 60 Pa.

Then, a quartz microbalance for measuring the deposition rate was positioned facing the target material on the plume axis, hence with θ equal to 0°, at a distance of 5 cm from the target material itself. A laser repetition frequency of 20 Hz was set. A deposition rate r curve was then acquired, as a function of the laser energy, E, and accordingly of the fluence, F, as represented in figure 2. Said curve was then interpolated with a linear function. The suitable fluence values for the deposition of the nanostructured tetrasilver tetroxide material were those of the lower end of the linear zone, with a deviation of less than 2%. The following Table 1 reports the data collected during the procedure for the determination of the linear function by interpolation of the deposition rate r curve as a function of the fluence F.

Table 1 — Deposition rate as a function of the fluence

The following are reported: in the first column, the energy of the laser pulse E, expressed in mJ; in the second column, the actual fluence F on the target material excluding the energy losses of the optical path, expressed in J/cm²; in the third, the deposition rate r expressed in ng/s; in the fourth, the linear interpolation (or rate fit); and on the fifth the deviation from the latter (Δ fit). The data of Table 1 are also reported in graphic form, in figure 2, where the" deposition rate r is represented as a function of the laser fluence F. The experimental measurements, the linear interpolation thereof and the percentage deviation of the measurements from the interpolating line are reported. The shaded band indicates the optimal zone. A working fluence of 1.565 J/cm² was therefore selected.

Subsequently, with a digital camera, images of the plume were acquired with different exposure times, as represented in figure Ib.

The microbalance was then removed and the substrate was positioned facing and parallel to the target material at a distance x_s of 5 cm. The distance x_p was measured perpendicular to the target, from the point of ablation to the point wherein the axis identified by x_s intersects the plume front, i.e. by measuring the black colour segment traced in figure Ib. In the present Example, x_p measured 5 cm.

The material of tetrasilver tetroxide nano-aggregates was allowed to deposit for a time 30 minutes.

In summary, the following process parameters were set:

Laser fluence: 1.565 J/cm²

Pulse repetition frequency: 20 Hz

Gas type: synthetic air (80% N₂, 20% O₂) Gas pressure: 60 Pa

Angle θ: 0°

Deposition distance, x_s: 5 cm

Distance x_p: 5 cm

Deposition duration: 30 min Based on such values, the ratio Ω was equal to 1.

The substrate was then removed from the vacuum chamber and subjected to a number of analyses. The thickness of the film deposited on said substrate was measured; such thickness was 940.8 ran. The film was then brought to the scanning electron microscope, where its surface and section were observed. An enlargement was acquired of the surface of the material thus obtained, which is reported in figure 3, and an enlargement of the section of the same material, which is represented in figure 5 A. From such figures, it was observed that the film was composed of nano-aggregates, in particular very well defined columnar nano-aggregates of around 50 nm section. The material of the film was then weighed: 82.4 μg had been obtained. The material of the deposited film was finally subjected to Raman analysis. In figure 4, the resulting Raman spectrum is reported. Such spectrum demonstrated that the material was pure Ag₄O₄.

EXAMPLE 2 - Preparation of a material OfAg₄O₄ nano-aggregates The procedure of Example 1 was repeated, setting the same parameters except for x_s which in this case was 6.5 cm. The increase of the distance x_s with respect to that of Example 1, all other parameters being the same, led to a decrease of the deposition rate r. Therefore, in order to once again obtain 82.4 μg of deposited material, the duration of the process was increased to 55 min. All of the other parameters being identical to those of Example 1, distance x_p was again 5 cm. The ratio Ω in this case was therefore equal to 1.3.

A tetrasilver tetroxide film was obtained with 892.6 nm thickness. The section, seen at the scanning electron microscope, is represented in figure 5B. It was observed, therefore, that with the increase of the Ω ratio at the same pressure, the morphology of the film was different from that of Example 1 (figure 5A). The nano-aggregate material of the present Example, while having a columnar structure as in Example 1 (section of the columns on average less than 50 nm), showed a more disordered morphology (the columns had an irregular surface and tended to branch during growth). The Example therefore showed how, by increasing the ratio Ω, the overall specific surface of the material Of Ag₄O₄ nano-aggregates was increased. EXAMPLE 3 - Preparation of a material OfAg₄O₄ nano-aggregates The procedure of Example 1 was repeated, setting the same parameters except for the pressure inside the vacuum chamber, which in this case was 150 Pa. The pressure increase led to a shortening of the plume, so that in this case x_p was 4.3 cm. The deposition distance, x_s, was 4.3 cm. In this case, the ratio Ω was equal to 1. Also in the present Example, 82.4 μg of Ag₄O₄ nano-aggregate material was deposited. A film of tetrasilver tetroxide 734.8 nm thick was obtained. The section of the obtained film, seen at the scanning electron microscope, is represented in figure 5C. It was observed, with respect to the material of Example h (figure 5A), that with the increase of the pressure in the vacuum chamber at the same Ω value, the film morphology resulted different. The material again had a columnar structure as in Example 1 (average section of the columns around 50 nm), but showed a slightly more disordered morphology (the columns had a more irregular surface) and the presence of a nonuniform growth over the entire surface.

EXAMPLE 4 Preparation of a material OfAg₄O₄ nano-aggregates The procedure of Example 3 was repeated, setting the same parameters except for x_s, which was 5.5 cm. Hence the ratio Ω was equal to 1.28. 82.4 μg Of Ag₄O₄ nano- aggregate material was deposited. A film of tetrasilver tetroxide 624.2 nm thick was obtained. The section, seen at the scanning electron microscope, is represented in figure 5D. It was observed that, with respect to the material of Example 2 (figure 5B), i.e. with the increase of the ratio Ω, and with respect to that of Example 3 (figure 5C), i.e. with the increase of pressure, the morphology of the film obtained in this case was considerable different. The material had nearly completely lost its columnar structure, to assume a more "cauliflower" morphology. It was no longer possible to easily identify an average section of the columns, but there was branching of about 20 nm size. The high structural disorder considerably increased its specific surface area. EXAMPLE 5 — Bacteriostaticity measurements and tests The evaluation of the antibacterial activity was undertaken through a bacteriostaticity test by employing gram-positive and gram-negative bacteria.

The object of such test was to observe the antibacterial activity of the material of Ag₄O₄ nano-aggregates according to the invention. In other words, the capacity of the material of the invention to inhibit bacterial proliferation was verified. A stationary-phase culture was prepared of the following bacteria and yeast:

- Pseudomonas aeruginosa Gram-negative pathogen

- Escherichia coli Gram-negative non-pathogen

- Enterococcus faecium Gram-positive pathogen

- Staphylococcus aureus Gram-positive pathogen

- Saccharomyces cerevisiae yeast in a complete medium (LB broth) (growth for 16-18 hours). The measurement of the growth of the strains was carried out by means of optical density evaluation (OD) at 600 nm (ODβoo)-

The yeast Saccharomyces cerevisiae was used as a control in order to verify the absence of activity on eukaryotic cells. The stationary-phase bacteria cultures and the yeast in LB broth were diluted so to obtain initial OD«κ) of about 0.05.

The bacteria were grown up to OD₆Oo of about 0.5.

The yeast Saccharomyces cerevisiae was grown up to an OD600 of about 0.9 - 1.0. The optimal growth temperatures were the following:

- 37°C per Streptococcus faecium, Staphylococcus aureus, Escherichia coli;

- 3O⁰C per Pseudomonas aeruginosa.

Once the desired optical density was reached, the bacteria were diluted in 2.5 ml of soft agar, so to obtain approximately 5 x 10⁵ cells. The soft agar containing the bacteria Escherichia coli was poured on eight plates of LB and left to solidify.

The soft agar containing the Staphylococcus aureus bacteria was poured on eight plates of LB and left to solidify.

The soft agar containing the bacterium Enterococcus faecium was poured on eight plates of LB and left to solidify.

The soft agar containing the bacterium Pseudomonas aeruginosa was poured on eight plates of LB and left to solidify.

The object of this passage was to obtain a uniform and homogenous growth of bacteria on the surface of the plate. It was not possible to use the soft agar technique for spreading yeast

Saccharomyces cerevisiae, since in these conditions a very poor growth is obtained. The yeast Saccharomyces cerevisiae was thus spread on plate (figure 7) by spatula.

Eight samples of the invention according to examples 1, 2, 3, 4 were separately prepared on eight cellulose discs, two for each example. At this point, each sample was deposited on four plates and the plates were incubated at the optimal grown temperatures, reported above. In figure 6, the four plates containing Escherichia coli, Staphylococcus aureus, Enterococcus faecium and Pseudomonas aeruginosa are represented, on which the sample of Example 1 was deposited.

Two discs of equal diameter were moreover prepared of a nanocrystalline metallic silver-based commercial product (active ingredient of the Acticoat of Smith & Nephew), on which an amount of silver is present equal to that present on the discs of examples 1, 2, 3, 4. For illustrating purposes, in figure 8 the plate is represented containing Escherichia coli on which the sample of the prior art was deposited. For the purposes of the present test, the Acticoat product was decomposed in order to separate and use the high density polyethylene mesh on which the constituent nanocrystalline metallic silver is deposited.

From the test, the formation of a transparent halo (zone of inhibition, ZOI) was observed, as represented in figure 6 for Example 1, due to the inhibition of the bacterial growth around samples bearing the material of Ag₄O₄ nano-aggregates. Said halo was visually evaluated, the extension of the same being proportional to the bacteriostatic properties of the samples.

The evaluation of the antibacterial activity was made after incubation at the relative optimal temperatures for a time of 16-18 hours, evaluating and measuring the inhibition hallow around each deposited disc. Inhibition zone measurements after 16-18 hours

Sample Sample Sample Sample ofEx. 1 ofEx. 2 ofEx. 3 ofEx. 4

E. coli 14 mm 14 mm 13 mm 12 mm 14 mm 13 mm 12 mm 12 mm

P. aeruginosa 17 mm 15 mm 14 mm 13 mm 16 mm 14 mm 14 mm 13 mm

E. faecium 21 mm 21 mm 22 mm 20 mm 22 mm 21 mm 22 mm 21 mm

S. aureus 14 mm 15 mm 15 mm 14 mm 15 mm 16 mm 14 mm 14 mm S. cerevisiae 0 0 0 0

0 0 0 0

nanocrystalline metallic silver (Acticoat)

E. coli 9 mm

9 mm

As is clear from the above tables, the inhibition zones obtained in the case of the samples according to the invention were significantly more extensive with respect to those obtained in the case of the sample of the prior art, while they were advantageously non-active towards the yeast Saccharomyces cerevisiae. In particular, the samples of the invention were advantageously very active against Enterococcus faecium. Therefore, the material of tetrasilver tetroxide nano-aggregates according to the invention shows high bacteriostatic activity. EXAMPLE 6 - Antibacterial activity measurements and tests

The test was carried out in order to test the antibacterial activity of the material of tetrasilver tetroxide nano-aggregates. In a liquid bacteria culture, a disc of cellulose was introduced on which nano-aggregate material was deposited according to Example 1.

The number of bacteria was monitored by means of optical density measurements (OD_OOO)- In order to measure the fraction of live bacteria over the total culture bacteria, samples were drawn at regular intervals. The drawn bacteria were plate-deposited and their biological activity was verified by means of counting the biologically-active colonies {Colony Forming Unit/ml, CFU/ml), that is those capable of reproducing themselves. The procedure for carrying out the antibacterial activity test in liquid is reported below.

Evaluation of the antibacterial activity of the sample according to Example 1 in liquid culture

Bacterial strain used Escherichia coli K12 (MG1655) Gram-negative non-pathogen Experimental procedure

- Preparation of a stationary-phase culture of the bacterial strain MG 1655 in complete medium (LB broth) (growth for 16-18 hours at 37°C). Measurement of the optical density (OD) at 600 nm (OD₆₀₀) 3 flasks were inoculated containing 30 ml of LB with the stationary-phase bacteria culture, so to obtain initial OD₆₀₀ ~ 0.05. The number of initial viable bacteria was titrated into the three flasks (colony forming unit CFU /ml).

The flasks 1, 2 and 3 were grown until OD₆oo ~ 0.1 was reached. The fractions were titrated corresponding to the viable counts (CFU/ml). After 30 minutes from the start of the test, a disc in accordance with Example 1 was added in flasks 2 and 3.

Flask No. 1 represented the blank, flasks 2 and 3 were treated with the sample according to Example 1, flask 2 being defined below Example 1-1 and flask 3 Example 1-2.

The growth was monitored every 30 minutes, measuring the OD₆₀₀ and the fractions corresponding to the viable counts were measured every 60 minutes (CFU/ml).

In tables 1 and 2 below, the values of ODβoo and CFU/ml are reported, while in figures 9A and 9B the corresponding graphs are illustrated.

Table 1

Table 2

As results from table 1 and the graph of figure 9A, the number of bacteria kept substantially constant after the introduction of the disc in the culture.

From table 2 and from the graph in figure 9B, it was observed that the number of biologically active colonies decreased with the change of exposure time to the material of the present invention. Another two flasks of MG 1655 were prepared wherein two discs of equal diameter were added of a nanocrystalline metallic silver-based commercial product (active ingredient of the Acticoat of Smith & Nephew), on which a silver quantity was present equal to that present on the discs of example 1. For the purposes of the present test, the Acticoat product was decomposed in order to separate and use the high density polyethylene mesh, on which the constituent nanocrystalline metallic silver is deposited. In such flanks, the same antibacterial effectiveness test was carried out as that described above, and the results are reported in the following table 3 and 4.

Table 3

Table 4

The trend of tables 2 and 4 are reported in figure 10. From figure 10, it is observed that the nano-aggregate material of Example 1 had significantly more effective antibacterial properties than the nanocrystalline metallic silver (active ingredient of the Acticoat of Smith & Nephew). While in 2 hours the active ingredient of the Acticoat induced only a small reduction of the active bacteria population (from an initial value of 3 x 10⁷ CFU/ml to a value in the range of 2.6 x 10⁷ and 8 x 10⁶ CFU/ml in 2 hours), the nano-aggregate material of Example 1 in the same range of time led a reduction of nearly 3 orders of magnitude (from an initial value of 3 x 10⁷ CFU/ml to a value in the range of 2 x 10⁴ - 4 x 10⁴ CFU/ml in 2 hours). In addition, even in the case of prolonged 5-hour exposure of the bacteria to the Acticoat active ingredient, the active bacteria population was not reduced beyond one order of magnitude (from 3 x 10⁷ to 2 x 10⁶ CFU/ml in 5 hours). Unlike the active ingredient of the Acticoat, the material of Ag₄O₄ nano- aggregates of Example 1 showed a reduction of the active bacteria concentration of over 3 orders of magnitude in only 4 hours (from 3 x 10⁷ to 1 x 10⁴ CFU/ml in 4 hours).

*****

From the above reported measurements, the improved effectiveness of the material of tetrasilver tetroxide nano-aggregates is showed with respect to nanocrystalline metallic silver, such as for example the Acticoat active ingredient. For the latter, in fact, inhibition zones were observed with over 30% lower area with respect to the inhibition zones observed for the material of the invention. Moreover, with the material of the invention, an antibacterial capacity was observed being greater by several orders of magnitude.

In considering the problems tied with the over-exposure of the organism to silver, such an effectiveness demonstrated by the material of the invention is an extremely important result for the purposes of the development of biomedical equipment or pharmaceutical products in general.

Moreover, the material of tetrasilver tetroxide nano-aggregates obtained according to the invention, unlike the Ag₄O₄ powders obtained chemically according to the prior art, is active without the use of any activating chemical agent and maintains its adhesion to the substrate even after prolonged use in liquid environment. This feature, together with the density and morphology control of the produced material by means of above-described suitable process parameters, allows a conveniently high control of the antibacterial activity over time.

From the examples reported above and from the attached figures, the surprising results achieved by the material of tetrasilver tetroxide nano-aggregates according to the invention are therefore evident. The recognition of the ratio Ω as the technical characteristic capable of leading to the formation Of Ag₄O₄ in nano-aggregates of desired density and morphology is extremely advantageous, particularly for medical applications on deep lesions or extensive skin infections to be treated in terms of contact area available between the lesion to be treated and the active material. In fact, the material of tetrasilver tetroxide nano-aggregates according to the invention proved to be a very effective antibacterial agent, thus suitable for treating severe lesions, and conveniently without any need to add an activating agent.

* * *

Claims

1. Process for preparing a material of tetrasilver tetroxide nano-aggregates, comprising the steps of: a) providing a silver target material in a vacuum chamber, b) providing a substrate in said vacuum chamber, c) delivering a pulsed laser radiation of wavelength of 100 to 1500 nm to the silver target material of step a), such as to generate a plasma plume by ablation of the target material, d) positioning the substrate of step b) at a distance x_s from the point of ablation from the target material of step a), being θ the angle from the plume axis to the axis identified by x_s itself; e) determining the distance x_p from the point of ablation from the target material of step a) to the point wherein the axis identified by the distance x_s intersects the front of the plume generated in step c); f) allowing nano-aggregates of tetrasilver tetroxide to deposit from the plasma plume of step c) on the substrate positioned in step d), characterised in that, in the vacuum chamber, a gas is present comprising at least 5% oxygen, the remaining being an inert gas, that the angle θ is of 0° to 50° and that the ratio

Ω between X₅ and X_p is of 0.7 to 1.4.

2. Process according to claim 1, wherein the ratio Ω is of 0.95 to 1.3.

3. Process according to claim 2, wherein the ratio Ω is about 1.

4. Process according to any one of the claims 1-3, wherein, in the vacuum chamber, the gas comprises an inert gas and at least 10% oxygen.

5. Process according to claim 4, wherein, in the vacuum chamber, the gas is 20% oxygen and 80% nitrogen.

6. Process according to any one of the claims 1-5, wherein, in the vacuum chamber, the pressure is of 1 Pa to 101325 Pa.

7. Process according to claim 6, wherein, in the vacuum chamber, the pressure is of 20 to 150 Pa.

8. Process according to claim 7, wherein, in the vacuum chamber, the pressure is of about 60 Pa.

9. Process according to any one of the claims 1-8, wherein in step c) the angle of incidence of the laser radiation on the surface of the target material is of 0° to 90°, preferably of 30° to 50°, more preferably it is about 40°.

10. Process according to any one of the claims 1-9, wherein the pulsed laser radiation of step c) has a fluence of 1 to 4 J/cm² and a pulse energy of 150 to 450 mJ.

11. Process according to any one of the claims 1-10, wherein the pulsed laser radiation is generated by an ultraviolet laser.

12. Process according to claim 11, wherein the pulsed laser radiation is generated by a KrF excimer laser (λ = 248 nm).

13. Process according to any one of the claims 1-12, wherein the angle θ is equal to about 0°, being the substrate positioned substantially facing the target material.

14. Process according to claim 13, wherein the angle θ is equal to 0°, being the substrate positioned substantially facing the target material and being the surface of the substrate itself substantially parallel to the surface of the target material.

15. Process according to claim 14, wherein in step d) the substrate and the target material are arranged facing and parallel to each other at a distance x_s of at least about 2 cm from each other.

16. Process according to claim 15, wherein in step d) the substrate and target material are arranged facing and parallel to each other at a distance x_s from about 3 to about 10 cm from each other.

17. Process according to any one of the claims 1-16, wherein step f) has a duration of not less than 1 minute.

18. Material of tetrasilver tetroxide nano-aggregates obtainable from the process according to any one of the claims 1-17.

19. Material of tetrasilver tetroxide nano-aggregates according to claim 18 wherein the nano-aggregates have size of 5 to 80 nm.

20. Material of tetrasilver tetroxide nano-aggregates according to claim 19 wherein the nano-aggregates size from 5 to 50 nm.

21. Material of tetrasilver tetroxide nano-aggregates according to claim 20 wherein the nano-aggregates size from 5 to 10 nm.

22. Film of material of tetrasilver tetroxide nano-aggregates according to any one of the claims 18-21".

23. Film of material of tetrasilver tetroxide nano-aggregates according to claim 22, wherein the thickness of said film is of 0.1 to 1 μm.

24. Material of tetrasilver tetroxide nano-aggregates according to any one of the claims 18-21 or film of material of tetrasilver tetroxide nano-aggregates according to any one of the claims 22-23 for use as medicament.

25. Use of a material of tetrasilver tetroxide nano-aggregates according to any one of the claims 18-21 or a film of material of tetrasilver tetroxide nano-aggregates according to any one of the claims 22-23 for preparing a medicament having antibacterial activity in the treatment of severe lesions.

26. Medical device comprising a material of tetrasilver tetroxide nano-aggregates according to any one of the claims 18-21 or a film of material of tetrasilver tetroxide nano-aggregates according to any one of the claims 22-23 and suitable medical grade elements.

27. Medical device according to claim 26, wherein said suitable medical grade elements are selected from the group consisting of one or more substrates and optionally one or more suitable excipients.