Patent Description:
The invention provides also synthesis methods for the production of ZnO nanostructures with silver coating, with nanometric SiO<NUM> layer interposition. State of the art.

At the state of the art, there are known zinc oxide nanostructures (ZnO) in the form of crystalline wurtzite, which can be synthetized in various shapes and dimensions through chemical or metallurgical processes.

The metallurgical processes typically used for ZnO synthesis in the wurtzite crystalline form guarantee optimal yields both from the qualitative and quantitative point of view, but they require sophisticated equipment and the relative costs are very high. The chemical processes are based on mechanical-chemical, sol-gel, hydro- or solvothermal synthesis, pyrolysis, sonochemical processes or on controlled precipitation.

Among these ones, the controlled chemical precipitation is among the simpler and more economical ones and allows to produce crystalline ZnO apt for applications in biological and bio-technological field (as anti-bacterial agent and natural photosensitizer), as well as in nanotechnologies field for the production of piezoelectric nanogenerators.

The process comprises two steps: the first one providing the reduction of a solution of a zinc salt (i.e. zinc sulfate, ZnSO<NUM>) by means of a reducer (typically sodium hydroxide, NaOH), thus obtaining a zinc oxide "precursor". In the second step, the precursor is subjected to a thermal treatment with the double function of converting the precursor (i.e. Zn(OH)<NUM>) in ZnO and of avoiding the formation of intermediate compounds which can nullify the purity, and generally the chemical-physical properties of the material, thus making it not suitable to be used in biological and bio-technological field.

The above described two steps process is regulated by various parameters of synthesis, and in particular:.

Each of these parameters influences the material final properties significatively, both in terms of crystallinity and purity, but also in terms of particles dimensions. The adjustment of such parameters allows to control the synthesis process very carefully and to obtain, as a function the chosen set-up, a material with determined chemical-physical properties and in particular a determined crystallinity and purity grade.

It is also known that the composition of the metal salt and/or the usage of possible additives can modify the shape and dimension of ZnO particles. More in detail, the effect of the additives used is very much different according to their chemical nature. For example, the inorganic additives are incorporated in the crystal lattice during the precipitations and this can cause disorder in the lattice (i.e. this can induce a low crystallinity and purity grade), while the organic compounds can be adsorbed on the crystalline face and can modify the growth rate (i.e. the particles dimensions).

In the document "Microwave-assisted synthesis, photocatalysis and antibacterial activity of Ag nanoparticles supported on ZnOs flowers" (<NPL>), heterogenous ZnO/Ag structures are described in which the zinc oxide, in wurtzite crystalline form, appears in clusters of many hundreds nanorods with a flower shape, the single nanorods being dimensioned about <NUM> and having a length equal to <NUM>-<NUM>. On these structures, Ag cubic nanoparticles are provided, with average dimension lower than <NUM>, and silver lower than <NUM>% by weight.

In the document <NPL>) there are described structures in which Ag nanocubes with dimensions equal to about <NUM> are distributed on nanostructures comprising a series of elements and compounds, among which zinc oxide. Ag is about <NUM>% by weight.

"<NPL>) describes ZnO nanotubes in the wurtzite crystalline form on which Ag nanoparticles are provided, with diameter about <NUM>, the ones next the other ones in order to cover completely the nanotube.

"<NPL>) discloses a simple large-scale approach to prepare ZnO/SiO<NUM>/Ag nanoparticles.

At the state of the art there are not known efficient and economical synthesis methods for the production of such nanostructures.

Therefore, the present invention provides nanostructures as defined in claim <NUM>, comprising a ZnO seed in its hexagonal crystalline form (or wurtzite) partially coated with silver in the form of nanoparticles, with a nanometric silicon dioxide layer interposed between the ZnO seed and the Ag nanoparticles coating (thus obtaining the ZnO@SiO<NUM>@Ag complex). Yet, the present invention provides synthesis methods as defined in claim <NUM> for the production of the just cited nanostructures.

Also disclosed is a method for the production of singlet oxygen <NUM>O<NUM> by means of the activation of the above described nanostructures by means of visible light.

The invention is applied in the pharmaceutical, bioengineering, bio-technological and advanced medical therapies fields. ZnO nanostructures functionalized with Ag·NPs (i.e. ZnO@Ag adducts which are however not according to the invention) are in fact characterized by excellent antibacterial, anti-inflammatory and anti-tumoral properties and can be conveniently and efficiently incorporated in protein-based (e.g. collagen), polysaccharide-based (e.g. hyaluronic acid) and/or inorganic-based (e.g. hydroxyapatite) preparations, of natural and/or synthetic origin, for the production of bioactive and "smart" materials and devices to be used in the above cited fields.

The present invention reaches the prefixed aims since it is a nanostructure comprising a zinc oxide seed (ZnO), in the wurtzite hexagonal crystalline form, surface-coated with silver nanoparticles (Ag), said silver nanoparticles being spaced to each other. The ZnO seed is in nanoflakes form, and it has characteristic dimensions of few hundreds of nanometers in length and few nanometers in thickness; according to the invention also said nanostructure comprises a nanometric silicon dioxide layer interposed between said zinc oxide seed and said silver nanoparticles coating.

Also disclosed is a synthesis method of nanostructures comprising a ZnO seed coated with silver nanoparticles, comprising the steps of:.

As yet said, the invention provides nanostructures comprising a zinc oxide seed, in the hexagonal crystalline form (i.e. wurtzite).

The wurtzite crystalline form is needed to guarantee that the ZnO intrinsic chemical-physical properties are preserved. In fact, to it the capacity of absorbing light is associated, the basic property of the applications of the nanostructures of interest. Moreover, thanks to the particular crystalline structure of the basic material (i.e. ZnO), as well as the peculiar architecture of the adduct/complex obtained, these structures show also particularly relevant pyroelectric and piezoelectric properties.

The ZnO nanostructures have the shape of nanoflakes with dimensions about <NUM> and a thickness between <NUM> and <NUM> and preferably equal to about <NUM>.

The nanoflakes form has allowed to optimize the anisotropic cover with Ag nanoparticles of the ZnO surface, which is a fundamental requirement for the desired technical effect.

According to the invention,
Ag NPs deposited on the surface have a substantially spherical shape, and an average dimension between <NUM> and <NUM>. They are adhered to the surface of the ZnO nanostructure and spaced to each other. According to the invention, between the single silver nanoparticles it is provided a distance about <NUM>-<NUM>. The spacing is a pre-requirement fundamental for the applications of the invention. The aggregation of many Ag NPs is absolutely to be avoided to allow the plasmonic excitation and so, again to maximize the light absorbance and to exploit the electronic communication between ZnO and Ag at best.

The surface coating of Ag nanoparticles has the function to improve a series of wurtzite intrinsic optical properties, thus making it an efficient antibacterial and anti-inflammatory agent, as well as a powerful photosensitizer for the production of oxygen free radicals, in primis the singlet oxygen, with a very greater efficiency than the ZnO as it is.

The nanostructures comprising ZnO seeds decorated with Ag·NPs, are characterized by excellent antibacterial, anti-inflammatory and anti-tumoral properties and can be conveniently and efficiently incorporated in protein-based (e.g. collagen), polysaccharide-based (e.g. hyaluronic acid) and/or inorganic-based (e.g. hydroxyapatite) preparations, of natural and/or synthetic origin, for the production of bioactive and "smart" materials and devices to be used in the pharmaceutical, biomedical and bio-technological fields.

The basic idea is to realize a ZnO-Ag interface or junction in order to exploit the photo-induced charge transfer from the semiconductor conduction band (i.e. ZnO) to the Fermi level of the metal (i.e. Ag).

Such charge passage induces a delay in the recombination of the (h+)/electron defect couple, with defects stabilization in valence band of ZnO and electrons photo-excited at Ag level. Such electrons reduce atmospheric O<NUM> to O<NUM><NUM>- which is in turn oxidized to <NUM>O<NUM> (singlet oxygen) by the defects stabilized at ZnO level. As it is well known, the singlet oxygen has a very important role in the photo-dynamic therapy of the cancer and other diseases. In fact, the oxygen free radicals, such as the singlet oxygen, can induce the death of pathological cells, since they are very powerful oxidant species.

The analyzed electronic transfer allows to improve remarkably a series of ZnO intrinsic properties in its wurtzite crystalline form, which, so modified, results a powerful antibacterial, anti-inflammatory and photosensitizer agent for the production of oxygen free radicals, in primis the singlet oxygen. The specific characteristic of Ag coating according to the invention, and in particular the spacing between nanoparticles, allow to optimize the photoexcitation effect, since the aggregation of Ag nanoparticles would alter the electronic communication between the semiconductor and the same Ag. The geometry obtained is in fact another fundamental requirement for the application of the invention.

According to the invention, the ZnO@Ag nanostructures contain further a nanometric silicon dioxide layer interposed between the zinc oxide seed and the silver nanoparticles coating, thus obtaining the ZnO@SiO<NUM>@Ag complex. According to the invention, the nanometric layer is about <NUM> thick.

The introduction of an insulating silicon dioxide layer (SiO<NUM>) between ZnO seed and Ag NPs allows to exploit the localized Surface Plasmonic Resonance phenomenon. In this way, in fact, the visible light can excite the surface plasmon associated with Ag nanoparticles, and this energy can be transferred to ZnO by Plasmon Induced Resonant Energy Transfer (PIRET). After PIRET, other electrons will be promoted in ZnO conduction band able to reduce further the atmospheric O<NUM> to O<NUM><NUM>-, thus increasing remarkably the production of singlet oxygen and other reactive oxygen species (or "ROS"). The role of the SiO<NUM> layer is to create a little insulating barrier which avoids the charge-transfer from the semiconductor (ZnO) towards the metal, thus favouring the plasmonic resonance effect and delaying the electronic recombination.

The very relevant result reached is the possibility to produce singlet oxygen and other ROS through simple irradiation of the ZnO@Ag@SiO<NUM> complexes in the visible range and not in UV.

As yet said according to the state of the art, the parameters of synthesis have a relevant effect on the characteristics of the nanostructures obtained. Therefore, the present invention provides a particular set-up of the parameters of synthesis, which allows to obtain nanostructures comprising ZnO seeds characterized by a high crystallinity and purity grade. In this case, the present invention is aimed at the definition of a particular thermal treatment to which the ZnO precursor (i.e. Zn(OH)<NUM>) has to be subjected to, defined by the temperature applied and the heating duration, which allows to obtain ZnO with high crystallinity and purity grade, particularly apt for applications in the above cited fields.

Such thermal treatment, and as a consequence the whole synthesis process, besides being more efficient in terms of purity and crystallinity grade than the final material (i.e. nanocrystalline ZnO in wurtzite form), compared to the chemical precipitation methods currently used, is also more economical, by providing the exposition of the material to definitely lower temperatures (i.e. <NUM> ± <NUM>) compared to these last ones (i.e. <NUM> ± <NUM>).

The process comprises two steps, the first one providing the reduction of a solution of zinc oxide (i.e. zinc sulfate, ZnSO<NUM>), by means a reducer (typically sodium hydroxide, NaOH), thus obtaining a zinc oxide "precursor" (i.e. zinc hydroxide, Zn(OH)<NUM>) followed by the precipitation of the same precursor inside the solution (reaction <NUM>).

In the second step, the precursor is subjected to a thermal treatment, with the double function of converting the precursor (i.e. Zn(OH)<NUM>) in ZnO and of avoiding the formation of intermediate compounds which can nullify the purity, and generally the chemical-physical properties of the material, thus making it not suitable to be used in biological and bio-technological field.

In particular, Zn(OH)<NUM> is heated at ambient pressure in a controlled way, and this allows to obtain highly pure, crystalline ZnO powders (reaction <NUM>).

ZnSO<NUM> + 2NaOH -> Na2SO<NUM> + Zn(OH)<NUM>     (reaction <NUM>).

Zn(OH)<NUM> + Q -> ZnO + H<NUM>O     (reaction <NUM>).

As a way of not limiting example, it was found that it is needed to fix the ZnSO<NUM> to NaOH molar ratio between <NUM> and <NUM> to obtain ZnO seeds in the wurtzite crystalline form about <NUM> and with nanoflakes form; that, to obtain the just described result, the treatment temperature is between <NUM> and <NUM>, and preferably about <NUM>, and that the treatment duration is preferably equal to at least <NUM>.

Concerning the silver nanoparticles (Ag·NPs), they are synthetized with a two steps chemical process, ad hoc developed in order to obtain the adduct.

The first step provides the synthesis and the deposition of a silver precursor (i.e. diamminesilver [Ag(NH<NUM>)<NUM>]+ on the ZnO seed surface. Specifically, the crystalline ZnO synthetized according to what yet described is contacted to silver nitrate (AgNOs) and ammonium hydroxide (NH<NUM>·OH) in strongly alkaline environment (pH > <NUM>) and under constant stirring. The reaction time is between <NUM> and <NUM> hours, and the reaction occurs at ambient pressure and temperature.

AgNOs and NH<NUM>·OH react by forming [Ag(NH<NUM>)<NUM>]+, which, being positively charge, binds to the zinc oxide surface (typically provided with net negative surface charge).

The deposition of the silver precursor on the ZnO seed surface allows that the precursor reduction, in the next step, occurs directly at contact to the ZnO seeds surface, thus avoiding the Ag·NPs homologous nucleation, i.e. the formation of not adhered Ag·NPs to ZnO seeds.

The second step consists in fact of the in situ precursor reduction, obtained by adding a strong reducer agent (e.g. sodium borohydride, NaBH<NUM>) at controlled concentration and speed.

The quantity of reducer to be used and its addition speed represent very important parameters for the careful control of Ag·nPs nucleation on the ZnO seed surface, and, so, of their shape and dimensions. Preferably, <NUM> of (<NUM>) NaBH<NUM> for each <NUM> reaction, in which Ag precursor (<NUM>) and ZnO (<NUM>) are dissolved, are added at <NUM>/min speed. The precursor reduction leads to the obtainment of crystalline ZnO nanostructures, surface-coated with silver nanoparticles.

The SiO<NUM> heterologous growth on ZnO seeds surface is carried out by means of a sol-gel process, starting from an organic precursor (e.g. tetraethyl orthosilicate, Si(OC<NUM>H<NUM>)<NUM>) which is hydrolyzed and condensed in alkaline environment (pH > <NUM>).

The SiO<NUM> shell thickness has to be about <NUM>, in order to guarantee the desired electronic effects. The chosen growth process is controlled by the precursor concentration, by the pH and reaction time, which is carried out at ambient temperature and pressure; it is important to find the optimal set-up of such parameters to control the coating thickness.

According to the invention, the reaction is carried out under stirring for <NUM> hours at ambient temperature and pressure, fixing the reagents molar ratio ZnO - tetraethyl orthosilicate - ammonium hydroxide equal to <NUM>:<NUM>, <NUM>:<NUM>.

According to the invention, the Ag nanoparticles are deposited only after SiO<NUM> layer interposition and grown in situ on the silica surface by means of the same method, previously described concerning bare ZnO seed.

Other characteristics and advantages of the invention will be clearer in light of the detailed description contained in the (preferred but not exclusive) embodiments reported in the next section and shown as a way of not limiting example with reference to the appended drawings.

It is clear the characteristic morphology of spherical AgNPs, with dimensions about <NUM>-<NUM> and spaced between <NUM> and <NUM>. Clearly, not all the nanoparticles have these dimensions and this distance, which have to be intended as average values.

A <NUM> solution of sodium hydroxide (NaOH) in ultrapure water (solution A1) is additioned to a <NUM> solution of zinc sulfate (ZnSO<NUM>) in water/isopropanol (solution B1). The reaction is carried out under strong stirring for <NUM> hours at ambient temperature. The white precipitate obtained is washed repeatedly with ultrapure water and separated by centrifugation, and finally oven dried. The parameters and the process steps are the following:.

A <NUM> solution of silver nitrate (AgNO<NUM>) in water (solution A2) is lead to pH <NUM> by addition of a solution of NH<NUM>·OH <NUM>% (solution B2). To this solution <NUM> of ZnO are added, synthetized as in example <NUM>, thus obtaining a new solution (solution C2). The reaction is carried out under strong stirring for <NUM> hours at ambient temperature. At the end of stirring, a <NUM> solution of sodium borohydride (NaBH<NUM>) (solution D2) is additioned to solution C2 at controlled speed, equal to <NUM>/min. The reaction is carried out for <NUM> at ambient temperature and pressure. The grey precipitate obtained is washed repeatedly with ultrapure water and separated by centrifugation, and finally oven dried. The parameters and the process steps are the following:.

A solution of NH<NUM>·OH <NUM>% in water (solution A3) is additioned to a second solution (solution B) <NUM> ZnO (prepared as in example <NUM>) and <NUM> TEOS in water/ethanol. The reaction is carried out under strong stirring for <NUM> hours at ambient temperature. The white precipitate obtained is washed repeatedly with ultrapure water and separated by centrifugation, and finally oven dried. The parameters and the process steps are the following:.

A <NUM> solution of silver nitrate (AgNOs) in water (solution A4) is lead to pH <NUM> by addition of a solution of NH<NUM>·OH <NUM>% (solution B4). To this solution <NUM> of ZnO@SiO<NUM> are added, synthetized as in example <NUM>, thus obtaining a new solution (solution C4). The reaction is carried out under strong stirring for <NUM> hour at ambient temperature. At the end of stirring, a <NUM> solution of sodium borohydride (NaBH4) (solution D4) is additioned to solution C4 at controlled speed, equal to <NUM>/min. The reaction is carried out for <NUM> at ambient temperature and pressure. The grey precipitate obtained is washed repeatedly with ultrapure water and separated by centrifugation, and finally oven dried. The parameters and the process steps are the following:.

Claim 1:
Synthesis method of nanostructures comprising a zinc oxide seed (ZnO) in the wurtzite hexagonal crystalline form and nanoflake shape with dimensions of <NUM> and <NUM> thickness, surface-coated with silver nanoparticles (Ag) of substantially spherical shape and average dimensions between <NUM> and <NUM>, said silver nanoparticles being adhered to the surface of said nanostructure, and being spaced to each other, further comprising a nanometric silicon dioxide layer of <NUM> interposed between said zinc oxide seed and said silver nanoparticles coating, the method comprising the steps of:
A. reduction of a solution of a zinc salt by means of a reducer, thus obtaining a zinc oxide precursor and precipitation of said precursor in the solution;
B. thermal treatment of said precursor at ambient pressure and temperature between <NUM> and <NUM>, thus obtaining ZnO seeds;
C. addition of silver nitrate (AgNOs) and ammonium hydroxide (NH<NUM>·OH) to said ZnO nanoparticles, under constant stirring at ambient pressure and temperature, thus obtaining [Ag(NH<NUM>)<NUM>]+ which, being positively charged, binds to the surface of said ZnO seeds;
D. reduction of [Ag(NH<NUM>)<NUM>]+ by means of the addition of a reducer which allows the formation of silver nanoparticles thus obtaining nanostructures comprising crystalline ZnO seeds, surface-coated with silver nanoparticles
characterized in that it further comprises, after step B and prior to step C, the step of B'. reaction of said ZnO seeds, obtained in step B, and tetraethyl orthosilicate and ammonium hydroxide so that the molar ratio ZnO - tetraethyl orthosilicate and ammonium hydroxide is equal to <NUM>:<NUM>, <NUM>:<NUM>, for two hours at ambient temperature and pressure.