WO2011045627A1

WO2011045627A1 - Viricide agents having nanostructured biocatalysts materials of titanium dioxide (tio2) and silicium dioxide (si02) with platinum and iridium modified and dispersed on the surface

Info

Publication number: WO2011045627A1
Application number: PCT/IB2009/007188
Authority: WO
Inventors: Tessy Maria Lopez Goerne
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

This invention is related to the synthesis of nanostructured inorganic materials with a general formula M_aL_bB_c0_2(b+c)(OH)x(PO₄)y(S0₄)_z where M is silicium, titanium or a mix of both, and L is iridium and B platinum, B+L<M, where b+c=1 and b is different of c. The particle size of the nanostructured matrix, acidity, mean pore size of the matrix and platinum, iridium or platinum-iridium particle size can be controlled since the synthesis. Nanostructured materials can be use as viricide.

Description

VIRICIDE AGENTS HAVING NANOSTRUCTURED BIOCATALYSTS MATERIALS OF TITANIUM DIOXIDE (TI0₂) AND SILICIUM DIOXIDE (SI0₂) WITH PLATINUM AND IRIDIUM MODIFIED AND DISPERSED ON THE

SURFACE. DESCRIPTION

This invention is related to de synthesis of nanostructured inorganic materials with a general formulation MaLbB_c02(b+c)(OH)_x(P04)y(S0₄)z where M is silicium, titanium or a mix of both, and L is iridium and B platinum, B+L<M, where b+c=1 and b is different of c. The particle size of the nanostructured matrix, acidity, mean pore size of the matrix and platinum, iridium or platinum-indium particle size can be controlled since the synthesis. Nanostructured materials can be use as viricide.

BACKGROUND FOR THE INVENTION

In the past decade, scientists' ability to manipulate materials at the molecular and atomic levels using nanotechnology has moved from science fiction into scientific fact. Now, nanotechnology is being developed to prevent, diagnose, and treat infectious diseases, with some products in or nearing clinical trial stage. Progress in this field is exponential ^{(1 6)}. Cross-disciplinary nanoscience and nanomedicine researches involving chemists, physicists, biologists and engineers is concerned about the need for developing environmentally friendly and sustainable methods for the synthesis of nanomaterials. There is a current drive to integrate all the green chemistry approaches to design environmentally benign materials and processes. Rapid developments are taking place in the synthesis of biocompatible mixed oxides or simple oxides metallic and bimetallic nanomaterials and their surface modification for bioactivity and nanomedical applications. Biosynthesis of nanoparticles as an emerging highlight of the intersection of nanotechnology and biotechnology has received increased attention due to a growing need to develop environmentally benign technologies in material syntheses. Biomolecules as reluctant are found to have a significant advantage over their counterparts as protecting agents ^{(7 13)}.

The properties of the materials can change notably when its particle size is reduced to particles in the scale of nanometers. In materials science "particle" is a general term to describe small solid objects with sizes ranging from atomic scale (10^"1° m) to the microscopic scale (10 ³ m). However, the particle size often lies between 10^"9-10^"5 m. Large particles (> 10^"6) are commonly called grains (i. e. zeolites, carbon, Raney metals) and small particles (<15 nm) are frequently added (metals) to mixed oxides, i.e. Ti02-Si0₂, S1O2 or TiO2^(14"20). All materials consist of grains (particles) formed by the agglomeration of nanoparticles (Figure 1 ).

In conventional materials the grains have a size ranging from 100 micrometers to millimeters (mm), while the Nanomaterials particles are in the order of a one billionth of a meter (10 ⁹). A nanometer is around the average diameter of human hair. The radius of an atom is 1 to 3 Angstrom (A) and a nanometer is equal to 10 A. Nanomaterials are rigid solid, resistant and are ductile at high temperatures, are resistant to degradation, erosion and corrosion, are also very chemically active. The physical and chemical properties of each nanomaterial or nanostructured material are determined by the type of interactions compounds that functionalized the nanoparticles; as well the electronic density and hydroxyl concentration in the net have an important role in the DNA cracking.

One of the areas in which nanoparticles have increased the importance is in the field of catalysis and biocatalysis, in order to obtain a distribution of particles with well defined shape and size to improve the catalytic activity. In particular, it is necessary to obtain highly dispersed particles in which most of the atoms are located on the surface. The structure includes a solid area, pore size, as well as the shape and volume of the pores. These parameters are important in catalysis and in biocatalysis, because they are responsible to increase the reaction rate. Although the catalytic activity can be related directly with its total contact area between the material and the reactive to catalyze, the determination of the area is generally considered to be an important requirement in the characterization of the biocatalysts. Furthermore, it is necessary to specify the nature of the structure of the pores as they control the transport of reagents and products in the catalytic reaction. In many cases, the course of the reaction is influenced by the size and shape of the pore but the particle size of the support and the metals are more definitively.

Titanium dioxide occurs in nature in three crystalline phases; anatase, rutile and brookite (Fig. 2). The anatase and brookite can transform into rutile at high temperatures. The anatase to rutile can be irreversibly transformed by heating. There are several factors that influence the phase shift, such as particle size, crystal morphology, but in particular influence of the poisoning ions to the network. The literature indicates that the three phases, the anatase has a great chemical stability, resistance to corrosion, is inert from biological agents and has high specific surface area. However, the commercial titania Is a mix (Degussa P25) and contains 60 to 80% anatase. The problem only to produce the anatase phase is due to the rutile phase is thermodynamically more stable. Therefore by the sol- gel process is possible to obtain pure anatase and pure rutile. The structure of anatase and rutile are tetragonal, whereas brookite is orthorhombic, each titanium atom is attached to 6 oxygen atoms almost equidistant and each oxygen atom is linked to three atoms of titanium.

By the other hand silica sol-gel is an amorphous material with high specific area and high concentration of hydroxyl groups in its surface. Recently, the need for disinfectants, bactericides and viricides with confirmed efficiency against various microorganisms like virus and bacteria has increased (Fig. 3).

This is related to emergence of new infections (e.g. HIV) and re-emergence of previously controlled infections due to drug resistance, environmental changes, and lifestyle alterations. Moreover, some infectious diseases can extend by use of new medical devices that cannot be sterilized by traditional techniques, such as heat treatment. Nanomedicine will have a profound impact on infectious disease in several ways, for improved diagnostics and detection, targeted therapies, and antibacterial or antiviral surfaces. Diagnostic technologies combine a recognition system and a detection system, from tiny cantilever that move on binding antigen to nanowires that detect current from immune-cell binding. For prevention, nanotechnology-based microbicides against HIV are now in early clinical trials. Laboratory studies on new vaccines against rotavirus, hepatitis B, tuberculosis, HIV, Influenza and antibacterial coatings for surfaces or materials look promising. Such coatings may "reduce the problem of bacterial or viral adhesion on hospital surfaces and have a beneficial impact on in-hospital transmission of multiresistant bacteria or virus that represents a major, unsolved problem. Sol-gel derived silica and titania have a specific interaction with many biological molecules, microbes, algae, cells and living tissue. The specific interactions mean that they differ from common reactions between non-viable materials and biomolecules or living tissues and the interactions are mostly beneficial from the viewpoint of biotechnical applications. Peptides and proteins may preserve their activity and bacteria, algae and cells may preserve their viability and viruses their infectivity as encapsulated in sol-gel derived silica. Silica and titania are known to form a direct bond with living tissue which can be utilized in the biomaterial applications. Other application areas of silica and titania are in biosensing, tissue engineering, gene therapy, controlled delivery of therapeutic agents and environmental protection*^21"30'. ADDITIONAL TITANIA, SILICA OR TIO₂-SIO₂ PATENTS USING SOL-GEL METHOD

U.S. Pat. No. 6 124 367. This patent protects reservoirs used in the Fischer Tropsch reactions from sintering by imparting a higher degree of mechanical strength to the reservoir. It incorporates S1O2 and Al₂0₃ into the reservoir and claims a rutile-anatase ratio of 1/9. It is a porous reservoir with either a spherical or a cylindrical shape. It is made by extrusion, spray drying or tableting.

U.S. Pat. No. 6117814. This patent describes a titania reservoir which also incorporates silica and alumina as a binder into the structure. The purpose of the binder is to impart better mechanical properties to the reservoir. The size range of this reservoir is from between 20 to 120 microns. The reservoir is approximately 50% binder, which is fabricated by a sol-gel process.

U.S. Pat No 6087405. This patent describes a reservoir to be used in a Fischer Tropsch gas synthesis reaction. The reservoir incorporates group VII metals into its structure. The rutile-anatase ratio in the structure is a distinguishing feature of this patent.

Centre National De La Recherche Scientifique. PWO/2003/064324. The invention relates to a titanium oxide-based polymer composition. The inventive composition comprises a TiOx(OH)y(H₂0)_z (x+y+z=3) titanium oxide-based polymer in the form of a gel or sol. Said polymer, which has a one-dimensional (1 D) structure, is made from concentrically-wound fibres having a periodicity, which is deduced from the spacing between said fibres, of between 3.5 A and 4 A. Each fibre comprises Ti0₆ octahedrons and each Τΐθε octahedron shares two opposite edges with two adjacent octahedrons (2 x 2.92 A) in order to form infinite chains which develop along the axis of a fibre. According to the invention, two adjacent chains form double lines as a result of the shared edges (2 x 3.27 A). The inventive polymer is suitable for use as a photosensitive element in a photovoltaic cell, such as a sunscreen for a window. COMMISSARIAT A L'ENERGIE ATOMIQUE. WO/2006/079757. Method of preparing stable oxide ceramic precursor sol-gel solutions based on lead, titanium, zirconium and lanthanide(s) and method of preparing said ceramic. The invention relates to a method of preparing a stable oxide ceramic precursor sol-gel solution based on lead, titanium, zirconium and lanthanide(s). The invention comprises the following successive steps consisting in: a) preparing a sol-gel solution by bringing a molecular lead precursor, a molecular titanium precursor, a molecular zirconium precursor and a molecular lanthanide precursor into contact with a medium comprising a diol solvent and optionally an aliphatic mono-alcohol; b) leaving the solution thus obtained to stand for a sufficient period of time in order to obtain a solution having an essentially-constant viscosity; and c) diluting the solution obtained in the preceding step with a diol solvent identical to that used in step a or a solvent that is miscible with said solvent, at a predetermined rate. The invention can be used to prepare an oxide ceramic material comprising lead, a lanthanide metal, titanium and zirconium.

Universidad Autonoma Metropolitans. WO/2007/141590. Sol-gel nanostructured titania reservoirs for use in the controlled release of drugs in the central nervous system and method of synthesis. The invention is related to a sol- gel nanostructured titania reservoir and its synthesis which is biocompatible with brain tissue. The pore size distribution, crystallite size and the extent of the crystalline phase distribution of anatase, brookite and rutile can be fully controlled. This device may be used to contain neurological drugs. It may be inserted directly into brain tissue for the purpose of the controlled time release of drugs over a period of from 6 months to three years.

W093/21969. Novel coating materials for biomedical applications, particularly for use on biomedical implants, the coating material containing gel-derived titania where the material is capable of inducing calcium phosphate formation onto its surface under in vitro conditions, e.g. in a simulated body fluid and/or under in vivo conditions, processes for the preparation of the coating materials as well as their use in biomedical implant technology.

OBJECTIVES

1. The development of nanostructured biomaterials for use like viricide. 2. Optimization of these nanomaterials to enable control of the following parameters: acidity of the support, BET area, pore size distribution, particle size, degree of functionalization, electronic density, dispersion of the metal or metals on the support.

3. Obtain high activity to break the C-C and C-N bonds of the proteins, RNA and DNA , that compound the virus specie. It will be important that the concentration of platinum and Iridium or platinum-iridium supported on the matrices have elevated dispersion to get high efficiency to cracking the C-C and C-N protein and nucleotides bonds.

4. The materials will consist of nanostructured titania, silica or mixed oxides titania-silica prepared using sol-gel methods. These materials are functionalized and have high dispersed Pt or Pt-lr with a small particle size (2- 10 nm).

INVENTION DETAILS

1. The present invention is related to novel nanostructured materials (silica, titania and titania-silica) synthesized by the sol-gel process. 2. This nanomaterials are gels partially hydroxylated, with a Ti:Si ratio between 0: 100 and 100:0.

3. These materials were prepared using the sol-gel process to synthesize glass and ceramic with a particle size between 1 -200 nm. 4. During the gelation and drying process, the temperature is controlled in order to stabilize the interactions within the network and attractions molecular Van der Waals type. If the gel does not have enough time to evaporate the liquid, properties change, i.e. could increase the particle size and not create sufficient Bronsted and Lewis acid sites in the network of the matrix. The resulting gels were dried from room temperature to 70°C using a rotary evaporator.

5. The nanoparticulate materials were functionalized with phosphate, ammonia, • carboxyl and hydroxyl groups, which are stable and linked to the surface after drying. Using ammonium sulphate, ammonium phosphate, phosphoric acid, sulfuric acid, GABA, acetate and acetilacetonate. 6. The polymerization continues for a long period of time after the gelacion. Both the titania (T1O2) and silica (S1O2) or a mixture of both (Ti02-Si02) are functionalized from the start of the reaction to achieve biocompatible materials.

7. In the nanostructured materials obtained have been replaced the Si or Ti atoms for platinum (Pt) and iridium (Ir) atoms from 0.01 % to 5%. In order to act as biocatalysts breaking C-C and C-N linkages of RNA and proteins of the virus. . The biocatalytic activity of nanostructured biomaterials depends on the particle size of titania or silica or mixed oxides, the particle size of iridium and platinum supported on the surface and the density of acid sites. . The electronic structure of nanostructured biocatalysts is controlled in order to generate point defects in the network of biocatalysts leads to free radicals that accelerate the cracking of C-C and C-N links in the RNA bases, and in the proteins of the virus.

10. Both the titania (Ti0₂) and silica (S1O2) or a mixture of both (Ti0₂-Si02) have hydroxyl, carboxyl, ammine, sulphate and phosphate functional groups linked to the nanoparticle surface to be recognized by the virus.

1 1 . The synthesis of pure titania, pure silica or a mixture of both is done at a pH between 2 and 12.

12. The synthesis of pure titania, pure silica or a mixture of both used silicon and titanium alkoxides as precursors. These alkoxides can be ethoxide, propoxide or butoxide of Ti or Si.

13. The synthesis of pure titanium, pure silica or a mixture of both is performed with a molar ratio alkoxide: H₂0 from 1 :4 to 1 :70.

14. The molar alkoxide: solvent from 1 :8 to 1 :70. The solvent used is polar or non- polar, aprotic or protic. 15. The synthesis of pure titanium, pure silica or a mixture of both is performed via sol-gel process with constant agitation at a temperature from ambient to 70°C. 16. The kinetics of the process of cracking links biocatalytic C-C and C-N show a direct dependence of the particle size from 1 nm to 200 nm.

17. The order of the kinetics reaction and the activity of biocatalysts depend of the platinum ligands, iridium ligands, specific area and mean pore size, acidity and electronic distribution of the matrix (Si0₂, Ti0₂ and Si0₂-Ti0₂).

18. The order of the kinetics reaction and the activity of biocatalysts depend or the virus conformation.

DETAILED DESCRIPTION OF THE METHOD.

Sol-gel process using metal alkoxides. At the functional group level, three reactions are generally used to describe the sol-gel process: hydrolysis, alcohol condensation, and water condensation. However, the characteristics and properties of a particular sol-gel inorganic network are related to a number of factors that affect the rate of hydrolysis and condensation reactions, such as, pH, temperature and time of reaction, reagent concentrations, catalyst nature and concentration, H₂0/M molar ratio (R), aging temperature and time, and drying. Of the factors listed above, pH, nature and concentration of catalyst, H₂O/M molar ratio (R), and temperature have been identified as most important. Thus, by controlling these factors, it is possible to vary the structure and properties of the sol-gel-derived inorganic network over wide ranges. For example, Sakka et al. observed that the hydrolysis of TEOS utilizing R values of 1-2 and 0.01 M HCI as a catalyst yields a viscous, spinnable solution. It was further shown, that these solutions exhibited a strong concentration dependence on the intrinsic viscosity and a power law dependence of the reduced viscosity on the number average molecular weight^{(31 34)}:

[n] = k(Mn)a (1 )

Values for a ranged from 0.5 to 1.0, which indicates a linear or lightly branched 5 molecule or chain.

Values of "a" in eq. 1 ranged from 0.1 to 0.5, indicating spherical or disk shaped particles. These results are consistent with the structures which emerge under the conditions employed by the Strober process, for preparing S1O2 powders. It was further shown that with hydrolysis under basic conditions and R values ranging I 0 from seven (7) to twenty-five (25), monodisperse, spherical particles could be produced.

Alcoholysis

Generally speaking, the hydrolysis reaction (Eq. 2), through the addition of water, replaces alkoxide groups (OR) with hydroxyl groups (OH). Subsequent ) 5 condensation reactions are made, involving the silanol groups (Si-OH) produce siloxane bonds (Si-O-Si) plus the by-products water or alcohol in the case of silica. Under most conditions, condensation commences before hydrolysis is complete. However, conditions such as, pH, H₂0/Si molar ratio (R), and catalyst can force completion of hydrolysis before condensation begins. Additionally, because water and alkoxides are immiscible, a mutual solvent is utilized. With the presence of this homogenizing agent, alcohol, hydrolysis is facilitated due to the miscibility of the alkoxide and water. As the number of siloxane bonds increases, the individual molecules are bridged and jointly aggregate in the sol. When the sol particles are aggregate, or inter-knit into a network, a gel is formed. Upon drying, trapped volatiles (water, alcohol, etc.) are driven off and the network shrinks as further condensation can occur. It should be emphasized, however, that the addition of solvents and certain reaction conditions may promote esterification and depolymerization reactions. The hydrolysis/condensation reaction follows two different mechanisms, which depend of the coordination of metallic central atom. When the coordination number is satisfied the hydrolysis reaction occurs by nucleophilic substitution (S_n)^(31"34):

O R O R

\

H _jO ⁺ I

R O M —O R R O M — O a*

I I I

H

Hydrolysis reaction via nucleophilic substitution (S_n). When the coordination number is major, the hydrolysis reaction takes place by nucleophilic addition:

O R O R O H

O R O R

\

R O M o R O M O H + O H

° ^R O R Hydrolysis reaction via nucleophilic addition (A_n).

These mechanisms need that the oxygen coordination is increased from 2 to 3, the additional bond generation involves one electron pair of the oxygen and the new bond can be equivalent to the other bonds. During the condensation step an enormous concentration of hydroxyl groups are formed. This OH can be linked between the metallic atoms or only be simple -OH ligand in the surface'^35"38'.

* · ^{4 *} * ·

6

Condensation step of the sol-gel method Acid-Catalyzed Mechanism

Under acidic conditions, it is likely that an alkoxide group is protonated in a rapid first step. Electron density is withdrawn from the silicon atom, making it more electrophilic and thus more susceptible to attack from water. This results in the formation of a penta-coordinate transition state with significant SN₂-type character. 13 The transition state decays by displacement of an alcohol and inversion of the silicon tetrahedron, using silica as example*^39"40*:

FAST

-Si-OR + H — Si-0-R

I H

Acid-Catalyzed HydrolvsisBased-Catalyzed MechanismBase-catalyzed hydrolysis of silicon alkoxides proceeds much more slowly than acid-catalyzed hydrolysis at an equivalent catalyst concentration. Basic alkoxide oxygens tend to repel the nucleophile, -OH. However, once an initial hydrolysis has occurred, following reactions proceed stepwise, with each subsequent alkoxide group more easily removed from the monomer then the previous one. Therefore, more highly hydrolyzed silicones are more prone to attack. Additionally, hydrolysis of the forming polymer is more sterically hindered than the hydrolysis of a monomer. Although hydrolysis in alkaline environments is slow, it still tends to be complete and irreversible. Thus, under basic conditions, it is likely that water dissociates to produce hydroxyl anions in a rapid first step. The hydroxyl anion then attacks the silicon atom. Again, an SN₂-type mechanism has been proposed in which the -OH displaces -OR with inversion of the silicon tetrahedron ^{( 1_ 3)}.

Base-Catalyzed Hydrolysis

EXAMPLE

Pt(NH₃)₄Cl2 was dissolved in ethanol and distilled water. The solution was stirred continuously under constant reflux. Following the complete dissolution of the salt, tetraethoxysilane (TEOS), γ-Aminobutyric acid (GABA), phosphoric acid and ammonium phosphate were added to the solution. The resulting sol was maintained under constant flux and continual stirring until the gel was formed. The evaporation of the solvent and water was performed at room temperature. The dry solid was crushed and subsequently used to perform characterization studies.

Infrared and Raman studies.

In the infrared transmittance spectrum (Fig 3), a band centered at 3667cm^"1 is observed. This band is assigned to an OH stretching vibration which is interacting with the Pt complex. In general this band is observed at 3700 cm^'1 on pure silica and it is due to the presence of terminal hydroxyl groups which give rise to both Lewis and Bronsted acid sites. The band centered at 3451 cm^'1 is due to OH stretching vibrations, which are incorporated into the framework of silica. The corresponding OH bending vibrations are centered at 1633 cm^"1. The infrared bands associated with the stretching vibrations of the amine groups are observed at 3230 cm^"1. These observations are consistent with the fact that the complex may have lost only one chlorine atom and that some decomposition of the complex has most likely occurred resulting in some PtO and supported coordinated Pt. In the low energy region of the spectrum, a broad band centered at 1095 cm^"1 with a shoulder at 1228 cm^"1 is observed. These vibrations are due to stretching overtone vibrations (-O-Si-O-). The platinum precursor used in the synthesis, resulted in several new features observed in the infrared spectrum. In particular an H-N-H deformation band centered at 1548 cm^"1 and an asymmetric stretching band at 3230cm^"1 are evident.

In recent years virus particle assembly, virus-cell interactions, and viral pathogenesis, approaches for the development of novel antiviral strategies or agents can be designed. Rotavirus is a genus of double-stranded RNA virus in the family Reoviridae (Double-stranded (ds) RNA viruses are a diverse group of viruses that vary widely in host range (humans, animals, plants, fungi, and bacteria), genome segment number (one to twelve), and virion organization (T- number, capsid layers, or turrets).

Influenza, commonly referred to as the flu, is an infectious disease caused by RNA viruses. The influenza A virus particle or virion is 80-120 nm in diameter and usually roughly spherical, although filamentous forms can occur. Unusually for a virus, the influenza A genome is not a single piece of nucleic acid; instead, it contains eight pieces of segmented negative-sense RNA (13.5 kilobases total), which encode 11 proteins (HA, NA, NP, M1 , M2, NS1 , NEP, PA, PB1 , PB1-F2, PB2). The best-characterised of these viral proteins are hemagglutinin and neuraminidase, two large glycoproteins found on the outside of the viral particles. The nanostructured and functionalized biocatalysts of this patent break the RNA bonds and the protein structure of the virus of this kind.

Methodology to cellular analysis.

Part i

Twelve samples of different nanostructured biocatalysts, are labeled with numbers for blind testing samples. In order to study the effect of different nanostructured materials on healthy cells MA104. This followed the steps:

1. Cell cultures were prepared on 4 plates of 48 wells (1.3X10⁵ cells per well). 2. We study the first 4 samples of nanostructured biocatalyst and prepared mixes with culture medium at a ratio 1 :30 (i.e. 1 g of sample per 30 ml culture medium) were placed in glass and were called M1a, M2a, M3a and M4a. M is the number that identifies the nanostructured biocatalyst; the letter "a" was added to identify the proportion 1 :30.

3. The preparations were sterilized at 120° C for 30 min.

4. Then, were put in repose until precipitation of agglomerate nanoparticles, we use the resultant suspension with the supernatant of nanoparticles. 5. This supernatant was placed in sample tubes, one for each. The supernatant 1 was filtered as reference in a filter paper of 0.2 μητι, trying to avoid a large agglomerates and visualize cells of the monolayer.

6. The 4 supernatants were neutralized with bicarbonate, to adapt to the environment of the cells. The amount required of HCO₃ in each culture, permit us to calculate the intrinsic acidity of the nanoparticles.

• The supernatant 1a was neutralized with 200 μΙ of bicarbonate.

• The supernatant 2a was neutralized with 1000 μΙ of bicarbonate.

• The supernatant 3a was neutralized with 400 μΙ of bicarbonate

• The supernatant 4a was neutralized with 400 μΙ of bicarbonate

The following step was made different dilutions in the culture medium with each one of the supernatants: S1a, 1 :1 , 1 :3, 1 :7, 1 :15, 1 :31 , 1 :63, 1 :127

1. Were placed 100 μΙ in each vial of 96 hollows of the 4 each plates previously cultivated.

2. Incubate 4 boxes, then observed and marked at different times.

3. Marking was performed with trypan blue (stains only dead cells in blue tone), which are observed under a microscope. Then remove the tinge. 4. A second crystal violet tinge were made (which fix the labeled cells to observe the morphology) using a microscope.

5. The crystal violet stains only cells that remain attached to the monolayer on the bottom of the well. When cells flow is indicative of losing their adhesion ability, i.e. they approach death. This method is very useful because there is a directly correlation between the amount of blue violet and healthy cells.

Conclusion of this step:

• "Healthy cells are not affected by the nanoparticles". Trypan blue tint was made known cell concentrations without nanoparticles. We used the same proportion to the nanoparticles.

• "The morphology of cells was not altered", however the required amounts of crystal violet were lower, due to pH, since the nanostructured biocatalysts have very high acidity in the atomic lattice point defects generated by the solid state structure.

• "Finally after 48 and 72 hours a healthy cells recount was made and the amount of living cells remained stable," concluding that the nanostructured biocatalyst do not damage healthy cells.

Part II

Once confirmed the viability of testing with the virus; the virus was incubated with nanoparticles and activated for one hour and then applied to healthy cells. Viruses with cells without nanoparticles were used as reference and to differentiate the activity of the infection with and without nanoparticles. Other controls of nanoparticles with cells were used to confirm the viability of these. Conclusion of this part.

• In control cells (without nanoparticles) had significantly greater amount of virus. The amount of virus varies with the concentration of nanoparticles added. · It was noted that the more diluted the nanoparticles are higher the death rate of cells infected by the virus. This is because at high concentrations of nanostructured biocatalyst, the nanoparticles are agglomerated and therefore the access to the capsid is difficult or impossible. However, it can be seen in Figure 6D, the particles surround the infected cells. This result is already cleared a significant conclusion because it is not near healthy cells.

See Figure 6.

REFERENCES

1. Maria Grazia Manera, Pantaleo Davide Cozzoli, Gabriella Leo, Maria Lucia Curri, Angela Agostiano, Lorenzo Vasanelli, Roberto Rella. Sensors and

Actuators B: Chemical, 126- 2, (2007) 562-572.

2. Si-Shen Feng, Lin Mei, Panneerselvan Anitha, Chee Wee Gan, Wenyou Zhou. Biomaterials, 30 -19 (2009) 3297-3306.

3. F. Ahmed, R.I. Pakunlu, G. Srinivas, A. Brannan, F. Bates, M.L. Klein, T. Minko and D.E. Discher. Mol. Pharm. 3 (2006) 340-350. 4. Zhenxin Wang, Lina Ma. Coordination Chemistry Reviews, 253, 1 1-12, (2009) 1607-1618. 5. Annamalai Pratheep Kumar, Dilip Depan, NamrataSingh Tomer, Raj Pal Singh. Progress in Polymer Science, 34 - 6 (2009) 479-515.

6. G.M. Whitesides, Nanoscience, nanotechnology, and chemistry, (2005), 172-179.

7. P. Mazzoldi, G. Mattei. Metal Nanoclusters in Catalysis and Materials Science, (2008) 269-291 . 8. Hui-Hsin Tseng, Ming-Chi Wei, Shao-Fan Hsiung, Chih-Wei Chiou, Chemical Engineering Journal, 150-1 (2009) 160-167.

9. Ali Alem, Hossein Sarpoolaky, Mehrdad Keshmiri. Ceramics International, Volume 35 - 5 (2009) 1837-1843. 10. Gregorio F. Ortiz, Hie Hanzu, Philippe Knauth, Pedro Lavela, Jose L. Tirado, Thierry Djenizian. Electrochimica Acta, 54, 17 (2009) 4262-4268.

1 1. Katarzyna Anna Michalow, Dmitry Logvinovich, Anke Weidenkaff, Martin Amberg, Giuseppino Fortunato, Andre Heel, Thomas Graule, Mieczyslaw Rekas, Catalysis Today, 144, 1 -2 (2009) 7-12. 12. Jian Wang, Yonglie Chao, Qianbing Wan, Zhimin Zhu, Haiyang Yu. Acta Biomaterialia, 5, Issue 5 (2009) 1798-1807.

13. King Lun Yeung, Wai Kin Leung, Nan Yao, Shengli Cao, Catalysis Today, 143, Issues 3-4 (2009) 218-224. 14. Edit Pal, Viktoria Hornok, Albert Oszko, Imre Dekany. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 340, Issues 1-3 (2009) 1 -9.

15. Jianshu Lu, Luciana Pitta Bauermann, Peter Gerstel, Ute Heinrichs, Peter Kopold, Joachim Bill, Fritz Aldinger. Materials Chemistry and Physics, 115, Issue 1 (2009) 142-146.

16. Rong R. Zhu, Shi L. Wang, Jun Chao, Dong Lu Shi, Rui Zhang, Xiao Y. Sun, Si D. Yao. Materials Science and Engineering: C, 29, Issue 3 (2009) 691 -696.

17. Andrea Mayer, Maria Vadon, Beate Rinner, Alexandra Novak, Reinhold Wintersteiger, Eleonore Frohlich. Toxicology, 258, Issues 2-3, (2009) 139-147 18. Amparo M. Gallardo-Moreno, Miguel A. Pacha-Olivenza, Laura Saldana, Ciro Perez-Giraldo, Jose M. Bruque, Nuria Vilaboa, M. Luisa Gonzalez-Martin. Acta Biomaterialia, 5, Issue 1 (2009) 181 -192.

19. Zouhir E. Allouni, Mihaela R. Cimpan, Paul J. Η0Ι, Tore Skodvin, Nils R.

Gjerdet. Colloids and Surfaces B: Biointerfaces, 68, Issue 1 (2009) 83-87. 20. Eric M.B. Brown, Tatjana Paunesku, AiGuo Wu, K. Ted Thurn, Benjamin Haley, Jimmy Clark, Taisa Priester, Gayle E. Woloschak. Analytical

Biochemistry, 383, Issue 2 (2008) 226-235.

21 . Willie E. Bawarski, Elena Chidlowsky, Dhruba J. Bharali, Shaker A. Mousa, Nanomedicine: Nanotechnology, Biology and Medicine, 4, Issue 4 (2008) 273-282. 22. T. Lopez, S. Recillas, P. Guevara, J. Sotelo, M. Alvarez, J.A. Odriozola. Acta Biomaterialia, 4, Issue 6 (2008) 2037-2044.

23. Paul Thevenot, Jai Cho, Dattatray Wavhal, Richard B. Timmons, Liping Tang Nanomedicine: Nanotechnology, Biology and Medicine, 4, Issue 3 (2008) 226- 236.

24. Eudald Casals, Socorro Vazquez-Campos, Neus G. Bastus, Victor Puntes, TrAC Trends in Analytical Chemistry, 27, Issue 8 (2008) 672-683.

25. Wei-hui Wu, Xun Sun, Ye-ping Yu, Jia Hu, Lei Zhao, Qian Liu, Yu-fen Zhao, Yan-mei Li. Biochemical and Biophysical Research Communications, 373, Issue 2 (2008) 315-318.

26. Lino Ferreira, Jeffrey M. Karp, Luis Nobre, Robert Langer. Cell Stem Cell, 3, Issue 2 (2008) 136-146.

27. Nuria Sanvicens, M. Pilar Marco. Trends in Biotechnology, 26, Issue 8, (2008) 425-433. 28. Laurent Le Guehennec, Marco-Antonio Lopez-Heredia, Benedicte Enkel, Pierre Weiss, Yves Amouriq, Pierre Layrolle. Acta Biomaterialia, 4, Issue 3 (2008) 535-543.

29. K. Byrappa, S. Ohara, T. Adschiri. Advanced Drug Delivery Reviews, 60, Issue 3, (2008) 299-327. 30. Xiaohua Liu, Haixin Bai, Lixue Zhang, Erkang Wang. Advances in Planar Lipid Bilayers and Liposomes, 7 (2008) 203-220. 31. Butler, G.B.; O'Driscoll, K.F.; Wilkes, G.L. JMS-Rev. Macromol. Chem. Phys. 1994, C34 (3), 325-373.

32. Yeager, H.J.; Eisenberg, A. in: Perfluorinated lonomer Membranes; eds. A. Eisenberg and H.L Yeager, ACS Symp. Ser. No.180, (American Chemical Society: Washington, DC, 1982) pp. 1-6, 41-63.

33. Mauritz, K.A.; Storey, R.F.; Jones, C.K. in: ACS Symp. Ser. No. 395;

(American Chemical Society: Washington, DC, 1989) 401-417.

34. Sondheimer, S.J.; Bunce, N.J.; Fyfe, C.A. JMS-Rev. Macromol. Chem.

Phys. 1986, C26(3), 353-413. 35. Hench, L.L.; West, J.K. Chem. Rev. 1990, 90, 35-40.

36. Lev, O. et al. Analytical Chemistry. 995, 67(1 ), 22A-30A.

37. C.J. Brinker and G.W. Scherer, Sol-Gel Science: The Physics and

Chemistry of Sol-Gel Processing (Academic Press, Inc.: New York, 1990).

38. Brinker, C.J.; Scherer, G.W. J. Non-Crystalline Solids. 1985, 70, 301 -322. 39. K.D. Keefer, ACS Advances in Chemistry Ser. No. 224, (American Chemical Society: Washington, DC, 1990) pp. 227-240.

40. M. Prassas and L.L. Hench, in: Ultrastructure Processing of Ceramics,

Glasses, and Composites; eds. L.L Hench and D.R. Ulrich, (John Wiley & Sons: New York, 1984) pp. 100-125. 41. Brinker, C.J. J. Non-Crystalline Solids. 1988, 100, 31 -50. 42. Sakka, S.; Kamiya, K. J. Non-Crystalline Solids. 1982, 48, 31.

43. S. Sakka, in: Better Ceramics Through Chemistry, eds. C.J. Brinker, D.E.

Clark, and D R. Ullrich, (Elsevier-North Holland: New York, 1984) p. 91.

44. Aelion, R.; Loebel, A.; Eirich, F. J. Am. Chem. Soc, 1950, 72, 5705-5712. 45. Dubois, E., Guyader, F.L., Haugarreau, L, Kopecka, H., Cormier, M.,

Pommepuy, M., 1997. Molecular epidemiological survey of rotaviruses in sewage by reverse transcriptase, seminested PCR and restriction fragment length polymorphism assay. Appl. Environ. Microbiol. 63, 1794-1800.

46. Estes, M.K., Graham, D.Y., Mason, B.B., 1981 . Proteolytic enhancement of rotavirus infectivity: molecular mechanisms. J. Virol. 39, 879-888.

47. Flewett, T.H., Woode, G.N., 1978. The rotaviruses. Arch. Virol. 57, 1-23.

48. Grinde, B., Jonassen, T.0., Ushijima, H., 1995. Sensitive detection of group A rotaviruses by immunomagnetic separation and reverse transcription-polymerase chain reaction. J. Virol. Methods 55, 327-338. 49. Horan, P.K., Wheeless, L.L., 1977. Quantitative single cell analysis and sorting. Science 198, 149-157.

50. Hoshino, Y., Kapikian, A.Z., 1994. Rotavirus antigens. In: Ramig, R.F. (Ed.), Current Topics in Microbiology and Immunology. Rotaviruses. Springer, New York, pp. 179-227.

Claims

1. A viricide agent comprising nanostructured biomaterials and a functional group linked to the nanoparticle surface.

2. The viricide agent of claim 1 wherein nanostructured biomaterials are

titania, silica or mixed oxides titania-silica with iridium, platinum or a mix of platinum and iridium.

3. The viricide agent of claim 1 wherein nanostructured biomaterials have a particle size of 1 nm to 200 nm

4. The viricide agent of claim 1 wherein the iridium, platinum or mix of

platinum and iridium are highly dispersed with a particle size of 2-10 nm.

5. The viricide agent of claim 1 wherein the functional group is selected from hydroxyl, carboxyl, ammine, sulphate and phosphate groups.