DESCRIPTION

FIELD OF THE INVENTION

This invention is related to the synthesis of nanostructured inorganic nanostructured and biocompatible biocatalysts defined as M _n O _2n - ₍ χ ₊ y _)/2 (OH) _v (Sθ ₄ )w(Pθ ₄ )χ(OR) _y (CI) _z where M stands for silicon, titanium or a mixture of both and R for an organic ligand, preferably C _n H _n+ i, either linear or branched to which Pt-based compounds, in II, III or IV oxidation state, having cytotoxic activity (i.e. cisplatin, carboplatin, Pt(IV) based drugs). These nanostructured biocatalysts are directly administered into the tumor. The matrix acidity, structure, electronic density, pore size distribution, matrix particle size, platinum particle size, platinum dispersion on the support (silica or titania), crystallite size and oxidation state of platinum are controlled. These anticancer biocatalyst formulations will be delivered directly into the tumor.

BACKGROUND FOR THE INVENTION Cancer is one of the leading causes of death all over the world. The treatment use is surgery, radiotherapy, chemotherapy or a combination of them. Chemotherapy uses chemical agents (anticancer drugs) to kill cancer cells, is one of the primary methods to cancer treatment. Unfortunately, most of the anticancer drugs have limited selectivity for cancer and are inherently toxic to both cancer and normal tissues. Like other cancer chemotherapeutic agents, compounds that exhibit high antitumor activity such as cis-platin are typically highly toxic. The main disadvantages of cis-platin are its extreme nephrotoxicity and neurotoxicity, which is an important limiting factor to use. Its rapid distributed via blood stream, with a circulation half life of only a few minutes, and its strong affinity to plasma proteins (Freise et al. 1982 Arch. Int. Pharmacodyn Ther. 258(2): 180-192). Other side effects of anticancer drugs include the decrease of white blood cells, red blood cells and platelets increasing the risk of infections, bruising and bleeding. Most important, conventional treatments may cause drug resistance and hence treatment failure (Pastan and Gottesman, 1991 , Gottesman 2002). A major mechanism of resistance is related to the P-glycoprotein pump located in cell membrane (Gottesman 2002) that binds drugs as they enter the plasma membrane transporting the drug out of the cells. As a consequence the effective drug concentration in the cytoplasm is well below the cell-killing threshold, resulting in a limited therapeutic efficacy.

The development of new methodologies that have higher drug selectivity for cancer and simultaneously reduce toxicity to healthy tissues is a major challenge in cancer treatment. The cancer cutoff size of cancer's blood capillaries (ca. 400-800 nm) allows extravasations of colloid particles to cancer tissues on the other hand as cancer tissues have fewer lymphatic capillaries drainage from these capillaries to healthy tissues is reduced causing the trapping of colloidal particles in cancer tissues, this is referred as the "enhanced permeability and retention effect" (Maeda et al. 2001 , Lukyanov et al. 2002). Nanoparticles fabricated by self-assembling of amphiphilic copolymers have been used as carriers for cis-platin (Yokoyama et al. 1996, Bogdanov et al. 1997). The use of inorganic oxides nanoparticles offers a suitable mean to deliver drugs to tissues or cells. Their submicrometric size favors the taken up by cells via endocytosis/phagocytosis, the hydrophilic character of their surfaces allows evading the recognition by the reticuloendothelial systems and their intrinsic stability prevents the breakage in the bloodstream. In addition to this, they may have high surface area a controlled pore size distribution and if required tailored surface acid-base properties for adapting them to site specificity.

State of the art research in the treatment of chronic diseases is based on the development of controlled release systems capable of delivering drugs rapidly and efficiently to where they are needed. A major requirement is that these devices should insure delivery and penetration of the drug to the active site. New nanostructured materials represent an efficient way to administer medications and biological products in future applications ^1"5 . Hydrogels based on n-isopropylacrilimide and methacrylic acids (MAA) have recently received considerable attention. This is due to their ability to swell in response to the stimulation of the medium ^6"8 . In the solid state, the existence of interpolymeric complexes in which monomers are linked together through hydrogen bonds has been observed. These linkages occur under acid conditions and are stabilized through hydrophobic interactions. This leads to a marked dependence on the pH of the medium in which swelling occurs. This swelling is also strongly dependent on the degree of cross-linking. The use of drug delivery by oral means has received considerable attention, particularly in cases in which activation is controlled by variations in the pH. Copolymers having a high concentration of N-isopropylacrilamide appear to be the most effective in enabling one to obtain different cut-off curves used in the drug model. ^12"15 In the majority of cases, which involve controlled drug release, the medication or other biological agent, is introduced into the interior of the reservoir normally known as the transporter. The transporter usually consists of a polymeric material. Under normal conditions the rate of drug release is controlled by the properties of the polymeric material which constitutes the transporter. However, other factors may also be rate determining. When these factors are taken into account, it may be possible to insure a slow, constant rate of drug delivery over extended periods of time. ^16'18 The use of these materials has lead to considerable advances in drug delivery when compared to systems currently in use. In conventional drug delivery systems, drug concentrations reach a maximum value only to decay, finally reaching a concentration, which requires the administration of another dose. Additionally, if the maximum drug concentration exceeds the safe level or if, alternatively it falls below the required dose, cyclic periods will occur during which the drug is not producing the desired effect. This is generally known as "variations in tisular exposure". When controlled drug release is used, it may be possible to maintain drug concentrations, which fall between the maximum allowed rate, and the minimum concentration at which the rate is effective ^19'21 .

When dealing with inorganic oxide nanoparticles, the sol-gel technique with or without the use of templates can be used as a good method by which the various solid phases can be controlled (T.Lopez et.al., Catalysis Today 35,293.1997 y otras mas). A greater degree of control can be achieved in comparison to other methods of synthesis. One can tailor make the reservoir to fit specific applications by using this method. Advances include: Superior homogeneity and purity; High solid acidity; High biocompatibility with any tissue; Better nano and microstructural control of the inorganic oxide matrices; Greater BET surface area; High dispersion of the platinum on the matrices; Improved thermal stability of the drugs attached to the transporter; Well-defined mean pore size distributions; Inorganic chain structures can be generated in solution; A finer degree of control over the hydroxylation of the transporter can be achieved.

The process of transporter fabrication has as an aim the optimization of the following variables: particle size, mean pore size, interaction forces and the degree of functionalization. It may also be desirable to modify the textural and electronic behavior of the transporter. Sol-gel technology is an important synthesis method by which the crystalline phases and particle size of inorganic hydrous oxides can be controlled. A sol is a fluid, colloidal dispersion of solid particles in a liquid phase where the particles are sufficiently small to stay suspended in Brownian motion. A "gel" is a solid consisting of at least two phases wherein a solid phase forms a network that entraps and immobilizes a liquid phase (ref). In the sol-gel process the dissolved or "solution" precursors can include metal alkoxides, alcohol, water, acid or basic promoters and on occasion salt solutions. Metal alkoxides are commonly employed as high purity solution precursors. When they react with water through a series of hydrolysis and condensation reactions they yield amorphous metal oxides or oxo-hydroxide gels. When the volatile alcohol molecules are removed the result is the formation of crystalline solid compounds. This solid can be modified by adding suitable amounts of desired molecules during the synthesis process whose amount an stability are determined by the stability constant (ref).

The materials that are used as colloid precursors can be metals, metal oxides, metal oxo-hydroxides or other insoluble compounds. The degree of aggregation or flocculation in the colloidal precursor can be adjusted in such a way that the pore size distribution can be controlled. Dehydration, gelation, chemical cross-linking and freezing can be used to form the shape and appearance of the final product. Some advantages using sol-gel technology include control over the purity of the alkoxide precursors, control over the homogeneity of the product, control over the evolution of the desired crystalline phases and most importantly, the reproducibility of the materials synthesized. The hydrolysis product is not fully hydrolyzed nor can it ever be a pure oxide. It can be in the form, M _n O _2n -(x+y) _/2 (OH) _x OR)y, M stands for silicon, titanium or a mixture of both and R for an organic fragment, preferably C _n H _n+I , either linear or branched, where n is the number of titanium atoms polymerized in the polymer molecule and x and y is the number of terminal OH and OR groups respectively. It is well known that some sol-gel structures attain their highest coordination state through intermolecular links (Sankar G., Vasureman S, and Rao C. N. R., J.Phys. Chem, 94,1879 (1988)y otras mas modemas). Because there are strong chemical interaction forces between the drugs and the inorganic nanoparticle transporter, it is possible to encapsulate a large amount of medication within the transporter.

Additional titania patents using sol-methods

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 SiO ₂ and AI ₂ O ₃ 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 No6 087405. 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.

Additional platinum-titanϊa patents using sol-gel methods

OBJECTIVES

1. The development of nanostructured materials for use as biocatalyst in treatment of cancer.

2. Obtain and optimize the nanostructure biocatalyst capable to kill maligned cells by means catalytic reaction.

3. Optimization of materials to enable control of the following parameters: pore size distribution, contact area, structure, electronic density, particle size, crystalline phase, degree of functionalization, diffusion, size of biocatalyst required to react with the cell, the drug, and release time for effective delivery 4. Obtain an effective nanoparticle to use in cancer therapy and to prevent a side effects on the blood stream, liver, intestine and kidneys.

DETAIL OF THE INVENTION

1. The present invention includes a novel nano-material (silica, titania and silica-titania) obtained by the sol-gel process to which platinum compounds are bound. 2. The support particle size ranges between 10 nm to 1 Dm.

3. The platinum metal is either bound as metallic nanoparticles or covalently bound platinum complexes. The metal nanoparticle size ranges from atomic dispersion to 100 nm.

4. This nanomaterial consists of partially hydrolyzed oxides having a Ti:Si range of compositions between (100:0 and 0:100). These materials were prepared using a sol-gel process, which has been used to synthesize ceramic and glass materials.

5. The titania, silica and titania-silica xerogels (100:0, 0:100) materials are found to be biocompatible with surrounding tissue. 6. The synthesis of the platinum containing drug is carried out by adding the platinum compound during the gelation process or by grafting the platinum compound to the sol-gel obtained oxides. The total amount of platinum can be as high as 10% by weight. 7. Mesoporous sol-gel oxides can be synthesized, in reactive (i.e. air, carbon dioxide, etc.) or inert atmosphere (i.e nitrogen, argon, etc.) at pH ranging from 2 to 12 using water: alkoxide ratio ranging from 2 to 64. Water, Ci to C ₅ primary, secondary or tertiary alcohols, acetyl acetone, acetone or a mixture alcohol-water or acetone- acetyl acetone was used as solvent for the synthesis.

8. The pH during the synthesis was fixed using HCI, H ₂ SO ₄ , H ₃ PO ₄ carboxylic acids (i.e. EDTA, acetic acid, D-amino butyric acid, glutamic acid, etc) or bases (i.e. amonium hydroxide, phenitoine, puric bases, pyrimidic bases, etc)

9. The gelation process was carried out from room temperature to 8O ⁰ C in the presence or absence of organic templates or modifiers (i.e. P 123, acetylacetone, CTAB, etc).

10. Platinum compound precursors are HaPtCI ₆ cis-Pt or PtAcAc or Pt(NHs) ₄ CI ₂ .

11. Pore volumes and pore diameters are not strongly affected by platinum compound loadings.

12. The administration form can be: a) nanoparticle suspension in physiological compatible fluids; b) extrudates, in this case biocompatible binders might be used (i.e. poly[bis(p- carboxypenoxy)]propane-sebacic acid, PLGA, methylcellulose,

PVP, etc); and c) implantable self-supported nanodevices. Detailed Description of the Synthesis Methods Used

Biocatalysts platinum compound-sol-gel synthesis. In the three-necked flask, a mixture consisting of deionized water, platinum compound, base or acid and solvent were refluxed. Prior to initiating the reflux, the pH of the solution was adjusted. In either case, the acid or the base was added drop by drop manner until the desired pH was obtained. The pH was monitored continually using a potentiometer throughout the entire process. Using a funnel, metal alkoxide or a mixture of metal alkoxides was added to the solution being refluxed. The dropwise addition was performed over a four-hour period in order to enhance nucleation and functionalization. Following the addition of the alkoxide, the colloidal suspension was refluxed over a period from 24 to 240 hours. Following this process, the samples were dried under vacuum conditions in a roto-vapor (10 ^~3 mm of Hg) in order to remove excess water and alcohol. Finally the samples were dried at 3O ⁰ C for 72 hours. In order to reach the final drying temperature of 3O ⁰ C, the temperature was increased at a rate of 0.25°C/min to 5C/min using a conventional furnace.

In the case of mesostructured oxides the synthesis procedure respected the known synthesis procedures for obtaining the adequate micelle concentration. Alternatively, the inorganic oxides were synthesized following the same procedure but in the absence of the platinum compound. Once the nanomaterial is obtained the desired amount of platinum is added by: a) A solution containing the platinum compound is added to the inorganic alkoxide in such a way that the solution volume matches the pore volume of the inorganic oxide. b) A solution containing the platinum compound is added to the inorganic alkoxide at pH above or below the isoelectric point of the surface. In every case, the pH is adjusted to either preserve or decompose the platinum compound. For example for grafting [Pt (NHs) ₄ ]CI ₂ to a titania surface, a chloride rich solution at low pH is used.

Example 1.

The sample was prepared as follow: Pt(NHs) ₄ CI ₂ was dissolved in ethanol and distilled water. The solution was stirred continuously under constant reflux. After the salt was completely dissolved, gamma-aminobutyric acid and TEOS were added. The resulting sol was maintained under constant flux and continuous stirring until the gel was formed. The evaporation of the solvent and water was performed at room temperature. The dry solid was crushed and later characterized.

Example 2.

The sample was prepared as follow: H ₂ PtCI ₆ .6H ₂ O was dissolved in distilled water. The solution was stirred continuously under constant reflux. After the salt was completely dissolved, ethanol, gamma-aminobutyric acid and TEOS were added. The resulting sol was maintained under constant flux and continuous stirring until the gel was formed. The evaporation of the solvent and water was performed at room temperature. The dry solid was crushed and later characterized.

In figure 1a, an x-ray diffraction pattern, (obtained using a Brucker D-5000 instrument equipped with Cu-Ka radiation with a wavelength of 1.5418 A (45kV and 4OmA)), in which an undefined broad band characteristic of amorphous silica is shown. Several small bands, which are reflections from the Pt (NHs) ₄ CI, centered at 12° and 24°(2 theta) are also observed. These results suggest that an OH group on the silica has been coordinated to Pt resulting in a square planar structure.

In the infrared transmittance spectrum showed in figure 1b,( the infrared spectra of the powdered samples was performed at room temperature using a Termo- Nicolet Nexus FT-IR spectrophotometer) , 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 hydroxy! groups which give rise to both Lewis and Brόnsted 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 has lost only one chlorine atom and that some decomposition of the complex has most likely occurred resulting in some PtO and supported metallic 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 (-0-Si-O-) vibrations. 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.

Figure 1 (a) X-ray diffraction pattern and, (b) FTIR spectrum of Pt/SiO ₂ - Pt(NHs) ₄ CI ₂

In figure 2, XPS and FTIR-Py (acidity measurements)

In the micrographs shown in figure 3 (Zeiss, model MM 910 transmission electron microscope operating at 100kv), the homogeneous morphology of the small agglomerates of spherical particles, around 30 nm in diameter, can be observed. On the left side of the figure the particle dimensions are clear. However, the right hand side gives a better idea of their distribution. Because Pt is very highly dispersed on the surface and has been cogelled with the tetraethoxysilane, it is not seen in the micrograph. Future studies using high resolution TEM will be focused on the identification of the Pt atoms on the support.

Figure 3 Transmission electron microscopy of the nanostructured particles, which comprise the PI- ⁷ SiO ₂ -Pt(NHs) ₄ Cl ₂ biocatalyst Histological studies using hematoxiline-eosine were performed on the tissue surrounding the trajectory of the injection of the suspension of Pt/SiO2-H ₂ PtCI ₆ nanoparticles Figure 4. The micrographs pertaining to this study tunel are shown in figure4. In Fig 4a, an interface clearly shows a line of demarcation between two zones, one in which the tumor cells are clearly visible and the other, visibly showing the cell damage. In figure 4b, a higher magnification is used to examine the damaged area. In figure 4c the absence of growth in the tumoral tissue is apparent. The white dots are DNA fragments. Figure 5 Photomicrographs of hematoxylin and eosin stained sections of (a) tumor treated with PIZSiO ₂ -Pt (NH ₃ ) ₄ CI2 nanoparticles, (b) higher amplification, and, (c) TUNEL analysis

Example 3.

To obtain 1 w/w % of platinum metal on TiO ₂ , 320 mg of Pt(NHs) ₄ CI ₂ XH ₂ O was incorporated to a mixture containing 190 ml_ of ethanol and 29 ml_ of deionized water, under constant stirring at 343K. This mixture was refluxed for 10 minutes at 343K prior to the addition of the titanium alcoxide. Then 69 ml_ of the TiO ₂ precursor, titanium n-butoxide, was added dropwise over a 4 h period. The resulting sols were maintained under constant stirring until gelation occurs. The total molar ratio water:alkoxide:alcohol was 8:1:16. Alter and aging period of 72 hours at room temperature xerogel samples were obtained by oven drying the obtained solids at 343K.

Table 1 shows the final volume of the tumours as a function of treatment. From this data it is clear that both the platinum coordination compound and the Tiθ2 carrier produce a significant reduction of the tumour volume. This effect is greatly enhanced in the case of the groups treated with the TiO ₂ and TiO ₂ -Pt nanodevices. In this later case, the tumour volume is just 44% of the volume achieved by the control group.

Table 1. Average tumour volume for the four designed groups of Wistar rats.

Treatment n Volume / cm ³

Control 2 20.9±4.9

TiO ₂ -Pt(NHs) ₄ CI ₂ 2 47.2±7.2

TiO ₂ -cisPt 1 26.7+4.9

SiO ₂ -Pt(NHs) ₄ CI ₂ 2 35.6±8.5

Example 3.

A 0.225 M solution of EO ₂₀ PO ₇ oEO ₂₀ was added HCI (4M) at 4O ⁰ C in constant stirring until the polymer was dissolved. Then, tetraethoxysilane (TEOS) was added drop by drop during two hours. The molar ratio in the solution was TEOS:OS:P123:HCI:H ₂ O equal 1 :0,017:6,3:200. The solution is maintained during 20 h, and then is dried at 9O ⁰ C 24 hours. The resultant solid was filtered and washed at 2 b. of pressure with a mixture of ethanol and water. The surfactant elimination take place using a Soxhlet reactor. The solvent is ethanol and is put in reflux during 24 hours. Then is washed and filtered. Finally is dried at 50°C/24 h.

The introduction of CISPLATIN ^® in the silica matrix is by impregnation: 100 g of silica are mixed with water and HCI 1M. The platinum salt is dissolved in the HCI solution (0,5% wt of Pt). The mixture was stirred at 30 ⁰ C, during 72 h. The sample is dried with vacuum and is maintained at ambient temperature.

SEM and TEM micrographs (Figures 5c y 5d) show that in the incorporation of CISPLATIN ^® process, microstructural variation in the silica matrix was not observed, due to a soft chemistry used in the synthesis.

In 5d figure is possible to see the existence of dark nodules on the silica surface, due to the continuous exposition to the electron flow in the microscope. Platinum has an oxidation state of (II) and favored the nanoparticles formation.

Example 1.

Poner en los reclamos menos de 10 %mol

The sample was prepared as follow: Pt(NHs) ₄ CI ₂ was dissolved in ethanol and distilled water. The solution was stirred continuously under constant reflux. After the salt was completely dissolved, gamma-aminobutyric acid and TEOS were added. The resulting sol was maintained under constant flux and continuous stirring until the gel was formed. The evaporation of the solvent and water was performed at room temperature. The dry solid was crushed and later characterized. The final platinum concentration was 1 mol%. In figure 1a, an x-ray diffraction pattern, (obtained using a Brucker D-5000 instrument equipped with Cu-Ka radiation with a wavelength of 1.5418 A (45kV and 4OmA)), in which an undefined broad band characteristic of amorphous silica is shown. Several small bands, which are reflections from the Pt (NH ₃ ) ₄ Cl ₂ , centered at 12° and 24°(2 theta) are also observed. These results suggest that an OH group on the silica has been coordinated to Pt resulting in a square planar structure.

In the infrared transmittance spectrum showed in figure 1b,( the infrared spectra of the powdered samples was performed at room temperature using a Termo-Nicolet Nexus FT-IR spectrophotometer) , 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 hydroxy! 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 has lost only one chlorine atom and that some decomposition of the complex has most likely occurred resulting in some Pt-OH. 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 are due to stretching (-0-Si-O-) vibrations. 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 the micrographs shown in figure 2 (Zeiss, model MM 910 transmission electron microscope operating at 100kv), the homogeneous morphology of the small agglomerates of spherical particles, around 30 nm in diameter, can be observed. On the left side of the figure the particle dimensions are clear. However, the right hand side gives a better idea of their distribution. Because Pt is very highly dispersed on the surface and has been cogelled with the tetraethoxysilane, it is not seen in the micrograph. Future studies using high resolution TEM will be focused on the identification of the Pt atoms on the support.

Glioma Induction. Model of malignant glioma C6 was used to evaluate the cytotoxic activity of the nanostructured biocatalysts. C6 glioma cells (benda et al. 1968), obtained from the American Tissue Culture Collection (Rockville, MD) were cultured under sterile conditions at 37 ⁰ C in a humid environment with 5% of CO ₂ in Ham F-10 medium supplemented with bovine foetal serum (2.5%) and horse serum (15%). After the cultures became confluent, the cells were washed with saline solution, harvested and counted; 1x10 ⁷ C6 cells were inoculate intraperitoneal^ in a male Wistar rat. Twenty days later, a large peritoneal tumor developed. The tumor was mechanically dissociated at 4 ⁰ C and 1x10 ^r cells, suspended in 500μL of saline solution, were inoculated subcutaneously into the left thighs of 12-week-old Wistar rats. A subcutaneous tumour developed in 80% of animals (Arrieta et al. 1998; Guevara and Sotelo 1999). Histological studies using hematoxiline-eosine were performed on the tissue surrounding the trajectory of the injection of the suspension of PtZSiO ₂ - Pt(NHs) ₄ CI ₂ nanoparticles. The micrographs pertaining to this study are shown in figure 3. In Fig 3a, an interface clearly shows a line of demarcation between two zones, one in which the tumor cells are clearly visible and the other, visibly showing the cell damage. In figure 3b, a higher magnification is used to examine the damaged area. In figure 3c the absence of growth in the tumoral tissue is apparent. The white dots are DNA fragments.

Example 2.

To obtain 1mol % of platinum metal on TiO ₂ , H ₂ PtCI ₆ xH ₂ O was incorporated to a mixture containing of ethanol and deionized water and γ-amminobutyric acid, under constant stirring at 343K. This mixture was refluxed for 10 minutes at 343K prior to the addition of the titanium alkoxide. Then titanium n-butoxide, was added dropwise over a 4 h period. The resulting sols were maintained under constant stirring until gelation occurs. The total molar ratio water:alkoxide:alcohol was 8:1 :16. Alter and aging period of 72 hours at room temperature xerogel samples were obtained by oven drying the obtained solids at 343K.

Table 1 shows the final volume of the tumours as a function of treatment. From this data it is clear that both the platinum coordination compound and the TiO ₂ carrier produce a significant reduction of the tumour volume. This effect is greatly enhanced in the case of the groups treated with the TiO ₂ and TiO ₂ -Pt nanodevices.

These nanostructured biocatalysts were characterized N2 adsorption- desorption isotherms at 77K for both biocatalysts are shown in Figure 1. H2PtCI6/SiO2 biocatalysts exhibit a type I isotherm, according to the IUPAC classification, characteristic of microporous materials. However, the presence of a broad knee at low relative pressures (below p/pθ < 0.4) clearly reflects the presence of a wide micropore size distribution. On the other hand, H ₂ PtCI ₆ ZTiO ₂ biocatalyst exhibits a type IV isotherm, characteristic of porous materials with bimodal pore size distribution (presence of both micro and mesopores). The presence of a hysteresis loop on the H2PtCI6/TiO2 biocatalysts above p/p _o ~O.4 reflects the capillary condensation of nitrogen molecules in the mesopores (figure 4). The BET surface area for both samples is 416 m ² /g and 250 m ² /g, for H ₂ PtCl6/Siθ2 and H ₂ PtCI ₆ ZTiO ₂ , respectively, whilst the micropore volume (Vo) is 0.14 and 0.08 ccZg, respectively. The average pore size is 1.4 and 3.1 nm for H ₂ PtCI ₆ ZSiO ₂ and H ₂ PtCI ₆ ZTiO ₂ , respectively, which is in agreement with the analysis done on their isotherms.

X-ray photoelectron spectroscopy (XPS). According to the XPS spectra, Pt nanoparticles are well-dispersed on the surface of the TiO ₂ support, as it can be inferred from the PtZTi atomic ratio (this ratio can be used as a rough estimation of the Pt dispersion on the surface of the catalyst). The Pt 4f level X-ray photoelectron spectra for H ₂ PtCI ₆ ZTiO ₂ biocatalyst shows the presence of two broad bands which corresponds to the Pt 4f ₇ / ₂ and Pt 4f _5/2 levels (see Figure 5). The binding energy (eV) corresponding to these two levels are 72.2 eV and 75.5 eV, respectively. According to the literature, the 4f _7/2 level of platinum in different oxidation states appears as follow: Pt ⁰ , 71.0-71.3 eV; K ₂ Pt ¹¹ CI ₄ , 72.8- 73.4 eV; K ₂ Pt ^lv CI ₆ , 74.1-74.3 eV ^! . Thus, the absence of any peak in the catalysts at a low binding energy (approx. 71.0 eV) clearly rules out the presence of metallic platinum. This observation confirms that the metal precursor and, more specifically, the oxidation state of the platinum precursor is preserved. Consequently, Pt(II) should be considered as the active compound for any further application. The morphology of the Pt nanoparticles and the oxide support was analyzed using transmission electron microscopy. Figure 6 shows TEM images of both (a) H ₂ PtCI ₆ /Ti0 ₂ and (b) H ₂ PtCVSiO ₂ biocatalysts. As expected, TiO ₂ support (Figure 6b) exhibits a high degree of crystallinity in accordance with previous observations using raman. Interestingly, a close inspection through the whole biocatalyst does not allow discerning any spot attributed to Pt nanoparticles. The absence of any observation attributed to the platinum nanoparticles is usually attributed to the lower density of the oxide nanoparticles (compared to the pure metal), together with the presence of spectators (organic precursor). In the case of the H ₂ PtCVSiO ₂ biocatalyst, the situation is different. TEM images clearly show the presence of Pt nanoparticles, thus confirming the presence of reduced metal species (Pt(O)). Additionally, although isolated Pt nanoparticles of around 8-10 nm can be observed, the main proportion of Pt is associated forming agglomerates of small nanoparticles (see Figure 6c and d). These agglomerates are responsible for the low Pt dispersion anticipated from the XPS measurements.

Example 3. EO2 ₀ PO ₇ 0EO2 ₀ 0,225 M was mixed with HCl (4M) and maintained at

37°C with reflux and continuous stirring until the polymer was completely dissolved. Then tetraethoxysilane (TEOS) was added. Molar composition TEOS:OS:P123:HCI:H ₂ O was 1:0,017:6,3:200.

The mixture was under reflux during 20 h and then is dried at 9O ⁰ C/ 24 h. Elimination of surfactant was carried out using Soxhlet extraction at 2 bar. Finally was dried at 50°C/12 h. CISPLATIN ^® was impregnated on the support using a solution of HCI (1M). The mixture was stirred at 30 ⁰ C during 72 h. Then the sample was dried at room temperature in roto-vapor.