Synthesis of nanocompounds comprising anatase-phase titanium oxide and compositions containing same for the treatment of cancer

Abstract

This invention relates to novel nanocompounds that are cytotoxic to tumor cells when combined with ultraviolet light, the nanocompounds comprising multilayered carbon nanotubes with anatase-phase titanium dioxide or anatase-phase titanium dioxide and folate. The invention also relates to a composition containing said nanocompounds and to a method for the treatment of cancer; comprising the administration of said composition in co-treatment with UV radiation. The invention further relates to a method for the synthesis of the nano-compounds.

Claims

1. A nano-compound comprising folate functionalized TiO.sub.2 multi-walled carbon nanotubes, wherein the TiO.sub.2 and the folate are covalently attached to the multi-walled carbon nanotubes.

2. The nano-compound of claim 1, wherein the TiO.sub.2 functionalized multi-walled carbon nanotubes have diameters of between 20 to 30 nm and length of between 1 to 30 m.

3. A pharmaceutical composition comprising the nano-compound from claim 2 and a pharmaceutically acceptable diluent.

4. A process for synthesis of the nano-compound of claim 1 that comprises the stages of: dispersing between 1 and 5% p/p of functionalized multi-walled carbon nanotubes in anhydrous C.sub.1-4 alcohol in an ultrasonic bath with a frequency of 20 kHz for a time of between 1 and 5 min at a temperature of between 10 and 20 C.; wherein said nanotubes have a length of between 1 and 30 m, a diameter of between 20 and 30 nm, a length of 10 to 30 m, ash content below 1.5%, and purity above 90% to form a mixture; adding titanium tetrabutoxide (Ti(OBu).sub.4) at 99% purity and shaking for 1 to 5 min at a temperature of between 10 and 20 C.; adding deionized distilled water at a rate of between 10 and 20 ml/min to obtain a solution of C.sub.1-3 alcohol:water with a concentration of between 70 and 90% of C.sub.1-3 alcohol and 10 to 30% of water by volume; allowing the mixture to rest in a desiccator at a temperature of between 10 and 20 C. for 24 to 96 h to permit a condensation reaction to occur; heating the mixture at a rate of between 0.01 and 0.1 C./min to a temperature of between 100 and 250 C. for 1 to 3 h; heating the mixture at a rate of between 0.01 to 0.1 C./min to a temperature of between 450 and 550 C. and maintaining this temperature for 1 to 3 h; cooling the mixture to a temperature of between 10 and 20 C. at a rate of between 100 to 150 C./min to produce TiO.sub.2-FMWNTs; separately mixing dicyclohexylcarbodiimide and N-hydroxysuccinimide in a 1:1 ratio at a concentration of 0.01 to 0.1 M in DMSO for a time of between 3 and 7 min in ultrasound equipment to form a mixture; combining TiO.sub.2-FMWNTs with the mixture of dicyclohexylcarbodiimide and N-hydroxysuccinimide made in the previous step; shaking the combination made in the above step in an ultrasonic bath for a time of between 30 and 90 min at a temperature of between 10 and 20 C.; adding folic acid to the mixture of TiO.sub.2-FMWNTs and dicyclohexylcarbodiimide and N-hydroxysuccinimide in DMSO; shaking the mixture of the above step in ultrasonic bath; centrifuging the mixture of the above step and removing the supernatant.

5. A method for the treatment of cancer comprising administering a therapeutically effective amount of the nano-compound from claim 1 concomitantly with UV-A type ionizing radiation in a localized manner over cancerous tissue.

6. A method for the treatment of cancer comprising administering a therapeutically effective amount of the nano-compound from claim 2 concomitantly with UV-A type ionizing radiation in a localized manner over cancerous tissue.

Description

BRIEF DESCRIPTION OF THESE FIGURES

(1) FIG. 1 shows a scheme of the temperature ramp used during the synthesis process of the invention to obtain Anatase phase TiO.sub.2.

(2) FIG. 2 shows the cytotoxicity of the nano-compounds upon a cervical cancer (HeLa) cell line. a) 20 min without UV, b) 20 min with UV, c) 40 min without UV, d) 40 min with UV.

(3) FIG. 3 presents the lack of cytotoxicity of the nano-compounds upon normal Chinese hamster ovary (CHO) cells. a) 20 min without UV, b) 20 min with UV, c) 40 min without UV, d) 40 min with UV.

OBJECTS OF THE INVENTION

(4) In a first object, the invention is related to the synthesis of a nano-compound that comprises functionalized multi-walled carbon nanotubes and Anatase phase TiO.sub.2 (TiO.sub.2-FMWNTs).

(5) In a second object, the invention reveals a nano-compound that comprises functionalized multi-walled carbon nanotubes, Anatase phase TiO.sub.2, and folate (TiO.sub.2-FMWNTs-Folate).

(6) In a third object, the invention reveals a composition containing the nano-compounds and a pharmaceutically acceptable diluent.

(7) In an additional object, the invention reveals a method for cancer treatment that comprises administration of a therapeutically effective amount of a nano-compound and irradiation of cancerous tissue with UV-A light.

DETAILED DESCRIPTION OF THE INVENTION

(8) In a first object, the invention divulges a synthesis process of a nano-compound that comprises functionalized multi-walled carbon nanotubes and Anatase phase TiO.sub.2 (FMWCNTs) that comprises the stages of: a) Dispersing in anhydrous C.sub.1-4 alcohol between 1 and 5 wt % of functionalized multi-walled carbon nanotubes (FMWCNTs), with diameters of 20 to 30 nm, length of 10 to 30 m, ash content below 1.5% in an ultrasonic bath for a time of between 1 and 5 min at temperature between 10 and 20 C. Where said nanotubes are in a proportion between 1 and 5% p/p with purity higher than 90%. b) Adding titanium tetrabutoxide (Ti(OBu).sub.4) 99% purity and agitating for 1 to 5 min at a temperature of between 10 and 20 C. c) Adding deionized distilled water at a rate of between 10 and 20 ml/min to obtain an ethanol:water solution with a composition in volume between 70 and 90% of ethanol and 10 to 30% of water. d) Allowing the solution to rest in a desiccator at a temperature of between 10 and 20 C. for 24 to 96 h to permit the condensation reaction. e) Subjecting the material to heating at a rate of between 0.01 to 0.1 C./min to a temperature of between 100 and 250 C. for 1 to 3 h. f) Heating the material at a rate of between 0.01 to 0.1 C./min to a temperature of between 450 and 550 C. and maintaining this temperature for 1 to 3 h. g) Cooling the material to a temperature of between 10 and 20 C. at a rate of between 100 to 150 C./min.

(9) In an additional aspect, the invention reveals a nano-compound that comprises functionalized multi-walled carbon nanotubes, Anatase phase TiO.sub.2 and folate (TiO.sub.2-FMWNTs-Folate) that comprises the stages of: a) Mixing dicyclohexylcarbodiimide and N-hydroxysuccinimide in a 1:1 ratio at a concentration of 0.01 to 0.1 M in DMSO for a time of between 3 and 7 min in ultrasound equipment. b) Adding to the mixture functionalized multi-walled carbon nanotubes (FMWCNTs) obtained in the previous process in a proportion of between 1 and 5 parts per part of the mixture from stage (a) and shaking in ultrasonic bath for a time of between 30 and 90 min at a temperature of between 10 and 20 C. c) Adding folic acid in a proportion of between 0.01 and 0.1 parts for one part of the functionalized multi-walled carbon nanotubes (FMWCNTs) and shaking in an ultrasonic bath for a time of between 30 and 60 min at a temperature of between 10 and 20 C. d) Centrifuging at between 800 and 1200 rpm for a time of between 30 and 60 min at a temperature of between 10 and 20 C. and removing the supernatant. e) Heating the material at a rate of between 1 to 5 C./min to a temperature of between 75 and 150 C. and maintain this temperature during 30 to 90 min, to, thus, obtain functionalized multi-walled carbon nanotubes-Folate (FMWCNTs-Folate). f) Dispersing in an anhydrous C.sub.1-4 alcohol of between 1 and 5 wt % of functionalized multi-walled carbon nanotubes-Folate (FMWCNTs-Folate), with diameters from 20 to 30 nm, length from 10 to 30 m, ash content below 1.5% in ultrasonic bath for a time of between 1 and 5 min at a temperature of between 10 and 20 C. Where said nanotubes are in a proportion of between 1 and 5% p/p with purity above 90%. g) Adding titanium tetrabutoxide (Ti(OBu).sub.4) of 99% purity and shaking for 1 to 5 min at a temperature of between 10 and 20 C. h) Adding deionized distilled water at a rate of between 10 and 20 ml/min to obtain an ethanol:water solution with a concentration of between 70 and 90% in volume of ethanol and 10 to 30% of water. i) Allowing the solution to rest in a desiccator at a temperature of between 10 and 20 C. for 24 to 96 h to permit the condensation reaction. j) Subjecting the material to heating at a rate of between 0.01 and 0.1 C./min to a temperature of between 100 and 250 C. for 1 to 3 h k) Heating the material at a rate of between 0.01 to 0.1 C./min to a temperature of between 450 and 550 C. and maintaining this temperature for 1 to 3 h. l) Cooling the material to a temperature of between 10 and 20 C. at a rate of between 100 to 150 C./min.

(10) These nano-compounds comprising functionalized multi-walled carbon nanotubes and Anatase phase TiO.sub.2 (FMWCNTs) and functionalized, Anatase phase TiO.sub.2 and folate (TiO.sub.2-FMWNTs-Folate) are cytotoxic against tumor cells in co-treatment with UV-A ultraviolet light, while not inducing cytotoxic effects upon normal cells with or without UV-A ultraviolet light co-treatment. Said cytotoxicity against tumor cell lines is evidenced in treatment times below 60 min and in synergy with UV-A light irradiation. Likewise, the nano-compounds object of the present patent are neither genotoxic nor mutagenic.

(11) In a third aspect, the invention refers to a pharmaceutical composition with cytotoxic activity for cancer treatment that comprises a functionalized multi-walled carbon nano-compound and Anatase phase TiO.sub.2 (FMWCNTs) and/or functionalized, Anatase phase TiO.sub.2 and folate (TiO.sub.2-FMWNTs-Folate) and one or more pharmaceutically acceptable excipients to adapt the pharmaceutical liquid, solid, or heterodisperse form.

(12) Said composition can be formulated with one or more pharmaceutically acceptable excipients for oral administration in pharmaceutical liquid or solid forms; for topical administration in heterodisperse forms (W/O creams, O/W creams, gels, and ointments, among others) and for parenteral or rectal administration. The compositions of the invention can be administered orally, rectally, parenterally, topically, intravaginally, bucally, or as a nasal or oral spray to humans and other mammals.

(13) An additional aspect divulges a method for cancer treatment that comprises administration of a therapeutically effective amount of a functionalized multi-walled carbon nano-compound and Anatase phase TiO.sub.2 (FMWCNTs) and/or functionalized, Anatase phase TiO.sub.2 and folate (TiO.sub.2-FMWNTs-Folate) concomitantly with irradiation with UV-A light in localized manner over cancerous tissue.

Example 1. Synthesis of Anatase Phase Titanium Dioxide

(14) To synthesize the TiO.sub.2 nanomaterial, the following procedure is carried out: add TBT (titanium tetrabutoxide) at 99% in anhydrous ethanol and shake in ultrasonic bath for 3 min at room temperature. Then, add water and shake for 10 min in ultrasound equipment. The percentages of the precursor (TBT) with the ethanol and water reagents are at 76.34%, 21.96%, and 1.7% in volume, respectively. The sol obtained is brought to a desiccator for 72 h, permitting the condensation reaction. Thereafter, it is dried at 100 C.; it is pre-calcined at 200 C. and calcined at 500 C., according to the diagram in FIG. 1, to obtain Anatase phase TiO.sub.2.

Example 2. Synthesis of Multi-Walled Carbon Nanotubes Comprising Anatase Phase Titanium Dioxide

(15) To obtain this nanomaterial of titanium dioxide-multilayered carbon nanotubes (TiO.sub.2-MWCNTs), of between 1 and 5 wt % of multi-walled carbon nanotubes (MWCNTs) were used without functionalizing, with diameters from 20 to 30 nm, length from 10 to 30 m, ash content below 1.5%, and purity above 95%. The proportion of nanotubes used in the samples is 2.1% in mass. The MWCNTs are dispersed in anhydrous ethanol (Mallinckrodt) in an ultrasonic bath for 3 min and at room temperature.

(16) Then, TBT (Aldrich 99%) is added and shaken for another 3 min; thereafter, deionized distilled water is added slowly (10 min). During the process, hydrolysis and poly-condensation are generated upon interaction of the precursor (TBT) with the ethanol and water reagents, in 76.34%, 21.96%, and 1.7% in volume, respectively. The sol obtained is brought to a desiccator for 72 h, permitting the condensation reaction. It is, then, dried at 100 C., pre-calcined at 200 C., and calcined at 500 C., according to the diagram in FIG. 1, to obtain Anatase phase TiO.sub.2.

Example 3. Synthesis of Functionalized Multi-Walled Carbon Nanotubes Comprising Anatase Phase Titanium Dioxide and Folate (Tio2-Fmwnts-Folate)

(17) To obtain this nanomaterial of functionalized multi-walled carbon nanotubes, Anatase phase TiO.sub.2, and folate (TiO.sub.2-FMWNTs-Folate), the material obtained in example 2 is used. Initially, 30 mL of dicyclohexylcarbodiimide and 30 mL of N-hydroxysuccinimide (Aldrich chemistry) are shaken in a 1:1 ratio with a concentration 0.05 M in DMSO for 5 min in ultrasound equipment. Add 1.5 g of the material from example 2 and agitate for 1 h in ultrasound; then, add 0.03 g of folic acid (FA) and agitate for 45 min in ultrasound. Thereafter, shaken on a plate for 6 h and centrifuged; remove the supernatant and dry for 1 h at 100 C. The end result is a nanomaterial of TiO.sub.2-FMWCNTs-Folate.

Example 4. Cytotoxic Activity of the Nano-Compounds of the Invention Against Tumor and Non-Tumor Cell Lines

(18) The cytotoxic activity was evaluated in two cell lines, one of (HeLa) human cervical neoplastic cells, and another of normal CHO cells obtained from the cell line bank of the ATCC. Cytotoxicity was evaluated through LDH determination, according to formula:

(19) $\%\mathrm{of}\mathrm{cytotoxicity}=\frac{\mathrm{Sample}\mathrm{Reading}\left(\mathrm{CEM}\right)-\mathrm{Lowcontrol}}{\mathrm{Highcontrol}-\mathrm{Lowcontrol}}$

(20) Where CEM is the calculation of the average absorbance values of the tests in triplicate.

(21) The treatments were evaluated in two concentration or dosage levels: 100 and 200 g/mL. The nano-compounds evaluated were: anatase phase titanium dioxide (A), multi-walled carbon nanotubes comprising anatase phase titanium dioxide (TiO.sub.2-MWCNTs), and (B) multi-walled carbon nanotubes comprising anatase phase titanium dioxide and folate (TiO.sub.2-FMWNTs-Folate) (C); triton X-100 at 2% was used as positive control.

(22) Treatments were evaluated with or without UV irradiation for a time of 20 to 40 min. FIG. 2 shows the cytotoxicity percentage of the HeLa cell line (dead cells with respect to the control) for each of the treatments and conditions evaluated. It is noted that the highest cytotoxicity percentage was 98.6% corresponding to the effect of the B1 nano-compound (TiO.sub.2-FMWCNT) at a concentration of 200 g/ml, exposed to UV radiation for 40 min. It can also be noted that the cytotoxic effect of the nano-materials increases over time (higher effect at 40 min); likewise, cytotoxicity increases through co-treatment with UV radiation.

(23) Similarly, it is noted that the C1 nano-compound (200 g/ml of TiO.sub.2-FMWCNT-folate) has an increase in the significant cytotoxic percentage when treatment time increases from 20 to 40 min without exposure to UV light.

(24) FIG. 3 shows that the cytotoxicity percentage values of these non-tumor CHO cells are negative, from where it is inferred that there is no cytotoxic effect of the nano-compounds upon normal CHO cells. It is also evidenced that no treatment below the treatment conditions is cytotoxic with or without exposure to UV light, which shows the specificity of the nano-compound's cytotoxic effect.

Example 5. Genotoxicity and Mutagenicity of the Invention's Nano-Compounds

(25) The genotoxicity tests were conducted with EBPI SOS-CHROMOTEST and mutagenicity tests were conducted with EBPI's Muta-ChromoPlate.

(26) The EBPI SOS-CHROMOTEST is a practical approach for detection of genotoxic activity and genotoxic materials in environments, like: water, sediments, air, chemicals, foods, cosmetics, and biological fluids. Genotoxic materials can be dangerous due to the ease with which they induce cancerous transformations to normal cells. The readings permitted concluding that the wells contain bacteria, but not genotoxins, evidencing production of enzymes not connected to activation of the SOS gene repair complex.

(27) The EBPI Muta-ChromoPlate kit is a convenient approach for detection of mutagenic activity and of mutagenic materials in environments, like: water, sediments, air, chemicals, food components, cosmetics, and biological fluids. Mutagenic materials can be dangerous due to the ease with which they induce cancerous transformations to normal cells.

(28) Results are shown in Table 1, where it is noted that none of the nano-materials of the invention induce significant mutagenotoxicity after 5 days of culture.

(29) TABLE-US-00001 TABLE 1 Mutagenotoxicity detected at days 3, 4, and 5 for the nano- compounds of the invention Treatment Day 3 Day 4 Day 5 Target 0 0 0 Background 5 9 11 Positive control 34 89 96 Anatase phase TiO.sub.2 10 12 13 TiO.sub.2-MWCNTs 7 15 16 TiO.sub.2-FMWNTs- 5 6 8 Folate

(30) Although the present invention has been described with the preferred embodiments shown, it remains understood that the modifications and variations that conserve the spirit and reach of this invention are understood within the reach of the claims included.