USE OF MUTANT P53 GENE TARGETED LEAD BORATE NANOPARTICLES IN CANCER TREATMENT AND PRODUCTION METHOD OF THESE NANOPARTICLES

Abstract

Use of nano-sized lead borate compounds for treatment purposes due to their selective anticancer activity on p53 mutant breast cancer cell line, T47D is disclosed. The method of synthesizing nano-sized lead borates of the present invention comprises the steps of preparing a borate buffer solution by sodium hydroxide and boric acid, dissolving lead nitrate (and preferably PEG) in distilled water by stirring, mixing the borate buffer solution with the lead nitrate (and preferably PEG) solution, washing the resulting solution with distilled water and drying to remove the impurities.

Claims

1. Nano-sized lead metaborate compounds, wherein the nano-sized lead metaborate compounds are used for a treatment of a cancer due to a selective toxic effect of the nano-sized lead metaborate compounds on cancer cells.

2. The nano-sized lead metaborate compounds according to claim 1, wherein, due to the selective toxic effect of the nano-sized lead metaborate compounds on a p53 mutant breast cancer cell line, T47D, the nano-sized lead metaborate compounds arc used for the treatment of cancerous cells of the T47D.

3. A method for synthesizing the nano-sized lead borate compounds according to claim 1, comprising the steps of dissolving and mixing sodium hydroxide and boric acid with each other in distilled water to obtain a borate buffer solution (a NaOH/H.sub.3BO.sub.3 buffer solution), dissolving lead nitrate in distilled water in a separate beaker by a stirring to obtain a lead nitrate solution, mixing the lead nitrate solution with the borate buffer solution to obtain a product, washing the product with distilled water to obtain a washed product and then drying the washed product to remove impurities.

4. The method for synthesizing the nano-sized lead borate compounds according to claim 3, wherein the method is applied at room conditions. 5. (Currently Amended) The method for synthesizing the nano-sized lead borate compounds according to claim 3, wherein the sodium hydroxide and the boric acid are mixed in a stoichiometric ratio of 1:2 in the distilled water.

6. The method for synthesizing the nano-sized lead borate compounds according to claim 3, wherein the borate buffer solution maintains a pH value of a reaction medium between 9 and 9.5

7. The method for synthesizing the nano-sized lead borate compounds according to claim 3, wherein, in the step of mixing the lead nitrate solution and the borate buffer solution, the lead nitrate solution and the borate buffer solution are stirred for 30 minutes at 2000 rpm under a mechanical stirrer.

8. The method for synthesizing the nano-sized lead borate compounds according to claim 3, wherein, in the step of washing the product and drying the washed product to remove the impurities, the product is washed 4 times with distilled water and then the washed product is dried at 60° C. for 24 hours.

9. The method for synthesizing the nano-sized lead borate compounds according to claim 3, wherein 10 mmol of the lead nitrate is dissolved in 20 ml of the distilled water.

10. The method for synthesizing the nano-sized lead borate compounds according to claim 3, wherein, in the case that the lead nitrate is dissolved in the distilled water, PEG (Polyethylene Glycol) is also dissolved in the distilled water together with the lead nitrate.

11. The method for synthesizing the nano-sized lead borate compounds according to claim 10, wherein the lead nitrate and the PEG (400 to 20000 Da) are dissolved in 200 ml of the distilled water in a stoichiometric ratio of 1:1.5.

12. The method for synthesizing the nano-sized lead borate compounds according to claim 3, wherein, due to the selective toxic effect of the nano-sized lead metaborate compounds on a p53 mutant breast cancer cell line, T47D, the nano-sized lead metaborate compounds are used for the treatment of cancerous cells of the T47D.

13. The method for synthesizing the nano-sized lead borate compounds according to claim 4, wherein, in the case that the lead nitrate is dissolved in the distilled water, PEG is also dissolved in the distilled water together with the lead nitrate.

14. The method for synthesizing the nano-sized lead borate compounds according to claim 5, wherein, in the case that the lead nitrate is dissolved in the distilled water, PEG is also dissolved in the distilled water together with the lead nitrate.

15. The method for synthesizing the nano-sized lead borate compounds according to claim 6, wherein, in the case that the lead nitrate is dissolved in the distilled water, PEG is also dissolved in the distilled water together with the lead nitrate.

16. The method for synthesizing the nano-sized lead borate compounds according to claim 7, wherein, in the case that the lead nitrate is dissolved in the distilled water, PEG is also dissolved in the distilled water together with the lead nitrate.

17. The method for synthesizing the nano-sized lead borate compounds according to claim 8, wherein, in the case that the lead nitrate is dissolved in the distilled water, PEG is also dissolved in the distilled water together with the lead nitrate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0041] “Use of mutant p53 gene targeted lead borate nanoparticles in cancer treatment and production method of these nanoparticles” developed to fulfill the objective of the present invention is illustrated in the accompanying figure, in which:

[0042] FIG. 1A is the graphical representation of the effect of variable concentrations of nano-sized lead borate on viability in final: (Healthy Control Keratinocyte) cells.

[0043] FIG. 1B is the graphical representation of the effect of variable concentrations of nano-sized lead borate on viability in T47D (Breast Cancer) cells.

[0044] FIG. 1C is the graphical representation of the effect of variable concentrations of nano-sized lead borate on viability in MCF7 (Breast Cancer) cells.

[0045] FIG. 1D is the graphical representation of the effect of variable concentrations of nano-sized lead borate on viability in A549 (Lung Cancer) cells.

[0046] FIG. 2 is a representation of the gene expression change of the T47D cell line having p53 mutation and the MCF7 cell line not having p53 mutation following treatment with lead nanoparticle via real-time polymerase chain reaction. (The results were determined upon being normalized in terms of the controls of the cells not involving nano-particle application and the expression levels of the GAPDH gene which can also be referred to as the guardian gene.)

[0047] The present invention relates to nano-sized lead metaborate compounds used for the treatment of cancer due to their selective toxic effect on cancer cells. Within the scope of the present invention, due to their selective anti-cancer effect (toxic effect) on the p53 mutant breast cancer cell line, T47D, nano-sized lead borate compounds are used for the purpose of treatment of these cancerous cells.

[0048] The synthesis of nano-sized (below 100 nm) lead borate compounds developed for use in the cancer treatment process In the scope of the invention is carried out under room conditions by means of the buffered precipitation method. The steps of this synthesis method are as follows: [0049] 4—Sodium hydroxide and boric acid are dissolved and mixed with each other in a stoichiometric ratio of 1:2 in distilled water, and the borate buffer (NaOHM.sub.3BO.sub.3 buffer solution) having a pH value of 9 to 9.5 and having the ability to maintain the pH value of the reaction medium within this range is prepared. [0050] 5—Lead nitrate (and preferably PEG (400 to 20000 Da)) is dissolved again in 200 ml of distilled water in a separate beaker in a stoichiometric ratio of 1:1.5, Lead nitrate (and preferably PEG) solution and borate buffer solution are stirred for 30 minutes at 2000 rpm under a mechanical stirrer. [0051] 6—The Obtained product is washed 4 times with distilled water and then dried at 60° C. for 24 hours to remove the impurities.

[0052] PEG is a biocompatible surfactant. In the method described above, it is also possible to carer out the reaction without the use of surfactants such as PEG, In this case, only 10 mmol of lead nitrate is dissolved in 20 ml of water in a beaker. The purpose of using PEG in the reaction is to obtain lead borate nanoparticles having smaller particle sizes.

[0053] The particle sizes of the nano-sized lead borate compounds synthesized according to the invention are below 100 nm. The particle sizes below 100 nm allow these compounds to be used in in vivo biological applications.

[0054] The synthesis method used is a method which is much easier, economical and suitable for fabrication in comparison to the hydrothermal/solvothermal and microwave precipitation synthesis methods due to the fact that it can be carried out without any need for high temperature, long reaction times, high pressure and any kind of irradiation.

[0055] PEG (Polyethylene glycol) added to the medium is a biocompatible surfactant. It allows the obtained particles to be obtained in smaller sizes (50 nm and below) and to be easily dispersed in water. In order to be able to use lead borate compounds in biological applications and to obtain smaller nanoparticles, PEGs and other biocompatible surfactants with different molecular weights can be used during or after the reaction.

[0056] This buffered-precipitation method for forming the nanoparticle according to the invention is a novel precipitation method for the synthesis of only metal borates obtained by improvement of certain parts of the conventional precipitation method previously known in the literature The most important feature of this method is the ability of the sodium hydroxide/boric acid buffer used as the borate source to maintain the pH value of the medium between 9 and 9.5 throughout the reaction. This synthesis method can be used not only to obtain lead borate but also to obtain all other (+2) and (+3) charged metal borates. The metals of the other metal borate compounds that can be synthesized using this synthesis method are as follows: Ca.sup.+2, Mn.sup.+2, Ni.sup.+2, Co.sup.+2, Cu.sup.+2, Zn.sup.+2, Sr.sup.+2, Ba.sup.+2, Sc.sup.+3, Fe.sup.+3, Cr.sup.+3, Al.sup.+3, Y.sup.+3, La.sup.+3, Ce.sup.+3, Pr.sup.+3, Nd.sup.+3, Sm.sup.+3, Eu.sup.+3, Gd.sup.+3, Tb.sup.+3, Dy.sup.+3, Ho.sup.+3, Er.sup.+3, Tm.sup.+3, Yb.sup.+3, Lu.sup.+3, Bi.sup.+3, Tl.sup.+3. The examples given here are examples of the other metal borate examples that can be obtained by this synthesis method.

Experimental Studies

Cytotoxicity Experiment

[0057] The effect of the prepared nanoparticles on cell viability was determined by using the MTS (material testing systems) method given in the literature. The molecules used in the product were prepared alone or in combination in the medium and applied on T47D (Breast Cancer), MCF7 (Breast Cancer), A549 (Lung Cancer) and Hacat (Healthy Control Keratinocyte) cells which were seeded on 96-well. culture plates by counting at a concentration of 4000 cells per each well. The response of the cells to toxicity of the molecules was determined by measuring cell viability for 3 days. Cell viability was determined by using a method called MTS which measures mitochondrial dehydrogenase enzyme activity of the cell. The MTS substance added onto the cells together with the medium results in colored formazan crystals formation as an indicator of cell viability. The resulting color change was evaluated based on the absorbance measurement by using ELISA plate reader. The obtained results were analyzed.

Real Time Polymerase Chain Reaction

[0058] Real-time polymerase chain reaction assay is performed to observe the changes in the gene levels of the cells treated with nanoparticles. These changes are both at morphological level and gene expression level. The primers that were used were designed using Primer BLAST software (The National Center for Biotechnology=NCBI). Total RNAs were isolated from the cells on which gel combination was applied and cDNA was synthesized. The synthesized cDNAs were mixed with primers in Fermentas Maxima SYBR Green mixture product such that the final volume will be 20 μl and the expression levels of the genes were analyzed by using BIO-RAD device.