USE OF NANO-SIZED LANTHANIDE BORATE (DYSPROSIUM BORATE AND ERBIUM BORATE) COMPOUNDS FOR WOUND HEALING PURPOSES AND PRODUCTION METHOD THEREOF

Abstract

Use of nano-sized lanthanide borate (erbium borate and dysprosium borate) compounds for wound treatment due to their significant level of wound healing effect on the cells is disclosed. In the scope of the invention, the synthesis of nanometer-sized erbium borate and dysprosium borate compound by buffered-precipitation method at room conditions and the use of these compounds in biological applications are discussed.

Claims

1. Nano-sized lanthanide borate compounds, wherein the nano-sized lanthanide borate compounds are used in a wound treatment due to a significant wound healing effect of the nano-sized lanthanide borate compounds on cells.

2. The nano-sized lanthanide borate compounds according to claim 1, wherein particle sizes of the nano-sized lanthanide borate compounds are below 50 nm.

3. The nano-sized lanthanide borate compounds according to claim 1, wherein the nano-sized lanthanide borate compounds are Erbium borate.

4. The nano-sized lanthanide borate compounds according to claim 1, wherein the nano-sized lanthanide borate compounds are Dysprosium borate.

5. A method for producing the nano sized lanthanide borate compounds according to claim 1, comprising the steps of preparing a borate buffer solution by dissolving and mixing sodium hydroxide and boric acid in distilled water, dissolving lanthanide nitrate in distilled water in a separate beaker to obtain a lanthanide nitrate solution, mixing the lanthanide nitrate solution with the borate buffer solution to obtain a product, washing and drying the product to remove impurities.

6. The method according to claim 5, wherein the sodium hydroxide and the boric acid are mixed in a stoichiometric ratio of 1:2.

7. The method according to claim 5, wherein the borate buffer solution maintains a pH value of a reaction medium between 9 and 9.5.

8. The method according to claim 5, wherein, in the step of mixing the lanthanide nitrate solution and the borate buffer solution, the the lanthanide nitrate solution and the borate buffer solution are stirred for 30 minutes at 2000 rpm under a mechanical stirrer.

9. The method according to claim 5, wherein the product is washed with the distilled water 4 times and then dried in an oven at 60° C. for 24 hours to remove the impurities.

10. The method according to claim 5, wherein 10 mmol of the lanthanide nitrate is dissolved in 20 ml of the distilled water.

11. The method according to claim 5, wherein, in a case that the lanthanide nitrate is dissolved in the distilled water, PEG (Polyethylene Glycol) is also dissolved in the distilled water together with the lanthanide nitrate.

12. The method according to claim 10, wherein the lanthanide nitrate and PEG (400 to 20000 Da) are dissolved in 200 ml of the distilled water in a stoichiometric ratio of 1:1.5.

13. The nano-sized lanthanide borate compounds according to claim 2, wherein the nano-sized lanthanide borate compounds are Erbium borate.

14. The nano-sized lanthanide borate compounds according to claim 2, wherein the nano-sized lanthanide borate compounds are Dysprosium borate.

15. The method according to claim 5, wherein particle sizes of the nano-sized lanthanide borate compounds are below 50 nm.

16. The method according to claim 5, wherein the nano-sized Ian borate compounds are Erbium borate.

17. The method according to claim 5, wherein the nano-sized lanthanide borate compounds are Dysprosium borate.

18. The method according to claim 6, wherein, in a case that the lanthanide nitrate is dissolved in the distilled water, PEG is also dissolved in the distilled water together with the lanthanide nitrate.

19. The method according to claim 7, wherein, in a case that the lanthanide nitrate is dissolved in the distilled water, PEG is also dissolved in the distilled water together with the lanthanide nitrate.

20. The method according to claim 8, wherein, in a case that the lanthanide nitrate is dissolved in the distilled water, PEG is also dissolved in the distilled water together with the lanthanide nitrate.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0014] “Use Of Nano-Sized Lanthanide Borate (Dysprosium Borate and Erbium Borate) Compounds For Wound Healing Purposes and Production Method Thereof” developed to fulfill the objective of the present invention is illustrated in the accompanying figures, in which;

[0015] FIG. 1 is a graphical representation of the effect of Dysprosium borate on the viability of the Hacat cell at different concentrations at 24 and 48 hours.

[0016] FIG. 2 is a graphical representation of the effect of Erbium borate on the viability' of the Hacat cell at different concentrations at 24 and 48 hours.

[0017] FIG. 3 shows the light microscopy images of the effect of Dysprosium Borate on wound closure. (The dark colors show the areas not containing cells. The image of the cells covering the empty area shows the wound closure potential of the cells.)

[0018] FIG. 4 shows the graphical representation of the effect of Dysprosium Borate on wound closure.

[0019] FIG. 5 shows the light microscopy images of the effect of Erbium Borate on wound closure. (The dark colors show the areas not containing cells. The image of the cells covering the empty area shows the wound closure potential of the cells.)

[0020] FIG. 6 shows the graphical representation of the effect of Dysprosium. Borate on wound closure.

[0021] FIG. 7 shows the graphical representation of the effects of Dysprosium and Erbium Borate application on the expression level of IL-6 gene.

[0022] FIG. 8 shows the graphical representation of the effects of Dysprosium and Erbium Borate application on the expression level of MMP-9 gene.

[0023] The present invention relates to the fact that nano-sized lanthanide borate (erbium borate and dysprosium borate) compounds have therapeutic feature due to their significant level of wound healing effect on the cells. The fact that particle sizes of erbium borate and dysprosium borate compounds are below 50 nm allows these compounds to be used in in vivo biological applications. The synthesized nano-sized erbium borate and dysprosium borate compounds have an amorphous structure.

[0024] In the scope of the invention, the synthesis of nanometer-sized lanthanide borate (erbium borate and dysprosium borate) compound by buffered-precipitation method at room conditions and the use of these compounds in biological applications are discussed. The process steps of this method are as follows: [0025] 5—Sodium hydroxide and boric acid substances were mixed with each other in a certain stoichiometric ratio (stoichiometric ratio of 1:2), dissolved in distilled water and a borate buffer with a pH value of 8 to 9.5 was prepared. [0026] 6—Lanthanide nitrate (Erbium nitrate or Dysprosium nitrate) and PEG (400 to 20000 Da) were dissolved again in a certain amount of distilled water in a separate beaker in an appropriate stoichiometric ratio. [0027] 7—Lanthanide nitrate (Erbium nitrate or Dysprosium nitrate)—PEG solution and Borate buffer solution were stirred for 30 minutes at 2000 rpm under a mechanical stirrer. [0028] 8—The obtained products were washed with distilled water 4 times and then dried in an oven at 60° C. for 24 hours to remove the impurities.

[0029] The nano-sized erbium borate and dysprosium borate compounds are synthesized using the buffered-precipitation synthesis method and this synthesis method utilizes the ability of NaOH/H.sub.3BO.sub.3 buffer solution to keep the pH of the reaction medium constant between 8 and 9.5. The synthesis method used is a method which is much easier, economical and suitable for fabrication in comparison to the hydrothermal and solid-state 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. 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 erbium borate and dysprosium 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.

[0030] PEG is a biocompatible surfactant. In the method described above, it is also possible to carry out the reaction without the use of surfactants such as PEG. In this case, only 10 mmol of lanthanide nitrate is dissolved in 20 ml of water in a beaker. The purpose of using PEG at the reaction is to obtain lead borate nanoparticles having smaller particle sizes. On the other hand, in the case that PEG is used, lanthanide nitrate and PEG (400 to 20000 Da) are dissolved in 200 ml of distilled water in a stoichiometric ratio of 1:1.5.

EXPERIMENTAL STUDIES

[0031] 4. Culturing of the Cells

[0032] Human skin keratinocyte cells (HACAT) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (Invitrogen) and 1% PSA (Biological Industries, Beit Haemek, Israel) at a temperature of 37° C. in cell culture incubators with 5% Ca.sub.2 medium. [0033] 5. Toxicity

[0034] After the cells were seeded in 96-well culture plates (Corning Glasswork, Corning, N.Y.) at 5000 cells/well in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (Invitrogen) and 1% PSA (Biological Industries, Beit Haemek, Israel), the viability levels of the cells were measured on day 1 and 2. Cell viability was measured by using 3-(4,5-di-methyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS)-method (CellTiter96 AqueousOne Solution; Promega, Southampton, UK). 10 μl MTS solution was added onto the cells within a 100 μl medium and they were incubated at 37° C. in dark for 2 hours. After the incubation process, cell viability was observed by performing absorbance measurement via ELISA plate reader (Biotek, Winooski, Vt.) device at 490 nm wavelength. [0035] 6. Wound Healing

[0036] The wound scratch method was used to determine the migration potential of human skin keratinocyte cells (HACAT). In this method, 24 hours after the cells were seeded in 24-well plates in three replicates at 10,000 cells per well, the cell monolayer was cut (slit) in a straight direction with the aid of a 200p1 pipette tip and the wound scratch closure was observed. After the slit was formed, the medium on the cells was discharged and non-toxic doses of Dysprosium and Erbium Borate according to the results obtained from the toxicology tests were added to the cells except the ones in the control group. The wound scratch closure was measured at hours 0 and 20 by using Carl Zeiss AxioVision Rel. 4.8 software program and the wound closure process was evaluated by comparing the Dysprosium and Erbium Borate administration groups and the control group.

REFERENCES

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