WO2022103364A1

WO2022103364A1 - Carbon nanoparticle synthesis method and use thereof for huvec cancer treatment

Info

Publication number: WO2022103364A1
Application number: PCT/TR2021/051158
Authority: WO
Inventors: Afsin KARIPER
Original assignee: Erciyes Universitesi
Priority date: 2020-11-11
Filing date: 2021-11-08
Publication date: 2022-05-19
Also published as: TR202018046A2

Abstract

The present invention relates to synthesis method of carbon nanoparticle solutions which can be produced in large quantities, used as carrier for drugs that are used in treatment process for cancer cells such as breast cancer (MCF-7) and human osteosarcoma (Saos-2) and which are also used for cancer treatment due to their ability to show cytotoxic effects against cancer cells in cell lines of human umbilical vein endothelial cells (HUVEC).

Description

CARBON NANOPARTICLE SYNTHESIS METHOD AND USE THEREOF FOR HUVEC CANCER TREATMENT

Field of the Invention

The present invention relates to a synthesis method of carbon nanoparticle solutions, which can be produced in large quantities, and used as carrier for drugs that are used in treatment process for cancer cells such as breast cancer (MCF-7) and human osteosarcoma (Saos-2), and which are also used for cancer treatment due to their ability to show cytotoxic effects against cancer cells in cell lines of human umbilical vein endothelial cells (HUVEC).

Background of the Invention

With the introduction of nanotechnology into our lives, progress has been made very rapidly in health, technique and technology. Besides being environmentally friendly materials, carbon nanoparticles are widely used especially as nanodots in fields such as fluorescence imaging (FL), two-photon FL (fluorescence imaging), Raman imaging, magnetic resonance imaging (MRI), tomography (CT), photoacoustic imaging (PAI), computed positron emission tomography/single photon emission computed tomography (PET/SPECT) and multimodal imaging. On the other hand, carbon nanotubes are used for energy storage thanks to their large surface areas and conductivity. In addition, carbon nanotubes also play a role in the repair of DNA. Carbon nanoparticles have recently led to a new technological era with their aromatic structures having conjugated double bonds being produced at nanoscale. In addition to this, carbon nanoparticles have been started to be widely used by researchers not only in imaging but also in drug release [1-2].

Until recently, the researchers have tried many ways to produce carbon nanoparticles. In the beginning, the basic logic was similar to that of metal nanoparticle production. In the production of metal nanoparticles, first a reducing reagent (such as NaBtU, L1AIH4) is used so that an ion pair layer is not formed around the metal, since the metal is positive valent. Additionally, it is surrounded by a reagent (such as trisodium citrate) by utilizing the core- shell relationship against the risk of oxidation in the aqueous environment [3]. The same approach was followed in the production of carbon nanoparticles. Around the carbon nanoparticles, a polymer such as Oxa (IV)COOH or Poly(N-isopropylacrylamide) is also used [4-5],

When researchers discovered the properties of carbon nanodots with sizes below 10 nm, they focused on carbon nanodots. The easiest way known so far is to produce carbon nanodots by treating lemon juice and onion juice in microwave [6-7], However, when this method is tested by other researchers, it is seen that its repeatability is low. On the other hand, other researchers resorted to extract the carbon nanoparticles in the solution by ensuring reverse diffusion of the sample taken from the microwave after the carbonization process under a certain pressure in the dialysis machine. As it can be seen, one -pot carbon nano production has been the focus of researchers [8-9], However, the fact that the use of a dialysis device leads to a method which is both expensive and requires a longer process prompts researchers to develop different methods.

In the literature, there are many publications regarding the use of carbon nano or carbon nanoparticles together with imaging techniques, as explained above. In the production methods disclosed in these publications, very expensive devices such as a ultra-centrifuge device are needed and mass production cannot be achieved with these devices. Furthermore, even though in the literature it is claimed that it can be produced in large quantities in the studies carried out with "one pot synthesis", the carbon nanodots that are produced are not stable and cannot be produced in large quantities [6-9]. The production carried out with the applications known in the state of the art allows limited production in very low amounts, it is not stable and also it cannot exhibit cytotoxicity towards cancer cells such as MCF.

Patent documents numbered US2019367368, CN108659834, CN104789216, TW201534321, KR101496697, CN105905882 and CN105802623 disclose several prior art applications for the synthesis of carbon nanodots.

Summary of the Invention

The objective of the present invention is to synthesize carbon nanoparticle solutions, which can be produced in high concentrations with low investment costs, and used as carriers for drugs that are used in treatment process for cancer cells such as breast cancer (MCF-7) and human osteosarcoma (Saos-2), and which can also be used in process of cancer treatment due to their cytotoxic effects against cancer cells in cell lines of human umbilical vein endothelial cells (HUVEC).

Another objective of the present invention is to provide the mass production of carbon nanoparticles produced by natural diffusion in the intestine or fibrous tissue.

Detailed Description of the Invention

Carbon Nanoparticle Synthesis Method And Use Thereof For HUVEC Cancer Treatment” developed to fulfill the objective of the present invention is illustrated in the accompanying figures, in which;

Figure 1- Graphical representation of the carbon nanoparticles synthesized within the scope of the invention through the FTIR spectrum, ((a). 4 hours; (b). 6 hours; (c). 12 hours)

Figure 2- Microscopic representation of SEM analysis of carbon nanoparticles synthesized within the scope of the invention, ((a). 4 hours; (b). 6 hours; (c). 12 hours) Figure 3- Graphical and tabular representation of Zetasizer analysis of carbon nanoparticles synthesized within the scope of the invention, ((a). 4 hours; (b). 6 hours; (c). 12 hours)

Figure 4- Graphical representation of the UV spectrum of carbon nanoparticles synthesized within the scope of the invention.

Figure 5- Graphical representation of the percentage (%) of viable cells after 72 hours in HUVEC and MCF7 cells incubated with carbon nanoparticles at various concentrations.

Figure 6- Graphical representation of the percentage (%) of viable cells after 72 hours in HUVEC and MCF7 cells incubated with 5FU-carbon nanoparticles at various concentrations.

Figure 7- Viability (%) of HUVEC, MCF-7 and Saos-2 cells incubated with drug-free carbon nanoparticles at various ratios for 72 hours. (*The drug-free carbon nanoparticles were diluted with medium in ratios of 1/2, 1/4, and 1/8.) Figure 8- Viability (%) of HUVEC (a), MCF-7 (b), Saos-2 (c) cells incubated with 5-FU loaded carbon nanoparticles and with various concentrations of free 5-FU for 72 hours. (* Free 5FU and nano 5FU concentrations were used at the same ratio in HUVEC, Saos-2 and MCF-7 cell lines.)

Figure 9- Schematic representation of diffusion of the caramelized solsolution mixture within the scope of the invention into distilled water by fixing the intestine or fibrous tissue, in which it is placed, on a fixed metal.

The method of synthesizing carbon nanoparticles according to the present invention, which can be produced in large quantities and can show cytotoxic effects against cancer cells, comprises the steps of

- gelation of chitosan by dissolving it together with glacial acetic acid in distilled water,

- caramelizing the obtained sol-gel in a microwave oven at 800 W power for 12 minutes,

- adding up to 200-250 ml of distilled water to the caramelized product, - introducing a 15 ml sample taken from the surface of the obtained aqueous solution with a dropper into the 3 -layer animal intestine or fibrous tissue obtained from the third layer of the animal skin, which was kept in an organic liquid for 1 day,

- tying both open sides of the intestine or fibrous tissue tightly with a string and attaching to a fixed metal,

- immersing the intestine or fibrous tissue into a beaker filled with approximately 15-20 ml of distilled water in such a way that its opening tied with the string does not contact the distilled water,

- drawing, with the help of a dropper, the carbon nanoparticles that diffuse into the distilled water in the beaker through the intestinal membrane or fibrous tissue in certain periods from 30 minutes to 48 hours,

- refilling the beaker with distilled water to replace each liquid drawn with a dropper,

- obtaining carbon nanoparticles which are the final product.

In one embodiment of the invention, chitosan by weight of 1:10 and glacial acetic acid by volume of 1:10 are added to the distilled water in the step of gelation by dissolving in distilled water. Preferably, 1 gram of chitosan and 1 ml of glacial acetic acid are dissolved in 10-20 ml of distilled water and thereby it is gelled.

In one embodiment of the invention, the organic liquid, in which the caramelized solution-sol mixture is placed, is preferably obtained by a mixture of acetone, ethanol, chloroform, and water at a ratio of 1:1: 1:1 by volume.

Within the scope of the invention, unlike the studies in the literature, carbon nanoparticles are obtained by gelling chitosan and caramelizing it in the microwave, and then, unlike the literature, by subjecting the carbon material that we have caramelized to natural diffusion in the animal intestinal membrane. Furthermore, it is also aimed to use these carbon nanoparticles as drugs directly in cancer treatment within the scope of the invention. Within the scope of the invention, a natural polymer such as chitosan is gelled and the gelling solution is treated with microwave. Carbon nanoparticles are obtained by diffusion of the obtained carbon material through the intestinal membrane or fibrous tissue. Although their sizes are not as small as carbon nanodots, it has been proven by the experimental studies conducted within the scope of the invention that carbon nanoparticles produced by the method according to the invention are more effective against the cancerous cells than carbon nanodots. In addition, within the scope of the experimental studies, the characterization of the carbon nanoparticles produced by the method according to the invention was carried out with FTIR, SEM, EDX, while the effect of carbon nanoparticles on cancer cells was examined with IC 50 tests.

Within the scope of the invention, a method suitable for mass production with high concentration and low investment cost has been developed. Therefore, the present method is both innovative according to the literature and it is different from other carbon nanoparticle production methods with respect to the obtained results. The effects achieved on cancer cells are also groundbreaking in this field. The reason why other carbon nanoparticles do not exhibit this effect is due to the lack of sufficient carboxylic groups in the structure after caramelization.

Carbon nanoparticles obtained by the synthesis method according to the invention can be used directly for cancer treatment due to their cytotoxic effect on cancerous cells, or they can be used so as to function as carriers for drugs used in the treatment of cancerous cells. The tests carried out within the scope of the invention were carried out in vitro, that is in the laboratory environment, and usability of the carbon nanoparticles as a carrier for drugs applied in the treatment of cancerous cells such as breast cancer (MCF-7) and human osteosarcoma (Saos- 2) due to their cytotoxic effect on cancerous cells and its usability for cancer treatment due to the fact that it shows cytotoxic effect on cancerous cells in human umbilical vein endothelial cells (HUVEC) cell lines have been shown with experimental studies.

EXPERIMENTAL STUDIES

Materials and Methods:

The surface images of the carbon nano materials were examined using a GEMINI 500 computer controlled digital transmission electron microscopy (TEM). Their elemental analysis was performed with an EDX device. Infrared spectrums of electrodes in solid powder form were measured with Perkin Elmer Spectrum 400 spectrometer device equipped with a DTGS detector at a resolution of 4 cm'¹ performing 10 scans. Nanoparticle sizes were measured at a temperature of 25°C on a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK). Ultra-distilled water was used as reference liquid.

1 g of chitosan was dissolved together with 1 mL of glacial acetic acid in 10-20 mL of distilled water and then it was gelled. The resulting sol-gel was caramelized in a microwave oven at 800 W for at least 12 minutes. Distilled water up to 200- 250 mL was added to the caramelized product. The 15 ml sample taken from the surface of the obtained solution with a dropper was introduced into the 3 -layer animal intestinal membrane or fibrous tissue which was previously kept in organic liquids such as acetone, ethanol, chloroform for 1 day. Both open sides of the intestine or fibrous tissue were tied tightly with a string and attached to a fixed metal. In a beaker filled with approximately 15-20 mL of distilled water, the intestine or fibrous tissue was attached to the metal in such a way that its opening, which was tied with a string, would not come into contact with distilled water (Figure 9). If the fibrous tissue or intestine is not attached to a fixed metal at both ends, the caramelized solution-sol mixture can flow into the distilled water below, and this mixture contains solid carbon particles in micron or even cm size. As a result of this, carbon nanoparticles cannot be produced. Carbon nanoparticles that diffused into the distilled water through the intestine or fibrous tissue were drawn with the help of a dropper at certain periods. The lower beaker was refilled with distilled water to replace each drawn liquid, and the times were scanned. By changing the diffusion time only, different sizes of carbon nanoparticles can be produced each time by drawing the water below at different times.

Cell culture Method

Cell culture and cytotoxicit test (MTT assay)

In this study, human breast cancer (MCF-7), human osteosarcoma (Saos-2) and human umbilical vein endothelial cells (HUVEC) cell lines obtained from the ATCC (American Type Culture Collection) were used. The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Sigma) supplemented with 10% heated-inactivated fetal bovine serum (FBS; Sigma) and 100 units/mL penicillin and 100 pg/mL of streptomycin (Sigma) in a humidified incubator 5% CO2 at 37°C atmosphere. The cell lines were passaged after reaching 80% monolayer confluency.

The cytotoxic effects of free-carbon nanoparticles, 5-FU loaded carbon nanoparticles and free 5FU were determined using the MTT method on the cells for 72 hours [15]. The MTT reduction characteristic of cells is a test that measures cell viability, staining intensity acquired at the end of MTT analysis demonstrates the correlation with the number of living cells. For this purpose, the cells were cultured in 96-welled plates with IxlO⁵ cells/ml. The cells grown in DMEM were exposed to increasing concentrations of each free carbon nanoparticles, 5FU loaded carbon nanoparticles and free 5FU. After 72 hours incubation, MTT solution (10 pl) was added at 37°C for 4 h and medium was taken away from the environment and cells were lyzed with 100 pl DMSO in which formazan crystals formed by MTT were dissolved. Absorbance was read in an enzyme-linked immunosorbent assay (EEISA) plate reader.

Results and Discussion: The FTIR spectrum of carbon nanoparticles is given in Figure 2. At 2976 cm' and 2914 cm'¹ -C-H aliphatic symmetric vibrations, at 2324 cm'¹ -C=O symmetric vibration signals, at 1712 cm'¹ C=O stretching vibrations, at 1400 cm'¹ -C-H bending vibrations, at 1224 cm'¹ -C-H stretching vibrations, at 1054 cm'¹ -C-H stretching band, at 885 cm ¹ ’de -C-H stretching vibrations corresponding to the plane were seen [10]. Apart from this, the vibration signals of the amine and hydroxyl groups of chitosan could be detected very weakly. No significant difference was observed in the FTIR spectra of the carbon nanoparticles obtained at different diffusion times.

While TEM images are given in Figure 3, zetasizer analyzes of carbon nanoparticles are given in Figure 4. According to these results, carbon nanoparticles below 100 nm were diffused in 4 hours, carbon nanodots below 5 nm were obtained in 6 hours, and carbon nanoparticles around 10 nm were obtained in 12 hours. TEM images confirm these claims. When examined carefully, it is seen that the particle sizes are below 10 nm in 6 hours of diffusion. Of course, since the samples for TEM analysis were prepared by dropping on the conductive layer and drying beforehand, and then coated with Au/Pt, agglomerations were seen. But still, the nanoparticles have distinguishable size. Diffusion time scanning was then tested up to 24, 36 and 48 hours; and only after these times, carbon nanoparticles with a size range of 300-500 nm were detected.

In Figure 5, especially the UV spectrum of the carbon nanodot obtained by diffusion for 6 hours is given. This ridge, which generally corresponds to 7t-7t* absorption transitions in the range of 240-270 nm in the literature, gave a small transition peak corresponding to the n— n* absorption transition around 332 nm in our study. The reason why the UV spectrum we obtained is different from the others is due to the starting material chitosan. It is also seen that after caramelization the vibration signals of functional groups such as chitosan -NH, - OH are quite weak in the FTIR spectrum. This is due to the fact that most of the - NH and -OH groups are detached from the structure during caramelization and - CH and -C=O functional groups are rather more dominant in the structure. However, such a peak at this point indicates that the produced carbon material is in nano size (<5nm). Normally, in order for adsorption to be observed at 240-270 nm, there must be -C=C groups present in the structure. It is possible to see such studies compatible with UV spectrum in the literature [11-13].

In the preliminary study that we carried out, MCF-7 (human breast cancer cell line) and HUVEC (Human Endothelial Cell line) were used. Cells were assessed after incubation with carbon nanoparticle for 72 hours. It is observed that the cell viability decreases as the concentration increases. As it is seen in the figure, it was more effective in MCF-7 cell line than in healthy cell. When we calculate the IC50 values from the figure, we found 43 pg/ml for MCF-7 and 55 pg/ml for HUVEC.

In the preliminary study, MCF-7 (human breast cancer cell line) and HUVEC (Human Endothelial Cell line) were used. Carbon nanoparticles were combined with 5 Flurasil-U. Cells were assessed after incubation with carbon nanoparticle for 72 hours. It is observed that the cell viability decreases as the concentration increases. It is observed that the efficiency of carbon nanoparticles applied with the combination is increased even more. When the IC 50 values are reviewed, it is seen to be 12 in MCF-7, while 21 pg/ml in HUVEC. The IC50 value was more effective, with a difference of about 50% with the living cell.

Table 1. IC50 values of the compounds:

IC50: Concentration of inhibition that kills 50% of cells

MCF7: Breast cancer cell line

HUVEC: Human Umbilical Vein Endothelial Cells (Healthy cell) In Figure 7, it is seen that drug-free nanoparticles are cytotoxic especially on MCF-7 and Saos-2 although there is no drug loaded with nanoparticles. On the other hand, it is effective on HUCEV cell lines of drug-free nanoparticles at higher rates than MCF-7 and Saos-2. As the drug-free nanoparticles ratio increases, cytotoxicity also increases. Drug-free nanoparticles are most cytotoxic on Saos-2 cell lines.

Table 2. Cytotoxicity of free-carbon nanoparticles, 5-FU loaded carbon nanoparticles and free 5-FU obtained by MTT on MCF-7, Saos-2 and HUVEC cell lines.

Cell Lines MCso (pg/ml) **ICso Carbon ratio

HUVEC Free 5FU 55

MCF7 Free 5FU 43

Saos-2 Free 5FU 21

HUVEC Carbon Nano 5FU 21 0,54-1/2

MCF7 Carbon Nano 5FU 12 0,25=1/4

Saos-2 Carbon Nano 5FU 23 0,43-1/2

HUVEC Drug-free Carbon nano 1

MCF-7 Drug-free Carbon nano 0,43-1/2

Saos-2 Drug-free Carbon nano 0,22-1/4

Cytotoxicity was assessed by MTT assay. After 72 hours, IC50 values were determined.

* IC50: Concentration that inhibited cell growth by 50%.

**The drug-free carbon nanoparticles were diluted with medium in ratios of 1/2, 1/4, and 1/8.

(HUVEC: Human umbilical vein endothelial cells, MCF-7: Human breast cancer cell line, Saos-2: Human osteosarcoma)

5FU loaded nanoparticles (IC50: 12 pg / ml) appear to be 3.5 times more effective than free 5FU (IC50: 43 pg / ml) on MCF-7 cell lines. Thus, a lower 5FU concentration is used in MCF-7 cells. This is a very important finding in terms of reducing the side effects of the drug. When IC50 values of 5FU loaded nanoparticles are compared between HUVEC and MCF-7 cells, it is seen that the IC50 value for HUVEC cells is approximately 2-times higher than MCF-7 cells at 21 pg/ml. This means that the concentration used for MCF-7 cells is much less toxic in healthy cells.

It is seen that free nanoparticles are more effective on Saos-2 cells than 5FU loaded nanoparticles. It is reported that Saos-2 cells show strong adhesion with carbon nanoparticles [14]. In addition, it is an important finding that carbon nanoparticles are effective without drug loading. When the effects of free nanoparticles on HUVEC and Saos-2 cells are compared, it is seen that the ratio of free nanoparticles used on HUVEC cells is at least 2 times more effective on Saos-2 (Table 2).

In conclusion;

As we mentioned before, the production of nanoparticles with chitosan has never been tried in the literature and its effects on cancer cells could not been determined. In general, in the studies carried out so far, it has been centrifuged by ultracentrifuge after carbonization process with ascorbic acid, lemon juice, or fruit skins. However, production cost is high, and carbon nanoparticles are produced in very low concentrations. On the other hand, carbon nanoparticles produced as a result of studies carried out without the requirement of ultracentrifuge in "one pot step" studies are not stable. In the studies, different results are obtained in each trial. In this study, we gelated chitosan, caramelized it in a microwave oven, and subjected it to natural diffusion through the animal intestine. Therefore, we have developed a method suitable for mass production with high concentration and low investment cost. Thus, the present method is both innovative according to the literature and it is different from other carbon nanoparticle production methods according to the obtained results. The effects achieved on cancer cells are also groundbreaking in this field. The reason why other carbon nanoparticles do not exhibit this effect is due to the lack of sufficient carboxylic groups in the structure after caramelization. REFERENCES

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Claims

CLAIMS A method of synthesizing carbon nanoparticles, which can be produced in large quantities and can show cytotoxic effects against cancer cells, comprising the steps of

- caramelizing the obtained sol-gel in a microwave oven,

- adding distilled water to the caramelized water,

- introducing the sample taken from the surface of the obtained aqueous solution inside animal intestine membrane or fibrous tissue in an organic liquid beforehand,

- tying both open sides of the intestine or fibrous tissue tightly with a string and attaching it to a fixed metal,

- immersing the intestine or fibrous tissue into a beaker filled with distilled water in such a way that its opening tied with the string does not contact the distilled water,

- drawing, with the help of a dropper, the carbon nanoparticles that diffuse into the distilled water in the beaker through the intestinal membrane or fibrous tissue,

- obtaining the carbon nanoparticles which are the final product. Method of synthesizing carbon nanoparticles according to claim 1, characterized by dissolving 1 gram of chitosan together with 1 ml of glacial acetic acid in 10-20 ml of distilled water and gelling it. Method of synthesizing carbon nanoparticles according to claim 1, characterized by caramelizing the obtained sol-gel in a microwave oven at 800 W power for 12 minutes. Method of synthesizing carbon nanoparticles according to claim 1, characterized by adding up to 200-250 ml of distilled water to the caramelized product. Method of synthesizing carbon nanoparticles according to claim 1, characterized by introducing the 15 ml sample taken from the surface of the obtained aqueous solution with a dropper into the 3 -layer animal intestine membrane or fibrous tissue obtained from the third layer of the animal skin, which was kept in an organic liquid for 1 day. Method of synthesizing carbon nanoparticles according to claim 5, characterized by using an organic liquid selected from a group comprising acetone, ethanol, chloroform and mixtures thereof. Method of synthesizing carbon nanoparticles according to claim 1, characterized by immersing the intestine into a beaker filled with approximately 15-20 ml of distilled water in such a way that its opening tied with the string does not contact the distilled water. Method of synthesizing carbon nanoparticles according to claim 1, characterized by drawing carbon nanoparticles with the help of a dropper at certain periods between 30 minutes and 48 hours in the step of drawing, with the help of a dropper, the carbon nanoparticles that diffuse into the distilled water in the beaker through the intestinal membrane or fibrous tissue. Carbon nanoparticles obtained with the method of synthesizing carbon nanoparticles according to any one of the preceding claims and used for cancer treatment due to their cytotoxic effects on cancerous cells.

10. Carbon nanoparticles according to claim 9, used for cancer treatment in human umbilical vein endothelium cells (HUVEC) cell lines due to their cytotoxic effects on cancerous cells. 11. Carbon nanoparticles obtained with the method of synthesizing carbon nanoparticles according to any one of the claims 1-8 and used to function as a carrier for drugs that are used in treatment of cancerous cells in breast cancer (MCF-7) and human osteosarcoma (Saos-2) cell lines.

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