Gamma irradiation of ocular melanoma and lymphoma cells in the presence of gold nanoparticles: in vitro study

Abstract The aim of this work was to determine whether conjugation of cultivated choroidal melanoma and Burkitt's lymphoma cells with gold nanoparticles (GNPs) is beneficial for these series of ocular cancer patients. GNPs are radiosensitizers and can sensitize tumors to radiotherapy.This application has been examined in several tumor types, but not in choroidal melanoma. This study shows the results of in vitro study on the choroidal melanoma and also Burkitt's lymphoma cells in the presence of GNPs during continuous gamma irradiation. Cytotoxicity of GNPs were assessed for five different concentrations then cultured melanoma and Burkitt's lymphoma cells were irradiated with a Gamma source in the presence and absence of NPs. Incubation of melanoma cells with GNP concentrations below 100 μg/ml, accompanied by gamma irradiation, increased cell death (P value = 0.016) . In the absence of irradiation, GNPs at these concentrations did not affect cultured melanoma cell metabolism. Reduced cell viability resulted from a significant increase in absorbed energy by the tumor. Moreover, GNP concentrations higher than 200 μg/ml induced cytotoxicity in melanoma cells. Cytotoxicity assay in GNPs‐loaded Burkitt's lymphoma cells showed a slight decrease in cell viability at 50 μg/ml and clear cytotoxicity at concentrations higher than 100 μg/ml (P value = 0.035). Concentration and proper injection doses of GNPs in sensitive tissues such as the human eye are important variables yet to be determined.This is the first report of choroidal melanoma dosimetry performed in the presence of GNPs and provides valuable insights into future therapeutic approaches. Further in vitro study with more different sizes and concentrations is needed to determine the optimum size and concentration before any clinical research in this regard.


| INTRODUCTION
Choroidal melanoma 1 is the most common eye cancer which originating from melanin-containing cells and has the highest rate of metastasis among intraocular tumors. [2][3][4][5] The most common method of treatment for choroidal melanoma is radiation therapy. 6 Experimental and Monte Carlo (MC) studies have investigated the dosimetry of choroidal melanoma using different radiotherapy sources. [7][8][9] In radiation therapy, X and gamma rays, and beta and alpha particles can be emitted from radiation device such as linear accelerators, sealed radioactive sources, or from radiolabeled substances. Plaque brachytherapy is the most widely applied technique for choroidal melanoma radiotherapy. Radioactive plaques are placed on the exterior of the tumor to deliver high-dose radiation to the tumor while reducing radiation exposure in surrounding healthy tissues. [10][11][12] However, healthy and tumor tissues follow the same specific algorithm of radiation dose absorption, which is a regular obstacle in the treatment of eye tumors.
Gold nanoparticles (NPs) have strong photoelectric absorption owing to their high atomic number and electron density, making them potential radiosensitizer. Secondary electrons and Auger electrons caused by gamma or x-ray irradiation can produce very high local ionization density. 13 Increasing the irradiation dose absorbed by the tumor reduces treatment time. Given that the tumor and nearby healthy tissues follow the same energy absorbance pattern, reduce treatment time lower the irradiation dose absorbed by healthy tissues and results in reduced adverse effects. However, if the NPs penetrate to the surrounding normal cells, the absorbed dose by these healthy cells will increase and this method does not benefit the ocular cancer patient. Our previous work demonstrated the proper distribution of GNPs within the eye tumor and showed that GNPs were not observed in extratumoral areas. 14 Previously, it has been shown that loading gold NPs into prostate, skin melanoma, and liver tumors, which are later exposed to irradiation, results in greater dose absorption within the tumor than the surrounding tissues. [15][16][17][18] In recent MC studies, 19,20  We performed an in vitro study on human choroidal melanoma and Burkitt's lymphoma cells and the comparison was made between them. To the best of our knowledge, this is the first time that choroidal melanoma dosimetry has been performed with experimental methods in the presence of GNPs.
In vitro analysis of the toxicity, radiosensitivity and the death of cells was performed by multiple MTT and cell death assay. 21,22 In the in vitro phase, cultured melanoma cells were incubated with GNPs and were irradiated with Gamma source to observe the induced effects of the radioactive source through measuring cell death. Cytotoxicity and optimum GNP concentrations were examined using five different GNP concentrations. The same analysis was performed on Burkitt's lymphoma cells using MTT assay. Furthermore, the response to radiation of GNP-loaded lymphoma and choroidal melanoma cells were studied through with cell death assay and compared.

2.A | Nanoparticle synthesis
Gold NPs were synthesized following the FERN method 23 by employing chloroauric acid (HAuCl4-gold halides) (Merck, Darmstadt, Germany), which was then reduced by sodium citrate. HAuCl4 compound was dissolved in an adequate volume of water, to obtain the desired 0.2145 M solution. The noted substance was heated, and a 1.26544 M sodium citrate was added.
The size of the resultant NPs can be controlled by the amount of added citrate. In this reaction, citrate is used to convert Au (III) to Au (I), and create the NPs. By shifting some factors such as temperature, and the ratio of the administered compounds, it is possible to control both the size, and the distribution of the NPs. [24][25][26] Transmission electron microscopy (TEM) was used to evaluate the morphology and size of synthesized gold NPs using a Zeiss-EM10C-80 KV electron microscope (Oberkochen, Germany) ( Fig. 1).
The size of gold NPs was determined by measuring the diameter of whole particles on the TEM image. The average size of these NPs was in the range of 50 nm and most were spherical in shape.
Absorption spectra and absorbance measurements were obtained with a PerkinElmer UV-visible spectrophotometer model Lambda 25  FBS, 100 U/ml penicillin, 100 lg/ml streptomycin, and 0.05% L-glutamine serum in a T75 flask. Cells were dispensed into 24-well tissue culture plates (1 ml containing 2 9 10 5 cell per well) and incubated at 37°C with 5% CO 2 . GNPs were added to the cells as detailed above.
Cytotoxicity induced by different concentrations of GNPs was measured using a multiple MTT assay test (a quantitative colorimetric method to determine cytotoxicity) in a multiwell plate. Cell viability, expressed as percentage relative to the untreated control (100% cell viability), was measured in triplicate.

2.C | Irradiation
Melanoma tumor or Burkitt's lymphoma cells were cultured in four 24-well tissue culture plates and 50, 100, 200, 400, or 600 lg/ml GNPs were added,;this experiment was performed in triplicate. The first wells in all four plates were the control samples and irradiated in the absence of NPs.
The plates were exposed to 30 Gy radiation using a Cobalt 60 machine, producing two gamma rays with energies of 1.17 and

| RESULTS
Melanoma cells were exposed to 50, 100, 200, 400, or 600 lg/ml GNPs and cell viability was measured and compared to evaluate the Apoptotic response of GNPs-loaded (50, 100, and 200 lg/ml) melanoma and Burkitt's lymphoma cells to irradiation was compared on the seventh day after 30 Gy irradiation (Fig. 8).

| DISCUSSION
Regarding the results of our experimental study (Fig. 7),  be that a different method of GNPs delivery is required for eye tumors. An in vivo study can provide the optimal conditions for determining the most suitable way to deliver NPs into eye tumors. The