Razi Journal of Medical Sciences

fa بررسی سمیت سلولی نانوذرات الماس بر روی سلول‌های سرطان سینه (MCF-7) و لنفوسیت و میانکنش آنها با آلبومین سرم انسانی با استفاده از روش‌های طیف‌سنجی Evaluation of Diamond Nanoparticles Cytotoxicity on Breast Cancer Cells (MCF-7) and Lymphocyte and their Interaction with Human Serum Albumin using Spectroscopic Methods فارماکولوژی Pharmacology پژوهشي Research زمینه و هدف: استفاده از نانومواد کربن، برای اهداف مختلف زیست‌پزشکی در زمینه‌های تشخیص و درمان کاربرد فراوانی دارد. این مطالعه به‌منظور بررسی نوع تعامل نانوذرات الماس با پروتئین آلبومین و همچنین بررسی سمیت سلولی نانوذرات الماس در سلول سرطان سینه (MCF-7) و سلول لنفوسیت انجام شد. روش کار: به‌منظور بررسی تعامل نانوذرات الماس با پروتئین آلبومین، تکنیک‌های طیف‌سنجی دورنگ نمایی دورانی، طیف‌سنجی Visible-UV و طیف‌سنجی فلورسانس طبق دستورالعمل صورت گرفت. به‌منظور بررسی سمیت نانوذرات بر روی سلول‌های سرطان سینه (MCF-7) و سلول لنفوسیت تست MTT، LDH، آپوپتوز مطابق دستورالعمل انجام شد. یافته‌ها: نتایج تست MTT  نشان داد بیشترین سمیت سلولی نانوذرات الماس بر روی سلول‌های سرطان سینه (MCF-7) در غلظت 50 و 100 میکروگرم در میلی‌لیتر است که میزان زنده ماندن سلول‌ها را به ترتیب به 2/53% - 3/49% (01/0p<) و 68.4% - 3/64% (001/0p<) نسبت به گروه کنترل کاهش داد. درحالی‌که تأثیری در کاهش میزان زنده ماندن سلول‌های لنفوسیت نشان نداد. نتایج تست LDH نشان داد که نانوذرات برخلاف سلول‌های لنفوسیت منجر به افزایش میزان نشت لاکتات دهیدروژناز و القاء آپوپتوز در سلول سرطان سینه (MCF-7) می‌شود. ‌پژوهش‌های طیف سنجی فلورسانس جابه‌جایی به سمت طول موج‌های کوتاه‌تر و کاهش شدت نشر ماکزیموم در اثر افزایش غلظت نانوذرات را نشان داد. این تغییر در ویژگی فلورسانس پروتئین آلبومین حاکی از این است که نانو ذرات الماس با پروتئین آلبومین کمپلکس تشکیل می‌دهد. دمای نیمه ذوب آلبومین در حالت آزاد برابر با 61 درجه سانتی گراد است و دمای نیمه ذوب در حضور نانوذرات الماس به 58 درجه سانتی گراد کاهش یافت. نتیجه‌گیری: نانوذرات الماس منجر به کاهش زنده ماندن سلول‌های سرطانی می‌شوند درحالی‌که تأثیری در کاهش زنده ماندن سلول‌های لنفوسیت نداشت. همچنین این نانوذرات با تغییر ویژگی‌های ساختاری پروتئین آلبومین منجر به تغییر عملکرد آن می‌شوند.   Background & Aims: In the last decade, diamond nanoparticles (DI-NPs) have gained considerable attention in both industries such as medicine and academia. Numerous studies have used various nanoparticles, including diamond nanoparticles, as drug carriers, but the direct effect of diamond nanoparticles on different cell lines, including breast cancer cells (MCF-7) and lymphocyte, is obscure. Therefore, in this study, we investigated and compared the effect of diamond nanoparticles on the cells of breast cancer cells (MCF-7) and human lymphocyte. Some authors have pointed to the destructive effect of DI-NPs on red blood cells. These studies revealed that the characteristics of blood cells, including red blood cells change during the incubation of whole human blood samples with the suspension of DI-NPs in vitro. Blood cell aggregation and their ability to deform are among the most considerable changes that these cells experience. The mechanisms responsible for this effect may be based on the interaction of Nanodiamonds with red blood cells directly or with plasma proteins such as albumin, the major molecular component of blood plasma, indirectly. Numerous studies have been conducted to investigate the effect of nanoparticles on the protein structure of albumin. However, a detailed study has not been performed to investigate the binding mechanisms and the exact interaction of DI-NPs with albumin protein, as well as to compare the effect of DI-NPs on healthy cells and cancer cells. Therefore, this study aimed to investigate the interaction of albumin protein with DI-NPs using spectroscopic methods and comparing the effect of DI-NPs on the viability of lymphocytes and cancer cells. Methods: Cells were cultured in a 96-well culture plate and treated with different concentrations of DI-NPs (0, 1, 10, 20, 50, 100 µg / ml) for 24 hours. Then, 10 µL of MTT solution were added to the cells. After four hours of holding in a 37 ° incubator, the reaction was stopped by adding DMSO solution, and finally, the adsorption rate was measured by an ELISA plate reader at 570 nm. Lactate dehydrogenase test: Cells were treated in a 24-well culture plate with specific concentrations of DI-NPs for 24 hours. The activity of the LDH enzyme in the supernatant and cell lysed was measured using the Pars Azmoun LDH diagnostic kit. Cells were cultured in a 96-well culture plate and treated with DI-NPs for 24 hours. Next, cells were precipitated and washed with PBS solution; after that 100 μl of cell solution with 1 μl of Acridine Orange-Ethidium Bromide dye, the mixture was kept at laboratory temperature for 5 minutes. In the final step, 10 µL of cell suspension were placed on a slide and examined by fluorescence microscopy. Fluorescence measurements were recorded on a fluorescence spectrophotometer. To do so, 2 μM protein solution concentration and different concentrations of DI-NPs (0, 1, 5, 10, 15, 20 μg/ml) were used. The excitation wavelength was set at 270 nm and the emission rate was measured in the range of 300-400 nm. Three sets of fluorescence measurements were recorded at 310, 298, and 315 ° K for different concentrations of DI-NPs. To record the Spectra, a spectropolarimeter was used, and in the ultraviolet region ranging from 190 nm to 260 nm measurements were performed. 2 micromolar protein solution concentration and different concentrations of nanoparticles (0, 1, 5, 10, 15, 20 μg/ml) were utilized. It is also worth mentioning, the experiments were done in the presence and absence of nanoparticles. Measurements were recorded after at least 2 minutes of incubation required for the interaction between DI-NPs and albumin protein. Results: After exposure of lymphocytes and breast cancer cells (MCF-7) to DI-NPs, a decrease in cell survival was observed in breast cancer cells (MCF-7), however, no significant reduction was observed in the viability of lymphocyte cells. When Nanoparticle cytotoxicity started at a concentration of 20 μg / ml, cell viability decreased by 25-21% (p<0.05) compared to the control group; accordingly, cytotoxicity on breast cancer cells increased with increasing nanoparticle concentration. At a concentration of 50 and 100 μg/ml, the survival rate of cancer cells decreased 53.2% - 49.3% (p<0.01) and 68.4% - 64.3% (p<0.001), respectively compared to the control group. Therefore, MTT test results confirmed a concentration-dependent decrease in cancer cell viability. The results also showed that the leakage of LDH enzyme from breast cancer cells (MCF-7) is concentration-dependent. For instance, at the concentrations of 20, 50 and 100 μg / ml the leakage rate of LDH enzyme increased 145.82 ± 10.56 % (p<0.05), 160.76 ± 12.34 % (p<0.01), and 170.46 ± 11.43 % (p<0.001) respectively compared to the control group. the highest rate of lactate dehydrogenase leakage and cell damage was observed at a concentration of 100 μg/ml (Figure 2). The outcomes of the Akredin Orange / Ethidium Bromide test demonstrated that DI-NPs lead to compaction and fragmentation of cellular DNA and induce apoptosis in cancer cells (MCF-7), while no significant effect was spotted on healthy cells. The fluorescence spectroscopy’s results showed a shift towards shorter wavelengths and a decrease in the maximum emission intensity due to the increase in nanoparticle concentration, indicating an increase in protein surface hydrophobicity and displacement of aromatic amino acids to albumin protein level due to increased nanoparticle concentration. The fluorescence spectrum of albumin protein at 298, 310, 315° K declined with increasing concentration of DI-NPs. Therefore, it can be inferred that the DI-NPs led to quenching and suppression of albumin protein. The binding mechanism between albumin protein and DI-NPs is presented using the Stern-Volmer diagram in Figure - 5. As shown in Table 1, while the temperature increases, the Stern Volmer constant decreases. The inverse relationship between temperature and Stern-Volmer constant implies a static bond between DI-NPs and albumin protein. Therefore, it can be concluded that DI-NPs are complexed with albumin protein and the type of quenching mechanism of albumin nanoparticles with albumin protein is static quenching. The CD spectrum of a protein shows information about the secondary structure of the protein. Figure 6 illustrates that the albumin protein has a peak at 208 and 222 nm, indicating the Alpha-Helix structure of the albumin protein. With the increasing concentration of DI-NPs, the structure of albumin protein changes to the random coil structure. Consequently, DI-NPs change the structure of albumin protein from alpha-helix to random coil. Although the melting point of albumin protein is 61°C (see Figure - 7), this temperature changed to 58°C in the presence of diamond nanoparticles, which indicates a decrease in albumin protein stability. Conclusion: In this study, breast cancer cell lines (MCF-7) and lymphocyte cell lines were used to evaluate the toxicity of diamond nanoparticles. The results showed that nanoparticles lead to increased leakage of lactate dehydrogenase in breast cancer cells (MCF-7); while, no enzyme leakage was observed in lymphocyte cells. The results of the apoptosis test confirmed the induction of cell apoptosis in cancer cells, whereas the induction of apoptosis in lymphocyte cells produced no effect. Additionally, the structure of albumin protein in the presence of DI-NPs was investigated by a series of techniques. UV-vis spectroscopy shows an obvious change in the secondary structure of albumin protein after binding to diamond nanoparticles. UV-vis and CD spectroscopy reveal that the structural properties of albumin protein are not preserved in the collection of DI-NPs with albumin protein. Fluorescence spectroscopy shows that the amino acid tryptophan in albumin protein is placed in a more hydrophobic environment in a set of DI-NPs with albumin protein, indicating that protein expansion is likely to occur. The results confirm that DI-NPs can greatly alter the structural properties of albumin protein in the collection of DI-NPs with albumin protein. Therefore, the use of DI-NPs in medical applications needs further study. نانوذرات الماس, آلبومین, سلول سرطان سینه (MCF-7), سلول لنفوسیت, سمیت سلول. Diamond nanoparticles, Albumin, Breast cancer cell (MCF-7), Lymphocyte cell. 1 13 http://rjms.iums.ac.ir/browse.php?a_code=A-10-6053-1&slc_lang=fa&sid=1 Seyyedeh Elaheh Mousavi سیده الهه موسوی semousavi@sina.tums.ac.ir 3900319475328460063406 3900319475328460063406 Yes Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran استادیار، گروه فارماکولوژی، دانشکده پزشکی، دانشگاه علوم پزشکی تهران، ایران Maryam Zivari fard مریم زیوری فرد MaryamZivari70@gmail.com 3900319475328460063407 3900319475328460063407 No Department of Applied Cell Science, School of Medicine, kashan University of Medical Sciences, Kashan, Iran دکتری تخصصی علوم سلولی کاربردی، گروه علوم سلولی کاربردی، دانشکده پزشکی، دانشگاه علوم پزشکی کاشان، کاشان، ایران Seyed Mehdi Rezayat سید مهدی رضایت rezayat@sina.tums.ac.ir 3900319475328460063408 3900319475328460063408 No Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran استاد، گروه فارماکولوژی، دانشکده پزشکی، دانشگاه علوم پزشکی تهران، ایران Farnoosh Attar فرنوش عطار F.attar@standard.ac.ir 3900319475328460063409 3900319475328460063409 No Department of Food Toxicology, Research Institute of Food Industries and Agricultural Products, Standard Research Institute, Karaj, Iran استادیار، گروه سم‌شناسی مواد غذایی، پژوهشکده صنایع غذایی و فرآورده‌های کشاورزی، پژوهشگاه استاندارد، کرج، ایران