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EXPERIMENTAL ARTICLES
UDC 615.277.3+615.012]-022.532-092.9 https://doi.org/10.15407/biotech13.03.045
PREVENTION OF CISPLATIN TOXICITY AGAINST NORMAL CELLS BY COMPLEXATION WITH C60 FULLERENE
S. V. Prylutska1
1.1. Grynyuk1 1Taras Shevchenko National University of Kyiv, Ukraine
T. D. Skaterna2
L. B. Drobot2 2Palladin Institute of Biochemistry
N. S. Slobodyanik1 of the National Academy of Sciences of Ukraine, Kyiv
O. P. Matyshevska2
E-mail: [email protected]
Received 23.02.2020 Revised 16.05.2020 Accepted 30.06.2020
The aim of this study was to evaluate the toxicity of noncovalent nanocomplex of C60 fullerene with cisplatin (C60-Cis-Pt) against normal cells. The toxicity of the C60-Cis-Pt nanocomplex compared to the free Cis-Pt was studied by estimating kidney human embryonic (HEK293) cells viability using MTT assay and rat erythrocytes resistance to acid haemolysis. It was shown that free 40 pM Cis-Pt changed the morphology and reduced the viability of HEK293 cells, as well as increased the number of haemolyzed erythrocytes compared to the control. According to the investigated parameters analysis no cytotoxic effects of C60-Cis-Pt nanocomplex was observed at Cis-Pt equivalent concentration. The prevention of Cis-Pt toxic action against normal cells by its complexation with C60 fullerene opens the prospect of nanostructure usage as an effective cytoprotector and a target carrier in tumor cells.
Key words: C60 fullerene, cisplatin, nanocomplex, HEK293 cells, cytotoxicity, erythrocytes, haemolysis.
The use of biologically active nanomaterials for targeted drug delivery, enhancement of the traditional anticancer drugs therapeutic efficacy and prevention of its side effects is a significant and complex problem of modern biotechnology. The representative of carbon nanostructures C60 fullerene is promising in this direction. It is a chemically stable, nanosized (0.72 nm), almost spherical and hydrophobic molecule that penetrates through biological membranes, localizes within cells [1-3]. As is known, chemical modification affects the physical, chemical and biological properties of C60 fullerene. Pristine C60 fullerene and its water-soluble derivatives do not cause toxic effects [1, 4, 5]. The accumulation of C60 fullerene in tumors of the liver, stomach, intestine, lungs, bones and its selective damaging effect on malignantly
transformed cells was detected [6-8]. The surface structure of C60 molecule with a system of double ^-conjugated electron-deficient bonds is unique and determines the properties of this nanostructure as an antioxidant (free radical scavenger) [9, 10] as well as its ability to generate reactive oxygen species (ROS) after UV-Vis light irradiation [11-14] that can be used in photodynamic therapy of tumors. Besides, C60 fullerene can form stable complexes with chemotherapeutic drugs [1517], that can be used to optimize their action.
The traditional broad-acting anticancer drug is cisplatin (cis-diaminodichloroplatinum, cis-[Pt(II)(NH3)2Cl2], Cis-Pt), the cytotoxic effect of which is caused by DNA damage and oxidative stress induction [18-22]. Despite the negative side effects of Cis-Pt, it is widely used in antitumor therapy. Clinical usage of
Cis-Pt might be increased with improving its selectivity, overcoming drug resistance and reducing toxicity.
To increase the effectiveness of Cis-Pt antitumor effect and minimize its side effects, a noncovalent nanocomplex of C60 fullerene with Cis-Pt (C60-Cis-Pt) was created [23]. Estimation of C60-Cis-Pt nanocomplex toxicity is an important prerequisite of its usage for biomedical purposes. Thus, the aim of this work was to estimate the toxic effect of free Cis-Pt against normal cells in comparison with C60-Cis-Pt nanocomplex.
Materials and Methods
Creation of C60-Cis-Pt nanocomplex. C60 fullerene aqueous colloid solution (C60FAS) (150 ug/ml, 2-10-4 M, purity 99.95%) was prepared at the Technical University of Ilmenau (Germany) as described in [24, 25]. C60 FAS is characterized by a high C60 fullerene concentration and stable up to 12 months at +4 °C.
To generate C60-Cis-Pt nanocomplexes, the C60 solution (150 ug/ml) and Cis-Pt (Sigma, USA) solution in 0.9% NaCl saline (150 ug/ml) were mixed in 1:1 volume ratio. The mixture was sonicated with ultrasound (22 kHz, 20 min) and stirred (400 rpm, 18 h). Final concentrations of C60 fullerene and Cis-Pt were 75 ug/ml (104 uM) and 75 ug/ml (250 uM), respectively. The stability of C60-Cis-Pt nanocomplexes in the aqueous medium was confirmed by the results of the dynamic light scattering technique [26]. The calculated dissociation constant for the obtained noncovalent C60-Cis-Pt nanocomplex is ~ 2 mM [27].
Cell culture. Non-tumor HEK293 (human embryonic kidney 293) cells were kindly supplied by the Bank of Cell Cultures and Transplantable Experimental Tumors of Ka-vetsky Institute of Experimental Pathology, Oncology, and Radiobiology of the National Academy of Sciences of Ukraine (Kyiv, Ukraine). Cells were maintained in DMEM (Sigma-Aldrich Co, Ltd, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich Co, Ltd, USA), 50 U/ml penicillin and 100 ug/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2. Cells were incubated for 24 h with or without free Cis-Pt or C60-Cis-Pt nanocomplex in Cis-Pt equivalent concentration.
Cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Sigma-Aldrich Co, Ltd, USA) reduction assay [28]. At indicated time
points of incubation 100 ul aliquots (10x 103 cells) were placed into the 96-well microplates Greiner (Sigma-Aldrich Co, Ltd, USA), 10 ul of MTT solution (4 mg/ml in phosphate-buffered saline (PBS)) was added to each well and the plates were incubated for another 2 h at 37 °C. Precipitates were dissolved with 100 ul of dimethyl sulfoxide (DMSO) (Sigma-Aldrich Co, Ltd, USA). Diformazan formation was determined by measuring absorption at 570 nm with a microplate reader uQuant (BioTEK, USA).
Curve fitting and calculation of the halfmaximal inhibitory concentration (IC50 value) were done using GraphPad Prism 7 (GraphPad Software Inc., USA). Briefly, individual concentration-effect curves were generated by fitting the logarithm of the tested compound concentration versus corresponding normalized percent of cell viability values using nonlinear regression.
Cells morphology was investigated using phase-contrast microscopy (Olympus CKX41SF, Japan). For light microscopy images an Olympus SP-500UZ (Indonesia) camera was used.
Erythrocytes haemolysis. Erythrocytes isolated from the heparinized rat blood, were incubated at 37 °C with or without C60-Cis-Pt nanocomplex. Erythrocytes haemolysis was induced by addition of hydrochloric acid to the final concentration of 0.001 N [29]. Measurements of the haemolysis dynamics were carried out for 2 min with a 10 s interval on the spectrophotometer (Scinco, Germany) at X = 630 nm.
All experiments with animals in this study were performed according to the Bio-Ethics Committee of the abovementioned institution.
Statistical analysis was performed using two-way ANOVA followed by post Bonferroni tests. The IC50 value was represented as M ± SD of more than four independent experiments. A value of P < 0.05 was considered statistically significant.
Results and Discussion
Viability and morphology of НЕК293 cells. Cytotoxic activity of C60-Cis-Pt nanocomplex against HEK293 cells in Cis-Pt equivalent concentrations of 5-40 uM in comparison with the free drug was studied by MTT test at 24 h of incubation. The viability of cells incubated without additions of C60 fullerene, Cis-Pt, or C60-Cis-Pt nanocomplex was taken as 100% (control).
No effect of C60 fullerene used alone in the range of 2.8-16.6 uM concentrations,
equivalent to those in C60-Cis-Pt nanocomplex, on HEK293 cells viability during the incubation period was detected (data are not presented). The calculated IC50 values for C60 fullerene (IC50 = 530 pM) (Table 1) action on HEK293 cells showed that it is a low toxic compound. These results are in a good agreement with the literature data. Thus, the toxic effect of pristine C60 fullerene against normal baby hamster kidney BHK-21 cells were observed only at high 440 pM concentration [30].
We have detected the cytotoxic effect of Cis-Pt against HEK293 cells at 40 pM concentration. Under the action of the drug at this concentration cell viability at 24 h was reduced by 28% compared to the control (Fig. 1). The decrease of HEK293 cells viability by 43% compared to the control under the action of 50 pM Cis-Pt was also demonstrated in [31]. The calculated IC50 value for Cis-Pt was shown to be 75 pM (Table 1).
Table 1. IC50 values for C60 fullerene, Cis-Pt and C60-Cis-Pt nanocomplex in НЕК293 cells
(M ± m, n = 6)
Compounds IC50 (дМ), 24 h
C60 fullerene 530 ± 43
Cis-Pt 75 ± 5.6
C60-Cis-Pt nanocomplex 90 ± 6.8*
Note: * — P < 0.05 in comparison with Cis-Pt.
Fig. 1. Viability of HEK294 cells treated with free Cis-Pt or C60-Cis-Pt nanocomplex in Cis-Pt equivalent concentrations at 24 h
of incubation (M ± m, n = 6): * P < 0.05 in comparison with control (untreated cells); # P < 0.05 in comparison with Cis-Pt
With the action of 5 pM C60-Cis-Pt nanocomplex the viability of HEK293 cells was increased by 20% compared to the control (Fig. 1), probably due to the initial adaptive response to the compound. C60-Cis-Pt nanocomplex at 40 pM Cis-Pt equivalent concentration inhibited HEK293 viability, but the toxic effect appeared to be only 18% as compared to control (Fig. 1). The calculated value of IC50 for C60-Cis-Pt nanocomplex was higher (90 pM) than that for free Cis-Pt (Table 1) confirming the decreased cytotoxicity of Cis-Pt against non-tumor cells at complexation with C60 fullerene.
Morphological studies showed that untreated (control) HEK293 cells formed elongated epithelioid structures and dense monolayer in some areas, a large number of intercellular contacts were observed. The cytotoxic effect of 40 pM Cis-Pt on HEK293 cells was evidenced by morphological changes of cells (Fig. 2, Table 2).
Most of HEK293 cells treated with 40 pM Cis-Pt were characterized by atypical morphology and smaller size (Table 2). No evident effect of C60-Cis-Pt nanocomplex in equivalent 40 pM Cis-Pt concentration on cells morphology was detected.
Therefore, the toxic effect of Cis-Pt on normal cells at complexation with C60 fullerene was reduced. The protective effects of C60 fullerene against the toxic effects of Cis-Pt may be due to the antioxidant properties of the carbon nanostructure [32, 33, 34]. We have previously shown that C60 fullerene prevented ROS production in thymocytes and prevented the decrease of thymocytes viability induced by Cis-Pt [35, 36].
Erythrocytes haemolysis. The use of platinum-based drugs in chemotherapy is limited due to its high haematotoxicity, that is the cause of haemolytic anemia and bone marrow disease [37]. So the search for erythrocyte protection pathways against drug damage is of current interest.
The effect of Cis-Pt nanocomplex at the level of cells plasma membrane was estimated by the dynamics of erythrocytes haemolysis, which reflects the dynamics of erythrocyte plasma membrane destruction and the release of haemoglobin into the environment. Erythrocytes haemolysis of the control (untreated red blood cells) was accelerated at 40 s after the treatment with haemolytic and reached the maximum at 60 s, the number of haemolyzed cells was 30 ± 2% (Fig. 3).
Haemolysis of erythrocytes treated with 40 pM Cis-Pt slightly slowed, however, the
and C60-Cis-Pt
Control
Cis-Pt
C60-Cis-Pt nanocomplex
Fig. 2. Microphotographs of HEK293 cells incubated for 24 h in the presence of free 40 pM Cis-Pt or C60-Cis-Pt nanocomplex
(phase-contrast microscopy, x 400)
Table 2. Morphological features in HEK293 cells at 24 h after action 40 pM Cis-Pt or C60-Cis-Pt nanocomplex (M ± m, n = 6)
Compounds Changes
Atypical cell morphology Smaller cell size Roud shape and nonadherent pattern
Cis-Pt ++++ ++ ++++
C60-Cis-Pt nanocom-plex + - +
Note: + few, ++ moderate, +++ severe, ++++ many.
Fig. 3. Erythrocytes haemolysis after action 40 pM Cis-Pt or C60-Cis-Pt nanocomplex (M ± m, n = 6): * P < 0.05 in comparison with control (untreated cells)
number of haemolyzed cells reached 55 ± 2% (Fig. 3), indicating on Cis-Pt-induced decrease of cells resistance to haemolysis. As it was shown in [38], Cis-Pt at 35 pM concentration caused morphological changes of human erythrocyte membrane by embedding into the inner layer of cells. The extranuclear mechanism of Cis-Pt cytotoxic action is associated with its high affinity to phosphatidylserine the inner monolayer of the erythrocyte plasma membrane [39].
C60 fullerene at 16.6 pM concentration equivalent to that in C60-Cis-Pt nanocomplex did not change parameters of erythrogram, indicating on the absence of haemolytic effect of carbon nanostructure (data are not presented). As it was shown by us previously [40, 41], the slight haemolytic activity of C60 fullerene was detected at concentrations > 50 pM.
No haemolytic effect was detected when erythrocytes were treated with C60-Cis-Pt nanocomplex at 40 pM Cis-Pt equivalent concentration. The parameters of erythrogramm were the same as in control (Fig. 3) indicating that C60 fullerene weakened Cis-Pt interaction with erythrocyte membrane and was able to increase the resistance of erythrocyte membrane to Cis-Pt induced haemolysis.
The data obtained showed that the cytotoxic effect of Cis-Pt against normal cells was prevented at complexation with C60 fullerene and that C60-Cis-Pt nanocomplex has a potential to be used for minimization of anticancer drug side effects.
This work was partially supported by the budget themes of Taras Shevchenko National University of Kyiv (State Registration Numbers 0119U100316 and 0119U100331).
The authors declare that they have no conflicts of interest.
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ЗАПОБ1ГАННЯ ТОКСИЧН1Й ДП ЦИСПЛАТИНУ ЩОДО НОРМАЛЬНИХ КЛ1ТИН ЗА КОМПЛЕКСОУТВОРЕННЯ 13 С60 ФУЛЕРЕНОМ
С. В. Прилуцька1,1.1. Гринюк1, T. Д. Скатерна2, Л. Б. Дробот2, М. С. Слободяник1, O. П. Матишевська2
1Ки!вський нащональний ушверситет iMeHi Тараса Шевченка, Украша 21нститут 6ioxiMiï iM. О. В. Палладша НАН Украши, Кшв
E-mail: [email protected]
Метою роботи було ощнити токсичшсть нековалентного нанокомплексу С60 фуле-рену з цисплатином (C60-Cis-Pt) щодо нор-мальних клiтин. Токсичнiсть C60-Cis-Pt нанокомплексу, порiвняно з в^ьним Cis-Pt, вивчали на клiтинах ембрюнально! нирки людини (HEK293), оцiнюючи ïx життездат-нiсть за допомогою МТТ-тесту та на еритро-цитах щура за !хньою стiйкiстю до кислотного гемолiзу. Було показано, що вiльний 40 мкМ Cis-Pt змшював морфологiю та знижував життездатнiсть кл^ин HEK293 на 28%, а також зб^ьшував кiлькiсть ге-молiзованиx еритроцийв на 25% порiвня-но з контролем. C60-Cis-Pt нанокомплекс за еквiвалентноï концентраци Cis-Pt не впливав на доойджуваш показники i не спричиняв цитотоксичних ефекйв. Запо-бiгання токсичнш дп Cis-Pt на нормальнi кл^ини за його комплексоутворення iз С60 фулереном вiдкривае перспективу викори-стання наноструктури як ефективного ци-топротектора i таргетного нойя у пухлинш клiтини.
Ключовi слова: С60 фулерен, цисплатин, нанокомплекс, НЕК293 клггини, цитотоксичнiсть, еритроцити, гемолiз.
ПРЕДОТВРАЩЕНИЕ ТОКСИЧЕСКОГО ДЕЙСТВИЯ ЦИСПЛАТИНА
НА НОРМАЛЬНЫЕ КЛЕТКИ ПУТЕМ КОМПЛЕКСООБРАЗОВАНИЯ С С60 ФУЛЛЕРЕНОМ
С. В. Прилуцкая1, И. И. Гринюк1, T. Д. Скатерна2, Л. Б. Дробот2, Н. С. Слободяник1, O. П. Матышевская2
1Киевский национальный университет имени Тараса Шевченко, Украина 2Институт биохимии им. А. В. Палладина НАН Украины, Киев
E-mail: [email protected]
Целью работы было оценить токсичность нековалентного нанокомплекса С60 фуллере-на с цисплатином (C60-Cis-Pt) относительно нормальных клеток. Токсичность C60-Cis-Pt нанокомплекса, в сравнении со свободным Cis-Pt, изучали путем оценки жизнеспособности клеток эмбриональной почки человека (HEK293) с помощью МТТ-теста и на эритроцитах крысы по их устойчивости к кислотному гемолизу. Было показано, что свободный 40 мкМ Cis-Pt вызывал морфологические изменения и снижение жизнеспособности клеток HEK293 на 28%, а также увеличение количества гемолизированных эритроцитов на 25% по сравнению с контролем. C60-Cis-Pt нанокомплекс в эквивалентной концентрации Cis-Pt не влиял на исследуемые показатели и не вызывал цитотоксических эффектов. Предотвращение токсического действия Cis-Pt на нормальные клетки при комплек-сообразовании с С60 фуллереном открывает перспективу применения наноструктуры как эффективного цитопротектора и таргетного носителя в опухолевые клетки.
Ключевые слова: С60 фуллерен, цисплатин, нанокомплекс, НЕК293 клетки, цитотоксич-ность, эритроциты, гемолиз.
