PACS numbers: 61. 46+w, 61.48+c, 61.48De, 87.15-v, 87.64-t

Impact of carbon nanomaterials on the formation of multicellular spheroids by tumor cells.

O.M.Yakymchuk1, O.M.Perepelytsina1, А.D.Rud2, I.M.Kirian2, M.V.Sydorenko1

1GO “Department for biotechnical problems of diagnostic Institute for problems of cryobiology and cryomedicine of NAS Ukraine”.2G.V. Kurdyumov Institute for Metal Physics of NAS Ukraine, Kiev, Ukraine.

Abstract This paper investigates the effect of different concentrations of nanostructured materials: fullerene-like (С60), onion-like carbon (OLC) and ultra dispersed diamonds (UDD) on the formation of multicellular spheroids (MS). Chemical composition and purity of nanomaterials is controlled by Fourier transform infrared spectroscopy (FT-IR). The strength and direction of the impact of nanomaterials on the cell population was assessed using microphotography of multicellular spheroids culture and Pearson's correlation coefficient. The results demonstrated that UDD and OLC reduced adhesion and cohesive ability of cells and stimulated generation of cell spheroids of ~ 3 ∙ 10 mm3 in significant amount. The fullerenes reduced in the main cell adhesion to substrate that led to formation of cell aggregates of ~ 5 ∙ 10-3 mm3. The results could be useful for achievement of the directed cell growth in three-dimensional culture.Keywords: ultra dispersed diamonds, fullerenes, onion-like carbon, multicellular spheroids.

IntroductionRecently, in literature biological and medical properties of nanostructured materials has attracted abundant interest

[1-5]. Creation of biologically similar nanodevices with essential qualities requires multidisciplinary research efforts that can be translated directly into new technologies and products for biomedical applications. The aim of our study is to compare the cytological influence of nanoscaled materials with different structure. In the study we used tree types of nanomaterials (NM): ultra dispersed diamonds (UDD), C60 fullerenes and onion-like carbon (OLC).

In our study we suggest that biological property of nanostructured materials is determined by their spatial structure, the presence of functional groups and the ability to aggregate. In this context we compared biological influence of NM with different physic-chemical characteristics.

Ultra dispersed diamonds [2] have unique physical and chemical properties: highly developed surface of particles (270 - 280 m2 /g), a large number of charged groups (carboxyl, carbonyl, hydroxyl, ester), carbon fragments and trace metals [6, 7]. In the literature, there are contradictory data about toxicity of the UDD and their possible applications in biology and medicine. For example, in [8-10] have been shown that intramuscular and subcutaneous injections of sterile nanodiamonds didn`t have destructive influence on the cells in the field of localization. Moreover, intravenous injection of UDD didn’t have significant changes in the nature and state of heart and other organs of experimental animals. Another authors reported about evidently anticancer activity and anti-radiation effect of nanodiamonds [11, 12]. However, there are some works in which a destructive effect of UDD on cells was observed [13]. Therefore, the reseach of the interaction of nanodiamonds with biological objects and compare their effects with biological properties of onion-like carbons and fullerenes is an important task for physics and biology research.

The fullerene molecule has a closed symmetric shape [14] which is completely unique for natural/biological molecules. The most stable and common compound of this class is the C 60 molecule. Diameter of C60 molecules is 0,708 nm is comparable with a diameter of α- helix polypeptide or steroid molecule [15]. The unusual structure of fullerenes causes their special properties, which allow us to consider these compounds as promising for use in biological objects [16, 17]. For example, fullerenes C60 are insoluble in polar solvents such as water or methanol, but effectively extracted into toluene and benzene. Due to the hydrophobicity the molecule of C60 can be integrated into biological membranes and localized near phospholipids and proteins of membrane. Using lipids membranes (phosphatidylcholine: cholesterol) C60

fullerenes can penetrate the lipid matrix and change membrane permeability [18, 19]. In addition, fullerenes exhibit renewable capacity, easily connecting to their molecules from one to six electrons, so they can act as antioxidants. Redox potential of fullerenes allows their using as adsorbents of free radicals including reactive oxygen ones [20], hyperproduction which causes much pathology.

A relatively new class of carbon compounds is onion-like carbon (OLC) – spherical nanoparticles composed of enclosed concentric graphite shells. They have different properties from those of other carbon nanostructures, such as graphite, nanotubes and nanodiamonds.. Several recent publications have shown that OLC can be used as components of nanocapsules for drug delivery. In this case the outer layers of graphite material provided protection of drugs and can serve as a basis for immobilization of necessary functional and receptor groups [5].

Thus, ultra diamonds, fullerenes C60 and onions are characterized by unique chemical, physical and spatial properties, which open prospects for their utilization in biological research.

Material and methods Getting carbon nanomaterials Onion-like carbon. Onion-like carbon materials were produced by high-voltage electrical discharge technique in the Institute of Pulse Research and Engineering of NAS of Ukraine. This method is based on exposure by periodic short current pulses of hydrocarbons – source of carbon [21]. During the processing, a destruction of hydrocarbon molecules into carbon clusters occurs what results in synthesis of different types of CNM in a process of their ultrafast cooling. After series of electrical discharges, the working liquid with colloidal solution of carbon nanoparticles is decanted from the

explosive chamber and centrifugated during 0,3-2 hours. The obtained material is exsiccated at the sparing temperatures (up to 500 K) with a purpose to form a dry powder.Fullerens C60 were produced in the Physical-Technical Institute of the Rub of the RAS by the traditional arc evaporation of graphite electrodes technology with following extraction by toluene in a Soxhlet apparatus.Ultra dispersed diamonds were produced by detonation of carbon-containing explosive by the commercial enterprise “Sinta”, Ukraine. Individual UDD particles have characteristic sizes of 4-6 nm, sizes of primary aggregates are ~ 20-30 nm with the specific surface area of ~550 m2/g.

Fullerene C60 UDD agglomerate Onion-like carbon

[22, 23]

[1, 24] [21]

Table1 The comparative table of the tree dimensional structure of the UDD, onion-like carbon and fullerenes C60 according to the literature.

Cell line Cell line of Human Caucasian breast adenocarcinoma (MCF-7) was used as experimental model of cell microaggregates. The line was purchased from Bank of cell lines and tissues of animals Kavetsky` Institute of experimental pathology, oncology and radiobiology NAS Ukraine. The cells were handled in standard tissue culture conditions (100% humidity, 5% CO2 in air; 37°C) under laboratory containment level 2.

FT-IR spectroscopy The chemical composition of the obtained nanomaterials were investigated by FT-IR spectroscopy. IR spectra were determined by FTS 7000e Varian FTIR spectrometer. Samples for analysis were prepared by grinding in a mill of a mixture of ~ 1 mg of nanomaterials and 150 mg of spectrally pure KBr. Samples were prepared by using a press with a pressure force of 3,0 – 3,5 ∙ 103 kg/cm2. The samples were dehydrated by heating at a temperature of 600 oC for 60 min. Pre-shot spectra of KBr were preliminary obtained, then they are subtracted from the spectra of the samples. All spectra of UDD, OLC and fullerenes C60 were recorded by the same technology (Table 1). All conditions of handling, sample preparation and compositions were similar for the all used carbon nanomaterials.

Preparation of stable suspensions of nanomaterials The colloidal suspension of NM was carried out in two stages. In the first stage, carbon nanomaterials have been subjected to ultrasonic treatment in Phosphate Saline Buffer (PBS) using an ultrasonic disperser UZDN - 2T. For all types of nanomaterials processing modes were I = 10 mA , R = 22 kHz, duration - 10 min. In the second stage, the resulting hydrosol was dispersed by the centrifugation at room temperature. The process includes several centrifugation cycles. So, the hydrophilic dissolving NM fraction was selected this way. Before adding to the suspension cultured cells, solutions of NM were sterilized by boiling during 30 min.

3 - D cell model system Spheroid (3-D) model system of MCF-7 cells was cultured by well-established method which was described in [26]. Briefly, cell suspension were counted using trypan blue and planted an equal number of cells (5 ∙ 10 4

cells/ml). The 3-D cell culture was maintained in RPMI medium with 10% FBS in standard conditions (100% humidity, 5% CO2 in air, 37°C). Generation of multicellular spheroids (MS) was performed by technology which was developed in our laboratory. It means cultivation of cells for 24 hours in 24 - well plates coated with 1% agar in culture medium with 0,24% of carboxy-methyl-cellulose. For investigation the dependence of the size and number of MS on the concentration and type of NM, MS was generated in the presence of various concentrations of UDD, OLC and C60. Before MS generation to the cell cultures NM solution in PBS was added to culture to the end concentration of 12,5; 25; 50; 100; 150 and 200 μg/ml. Further cultivation was conducted during 48 hours at a constant rotation of plates. At the next stage micro photo images were taken by “dark field” method. A total was done 120 images. Then, the volume of all MS, which were on the files, was calculated. We used the formula of Rolf B'yerkviha: V = 0,4 * a * b, where a and b - the geometric sizes of the spheroids [27].

Statistical analysis, Pearson's coefficient For statistical analysis all cell aggregates were sorted into groups according to size from 1 ∙ 10-4 mm3 to 1 ∙ 10-2 mm3 with step in 1 ∙ 10-3 mm3 and the estimated number on MS in each group and median of MS volume for each group. For micro statistic assay normally distributed random variables we used the Student` coefficient for small population. To determine the relationship between concentration of NM and response of experimental biological systems Pearson`s correlation coefficient was used. It was calculated for the median size of the cell spheroid culture and the concentration of nanostructured materials (UDD, C60, OLC) by formula [28]. The correlation coefficient ranges from – 1(inverse relation) to +1(direct relation). Thus for independent parameters it is 0, and for closely related approaches to the module unit.

Results Chemical properties of nanomaterials

The chemical structure and purity of nanostructured materials which will be used in experiments was determined by Infrared Fourier Transform Spectroscopy (FTIR). Results were compared with literature data of FTIR spectra (Table 2.)

Chemical groups The wavelength of the absorption peaks, cm-1

O-H free, O-H 3573, very weak-NH2, = NH, >NH 3373 - 3432, very broadC – H (symmetric) 3006 – 2854, very weakC – H (asymmetric) 2851, very weakR2 - C2O3 1717, very broad-NH2, >C=C< 1619 - 1654, very broad5-, 6- гранні циклічні вуглеводні 1176 - 1452, broadC-OH, absorbed CO, CO2 1102,2 , 1247, mediumAromatic anhydrites 1800 - 1000, very broadC-H aromatic 792 - 920, weakRadial movement of carbon atoms 527- 576, very broad

Table 2 Compliance of FTIR analysis-based absorption peaks to types of chemical bonds in carbon nanomaterials [1, 29, 30].

The results of FTIR assay were demonstrated on Fig.1-3. The main features of UDD and OLC (Fig. 1, 2) correspond to the groups O−H stretched (3600 - 3200 cm- 1) and the amplitude of vibrations at 1619 cm-1 decreased. The amino group (−NH2) showed a strong signal at 3431 and 3411 cm -1. Also in these samples low vibration peaks of methyl (at 2924 - 2913 cm-1), methylene (at 2845 - 2851 cm-1) and aromatic C − H groups (792 cm-1) are observed. More that that, there are quite intense peaks of carbohydrate linkages in the region of 1717 cm -1, it indicated the presence of carboxyl groups. There was also a shift and decrease in intensity of the C−O covalent band vibrations, which were fixed at 1619 and 1653 cm-1. Meanwhile, some carbonyl groups can emerge from adsorbed CO and CO2 (at 1102 and 1247 cm-1, respectively) in UDD specters. The appearance of vibration at 1359 cm -1 is evidence of presence in UDD of cyclic carbon groups.

Figure1 FTIR spectra of UDD.

Figure 2 FTIR spectra of onion-like carbon.

Figure 3 FTIR spectra of fullerenes C60.

Strong signals in the area of wave number 1182-1427 cm-1 were observed for fullerenes C60, which can be attributed to presence of a large fraction of carbon cyclic pentagonal compounds (Fig. 3). In addition, according to the obtained spectra fullerenes C60 have a small amount of absorbed or covalent chemical additives. Instead, there is a predictable strong peak of initial radial movement of carbon atoms (at 527 - 576 cm-1) and aromatic C - H groups’ vibration from 792 to 920 cm-1. Our spectra were similar to those presented by other authors [1, 24, 25, 29, 30], which allowed us to conclude that compliance with the chemical structure of the samples, their proper degree of purification are relevant for use in further biological studies.

Effect of nanostructured materials for formation of multicellular spheroidsFor the first step we generated multicell spheroids in the presence of the same concentrations of the UDD, OLC

and C60. Then we determined the dependence of the size and number of multicellular aggregates on the concentration and type of nanostructured materials. All cultures of cell spheroids were photographed and processed in the same way (Fig.4).

Figure 4 Images of MS culture in presence of NM in concentration 25 and 200µg/ml

In particular, all cell aggregates were sorted into groups according to volume values: from 10-4 mm3 to 10-2 mm3

with step of 10-3 mm3. After that, number of cell aggregates in each group were estimated (Fig.5), medians of MS volume in each group were calculated and compared with concentration of nanomaterials (Fig.6).

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S

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Figure 5 Dependence of the MS number and volume from co-cultured concentration and sort of NM: (a) - 25 µg/ml of NM, (b) - 200 µg/ml of NM.

So, it was found that the presence of OLC in culture medium leads to decreasing the size of cell spheroids and increasing their number. The same trend was observed in the culture with UDD. So at concentration 20 µg/ml of NM, the largest number of MS in 5 fields of view was observed in culture with UDD and OLC - 47 and 48 aggregates respectively. In the presence of C60 were formed 27 MS, in control culture - only 22 MS (Fig.5a). Along with increasing concentrations of NM this trend continued. And at 200 µg/ml of NM the number of MS in culture with UDD and OLC in 3 times is more than in control samples and reached 59 and 60 MS (Fig.5b). Increasing concentration of C60 to 200 µg/ml didn’t correlate with increasing number of MS (Fig.5b). Perhaps, OLC and UDD acted as modifiers of adhesive properties of surface in tumor cells culture, reducing not only the cells adhesion to the substrate, but also cohesion between the cells themselves. This assumption is supported by decreasing an average volume of cell spheroids with increasing concentration of OLC and UDD (Fig.6). Perhaps, that why we observed a very small number of cell spheroids larger than 1 ∙ 10-3 mm3 in co-culture with UDD and OLC (Fig.5), even compared with the control. The opposite results we obtained if tumor cells were co-cultured with fullerene C60. After increasing concentration of C60 we observed the increaseing of the number of spheroids and average volume larger than 3 ∙ 10-3 mm3, of cell spheroids (Fig.5, 6). Thus, the effect of C60 was manifested as increasing of the number of cell spheroids, so also in stimulating cohesive in cell populations. The same takes place as with small concentrations of C60 (12,5 – 50 µg/ml) - average volume of cell spheroids was ranged from 3 to 4,5 ∙ 10 -3 mm3. Increasing concentrations of C60 to 100 - 200 µg/ml resulted in increasing average volume of cell spheroids to 5 ∙ 10-3 mm3

(Fig. 6).

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Figure 6 The dependence of the median of MS volume and nanostructured materials concentration. OLC – onion-like carbon, UDD – ultra dispersed diamonds, C60 – fullerenes C60.

At the last stepage, the correlation between the number and size of cell aggregates and the concentration of experimental nanomaterials was analyzed using Pearson's coefficient. The obtained results are shown in Table 3. We demonstrate a positive correlation between spheroids sizes and concentration of C60 which is confirmed by the statistical analysis: the Pearson` coefficient for C60 is 0,5. At the same time, inverse correlation between the sizes of cell aggregates and concentration is observed for OLC and UDD – 0,84 and 0.74, respectively (Table 3). These effects are also confirmed by the ratio of the cell aggregates size in control samples and after incubation with NM. In Table 3 the results in the smallest and the largest concentration of NM are shown.

Co-cultured nanomaterials,

Median of V,Е-03 мм3, 25,0 µg/ml Vеx/Vc, %

Median of V,Е-03 мм3,

200,0 µg/ml Vеx/Vc, %

Pirson` coefficient correlation

Control 2,65±0,06    

Fullerens, С60 3,54±0,27 133,6 4,79±0,22 180,8 0,50

OLC 3,17±0,29 119,6 1,42±0,10 53,6 -0,85

UDD 2,27±0,08 85,7 1,47±0,09 55,5 -0,74

Table 3 The determination the interrelation between concentration of nanostructured materials and sizes of MS by the Pearson correlation coefficient.

The main goal of our study was to find connection between micro-structure of nanomaterials and cell ability to form multicellular aggregates. The results obtained suggested that NM have strong influence on cell motility, migration to suspension and formation of multicellular aggregates. Other authors [31, 32] define the predominant relation between cell/substrate and cell-cell interaction, substrate flexibility, rigidity and cell motility, proliferation and migration. Our results have support this hypothesis. According to our data, we assume that UDD, OLC, C60 can decrease cell adhesion to substrate by changing conformation of protein complex focal adhesive contacts such as integrins, talin, α-actin, filamin, vinculin, junction filaments [33-35]. This effect can be realized via chemical covalent bonds with charged residues of the proteins or by the physical presence of carbon aggregates in the conformation structure of adhesion proteins complex. At the same time impact of UDD and OLC was differs from that of C 60. Our results demonstrated that UDD and OLC modulated as cell-substrate as cell-cell adhesive ability. C60 influenced only on adhesion to substrate. Reason for this phenomenon may be different size, structure and chemical properties of nanoaggregates UDD, OLC and C60. Undoubtedly it led to change in cells proliferation, differentiation, gene expression and cell survival.

Interestingly that in nanobiotechnology there are study of usage nanomaterials as artificial extracellular matrix, scaffold and substrate [36, 37]. Most of them deal with monolayer cell culture. In this time our investigation focuses on tree-dimentional cell culture that needs specific condition for caring on and assay. That`s why elaborated responses reported in our work contribute suspension cell growth and may be useful for developing tissue engineering in 3-D culture.

Conclusion

Thus, as a result of our work it was found that carbon nanomaterials - onion-like carbon, ultra dispersed diamonds and fullerenes C60, impact on cells adhesion and cohesion in culture and formation of multicellular aggregates. NM can determine the further development of the cell population. OLC and UDD reduce cell adhesion to the substrate and cohesion between cells and stimulate increasing the number of small cell spheroids. Fullerenes C60, opposite, create conditions for formation cell aggregates large, up to 5 ∙ 10-3 mm3. These conclusions are supported by statistical calculations of Pearson` correlation coefficient, which is -0,84 for OLC, -0,74 for UDD and +0,5 for C 60. Obviously, through modification of the physicochemical properties of nanomaterials, we have possibility to influence on cell growth, depending on the tasks of biotechnology. The significance of our dates is in discover of fundamental properties of NM which could be use as efficient platform for various biomedical application such as stimulation directed growth of a cell population (tissue engineering) and cell spheroid large volume (angiogenesis) or, conversely, to stimulate apoptotic processes, reducing the survival of the cell population.

References

[1] A. M. Schrand, S.A. Hens Ciftan, O. A. Shenderova, Critical Reviews in Solid State and Materials Sciences, 34:18 (2009).[2] A. Krueger, J. Stegk, Y. Liang, L. Lu, and G. Jarre, Langmuir, 24:4200 (2008). [3] A.W. Jensen, S.R. Wilson, D.I. Schuster, Bioorg. Med. Chem, 4:767 (1996).[4] S. Yamago, H. Tokuyama, E. Nakamura, et al, Chem. Biol.,2:385 (1995).[5] Dr. De-en Jiang, Guang Feng and Peter T. Cummings, J. Chem. Theory Comput.,8:1058 (2012). [6] S. Osswald, G. Yushin, V. Mochalin, et al, J. Am. Chem. Soc., 128:11635 (2006). [7] V. Pichot, M. Comet, E. Fousson, C. Baras, A. Senger, et al, Diamond Relat. Mater., 17:13(2008).[8] M. Baidakova and A. Vul, J. Physics D-Appl. Physics, 40:6300 (2007).[9] A.S. Chiganov, Phys. Solid State, 46:620 (2004). [10] V.Y. Dolmatov, M.V. Veretennikova, V.A. Marchukov, Phys. Solid State, 46:611 (2004). [11] I.S. Larionova, I.N. Molostov, L.S. Kulagina, Method of purification of synthetic ultradispersed diamonds (RU Patent(1999) 2168462).[12] E.V. Pavlov and J.A. Skrjabin, Method for removal of impurities of non-diamond carbon and device for its realization (RU Patent (1994) 2019502). [13] A.P. Kaplun, D.A.Bezrulov, et al., Nanotechnology-medicine,10, № 2:3 (2007).[14] Aleksandr V. Eletslii, Physics-Uspechi, 38, №9:935 (1995).[15] A.W. Jensen, S.R. Wilson, D.I. Schuster, Bioorg. Med. Chem.,4:767 (1996).[16] R.S. Ruoff, D.S. Tse, M. Malhotra, D.C. Lorents, J. Phys. Chem., 97:3379 (1993). [17] N. Sivaraman, R. Dhamodaran, I. Kaliappan, et al, J. Org. Chem., 57:6077 (1992).[18] S.V. Prylutska, U. Ritter, P. Scharff, et al., Mol. Crys. Liq. Crys., 468:617 (2007).[19] N. Levi, R. Hantagan, M. Lively, et al, J. of Nanobiotechnology,4:14 (2006).[20] A.Djordjevic, G.Bogdanovic , S.Dobric., J BUON.,11, № 4:391 (2006).[21] Rud А.D., Kuskova N.I., Ivaschuk L.I., et al., Nanotechnology and nanomaterials, 5:99 (2011). [22] A. Goel, Jack B. Howard, John B. Vander Sande, Carbon , 42:1907 (2004).[23] Nicole Levi, Roy R Hantgan, et al, Journal of Nanobiotechnology, 4:14 (2006).[24] V.Yu Dolmatov, Russ Chem Rev, 76, № 4:375 (2007).[25] P.Ganesh, P.Kent, and V.Mochalin, Journal of Applied Physics, 110:73506 (2011).[26] L.V.Garmanchuk, E.M.Perepelytsina, et al., Cytology and Genetics, 43, №5:305 (2009) [27] R. Bjerkvig, Spheroid culture in cancer research, (CRC Press, London, 1992), p.335.[28] Под ред. Р.А. Шмойловой, Общая теория статистики (Москва: Финансы и Статистика: 2002).[29] S. Iglesiai-Groth, F. Cataldo, A. Manchado, Mon. Not. R. Astron. Soc., 4:1 (2011).[30] Chia-Chen Li, Chun-Lung Huang, Colloids and Surfaces A: Physicochem. Eng. Aspects, 353: 52 (2010).[31] Dikta Raz-Ben, Hanoch Daniel Wagner, Adv. Material, 18: 1537 (2006).[32] Robert J. Pelham, Jr, Yu-Li Wang, Proc. Natl. Sci. USA, 94: 13661 (1997).[33] Valerie Petit, Jean-Paul Thiery, Biology of the cell, 92: 477 (2000).[34] Michaele A. Wozniak, K. Modzelewska, L. Kwong et al., Biochimica et biophysica Acta, 1692: 103 (2004).[35] Yu Q., Yang Q., Cell Biology international, 33: 78 (2009).[36] Ziegler K.J., et al, Trends in Biotechnol, 23, №9: 440 (2005). [37] Ajayan P.M., Zhou O.Z., Carbon, 3: 389 (2005).