In Vitro Study of Influence of Au Nanoparticles on HT29 and SPEV Cell Lines

In Vitro Study of Influence of Au Nanoparticles on HT29 and SPEV Cell Lines Cell culture models are excellent tools for potential toxicity of nanoparticles and fundamental investigations in cancer research. Thus, information about AuNP potential toxicity and effects on human health is necessary for the use of nanomaterials in clinical settings. The aim of our research is to examine the effects of AuNPs on the epithelial origin cell lines: continuous and oncogenic. Embryonic porcine kidney epithelial inoculated (SPEV) cell line and colorectal carcinoma cell line (HT29) were used. In the test cultures, the cell proliferation, necrosis/apoptosis, and multicellular spheroids generation were evaluated. We demonstrated that AuNP concentrations of 6–12 μg/ml reduced the proliferation of SPEV and HT29 cells and increased the cell number at early and late stages of apoptosis and necrosis. It was shown that small concentrations of AuNPs (1–3 μg/ml) stimulate multicellular spheroid formation by HT29 and SPEV cells. However, higher AuNP concentrations (6–12 μg/ml) had both cytotoxic and anti-cohesive effects on cell in suspension. The large sensitiveness to the action of AuNPs was shown by the line of HT29 (6 μg/ml) as compared to the SPEV cells (12 μg/ml). This experimental study of the effect of AuNPs on SPEV and HT29 cell lines will justify their further application in AuNP-mediated anticancer treatment. PACS: 61.46+w, 61.48+c, 61.48De, 87.15−v, 87.64−t Background etc. [12–14]. Thus, information about their potential tox- Production and investigation of the gold nanoparticles icity and effects on human health is necessary for the use (AuNPs) have not only high potential for wide thera- of nanomaterials in clinical settings. peutic application of gold [1, 2] but also made them suit- Currently, despite the great success in the targeted able for specific biomedical applications such as target therapy, the problem of selective delivery of AuNPs in therapies [3, 4]. Recent reports have demonstrated that the target tissue remains unsolved. Some studies have the use of AuNPs provides an opportunity for novel an- noted different rates of uptake of NPs by epithelial cells titumor therapies with a reduced risk for development of of different origin [15, 16]. Yet, investigations to explain resistance. Thus, several studies have proven nanoparti- this phenomenon are lacking even though they may help cle antitumor activity against breast, liver, gastric, colon, to achieve tissue-selective targeting of AuNPs. Anatom- lung cancer [5, 6]. ical or physiological differences between different epithe- It is known that nanoparticles (NPs) can modulate cell lia could explain differences in AuNPs uptake and fate, induce or prevent mutations, initiate cell–cell com- transport rates. In particular, the rate of uptake may be munication, and modulate cell structure [7, 8]. In addition, influenced by the plasma membrane properties of the AuNPs have advantages over other metal NPs due to their cells and the binding of nanoparticles to cell surface biocompatibility and antitumor activity [8–12]. The cyto- glycoproteins and proteoglycans, as well as the cells’ toxic and genotoxic effects of AuNPs are associated with capacity for vesicular transport [17]. Thus, taking into their shape, size, charge, concentration, interaction time, account the impossibility of exclusively selective inter- action of nanoparticles with target cells, the comparative study of the features of their interaction with normal * Correspondence: volkovana781@gmail.com and oncogenic cells in order to avoid undesirable conse- Institute for Problems of Cryobiology and Cryomedicine, National Academy of Science of Ukraine, Pereyaslavskaya str., 23, Kharkiv 61015, Ukraine quences of cancer therapy is topical [8–10]. Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 2 of 9 Although in vivo models are valuable for evaluating Culture of HT29 Cells biological toxicity of nanoparticles, cell culture models HT29 cells were cultured in plastic flasks (Nunc, are highly useful for preclinical physiological and toxico- Denmark) in RPMI-1640 medium (Sigma, USA) with logical studies. Currently, cell cultures are widely used in 10% FCS (v/v) (HyClone, USA) supplemented with various fields of biology, medicine, veterinary medicine, 2 mM L-glutamine (Sigma, USA) and 40 mg/ml genta- and biotechnology. The use of cell cultures allows to mycin (Sigma, USA) under standard conditions (5% explore biological processes that are difficult and some- CO , 95% humidity) [21]. The optimal cell density was 4 2 times impossible, to study at the level of organisms. An 0.5–4.0 × 10 cells /cm . The cells were kindly provided important role of cell cultures is played in biotechnology to us by the Bank of Cell Lines from Human and Animal in the production of many vaccines, test systems, and Tissue of RE Kavetsky Institute of Experimental Path- biologically active substances. Cell cultures are used to ology, Oncology and Radiobiology NAS of Ukraine. diagnose diseases of various etiologies, as test objects when testing new pharmacological, therapeutic, and Manipulations with AuNPs cosmetic agents, as well as food additives [18]. AuNPs were kindly provided by the Institute of Bio- In this work on cell culture models, we tried to examine chemistry and Physiology of Plants and Microorganisms, the features of effects of AuNPs of the epithelial cells of Russian Academy of Sciences. AuNPs were synthesized continuous and oncogenic cell line origin. Monolayer cul- by citrate method [22]. The average size of nanoparticles ture of epithelial cells SPEV (embryonic porcine kidney was 15 nm. The initial concentration of gold was 57 μg/ epithelial inoculated line) and HT-29 (colon carcinoma ml. The results of dark field electron microscopy, image cell line) cells can be considered as a model of normal and of 15 nm AuNPs, and extinction spectra of 15 nm cancer epithelial tissues when anti-tumor therapy with AuNPs (b) are shown in Fig. 1 (Fig. 1a; note––diagram AuNPs is applied. Several traditional cytotoxicity assays, of the size distribution) [23]. AuNPs were introduced in including the adhesion, proliferation, necrosis/apoptosis, cells by passive diffusion at 37 °C. The investigated con- and multicellular spheroids were employed to validate the centrations were 1, 3, 6, and 12 μg/ml. Cells without cell cytotoxicity of AuNPs. AuNPs under the same conditions were taken as control ones. Methods Culture of SPEV Cells Adhesion and Proliferation Cells SPEV cells were cultured in plastic flasks in DMEM Morphofunctional state of cell cultures was judged by (Sigma, USA) with 5% FCS (v/v) (HyClone, USA) sup- adhesive properties and proliferative activity. The adhe- plemented with penicillin/streptomycin (PAA, Austria) sive properties of SPEV and HT29 cells were visually and amphotericin B (5 μg/ml) (5% CO , 95% humidity) evaluated using an inverted microscope; the numbers of as reported by [19]. Seeding concentration was 0.5– adhered and flattened cells were counted 30, 60, 120, 4 2 2×10 cells/cm . Culture medium was replaced every 180, and 1440 min after culturing beginning. 2 days. Cells were passaged at 100% confluence [20]. The proliferation dynamics of SPEV and HT29 cells SPEV cell line grew and preserved initial morphological was studied for 1–4 days. To examine the increment of structure of monolayer during serial passages without cell number in the studied cultures to the investigation evidence of cell degeneration in culture. terms, they were enzymatically (1:1 (0.25% trypsin Fig. 1 Results of dark field electron microscopy, a image of 15 nm AuNPs (note––diagram of the size distribution), b extinction spectra of 15 nm AuNPs Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 3 of 9 solution: EDTA, PAA)) detached from plastic and the adhesion terminated, cells became flattened and gained number of cells was counted. The total number of appropriate morphology. Adhesive properties of SPEV cultured cells was counted by the traditional method in cells are presented in Fig. 2. Goryaev’s chamber. After 1 h cultivation of SPEV cells with AuNPs at 1, 3, and 6 μg/ml, the number of adhered cells was lower ver- Apoptotic/Necrotic Processes sus the control value. The percentage of flattened cells Apoptotic and necrotic processes in SPEV and HT29 in samples with AuNPs for these concentrations did not cells exposed to AuNPs were investigated in 4 days with significantly differ from the control. Adhesion was slo- Annexin-V (BD, USA), 7-Amino-Actinomycin (7AAD) wed down after 1 h incubation with AuNPs at 12 μg/ml. (BD) dyes using a FACS Calibur Becton-Dickinson. The The number of adhered cells per squared centimeter results were analyzed with WinMDI v.2.8 software. was reduced by 1.8 times versus the control. This ten- dency in adhesion persisted for all the test periods. After Multicellular Spheroids 24 h of observation, the number of adhered cells was The multicellular spheroids (MSs) were generated to es- lower versus the control by 1.3 times. At the same time, timate the in vitro impact of AuNPs on migration and incubation of AuNPs at small concentrations (1 and aggregation potential of investigated cells. Spheroid (3- 3 μg/ml with tumor cells (HT29) had no significant ef- D) model system of SPEV and HT29 cells was cultured fect on the amount of adhesive cells. Increasing of AuNP by the conventional method, which was reported by [24] concentration to 6 and 12 μg/ml lead to decreasing the and modified in our laboratory [25]. Briefly, cell suspen- number of tumor cells in the adhesive fraction in 1.16 sion were counted using trypan blue and equal numbers and 1.28 times, respectively, (Fig. 3). The obtained data 4 2 of cells (5 × 10 cells/cm ) were planted in full supple- can be influenced by several processes. The one is the mented culture medium. MS generation was achieved by cytostatic/cytotoxic effect of AuNPs on the adhesion handling cell culture with 0.24% carboxy-methyl- fraction of both tumor and embryonic cell lines, which cellulose (CMC) in 24-well plates coated with 1% agar leads to cell death, transition to apoptosis, or necrosis. with rotation (80 rpm) for 24 h. After that, 3-D cell cul- The other process is the reduction of cell adhesion, ture was maintained in standard conditions. To investi- under the influence of AuNPs and transfer of cells into gate dependence of the size and number of MSs on the the suspension fraction. Notably, both processes can be AuNP concentration, MSs were generated in the pres- realized simultaneously, and each one can contribute to ence of AuNPs. Further cultivation was conducted for the decrease in the number of living cells in the adhe- 48 h with constant rotation of plates. At the next stage, sion fraction. micro photo images were made by dark field method with a Carl Zeiss Stemi 2000 microscope. MS morph- Effect of AuNPs on Proliferation of SPEV and HT-29 Cells ology was studied with the help of Axio Vision Release The effect of AuNPs within the concentration range of 4.7 program (Zeiss). This program allows measuring the 1–12 μg/ml on proliferative processes in SPEV cell geometric dimensions of cell aggregates. Then, the vol- culture was studied (Fig. 4). On days 2–4 of culturing ume of all MSs, which were in the files, was calculated. with AuNPs at 1, 3, and 6 μg/ml, the cell number did It was used with the following formula: V = 0.4 × a × b , not significantly differ from the control. On day 4 of where a and b––geometric diameters of MSs [24]. For statistical analysis, all cell aggregates were grouped by −4 3 −2 3 size from 1 × 10 mm to 1 × 10 mm with increment −3 3 of 1 × 10 mm . The MS number and median of MS volumes were estimated for each group. Statistical Analysis A single-factor analysis of variance and Student’s t test were used for statistical processing of the data with the software package Statistica 8. The significance threshold was 0.05. The results are presented as means and stand- ard errors (M ± SE). Results Effect of AuNPs on Adhesion of SPEV and HT-29 Cells Fig. 2 Dynamics of adhesion of SPEV cells after exposure of AuNPs, Cell adhesion is an indicator of functional state of cells, *p ≤ 0.05 is significant versus with the control and it is necessary for further growth of culture. When Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 4 of 9 Fig. 5 Proliferation of HT29 cells after exposure of AuNPs, *p ≤ 0.05 Fig. 3 Dynamics of adhesion of HT29 cells after exposure of AuNPs, is significant versus with the control *p ≤ 0.05 is significant versus with the control culturing with AuNPs at 3 and 6 μg/ml, this index number was lower than the control in 1.33 and 1.44 decreased by 1.15 and 1.23 times, respectively, as com- times, respectively. pared with the control. Reduction in the cell number by 1.5 times (days 2 and 3) and by 1.15 times on day 4 of Effect of AuNPs on Apoptotic/Necrotic Processes in SPEV culturing with AuNPs at 12 μg/ml was observed in SPEV and HT-29 Cells culture versus the control. Thus, the AuNP concentra- SPEV and HT-29 cells in the presence of AuNPs were tion, 12 μg/ml, slowed down cell growth within the ob- cultured for 4 days under the standard conditions. The served time period. culturing of SPEV and HT29 cells with AuNPs at 1 and The effect of AuNPs at concentrations from 1 to 3 μg/ml and the indexes of apoptotic/necrotic processes 12 μg/ml on the number of HT 29 cells in a monolayer did not significantly differ from the control (Tables 1 culture is shown in Fig. 5. During the first 3 days of in- and 2). cubation, the number of cells in the control and in the Culturing with AuNPs at 6–12 μg/ml increased the presence of AuNPs was not statistically different. On the percentage of Annexin V+/7AAD+, Annexin V−/7AAD 4th day of cultivation, it was noted a dose-dependent de- +, and Annexin V+/7AAD-cells as well as reduced the creasing of the number of cells in 2D culture. So, after percentage of alive cells. The number of Annexin V + + 4 days of cultivation, for low concentrations of AuNPs /7AAD SPEV cells was higher than the control value (1 and 3 μg/ml), the number of HT 29 cells is not sig- by 7.8 ± 0.7% (p ≤ 0.05) with 12 μg/ml of AuNPs. The + + nificantly different in comparison with control. But at number of Annexin V /7AAD HT 29 cells was higher higher AuNP concentrations (6–12 μg/ml), HT29 cell than the control value by 3.2 ± 0.4% (p ≤ 0.05) with 6 μg/ml of AuNPs and by 4.8 ± 0.6% (p ≤ 0.05) with 12 μg/ml of AuNPs. Effect of AuNPs on Generation of Multicellular Spheroids from SPEV and HT29 Cells To determine the dependence of size and number of multicellular spheroids (MSs) on AuNP concentration, MSs were generated at various concentrations of AuNPs during 48 h. Our data demonstrated the variety ability of HT29 and SPEV cells to form multicellular spheroids under the same conditions of the microenvironment (Figs. 6 and 7). So, if the control samples of HT29 cells for 48 h −3 3 formed spheroids in average volume 5.19 × 10 mm , the average volume spheroid of SPEV cells was 0.79 × 10 −5 3 mm . At the same time, the influence of AuNPs on Fig. 4 Proliferation of SPEV cells after exposure of AuNPs, *p ≤ 0.05 the HT29 and SPEV cells had the same trend. The pres- is significant versus with the control ence of AuNPs in the cell microenvironment stimulated Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 5 of 9 Table 1 Cytofluorimetric analysis of SPEV cells after 4 days culturing with AuNPs, staining with Annexin V and 7AAD + − + + − − − + Sample/region Annexin V /7AAD Annexin V /7AAD Annexin V /7AAD Annexin V /7AAD Control 3.4 ± 0.4 4.5 ± 0.7 91.6 ± 1.0 0.5 ± 0.1 SPEV + Au_NPs_1 μg/ml 3.5 ± 0.6 4.4 ± 0.8 91.4 ± 1.2 0.7 ± 0.2 SPEV + Au_NPs_3 μg/ml 3.7 ± 0.5 3.6 ± 0.8 91.0 ± 1.1 1.7 ± 0.1 SPEV + Au_NPs_6 μg/ml 3.9 ± 0.6 10.1 ± 0.5* 84.6 ± 1.2* 1.4 ± 0.2* SPEV + Au_NPs_12 μg/ml 5.1 ± 0.8* 12.5 ± 0.7* 81.1 ± 1.2* 1.3 ± 0.1* Note*p ≤ 0.05 is significant versus with the control the formation of multicellular spheroids in both cultures. which reduced cell numbers in the adhesive fraction and Thus, when the concentration of AuNPs was 1 and the number of MSs in suspension. Previously, the authors 3 μg/ml volume of MSs for SPEV increased by 9.7 and reported that carbon nanoparticles reduce the adhesion of 7.4 times, respectively, compared with the control (Fig. cells to the substrate, stimulate cell transfer into the sus- 6), the same AuNP concentrations also stimulated an in- pension, and leaded to formation of multicellular spher- creasing volume of MSs for HT29 by 1.4 and 1.2 times, oids [25, 26]. In the literature, there are data about AuNP respectively (Fig. 7). ability to break the structure of actin/myosin microfila- Further increasing of AuNP concentration leaded to ments and decrease cell proliferation, adhesion, and differ- decreasing average volume of MSs in both cultures. The entiation [27]. Our data confirmed this assumption. elevation in AuNP concentration from 1 to 12 μg/ml de- −3 3 creased the volume of HT29 MSs from 7.18 × 10 mm Discussion −3 to 4.24 × 10 , in 1.69 times, according to the control. We evaluated the effects of AuNPs on proliferation, ne- As for SPEV, when the concentration of AuNPs was crosis/apoptosis, and formation of multicellular spher- increased from 1 to 12 μg/ml, the volume of MSs oids of the epithelial cells continuous and oncogenic cell −5 3 decreased from 7.69 to 4.58 × 10 mm , in 1.68 times, line origin. It was shown that the AuNPs at 6–12 μg/ml according to control. However, increasing AuNP con- reduced the number of SPEV and HT29 cells and in- centration coincidences with reduction in volume of creased the cell number at early and late stages of apop- MSs and correlated with increases in the number of tosis and necrosis. The small concentrations of AuNPs spheroids in culture of HT29 cells (Figs. 6 and 7). The stimulate formation of multicellular spheroids by HT29 number of HT29 MSs increased from 3 to 10 per field and SPEV cells. However, higher AuNP concentrations of view at AuNP concentration from 1 to 12 μg/ml. At had both cytotoxic and anti-cohesive effects on cell in the same time, the number of SPEV MSs decreased from suspension. The large sensitiveness to the action of 32 to 19, respectively. AuNPs was shown by the line of HT29 (6 μg/ml) as The obtained data (Figs. 6 and 7) demonstrate that compared to the SPEV cells (12 μg/ml.) AuNPs are capable of influencing cohesive interactions The effects of AuNPs on cellular morphology and in the cell-to-cell system. Our data show that small con- cytoskeleton have only recently received more attention, centrations of AuNPs (1–3 μg/ml) stimulated the forma- and the underlying mechanism and forthcoming conse- tion of multicellular spheroids of both embryonic and quences have not been investigated in depth [28–30]. In tumor cells. However, higher AuNP concentrations (6– this regard, it is important for all novel AuNP types to 12 μg/ml) had both cytotoxic and anti-cohesive effects evaluate their endocytic uptake pathway and intracellular on cell in suspension. This process contributed to for- localization as a function of time. For different types of mation of a larger number of HT29 MSs with the de- AuNPs, the effects have been described to be dependent creased average volume. As for SPEV, the high on intracellular AuNP concentration and to be transient, concentration of AuNPs may have a cytostatic effect where after recurrent cell divisions, the intracellular Table 2 Cytofluorimetric analysis of HT29 cells after 4 days culturing with AuNPs, staining with Annexin V and 7AAD + − + + − − − + Sample/region Annexin V /7AAD Annexin V /7AAD Annexin V /7AAD Annexin V /7AAD Control 3.1 ± 0.5 4.6 ± 0.6 91.8 ± 0.9 0.5 ± 0.2 HT29 + Au_NPs_1 μg/ml 2.9 ± 0.7 3.8 ± 0.5 92.8 ± 1.1 0.5 ± 0.3 HT29 + Au_NPs_3 μg/ml 3.0 ± 0.6 3.9 ± 0.7 92.5 ± 1.2 0.6 ± 0.2 HT29 + Au_NPs_6 μg/ml 4.1 ± 0.5* 7.7 ± 0.4* 87.3 ± 1.5* 0.9 ± 0.3* HT29 + Au_NPs_12 μg/ml 5.3 ± 0.9* 9.2 ± 0.8* 84.0 ± 1.3* 1.2 ± 0.2* Note *p ≤ 0.05 is significant versus with the control Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 6 of 9 Fig. 6 Number and volume of MS cells SPEV after incubation with AuNPs, #p ≤ 0.01 (for number MSs); **p ≤ 0.01 (for volume of MSs) AuNP concentrations decrease exponentially and the ef- cell types, such as A549 human carcinoma lung cells fects are no longer observed. Also, possible endosomal [32]. AuNPs have also been described to have a escape of the AuNPs must be assessed. As cytoskeleton concentration-dependent effect on the actin fibrils of defects have been described to be clearly dependent on human dermal fibroblasts [33, 34]. Mironava et al. [35, AuNP concentrations, a wide concentration range of 36] further showed the cytoskeleton filaments to be dis- particles should be tested in order to try and assess the rupted as a function of AuNP exposure time, concentra- maximal cellular loading capacity without any effects. tion, and size of the NPs although actin or β-tubulin Furthermore, as the cytoskeleton is also involved in protein expression levels were not affected. many intracellular signaling pathways, it remains to be The cell type used is also of great importance as differ- investigated whether the AuNPs induced cytoskeletal ent cell types, even when closely related, can react quite disruption leads to secondary effects [31]. differently for the same type of nanomaterials [37, 38]. As NPs have certain physical dimensions, the intracel- Preferably, those cell types which are most involved in lular volume they occupy can lead to alterations in cellu- the (future) biomedical applications of the NPs should lar morphology or affect the structure of the cellular be tested (e.g., epithelial, endothelial cells), or multiple cytoskeleton network [28, 29, 31]. The later effects can cells which are derived from the different germ layers. also be due to the high demands of the NP pose on the When investigating cytotoxic effects, the use of cancer cellular endocytic manner. AuNPs have been described cell types should be minimized, as these can lead to ab- to have a profound effect on the morphology of several errant results [39]. Cancer cells have several specific Fig. 7 Number and volume of MS cells HT29 after incubation with AuNPs. #p ≤ 0.01 (for number MSs); **p ≤ 0.01 (for volume of MSs) Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 7 of 9 characteristics and altered intracellular signaling path- be realized until the aggregates forming central necrosis, ways which are destined to upregulate proliferation and due to limited cell mass growth or spontaneous differenti- maintain cell viability, which will make them less prone ation of embryonic cells. to some NP-mediated cytotoxic effects. In literature, there is information about interaction In our opinion, binding of AuNPs to surface functional AuNPs with colon cancer cell line and embryonic cell groups (e.g., transmembrane proteins) of cells can be re- lines [46, 47]. According these data, exposure to even versible or irreversible, resulting in temporary or perman- very low concentrations of AuNPs may have a damaging ent structural injuries [40, 41]. Potential implications of effect on the Human Embryonic Neural Precursor Cells changes in biomechanical properties (e.g., hardness and and HT29 by stressing cell proliferation, differentiation, elasticity), adhesiveness, and surface electrical properties and apoptotic cell death. of cells are perceivable. Thus, changes in hardness or elas- There are published data that the effect of AuNPs is ticity are likely to influence the surface structural flexibil- based on G0/G1 phase accumulation, S and G2/M phase ity, production of mechanical energy for cell division, and depletion, as well as on reduced ATP levels in human cell motility. As for adhesiveness, the cell microenviron- oral squamous carcinoma cells (HSC-3) [48]. Cell cycle ment is normally composed of extracellular matrix with regulation may be addressed by violation of focal con- specific molecules that allow cells to adhere to their sur- tacts of cells with substrate and cell transfer to sus- roundings [42]. Surface charge undoubtedly plays an im- pended fraction in 2D culture and inhibition cell-to-cell portant role in interactions between cells and their contacts in gap junction in 3D culture [48–50]. Due to surroundings. nanosize of AuNPs (near 15 nm), it cannot be centers of The other authors also reported of the NPs are prefer- cohesion for cells. At the same time, intercalation of entially localized in mitochondria and cause oxidative AuNPs into cell membrane [51], influence on cell mem- stress as well as potentiate structural damage [40]. A re- brane AuNP zeta potential [32], and influence on forma- cent article by Pan et al. describes that 1.4 nm AuNPs tion cell-to-cell/cell-to-surface contacts obviously can induce necrosis via oxidative stress and mitochondrial trigger mechanism for necrosis/apoptosis, cytotoxic ef- damage in Hela cells [43]. Accumulation of nanoparti- fect, and cell cycle arrest. Violation of focal contacts of cles in cell medium upon biodegradation is unsafe be- cells with substrate and cell transfer to suspended frac- cause it may disrupt organelles and even cause genetic tion is a way of cell cycle regulation [48, 49]. Small con- mutations. centrations of AuNPs exerted no statistically significant Changes occurring in cells during apoptosis are similar cytotoxic effect on cells. However, higher AuNP concen- for most of cell types. In apoptotic cells, there are trations had both cytotoxic and anti-cohesive effects on changes of lipid composition of plasma membrane: cell in suspension. This process contributed to formation phosphatidyl serine transfers from cytoplasmic part of of a larger number of MSs with the decreased average bilayer to outer side, causing caspase cascade activation, volume. We suppose that AuNP wedge into cohesive chromatin condensation, and disorder of electron trans- contacts of cells and compromise them. Thus, our ex- port chain in mitochondria and eventually arresting ATP periments on the effects of AuNPs on SPEV and HT29 synthesis. Programmed cell death can be triggered by cell lines support their further application in develop- receptor-mediated physiological stimuli resulted from ment of AuNP-mediated cancer therapies. genetic disorders, exposure to chemical or physical fac- Although future studies will be necessary to confirm tors as well as by other changes in cells. We observed anti-cancer effects on the in vivo animal studies. Never- this effect is with 6–12 μg/ml of AuNPs. theless, our deep conviction is that if we know the na- Multicellular aggregates (spheroids, embryoid body) ture of substance and its possible negative influence, we represent an intermittent level between monolayer grow- are able to avoid the detrimental effects of AuNP and ing cells and tissue culture. Spheroids are objective model use their positive biotechnological potential. Our investi- of the cell three-dimensional growth and organization, the gation could be applied quite reliably in effective mate- cell-to-cell interactions and influence of microenviron- rials for anti-cancer treatment context with maximum mental conditions, for example, AuNP concentration, on advantage for medicine. intensiveness of proliferation as well as on cell adhesive- ness and formation of microaggregates. MS formation is a Conclusions well-established culture method as for tumor as for em- Our results support the notion that AuNPs induce dose- bryonic cell lines [24, 44, 45]. In our work, formation and dependent cytotoxicity in SPEV and HT29 cells. Further- growth of spheroids is achieved by adding CMC as part of more, this report for the first time demonstrates that artificial extracellular matrix and surface coating by 1% 15 nm AuNPs in concentrations of 6–12 μg/ml reduced agar which inhibited cell adhesion to surface and stimu- the proliferation of SPEV and HT29 cells and increased lated cell aggregation. At these conditions, MS culture can the cell number at early and late stages of apoptosis and Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 8 of 9 necrosis. Also, it was shown that small concentrations of 9. Xiang-Yu S, Liu P-D, Wu H, Ning G (2014) Enhancement of radiosensitization by metal-based nanoparticles in cancer radiation therapy. Cancer Biol Med AuNPs (1–3 μg/ml) stimulate formation of multicellular 11(2):86–91 spheroids. However, higher AuNP concentrations had 10. 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In Vitro Study of Influence of Au Nanoparticles on HT29 and SPEV Cell Lines

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Materials Science; Nanotechnology; Nanotechnology and Microengineering; Nanoscale Science and Technology; Nanochemistry; Molecular Medicine
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Abstract

Cell culture models are excellent tools for potential toxicity of nanoparticles and fundamental investigations in cancer research. Thus, information about AuNP potential toxicity and effects on human health is necessary for the use of nanomaterials in clinical settings. The aim of our research is to examine the effects of AuNPs on the epithelial origin cell lines: continuous and oncogenic. Embryonic porcine kidney epithelial inoculated (SPEV) cell line and colorectal carcinoma cell line (HT29) were used. In the test cultures, the cell proliferation, necrosis/apoptosis, and multicellular spheroids generation were evaluated. We demonstrated that AuNP concentrations of 6–12 μg/ml reduced the proliferation of SPEV and HT29 cells and increased the cell number at early and late stages of apoptosis and necrosis. It was shown that small concentrations of AuNPs (1–3 μg/ml) stimulate multicellular spheroid formation by HT29 and SPEV cells. However, higher AuNP concentrations (6–12 μg/ml) had both cytotoxic and anti-cohesive effects on cell in suspension. The large sensitiveness to the action of AuNPs was shown by the line of HT29 (6 μg/ml) as compared to the SPEV cells (12 μg/ml). This experimental study of the effect of AuNPs on SPEV and HT29 cell lines will justify their further application in AuNP-mediated anticancer treatment. PACS: 61.46+w, 61.48+c, 61.48De, 87.15−v, 87.64−t Background etc. [12–14]. Thus, information about their potential tox- Production and investigation of the gold nanoparticles icity and effects on human health is necessary for the use (AuNPs) have not only high potential for wide thera- of nanomaterials in clinical settings. peutic application of gold [1, 2] but also made them suit- Currently, despite the great success in the targeted able for specific biomedical applications such as target therapy, the problem of selective delivery of AuNPs in therapies [3, 4]. Recent reports have demonstrated that the target tissue remains unsolved. Some studies have the use of AuNPs provides an opportunity for novel an- noted different rates of uptake of NPs by epithelial cells titumor therapies with a reduced risk for development of of different origin [15, 16]. Yet, investigations to explain resistance. Thus, several studies have proven nanoparti- this phenomenon are lacking even though they may help cle antitumor activity against breast, liver, gastric, colon, to achieve tissue-selective targeting of AuNPs. Anatom- lung cancer [5, 6]. ical or physiological differences between different epithe- It is known that nanoparticles (NPs) can modulate cell lia could explain differences in AuNPs uptake and fate, induce or prevent mutations, initiate cell–cell com- transport rates. In particular, the rate of uptake may be munication, and modulate cell structure [7, 8]. In addition, influenced by the plasma membrane properties of the AuNPs have advantages over other metal NPs due to their cells and the binding of nanoparticles to cell surface biocompatibility and antitumor activity [8–12]. The cyto- glycoproteins and proteoglycans, as well as the cells’ toxic and genotoxic effects of AuNPs are associated with capacity for vesicular transport [17]. Thus, taking into their shape, size, charge, concentration, interaction time, account the impossibility of exclusively selective inter- action of nanoparticles with target cells, the comparative study of the features of their interaction with normal * Correspondence: volkovana781@gmail.com and oncogenic cells in order to avoid undesirable conse- Institute for Problems of Cryobiology and Cryomedicine, National Academy of Science of Ukraine, Pereyaslavskaya str., 23, Kharkiv 61015, Ukraine quences of cancer therapy is topical [8–10]. Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 2 of 9 Although in vivo models are valuable for evaluating Culture of HT29 Cells biological toxicity of nanoparticles, cell culture models HT29 cells were cultured in plastic flasks (Nunc, are highly useful for preclinical physiological and toxico- Denmark) in RPMI-1640 medium (Sigma, USA) with logical studies. Currently, cell cultures are widely used in 10% FCS (v/v) (HyClone, USA) supplemented with various fields of biology, medicine, veterinary medicine, 2 mM L-glutamine (Sigma, USA) and 40 mg/ml genta- and biotechnology. The use of cell cultures allows to mycin (Sigma, USA) under standard conditions (5% explore biological processes that are difficult and some- CO , 95% humidity) [21]. The optimal cell density was 4 2 times impossible, to study at the level of organisms. An 0.5–4.0 × 10 cells /cm . The cells were kindly provided important role of cell cultures is played in biotechnology to us by the Bank of Cell Lines from Human and Animal in the production of many vaccines, test systems, and Tissue of RE Kavetsky Institute of Experimental Path- biologically active substances. Cell cultures are used to ology, Oncology and Radiobiology NAS of Ukraine. diagnose diseases of various etiologies, as test objects when testing new pharmacological, therapeutic, and Manipulations with AuNPs cosmetic agents, as well as food additives [18]. AuNPs were kindly provided by the Institute of Bio- In this work on cell culture models, we tried to examine chemistry and Physiology of Plants and Microorganisms, the features of effects of AuNPs of the epithelial cells of Russian Academy of Sciences. AuNPs were synthesized continuous and oncogenic cell line origin. Monolayer cul- by citrate method [22]. The average size of nanoparticles ture of epithelial cells SPEV (embryonic porcine kidney was 15 nm. The initial concentration of gold was 57 μg/ epithelial inoculated line) and HT-29 (colon carcinoma ml. The results of dark field electron microscopy, image cell line) cells can be considered as a model of normal and of 15 nm AuNPs, and extinction spectra of 15 nm cancer epithelial tissues when anti-tumor therapy with AuNPs (b) are shown in Fig. 1 (Fig. 1a; note––diagram AuNPs is applied. Several traditional cytotoxicity assays, of the size distribution) [23]. AuNPs were introduced in including the adhesion, proliferation, necrosis/apoptosis, cells by passive diffusion at 37 °C. The investigated con- and multicellular spheroids were employed to validate the centrations were 1, 3, 6, and 12 μg/ml. Cells without cell cytotoxicity of AuNPs. AuNPs under the same conditions were taken as control ones. Methods Culture of SPEV Cells Adhesion and Proliferation Cells SPEV cells were cultured in plastic flasks in DMEM Morphofunctional state of cell cultures was judged by (Sigma, USA) with 5% FCS (v/v) (HyClone, USA) sup- adhesive properties and proliferative activity. The adhe- plemented with penicillin/streptomycin (PAA, Austria) sive properties of SPEV and HT29 cells were visually and amphotericin B (5 μg/ml) (5% CO , 95% humidity) evaluated using an inverted microscope; the numbers of as reported by [19]. Seeding concentration was 0.5– adhered and flattened cells were counted 30, 60, 120, 4 2 2×10 cells/cm . Culture medium was replaced every 180, and 1440 min after culturing beginning. 2 days. Cells were passaged at 100% confluence [20]. The proliferation dynamics of SPEV and HT29 cells SPEV cell line grew and preserved initial morphological was studied for 1–4 days. To examine the increment of structure of monolayer during serial passages without cell number in the studied cultures to the investigation evidence of cell degeneration in culture. terms, they were enzymatically (1:1 (0.25% trypsin Fig. 1 Results of dark field electron microscopy, a image of 15 nm AuNPs (note––diagram of the size distribution), b extinction spectra of 15 nm AuNPs Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 3 of 9 solution: EDTA, PAA)) detached from plastic and the adhesion terminated, cells became flattened and gained number of cells was counted. The total number of appropriate morphology. Adhesive properties of SPEV cultured cells was counted by the traditional method in cells are presented in Fig. 2. Goryaev’s chamber. After 1 h cultivation of SPEV cells with AuNPs at 1, 3, and 6 μg/ml, the number of adhered cells was lower ver- Apoptotic/Necrotic Processes sus the control value. The percentage of flattened cells Apoptotic and necrotic processes in SPEV and HT29 in samples with AuNPs for these concentrations did not cells exposed to AuNPs were investigated in 4 days with significantly differ from the control. Adhesion was slo- Annexin-V (BD, USA), 7-Amino-Actinomycin (7AAD) wed down after 1 h incubation with AuNPs at 12 μg/ml. (BD) dyes using a FACS Calibur Becton-Dickinson. The The number of adhered cells per squared centimeter results were analyzed with WinMDI v.2.8 software. was reduced by 1.8 times versus the control. This ten- dency in adhesion persisted for all the test periods. After Multicellular Spheroids 24 h of observation, the number of adhered cells was The multicellular spheroids (MSs) were generated to es- lower versus the control by 1.3 times. At the same time, timate the in vitro impact of AuNPs on migration and incubation of AuNPs at small concentrations (1 and aggregation potential of investigated cells. Spheroid (3- 3 μg/ml with tumor cells (HT29) had no significant ef- D) model system of SPEV and HT29 cells was cultured fect on the amount of adhesive cells. Increasing of AuNP by the conventional method, which was reported by [24] concentration to 6 and 12 μg/ml lead to decreasing the and modified in our laboratory [25]. Briefly, cell suspen- number of tumor cells in the adhesive fraction in 1.16 sion were counted using trypan blue and equal numbers and 1.28 times, respectively, (Fig. 3). The obtained data 4 2 of cells (5 × 10 cells/cm ) were planted in full supple- can be influenced by several processes. The one is the mented culture medium. MS generation was achieved by cytostatic/cytotoxic effect of AuNPs on the adhesion handling cell culture with 0.24% carboxy-methyl- fraction of both tumor and embryonic cell lines, which cellulose (CMC) in 24-well plates coated with 1% agar leads to cell death, transition to apoptosis, or necrosis. with rotation (80 rpm) for 24 h. After that, 3-D cell cul- The other process is the reduction of cell adhesion, ture was maintained in standard conditions. To investi- under the influence of AuNPs and transfer of cells into gate dependence of the size and number of MSs on the the suspension fraction. Notably, both processes can be AuNP concentration, MSs were generated in the pres- realized simultaneously, and each one can contribute to ence of AuNPs. Further cultivation was conducted for the decrease in the number of living cells in the adhe- 48 h with constant rotation of plates. At the next stage, sion fraction. micro photo images were made by dark field method with a Carl Zeiss Stemi 2000 microscope. MS morph- Effect of AuNPs on Proliferation of SPEV and HT-29 Cells ology was studied with the help of Axio Vision Release The effect of AuNPs within the concentration range of 4.7 program (Zeiss). This program allows measuring the 1–12 μg/ml on proliferative processes in SPEV cell geometric dimensions of cell aggregates. Then, the vol- culture was studied (Fig. 4). On days 2–4 of culturing ume of all MSs, which were in the files, was calculated. with AuNPs at 1, 3, and 6 μg/ml, the cell number did It was used with the following formula: V = 0.4 × a × b , not significantly differ from the control. On day 4 of where a and b––geometric diameters of MSs [24]. For statistical analysis, all cell aggregates were grouped by −4 3 −2 3 size from 1 × 10 mm to 1 × 10 mm with increment −3 3 of 1 × 10 mm . The MS number and median of MS volumes were estimated for each group. Statistical Analysis A single-factor analysis of variance and Student’s t test were used for statistical processing of the data with the software package Statistica 8. The significance threshold was 0.05. The results are presented as means and stand- ard errors (M ± SE). Results Effect of AuNPs on Adhesion of SPEV and HT-29 Cells Fig. 2 Dynamics of adhesion of SPEV cells after exposure of AuNPs, Cell adhesion is an indicator of functional state of cells, *p ≤ 0.05 is significant versus with the control and it is necessary for further growth of culture. When Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 4 of 9 Fig. 5 Proliferation of HT29 cells after exposure of AuNPs, *p ≤ 0.05 Fig. 3 Dynamics of adhesion of HT29 cells after exposure of AuNPs, is significant versus with the control *p ≤ 0.05 is significant versus with the control culturing with AuNPs at 3 and 6 μg/ml, this index number was lower than the control in 1.33 and 1.44 decreased by 1.15 and 1.23 times, respectively, as com- times, respectively. pared with the control. Reduction in the cell number by 1.5 times (days 2 and 3) and by 1.15 times on day 4 of Effect of AuNPs on Apoptotic/Necrotic Processes in SPEV culturing with AuNPs at 12 μg/ml was observed in SPEV and HT-29 Cells culture versus the control. Thus, the AuNP concentra- SPEV and HT-29 cells in the presence of AuNPs were tion, 12 μg/ml, slowed down cell growth within the ob- cultured for 4 days under the standard conditions. The served time period. culturing of SPEV and HT29 cells with AuNPs at 1 and The effect of AuNPs at concentrations from 1 to 3 μg/ml and the indexes of apoptotic/necrotic processes 12 μg/ml on the number of HT 29 cells in a monolayer did not significantly differ from the control (Tables 1 culture is shown in Fig. 5. During the first 3 days of in- and 2). cubation, the number of cells in the control and in the Culturing with AuNPs at 6–12 μg/ml increased the presence of AuNPs was not statistically different. On the percentage of Annexin V+/7AAD+, Annexin V−/7AAD 4th day of cultivation, it was noted a dose-dependent de- +, and Annexin V+/7AAD-cells as well as reduced the creasing of the number of cells in 2D culture. So, after percentage of alive cells. The number of Annexin V + + 4 days of cultivation, for low concentrations of AuNPs /7AAD SPEV cells was higher than the control value (1 and 3 μg/ml), the number of HT 29 cells is not sig- by 7.8 ± 0.7% (p ≤ 0.05) with 12 μg/ml of AuNPs. The + + nificantly different in comparison with control. But at number of Annexin V /7AAD HT 29 cells was higher higher AuNP concentrations (6–12 μg/ml), HT29 cell than the control value by 3.2 ± 0.4% (p ≤ 0.05) with 6 μg/ml of AuNPs and by 4.8 ± 0.6% (p ≤ 0.05) with 12 μg/ml of AuNPs. Effect of AuNPs on Generation of Multicellular Spheroids from SPEV and HT29 Cells To determine the dependence of size and number of multicellular spheroids (MSs) on AuNP concentration, MSs were generated at various concentrations of AuNPs during 48 h. Our data demonstrated the variety ability of HT29 and SPEV cells to form multicellular spheroids under the same conditions of the microenvironment (Figs. 6 and 7). So, if the control samples of HT29 cells for 48 h −3 3 formed spheroids in average volume 5.19 × 10 mm , the average volume spheroid of SPEV cells was 0.79 × 10 −5 3 mm . At the same time, the influence of AuNPs on Fig. 4 Proliferation of SPEV cells after exposure of AuNPs, *p ≤ 0.05 the HT29 and SPEV cells had the same trend. The pres- is significant versus with the control ence of AuNPs in the cell microenvironment stimulated Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 5 of 9 Table 1 Cytofluorimetric analysis of SPEV cells after 4 days culturing with AuNPs, staining with Annexin V and 7AAD + − + + − − − + Sample/region Annexin V /7AAD Annexin V /7AAD Annexin V /7AAD Annexin V /7AAD Control 3.4 ± 0.4 4.5 ± 0.7 91.6 ± 1.0 0.5 ± 0.1 SPEV + Au_NPs_1 μg/ml 3.5 ± 0.6 4.4 ± 0.8 91.4 ± 1.2 0.7 ± 0.2 SPEV + Au_NPs_3 μg/ml 3.7 ± 0.5 3.6 ± 0.8 91.0 ± 1.1 1.7 ± 0.1 SPEV + Au_NPs_6 μg/ml 3.9 ± 0.6 10.1 ± 0.5* 84.6 ± 1.2* 1.4 ± 0.2* SPEV + Au_NPs_12 μg/ml 5.1 ± 0.8* 12.5 ± 0.7* 81.1 ± 1.2* 1.3 ± 0.1* Note*p ≤ 0.05 is significant versus with the control the formation of multicellular spheroids in both cultures. which reduced cell numbers in the adhesive fraction and Thus, when the concentration of AuNPs was 1 and the number of MSs in suspension. Previously, the authors 3 μg/ml volume of MSs for SPEV increased by 9.7 and reported that carbon nanoparticles reduce the adhesion of 7.4 times, respectively, compared with the control (Fig. cells to the substrate, stimulate cell transfer into the sus- 6), the same AuNP concentrations also stimulated an in- pension, and leaded to formation of multicellular spher- creasing volume of MSs for HT29 by 1.4 and 1.2 times, oids [25, 26]. In the literature, there are data about AuNP respectively (Fig. 7). ability to break the structure of actin/myosin microfila- Further increasing of AuNP concentration leaded to ments and decrease cell proliferation, adhesion, and differ- decreasing average volume of MSs in both cultures. The entiation [27]. Our data confirmed this assumption. elevation in AuNP concentration from 1 to 12 μg/ml de- −3 3 creased the volume of HT29 MSs from 7.18 × 10 mm Discussion −3 to 4.24 × 10 , in 1.69 times, according to the control. We evaluated the effects of AuNPs on proliferation, ne- As for SPEV, when the concentration of AuNPs was crosis/apoptosis, and formation of multicellular spher- increased from 1 to 12 μg/ml, the volume of MSs oids of the epithelial cells continuous and oncogenic cell −5 3 decreased from 7.69 to 4.58 × 10 mm , in 1.68 times, line origin. It was shown that the AuNPs at 6–12 μg/ml according to control. However, increasing AuNP con- reduced the number of SPEV and HT29 cells and in- centration coincidences with reduction in volume of creased the cell number at early and late stages of apop- MSs and correlated with increases in the number of tosis and necrosis. The small concentrations of AuNPs spheroids in culture of HT29 cells (Figs. 6 and 7). The stimulate formation of multicellular spheroids by HT29 number of HT29 MSs increased from 3 to 10 per field and SPEV cells. However, higher AuNP concentrations of view at AuNP concentration from 1 to 12 μg/ml. At had both cytotoxic and anti-cohesive effects on cell in the same time, the number of SPEV MSs decreased from suspension. The large sensitiveness to the action of 32 to 19, respectively. AuNPs was shown by the line of HT29 (6 μg/ml) as The obtained data (Figs. 6 and 7) demonstrate that compared to the SPEV cells (12 μg/ml.) AuNPs are capable of influencing cohesive interactions The effects of AuNPs on cellular morphology and in the cell-to-cell system. Our data show that small con- cytoskeleton have only recently received more attention, centrations of AuNPs (1–3 μg/ml) stimulated the forma- and the underlying mechanism and forthcoming conse- tion of multicellular spheroids of both embryonic and quences have not been investigated in depth [28–30]. In tumor cells. However, higher AuNP concentrations (6– this regard, it is important for all novel AuNP types to 12 μg/ml) had both cytotoxic and anti-cohesive effects evaluate their endocytic uptake pathway and intracellular on cell in suspension. This process contributed to for- localization as a function of time. For different types of mation of a larger number of HT29 MSs with the de- AuNPs, the effects have been described to be dependent creased average volume. As for SPEV, the high on intracellular AuNP concentration and to be transient, concentration of AuNPs may have a cytostatic effect where after recurrent cell divisions, the intracellular Table 2 Cytofluorimetric analysis of HT29 cells after 4 days culturing with AuNPs, staining with Annexin V and 7AAD + − + + − − − + Sample/region Annexin V /7AAD Annexin V /7AAD Annexin V /7AAD Annexin V /7AAD Control 3.1 ± 0.5 4.6 ± 0.6 91.8 ± 0.9 0.5 ± 0.2 HT29 + Au_NPs_1 μg/ml 2.9 ± 0.7 3.8 ± 0.5 92.8 ± 1.1 0.5 ± 0.3 HT29 + Au_NPs_3 μg/ml 3.0 ± 0.6 3.9 ± 0.7 92.5 ± 1.2 0.6 ± 0.2 HT29 + Au_NPs_6 μg/ml 4.1 ± 0.5* 7.7 ± 0.4* 87.3 ± 1.5* 0.9 ± 0.3* HT29 + Au_NPs_12 μg/ml 5.3 ± 0.9* 9.2 ± 0.8* 84.0 ± 1.3* 1.2 ± 0.2* Note *p ≤ 0.05 is significant versus with the control Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 6 of 9 Fig. 6 Number and volume of MS cells SPEV after incubation with AuNPs, #p ≤ 0.01 (for number MSs); **p ≤ 0.01 (for volume of MSs) AuNP concentrations decrease exponentially and the ef- cell types, such as A549 human carcinoma lung cells fects are no longer observed. Also, possible endosomal [32]. AuNPs have also been described to have a escape of the AuNPs must be assessed. As cytoskeleton concentration-dependent effect on the actin fibrils of defects have been described to be clearly dependent on human dermal fibroblasts [33, 34]. Mironava et al. [35, AuNP concentrations, a wide concentration range of 36] further showed the cytoskeleton filaments to be dis- particles should be tested in order to try and assess the rupted as a function of AuNP exposure time, concentra- maximal cellular loading capacity without any effects. tion, and size of the NPs although actin or β-tubulin Furthermore, as the cytoskeleton is also involved in protein expression levels were not affected. many intracellular signaling pathways, it remains to be The cell type used is also of great importance as differ- investigated whether the AuNPs induced cytoskeletal ent cell types, even when closely related, can react quite disruption leads to secondary effects [31]. differently for the same type of nanomaterials [37, 38]. As NPs have certain physical dimensions, the intracel- Preferably, those cell types which are most involved in lular volume they occupy can lead to alterations in cellu- the (future) biomedical applications of the NPs should lar morphology or affect the structure of the cellular be tested (e.g., epithelial, endothelial cells), or multiple cytoskeleton network [28, 29, 31]. The later effects can cells which are derived from the different germ layers. also be due to the high demands of the NP pose on the When investigating cytotoxic effects, the use of cancer cellular endocytic manner. AuNPs have been described cell types should be minimized, as these can lead to ab- to have a profound effect on the morphology of several errant results [39]. Cancer cells have several specific Fig. 7 Number and volume of MS cells HT29 after incubation with AuNPs. #p ≤ 0.01 (for number MSs); **p ≤ 0.01 (for volume of MSs) Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 7 of 9 characteristics and altered intracellular signaling path- be realized until the aggregates forming central necrosis, ways which are destined to upregulate proliferation and due to limited cell mass growth or spontaneous differenti- maintain cell viability, which will make them less prone ation of embryonic cells. to some NP-mediated cytotoxic effects. In literature, there is information about interaction In our opinion, binding of AuNPs to surface functional AuNPs with colon cancer cell line and embryonic cell groups (e.g., transmembrane proteins) of cells can be re- lines [46, 47]. According these data, exposure to even versible or irreversible, resulting in temporary or perman- very low concentrations of AuNPs may have a damaging ent structural injuries [40, 41]. Potential implications of effect on the Human Embryonic Neural Precursor Cells changes in biomechanical properties (e.g., hardness and and HT29 by stressing cell proliferation, differentiation, elasticity), adhesiveness, and surface electrical properties and apoptotic cell death. of cells are perceivable. Thus, changes in hardness or elas- There are published data that the effect of AuNPs is ticity are likely to influence the surface structural flexibil- based on G0/G1 phase accumulation, S and G2/M phase ity, production of mechanical energy for cell division, and depletion, as well as on reduced ATP levels in human cell motility. As for adhesiveness, the cell microenviron- oral squamous carcinoma cells (HSC-3) [48]. Cell cycle ment is normally composed of extracellular matrix with regulation may be addressed by violation of focal con- specific molecules that allow cells to adhere to their sur- tacts of cells with substrate and cell transfer to sus- roundings [42]. Surface charge undoubtedly plays an im- pended fraction in 2D culture and inhibition cell-to-cell portant role in interactions between cells and their contacts in gap junction in 3D culture [48–50]. Due to surroundings. nanosize of AuNPs (near 15 nm), it cannot be centers of The other authors also reported of the NPs are prefer- cohesion for cells. At the same time, intercalation of entially localized in mitochondria and cause oxidative AuNPs into cell membrane [51], influence on cell mem- stress as well as potentiate structural damage [40]. A re- brane AuNP zeta potential [32], and influence on forma- cent article by Pan et al. describes that 1.4 nm AuNPs tion cell-to-cell/cell-to-surface contacts obviously can induce necrosis via oxidative stress and mitochondrial trigger mechanism for necrosis/apoptosis, cytotoxic ef- damage in Hela cells [43]. Accumulation of nanoparti- fect, and cell cycle arrest. Violation of focal contacts of cles in cell medium upon biodegradation is unsafe be- cells with substrate and cell transfer to suspended frac- cause it may disrupt organelles and even cause genetic tion is a way of cell cycle regulation [48, 49]. Small con- mutations. centrations of AuNPs exerted no statistically significant Changes occurring in cells during apoptosis are similar cytotoxic effect on cells. However, higher AuNP concen- for most of cell types. In apoptotic cells, there are trations had both cytotoxic and anti-cohesive effects on changes of lipid composition of plasma membrane: cell in suspension. This process contributed to formation phosphatidyl serine transfers from cytoplasmic part of of a larger number of MSs with the decreased average bilayer to outer side, causing caspase cascade activation, volume. We suppose that AuNP wedge into cohesive chromatin condensation, and disorder of electron trans- contacts of cells and compromise them. Thus, our ex- port chain in mitochondria and eventually arresting ATP periments on the effects of AuNPs on SPEV and HT29 synthesis. Programmed cell death can be triggered by cell lines support their further application in develop- receptor-mediated physiological stimuli resulted from ment of AuNP-mediated cancer therapies. genetic disorders, exposure to chemical or physical fac- Although future studies will be necessary to confirm tors as well as by other changes in cells. We observed anti-cancer effects on the in vivo animal studies. Never- this effect is with 6–12 μg/ml of AuNPs. theless, our deep conviction is that if we know the na- Multicellular aggregates (spheroids, embryoid body) ture of substance and its possible negative influence, we represent an intermittent level between monolayer grow- are able to avoid the detrimental effects of AuNP and ing cells and tissue culture. Spheroids are objective model use their positive biotechnological potential. Our investi- of the cell three-dimensional growth and organization, the gation could be applied quite reliably in effective mate- cell-to-cell interactions and influence of microenviron- rials for anti-cancer treatment context with maximum mental conditions, for example, AuNP concentration, on advantage for medicine. intensiveness of proliferation as well as on cell adhesive- ness and formation of microaggregates. MS formation is a Conclusions well-established culture method as for tumor as for em- Our results support the notion that AuNPs induce dose- bryonic cell lines [24, 44, 45]. In our work, formation and dependent cytotoxicity in SPEV and HT29 cells. Further- growth of spheroids is achieved by adding CMC as part of more, this report for the first time demonstrates that artificial extracellular matrix and surface coating by 1% 15 nm AuNPs in concentrations of 6–12 μg/ml reduced agar which inhibited cell adhesion to surface and stimu- the proliferation of SPEV and HT29 cells and increased lated cell aggregation. At these conditions, MS culture can the cell number at early and late stages of apoptosis and Pavlovich et al. Nanoscale Research Letters (2017) 12:494 Page 8 of 9 necrosis. Also, it was shown that small concentrations of 9. Xiang-Yu S, Liu P-D, Wu H, Ning G (2014) Enhancement of radiosensitization by metal-based nanoparticles in cancer radiation therapy. Cancer Biol Med AuNPs (1–3 μg/ml) stimulate formation of multicellular 11(2):86–91 spheroids. However, higher AuNP concentrations had 10. 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Nanoscale Research LettersSpringer Journals

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