MTT assay dataset of polyethylenimine coated graphenoxide nanosheets on breast cancer cell lines (MCF7,MDA-MB-231,MDA-MB-468)

Abstract

Graphen oxide has emerged as a promising tool in medical biotechnology due to its outstanding properties applicable in several fields as well as cell imaging, drug and gene delivery. Monolayer structure and high surface area of Graphen benefits elevated loading capacity of drugs rather than other nanomaterials. However Graphen oxide in physiological solutions has unfavourable reactions which confine it’s application in biomedical field without additional surface functionalization. Coating of graphenoxide by polyethylenimine is an approach to enhance biocompatibility of graphen oxide and also provides desirable physicochemical features for oligonucleotides delivery. The data presented here is related to graphenoxide-PEI characterisation and it’s cytotoxicity assay on variouse breast cancer cell lines including MDA-MB-468 and MDA-MB-231 and MCF7 by MTT assay.

Keywords

Graphen oxide PEI MTT assay MDA-MB-468 MDA-MB-231 MCF7

1. Introduction

Nanotechnology is a 21st-century new science that examines the effects and applications of materials on a scale of 1 to 100 nm. As materials shrink to sizes 1–100 nm, their magnetic, electrical, optical, and mechanical properties have changed drastically, making new materials and devices more widely used. Significant advances in nanotechnology in recent years have shown that nanoparticles can be used as a suitable tool for diagnostic, therapeutic, and drug delivery purposes [1, 2, 3, 4]. Since nanoparticles are easy to manipulate and manufacture with their desired physical and chemical dimensions and properties, most of the third millennium researches have focused on the application of nanoparticles in various aspects of medical, industrial, agricultural, defense, nutrition, clothing, etc. [5, 6, 7, 8]. For the first time, a carbon shell in micrometer-size and as big as an atom was separated from the graphite surface [8]. This breakthrough made an evolution in nanotechnology as many scientists investigated the properties and capabilities of this two-dimensional crystal of honeycomb-shaped carbon atoms. Monolayer graphene with all atoms on the surface has unique properties including mechanical strength (1.1 trapascal), thermal conductivity (5000 Wm/k) and extremely high surface area (2630 m ${}^{2}$ /g). Graphene also has a high electrical conductivity due to the high electron mobility (200,000 cm ${}^{2}$ /Vs). The strong sigma bonds between the carbon atoms have led to the extremely high mechanical strength of the graphene, with the ultimate tensional stability of 130 GPa, which is actually about three hundred times stronger than aramid and steel. Additionally, graphene has a high surface area which plays a decisive role in the measurement of the reactivity and its therapeutic effects and toxicity [9, 10]. The number of graphene layers plays an important role in determining their physical, chemical and biological properties, with monolayer, bilayer and multilayer graphene having different biological effects. Graphene has many derivatives including graphene oxide, reduced graphene oxide, graphene nano-strips, graphene nano oxide, each having unique properties. There are active oxygen groups such as carboxylic acid at the edge and epoxy and hydroxyl at the graphene oxide surface [11, 12, 13]. These functional groups cause compounds such as dextran and polyacrylic acid to be covalently bond to graphene oxide through these functional groups. Some surfactants and polymers also have the ability to bind non-covalently to graphene oxide, which is often performed in the form of hydrophobic, pie-pie and electrostatic interactions. Also, some biomolecules such as DNA and siRNA and proteins can bind to graphene oxide. Therefore, the binding of different functional groups, polymers and drugs to graphene oxide surface is easily accomplished, which increases its scope of application in various medical and industrial fields [14, 15, 16, 17].

The unique properties of graphene and its appeal have made it to be at the center of nanomedical scientists’ attention today. Extensive studies have been conducted on the diagnostic and therapeutic applications of these nanomaterials. The high surface area of graphene oxide and the functional groups present on its surface provide suitable conditions for binding, transporting, and delivering drugs and biological molecules (genes and proteins) to the target tissue or cell. On the other hand, binding of different markers to the surface of these nanomaterials has led to their wide application in the detection and imaging of cancerous tissues. Because graphene oxide has a high absorption in the near-infrared region, it is nowadays used for the treatment of cancer by photo-thermal therapy [18, 19].

Since nanoparticles are capable of transporting drugs to all parts of the body, nanoparticles have been considered as a suitable drug delivery tool in recent decades. In 2008, the first drug research to treat cancer using graphene oxide was conducted. During these investigations, chemotherapy-specific drugs such as comptutcin, doxorubicin, and SNC-8 were mounted on graphene oxide nanoparticles and the anticancer properties of the graphene oxide-drug complex were investigated [20, 21]. The results showed that the graphene oxide-drug complex was more anticancer than either drug or graphene oxide alone. In some cases, to target the graphene-oxide complex, the surface of the graphene oxide was either pegylated or functionalized with a specific ligand for cancer cells. One of the major problems in the field of gene therapy today is the lack of transporter carriers that can specifically transduce DNA, siRNA and aptamer molecules into the target tissue. Studies in the field of gene therapy have shown that graphene oxide can act as a carrier for delivering siRNA and aptamer to cancer cells that specifically kills the target cancer cells [22, 23]. Various modifications were made to the graphene oxide surface, such that in some cases the nanoparticle surface was coated with polyethylene imine or polyethylene glycol or the drug was synthesized with doxorubicin [24, 25]. The combination of these strategies eventually led to the development of multipurpose graphene oxide to kill cancer cells.

Since the reduced graphene oxide is more absorbed in the near-infrared range than the graphene oxide, in subsequent studies the surface of the reduced graphene oxide was coated with polyethylene glycol polymer and then the peptide with arginine, glycine and aspartic acid (RGD) sequence was attached to it [26, 27].

In this study, graphene oxide conjugated positively with PEI 25 K, which led to the possibility for the siRNA to sit on its substrates due to electrostatic interactions induced by the cationic polymer. Addressing cell efficiencies of GO-PEI-25 K. This experiments show that grafting PEI to GO reduced the cytotoxicity of MDA-MB-468 and MDA-MB-231 and MCF7 cell lines.

2. Materials and methods

2.1 Preparation of GO-PEI

Graphen oxide powder was purchased from Nanos-any corporation. Coating of graphenoxide with PEI 25 K (branched, average MW $\sim$ 25,000 by LS, average Mn $\sim$ 10,000 by GPC, Sigma-Aldrich, St. Louis, MO, USA) was conducted approximately. We gently added a PEI solution (1 mg/ml) to a GO solution (about 0.1 mg/ml) on a magnetic stirrer during 30 minutes followed by 10 minutes ultrasonication and then the combination was stirred over night. The solution was rinsed several times with deionized water (DI) by centrifugation and finally re-diffused in DI-water.

2.2 DLS and FTIR

Graphen-oxide nanosheets functionalized by coating with branched PEI 25KD and characterized by FT-IR and DLS. cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) with 10% Fetal bovine serum (FBS, Gibco) in a humid atmosphere of 5% CO ${}_{2}$ at 37 ${}^{\circ}$ C.

DLS test was done in pastor institute of Iran, FT-IR was performed in cofacility lab of National institute of Genetic engineering and biotechnology (NIGEB) [39].

2.3 Cell culture

Mda-mb-231 and mda-mb-468 and Mcf7 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) with 10% Fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin in a humid atmosphere of 5% CO ${}_{2}$ at 37 ${}^{\circ}$ C.

2.4 MTT assay

MTT assay was done to determine cytotoxicity effect of different concentrations of GO-PEI nanoparticles (0.1, 1, 10. 100 mg/L) on mda-mb468 and mda-mb-231 and MCF7 cell lines in three time points (24, 48, 72 hours). Data provided here implemented by cell culture condition and coating of graphenoxide was done slowly by adding PEI. Cytotoxicity of GO-PEI assaied by MTT method and analyzed by Graph Pad prism8.0.2.

The cytotoxicity of nanoparticles on the named cell lines was evaluated by MTT assay. 6000 cells per well seeded into 96 well plates and cultured for 24 hours with 100 $\mu$ l complete DMEM. Different concentration of nanocomplexes added to the wells and incubated separately for another 24, 48, 72 hours, then the medium was replaced with fresh medium and 20 $\mu$ l of MTT solution (5 mg/ml) and incubated for further 4 hours. Then the medium was removed and 100 $\mu$ l DMSO added into the wells to dissolve the formazan crystals and the absorbtion data at 570 nm recorded by a microplate photometer (labsystem multiscan).

2.5 Statistical analysis

Data obtained from MTT assay were analyzed by unpaired t-test analysis using Graph Pad Prism version 8.0.2. data were analyzed by mean value with its standard deviation (Mean $\pm$ SD). P values $\leqslant$ 0.05 were considered statistically significant.

2.6 Gel retardation assay

Loading of siRNA on GO-PEI was performed with a series of math ratios (GO-PEI: siRNA) 0:1, 3:1, 6:1, 12:1, 24:1, 30:1, 35:1, 40:1, 50:1, 55:1, 60:1, 65:1, respectively. Different amounts of GO-PEI solution were mixed with 10 pico mole siRNA ( $\sim$ 0.125 $\mu$ g) in equal volumes followed by 30 minutes incubation in room temperature. Then 8 $\mu$ l from each solution was mixed with 2 $\mu$ l loading dye and transferred to the wells of 1% gel electrophoresis with TBE buffer at 150 V for 30 minutes and visualized by EtBr staining.

3. Results

This article contains the characterization data of Go-PEI using dynamic light scattering (DLS) for size distribution and zeta potential measurement. FT-IR confirms coating of graphenoxide with PEI properly. Graphenoxide coated with polyethylenimine was characterized by Dynamic light scattering (DLS) and FT-IR.

Cytotoxicity assay of GO-PEI on mda-mb-468 and mda-mb-231 two triple negative breast cancer cell line and MCF7 was done using MTT assay. Figures 1–3 demonstrated the MTT assay results in three different time points (24 h, 48 h, 72 h) for MDA-MB-231, MDA-MB-468 and MCF7 cell lines respectively.

Figure 1.

MTT assay of GO-PEI in MDA-MB-231cell line, student’s t-test analysis was used to determine statistically significant differences of cell viability between various concentrations of GO-PEI treated groups. Cell viability was significantly ( $P$ value $<$ 0.05) reduced in 100 mg/L GO-PEI treated groups in 24 and 48 hours after treatment but in 72 hours after treatment we observe no cytotoxicity effect.

Figure 2.

MTT assay of GO-PEI in MDA-MB-468 cells, student’s t-test analysis was performed and revealed no significant cytotoxicity effect in 24, 48, and 72 hours after treatment by the determined concentrations of GO-PEI up to 100 mg/L.

Figure 3.

MTT assay of GO-PEI in MCF7 cells, student’s t-test analysis showed no cytotoxicity effect of GO-PEI after 24, 48, and 72 hours from treatment.

Gel retardation assay indicated that PEI coated Graphen oxides are able to condense siRNAs strongly. Mobility of siRNAs was retarded when math ratios of GO-PEI to siRNA was higher than 60:1 in this experiment. So GO-PEI could be used as a carrier for siRNA transfection (Fig. 4).

Figure 4.

Gel retardation assay of GO-PEI/siRNA complexes at various math ratios. Ratios of each lane is shown on top of the wells, first lane in each gel is naked siRNA (without GO-PEI) as control.

4. Discussion

The effects of graphene oxide on the cell are influenced by various factors such as concentration and physical and chemical properties of graphene oxide, cell type, and duration of graphene oxide incubation with the cell. Graphene functionalized with amine groups is known to be one of the most biocompatible types of graphene because it does not have the effects of hemolysis and thrombosis [28]. Studies have shown that graphene oxide has no detrimental effect on A549 cells and only increases ROS concentrations at concentrations above 50 $\mu$ g/ml [29]. Graphene oxide with a low substrate destroys the mitochondria of neuronal cells. These compounds are less toxic when they are carboxylated or resuspended. Magnetized graphene oxide also showed low toxicity to Hela cells, as the toxicity of these nanoparticles against the cell depends on their concentration [30]. The addition of dot quantum to graphene oxide increased the biodegradability of these nanosheets. Scientists believe that the toxicity of the nanoparticles to the cell is mainly due to the direct interaction between the nanoparticles and the cell wall that results in physical destruction of the cell. Binding of graphene oxide nanosheets to the cell surface is accomplished by electrostatic interaction between the graphene negative groups and the phosphatidylcholine positive groups. The interaction of the nanoparticles with the cell also increases the cellular uptake of the nanoparticles, which eventually activates some signaling pathways, destroys the nucleus and disrupts the metabolic pathways [25, 31]. Because different cells have unique properties, each showed a different sensitivity or resistance to graphene oxide. For example, PANC-1 cells were more sensitive to graphene than Hela cells [32].

Studies have shown that the number of graphene oxide layers has a significant effect on its physical, chemical and physiological properties. Before examining the therapeutic efficacy and toxicity of graphene nanosheets, the physical, chemical and number of layers of these nanosheets should be thoroughly investigated in terms of size and thickness [33]. Manufacturing of monolayer graphene oxide nanosheets is extremely difficult and technically sophisticated. The presence of two or more layers of graphene oxide in the final solution made is inevitable, so methods of fabricating graphene oxide nanosheets should always be carried out in such a way as to minimize the amount of nanosheets of two or more layers. One of the most important challenges in the nanomedicine world has always been the contradictory results about the physiological effects or toxicity of nanoparticles/nanosheets. One of the most important ways to reduce the contradictory results is to fully identify the nature of the nanomaterials, as there is a belief that some contradictory reports are due to the lack of accurate and complete recognition of the nanomaterials. On the other hand, the biological responses of the body (in vivo) and cells (in vivo and in vitro) depend on the chemical nature of the nanoparticles/nanosheets. Extensive analyzes including AFM, XPS and Raman imaging have been performed, all of which confirmed the monolayer and purity of the graphene oxide nanosheets. Identification of the physical and chemical properties of nanomaterials is essential because the physical and chemical properties of nanomaterials play an important role in the formation of the corona protein. Competitive and yet dynamic process of adsorption of plasma proteins on the nanomaterial surface depend on various factors such as nature, size, shape, crystallinity, surface area, electrical charge, electron transferability, hydrophobicity, surface functional groups and protein/nanoparticle ratio [34, 35, 36].

The physical and chemical properties of the nanoparticles, such as their size and surface electric charge, play decisive roles in the extent and type of nanoparticle interaction with the cell. Obviously, upon entry of the nanoparticles into the surrounding plasma, they are coated with plasma proteins and the surface size and charge of the nanoparticle complex is changed [37]. Researches have shown that most of the therapeutic/toxic effects of nanoparticles on cells occur after they are absorbed by the cell and are incorporated into the cell. Therefore, evaluation of cellular uptake of nanoparticles is one of the important issues that should always be evaluated in nanomedical studies. Recent studies have shown that the graphene oxide complex (small and large in size) enters the lysosome upon entry into the cell [38].

Data provided here indicates physicochemical characters of GO-PEI. Graphenoxide nanosheets are remarkable because of their unique properties such as exceptional physicochemical features and high surface/ volume ratio which is a beneficent to use as a drug or gene carrier.

5. Conclusion

The conjugation of graphenoxide with PEI is a promising approach to optimize gene delivery and enhance its biocompatibility. Data presented here shows cytotoxicity effect of various dilutions of GO-PEI on breast cancer cell lines (mda-mb-468, mda-mb-231, MCF7), which are useful to other researchers to apply that in case of their own purposes. GO-PEI could be used as a cheap, safe and efficient vector to transfer plasmids or siRNAs into the cells by other researchers.

Footnotes

Acknowledgments

This work was supported by a research (grant 732) from National Institute of Genetic Engineering and Biotechnology of Iran.

References

Ferrari

, Cancer nanotechnology: opportunities and challenges, Nature Reviews Cancer 5(161) (2005).

Peer

et al., Nanocarriers as an emerging platform for cancer therapy, Nature Nanotechnology 2(751) (2007).

Schroeder

et al., Treating metastatic cancer with nanotechnology, Nature Reviews Cancer 12(39) (2012).

Wagner

Dullaart

Bock

A.K.

and Zweck

, The emerging nanomedicine landscape, Nature Biotechnology 24(1211) (2006).

Scott

and Chen

, Nanoscale science and engineering for agriculture and food systems, Industrial Biotechnology 9 (2013), 17–18.

Sozer

and Kokini

J.L.

, Nanotechnology and its applications in the food sector, Trends in Biotechnology 27 (2009), 82–89.

Cushen

Kerry

Morris

Cruz-Romero

and Cummins

, Nanotechnologies in the food industry-Recent developments, risks and regulation, Trends in Food Science & Technology 24 (2012), 30–46.

Novoselov

K.S.

et al., Electric field effect in atomically thin carbon films, Science 306 (2004), 666–669.

Prasher

, Graphene spreads the heat, Science 328 (2010), 185–186.

10.

Balandin

A.A.

et al., Superior thermal conductivity of single-layer graphene, Nano Letters 8 (2008), 902–907.

11.

Compton

O.C.

and Nguyen

S.T.

, Graphene oxide, highly reduced graphene oxide, and graphene: Versatile building blocks for carbon-based materials, Small 6 (2010), 711–723.

12.

Chen

Feng

and Li

, Graphene oxide: preparation, functionalization, and electrochemical applications, Chemical Reviews 112 (2012), 6027–6053.

13.

Jung

et al., Reduction kinetics of graphene oxide determined by electrical transport measurements and temperature programmed desorption, The Journal of Physical Chemistry C 113 (2009), 18480–18486.

14.

Zhang

et al., Enhanced chemotherapy efficacy by sequential delivery of siRNA and anticancer drugs using PEI-grafted graphene oxide, Small 7 (2011), 460–464.

15.

et al., Protein corona-mediated mitigation of cytotoxicity of graphene oxide, ACS Nano 5 (2011), 3693–3700.

16.

Cai

and Song

, Recent advance in functionalized graphene/polymer nanocomposites, Journal of Materials Chemistry 20 (2010), 7906–7915.

17.

Feng

and Liu

, Graphene in biomedicine: opportunities and challenges, Nanomedicine 6 (2011), 317–324.

18.

et al., A functionalized graphene oxide-iron oxide nanocomposite for magnetically targeted drug delivery, photothermal therapy, and magnetic resonance imaging, Nano Research 5 (2012), 199–212.

19.

Wang

et al., Gold nanoclusters and graphene nanocomposites for drug delivery and imaging of cancer cells, Angewandte Chemie International Edition 50 (2011), 11644–11648.

20.

Zhang

Xia

Zhao

Liu

and Zhang

, Functional graphene oxide as a nanocarrier for controlled loading and targeted delivery of mixed anticancer drugs, Small 6 (2010), 537–544.

21.

Yang

et al., High-efficiency loading and controlled release of doxorubicin hydrochloride on graphene oxide, The Journal of Physical Chemistry C 112 (2008), 17554–17558.

22.

Bates

and Kostarelos

, Carbon nanotubes as vectors for gene therapy: Past achievements, present challenges and future goals, Advanced Drug Delivery Reviews 65 (2013), 2023–2033.

23.

Shen

Zhang

Liu

and Zhang

, Biomedical applications of graphene, Theranostics 2(283) (2012).

24.

Zhang

et al., Synergistic effect of chemo-photothermal therapy using PEGylated graphene oxide, Biomaterials 32 (2011), 8555–8561.

25.

Zhang

et al., Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells, ACS Nano 4 (2010), 3181–3186.

26.

Yang

et al., Multimodal imaging guided photothermal therapy using functionalized graphene nanosheets anchored with magnetic nanoparticles, Advanced Materials 24 (2012), 1868–1872.

27.

Robinson

J.T.

et al., Ultrasmall reduced graphene oxide with high near-infrared absorbance for photothermal therapy, Journal of the American Chemical Society 133 (2011), 6825–6831.

28.

Donaldson

et al., Nanoparticles and the cardiovascular system: A critical review, Nanomedicine 8 (2013), 403–423.

29.

Chang

et al., In vitro toxicity evaluation of graphene oxide on A549 cells, Toxicology Letters 200 (2011), 201–210.

30.

Yang

et al., Multi-functionalized graphene oxide based anticancer drug-carrier with dual-targeting function and pH-sensitivity, Journal of Materials Chemistry 21 (2011), 3448–3454.

31.

Jastrzębska

A.M.

Kurtycz

and Olszyna

A.R.

, Recent advances in graphene family materials toxicity investigations, Journal of Nanoparticle Research 14(1320) (2012).

32.

Mao

et al., Hard corona composition and cellular toxicities of the graphene sheets, Colloids and Surfaces B: Biointerfaces 109 (2013), 212–218.

33.

Shih

C.-J.

et al., Bi-and trilayer graphene solutions, Nature Nanotechnology 6(439) (2011).

34.

Mahmoudi

et al., Irreversible changes in protein conformation due to interaction with superparamagnetic iron oxide nanoparticles, Nanoscale 3 (2011), 1127–1138.

35.

Nel

A.E.

et al., Understanding biophysicochemical interactions at the nano–bio interface, Nature Materials 8(543) (2009).

36.

Lynch

and Dawson

K.A.

, Protein-nanoparticle interactions, Nano Today 3 (2008), 40–47.

37.

Lundqvist

et al., Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts, Proceedings of the National Academy of Sciences 105 (2008), 14265–14270.

38.

et al., Size-dependent cell uptake of protein-coated graphene oxide nanosheets, ACS Applied Materials & Interfaces 4 (2012), 2259–2266.

39.

Sadeghi

et al., Dataset on cytotoxicity effect of polyethylenimine-functionalized graphene oxide nanoparticles on the human embryonic carcinoma stem cell, NTERA2 cell line, Data in Brief 26 (2019), 1–6.