NaB integrated graphene oxide membranes for enhanced cell viability and stem cell properties of human adipose stem cells

Abstract

Here we present the integration of boron (NaB) with graphene oxide (GO) to develop a new class of membranes which are biocompatible and cost-effective for cell and tissue culture studies. Ethanol (EtOH) assisted the uniform dispersion of GO flakes on top of a glass substrate. We investigated the effect of a GO + NaB membrane on the growth and proliferation of hASCs. hASCs showed better cell viability on NaB integrated GO membranes compared to their respective controls. The concentrations of NaB and GO are 0.02% and 1/20 of stock (0.024%) respectively. To our knowledge this is the first time that enhanced cell proliferation and attachment ability of hASCs with NaB integrated GO membranes has been observed. Our study provides a platform for the development of 3D-GO scaffold systems combined with NaB in tissue engineering.

---

Introduction

Stem cells have emerged as a promising prospect in various aspects of therapeutics.^1,2 Research on stem cells has seen a leap in recent years due to their role in regenerative medicine.³ A specific kind of stem cell, known as human adipose-derived stem cells (hASCs), are extensively proliferative, undergo multilineage differentiation along with classical mesenchymal lineages which include adipogenesis.⁴ The advantage of using human adipose derived stem cells is due to their high proliferation rate (∼1 × 10⁶/200 ml lipoaspirate) and capacity to differentiate into various cell lineages such as adipogenic, osteogenic, chondrogenic, hepatic, neurogenic and myogenic.⁵ hASCs are actively used to regenerate various tissues in vitro due to their multipotency, which makes them ideal for tissue regeneration in order to treat organ/tissue defects. However, the production of in vitro engineered tissue depends on the capacity of the stem cells to migrate, proliferate and differentiate in culture.⁶ Therefore, in order to provide the space and facilitate migration and differentiation of the stem cells, various bio scaffolds have been used.⁴ Of importance is the use of titanium rough/smooth surfaces for orthopedic implants for increased osteogenesis.⁵

Stem cells growth relies on interactions with growth factors and cell matrix adhesions. These are important in the microenvironment to regulate cell survival, cell differentiation and cell renewal. These factors are particularly important for conditioning and strengthening the stem cells further to be utilized in therapeutic applications. Growth factors may be added externally to the cell culture or are secreted by stem cells and are potent regulators of the cell development and fate of the stem cells and neighboring niche cells with respect to time and space. In order to control the niche interactions in 2D space, micro patterns of extracellular matrix (ECM) islands are introduced which hinder the diffusion of growth factors between islands and the altering effect of cell contacts and matrix. In human embryonic stem cells (hESCs), ECM islands are made by microstamping on a substrate thus providing a minimal island size for maintaining and expanding the pluripotent state. Similarly, stem cells can also be deposited into synthetic mimics which can serve as cell adhesion matrices in place of animal derived matrix products and non-human niche cells. Studies with acrylate-based polymers and hESCs showed that the polymers combined with soluble factors affect the cell attachment, cell proliferation and lineage induction.^7,8 One such 2D material recently used as a substrate for cell adhesion is graphene oxide.⁶

One of the main concerns in stem cell research is the viability and self-differentiation capability of stem cells in laboratory conditions. After carbon, boron is the most complex and intriguing element with unique chemistry⁹ and low abundance compared to other essential elements like hydrogen, carbon, nitrogen etc.¹⁰ It has been decades since it was realized that boron is one of the essential elements for a normal plant growth¹¹ and its role was evident when it was realized that boron and calcium metabolism is important in cell signalling.¹⁰ Boron has also been associated with several important functions within the animal kingdom like brain function and bone metabolism¹² though its actual mechanism is not completely understood yet. In keratinocytes, derivatives of boron are shown to increase the extracellular matrix turnover and cell migration by the increase in matrix metalloproteinase expression.^13,14 This demonstrates a promising prospect in wound healing. A recent in vitro study has shown that the derivatives of boron particularly sodium pentaborate pentahydrate (NaB) can augment the differentiation of human tooth germ stem cells (hTGSCs).¹⁵ Several other recent finding also implicate that boron is also a stem cell growth factor because of its role in diverse processes such as hormone production, ion transport, calcium metabolism and thus bone growth. Recently it was also shown that boron increases the viability of mesenchymal stem cells.¹⁶

Graphene oxide is generally prepared by modified Hummer's method¹⁷ though there are several other improved methods like Marcano's¹⁸ where the graphene is more oxidized. Graphene oxide is also commercially available either in a dispersed or powder form which makes it easier to deposit on a desired substrate in its native state, nevertheless, acquiring a uniform deposition has been a problem. Brief literature study suggests that uniform GO films can be deposited by using an appropriate solvent in specific concentrations (wt%). Few of the identified methods are deposition by ethanol assistance,¹⁹ using solvents like NMP and Ethyl acetate²⁰ to organize graphene flakes in to layers, electrolytic deposition using NaCl²¹ or by using a cross-linking agent.²² In this study the above mentioned solvents were used (except cross-linking agent) to realize the uniform deposition of GO layer on top of a glass substrate further choosing the best solvent after initial characterization.

Stem cell research is an extensively studied area where constant efforts are being applied to improve tissue engineering for regenerative medicine. Researchers mainly focussed on enhanced cell adhesion, differentiation and migration on various bio-scaffolds. Graphene oxide has emerged as a novel 2D nano-scaffold material having non-cytotoxic effects on cells and serving as an excellent cell adhesion layer.

In our study we show the parameters for realizing uniform GO films on a glass substrate by depositing graphene oxide in combination with certain solvents like ethanol, ethyl acetate, NaCl, and NMP. Two different concentrations of GO were used (1/20 (0.024%) and 1/40 (0.012%) from the stock) and the membranes were prepared by drop casting method. After initial optical and SEM characterization of the fabricated membranes it was realized that the samples with ethanol provide a uniform GO membrane on top of the glass surface. Further experiments were continued with the membranes prepared with GO + ethanol. Sodium pentaborate pentahydrate (NaB) as a boron source was integrated with GO membranes thereby developing a ready-made, bio-compatible and cost effective platform for cell and tissue cultures.

Experimental

Materials and methods

Highly concentrated graphene oxide (497 mg/100 ml) was purchased from Graphene Supermarket and different dilutions of this stock solution were prepared. Laboratory grade NMP (N-methyl-2-pyrrolidinone), NaCl, ethanol (EtOH) and ethyl acetate were used as solvents in order to realize the best solvent for a uniform GO film formation. Sodium pentaborate pentahydrate (NaB) was acquired from National Boron Research Institute-BOREN (Ankara, Turkey).

Preparation of graphene oxide membranes

Two different concentrations of GO solutions were prepared (1/20 (0.024%) and 1/40 (0.012%) from the stock GO solution) with di-water as a solvent and were sonicated for 1 h prior to every usage. Initially GO membranes were fabricated by spray drying and spin coating methods only to realize that the membranes were non-uniform in GO dispersion. Further, by using drop casting method along with the addition of few drops of volatile and organic liquids, we were successful in achieving uniform GO membranes.

Note: just by using GO solution we couldn't achieve uniform membranes by drop casting due to the stacking of GO flakes. Addition of one of these four solvents reduces the surface tension and in turn helps in uniform distribution of the GO flakes resulting in a much uniform GO membrane.

Choosing a right solvent

The first goal was to choose a right solvent that facilitates uniform distribution of GO flakes on the glass substrate. Prior to drop casting, glass slides were cleaned thoroughly with acetone and rinsed in di-water. Few drops of GO solution along with few drops of NaCl, EtOH, ethyl acetate and NMP were casted on separate glass slides and left over night for drying followed by heat treatment at 80 °C for 1 hour. The ratio of GO to one of these 4 solvents is in the range of 1 : 1 to 1 : 4 which needs certain optimization as the uniform spreading of GO flakes also depends on the external conditions such as cleanliness of the glass cover slip. Though all the samples seem to dry within minutes (at room temperature), the sample with NMP takes hours. After optical and SEM characterization of the fabricated membranes it was realized that the samples with ethanol provide uniform GO membrane. Further experiments were continued with the membranes prepared with GO + ethanol.

Choosing right GO concentration

Initially the samples prepared with GO + EtOH were used to check the stem cell viability and it was realized that 1/40 GO concentration was too low for the viability of the cells (data not presented). Better results were observed with the samples prepared from 1/20. Further experiments were carried out with 1/20 GO concentration.

Sample preparation: NaB

Sodium pentaborate pentahydrate (NaB) solution was prepared by dissolving NaB in the culture medium at a 0.1 g ml⁻¹ stock solution. The main stock was subsequently diluted to lower concentrations in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA).

Integration of NaB with GO membranes

The main idea behind integration is to facilitate and study stem cell viability and regeneration on a solid surface rather than in a suspended form. Higher concentrations of NaB were found to be toxic for certain type of stem cells.¹³ Long term stem cell viability was studied on these samples. The above mentioned NaB solution was mixed with GO solution and left for ultra-sonication for an hour followed by drop casting (initially, standard concentration of NaB was dissolved in 10 ml of di-water followed by adding 40 ml of 1/20 dilution of GO).

Glass slides were drop-casted with EtOH + GO + NaB. It was observed that first depositing 8 drops of EtOH all over the slide and afterwards placing 0.4–0.5 ml of GO + NaB resulted in more homogenous deposition (the sample size is 2 × 2 cm). These samples were used to analyze the stem cell viability, growth and long term toxicity.

Isolation of hASCs (human adipose stem cells)

Adipose cells obtained from breast tissues of healthy patients under general anesthesia were processed with collagenase enzyme as defined by Borges-Silva et al. Following to degradation of fat tissue by collagenase, samples were exposed to centrifugation at 250 rpm for 5 minutes. Pelleted cells were plated in six well plates (BIOFIL, TCP, Switzerland) and grown to confluency in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) PSA (10.000 units per ml penicillin, 10.000 μg ml⁻¹ streptomycin, 25 μg ml⁻¹ amphotericin B) (Invitrogen, Gibco, UK). Thereafter, the cells were trypsinized using 0.25% (v/v) trypsin/EDTA (Invitrogen, Gibco, UK). Medium was added to detached cells to inhibit the activity of trypsin followed by centrifugation at 300 × g for 5 minutes at room temperature. Further, the pellet was dissolved in fresh medium and seeded on a T-75 flask (Zelkultur Flaschen, Switzerland). The cells were maintained at 37 °C and 5% CO₂ in a humidified incubator. Cells from passages 3–4 were used in all experiments.

Characterization of hASCs

Isolated hASCs (passage 3) were characterized for their mesenchymal cell surface profile. As described above the cells were trypsinized and incubated with primary antibodies which were prepared in PBS. For characterization, primary antibodies against CD14 (ab82434), CD31 (ab27333), CD34 (ab18227), CD44 (ab58754), CD45 (ab134202), CD73 (ab157335), CD90 (ab95700), CD105 (ab53321), integrin beta 1 (ab27314) (Abcam, UK)) and CD29 (Zymed, San Francisco, CA, USA) were used. Flow cytometry analysis of the cells was conducted using Becton Dickinson FACS Calibur Flow Cytometry system (Becton Dickinson, San Jose, CA, USA).

Concentration dependent cytotoxicity of hASCs on GO

Characterized hASCs were seeded on GO coated plates. Cell viability was intended to measure on the 1^st, 2^nd and 3^rd days using MTS assay (CellTiter 96 Aqueous One Solution, Promega, UK). For each sample 3 plates were coated for each time point for the cell viability assay. Cells were seeded on GO + NaB coated plates (20 000 cells per well) in 24-well plates followed by incubation for 3 days. On the evaluation day, MTS (3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium) solution was prepared according to the manufacturer's instructions and cells were treated with the MTS solution. The plates were incubated for 2 hours in the dark followed by transferring 100 μl of MTS solution of each well to a 96-well plate. Cell viability depending on the toxicity of universal, bulk-fill and flowable composites was measured by ELISA plate reader at 490 nm absorbance (Biotek, USA).

Statistical analysis

All the data shown here is mean ± standard errors. Graphics were drawn using GraphPad Prism 5 software. Statistical analysis of the results was performed by one-way ANOVA followed by multiple-comparison Tukey's test using GraphPad Prism 5 software. Statistical significance was determined at p < 0.05.

Further, same above mentioned concentration and volume of growth factors and sterile tissue culture dishes were used to prepare large area NaB integrated GO membranes. Exact volumes that were used are listed below in the table (Table 1).

Table 1 Volumes and ratios of GF's, EtOH and GO used for the fabrication of large area membranes

Chemical	Chemical volume used	Ethanol	Ratio
GO	50 ml	35 ml	1.43 : 1
NaB	50 ml	80 ml	0.63 : 1

---

Results

Following up the integration of NaB with GO solution, membranes were casted by controlled drop casting method. Fig. 1a shows the optical images of the casted GO membrane with the aid of different solvents. As can be seen from the pictures, GO + ethanol shows more uniform layer compared to the other three membranes. In the case of bare GO and GO + ethylacetate, GO flakes can be seen more concentrated within the center of the glass substrate as compared to GO + ethanol. In the case of GO + NaCl though the GO flakes were uniformly distributed a lot of voids within the membrane can be observed dude to the aggregation of salt crystals.

Fig. 1 (a) Optical images of the fabricated membranes with GO and GO + ethyl acetate/EtOH and NaCl (b) SEM image of the sample prepared with GO + EtOH showing a continuous membrane.

Fig. 1b shows the SEM images of the fabricated GO membrane with integrated NaB (there is no visible difference in the top view of the bare GO and NaB integrated membranes). The membranes were drop casted on a cover slip and the continuity without voids can be observed.

FTIR (Fourier transform infrared spectroscopy)

The samples were completely air dried prior to FTIR analysis. From the spectra shown in Fig. 2, it can be observed that the peak at 1712 cm⁻¹ arises from GO. This is a characteristic peak of GO along with a broad spectrum between 3000 cm⁻¹ and 3650 cm⁻¹ indicating well dispersion of GO. The band at 1712 cm⁻¹ is attributed to C=O. The broad band between 3000 cm⁻¹ and 3650 cm⁻¹ attributes to O–H functional group stretching from graphene oxide surface. The peak at 1379.45 cm⁻¹ which arises from nitrate anion's ν3 stretching mode is due to the presence of NaB which is specific for the chemical structure.

Fig. 2 FTIR spectrum for bare GO and NaB + GO.

The slight shift of the peaks that is observed from the spectrum is because of the UV sterilization of GO substrates that was carried out prior to seeding the cells. The shift and the increase in absorption indicates slight de-oxygenation of GO due to UV irradiation.²³

Stem cell properties of hASCs seeded on GO + NaB membrane

In this study flow cytometry was used to detect surface markers from the cell culture derived from adipose tissue. The results showed that the hASCs used in this study has strong adhesion to plastic surface in standard culture condition; furthermore, cell population was positive for mesenchymal stem cell surface markers CD29, CD44, CD73, CD90, CD105 and negative for hematopoietic stem cell surface markers CD14, CD34, CD45 and endothelial marker CD31 (Fig. 3).

Fig. 3 Mesenchymal stem cell characterization of human adipose stem cells at passage 3 seeded on TCP, GO and NaB + GO flasks. Major population of cells maintained the characteristics of stem cells. Flow cytometry analysis data shows the positive mesenchymal stem cell surface markers CD29, CD44, CD73, CD90 and CD105 whereas the negative endothelial stem cell surface markers are CD31 and hematopoietic stem cell surface markers CD45, CD34 and CD14 (NC, Negative Control; TCP, Tissue Culture Plate; GO, Graphene Oxide; NaB, sodium pentaborate pentahydrate).

Characterization of GO + NaB coated membrane

A variety of test methods are used in general to determine the cytotoxicity of the materials. However, the results of such evaluations are dependent not only on the tested material but also on the test method.²⁴ In the present study, the cytotoxic effect of NaB integrated GO membranes was investigated with MTS assay²⁵ by measuring cell viability on the 3^rd, 8^th and 13^th days. MTS data indicated that cells seeded on NaB + GO membranes prepared with EtOH are more viable compared to the cells on bare GO coated membranes (Fig. 4a). Long term MTS data also suggests the increase in cell viability when the cells were seeded on NaB + GO coated membrane (Fig. 4b). This proves that NaB + GO coated membranes were not toxic and proliferative as expected.

Fig. 4 Effect of NaB integrated GO membranes on isolated human adipose stem cells (hASCs) (a) short-term cell viability of hASCs seeded on NaB + GO prepared with NaCl, GO prepared with NaCl, NaB + GO Prepared with EtOH, GO prepared with EtOH, TCP as Negative Control (NC) (b) long-term cell viability on 3^rd, 8^th, and 13^th days.

Discussion

The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy established three criteria to define human MSCs: adherence to plastic, express specific surface markers and differentiate variety of cells types including; osteo-, chondro- and adipo-genic cells.²⁶ According to the criteria, surface markers should be negative for the hematopoietic stem cell surface markers such as CD14, CD34, and CD45 and positive for the MSCs surface markers such as CD29, CD44, CD105, CD73, and CD90.

It can also be observed that the morphology of the hASCs were still the same while the cells were seeded on NaB + GO coated membranes as shown in Fig. 5a. The oxidant-sensitive dye DCFH-DA was utilized for monitoring the ROS level in a ROS assay kit. Cells (∼10⁵ cells per ml) were incubated with DCFH-DA (0.1 mM) for 24 h and washed thrice with PBS followed by treatment with different concentrations of rGONRs for another 24 h. The fluorescence intensity (indicating the ROS level) of the cells was monitored at various exposure periods. The ROS levels were expressed as the ratio of fluorescence intensity of each sample to the intensity of a control sample containing no cells. All the measurements were carried out without exposure to light. ROS levels on stem cells were still same when cells were seeded on NaB + GO coated membranes as shown in Fig. 5b.

Fig. 5 (a) Basal photomicrographic representation of hASCs morphology (b) ROS level changes of hASCs seeded on TCP as NC, GO and NaB + GO flasks. PC (ROS positive).

Cell differentiation mainly depends on the substrate morphology and substrate stiffness. The observed mechanism can be seen as an interplay of the interactions between GO and the cells, GO and the substrate and GO and NaB. The mechanical, chemical and electrical properties also play an important role which makes it more complicated to single out a unique property for the viability and differentiation of stem cells on NaB integrated GO substrates.

Graphene oxide is hydrophilic which means an easy dispersability in water. The flake size of the used GO is in the range of 0.5 to 5 μm with atleast 60–70% being one atomic layer. A single GO flake contains both pristine regions and oxidized regions in the ratio of 1 : 5 respectively where the oxidized region contain large amount of oxygen containing functional groups.^27,28 The final thickness of the fabricated GO membrane is approximately 1 to 1.3 μm. Under dry conditions the interlayer space between GO sheets is calculated to be approximately 6–7 Å.²⁹ This means that NaB is quite well integrated within the GO membranes and any external effects can be ruled out.

As mentioned by Nayak et al.,³⁰ cell proliferation and viability increased on the substrates with wrinkles and ripples compared to the ones without, though there has been a limitation as in the case of Dulgar Tulloch et al., where the grain size on ceramics played an important role in the differentiation of stem cells.³¹ It has been observed that stem cell differentiation mainly takes place when the surface morphology is disordered.³¹ If we consider our case with GO + NaB membranes, there are a lot of ripples and wrinkles within the membrane (Fig. 6) which account for our results though the mechanism is unclear at macroscopic level and how this disordered morphology is helping for the viability and differentiation. Dalby et al.³² has also suggested that these disorders could play a role in protein adsorption, adhesion, proliferation and differentiation.

Fig. 6 SEM images of surface morphology of GO + NaB membranes where the ripples and wrinkles are clearly visible.

Another factor that effects the cell proliferation is the stiffness and strain of the substrate. Cells can mechanically sense underlying layers up to several tens of nanometres.³⁰ The high Young's modulus and flexibility of graphene contributes to the cell differentiation.³³ Graphene can sustain lateral stress³⁰ which is another ability that provides the required local cytoskeleton tension which inturn generates strong anchor points and helps in unfolding and conformational change of the protein.³⁴ The particle size, surface charge and oxygen content plays an important role in the functioning of these bio-compatible substrates.³⁵ It has also been realized that the aggregated GO flakes show more compatibility to the viability and differentiation of the cells compared to the small or non-aggregated flakes due to the high electrostatic interactions.³⁶

Boron, a well-known semi-metal is actually known as a vital micronutrient for plant metabolism. Boron in optimum concentrations has been shown to be essential for bone regeneration in animals and for gestational development in some aquatic species. Boron deprivation is also known to cause metabolic disruptions in mammalian body.³⁷ High concentrations of boron accumulation in the mammalian body can cause serious physiological and metabolic disruptions.³⁸ The most prominent in vitro effect of boron till date that has been observed is its role in the differentiation of progenitor and mesenchymal stem cells in to osteogenic cell lineage.^13,39

Boron helps in maintaining the membrane integrity and stability within plant cells as has been mentioned in the literature^40–42 which can also correlate to our study here.¹⁶ Boron helps in stimulating the proliferation process though the exact mechanism is not completely known. One explanation is the possible calcium deposition that is responsible for the mineralization of extracellular matrix.¹³ By observing this promising effect of boron in the field of stem cell technology, researchers have been directed to develop a biocompatible scaffold system for regenerative medicine. It is important to develop a biocompatible, mechanically strong, anti-microbial and cost effective scaffold system, when implanted can induce cell differentiation and tissue regeneration to replace the degrading tissues over time. Our efforts with graphene oxide come in this line as GO fulfils the required aspects and there by introducing boron only makes it a more viable material in regenerative medicine. Our initial results show that low concentrations of boron integrated scaffolds can be used for stem cell differentiation without any toxicity. It is also quite important to tune the dosage for better results. Further studies are underway to study the exact underlying molecular mechanism on how boron facilitates the whole regenerative process.

Conclusion

In conclusion growth factor (NaB) integrated GO membranes were casted on a glass substrate prior to which the uniform deposition of GO flakes was tested with different solvents. EtOH + GO was realized to have uniform flake distribution compared to other solvents further integrating NaB with EtOH + GO. By integrating boron with GO a new substrate with a more favourable morphology has been developed. hASC's on these substrates show better viability and proliferation compared to control substrates. Long term viability of hASC's on these membranes was successfully tested. This presented study suggests that GO + NaB coated membranes, specifically with the aid of EtOH can be beneficial for the future stem cell related studies providing an easily-accessible, bio-compatible and economically viable ready-made scaffolds which can play an important role in regenerative medicine.

Acknowledgements

The authors would like to thank EC-Marie Curie Co-fund Circulation Scheme, TUBITAK (project no. 114C032) and VINNOVA (28240005) for providing financial support.

Notes and references

1. M. F. Corsten and K. Shah, Lancet Oncol., 2008, 9, 376–384.
2. K. Shah, Adv. Drug Delivery Rev., 2012, 64, 739–748.
3. A. K. Teo and L. Vallier, Biochem. J., 2010, 428, 11–23.
4. B. A. Bunnell, M. Flaat, C. Gagliardi, B. Patel and C. Ripoll, Methods, 2008, 45, 115–120.
5. G. Gastaldi, A. Asti, M. F. Scaffino, L. Visai, E. Saino, A. M. Cometa and F. Benazzo, J. Biomed. Mater. Res., Part A, 2010, 94, 790–799.
6. W. C. Lee, C. H. Lim, H. Shi, L. A. Tang, Y. Wang, C. T. Lim and K. P. Loh, ACS Nano, 2011, 5, 7334–7341.
7. M. F. Brizzi, G. Tarone and P. Defilippi, Curr. Opin. Cell Biol., 2012, 24, 645–651.
8. D. E. Discher, D. J. Mooney and P. W. Zandstra, Science, 2009, 324, 1673–1677.
9. N. N. Greenwood and A. Earnshaw, Chemistry of the Elements, Pergamon Press, New York, 1984.
10. L. Bolanos, K. Lukaszewski, I. Bonilla and D. Blevins, Plant Physiol. Biochem., 2004, 42, 907–912.
11. K. Warington, Ann. Bot., 1923, 37, 629–672.
12. F. H. Nielsen and S. L. Meacham, J. Evidence-Based Complementary Altern. Med., 2011, 16, 169–180.
13. P. N. Tasli, A. Dogan, S. Demirci and F. Sahin, Biol. Trace Elem. Res., 2013, 153, 419–427.
14. S. Demirci, A. Doğan, E. Karakuş, Z. Halıcı, A. Topçu, E. Demirci and F. Sahin, Biol. Trace Elem. Res., 2015, 168, 169–180.
15. P. N. Tasli, A. Dogan, S. Demirci and F. Sahin, Cytotechnology, 2015, 68, 1–11.
16. S. Demirci, A. Dogan, B. Sisli and F. Sahin, Cryobiology, 2014, 68, 139–146.
17. W. S. Hummers and R. E. Offeman, J. Am. Chem. Soc., 1958, 80, 1339.
18. D. C. Marcano, D. V. Kosynkin, J. M. Berlin, A. Sinitskii, Z. Sun, A. Slesarev, L. B. Alemany, W. Lu and J. M. Tour, ACS Nano, 2010, 4, 4806–4814.
19. J. Huang, M. Larisika, W. H. Derrick Fam, Q. He, M. A. Nimmo, C. Nowak and I. Y. Alfred Tok, Nanoscale, 2013, 5, 2945–51.
20. S. Jongwon, Y. Je Moon, Y. Taeyeong, K. Pilnam, L. K. Eun, L. W. Jun, R. Ryong, P. J. David, Y. Gi-Ra and K. S. Ouk, Nano Lett., 2014, 14, 1388–1393.
21. I. Chowdhury, M. C. Duch, N. D. Mansukhani, M. C. Hersam and D. Bouchard, Environ. Sci. Technol., 2014, 48, 961–969.
22. H. Wu, B. Tang and P. Wu, J. Membr. Sci., 2010, 362, 374–383.
23. S. Bittolo Bon and S. Valentini, http://www.arxiv.org/abs/1010.2108.
24. D. Baksh, L. Song and R. S. Tuan, J. Cell. Mol. Med., 2004, 8, 301–316.
25. G. Ergun, F. Egilmez and I. Cekic-Nagas, Med. Oral Patol. Oral Cir. Bucal, 2011, 16, 252–259.
26. E. P. Guven, M. E. Yalvac, M. B. Kayahan, H. Sunay, F. Sahın and G. Bayirli, J. Appl. Oral Sci., 2013, 21, 351–357.
27. A. Buchsteiner, A. Lerf and J. Pieper, J. Phys. Chem. B, 2006, 110, 22328–22338.
28. K. Erickson, R. Erni, Z. Lee, N. Alem, W. Gannett and A. Zettl, Adv. Mater., 2010, 22, 4467–4472.
29. D. An, L. Yang, T.-J. Wang and B. Liu, Ind. Eng. Chem. Res., 2016, 55, 4803–4810.
30. T. R. Nayak, H. Andersen, V. S. Makam, C. Khaw, S. Bae, X. Xu, P.-L. R. Ee, J.-H. Ahn, B. H. Hong, G. Pastorin and B. Ozyilmaz, ACS Nano, 2011, 5, 4670–4678.
31. A. J. Dulgar-Tulloch, R. Bizios and R. W. Siegel, J. Biomed. Mater. Res., Part A, 2008, 90A, 586–594.
32. M. J. Dalby, N. Gadegaard, R. Tare, A. Andar, M. O. Riehle, P. Herzyk, C. D. W. Wilkinson and R. O. C. Oreffo, Nat. Mater., 2007, 6, 997–1003.
33. C. Lee, X. Wei, J. W. Kysar and J. Hone, Science, 2008, 321, 385–388.
34. G. C. Reilly and A. J. Engler, J. Biomech, 2010, 43, 55–62.
35. K. H. Liao, Y. S. Lin, C. W. Macosko and C. L. Haynes, ACS Appl. Mater. Interfaces, 2011, 3, 2607–2615.
36. A. M. Pinto, I. C. Goncalves and F. D. Magalhaes, Colloids Surf., B, 2013, 111, 188–202.
37. H. Apdik, A. Dogan, S. Demirci, S. Aydin and F. Sahin, Biol. Trace Elem. Res., 2015, 165, 123–130.
38. F. U. Meryem Eren, K. G. Berrin and A. Atasever, Asian J. Anim. Vet. Adv., 2012, 7, 1079–1089.
39. X. Ying, S. Cheng, W. Wang, Z. Lin, Q. Chen, W. Zhang, D. Kou, Y. Shen, X. Cheng and F. A. Rompis, Biol. Trace Elem. Res., 2011, 144, 306–315.
40. C. Dordas and P. H. Brown, Plant Soil, 2005, 268, 293–301.
41. H. E. Goldbach, L. Huang and M. A. Wimmer, Adv. Plant Anim. Boron Nutr., Springer, 2007, pp. 3–25.
42. M. A. Wimmer, G. Lochnit, E. Bassil, K. H. Mühling and H. E. Goldbach, Plant Cell Physiol., 2009, 50, 1292–1304.

---

Footnote

† Nanotechnology Research and Application Center (SUNUM), Sabanci University, Orhanli/Tuzla, Istanbul 34956, Turkey.

---