Synthesis of polythiophene nanoparticles by surfactant-free chemical oxidative polymerization method: Characterization, in vitro biomineralization, and cytotoxicity evaluation

Journal of Industrial and Engineering Chemistry 77 (2019) 243–252

Contents lists available at ScienceDirect

Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec

Synthesis of polythiophene nanoparticles by surfactant-free chemical oxidative polymerization method: Characterization, in vitro biomineralization, and cytotoxicity evaluation Deval Prasad Bhattaraia,b , Ganesh Prasad Awasthia , Bikendra Maharjana , Joshua Leea , Beom-Su Kimc,**, Chan Hee Parka,d,* , Cheol Sang Kima,d,* a

Department of Bionanosystem Engineering, Graduate School, Chonbuk National University, Jeonju 561-756, Republic of Korea Department of Chemistry, Amrit Campus, Tribhuvan University, Kathmandu, Nepal c Carbon Nano Convergence Technology Center for Next Generation Engineers (CNN), Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do 54896, Republic of Korea d Division of Mechanical Design Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 13 December 2018 Received in revised form 21 April 2019 Accepted 23 April 2019 Available online 28 April 2019

Herein, we report the synthesis of polythiophene nanoparticles (PTh-NPs) by surfactant-free chemical oxidative polymerization of thiophene at 37
C using ammonium persulphate as oxidant. PTh-NPs synthesized without surfactant were compared to those with surfactant in terms of surface morphology, crystallinity, cytotoxicity and some other aspects. Thermogravimetric analysis showed a good thermal stability of as-synthesized PTh-NPs. In vitro biomineralization revealed the nucleation of calcium and phosphate onto the NPs. Cytotoxicity of PTh-NPs was evaluated by measuring cell viability of preosteoblast MC3T3-E1 and PC12 cell lines. PTh-NPs synthesized without using surfactant exhibited better cell viability compared to those with surfactant. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.

Keywords: Polythiophene nanoparticle Chemical polymerization Biomineralization MC3T3-E1 cell PC12 cell Cytotoxicity

Introduction Recently, conjugated polymer nanoparticles are being used in various fields of research such as optoelectronics, photoluminescence, bio-imaging, bio-sensing, nanomedicine, and drug delivery due to their tunable photophysical properties, non-toxicity, and biocompatibility [1–4]. Contiguous sp2 hybridized carbon centers in conducting polymer backbone induce delocalized set of orbitals imparting conjugated polymer structure capable of spacing free movement of electrons for electrical conductivity [5,6]. Polyaniline, polypyrrole, polythiophene and synthetically fused variants thereof are commonly used conducting polymers in a wide range of physicochemical and biomedical applications [7–10]. Thiophene is a sulphur containing, five-membered, heterocyclic compound with 6p-electron motifs which on polymerization gives

* Corresponding authors at: Department of Bionanosystem Engineering, Graduate School, Chonbuk National University, Jeonju 561-756, Republic of Korea. ** Corresponding author. E-mail addresses: [email protected] (B.-S. Kim), [email protected] (C.H. Park), [email protected] (C.S. Kim).

a p-conjugated heterocyclic polymer, polythiophene [11]. Polythiophene (PTh) and its derivatives are promising members of conducting polymer family due to the presence of delocalized p-electrons along the polymer backbone [12]. Some outstanding properties such as good biocompatibility, chemical and environmental stabilities, tunable particle size and morphology, facile route of synthesis and ease of derivatization of PTh have driven their great promises in a wide range of applications such as in sensors, organic photovoltaics, organic light emitting diodes (OLED), supercapacitor, and biomedical applications [13]. Some commonly deployed approaches for the synthesis of PTh include interfacial polymerization [14], template synthesis, selfassembly, chemical polymerization [15], gamma radiation-induced chemical oxidative polymerization [16], electrochemical polymerization [17–19], UV-irradiation [20], and so forth. Research findings have revealed that the morphology and physicochemical properties of PTh depends upon the mode of synthesis. Cheong et al. have reported the water dispersible poly(thiophene-co-3thiopheneacetic acid) nanoparticles synthesis by FeCl3/H2O2 catalyzed oxidative polymerization without using surfactant [21]. Ambade et al. have carried out a controlled growth of PTh nanofibers in TiO2 nanotube arrays by electrochemical

https://doi.org/10.1016/j.jiec.2019.04.045 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.

244

D.P. Bhattarai et al. / Journal of Industrial and Engineering Chemistry 77 (2019) 243–252

polymerization for supercapacitor applications [17]. Zong, et al. have shown that PTh synthesized at low temperature would have long and ordered polymer chain due to highly ordered p–p stacking and long conjugation length. They also mentioned that PTh synthesized at higher temperature possesses more structural defects [22]. The oxidative chemical polymerization method outweighs the electrochemical polymerization and other routes of PTh synthesis in view of bulk mass production. Therefore, chemical polymerization might be an appropriate method of polymerization where large-scale production turns out to be a prime concern rather than mere need of extreme conductivity. Many efforts have been made in the fabrication of PTh endowed with different geometries and morphologies such as PTh-nanoparticles, PTh-nanorods, PTh-nanowires, etc. [16]. These morphologies with different shape and size exhibit their great potentials for a variety of applications including tissue engineering where the cellular response within the niche of nanomaterials depends upon the physicochemical properties (shape, size, crystallinities) of nanomaterials, types of dopants, and surface functionality (cationic, anionic or neutral functional group) of that substrate [23]. This necessitates a detailed analysis of nanoparticles, their effect on cellular functions for the selection of appropriate candidate in support of tissue regeneration. Conducting polymers deserve its electrically controllable potential in a range of physical and chemical properties as well as promoting cell growth, adhesion and proliferation at polymer-tissue interface via electrical stimulation [24]. Electrical stimuli could be responded by some special cell-types such as neurons, osteoblasts, fibroblasts, and skeletal myoblasts (SMs) evolving a promising path for matching the special medical applications [25]. Furthermore, electrical conductivity in addition to composition, structure, surface characteristics such as surface roughness, surface charges, surface geometry (1D,2D geometry), surface topography, and surface chemistry of polymer can all affect the cellular interactions and corresponding responses with the substrates. The cellular response of nanomaterials is size-dependent and is associated with alveolo-capillary translocation via endocytosis or transcytosis or other cellular mechanisms [26]. Electrically responsive tissues sense electrical charges to stimulate proliferation or differentiation of various types of cells that are essential for cell imaging and other biological applications [27]. Interaction between the surface of nanomaterials and cells determines the fate of biocompatibility or cytotoxicity of the substrate. Toxic effects of nanomaterials upon long-term exposure into the body administration is a crucial factor to be evaluated to ensure its safe therapeutic applications [28]. Therefore, the first and foremost thing before stepping into biomedical applications of as-synthesized nanomaterials is to carry out its systematic and precise toxicity assessments [29]. Previously, conjugated conducting polymers were predominantly used in electronics and its related fields. However, with the advances in research findings, keen interests in these materials are expanding into bio-applications including tissue engineering. Pristine polythiophene or its derivatives or composites with different geometries and size domains have been reported for their uses in bio-applications [30,31]. Adhikari, et al. have developed gold nanoparticle-polythiophene composites and exhibited broadspectrum bactericidal activity [28]. Rincon, et al. have studied the fate of pre-osteoblast MC3T3-E1 cells on poly(3-octylthiophene) thin film and concluded the viability of the substrate for osteoblast attachment and proliferation [1]. Toxicity of nanomaterials can be assessed by varieties of methods, including the measurement of changes in cell morphology, cell viability, metabolic activities, and oxidative stress [32]. Basically, cytotoxicity of nanomaterials is measured by studying their cell morphology and cell viability under different concentration of the substrate.

In these perspectives, to avoid the potential toxicity and influence on physicochemical properties of substrate due to remnants of surfactants, we designed the formulation of PThNPs under a surfactant-free condition expecting its significant advantages of purity concern, low cost factor, and simple research design. The physicochemical properties of pristine PTh-NPs prepared under surfactant-free condition could act as a benchmark in the design and formulation of biomaterials such as bone tissue scaffolds, drug carrier or in derivatization of the nanoparticles. In this work, PTh-NPs were synthesized by chemical oxidative polymerization in presence of ammonium persulphate oxidant under surfactant-free condition. Furthermore, PTh-NPs synthesized without surfactant were compared to those with surfactant in some aspects to assess the benefits of surfactant-free route adoption. The cytotoxicity assessment was performed by studying cell viability and crystal violet cell staining of PTh-NPs treated MC3T3-E1, and PC12 cells compared to control phosphate buffer saline (PBS, pH 7.4). Experimental section Materials Thiophene (purity 99%) was purchased from Aldrich (Korea), ammonium persulfate (APS, purity >98.0%) was purchased from Dae Jung (Korea), sodium dodecyl sulphate (SDS, assay 34.5–38.5%) was purchased from Junsei, and acetonitrile (purity >99.5%) was purchased from Samchun (Korea). All materials and reagents were of analytical grade, and were used as received without any further purification. Preparation of polythiophene nanoparticles (PTh-NPs) In this work, 20 mL of 0.5 M ammonium persulphate solution was percolated into a vessel containing 30 mL of 0.5 M thiophene in 0.1 M acetonitrile solution with constant magnetic stirring. Then the vessel containing solution was left standing in a water bath maintained at 37
C for 1 week. The brownish black colored polythiophene (PTh) was extracted from the solution. The content was washed with methanol to remove unreacted acetonitrile and thiophene followed by washing with deionized water several times. For comparative study of PTh synthesized without surfactant to those with surfactant, 0.912 g sodium dodecyl sulphate (SDS) was added as surfactant in addition to the aforementioned composition of PTh synthesis. Then polymerization followed by successive purification processes were carried out under identical conditions. Hereafter, the polythiophene synthesized using SDS is termed as PTh-NPs/SDS. The PTh was dried in vacuum at 60
C for 12 h to achieve moisture free powdered polythiophene. Assynthesized PTh substrates were used for characterization and various tests. Physicochemical characterization Structural and surface morphology of as-synthesized PThNPs and PTh-NPs/SDS were studied using Field-emission scanning electron microscopy (FE-SEM, Carl Zeiss Supra-40 VP, Germany) at an accelerating voltage of 5.0 kV. Particle dimensions of various images of the same sample were measured using Image J software (NIH, USA) to obtain average dimension. Transmission electron microscopy (Bio-TEM, Hitachi H-7650, Japan) was used to observe the morphology of assynthesized PTh-NPs under an accelerating voltage of 100 kV. For this analysis, samples were prepared on C-flatTM carbon grids (Electron Microscopy Science, Hatfield, PA, USA). X-ray powder diffraction (XRD) patterns of samples were obtained

D.P. Bhattarai et al. / Journal of Industrial and Engineering Chemistry 77 (2019) 243–252

using Rigaku X-ray diffractometer (Japan) with Cu-Kα (l = 1.54 Å) radiation over Bragg’s angle (2u) of 5–90
. Polymerization of thiophene into polythiophene nanoparticles (PTh-NPs) was studied using Spectrum GX Fourier transform infra-red (FT-IR) spectroscope (Perkin Elmer, USA). FT-IR spectra were measured in the range of 4000–400 cm–1. Zeta potential and polydispersity index (P.I.) measurements of PTh-NPs and PTh-NPs/SDS were performed in distilled water using Zetasizer (Otsuka Zetasizer ELSZ-100 series, Japan). Substrate weight loss and heat flow as a function of temperature were studied using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), respectively. Thermal stability of PTh-NPs was studied using Q50 TGA device (TA instruments, USA). The sample under study was heated from 0 to 800
C at a heating rate of 10
C/min. Similarly, DSC was carried out using TMK 0017 universal analysis (TA Instruments, USA) to study the thermal behavior of the sample. The sample was scanned from –80 to 300
C under nitrogen gas flow at 50 mL/min for all runs. For the measurement of TGA/DSC, 12.5 mg of the sample was taken onto an aluminum pan. The initial scan was taken from 50 to 100
C to remove the effect of thermal history followed by cooling to room temperature. Ultra-violet absorption of PTh-NPs was studied using ultraviolet (UV)-Visible (Vis)-Near infra-red (NIR) spectroscopy (Scinco, USA) from 600 nm to 200 nm. A quartz cuvette with 1-cm path length was used to study the UV absorption of the sample.

245

Biomineralization study Bioactivity of as-synthesized PTh-NPs for bone tissue engineering application was investigated by in vitro biomineralization test. For this work, simulated body fluid (SBF) solution having pH 7.4 was prepared by dissolving the whole content of Hank’s balanced salt (Sigma-Aldrich, H2387) with anhydrous magnesium sulphate (0.097 g.), anhydrous sodium bicarbonate (0.350 g.), and anhydrous calcium chloride (0.185 g.) in distilled water to make oneliter solution [33]. The solution was filtered through a filter with 0.20 mm pore size (Minisart1 filter, non-pyrogenic, Sartorius stedim biotech) and was stored in cold (4
C). The solution was used for not more than one month after preparation. Three samples, each having one gram of PTh-NPs in 10 mL of as-prepared SBF solution was incubated at 37
C for five, ten and fifteen days respectively. The SBF solution was replaced by fresh SBF solution at every 24 h. After retrieving the sample from incubation at five, ten and fifteen days, each sample was washed with deionized water to remove loosely held residual mineral onto the surface of substrate and was kept dry. Cytotoxicity assay Mouse pre-osteoblast cell line (MC3T3-E1, newborn mouse calvarial derived cell line) and PC 12 cells (cell line derived from

Fig. 1. (A) FE-SEM (B) EDX, (C) Bio-TEM image, and (D) Bio-TEM image focused (few particles) of PTh-NPs. EDX mapping depicts the presence of carbon and sulphur as constituent elements of polythiophene nanoparticles. Spherical/oval PTh-NPs were observed in FE-SEM image which was supported by Bio-TEM characterization.

246

D.P. Bhattarai et al. / Journal of Industrial and Engineering Chemistry 77 (2019) 243–252

pheochromocytoma of rat adrenal medulla) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and used for cytotoxicity assay. The cells were grown in DMEM medium (Gibco-BRL, Gaithersburg, MD, USA) contained with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified CO2 incubator at 37
C. To evaluate cytotoxicity of PTh-NPs, MC3T3-E1 and PC12 cells (1 104 cells) were seeded in 96-well tissue culture plates and grown for 24 h, respectively. After cell culture media was refreshed, PTh-NPs with different concentration (10–200 mg/mL) were treated with cells. After treatment for 24 h, cell viability was evaluated using CCK8 assay according to the manufacture’s protocol (Dojinodo Molecular Technologies, Rockville, MD, USA). Mean and standard deviation values were calculated and as-obtained results were expressed in terms of percentage measurement. For crystal violet staining, PThNPs treated cells were fixed with 10% methanol and stained with 0.1% crystal violet solution for 10 min. After staining, cells were washed with PBS and observed under inverted microscope (DM IL LED; Leica Microsystems, Wetzlar, Germany). To compare the cytotoxicity between PTh-NPs and PTh-NPs/ SDS, CCK-8 assay was performed. Briefly, MC3T3-E1 and PC12 cells (1 104 cells) were seeded in 96-well plates, respectively. After 24 h of cultivation, 50 mg/mL of PTh-NPs and PTh-NPs/SDS were treated with cells and cell viability was evaluated at 24 h of cultivation. In the comparative cytotoxicity experiment, the 50 mg/ mL concentration was fixed at an optimal concentration, because it was a non-toxic concentration in both of MC3T3-E1 and PC12 cells. Results and discussion Physicochemical characterization Field Emission-Scanning Electron Microscopy: Structural and surface morphology of as-synthesized PTh-NPs and PTh-NPs/SDS were studied with the help of FE-SEM which clearly showed the spherical shape of the particles (Fig. 1A and S3). The average diameter of PTh-NPs was found to be 48.76  18.67 nm (n = 100) on the basis of measurement from Image J software (NIH, USA). However, some nucleated nanoparticles manifested a bit larger particle size, in some cases. The elemental analysis of the PTh-NPs was ascertained from energy-dispersive X-ray spectroscopy (EDX) analysis (Fig. 1B). The presence of carbon and sulphur in EDX mapping indicates these elements as constituents of PTh-NPs. The Bio-TEM image (Fig. 1(C, D)) of the PTh-NPs has shown the image of PTh-NPs in support of FE-SEM image. The Bio-TEM images also reveal the spherical as well as oval shaped PTh-NPs. Polymerization of thiophene is appreciable onward from about room temperature to as high as 60–70
C (boiling point of thiophene monomer 84
C) in agreement with many research findings [21,34,35]. Synthesis of polythiophene at higher temperature are prone to structural defects and formation of, possibly, larger particles size while those at relatively lower temperature are too slow for quantitative yield. After some pilot scale experiments, polymerization of thiophene was carried out at 37
C in view of minimizing structural defects and maximizing quantitative yield by preserving nano dimension of particles (Fig. S1). We examined the FE-SEM images of PTh-NPs for different duration (Fig. S2) of polymerization where formation of nanoparticles was obvious. However, the quantitative yield was found to be more with the increase of polymerization time. Furthermore, to examine the effect of surfactant in the synthesis of PTh-NPs, we compared the PTh-NPs and PTh-NPs/SDS in various aspects including FE-SEM image (Fig. S3). Uses of SDS surfactant, an anionic surfactant, manifested the formation of more spherical and homogenous polythiophene nanoparticles. Other aspects of PTh-NPs and PThNPs/SDS are discussed in coming sections.

Fig. 2. X–ray diffraction image of PTh-NPs and PTh-NPs/SDS.

X-Ray Diffractometry: The crystallinity/amorphous characters of as-fabricated PTh-NPs and PTh-NPs/SDS were evaluated by Xray diffraction measurement (Fig. 2). Both types of NPs exhibited almost similar crystalline behavior but PTh-NPs/SDS exhibited slightly higher peak intensity. In both cases, one major broad diffraction peak was observed at 2u value of 20–30
(centered ~24.20
). The chain-to-chain stacking distance of 2u value around 24.20
may have resulted due to the amorphously packed polythiophene main chain [36,37]. Fourier Transform Infra-Red spectroscopy: The polymerization of thiophene into polythiophene was ascertained by FT-IR spectroscopy by measuring the absorption peaks of the sample within the spectral range from 4000 to 400 cm–1 (Fig. 3(A), and (B)). The broad absorption peak at 3403 cm–1 corresponds to the OH stretching vibration of adsorbed water molecules [38,39]. Other IR peaks in the range of 3100–2800 cm–1 (peaks at 2959 and 2913 cm–1) attribute to the C H stretching vibration [16,36]. More pronounced absorption peak in PTh–NPs/SDS in the range of 3100–2800 cm–1 could be assigned to the aliphatic tail of SDS [35]. The large descending spectral region of 3700–2750 cm–1 was attributed due to free electron conduction in the doped state of the polymer [36,40]. The strong absorption peak at 1678 cm–1 corresponds to the aromatic C¼C stretching bond in polythiophene. The IR range of 1500– 600 cm–1 is the fingerprint region of PTh where many absorption peaks were observed [16]. IR band at 1475–1375 cm–1 (peak at 1407) [21], and around 704 cm–1 correspond to αC αC (C2 C5), C S bond in polythiophene, respectively [36]. Peak at 1217 cm–1 ascribes C C stretching vibration, peak at 1078 cm–1 ascribes stretching vibration of in-plane  C H bond, peak at 1045 cm–1 ascribes C S bond in polythiophene [41]. Similarly, the peaks observed at 1200– 1050 cm–1 corresponds to thiocarbonyl group (C¼S) group of polythiophene ring [42–44]. The IR absorption peaks for PTh-NPs and PTh-NPs/SDS were almost similar but with some difference in peak intensities. For instance, IR absorption of PTh-NPs/SDS at 1200– 1050 cm–1 exhibited more intense peak compared to PTh-NPs. The band around 800 cm–1 assigns for C S stretching vibration [35]. Similarly, increased peak intensity of PTh-NPs/SDS at 730–695 cm–1 (peak at 704) could be associated to the  CH2 bending of remnants of surfactant (dodecyl sulphate moieties) in addition to C S bond in polythiophene [37]. These FT-IR absorption peaks of as-synthesized substrates indicated the successful synthesis of polythiophene by chemical polymerization of thiophene under the aforementioned conditions. Zeta potential measurement Zeta potentials of the as-synthesized NPs were measured to study the electrophoretic mobility. The zeta potential recorded for

D.P. Bhattarai et al. / Journal of Industrial and Engineering Chemistry 77 (2019) 243–252

247

Fig. 3. FT-IR spectra of (a) PTh-NPs and (b) PTh-NPs/SDS. Figure (A) shows the FT-IR spectra within 4000–400 cm1 range and figure (B) mainly focuses the FT-IR spectra of PTh–NPs and PTh-NPs/SDS in finger print region.

PTh-NPs and PTh-NPs/SDS in aqueous solution were nearly –31.27 and –40.96 mV, respectively. The difference in zeta potential value could presumably be attributed to the difference in anionic charge adsorption onto the NPs. Sodium dodecyl sulphate is anionic surfactant and triggers the adsorption of anionic moieties onto the nanoparticle surface. The shifting of zeta potential towards more negative value correlates the further adsorption of anionic charge onto the PTh-NPs. The higher magnitude of zeta potential value signifies more stability of particles. Zeta potential of at least 30 mV (or, –30 mV) is expected to be good for a stable dispersion [45,46]. In this work, zeta potential values of PTh-NPs and PTh-NPs/SDS signifies the stable dispersion. The conductivity data recorded for PTh-NPs and PTh-NPs/SDS are near to agreement to the work of Jeon et al. [47] (Table 1). Thermogravimetric and Differential Scanning Calorimetry: Thermogravimetric analysis (TGA) and derivative thermogravimetric (DTG) curves were used to study the thermal behaviors such as weight loss, residue content and decomposition pattern of PTh-NPs as a function of temperature. Information relating to the thermal behavior of a substrate gives an idea in pre-formulation, processing, and modification stage of the substrate to estimate the potential physical and chemical interactions among the relevant chemicals. The TGA/DSC thermogram (Fig. 4A, and B) of assynthesized PTh-NPs revealed that only 3.39% weight loss of PTh occurred at 100
C which might be attributed to the removal of moisture, volatile components, adsorbed monomer molecule (boiling point of thiophene: 84
C), and dedoping of ions or molecules onto the sample. Major weight loss began after 197
C, retaining 44.49% and 36.31% residue at 600 and 795
C respectively. Though, no significant weight loss was observed up to 197
C, dedoping of ions could be associated with the loss of some important properties such as conductivity and surface charge. To account for the study of probable loss of doped ions, we did FESEM/EDX mapping (Fig. 4 C and D) of the dried PTh samples at two different temperatures of 60
C (normal drying temperature) and 120
C (temperature fairly above the boiling point of water and thiophene monomer) respectively. No contrast FE-SEM images of the samples revealed no structural deformation of polymer up to

Table 1 Different electro kinetic properties of PTh-NPs and PTh-NPs/SDS in aqueous media (n = 4). SN

Properties

PTh–NPs

PTh–NPs/SDS

1 2 3

Zeta potential (mV) Conductivity (mS/cm) Polydispersity index

–31.27  0.37 0.082  0.03 0.240  0.01

–40.96  0.70 0.021  0.01 0.303  0.01

that temperature. However, EDX mapping showed remarkable change in elemental compositions. Heating of PTh polymer from 60
C to 120
C showed decreased nitrogen and oxygen content but increased sulphur content. The decreased oxygen content could be associated with the loss of moisture (H2O) and oxygenated doped ions such as persulphate (source; ammonium persulphate). The decreased nitrogen content in the EDX mapping could be associated with the loss of nitrogenated ions such as ammonium ions (source, ammonium persulphate) and nitrile ions (source, acetonitrile). The increased sulphur content could be associated with the retention of sulphur in polythiophene chain (retention of sulphur could outweigh the minor loss of sulphur from dedoping of persulphate ions and thiophene monomer). Properties such as conductivity and zeta potential are related to the doped ions as evidenced from the zeta potential and conductivity measurement. That is why, diminish of conductivity and surface charge might be possible as a result of dedoping of some sorts of ions. Basically, the thermogram showed a single stage major mass loss occurrence with some other small mass loss episodes. The substantial weight loss started after 197
C exhibiting maximum rate of weight loss at 235
C as indicated by the upsurge peak in derivative thermogram and almost exponential thermal degradation thereafter. The TGA curve assigns that nearly 50% mass retention of the substrate occurred at 505
C in the study of thermal behavior as a function of temperature. These results showed the remarkable thermal stability of polythiophene in view of bio-application. The differential scanning calorimetry (DSC) curve (Fig. 4B) showed the glass transition temperature (Tg) of PTh as 51.06
C. Tg of a substance signifies an onset temperature for translation motion of chain segment in a polymer. Lower degree of polymerization leads to lower molecular mass corresponding to a lower Tg value for the polymer. With the progress of heating, one endothermic peak was observed at 257.84
C in the DSC thermogram. An endothermic transition peak observed at 310
C might be associated to crystallization. It could be due to ordering of chains in PTh. Upon progress of heating, endothermic peaks were observed in different steps corresponding to melting of crystalline phase relevant to the size and perfection of crystalline lamellae [48]. A Tg value well above room temperature defined a rigid structural property of as-synthesized substrate [49]. Ultra-Violet visible (UV–vis) spectra Herein, UV–vis absorption spectra were studied to avail more information about electronic structure of the conjugated polymers. In aqueous medium, PTh-NPs showed two broad absorption peaks of UV–vis spectra (Fig. 5) at 240 nm (UV region), and at 409 nm

248

D.P. Bhattarai et al. / Journal of Industrial and Engineering Chemistry 77 (2019) 243–252

Fig. 4. (A) TGA and DTGA (B) DSC curves of PTh-NPs. TGA (curve ‘a’) and DTGA (curve ‘b’) exhibited a good thermal stability of PTh-NPs. DSC image exhibited the glass transition temperature (Tg) 51.06
C. FE-SEM image of PTh-NPs heated at (C) 60
C (D) 120
C with corresponding EDX spectra. EDX spectra reveal that heating of PTh-NPs at 120
C could be associated with the loss of some doped ions.

polymer synthesis such as temperature, concentration, mode of synthesis, and so on [51]. Biomineralization study

Fig. 5. UV-Vis spectra of PTh–NPs. From UV–vis spectra two absorption peaks at 240 nm (UV region) and 409 nm (Visible region) indicate p–p stacking conformation imparting planar arrangement in PTh-NPs.

(Visible region). These UV–vis absorption bands are associated with p–p stacking conformation imparting planar arrangement in PTh and their resulting morphology [50]. Such conformations and conjugation length are the function of ambient condition of

Biomineralization is one of the most important indicators for the application of a substance in bone tissue engineering. When substrates for bone tissue application are introduced into the body administration, mineralization takes place over the substrate mimicking the microstructure of native bone. Calcium, phosphate, and carbonate are the most common minerals being deposited during mineralization process. Hydroxyapatite (HA) and fitricalcium phosphate (fi-TCP) are the most common varieties of calcium phosphate deposition. More often, the atomic ratio of calcium to phosphorus is measured to assess the microstructure similarity to hydroxyapatite composition of natural bone. Fig. 6 shows FE-SEM, EDX and FT-IR of biomineralized PTh-NPs for 15 days. FE-SEM image and EDX mapping revealed the minerals coating over the PTh-NPs with distinct particles size. The average size of biomineralized polythiophene particles evaluated was 164.62  49.62 nm (n = 100). This size is roughly three times the original size of PTh-NPs used. The EDX mapping of the incubated PTh-NPs substrate showed the presence of carbon (C), phosphorus

D.P. Bhattarai et al. / Journal of Industrial and Engineering Chemistry 77 (2019) 243–252

249

Fig. 6. Biomineralization study. (A) FE-SEM image (B) FT-IR spectra of biomineralized PTh-NPs and (C) EDX mapping. The FE-SEM images show the mineralization of PTh-NPs consequently increasing the particle size. The FT-IR spectra reveal the nucleation of hydroxyl, carbonate and phosphate groups over the NPs.

(P), sulphur (S), and calcium (Ca) as major elements where carbon and sulphur are inherent constituents of polythiophene while calcium and phosphorus are the constituents of biomineralized salts over the surface of the PTh-NPs substrate. Though the size of biomineralized PTh-NPs recorded was roughly three times the size of the original PTh-NPs used, EDX reports depict relatively low weight percentages deposition of phosphorus and calcium. This anomaly could be associated with the aggregation of polythiophene nanoparticles in addition to the slow deposition of biominerals such as calcium phosphates or hydroxyapatites over these aggregated particles during the course of biomineralization. The aggregation propensity of PTh-NPs can be explained on the basis of zeta potential and polydispersity index values. The zeta potential exhibited by PTh-NPs was –31.27 mV and polydispersity index recorded was 0.240. These data suggest that PTh-NPs exhibit moderate stability. PTh-NPs are likely to be aggregated to some extent in addition to deposition of mineral salts when immersed into SBF solution maintained at 37
C incubation temperature. The calcium to phosphorus molar ratio of the deposited mineralized salt in fifteen days was calculated to be 1.42 which is near to the standard value 1.67 [52]. This shows a good proportion of calcium phosphorus deposition over the nanoparticles. Further attempts were made to ensure the nucleation of phosphate, hydroxyl, and carbonate moieties in the mineralization process by FT-IR (Fig. 6B). The broad peak around 3433.27 cm–1 corresponds to the stretching vibration of  OH moieties in HA. The peak at 1659.67 cm–1 (Biomineralized PTh-NPs) indicates the slight shifting of the peak from 1678 cm–1 (C¼C bond in polythiophene chain) towards 1637.39 cm–1 (C¼O peak of fi-TCP) and peak at 1418 cm–1 indicates the absorbed carbon dioxide molecules. The peak at 1133.36 cm–1 corresponds to the stretching mode of P O bond in PO43– –1 moieties. The solder peak at 872.68 cm corresponds to the HPO42– moieties which could be formed by protonation of

phosphate moieties in aqueous media [33]. Oxidative polymerization of thiophene into polythiophene develops a net surface positive charge which can develop coulombic forces of attraction towards anionic moieties to impart surface negative charge. Furthermore, primarily doped anionic moieties and lone pair electrons of sulphur in thiophene molecules could assist in the exertion of coulombic forces of attraction towards cationic moieties such as calcium. Such types of coulombic forces of attraction lead to biomineralization over the substrate. Presence of calcium and phosphorus, known from EDX mapping, as well as phosphate moieties, known from FT-IR, showed the nucleation of calcium phosphate over the substrate during SBF incubation. Though there are many pre-requisites for a substance to be used as a biomaterial in bone tissue engineering, the deposition of calcium and phosphate group over the substrate is a good support for in vitro cell attachment as well as production of extracellular matrix (ECM). In addition, the nucleation of calcium, hydroxyl, carbonate and phosphate moieties might increase the hydrophilicity and bone tissue formation. This result showed that PTh-NPs could support the biomineralization though its efficiency has yet to be improved for more performance. Cytotoxic evaluation The in vitro cytotoxicity of PTh-NPs was investigated using MC3T3-E1 cells and PC12 cells. Herein, cytotoxicity data are presented in terms of cell viability of % control (PBS, pH 7.4). For further confirmation of cytotoxicity on each kind of cells, we observed the PTh-NPs treated cells using crystal violet staining. Study on MC3T3-E1 cells: The results of cytotoxicity assay of PTh-NPs on pre-osteoblast cells after 24 h treatment with reference to phosphate buffer saline (PBS, pH 7.4) as control is shown in the Fig. 7(A) and crystal violet cell staining of PTh-NPs

250

D.P. Bhattarai et al. / Journal of Industrial and Engineering Chemistry 77 (2019) 243–252

Fig. 7. Cytotoxic evaluation of PTh-NPs with reference to MC3T3-E1 cell line. (A) CCK-8 assay. (B) Inverted microscopy images of crystal violet staining. CCK-8 result assured that when concentration of PTh-NPs is 25 mg/mL the cell viability was optimum. Thereafter increasing amount of NPs showed decreasing cell viability and cytotoxicity effect was statistically significant at 200 mg/mL PTh-NPs concentration as compared to control (PBS, pH 7.4) as shown by the CCK-8 result and microscopy images of crystal violet staining. In this study, all the data were observed at p* < 0.01 with student t-test. The cytotoxicity assay was performed in triplicate (n = 3).

treated MC3T3-E1 cells is shown in Fig. 7(B). PTh-NPs did not show any cytotoxicity on MC3T3-E1 cells at 10–100 mg/mL concentration, rather cell proliferation slightly increased. However, treatment with 200 mg/mL of PTh-NPs had a statistically significant cytotoxicity (78  3.8% cell viability of control) on MC3T3-E1 cells (p** < 0.01) (Fig. 7A). For more confirmation of cytotoxic assay on MC3T3-E1, we observed the PTh-NPs treated MC3T3-E1 cells using crystal violet staining. Crystal violet stains nuclei a deep purple color and assists in their visualization. A number of cells were found dead and detached at 200 mg/mL of PTh-NPs in MC3T3-E1 cells (Fig. 7B). These crystal violet stained results were consistent with the quantitative CCK-8 results. Study on PC12 cells: The results of cytotoxicity assay of PTh-NPs on nerve cells after 24 h treatment with reference to phosphate buffer saline (PBS, pH 7.4) as control is shown in the Fig. 8(A) and crystal violet cell staining of PTh-NPs treated PC12 cells is shown in Fig. 8(B). With PTh-NPs treated PC12 cell, PTh-NPs showed a cytotoxicity at 100 mg/mL (86.4  1.8% viability of control) and 200 mg/mL (77.6  8.6% of viability), respectively (Fig. 8A). For more confirmation of cytotoxic assay on PC12 cells, we observed the PTh-NPs treated PC12 cells using crystal violet staining. The crystal violet stains nuclei a deep purple color and assisted in their visualization. A number of cells were found dead and detached at 100 and 200 mg/mL of PTh-NPs in PC12 cells (Fig. 8B). These crystal violet stained results were consistent with the quantitative CCK-8 results. Comparative cytotoxicity assay of PTh-NPs and PTh-NPs/SDS on MC3T3-E1 and PC12 cell lines: For comparative cytotoxic

evaluation, each 50 mg/mL concentration of PTh-NPs and PThNPs/SDS was fixed at an optimal concentration based on the aforementioned results on MC3T3-E1 and PC12 cells. In this study, PTh-NPs did not show any cytotoxicity on MC3T3-E1 cells, but when treated with PTh-NPs/SDS, the cell viability markedly decreased to 81.9% when compared with controls (Fig. 9A and B). Moreover, the cytotoxic effect of PTh-NPs/SDS was also observed in PC12 cells, and the viability was decreased to 87.9%. These results show that PTh-NPs prepared using SDS surfactant have a cytotoxicity. The cytotoxicity effect might be associated with the surface characteristics and remnant of surfactant onto the nanoparticles. Some research findings also have reported the cellular toxicity of SDS [53]. Sayed et al. mentioned the toxic effect of SDS reporting its hepatic and renal dysfunction on animal model [54]. In this context, PTh-NPs synthesized without surfactant exhibited less cytotoxicity towards the MC3T3-E1 and PC12 cells. Cytotoxicity/cell viability of a substrate depends upon morphology, surface composition, shape, size, and concentration (dose response). Cell viability, sometimes, has different results depending upon the types of cell lines [55,56]. Herein, concentrationdependent and cell lines-dependent cytotoxic evaluation of PThNPs were studied. Results showed that PTh-NPs at 200 mg/mL, and 100 mg/mL concentration exhibited statistically significant toxicity on pre-osteoblast MC3T3-E1 and PC12 cells (cell viability measured in terms of % of control), respectively. Cell viability was significantly higher than 100% when the concentration of PThNPs was up to 100 mg/mL and 50 mg/mL towards MC3T3-E1 and PC12 cell lines treatment, respectively. In this study, we

Fig. 8. Cytotoxic evaluation of PTh-NPs with reference to PC12 cell line. (A) CCK-8 assay. (B) Inverted microscopy images of crystal violet staining. CCK-8 result assured that, the cell viability was not significantly affected up to 50 mg/mL concentration of PTh-NPs but after then the increased concentration of NPs showed the increased cytotoxicity level and at 200 mg/mL concentration, NPs showed the significant cytotoxicity as compared to control (PBS, pH 7.4) which was also supported by the inverted microscopy image results. In this study, all the data were observed at p* < 0.01 with student t-test. The cytotoxicity assay was performed in triplicate (n = 3).

D.P. Bhattarai et al. / Journal of Industrial and Engineering Chemistry 77 (2019) 243–252

251

Fig. 9. Cytotoxic evaluation of PTh-NPs and PTh-NPs/SDS (50 mg/mL concentration) with reference to MC3T3-E1 and PC12 cell line. CCK-8 assay for (A) MC3T3-E1 cells, and (B) PC12 cells. CCK-8 result assured that, PTh-NPs/SDS exhibited significant cytotoxicity as compared to PTh-NPs and control (PBS, pH 7.4). In this study, all the data were observed at p* < 0.01 with student t-test. The cytotoxicity assay was performed in triplicate (n = 3).

represented the cell viability results as a % of control, the value more than 100% means that the cell proliferation was induced by PTh-NPs. Though this study did not directly focus on cellular mechanism of cell proliferation, our result on increased cell proliferation might be due to the effect of PTh material properties on biological behavior like cell attachment and spreading besides other physicochemical properties mentioned above [57]. More specifically, 100 mg/mL concentration of PTh-NPs exhibited cytotoxicity on the neural cells but increased the cell proliferation on pre-osteoblast cells. Furthermore, the level of cytotoxicity of PThNPs on both types of cells was nearly same at a concentration of 200 mg/mL (78  3.8% viability of control on MC3T3-E1 cells, and 77.6  8.6% viability of control on PC12 cells). With regards to the assay, PTh-NPs showed more compatibility on pre-osteoblast MC3T3-E1 cells than that on PC12 cells. Based on the outcomes from these two cell lines, this result showed a better cytocompatibility of PTh-NPs as compared to the result observed by other groups on similar conductive polymer, polypyrrole nanoparticles (polypyrrole nanoparticles showed dose-dependent (5–15 mg/mL) cytotoxicity on fibroblast cells) [58]. This result shows insights into, mainly, two things: concentration-dependent cytotoxicity, and cell-type-dependent cytotoxicity. The fundamental discussion for cytotoxicity of biomedical polymer onsets from the interaction between polymer surface and cells. Proteins are regarded as fundamental actor in the mediation of polymer-cell interactions. Therefore, it is the chemistry of the protein associated with a particular cell which determines the fate of cell-polymer interaction concluding cytocompatibility or cytotoxicity [59]. In a particular set of cell-NPs interaction, deposition of NPs at high concentration may lead to metabolic disturbance and toxicological outcomes, ultimately leading to cell death. AshaRani et al. also reported similar types of results in their research findings [32]. In this work, 100 mg/mL concentration of PTh-NPs exhibited cell proliferation on pre-osteoblast cells while statistically significant cytotoxicity on PC12 cells. This revealed the cell-specific response of PTh-NPs. It regards that what a cell sees as it faces with NPs should likely depend on the cell lines which is in agreement with the finding of others’ work [60]. The cytotoxicity response of the different target cells could be associated with the different cell physiology and cell functioning. Different types of cell respond to the foreign objects differently, and influence the NPs uptake accordingly resulting in the different fate of physiological action. On the basis of the results observed for pre-osteoblast MC3T3-E1 cells, and PC12 cells, 50 mg/mL concentration of PTh-NPs could be a limiting upper concentration (cell viability of MC3T3-E1-, and PC12 cells are 109.50  5.49 and, 98.77  3.69 in terms of percentage of control).

For the application of biomedical substrates, one of the major concerns from clinical applications point of view is that physiological systems work in co-relation rather than independently. For instance, brain-derived neurotrophic factor (BDNF) promotes human bone mesenchymal stem cells (hBMSCs) osteogenesis and neurogenesis developing a fundamental interconnection between bone tissues and nerve tissues [61]. Neurovascularized bone tissue scaffolds can mimic the natural skeletal tissues to recapitulate intricate micro-environment of bone tissue [62]. In this context, cytotoxic evaluation of the substrate with respect to bone cells as well as neural cells is more informative rather than an assay of either type only. Our findings of cell viability results on pre-osteoblast cells and neuronal cells support the application of PTh-NPs for bone tissue engineering. This argument is further supported by nucleation of mineralized salt in biomineralization assay. However, further optimization and a number of successive tests are always open to research to reach up to the clinical application phase. Conclusions In this study, PTh-NPs were successfully synthesized by controlled oxidative chemical polymerization method under surfactant-free conditions. As-synthesized PTh-NPs exhibited better cell viability compared to those with surfactant. Cell viability assay suggested the potential application of assynthesized NPs for bone tissue engineering. This type of nanoparticles can be immobilized on an implant surface or can be incorporated into membranes or can be used locally in the improvement of bone tissue regeneration, or to enhance osseointegration of implants. Furthermore, this type of nanoparticles might be useful in the treatment of systemic bone diseases like osteoporosis. The results showed that as-synthesized PTh-NPs can support growth of both MC3T3-E1 and PC12 cells. Since as-synthesized PTh-NPs could support the cytocompatibility for both bone cells and nerve cells, it can be a candidate for bone tissue engineering application. Though nanoparticles alone might not be effective in its usual form for the fabrication of a bone tissue scaffold, it can be used as a part of composite material to develop desirable physicochemical properties, cell labeling, drug delivery, and gene delivery. Bioactive molecules can be incorporated into such types of nanoparticles to influence osteoblast differentiation. This finding opens a new avenue in fundamental understanding on concentration-dependent and cell-type-dependent cytotoxicity of PTh-NPs. Further optimization, development, and extension of the present work would be considered in future to design the functionalized polythiophene applicable for specific applications.

252

D.P. Bhattarai et al. / Journal of Industrial and Engineering Chemistry 77 (2019) 243–252

Acknowledgement This research was supported by grant from the Basic Science Research Program through National Research Foundation of Korea (NRF) by Ministry of Education, Science and Technology (Project no. 2016R1A2A2A07005160). And also, partially supported by the program for fostering next-generation researchers in engineering of National Research Foundation of Korea (NRF) funded by the Ministry of Science, (Project no. 2017H1D8A2030449). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jiec.2019.04.045. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]

C. Rincón, J.C. Meredith, Macromol. Biosci. 10 (2010) 258. D. Tuncel, H.V. Demir, Nanoscale 2 (2010) 484. J. Pecher, S. Mecking, Chem. Rev. 110 (2010) 6260. S.J. Lee, J.M. Lee, H.-Z. Cho, W.G. Koh, I.W. Cheong, J.H. Kim, Macromolecules 43 (2010) 2484. H.S. Nalwa, Handbook of Nanostructured Materials and Nanotechnology, FiveVolume Set, Academic Press, 1999. K. Uleanya, A. Eboatu, Pac. J. Sci. Technol. 15 (2014) 200. D.P. Bhattarai, A.P. Tiwari, B. Maharjan, B. Tumurbaatar, C.H. Park, C.S. Kim, J. Colloid Interface Sci. 534 (2019) 447. E. Smela, Adv. Mater. 15 (2003) 481. T. Nezakati, A. Seifalian, A. Tan, A.M. Seifalian, Chem. Rev. 118 (2018) 6766. M. Gajendiran, J. Choi, S.-J. Kim, K. Kim, H. Shin, H.-J. Koo, K. Kim, J. Ind. Eng. Chem. 51 (2017) 12. P.A. Peart, L.M. Repka, J.D. Tovar, Eur. J. Org. Chem. 2008 (2008) 2193. M. Jaymand, R. Sarvari, P. Abbaszadeh, B. Massoumi, M. Eskandani, Y. BeygiKhosrowshahi, J. Biomed. Mater. Res. A 104 (2016) 2673. M. Hatamzadeh, M. Jaymand, RSC Adv. 4 (2014) 16792. C. Bora, R. Pegu, B.J. Saikia, S.K. Dolui, Polym. Int. 63 (2014) 2061. H. Guo, H. Zhu, H. Lin, J. Zhang, L. Yu, J. Disper. Sci. Technol. 29 (2008) 706. M.R. Karim, K.T. Lim, C.J. Lee, M.S. Lee, Synth. Met. 157 (2007) 1008. R.B. Ambade, S.B. Ambade, N.K. Shrestha, R.R. Salunkhe, W. Lee, S.S. Bagde, J.H. Kim, F.J. Stadler, Y. Yamauchi, S.-H. Lee, J. Mater. Chem. A 5 (2017) 172. X. Wang, G. Shi, Y. Liang, Electrochem. Commun. 1 (1999) 536. S. Aeiyach, E.A. Bazzaoui, P.-C. Lacaze, J. Electroanal. Chem. 434 (1997) 153. Y. Yagci, F. Yilmaz, S. Kiralp, L. Toppare, Macromol. Chem. Phys. 206 (2005) 1178. H.W. Ryu, Y.S. Kim, J.H. Kim, I.W. Cheong, Polymer 55 (2014) 806. X. Zong, X. Miao, S. Hua, L. An, X. Gao, W. Jiang, D. Qu, Z. Zhou, X. Liu, Z. Sun, Appl. Catal. B Environ. 211 (2017) 98. W.-K. Oh, S. Kim, M. Choi, C. Kim, Y.S. Jeong, B.-R. Cho, J.-S. Hahn, J. Jang, ACS Nano 4 (2010) 5301. A.-D. Bendrea, L. Cianga, I. Cianga, J. Biomater. Appl. 26 (2011) 3. Z.-B. Huang, G.-F. Yin, X.-M. Liao, J.-W. Gu, Front. Mater. Sci. 8 (2014) 39.

[26] A. Nel, T. Xia, L. Mädler, N. Li, Science 311 (2006) 622. [27] L. Cianga, A.-D. Bendrea, N. Fifere, L.E. Nita, F. Doroftei, D. Ag, M. Seleci, S. Timur, I. Cianga, RSC Adv. 4 (2014) 56385. [28] M.D. Adhikari, S. Goswami, B.R. Panda, A. Chattopadhyay, A. Ramesh, Adv. Healthc. Mater. 2 (2013) 599. [29] Y.S. Jeong, W.-K. Oh, S. Kim, J. Jang, Biomaterials 32 (2011) 7217. [30] M.R. Karim, C.J. Lee, M.S. Lee, J. Polym. Sci. A Polym. Chem. 44 (2006) 5283. [31] U. Baig, M.A. Gondal, M.F. Alam, M. Alam, W.A. Wani, H. Younus, Int. J. Polym. Mater. Polym. Biomater. 66 (2017) 243. [32] P.V. AshaRani, G. Low Kah Mun, M.P. Hande, S. Valiyaveettil, ACS Nano 3 (2009) 279. [33] D.P. Bhattarai, S. Shrestha, B.K. Shrestha, C.H. Park, C.S. Kim, Chem. Eng. J. (2018). [34] Z. Wang, Y. Wang, D. Xu, E.S.-W. Kong, Y. Zhang, Synth. Met. 160 (2010) 921. [35] K. Kadac, J. Nowaczyk, J. Appl. Polym. Sci. 133 (2016). [36] O. Zabihi, A. Khodabandeh, S.M. Mostafavi, Polym. Degrad. Stab. 97 (2012) 3. [37] S. Iqbal, J. Shah, R.K. Kotnala, S. Ahmad, J. Alloys Compd. 779 (2019) 487. [38] K. Wu, J. Zhao, R. Wu, B. Ruan, H. Liu, M. Wu, J. Electroanal. Chem. 823 (2018) 527. [39] S.R.P. Gnanakan, M. Rajasekhar, A. Subramania, Int. J. Electrochem. Sci. 4 (2009) 1289. [40] S.J. Lee, J.M. Lee, I.W. Cheong, H. Lee, J.H. Kim, J. Polym. Sci. A Polym. Chem. 46 (2008) 2097. [41] R. Liu, Z. Liu, Chin. Sci. Bull. 54 (2009) 2028. [42] M.G. Han, S.H. Foulger, Adv. Mater. 16 (2004) 231. [43] Y.A. Udum, K. Pekmez, A. Yıldız, Synth. Met. 142 (2004) 7. [44] F. Mohammad, J. Phys. D Appl. Phys. 31 (1998) 951. [45] C. Jacobs, O. Kayser, R.H. Müller, Int. J. Pharmaceut. 196 (2000) 161. [46] J.D. Clogston, A.K. Patri, in: S.E. McNeil (Ed.), Zeta Potential Measurement, Humana Press, Totowa, NJ, 2011 p. 63. [47] S.S. Jeon, S.J. Yang, K.-J. Lee, S.S. Im, Polymer 51 (2010) 4069. [48] F. Senatov, K. Niaza, M.Y. Zadorozhnyy, A. Maksimkin, S. Kaloshkin, Y. Estrin, J. Mech. Behav. Biomed. Mater. 57 (2016) 139. [49] D.S. Kelkar, A.B. Chourasia, Indian J. Phys. 86 (2012) 101. [50] Y. Yao, J. Gao, F. Bao, S. Jiang, X. Zhang, R. Ma, RSC Adv. 5 (2015) 42754. [51] R. Yang, S. Wang, K. Zhao, Y. Li, C. Li, Y. Xia, Y. Liu, Polym. Sci. B 59 (2017) 16. [52] Y. Song, S. Zhang, J. Li, C. Zhao, X. Zhang, Acta Biomater. 6 (2010) 1736. [53] J.C. Garay-Jimenez, A. Young, D. Gergeres, K. Greenhalgh, E. Turos, Nanomed. Nanotechnol. Biol. Med. 4 (2008) 98. [54] A.E.-D.H. Sayed, M.M.N. Authman, Ecotoxicol. Environ. Saf. 163 (2018) 136. [55] K.B. Riaz Ahmed, A.M. Nagy, R.P. Brown, Q. Zhang, S.G. Malghan, P.L. Goering, Toxicol. In Vitro 38 (2017) 179. [56] J. Jeevanandam, A. Barhoum, Y.S. Chan, A. Dufresne, M.K. Danquah, Beilstein J. Nanotechnol. 9 (2018) 1050. [57] D.-F. Li, H.-J. Wang, J.-X. Fu, W. Wang, X.-S. Jia, J.-Y. Wang, J. Phys. Chem. B 112 (2008) 16290. [58] A. Vaitkuviene, V. Kaseta, J. Voronovic, G. Ramanauskaite, G. Biziuleviciene, A. Ramanaviciene, A. Ramanavicius, J. Hazard. Mater. (2013) 250–251 167–174. [59] H. Chen, L. Yuan, W. Song, Z. Wu, D. Li, Prog. Polym. Sci. 33 (2008) 1059. [60] S. Laurent, C. Burtea, C. Thirifays, U.O. Häfeli, M. Mahmoudi, PLoS One 7 (2012) e29997. [61] D. Bhattarai, L. Aguilar, C. Park, C. Kim, Membranes 8 (2018) 62. [62] A. Marrella, T.Y. Lee, D.H. Lee, S. Karuthedom, D. Syla, A. Chawla, A. Khademhosseini, H.L. Jang, Mater. Today 21 (2018) 362.