Biocompatibility and antioxidant activity of polypyrrole nanotubes

Synthetic Metals 189 (2014) 119–125

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Biocompatibility and antioxidant activity of polypyrrole nanotubes J. Upadhyay a , A. Kumar a,∗ , B. Gogoi b , A.K. Buragohain b,c a

Department of Physics, Tezpur University, Napaam, Tezpur, 784028, Assam, India Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Tezpur, 784028, Assam, India c Dibrugarh University, Dibrugarh, 786004, Assam, India b

a r t i c l e

i n f o

Article history: Received 19 October 2013 Received in revised form 6 December 2013 Accepted 7 January 2014 Keywords: Polypyrrole nanotubes Antioxidant activity Biocompatibility High resolution transmission electron microscopy Haemolysis

a b s t r a c t In the present study antioxidant activity of polypyrrole nanotubes of varying diameter has been investigated. The haemolysis prevention efficiency of the polypyrrole nanotube having highest antioxidant activity has also been studied. Polypyrrole nanotubes have been prepared by chemical polymerization method using FeCl3 as oxidant with methyl orange (MO) and Cetyl trimethylammonium bromide (CTAB). Structural properties of polypyrrole nanotubes with varying CTAB concentrations have been studied by X-ray diffraction pattern and high resolution transmission electron microscopy (HRTEM). The HRTEM micrographs of polypyrrole nanotubes show significant decrease in diameter upon increase in CTAB concentration. Micro-Raman spectroscopy has been performed to understand the polymer conformation. We demonstrate the antioxidant activity of polypyrrole nanotubes by DPPH free radical assay. The biocompatibility of the polypyrrole nanotubes have been investigated via haemolysis assay. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The past decade has witnessed rapid growth in research on conducting polymer nanostructures motivated by their exceptional electrochemical and optical properties which arise due to their highly п-conjugated polymeric chains [1]. Owing to their good thermal stability and facile synthesis the conducting polymers have been receiving significant attention [2]. The important properties of the conducting polymers stimulated research for exploring these polymers for use in optical and electronic nanodevices and as interesting material for biosensing applications [3]. Considerable efforts have been made to explore the possibility of improved properties by fabricating different nanostructures of conducting polymers. Among the conducting polymers, polypyrrole is by far the most extensively studied in recent years. The intense focus on polypyrrole is attributed to its excellent redox activity, good thermal stability, biocompatibility and low toxicity [4]. Polypyrrole has emerged as a promising material for several applications including batteries, supercapacitors, sensors, light emitting diodes, conductive fabrics and newly in biomedical field as antioxidant or antimicrobial agent [5,6]. Reactive oxygen and nitrogen species are produced in the cell of human body as a consequence of certain undesirable stimuli. This leads to the imbalance between the oxidative and the antioxidant system resulting in tissue damage. This is known as oxidative

∗ Corresponding author. Tel.: +91 3712275553; fax: +91 3712267006. E-mail addresses: [email protected], ask [email protected] (A. Kumar). 0379-6779/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.synthmet.2014.01.004

stress [7]. Oxidative stress has harmful effects on the aging process and is responsible for several diseases like cancer, cardiovascular disease, neurodegenerative disorder, diabetes, etc. Reactive oxygen species, such as superoxide anion (• O2 − ), hydroxyl radical (OH• ) and hydrogen peroxide (H2 O2 ) originating from normal metabolic processes are responsible for the peroxidation of membrane lipids which lead to the accumulation of lipid peroxides [8]. Likewise free radicals also cause food degradation. Lipid oxidation by free radicals during storage or food processing is one of the key causes of deterioration of foods [9]. Antioxidants are added to food containing unsaturated fat to increase its shelf life. Polypyrrole can be switched on in different oxidation states, which motivates the use of polypyrrole as antioxidant material. It is understood that electron rich polypyrrole can donate electron to the free radical and is thus capable of inhibiting oxidative degradation. The pronounced antioxidant activity can be achieved by nanostructuring of materials with higher surface to volume ratio with consequent larger active sites for the free radicals. Several new approaches have been considered in the field of free radicals/antioxidant for the improvement in food storage as well as human health. Many novel approaches are made to enhance the antioxidant activity of conducting polymers and significant findings have come to light in recent few years [10]. Gizdavic-Nikolaidis et al. [11] reported microwave assisted synthesis of copolymers of aniline and 2-aminobenzoic acid or 2-aminosulfonic acid for antioxidant applications. The effects of thermal treatment on antioxidant activity of polyaniline have been investigated by Ashveen V. and his group [12]. The radical scavenging activity of polyaniline decreases slowly upto 200 ◦ C,

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beyond which a rapid fall is observed. This rapid decrease in the antioxidant property of polyaniline is attributed to the oxidation of polymer during heating run. Chu et al. [13] investigated the ABTS•+ scavenging activity of polypyrrole, polyaniline and poly(3,4-ethylenedioxythiophene) powders. They have reported that conducting polyaniline showed best antioxidant activity followed by polypyrrole and poly(3,4-ethylenedioxythiophene). The greater radical scavenging activity of polyaniline and polypyrrole than that of poly(3,4-ethylenedioxythiophene) is attributed to the presence of N-H group in their structure [13]. In the present study the effect of diameter on the antioxidant activity of polypyrrole nanotubes has been investigated. Polypyrrole nanotubes of varying diameters have been synthesized by the reactive self degrade methyl orange–ferric chloride (MO-FeCl3 ) template method in the presence of cationic surfactant CTAB. The antioxidant activity of polypyrrole nanotubes of different diameter have been investigated by DPPH free radical method. Haemolysis assay is employed to assess and compare the human red blood cell compatibility with the polypyrrole nanotubes that exhibit best antioxidant activity. 2. Experimental 2.1. Synthesis of polypyrrole nanotubes Polypyrrole nanotubes have been synthesized by the reactive self degrade MO-FeCl3 template method discussed elsewhere [14]. MO-FeCl3 complex acts as template in the formation of polypyrrole nanotubes which degrades automatically during polymerization. Polymerization of pyrrole takes place over the template due to the presence of FeCl3 (oxidant). CTAB controls the diameter of nanotubes by partially solubilising the template [15]. Polymerization was carried out for 24 h at room temperature. The resultant product was washed with ethanol several times to remove residual reagents. Finally the product was vacuum dried at room temperature for 48 hrs. 2.2. Apparatus The structural morphology of polypyrrole nanotubes was visualized by using a HRTEM model JEOL JEM 2100 at an accelerating voltage of 200 kV. For this purpose a drop of prepared sample was placed on a copper grid following solvent evaporation in ambient air at room temperature. X-ray diffractograms were recorded using Rigaku miniflex X-ray diffractometer with Cu K␣ radia˚ The scan rate, accelerating voltage and current tion ( = 1.5406 A). were kept at 5◦ /min, 30 kV and 15 mA, respectively during the experiment. Micro- Raman spectra were recorded in the range 600–1800 cm−1 using Renishaw in-via spectrometer (Rensihaw, Wotton-under-Edge, UK) at a resolution of 0.3 cm−1 . Ar+ ion laser of 514.5 nm wavelength was used as an excitation source. The UV measurements for both antioxidant and biocompatibility study were carried out using Thermo Scientific spectrometer model UV10. 2.3. Measurement of antioxidant activity Antioxidant activity was measured by using the DPPH• free radical method of Serpent et al. [16]. In a typical procedure, an amount of 0.2–0.6 mg of polypyrrole nanotubes were transferred to a test tube and the reaction was started by adding 3 ml of 100 ␮M DPPH• solution in methanol. The reaction mixture was vortexed for 45 s and stored in dark for the next 20 min and scanning was performed. All measurements for constant amount of 0.4 mg of polypyrrole samples were performed exactly 40 min after the mixing. The time

dependent tests were carried out by employing 0.4 mg of polypyrrole sample to the DPPH solution and the absorbance was recorded within the time limit of t = 0–180 min. The scavenging efficiency of DPPH free radicals was calculated as % of free radical scavenging by the following relation:



DPPH• scavenging(%) = 1 −

AS AB



× 100

(1)

where, AS is the absorbance of DPPH with sample and AB is the absorbance of DPPH without sample. 2.4. Haemolysis assay The degradation of red blood cell (RBC) membrane against polypyrrole nanotubes was investigated with the help of haemolysis assay using the method by Zhu et al. [17]. Blood was collected into heparinized tube containing 4% Sodium citrate and then centrifuged at 3000 rpm at 4 ◦ C for 20 min. Then erythrocytes were washed twice with a large volume of phosphate saline buffer (PBS, pH 7.4). 5% packed erythrocytes were gently resuspended with PBS. Different concentrations (1.25 mg/ml, 2.5 mg/ml, 5 mg/ml, and 10 mg/ml) of the nanotubes with 2 mM CTAB, dissolved in PBS followed by sonication. Triton X-100 was used as the positive control capable of damaging the red blood cells causing haemolysis and PBS is used as negative control. 100 ␮l of the dissolved sample was mixed with 1900 ␮l of haematocrit in different microfuge tubes and incubated at 37 ◦ C for 1 h. RBC cells were subsequently placed in an ice bath for 60 s followed by centrifuging at 3000 rpm for 5 min at 4 ◦ C. Supernatants were used for determining the free haemoglobin concentration as a measure of haemolysis by taking absorbance at 540 nm [18]. The % haemolysis was calculated as follows: Haemolysis percentage =

AS − AN × 100 AP − AN

(2)

where, AS , AP and AN are the absorbance of the sample, positive control and negative control, respectively. 3. Results and discussion 3.1. Morphological studies The HRTEM images depicting the tubular morphology of polypyrrole are illustrated in Fig. 1. The average diameter of the nanotubes without CTAB has been measured around 140 nm. It is apparent from the figure that average diameter of the nanotubes decreases continuously with increase in CTAB concentration and is found to be 120 nm and 90 nm with 1 mM and 2 mM CTAB concentration, respectively. The MO-FeCl3 template that forms due to the suppression of the electrostatic repulsive force between negatively charged MO aggregates in solution directs the growth of polypyrrole nanotubes and degrades automatically due to the reduction of oxidising cations. CTAB, a cationic surfactant, adsorbs on the surface of MO-FeCl3 template due to its amphiphilic character with hydrophilic group pointing towards the aqueous solution. The hydrophobic groups of CTAB point towards the MO-FeCl3 template partially solubilising it resulting in decrease in the diameter of the template, which in turn reduces the diameter and surface area of the nanotubes [15]. The diameter distribution of the polypyrrole nanotubes with different CTAB concentration has been depicted using histograms and a Gaussian distribution with standard deviation of the diameters shown in Fig. 2. 3.2. XRD analysis The XRD pattern of polypyrrole nanotubes (Fig. 3) shows a broad hump that ranges from 2 = 20◦ –30◦ in addition of two sharp peaks

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Fig. 1. High resolution transmission electron micrographs of polypyrrole nanotubes with different CTAB concentrations: (a) without CTAB, (b) 1 mM CTAB and (c) 2 mM CTAB.

at 2 values of 19.5◦ and 23◦ , respectively. This broad peak is a characteristic of amorphous polypyrrole and is attributed to the ␲–␲ interaction of partial polypyrrole chains similar to that of aromatic groups [19]. With increase in CTAB concentration this peak slightly shifted towards lower diffraction angle. The diffraction peaks at 2 values of 26.11 ± 0.2, 25.12 ± 0.25 and 24.1 ± 0.18 corre˚ sponding to d spacing of 3.41 ± 0.02, 3.54 ± 0.04 and 3.69 ± 0.03 A, respectively. Interestingly, the appearance of sharp peak at 19.5◦ corresponding to d-spacing of 4.56 A˚ indicates considerable degree of crystallinity in the polymer chain. The intensity of this peak decreases with increase in CTAB concentration with no change in 2 position implying the decrease in crystallinity. This decrease in peak intensity is attributed to the fact that addition of CTAB prevents the polymer chain organisation as the polymerization takes place within the hydrophobic micelles of CTAB. The broad diffraction peak centred on 2 = 25◦ , has been analysed to determine the domain length (L), strain (ε) and d-spacing which are presented in Table 1. The single line approximation has been employed to compare these contributions. This involves the extraction and analysis of Gaussian (ˇG ) and Lorentzian (ˇL ) component of integral breadth of a single Bragg peak corrected for instrumental broadening [20]. The domain length and microstrain have been calculated using the value of ˇL (equation (3)) and ˇG (equation (4)), respectively. The table shows that the domain length decreases while microstrain increases with increase in CTAB concentration, which suggests the enhancement of degree of disorder in the polymeric system with increase in CTAB concentration.

Table 1 Domain length (L), strain (ε) and d-spacing of polypyrrole nanotubes with different CTAB concentrations. Sample

´˚ Domain length (A)

Strain (%)

´˚ d spacing(A)

0 mM CTAB 1 mM CTAB 2 mM CTAB

6.07 5.13 4.64

14.13 14.79 16.1

1.75 1.83 1.92

Domain length, L =

Strain, ε =

 ˇLf cos

f ˇG

4 tan 

(3)

(4)

3.3. Micro-Raman spectroscopy Molecular structure and properties of polypyrrole nanotubes have been investigated by micro-Raman spectroscopy. Fig. 4 exhibits micro-Raman spectra of polypyrrole nanotubes in the range of 600–1800 cm−1 . The characteristic bands at 1590 and 1489 cm−1 are attributed to the C C stretching of oxidised (doped) and reduced (dedoped) polypyrrole, respectively [21]. The strong band at 1386 cm−1 is assigned to the asymmetric C N stretching of the oxidized polypyrrole [22]. C H in plane stretching peak appears at 1265 cm−1 . The peaks at 930 and 980 cm−1 are the ring deformation peaks [23]. The ratio of integrated intensities of the bands at 1590 and 1489 cm−1 (I1590 /I1489 ) cm−1 has been often used to determine the conjugation length in polypyrrole [21]. Conjugation length is generally used to explain the polymer segment along which delocalization of п-electrons takes place. The ratio (I1590 /I1489 ) of the band intensities has been calculated to be 1.84, 1.80 and 1.61 for polypyrrole nanotubes without CTAB and with 1 mM and 2 mM CTAB concentration, respectively. The decrease in intensities ratio with increase in CTAB concentration suggests the decrease in the conjugation length of polypyrrole nanotubes, which is consistent with the XRD results. 3.4. Antioxidant assay The UV-visible absorbance spectra of DPPH free radical with 0.4 mg of polypyrrole nanotubes with different CTAB concentrations is presented in Fig. 5. DPPH free radical shows absorbance band at 516 nm which is due to the ␲–␲* energy transition in DPPH

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Fig. 3. X-ray diffraction pattern of polypyrrole nanotubes (a) without CTAB, with (b) 1 mM and (c) 2 mM CTAB concentration, respectively.

[24]. It is apparent from the figure that the absorbance at 516 nm which is initially approximately at 0.5 units decreases on addition of the polypyrrole sample. This decline of the band intensity is attributed to the neutralization of DPPH free radicals by polypyrrole. The free radical scavenging activity of polypyrrole is basically due to the presence of N-H groups in the structure, which has low bond dissociation energy of 77–80 kcal mol−1 [25]. The antioxidant activity of polypyrrole nanotubes may be due to the transfer of electron from the N-H group to the odd electron located at the nitrogen atom in DPPH resulting in decrease in the band intensity at 516 nm. The reaction mechanism in polypyrrole and DPPH free radical is given as, DPPH + H+ + e− → DPPHH

Fig. 2. Diameter distribution of the polypyrrole nanotubes with different CTAB concentrations: (a) without CTAB, (b) 1 mM CTAB and (c) 2 mM CTAB.

(5)

where H+ and e− are the proton and electron originating from the polypyrrole. Percentage of DPPH scavenging activity of polypyrrole nanotubes of varying diameters with different weight has been calculated using the eq. (1) and presented in Fig. 6. It is noticeable from the figure that the scavenging activity increases with increasing amount of polypyrrole samples. This result is attributed to the availability of increased surface area with increase in the amount of polypyrrole that can scavenge more free radicals [26]. From the plot it is also clear that surface area of the nanotubes plays an important factor in the radical scavenging activity which is directly related to the amount of CTAB in the sample. It has been observed that with the increase in CTAB concentrations the scavenging activity is increased. This result is attributed to the formation of polypyrrole nanotubes with smaller diameter with increase in CTAB concentration giving rise to the increase in surface to volume ratio. Due to the smallest diameter of 2 mM CTAB sample, more nanotubes are available as compared to that of the higher diameter samples for equal amount, giving rise to larger number of reactive sites resulting in the maximum antioxidant activity. Polypyrrole owing to its redox active nature is capable of scavenging free radicals and with decrease in nanotube diameter more active sites per unit volume are available which can neutralize the free radicals and act as effective radical scavenger. Consequently polypyrrole nanotubes with average diameter of ≈90 nm exhibit higher antioxidant activity as compared to that of nanotubes having higher average diameters of 120 and 140 nm. The time dependent free radical scavenging activity of polypyrrole nanotubes is presented in Fig. 7. Polypyrrole nanotubes show a rapid scavenging of free radicals within the first 10 to 15 min

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Fig. 4. Micro Raman spectra of polypyrrole (a) without CTAB, with (b) 1 mM and (c) 2 mM CTAB concentration, respectively.

Fig. 5. UV-Vis absorption of blank DPPH free radicals and with 0.4 mg of polypyrrole nanotubes with different CTAB concentration.

and then decreases progressively with time suggesting that both faster and slower processes are involved in the scavenging mechanism. Within the first 10 min, the scavenging activity sharply rises to 27.25, 39.9 and 44.3% for nanotubes without CTAB, with 1 mM and 2 mM CTAB concentration, respectively. Initially, hydrogen rich polypyrrole donates hydrogen atom quickly to the DPPH free radicals leading to the faster scavenging reactions. Subsequently, the polypyrrole nanotubes that initially react with the DPPH free radicals act as intermediator via cross-linking or there could be

Fig. 6. DPPH scavenging activity of 0.2–0.6 mg polypyrrole with different CTAB concentration.

different oxidation states that lead to the slower scavenging process [25]. 3.5. Haemolysis assay Initial evaluation of in-vitro biocompatibility of polypyrrole nanotubes has been carried out via haemolysis test and the results obtained are plotted in Fig. 8. The evaluation of erythrocyte induced haemolysis has been considered as the reliable measurement for

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where, x1 , x2 , . . .., xi are the observed values of the absorbance, x¯ is the mean value of these observations and N is the number of times the experiments were carried out which is 2 in the present case. It is observed from the plot that polypyrrole nanotubes show overall less haemolysis activity and with increase in concentration, the haemolysis activity increases due to the cytotoxicity of polypyrrole. At higher concentrations polypyrrole is cytotoxic and causes haemolysis [28,29]. The biocompatibility of chemically synthesized polypyrrole nanoparticles has been investigated in mice in-vivo and reported that polypyrrole nanoparticles do not show any cytotoxic effects on mouse peritoneum cells. Furthermore they did not detect any allergic response or major changes in spleen, kidney and liver. The immune-related haematological parameters or inflammation symptoms were reported within the standard limit in the peritoneum after a period of six weeks [30]. It has been reported that 5% haemolysis is permissible for biomaterials and polypyrrole nanotubes show haemolysis below 5% upto a concentration of 2.5 mg/ml [31]. Therefore the synthesized polypyrrole nanotubes are biocompatible at lower concentrations.

Fig. 7. Time dependent antioxidant activity of polypyrrole nanotubes.

4. Conclusions

Fig. 8. Percentage of haemolysis of RBC in the presence different amount of polypyrrole nanotubes with 2 mM CTAB concentration.

estimating biocompatibility of the materials [27]. The optical density at 540 nm and percent haemolysis for different amounts of polypyrrole sample showing the best antioxidant activity have been calculated using eq. (2) and are given in Table 2. All the experiments were repeated twice to ensure uniform results and the standard deviation (SD) have been calculated by using the eq. (6).

 SD =

1

N

(N − 1)

i=1

(xi − x)

2

(6)

Table 2 Absorbance and %haemolysis of blood by polypyrrole nanotubes presented in the form of mean value ± standard deviation. Sample

Optical density at 540 nm

Hemolysis (%)

Triton X-100 PBS 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml

3.9855 ± 0.0007 0.0841 ± 0.0011 0.1354 ± 0.0016 0.2073 ± 0.0094 0.3105 ± 0.022 0.561 ± 0.01025

+ve control −ve control 1.31 ± 0.2334 3.15 ± 0.2334 5.8 ± 0.3394 12.22 ± 0.2545

In summary, we have synthesized polypyrrole nanotubes through chemical polymerization method in presence of reactive self degrade MO-FeCl3 template. Polypyrrole nanotubes of smaller diameter are obtained at higher CTAB concentration. This is attributed to the partial solubilisation of MO-FeCl3 template by the cationic surfactant CTAB. Domain length and microstrain calculations reveal the decrease in domain length with increase in CTAB concentration, while microstrain increases from 14.13 to 16.1%. The decrease in band intensity ratio (I1590 /I1489 ) with increase in CTAB concentration suggests the decrease in conjugation length in the polymeric system. Enhancement in antioxidant activity has been observed with decrease in nanotubes diameter which can be attributed to the increased surface area per unit volume of the nanotubes. Time dependent antioxidant activity reveals the existence of both faster and slower processes in the scavenging mechanism. Haemolysis test proves that polypyrrole nanotubes are biocompatible upto a concentration 2.5 mg/ml. In the context of the results obtained in the study, the polypyrrole nanotubes may be forwarded as an interesting candidate material for application especially in the biomedical field. References [1] Y. Cao, A.E. Kovalev, R. Xiao, J. Kim, T.S. Mayer, T.E. Mallouk, Electrical transport and chemical sensing properties of individual conducting polymer nanowires, Nano Lett. 8 (2006) 4653–4658. [2] M. Han, Y. Chu, D. Han, Y. Liu, Fabrication and characterizations of oligopyrrole doped with dodecylbenzenesulfonic acid in reverse microemulsion, J. Collids Sci. 296 (2006) 110–117. [3] H. Yoon, J.-H. Kim, N. Lee, B.-G. Kim, J. Jang, A novel sensor platform based on aptamer-conjugated polypyrrole nanotubes for label-free electrochemical protein detection, Chem. Bio. Chem. 9 (2008) 634–641. [4] S. Geetha, C.R.K. Rao, M. Vijayan, D.C. Trivedi, Biosensing and drug delivery by polypyrrole, Anal. Chimica. Acta 568 (2006) 119–125. [5] L.-X. Wanga, X.-G. Lia, Y.-L. Yang, Preparation, properties and applications of polypyrroles, React. Funct. Polym. 47 (2001) 125–139. [6] T. Ahuja, I.A. Mir, D. Kumar, Rajesh, Biomolecular immobilization on conducting polymers for biosensing applications, Biomaterial 28 (2007) 791–805. [7] Q. Liu, H. Liu, Z. Yuan, D. Wei, Y. Ye, Evaluation of antioxidant activity of chrysanthemum extracts and tea beverages by gold nanoparticles-based assay, Colloids Surf. B: Biointerf. 921 (2012) 348–352. [8] N. Singh, P.S. Rajini, Free radical scavenging activity of an aqueous extract of potato peel, Food Chem. 85 (2004) 611–616. [9] R. Amarowicz, R.B. Pegg, P. Rahimi-Moghaddam, B. Barl, J.A. Weil, Free-radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies, Food Chem. 84 (2004) 551–562. [10] A.V. Nand, S. Ray, A.J. Easteal, G.I.N. Waterhouse, M. Gizdavic-Nikolaidis, R.P. Cooney, J. Travas-Sejdic, P.A. Kilmartin, Factors affecting the radical scavenging activity of polyaniline, Synth. Met. 161 (2011) 1232–1237.

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