Antifungal activity of nanostructured polyaniline combined with fluconazole

Antifungal activity of nanostructured polyaniline combined with fluconazole

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j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 6 e3 1

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/JOPR

Original Article

Antifungal activity of nanostructured polyaniline combined with fluconazole Pavithra Yehgambaram a,b, R.G.S.V. Prasad a,c,*, Venkata Srinivas Jakka a, R.S.L. Aparna a, A.R. Phani c a

School of Pharmacy, Masterskill University College of Health Science, Kualalumpur 43200, Malaysia Faculty of Science, Technology and Engineering, La Trobe University, Bendigo, Victoria 3552, Australia c Nano-Research for Advanced Materials and Technologies, 70, 2nd Main, 3rd Stage, Vinyaka Layout, Vijayanagar, Bangalore 5600040, Karnataka, India b

article info

abstract

Article history:

Conductive polymers are gaining much interest in field of pharmacy and biomedicine.

Received 9 August 2012

Polyaniline (PANi) is one of such conducting polymer belonging to semi-flexible rod polymer

Accepted 3 November 2012

family. There are some potential applications of PANi in biosensors, biomedical applications, drug delivery and tissue engineering because of its excellent stability, high conductivity and

Keywords:

easy mode of synthesis. Nanofibres of polyaniline combined with fluconazole are prepared by

Polyaniline

simple and cost effective sol-gel method using D-10-camphorsulfonic acid (D-CSA) as a dopant

Antifungal activity

and as a surfactant, and ammonium persulfate as the oxidant. The synthesized nano-

Nanofibers

structured material was dissolved in dimethylsulfoxide at different concentrations and tested

Sol-gel process

for its antifungal properties against Candida albicans (ATCC 140503), Candida tropicalis (ATCC 13803) and Candida krusei (ATCC 34135). The results showed that, compared to nanofiber structured conducting PANi, polyaniline doped with fluconazole have shown higher antifungal activity on all the species tested. It is very much evident that PANi doped fluconazole has considerable enhanced antifungal activity. C. tropicalis (ATCC 13803) is more susceptible than C. albicans (ATCC 140503) and C. krusei (ATCC 34135). Structural and morphological properties of PANi with fluconazole nanofibers were evaluated by SEM and FTIR. Copyright ª 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

1.

Introduction

National Nanotechnology Initiative (NNI) define nanotechnology as the consumption of structures with at least one dimension of nanometer size for the production of materials, systems or devices with initially or extensively improved properties due to their nano size. Since nano-particles have high surface energy and a large surface area-to-volume ratio,

it can provide high durability for fabrics, at the same time presenting good affinity for fabrics and enhance durability of the function. Nano-Tex known as a secondary of the US-based Burlington Industries have done the earliest work on nanotextiles.1 To apply nano-particles onto textiles, the most frequently used technique is coating. Textiles are generally composed of nano-particles; a surfactant, ingredients and a carrier medium to entrap the nano-particles.2

* Corresponding author. Nano-Research for Advanced Materials and Technologies, 70, 2nd Main, 3rd Stage, Vinyaka Layout, Vijayanagar, Bangalore 5600040, Karnataka, India. E-mail address: [email protected] (R.G.S.V. Prasad). 0974-6943/$ e see front matter Copyright ª 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2012.11.009

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Spraying, transfer printing, washing, rinsing and padding are the several methods can apply coating onto fabrics. The method used for this is padding.3 By using a padder, the nanoparticles are attached to the fabrics which is adjusted to suitable pressure and speed, followed by curing and drying. Textiles are omnipresent to us, covering our skin and environments by not only giving protective shield but they also serve artistic appeal and cultural value. Smart clothes were created from intelligence to textiles which are added from advances in material science. They have fascinated because of their potential applications such as in dust and germ free clothing,4 cooling systems,5 electrotherapy,6 heat generation,7 health monitoring shirts, drug delivery,8 data transfer in clothing, electro chromic display, sensors and military applications like stealth technology. This smart textiles can be differentiated into three subtypes,9 acting as sensors where as active smart textiles can sense and react to the stimuli from the environment, and have an actuator function and very smart textiles, having the reward to alter their behavior to the situations where else passive smart textiles can only sense the environment. Furthermore, for the development of smart nanotextiles there are some suitable materials such as inherently conducting polymers (ICPs), carbon nanotubes (CNT) and a number of materials in the form of nano-particles or nanofibers.10 A type of ionic electro active polymer which changes the shape by mobility or diffusion of ions and conjugated substances defined as inherently conductive polymers.11 Polyacetylene, polypyrrole, polyaniline and polythiophene are usually used ICPs12 but Polyaniline (PANi) is one of the most commonly studied ICP. It has three possible oxidation states and is relatively steady in the environment.10 In smart nanotextiles, especially polyaniline and polypyrrole may have a vital role in remote monitoring those undergoing rehabilitation or chronically ill patients. Besides that, to build up materials with motor functions a combination of ICP actuators in textiles can be used.10 ICPs can also mimic and increases the sensory system of the skin by sensing external stimuliincluding proximity, touch, pressure, temperature, and chemical or biological substances.3 Studies have been done by using anti-bacterial agents in textiles such as, nano-sized silver,13 titanium dioxide14 and zinc oxide.15 The number of particles per unit area is increased with the use of nano-sized particles, so can maximize the anti-bacterial effects. A very big relative surface area can be caused by the nano-sliver particles. So, this will leads to rise in their contact with bacteria or fungi. Furthermore, greatly improving their antimicrobial efficiency which is usually applied to socks in order to prohibit the growth of bacteria. Synthetic compounds that have one or more azoles rings with three nitrogen atoms in the five membered rings known as antifungal triazoles. They primarily act by inhibiting CYP450-dependent conversion of lanosterol to ergosterol which leads to an accumulation of toxic 14-amethylsterols, which alters the function and cell membrane properties leading to the inhibition of replication and cell growth.16 The antifungal triazole which is used in this study is fluconazole. Treatment of candidemia over the past decade has been increased considerably by the introduction of fluconazole.17 In order to widen its antifungal spectrum of activity and to enhance its in vitro potency, fluconazole’s chemical structure has been modified.18 It has unique pharmacokinetics

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with a long half-life, good water solubility, low molecular weight, weak protein binding, and a high level of cerebrospinal fluid penetration. It has been effective in treating both superficial19 and systemic Candida infections.20 The development of resistant strains of Candida after use of fluconazole as primary therapy or as a prophylactic agent for superficial candidosis that have been documented in several other reports. Basically, fluconazole thought to be fungistatic rather than fungicidal in standard in vitro susceptibility tests. In present study, we prepared nanofibers of PANi and PANi with fluconazole by simple and cost effective sol-gel process and investigate its enhanced antifungal activity on various candida species. Structural and morphological properties of PANi doped fluconazole will be evaluated by SEM and FTIR.

2.

Materials and methods

2.1.

Chemicals

Aniline, ammonium persulfate, camphor sulphonic acid and fluconazole obtained from Sigma Aldrich with 99.5% purity. Methanol, barium chloride, sulfuric acid, acetone and dimethlysulfoxide were reagent grade. Sabouraud agar and Nutrient broth were obtained from HiMedia.

2.2.

Fungal organisms

Candida albicans (ATCC 140503), Candida krusei (ATCC 34135) and Candida tropicalis (ATCC 13803) used in this study were purchased from ATCC.

2.3.

Preparation of PANi with fluconazole

Required quantity of fluconazole was dissolved in acetone and was mixed for 30 min. Aniline (An) monomer was distilled under reduced pressure. D-CSA as the dopant and ammonium persulfate ((NH4)2S2O8, APS) as the oxidant were used as received without further treatment. PANIe(D-CSA) nanofibres were prepared by oxidative polymerization of aniline at 0e5  C (ice bath) using ammonium persulfate (APS) as the oxidant in the presence of D-CSA. A typical polymerization process of PANIe(D-CSA), briefly of aniline was been transferred to 100 ml beaker containing 10 ml of deionized water. The beaker was kept in ice bath (0e5  C) and the contents were stirred for 5 min. The equivalent moles of ammonium persulfate were dissolved in 10 ml of deionized water. The beaker was kept in ice bath (0e5  C) and the contents were stirred for 5 min. D-CSA and transferred into a 100 ml beaker containing 10 ml of deionized water and the contents were stirred for 5 min till a clear and homogeneous solution is obtained and added with fluconazole solution. After that the surfactant has been added to the monomer drop wise with constant stirring at 0e5  C. After the addition of surfactant, oxidant has been added to the monomer contents drop by drop under constant stirring and temperature of 0e5  C. After the addition of oxidant the contents color had slowly changed to dark green color indicating the polymerization of aniline to polyaniline. The final contents have been stirred for 10 min and kept in refrigerator at 0  C for 24 h. After that the contents were filtered by washing with deionized water

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for several times till all unreacted surfactant is washed. Finally washed with methanol to terminate polymerization. The dark green colored precipitate was dried overnight at 100  C.Similarly pure PANi is also prepared without adding fluconazole.

2.4.

Antifungal tests

Antifungal activity for PANi and PANi combined with fluconazole nanofibres was performed by agar diffusion method in Sabouraud agar. Sabouraud agar was prepared as per the manufacturer protocol. The agar medium was sterilized in aquilots of 15 ml at a pressure of 15 lbs for 15 min. This agar medium was transferred into sterilized petri dishes in a laminar air flow unit and allowed to solidify. After solidification of the media, a 24 h culture of each organism was standardized to 0.5. McFarland standard was cultivated as lawn culture by spreading the organism on the agar media using sterile cotton swab. Cup plate method was used to test the antifungal activity by using sterile bore with the diameter of 9 mm. Four different concentrations were prepared such as 10 mg/ml, 5 mg/ml, 2.5 mg/ml and 1.25 mg/ml of PANi and PANi doped fluconazole in dimethylsulfoxide solution. To this media, 100 ml of respective dilution were added using micropipette and incubated for 2 days at 37  C in the incubation chamber. Average zone diameters were measured after repeating the experiment for three times.

3.

Results and discussion

3.1.

Scanning electron microscopy

The prepared PANI combined with fluconazole nanofibers were studied by SEM The morphological structure of the synthesized PANI doped fluconazole nanofibers was identified

by scanning electron microscope (SEM). A fixed working distance of 5 mm and a voltage of 5e25 kV were used. Normally, sample preparation for the SEM measurement will be carried out inside the glove box by covering the sample holder with parafilm for minimal exposure to oxygen while transferring it to the secondary emission chamber. First of all, we investigated the influence of the parameters such like ratio of oxidant to monomer, the concentration of the surfactant, aging temperature and time and reaction temperature on the fiber formation of PANI doped fluconazole to discover the optimal conditions for the formation of PANI doped fluconazole nanofiber structure. It was found that the reaction temperature and to some extent aging temperature and time strongly affect the microstructure and the formation probability of PANI doped fluconazole nanofibers. In all the cases we have obtained nanofiber like structures but with different lengths and diameter. The SEM image of PANI doped nanofibers which shown in Fig. 1 which indicates the nanofiber diameter about 10 nm. And also the nanofibers are hallow in nature. The magnifications of the sample were reported in order of a, b and c

3.2.

Antifungal activity

All the fungi, C. albicans (ATCC 140503), C. tropicalis (ATCC 13803) and C. krusei (ATCC 34135) successfully showed consistent zones of inhibitions to PANI and PANI doped with fluconazole. As the concentration of PANI and PANI doped with fluconazole increased, the susceptibility also increased for all the fungi. The Fig. 2a shows inhibitory concentration of PANI on C. tropicalis (ATCC 13803). There is no inhibitory zone of PANI in DMSO which acts as a control. But there is an inhibitory zone of 7 mm for concentration of 1.25 mg/ml, 8 mm for concentration of 2.5 mg/ml, 9 mm for concentration of 5.0 mg/ml and 11 mm for concentration of 10 mg/ml. From this

Fig. 1 e Scanning electron microscopy of prepared PANI doped with fluconazole. (a) Shows PANI doped fluconazole nanofibers at a magnification of 10,000, (b) magnification of 25,000, (c) magnification of 50,000.

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Fig. 2 e Photographic image of antifungal activity of PANI on C. tropicalis. (a) Shows antifungal activity of PANI on C. tropicalis (ATCC 13803) with DMSO, conc. 10 mg/ml , 5 mg/ml, 2.5 mg/ml and 1.25 mg/ml. (b) Shows antifungal activity of PANI doped fluconazole on C. tropicalis (ATCC 13803) with conc. 10 mg/ml , 5 mg/ml , 2.5 mg/ml and 1.25 mg/ml.

we can assume that the minimum inhibitory concentration (MIC) of PANI for C. tropicalis (ATCC 13803) is 1.25 mg/ml. The Fig. 2b shows inhibitory concentration of PANI doped with fluconazole on C. tropicalis (ATCC 13803). Inhibitory zone of 9 mm for concentration of 1.25 mg/ml, 10 mm for concentration of 2.5 mg/ml, 11 mm for concentration of 5.0 mg/ml and 13 mm for concentration of 10 mg/ml. From this we can assume that the minimum inhibitory concentration (MIC) of PANI doped fluconazole for C. tropicalis (ATCC 13803) is 1.25 mg/ml. Furthermore, it shows the enhanced antifungal activity of PANI doped fluconazole nanofibers. Fig. 3a shows the antifungal activity of PANI and PANI doped fluconazole against C. albicans (ATCC 140503). C. albicans is more susceptible with their average zone diameters of 10.67 mm at 10 mg/ml concentration for PANI and average zone diameters of 13.00 mm at 10 mg/ml concentration for PANI doped with

fluconazole. The difference in average zone of inhibition diameter for PANI and PANI doped with fluconazole was also noted to be greatest at 5 mg/ml which was measured to be 2.66 mm. The difference in average zone of inhibition diameter for concentrations of 1.25 mg/ml, 2.5 mg/ml and 10 mg/ml were measured to be almost similar, ranging from 2.00 mm to 2.33 mm. As the concentration increases, the average zone of inhibition in diameter increases. It is also proven that there is enhanced antifungal activity of PANI doped fluconazole compare to PANI alone. Fig. 3b shows the antifungal activity of PANI and PANI doped fluconazole against C. tropicalis (ATCC 13803). PANI and PANI doped fluconazole showed considerable antifungal activity on all the concentrations tested. C. tropicalis is more susceptible with their average zone diameters of 12.00 mm at 10 mg/ml concentration for PANI and average zone diameters of 13.33 mm at 10 mg/ml concentration for PANI doped with fluconazole. As we can see Fig. 3b, the

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Fig. 3 e (a) Antifungal activity of PANI and PANI doped fluconazole against C. albicans. (b) Antifungal activity of PANI and PANI doped fluconazole against C. tropicalis. (c) Antifungal activity of PANI and PANI doped fluconazole against C. krusei.

candida is less susceptible when the concentration is low that is 1.25 mg/ml so there is less zone of inhibition for both PANI and PANI doped with fluconazole. The difference in average zone of inhibition diameter for PANI and PANI doped with fluconazole was also noted to be greatest at 10 mg/ml which was measured to be 1.33 mm. The difference in average zone of inhibition diameter for concentrations of 1.25 mg/ml, 2.5 mg/ml and 5 mg/ml were measured to be almost similar, ranging from 0.66 mm to 1.00 mm. It shows a steady increase in the difference in average zones of inhibition diameter. As the concentration increases, the average zone of inhibition in diameter increases. It is also proven that there is enhanced antifungal activity of PANI doped fluconazole compared to PANI alone. Fig. 3c shows the antifungal activity of PANI and PANI doped fluconazole against C. krusei (ATCC 34135). Besides that, the table shows the mean value of zones of inhibition for this particular candida. PANI and PANI doped fluconazole showed considerable antifungal activity on all the concentrations tested. C. krusei are more susceptible with their average zone diameters of 11.33 mm at 10 mg/ml concentration for PANI and average zone diameters of 13.33 mm at 10 mg/ml concentration for PANI doped with fluconazole. As we can see Fig. 3c, the candida is less susceptible when the concentration is low that is 1.25 mg/ml so there is less zone of inhibition for both PANI and PANI doped with fluconazole. The difference in average zone of inhibition diameter for PANI and PANI doped with fluconazole was also noted to be greatest at 10 mg/ml which was measured to be 2.00 mm. The difference in average

zone of inhibition diameter for concentrations of 1.25 mg/ml, 2.5 mg/ml and 5 mg/ml were measured to be almost similar, ranging from 1.00 mm to 1.34 mm. But there is a sudden decrease and rise in the difference in average zones of inhibition diameter. There are no changes in the difference in average zone of inhibition diameter at the concentrations of 2.5 mg/ml and 5.00 mg/ml. It is also proven that there is enhanced antifungal activity of PANI doped fluconazole compared to PANI alone. Based on the above discussion, it is very much evident that PANI doped fluconazole has got enhanced antifungal activity for all the candidas compared to PANI alone. But C. tropicalis (ATCC 13803) showed greater activity compared to C. albicans (ATCC 140503) and C. krusei (ATCC 34135). However continuous trials should be carried out in order to make this finding more established.

4.

Conclusion

In this research, we have synthesized Polyaniline and PANI with fluconazole about 100e150 nm in diameter by a simple and cost effective sol-gel process. The prepared PANI and PANI doped fluconazole nanofibers were characterized by SEM. The PANI and PANI doped fluconazole in dimethysulfoxide solvent under different concentrations have shown enhanced antifungal activity on various fungi tested. The results showed that compared to nanofiber structured conducting PANI, polyaniline doped with fluconazole have shown higher antifungal activity on all the species tested. It is very

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much evident that PANI doped fluconazole has got enhanced antifungal activity. It is also shows greater activity on C. tropicalis (ATCC 13803) compared to C. albicans (ATCC 140503) and C. krusei (ATCC 34135). This investigation will have many potential applications where smart nanotextiles may give impact on our lifestyles like antifungal fabrics where polyaniline is one of the ingredients.

Conflicts of interest All authors have none to declare.

Acknowledgments The authors are grateful to the management of the Masterskill University of Health Science, Malaysia for promoting research and providing financial support in carrying out this investigation and Nano-RAM Technologies, Bangalore, Karnataka State, India for their technical support.

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