- Email: [email protected]
Production of poly (ε-caprolactone) Antimicrobial Nanofibers by Needleless Alternating Current Electrospinning
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 17 (2019) 1100–1104
www.materialstoday.com/proceedings
RAMM 2018
Production of poly (ε-caprolactone) Antimicrobial Nanofibers by Needleless Alternating Current Electrospinning S. Manikandan*, M. Divyabharathi, K. Tomas, P. Pavel, L. David Faculty of Textile Engineering, Technical University of Liberec, Studentska 2, 461 17 Liberec, Czech Republic
Abstract The present study is focused on the fabrication of antimicrobial polycaprolactone (PCL) nanofibers using uncommon needleless and collector less alternating current (AC) electrospinning. The PCL polymeric solutions (6 & 12 wt.%) were prepared using 1:1 acetic acid and formic acid. The antimicrobial agent chlorhexidine (CHX) was added to the PCL precursor solution at 1 wt%. The PCL/CHX solutions (6 &12 wt.%) were electrospinnable at the applied AC potential of 25kV and 35kV rms. The surface morphological investigation of PCL/CHX antimicrobial nanofibers confirmed that smooth beadless nanofibers have been obtained. Fiber diameter and viscosity were increasing with respect to PCL/CHX concentrations. © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the 6th International Conference on Recent Advances in Materials, Minerals & Environment (RAMM) 2018. Keywords: Alternating current electrospinning; Polycaprolactone; Chlorhexidine.
* Corresponding author. Tel.: +420 77 436 4937. E-mail address: [email protected] 2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the 6th International Conference on Recent Advances in Materials, Minerals & Environment (RAMM) 2018.
S. Manikandan et al./ Materials Today: Proceedings 17 (2019) 1100–1104
1101
1. Introduction Electrospinning is a cost-effective and relatively simple method used to fabricate the uniform endless nano and microfibers from synthetic and natural polymeric solutions or melts. Nanofibers have increasingly found application in a broad range of fields such as tissue engineering, wound dressing, drug delivery, energy storage, etc [1]. Bacterial contamination on the biomaterial is one of the main problems in the biomedical field, especially in wound dressing application. Bacterial contamination in the biomaterial causes the local inflammatory reaction because it triggers the immune system, that is inadequate to contrast the infection [2]. To overcome this problem, antimicrobial nanofibers have to be prepared. PCL is a biocompatible and biodegradable aliphatic polyester which is used in many fields notably in biomedical fields, in which some particular attention has been devoted to antimicrobial PCL nanofibers for wound dressing. It is also used as surgical sutures, drug delivery systems and three dimensional scaffolds in tissue engineering. The antimicrobial agent CHX is active to both Gram-positive and Gram-negative bacteria. In order to prepare antimicrobial nanofibers, the biocides CHX was dissolved in PCL precursor solution prior to the electrospinning process. Direct addition of CHX to the precursor solution is technologically much easier than the subsequent post treatment of electrospun nanofibers [3]. Since PCL/CHX nanofibers and monofilaments have been fabricated using direct current (DC) electrospinning and other methods [4]. This is the first time PCL/CHX electrospun nanofibers were fabricated by uncommon needleless and collector less alternating current (AC) electrospinning using green solvents. The main advantage of this technology are that, it is more productive, has simpler experimental setup than the common DC electrospinning and this technology can easily be combined with other technologies due to the absence of grounded collector [5]. In this study, antimicrobial nanofibers were fabricated from PCL/CHX precursor solution using AC electrospinning. It has been found that addition of CHX to the PCL precursor solution not only improved the spinnability, but also had a positive effect on fiber morphology, fiber diameter and precursor solution viscosity. 2. Materials & Methods 2.1. Materials PCL with molecular average weight [Mn] of 80,000, density 1.145 g/ml (melting point 60°C) and CHX chemical formula: C22H30Cl2N10. (C2H4O2)2 (melting point 155°C) were purchased from Sigma Aldrich. Formic acid and acetic acids were purchased from Penta (98% p.a). 2.2. Methods 2.2.1. Preparation of precursor solution PCL was dissolved in 1:1 acetic acid and formic acid at the room temperature in order to prepare 6 wt.% & 12 wt.% precursor solution. Antimicrobial agent CHX was added to the precursor solutions at 1 wt.% at the solid state and then the precursors solution was stirred overnight using a magnetic stirrer at room temperature. 2.2.2. Preparation of Alternating Current Electrospinning Fabrication of PCL and PCL/CHX precursor nanofibers was carried out using uncommon needleless and collector less AC electrospinning. The electrospinning system can produce the AC potential up to 40 kV rms and operate at 60 Hz. In this system fiber collector was not necessary because of the corona wind which was lead the flow of generating fibers plume after the increment of AC potentials to polymeric solution [5,6]. For AC spinnability testing, 0.3-0.6 ml of precursors solution was placed on the top hexagonal shaped metallic electrode that had a diameter of 13mm and applied potentials of 25 kV and 35 kV rms. Propagating fibers were collected in a plain sheet using a non-conducting stick.
1102
S. Manikandan et al./ Materials Today: Proceedings 17 (2019) 1100–1104
2.2.3. Characterization of polymeric solution & fibers The viscosity of PCL and PCL/CHX precursor solutions were examined using a HAAKE RotoVisco 1 from Thermo Scientific, USA, paired with Rheowin 4 Job and Data manager software. Before each sample test, the RotoVisco was programmed to calibrate the null point. After the calibration, a drop of (0.5-0.8 ml) solution was placed on the RotoVisco solution platform, and viscosity values were obtained over a period of 240s and at the shear rate of 500-750 second-1. The recorded viscosity values and graphs were obtained from Rheowin 4 Data manager for further analysis. The surface morphology and shape of the PCL and PCL/CHX fibers were examined using VEGAS scanning electron microscopy (SEM) from Tescan, Czech Republic. Samples were coated with 7-10 nm of gold sputter to overcome the charging. The SEM micrographs were captured using secondary electron mode, with an accelerating voltage of 30 kV rms. Fiber diameter has been investigated using Image J software with 200 measurements on each sample. 3. Results and discussion The plumes of PCL/CHX fibers formation during the AC electrospinning process is displayed in Fig. 1(a). Spinnability of PCL and PCL/CHX precursor solution depended strongly on precursor concentration and applied AC potential. (a)
(c)
(b)
(g)
(h)
(e)
(d)
(g)
(f)
(h)
Fig. 1. Camera image of fiber plume and SEM images of PCL &PCL/CHX fibers (a) camera image of PCL/CHX nanofiber plume from the electrode at 35 kV rms, (b) 6 wt.% of PCL at 25 kV; (c) 6 wt.% of PCL at 35 kV; (d) 12 wt.% of PCL at 35 kV; (e) 6 wt.% of PCL/1wt% CHX at 25 kV; (f) 6 wt.% of PCL/1wt% CHX at 35 kV; (g) 12 wt.% of PCL/1 wt.% of CHX at 25 kV and (h) 12 wt.% of PCL/1 wt.% of CHX at 35 kV.
S. Manikandan et al./ Materials Today: Proceedings 17 (2019) 1100–1104
1103
Both precursor solutions were not spinnable at less than 25 kV rms. The critical applied AC potential for PCL and PCL/CHX solutions were 25 kV rms. The precursor PCL polymeric solution from 6 wt.% was AC spinnable at 25 kV and 35 kV rms. The PCL precursor solution concentration of 12 wt.% was not spinnable at 25 kV rms due to high viscosity and only spinnable at a high electric potential of 35 kV rms. The PCL/CHX precursor solutions concentrations 6 wt.% and 12 wt.% were AC spinnable at 25 kV and 35 kV rms. The SEM images proved that the addition of CHX to the PCL precursor solution strongly affects the fiber morphology and which improve the spinnability of precursor solution (Fig. 1(b)-(h)). The 6 wt.% of PCL at the applied potential of 25 kV and 35 kV rms, beads were observed with nanofibers (Fig. 1(b)&(c)). In the case of 12 wt.% of PCL at 35 kV rms, beadless fibers were observed (Fig. 1(d)). The 6 wt.% of PCL/CHX at 25 kV and 35 kV rms, beadless smooth fibers were obtained (Fig. 1(e)&(f)). The 12 wt.% of PCL/CHX at 25 kV and 35 kV rms, thicker beadless fibers were observed (Fig. 1(g)&(h)).
1000
Fiber diameter [nm]
25 kV 800
35 kV
600
400
200
0
6 wt% of PCL
12 wt% of PCL
6 wt% of PCL/CHX
12 wt% of PCL /CHX
Fig. 2. The PCL and PCL/CHX fiber diameter at 25 kV and 35 kV rms.
The effect of PCL concentration and 1 wt.% of CHX addition on PCL polymeric solution viscosity were showed in Table. 1. The resulting fiber diameters for applied AC potential were summarized in Fig. 2. Viscosity of PCL solution concentration was increased with respect to PCL concentration from 0.114±0.003 Pa. s at 6 wt.% to 0.946±0.005 Pa. s at 12 wt.%. Addition of 1wt% CHX to the 6 & 12 wt.% of PCL decreased the solution viscosity and increased the fiber diameter. Table 1. PCL and PCL/CHX solution viscosity. Precursor solution
Viscosity
concentration
[Pa.s]
PCL (6 wt.%)
0.114±0.003
PCL (12 wt.%)
0.946±0.005
PCL/CHX (6/1 wt.%)
0.093±0.003
PCL/CHX (12/1 wt.%)
0.666±0.008
1104
S. Manikandan et al./ Materials Today: Proceedings 17 (2019) 1100–1104
PCL fiber diameter was strongly depended on the precursor and additive concentration. There was not observed any significant differences in the both PCL and PCL/CHX electrospun diameter at 25 kV and 35 kV rms. PCL/CHX electrospun diameter was greater than that of PCL electrospun. 4. Conclusions Rapid fabrication of PCL/CHX antimicrobial nanofibers has been achieved using AC electrospinning for the first time. Addition of 1 wt.% CHX to the 6 wt.% and 12 wt.% of PCL solutions improved the fiber morphology from beaded compounded fibers to uniform smooth fibers. Viscosity and the fiber diameter were increased with respect to the precursor concentration. Further work will extend towards the analyze of the antimicrobial activity of AC electrospun material. Acknowledgements The authors would like to thank The Ministry of Education, Youth and Sports of Czech Republic and the student grant contest of the Technical University of Liberec, number SGS 21253 for providing financial support. References [1] [2] [3] [4] [5] [6]
Z. M. Huang, Y.Z. Zhang, M. Kotaki, S. Ramakrishna, Comps. Sci. technol. 63 (2003) 2223–2253. B. D. Masini, D.J. Stinner, S.M. Waterman, J.C. Wenke, J. surg. Educ. 68 (2011) 101-104. P. Rysanek, M. Maly, P. Capkova, M. Kormunda, Z. Kolska, M. Gryndler, O. Novak, L. Hocelikova, L. Bystriansky, M. Munzarova, J. Polym. Res. 24 (2017) 208-218. R. Scaffaro, L. Botta, M. Sanfilippo, G. Gallo, G. Palazzolo, A.M. Puglia, Appl. Microbiol. Biotechnol. 97 (2013) 99-109. P. Pokorny, E. Kostakova, F. Sanetmik, P. Mikes, J. Chvojka, T. Kalous, M. Bilek, K. Pejchar, J. Valtera, D. Lukas, Phys. Chem. Phys. 16 (2014) 26816-26822. C. Lawson, A. Stanishevsky, M. Sivan, P. Pokorny, D. Lukas, J. Appl. Polym. Sci. 133 (2016) 43232-43238.
ScienceDirect Materials Today: Proceedings 17 (2019) 1100–1104
www.materialstoday.com/proceedings
RAMM 2018
Production of poly (ε-caprolactone) Antimicrobial Nanofibers by Needleless Alternating Current Electrospinning S. Manikandan*, M. Divyabharathi, K. Tomas, P. Pavel, L. David Faculty of Textile Engineering, Technical University of Liberec, Studentska 2, 461 17 Liberec, Czech Republic
Abstract The present study is focused on the fabrication of antimicrobial polycaprolactone (PCL) nanofibers using uncommon needleless and collector less alternating current (AC) electrospinning. The PCL polymeric solutions (6 & 12 wt.%) were prepared using 1:1 acetic acid and formic acid. The antimicrobial agent chlorhexidine (CHX) was added to the PCL precursor solution at 1 wt%. The PCL/CHX solutions (6 &12 wt.%) were electrospinnable at the applied AC potential of 25kV and 35kV rms. The surface morphological investigation of PCL/CHX antimicrobial nanofibers confirmed that smooth beadless nanofibers have been obtained. Fiber diameter and viscosity were increasing with respect to PCL/CHX concentrations. © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the 6th International Conference on Recent Advances in Materials, Minerals & Environment (RAMM) 2018. Keywords: Alternating current electrospinning; Polycaprolactone; Chlorhexidine.
* Corresponding author. Tel.: +420 77 436 4937. E-mail address: [email protected] 2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the 6th International Conference on Recent Advances in Materials, Minerals & Environment (RAMM) 2018.
S. Manikandan et al./ Materials Today: Proceedings 17 (2019) 1100–1104
1101
1. Introduction Electrospinning is a cost-effective and relatively simple method used to fabricate the uniform endless nano and microfibers from synthetic and natural polymeric solutions or melts. Nanofibers have increasingly found application in a broad range of fields such as tissue engineering, wound dressing, drug delivery, energy storage, etc [1]. Bacterial contamination on the biomaterial is one of the main problems in the biomedical field, especially in wound dressing application. Bacterial contamination in the biomaterial causes the local inflammatory reaction because it triggers the immune system, that is inadequate to contrast the infection [2]. To overcome this problem, antimicrobial nanofibers have to be prepared. PCL is a biocompatible and biodegradable aliphatic polyester which is used in many fields notably in biomedical fields, in which some particular attention has been devoted to antimicrobial PCL nanofibers for wound dressing. It is also used as surgical sutures, drug delivery systems and three dimensional scaffolds in tissue engineering. The antimicrobial agent CHX is active to both Gram-positive and Gram-negative bacteria. In order to prepare antimicrobial nanofibers, the biocides CHX was dissolved in PCL precursor solution prior to the electrospinning process. Direct addition of CHX to the precursor solution is technologically much easier than the subsequent post treatment of electrospun nanofibers [3]. Since PCL/CHX nanofibers and monofilaments have been fabricated using direct current (DC) electrospinning and other methods [4]. This is the first time PCL/CHX electrospun nanofibers were fabricated by uncommon needleless and collector less alternating current (AC) electrospinning using green solvents. The main advantage of this technology are that, it is more productive, has simpler experimental setup than the common DC electrospinning and this technology can easily be combined with other technologies due to the absence of grounded collector [5]. In this study, antimicrobial nanofibers were fabricated from PCL/CHX precursor solution using AC electrospinning. It has been found that addition of CHX to the PCL precursor solution not only improved the spinnability, but also had a positive effect on fiber morphology, fiber diameter and precursor solution viscosity. 2. Materials & Methods 2.1. Materials PCL with molecular average weight [Mn] of 80,000, density 1.145 g/ml (melting point 60°C) and CHX chemical formula: C22H30Cl2N10. (C2H4O2)2 (melting point 155°C) were purchased from Sigma Aldrich. Formic acid and acetic acids were purchased from Penta (98% p.a). 2.2. Methods 2.2.1. Preparation of precursor solution PCL was dissolved in 1:1 acetic acid and formic acid at the room temperature in order to prepare 6 wt.% & 12 wt.% precursor solution. Antimicrobial agent CHX was added to the precursor solutions at 1 wt.% at the solid state and then the precursors solution was stirred overnight using a magnetic stirrer at room temperature. 2.2.2. Preparation of Alternating Current Electrospinning Fabrication of PCL and PCL/CHX precursor nanofibers was carried out using uncommon needleless and collector less AC electrospinning. The electrospinning system can produce the AC potential up to 40 kV rms and operate at 60 Hz. In this system fiber collector was not necessary because of the corona wind which was lead the flow of generating fibers plume after the increment of AC potentials to polymeric solution [5,6]. For AC spinnability testing, 0.3-0.6 ml of precursors solution was placed on the top hexagonal shaped metallic electrode that had a diameter of 13mm and applied potentials of 25 kV and 35 kV rms. Propagating fibers were collected in a plain sheet using a non-conducting stick.
1102
S. Manikandan et al./ Materials Today: Proceedings 17 (2019) 1100–1104
2.2.3. Characterization of polymeric solution & fibers The viscosity of PCL and PCL/CHX precursor solutions were examined using a HAAKE RotoVisco 1 from Thermo Scientific, USA, paired with Rheowin 4 Job and Data manager software. Before each sample test, the RotoVisco was programmed to calibrate the null point. After the calibration, a drop of (0.5-0.8 ml) solution was placed on the RotoVisco solution platform, and viscosity values were obtained over a period of 240s and at the shear rate of 500-750 second-1. The recorded viscosity values and graphs were obtained from Rheowin 4 Data manager for further analysis. The surface morphology and shape of the PCL and PCL/CHX fibers were examined using VEGAS scanning electron microscopy (SEM) from Tescan, Czech Republic. Samples were coated with 7-10 nm of gold sputter to overcome the charging. The SEM micrographs were captured using secondary electron mode, with an accelerating voltage of 30 kV rms. Fiber diameter has been investigated using Image J software with 200 measurements on each sample. 3. Results and discussion The plumes of PCL/CHX fibers formation during the AC electrospinning process is displayed in Fig. 1(a). Spinnability of PCL and PCL/CHX precursor solution depended strongly on precursor concentration and applied AC potential. (a)
(c)
(b)
(g)
(h)
(e)
(d)
(g)
(f)
(h)
Fig. 1. Camera image of fiber plume and SEM images of PCL &PCL/CHX fibers (a) camera image of PCL/CHX nanofiber plume from the electrode at 35 kV rms, (b) 6 wt.% of PCL at 25 kV; (c) 6 wt.% of PCL at 35 kV; (d) 12 wt.% of PCL at 35 kV; (e) 6 wt.% of PCL/1wt% CHX at 25 kV; (f) 6 wt.% of PCL/1wt% CHX at 35 kV; (g) 12 wt.% of PCL/1 wt.% of CHX at 25 kV and (h) 12 wt.% of PCL/1 wt.% of CHX at 35 kV.
S. Manikandan et al./ Materials Today: Proceedings 17 (2019) 1100–1104
1103
Both precursor solutions were not spinnable at less than 25 kV rms. The critical applied AC potential for PCL and PCL/CHX solutions were 25 kV rms. The precursor PCL polymeric solution from 6 wt.% was AC spinnable at 25 kV and 35 kV rms. The PCL precursor solution concentration of 12 wt.% was not spinnable at 25 kV rms due to high viscosity and only spinnable at a high electric potential of 35 kV rms. The PCL/CHX precursor solutions concentrations 6 wt.% and 12 wt.% were AC spinnable at 25 kV and 35 kV rms. The SEM images proved that the addition of CHX to the PCL precursor solution strongly affects the fiber morphology and which improve the spinnability of precursor solution (Fig. 1(b)-(h)). The 6 wt.% of PCL at the applied potential of 25 kV and 35 kV rms, beads were observed with nanofibers (Fig. 1(b)&(c)). In the case of 12 wt.% of PCL at 35 kV rms, beadless fibers were observed (Fig. 1(d)). The 6 wt.% of PCL/CHX at 25 kV and 35 kV rms, beadless smooth fibers were obtained (Fig. 1(e)&(f)). The 12 wt.% of PCL/CHX at 25 kV and 35 kV rms, thicker beadless fibers were observed (Fig. 1(g)&(h)).
1000
Fiber diameter [nm]
25 kV 800
35 kV
600
400
200
0
6 wt% of PCL
12 wt% of PCL
6 wt% of PCL/CHX
12 wt% of PCL /CHX
Fig. 2. The PCL and PCL/CHX fiber diameter at 25 kV and 35 kV rms.
The effect of PCL concentration and 1 wt.% of CHX addition on PCL polymeric solution viscosity were showed in Table. 1. The resulting fiber diameters for applied AC potential were summarized in Fig. 2. Viscosity of PCL solution concentration was increased with respect to PCL concentration from 0.114±0.003 Pa. s at 6 wt.% to 0.946±0.005 Pa. s at 12 wt.%. Addition of 1wt% CHX to the 6 & 12 wt.% of PCL decreased the solution viscosity and increased the fiber diameter. Table 1. PCL and PCL/CHX solution viscosity. Precursor solution
Viscosity
concentration
[Pa.s]
PCL (6 wt.%)
0.114±0.003
PCL (12 wt.%)
0.946±0.005
PCL/CHX (6/1 wt.%)
0.093±0.003
PCL/CHX (12/1 wt.%)
0.666±0.008
1104
S. Manikandan et al./ Materials Today: Proceedings 17 (2019) 1100–1104
PCL fiber diameter was strongly depended on the precursor and additive concentration. There was not observed any significant differences in the both PCL and PCL/CHX electrospun diameter at 25 kV and 35 kV rms. PCL/CHX electrospun diameter was greater than that of PCL electrospun. 4. Conclusions Rapid fabrication of PCL/CHX antimicrobial nanofibers has been achieved using AC electrospinning for the first time. Addition of 1 wt.% CHX to the 6 wt.% and 12 wt.% of PCL solutions improved the fiber morphology from beaded compounded fibers to uniform smooth fibers. Viscosity and the fiber diameter were increased with respect to the precursor concentration. Further work will extend towards the analyze of the antimicrobial activity of AC electrospun material. Acknowledgements The authors would like to thank The Ministry of Education, Youth and Sports of Czech Republic and the student grant contest of the Technical University of Liberec, number SGS 21253 for providing financial support. References [1] [2] [3] [4] [5] [6]
Z. M. Huang, Y.Z. Zhang, M. Kotaki, S. Ramakrishna, Comps. Sci. technol. 63 (2003) 2223–2253. B. D. Masini, D.J. Stinner, S.M. Waterman, J.C. Wenke, J. surg. Educ. 68 (2011) 101-104. P. Rysanek, M. Maly, P. Capkova, M. Kormunda, Z. Kolska, M. Gryndler, O. Novak, L. Hocelikova, L. Bystriansky, M. Munzarova, J. Polym. Res. 24 (2017) 208-218. R. Scaffaro, L. Botta, M. Sanfilippo, G. Gallo, G. Palazzolo, A.M. Puglia, Appl. Microbiol. Biotechnol. 97 (2013) 99-109. P. Pokorny, E. Kostakova, F. Sanetmik, P. Mikes, J. Chvojka, T. Kalous, M. Bilek, K. Pejchar, J. Valtera, D. Lukas, Phys. Chem. Phys. 16 (2014) 26816-26822. C. Lawson, A. Stanishevsky, M. Sivan, P. Pokorny, D. Lukas, J. Appl. Polym. Sci. 133 (2016) 43232-43238.