Macromolecular Research, Vol. 22, No. 2, pp 139-145 (2014) DOI 10.1007/s13233-014-2017-x

www.springer.com/13233 pISSN 1598-5032 eISSN 2092-7673

Bactericidal Efficacy of Electrospun Rosin/Poly(ε-caprolactone) Nanofibers Rajkumar Nirmala*,1, Woo-il Baek1, Rangaswamy Navamathavan2, Tae Woo Kim1, Duraisamy Kalpana3, Mira Park1, Hak Yong Kim*,1, and Soo-Jin Park4 1

Department of Organic Materials and Fiber Engineering, Chonbuk National University, Jeonbuk 561-756, Korea 2 School of Advanced Materials Engineering, Chonbuk National University, Jeonbuk 561 756, Korea 3 Department of Forest Science and Technology, Institute of Agricultural Science and Technology, Chonbuk National University, Jeonbuk 561-756, Korea 4 Department of Chemistry, Inha University, Incheon 402-751, Korea Received June 10, 2013; Revised September 2, 2013; Accepted September 8, 2013 Abstract. Poly(ε-caprolactone) (PCL)-containing rosin nanofibers were prepared via electrospinning technique for biomedical applications. To improve the biocompatibility properties, rosin was blended into PCL to prepare nanofibers. Nanofibers mats were prepared with different concentrations of rosin. The morphology, structure, and thermal properties of the resultant PCL/rosin nanofibers were analyzed using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and thermogravimetry (TGA) analysis. SEM images revealed that the nanofibers were well-oriented and had good incorporation of rosin. FTIR results indicated the various bonding groups of PCL/rosin nanofibers with stable structure. TGA analysis revealed that the onset degradation temperature was decreased with increasing rosin content in the nanofibers. The bactericidal activity of PCL/rosin nanofibers was also investigated. These results indicate that the PCL blended with rosin nanofibers can be utilized as a promising candidate material for many biomedical applications. Keywords: poly(ε -caprolactone)/rosin, electrospinning, nanofibers, bactericidal.

Introduction

identified as a candidate for various biomedical applications because their size and physical geometry are very similar to those of the natural extracellular matrix.8-11 This inexpensive, scalable method can also yield flexible composite nanofiber mats which can be utilized to fit virtually any surface. Therefore, the nanofibers have been identified as ideal biomedical materials. In particular, poly(ε-caprolactone) (PCL) is a biodegradable aliphatic polyester, which has been regarded as nontoxic and tissue compatible.12,13 So far, variety of composite nanofibers, such as poly(ε-caprolactone)/tricalcium phosphate,14 PCL/hydroxyapatite,15 poly(ε-caprolactone)/poly(trimethylene carbonate),16 and chitosan-PCL17,18 have been successfully electrospun, and explored for potential biomedical applications. Recently, incorporation of metal nanoparticles into polymer nanofibers has attracted a great deal of attention in biomedical applications because the resulting fibers have very strong bactericidal activity.4,19,20 Intensive research based on antibacterial agents continues to be a pressing necessity as both microbial resistance and infectious diseases remain. Microorganisms continue to contaminate the surfaces of medical device, hospital and dental equipment, water purification systems, food packing and textiles.21-23 Therefore, there is a strong demand for the development of a broad

Electrospinning has been extensively considered as a unique and facile technique for producing ultrafine and continuous sub-micron fibers and/or nanofibers. Ultrafine nanofiber scaffolds prepared by the electrospinning of a polymer solution have been broadly studied because of their unique properties such as high surface area-to-volume ratio, high porosity and fully interconnected pore networks.1-5 Over the past decade, electrospun nanometer- to sub-micrometer-sized polymer nanofibers have attracted much attention in both research and commercial purpose. In the electrospinning process, a strong electrostatic field was applied to the polymer solution. The charged polymeric particle was attached to the collector. The jet formed was attracted towards the counter electrode and the solvent was evaporated and the non-woven fiber mats was collected on the collector. During jet-flying in the air, solvent evaporation leaves behind ultrathin fibers.6,7 Recently, electrospinning of biologically significant polymers have increased since the electrospun membranes were *Corresponding Authors. E-mails: [email protected] or [email protected] The Polymer Society of Korea

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spectrum of bactericidal materials that can deliver those agents. In this context, the natural materials have attracted considerable attention as they are biodegradable and biocompatible. By utilizing polymers (PCL), it is possible to obtain composite nanofibers structures which maximize the bactericidal properties of active agents, such as rosin. Especially, rosin is a natural polymer obtained from pine trees, and its derivatives have attracted much interest in the field of pharmaceutical applications.24-26 There have been several reports available based on the rosin and their derivation which was synthesized in the form of coating, films, nanoparticles.27-30 To our knowledge, electrospinning of pure rosin in the form of nanofibers morphology has not previously been reported. In our recent report, we have successful electrospun the rosin as fibers by optimizing the various experimental parameters during electrospinning process.31,32 However, there were no reports available based on electrospun rosin blended PCL composite nanofibers. Herein, for the first time, we report the preparation of PCL/rosin blended nanofibers via electrospinning and studies their bactericidal activities. Both PCL and rosin are considered to be biodegradable, multi-functional and technologically important materials. Therefore, we believe that the combination of these PCL/ rosin components in the form of nanofiber morphology can be conveniently utilized for various technological applications. Furthermore, the incorporation of rosin into PCL nanofibers can also be achieved by appropriate blending of polymer solutions via electrospinning method. Therefore, we performed the bactericidal analyses of rosin blended PCL nanofibers to exploit their features to be utilized for the biomedical applications. In this study, we report the preparation of rosin blended PCL nanofibers via electrospinning technique and analyzed their bactericidal activities. The rosin blended PCL nanofibers with different wt% of rosin were electrospun. The resultant nanofibers were characterized by means of scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimentry (DSC) and thermogravimetry (TGA). Further, the bactericidal activity of PCL/rosin nanofibers was also investigated. These results indicated that the PCL blended rosin nanofibers can be utilized as promising candidate materials for many biomedical applications.

Experimental Materials. PCL (MW=80,000) was purchased from Sigma Aldrich, USA. Dichloromethane (MC) and N,N-dimethyl formamide (DMF, analytical grade, Showa, Japan) were used as solvents without further purification. Rosin (low molecular weight) used in this research was obtained from Laton Korea Co., South Korea. All the above chemical reagents used were of analytical grade and used without further purification. Staphylococcus aureus (KCCM 11256), Escherichia 140

coli (KCCM 11234), Salmonella typhimurium (KCCM 11862) and Klebsiella pneumoniae (KCCM 35454) were purchased from Korean Culture Centre of Microorganisms (KCCM). These three pathogenic micro-organisms were used as the model bacteria for the disc diffusion susceptibility test. For the bactericidal activity measurement, Mueller-Hinton broth (MHB) & Mueller-Hinton agar (MHA) (Difco, Sparks, Md., USA) were used. Electrospinning. PCL solution 8.5 wt% was prepared by dissolving PCL in MC and DMF at the ratio of 80:20. The 60% (w/w) rosin solutions were prepared in DMF at the same volumetric ratio. PCL with 10, 20, and 50 wt% of rosin polymer solutions were used to prepare the nanofiber mats. The prepared solutions were vigorously stirred for 24 h at room temperature without any treatment. We have prepared the polymer solutions with various rosin concentration to check the electrospinnability and then we used the optimized parameters for obtaining the nanofiber mats. After that the polymer solution was loaded into a 5 mL plastic syringe with 1 mm diameter wire needle on syringe pump (KD scientific Co., USA) with a flow rate of 1 μL/min by applying 15 kV. A high voltage power supply (CPS-60 k02v1, Chungpa EMT, Co., Korea) was used to produce electrically charged liquid-jet from solutions. The positively and negatively charged electrodes of the power supply were directly connected to the needle of solution and the collector which is a metallic rotating drum covered with aluminum foil by using a copper wire, and the distance between the needle and the metallic drum was fixed at 15 cm. The rotating speed of the collector was kept at 150 rpm. All experiments were conducted at room temperature. The resultant fiber mats were initially dried for 24 h at 50 oC under vacuum and utilized for the further characterizations. Characterizations. The electrospun PCL/rosin nanofibers were observed by scanning electron microscopy (SEM, JSM5900, Jeol, Japan). Structural characterization was carried out by X-ray diffraction in a Rigaku X-ray diffractometer operated with CuKα radiation (λ=1.540 Å). The bonding configurations of the samples were characterized with Fourier transform infrared (FTIR) spectroscopy. Differential scanning calorimetry (DSC, Perkin-Elmer, USA) characterizations were performed for the PCL/rosin nanofibers under nitrogen ambient with a flow rate of 20 mL/min. The samples was heated from room temperature to 250 oC at a scanning rate of 10 oC/min. Thermogravimetric analysis (TGA, Perkin-Elmer, USA) was carried out under nitrogen with a flow rate of 20 mL/min. The samples were heated from 30 to 800 oC at a rate of 10 oC/min and the differential TGA graph was recorded. Mechanical properties were measured with a universal testing machine (AG-5000G, Shimadzu, Japan), under a crosshead speed of 10 mm/min at room temperature. The samples were prepared in the form of standard dumbbell shapes according to ASTM Standard D 638 via die cutting from the electrospun PCL/rosin nanofibers mats and Macromol. Res., Vol. 22, No. 2, 2014

Bactericidal Efficacy of Electrospun Rosin/Poly(ε-caprolactone) Nanofibers

tested in the machine direction. Bactericidal Activity by the Disc Diffusion Susceptibility Test. The disc diffusion susceptibility test for Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus was performed on the MHA plate at the temperature of 37 oC. The MHB was containing 1.5×106 colony-forming units (cfu) of bacteria. Then, each bacteria was lawn cultured on the MHA Petri plate by using a sterile cotton swab and the PCL/rosin nanofibers impregnated paper discs were kept at uniform distance and then incubated overnight at 37 oC. The zone of inhibition was observed after 24 h of incubation and the diameter of the zone was measured.

Results and Discussion Figure 1 shows the low (left side) and high (right side) magnification SEM images of electrospun PCL/rosin nanofibers with different rosin concentration of 0, 10, 20, and 50 wt%. It is seen that the synthesized PCL/rosin nanofibers were continuous over long distances without any beads. These rosin fibers were observed to be spherical morphology and

Figure 1. SEM images of electrospun PCL/rosin nanofibers with different rosin concentrations; (a) 0, (b) 10, (c) 20, and (d) 50 wt%. The labels 1 and 2 show the low and high magnification SEM images. Macromol. Res., Vol. 22, No. 2, 2014

highly dispersed with diameter in the range between few hundred nanometers to few microns. Although the electrospun PCL/rosin nanofibers were observed to be aberrant morphologies and thickness, it was possible to electrospin the solution in the form of fiber morphology. As the rosin concentration increases from 10 to 50 wt%, the resultant diameter of the nanofibers was gradually increased. The spherical morphology of the nanofibers became flat and led to film like appearance with elongated holes. Further, it was observed that the polymer solution couldn’t electrospinnable for the rosin concentration beyond 50 wt%. This result is attributed to the increase of viscosity with increasing rosin concentration as investigated in our previous report.31 The viscosity of the polymer solution is gradually increased with increasing rosin concentration. XRD analysis was used to investigate the phase structures of the electrospun PCL/rosin nanofibers. The XRD diffractograms of the electrospun PCL/rosin nanofibers with different rosin concentrations are shown in Figure 2. The XRD data of pristine rosin and pristine PCL nanofibers exhibited diffraction peak centered at 2θ =15o and 20o, respectively, due to their amorphous nature of nanofibers structure. No significant diffraction peaks of any other phases or impurities can be detected in the XRD patterns, which indicate the successful formation of PCL/rosin nanofibers. Furthermore, no diffraction peaks assigned to the rosin was found in the nanofibers, which could be attributed to the small amount of rosin blending (maximum 50 wt%) and their highly dispersion in the nanofibers. The XRD data confirmed the formation of electrospun PCL/rosin nanofibers. The structural configurations of electrospun PCL/rosin nanofibers were characterized by using FTIR spectroscopy. The FTIR spectroscopy is an important analysis that reflects the interactions between the PCL and rosin nanofibers. Figure 3 shows the FTIR spectra of the electrospun PCL/rosin nanofibers with different rosin concentrations. A significant

Figure 2. XRD patterns of electrospun PCL/rosin nanofibers with different rosin concentrations. 141

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Figure 3. FTIR spectra of electrospun PCL/rosin nanofibers with different rosin concentrations.

change was observed in the region 1730-1200 cm-1 where the pristine PCL showed only one band at 1740 cm-1, but splits to two bands in the nanofibers. Moreover, it could be observed that with the increasing content of rosin, the relative strength of peaks in the region 1730-1200 cm-1 which belongs to PCL decreased due to the interaction of rosin. The characteristic transmittance peaks of the electrospun PCL/ rosin nanofibers can be assigned as the following; 2950, 2909, and 1740 cm-1 were assigned to the stretching vibrations of -CH2- bonds and vibration of -C=O bonds, respectively.16,33 Almost no changes in the positions of these peaks were noted. Furthermore, there were no chemical active groups such as -OH and -NH2 exist in the structure both PCL and rosin that can create a hydrogen bond forming environment. These FTIR spectra results confirmed the successful preparation of PCL/rosin nanofibers via electrospinning. The changes in crystallinity of electrospun PCL/rosin nanofibers upon thermal treatment were investigated by DSC at a heating rate of 10 oC/min. Figure 4 shows the DSC traces of electrospun PCL/rosin nanofibers with different rosin concentrations. As expected, the pristine PCL nanofibers showed a single melting peak at 60 oC and that of pristine rosin was not detected, as shown in Figure 4(a). The melting peaks of PCL/rosin composite nanofibers were observed to be decreased with increasing rosin content in the composite nanofibers. These results suggested that PCL and rosin were existed in a phase separated form in the nanofibers structure. The crystallization temperatures of the pristine PCL and PCL/rosin electrospun nanofibers were 24 and 19 oC, respectively, as shown in Figure 4(b). A noticeable change in the crystallization temperature (about 5 oC) was observed for pristine PCL and rosin blended PCL nanofibers. This may be attributed to the different types of nucleation and growth of crystallization of the constituents of composite materials.34 Figure 5 shows the TGA analyses of electrospun PCL/rosin nanofibers with different rosin concentrations. The TGA results 142

Figure 4. DSC traces of electrospun PCL/rosin nanofibers with different rosin concentrations; (a) heating and (b) cooling, cycle.

showed that the pristine PCL and rosin nanofibers decomposed in a single step, whereas the PCL/rosin nanofibers showed two step degradations. The onset of decomposition of the pristine PCL and rosin nanofibers were of 395 and 225 oC, respectively, and for the PCL/rosin nanofibers with different rosin concentrations were found to be in the range of 250-370 oC, as shown in Figure 5(a). On the other hand, as expected, there was a slight change in the residual weight due to the utilized different rosin concentration. As shown in the first derivative data in Figure 5(b), a single sharp peak appeared at 250 and 400 oC for the pristine rosin and pristine PCL nanofibers, respectively. However, two distinct peaks were observed for the PCL/rosin nanofibers in the range of 275-400 oC. The thermograms of the electrospun PCL/rosin nanofibers with different rosin concentrations demonstrated negligible differences in the thermal stabilities. Figure 6 shows the mechanical properties of the PCL/rosin nanofibers with different rosin concentrations. As shown in the stress - strain curve, the yield stress of the pristine PCL Macromol. Res., Vol. 22, No. 2, 2014

Bactericidal Efficacy of Electrospun Rosin/Poly(ε-caprolactone) Nanofibers

Figure 6. Stress versus strain curves of electrospun PCL/rosin nanofibers with different rosin concentrations.

Figure 5. (a) TGA traces of electrospun PCL/rosin nanofibers with different rosin concentrations and (b) First derivative TGA of electrospun PCL/rosin nanofibers with different rosin concentrations.

nanofibers (11.4 MPa) was found to be higher than that of the blended nanofibers. However, the yield stress of PCL/ rosin nanofibers was found to be decreased with increasing rosin concentrations. The yield stress of the PCL/rosin nanofibers with 50 wt% rosin was about 2.1 MPa. On the other hand, it is interesting to observe that the Young’s modulus of the blended nanofibers was enhanced with increasing rosin content in the nanofibers (Table I). Smith et al., have reported the detailed tribological behavior of rosin materials.35 Apart from that there are few reports available based on the mechanical properties of rosin mixed with some composite materials.36-39 Recently, Liu et al., reported the improvement

of mechanical properties of rosin-based waterborne polyurethanes by the addition of cellulose nanocrystals as nanofillers in these materials.40 Their results showed that the tensile strength of the composite films were increased from 28.2 to 52.3 MPa with increasing cellulose nanocrystals amount from 0 to 20 wt%. However, there were no reports available based on the mechanical behavior of PCL/rosin nanofibers. The mechanical properties of a nanofibers mat will influence the mechanical environment such as bactericidal component of wound dressing products. Hence, it is necessary to fabricate mechanically stable nanofibers. Thus, the PCL/ rosin nanofibers prepared by electrospinning process are believed to have great potential in the biomedical applications in which the combined advantage of biodegradable electrospun PCL nanofibers with improved mechanical properties by adding rosin. The effect of bactericidal activity of electrospun PCL/rosin nanofibers in the culture media was studied by the disc diffusion susceptibility test. Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae bacterial strains were chosen as causative organisms of many device related infections. The quantitative evaluation of bactericidal activity of the PCL/rosin nanofibers was examined. More pronounced bactericidal effect was observed for the pristine rosin nanofibers, as shown in Figure 7(a). On the other hand, no bactericidal activity was detected for the pristine PCL nanofibers as shown in Figure 7(b), which was the

Table I. Mechanical Properties of Electrospun PCL/Rosin Composite Nanofibers Young’s Modulus (MPa)

Tensile Strength (MPa)

Strain at Break (%)

PCL

8.7

11.1

671.6

PCL+10 wt% Rosin

13.3

10.1

446.3

PCL+20 wt% Rosin

15.6

4.5

189.2

PCL+50 wt% Rosin

18.6

2.5

143.3

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blended with rosin nanofibers can be utilized as promising candidate materials for many biomedical applications. Acknowledgments. This research was financially supported by the Ministry of Education, Science Technology (MEST) and National Research Foundation of Korea (NRF) through the Human Resource Training Project for Regional Innovation (2012H1B8A2025931). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012R1A2A2A01046086). Figure 7. Bactericidal activity of electrospun (a) pristine rosin nanofibers with (1) Staphylococcus aureus, (2) Salmonella typhimurium, (3) Escherichia coli, and (4) Klebsiella pneumonia, and (b) PCL/rosin nanofibers with Staphylococcus aureus for (1) pristine PCL and (2) with rosin concentration of 50 wt%.

actual diameter of the nanofibers disc (6 mm). The test was repeated and the results were found to be almost same. The plates were checked for the prolonged incubation time to check the efficiency of the PCL/rosin nanofiber release. As observed from the disc diffusion test, the efficacy of the PCL/rosin nanofibers with rosin content of 50 wt% showed significant bactericidal activity when compared to that of other samples. The disc diffusion susceptibility tests clearly indicate the relationship between the bactericidal activity (inhibition efficiency) of rosin and surface characteristics of the bacterial cell wall. As far as bactericidal activity is concerned, the use of nanofiber morphology can advantageous due to their huge aspect ratio which can lead direct interaction with cells and inhibit the bacterial growth. Thus, we demonstrate that the electrospun PCL/rosin nanofibers are predicted as a desirable candidate to be utilized for excellent bactericidal filters and also wound healing agents.

Conclusions The PCL containing rosin nanofibers was successfully prepared via an electrospinning technique for the biomedical applications. To improve the biocompatibility properties, rosin was blended in to PCL to prepare nanofibers. The nanofibers mats with different concentration of rosin were prepared. The resultant nanofibers were characterized by SEM, XRD, FTIR, DSC, and TGA analysis. SEM images revealed that the nanofibers were well-oriented and had good incorporation of rosin. FTIR results indicated that the various bonding groups of PCL/rosin nanofibers with stable structure. TGA analysis revealed that the onset degradation temperature was decreased with increasing rosin content in the nanofibers. Further, the bactericidal activity of PCL/rosin nanofibers was also investigated. Our results demonstrate that the feasibility of producing PCL/rosin nanofibers by using electrospinning technique. These results indicated that the PCL 144

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