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Nanofibers as Carrier Systems for Antimicrobial Microemulsions. Part I: Fabrication and Characterization C. Kriegel,† K. M. Kit,‡ D. J. McClements,† and J. Weiss*,§ Department of Food Science, UniVersity of Massachusetts, Chenoweth Laboratory, 100 Holdsworth Way, Amherst, Massachusetts 01003, Department of Materials Science and Engineering, UniVersity of Tennessee, 434 Dougherty Engineering Building, KnoxVille, Tennessee 37996, and Department of Food Science and Biotechnology, UniVersity of Hohenheim, Garbenstr. 25, 70599 Stuttgart, Germany ReceiVed September 17, 2008. ReVised Manuscript ReceiVed October 31, 2008 Antimicrobial nanofibers were prepared by solubilizing an antimicrobial essential oil (eugenol; 0.75-1.5 wt %) in surfactant micelles (Surfynol 465; 5-10 wt %) to form eugenol-containing microemulsions. Microemulsions were mixed with a nonionic synthetic polymer (poly(vinyl alcohol), PVA; Mw ) 130 kDa, degree of hydrolysis ≈ 87%) and solutions subjected to electrospinning to induce nanofiber formation. Solution properties, fiber morphology, and composition of nanofibers were determined. The surface conductivity and viscosity of the polymer solutions increased, while surface tension decreased as both surfactant and eugenol concentration increased. Material deposited on the collector plate consisted primarily of nanofibers with a circular cross section with some surface roughness, although some bead defects were observed. The mean fiber diameters ranged from 57 to 126 nm with fibers having a broad diameter distribution (10-280 nm). The mean diameter of the nanofibers decreased with increasing surfactant concentration and decreasing eugenol concentration. Transmission electron microscopy indicated that microemulsion droplets were homogenously dispersed throughout the nanofibers. Results suggest that electrospun nanofibers may serve as carrier vehicles for microemulsions containing solubilized lipophilic functional compounds such as bioactives, antimicrobials, antioxidants, flavors, and pharmaceuticals.
Introduction Electrospinning is an emerging technology to produce ultrafine polymer fibers (“nanofibers”) by applying a strong electrical field to a polymer solution or melt, that is pumped through a small capillary orifice.1 If a critical voltage is exceeded, a jet composed of polymer and solvent is ejected from the tip of the capillary and accelerated toward a grounded target. As the solvent in the jet gradually evaporates, the polymer concentration increases until ultrafine solid nanofibers are formed that can be collected on a collector plate as a nonwoven mesh or membrane with fiber diameters typically ranging between 10 and several 100 nm. Nanofibers have remarkable physicochemical properties that include an extremely large surface-to-mass ratio, a high porosity, and a superior mechanical performance.2,3 This has given rise to nanofibers being explored for use in a wide variety of applications including tissue engineering, wound healing, drug delivery, medical implants, dental application, biosensors, military protective clothing, filtration media, and industrial applications.4-6 Nevertheless, despite their remarkable properties, further functionalization of nanofibers by spinning of polymer blends, coaxial spinning of two polymer solutions, coating of fibers, or inclusion * Corresponding author. E-mail:
[email protected]. Telephone: (+49) 711 459 24415. Fax: (+49) 711 459 23233. † University of Massachusetts. ‡ University of Tennessee. § University of Hohenheim. (1) Greiner, A.; Wendorff, J. H. Angew. Chem., Int. Ed. 2007, 46(30), 5670– 5703. (2) Kim, J. S.; Reneker, D. H. Polym. Compos. 1999, 20(1), 124–131. (3) Frenot, A.; Chronakis, I. S. Curr. Opin. Colloid Interface Sci. 2003, 8(1), 64–75. (4) Burger, C.; Hsiao, B. S.; Chu, B. Annu. ReV. Mater. Res. 2006, 36, 333– 368. (5) Buschle-Diller, G.; Hawkins, A.; Cooper, J. Electrospun Nanofibers from Biopolymers and their Biomedical Applications. In Modified Fibers with Medical and Speciality Applications; Edwards, J. V., Ed.; Springer: Dordrecht, The Netherlands, 2006; pp 67-80. (6) Chen, Z. G.; Mo, X. M.; Qing, F. L. Mater. Lett. 2007, 61(16), 3490–3494.
of functional components within fibers may further broaden the range of applications in which these nanofibers are currently used.7 Surface active agents (surfactants) contain both hydrophilic and hydrophobic moieties that enable them to form association colloids when dispersed in a solvent and to absorb to various interfaces.8,9 Association colloids such as spherical or wormlike micelles are capable of solubilizing lipophilic compounds within their hydrophobic interior.10-15 The resulting structures are referred to as microemulsions or swollen micelles and are aggregates of surfactant monomers that contain an additional “solubilized” third component.16 Microemulsions are transparent, optically isotropic, homogeneous, and thermodynamically stable mixtures of water, lipids, surfactants, and in some cases cosurfactants in appropriate amounts, with particle sizes ranging from 10 to 100 nm.17 Solubilization of lipophilic compounds in surfactant micelles increases the concentration of the lipophilic compound in the solvent phase which can lead to improvements in their functional properties such as, for example, bioavailability, (7) Kriegel, C.; Arecchi, A.; Kit, K.; McClements, J.; Weiss, J. Crit. ReV. Food Sci. Nutr. 2008, 48(8), 775–797. (8) Goodwin, J. Colloids and Interfaces with Surfactants and Polymers - An Introduction; Wiley Interscience: West Sussex, England, 2004. (9) Lange, K. R. Surfactants a practical handbook; Hanser Publishers: Munich, 1999. (10) Stauffer, D.; Jan, N.; He, Y.; Pandey, R. B.; Marangoni, D. G.; Smithpalmer, T. J. Chem. Phys. 1994, 100(9), 6934–6943. (11) Weiss, J.; Cancelliere, C.; McClements, D. J. Langmuir 2000, 16(17), 6833–6838. (12) Weiss, J.; Coupland, J. N.; Brathwaite, D.; McClements, D. J. Colloids Surf., A 1997, 121, 53–60. (13) Weiss, J.; Coupland, J. N.; McClements, D. J. J. Phys. Chem. 1996, 100, 1066–1071. (14) Weiss, J.; Herrmann, N.; McClements, D. J. Langmuir 1999, 15, 6652– 6657. (15) Weiss, J.; McClements, D. J. Langmuir 2000, 16(14), 5879–5883. (16) Vandamme, T. F. Prog. Retinal Eye Res. 2002, 21(1), 15–34. (17) Sharma, M. K.; Shah, D. O. ACS Symp. Ser. 1985, 272, 1–18.
10.1021/la803058c CCC: $40.75 2009 American Chemical Society Published on Web 12/23/2008
Eugenol Microemusions in Electrospun PVA Nanofibers
antimicrobial, or antioxidant activities.18 For example, Gaysinksy et al. demonstrated in a number of studies that encapsulation of lipophilic antimicrobials in microemulsions can greatly enhance their activity against Gram positive and Gram negative foodborne pathogens due to an increased interaction with bacterial surfaces.19-22 Numerous studies have discussed the various interactions that may occur when surfactants and polymers are mixed. These interactions may influence key characteristics of both polymer and surfactants such as, for example, critical micelle concentration, critical aggregation number, structure of micelles, and molecular configuration of polymers.23-26 In the context of electrospinning, polymer-surfactant interactions may alter the rheology, surface tension, and conductivity, three of the most critical factors in the successful preparation of nanofibers by electrospinning.27 Thus, addition of surfactants to polymers may modulate the electrospinning process. For example, small amounts of nonionic surfactant have been reported to decrease the onset voltage and improve the reproducibility of the electrospinning process.28 In a previous study, we reported that type as well as concentration of surfactants modulated the electrospinning process, leading to the fabrication of nanofibers that varied in fiber morphology.29 Moreover, electron microscopy images of electrospun fibers from micellar solutions of DTAB and Brij 35 containing a chitosan-PEO blend suggested that micelles remained intact and were either evenly dispersed throughout the fiber or accumulated at surfaces depending on the charge of micelles. To the best of our knowledge, this was the first study on electrospinning of polymeric nanofibers in the presence of micellar structures. In this study, we hypothesize that nanofibers containing swollen micelles may be fabricated by electrospinning microemulsions formed with a lipophilic antimicrobial phytophenol (eugenol) and a Gemini surfactant (Surfynol 465) dispersed in polymer solutions (poly(vinyl alcohol), PVA). The resulting nanofibers are expected to exhibit antimicrobial activities, since eugenol is a potent antibacterial and antifungal phenolic compound that is the predominant constituent of clove essential oil (Syzygium aromaticum).30 We suggest that the fine porous structure of nanofibers allows for a rapid release of the encapsulated compound.31 The objectives of this two part study were to test this hypothesis by electrospinning blends of microemulsions and a matrix polymer. In this part of the study, we determined the optimal manufacturing conditions to produce microemulsioncontaining fibers by electrospinning polymer blends in the presence of microemulsions and characterized fiber morphology as a function of solution composition. In the forthcoming second (18) Weiss, J.; McClements, J.; Takhistov, P. Food Aust. 2007, 59(6), 274– 275. (19) Gaysinsky, S.; Davidson, P. M.; Bruce, B. D.; Weiss, J. J. Food Prot. 2005, 68(12), 2559–2566. (20) Gaysinksy, S.; Davidson, P. M.; Bruce, B. D.; Weiss, J. J. Food Prot. 2005, 68(7), 1359–1366. (21) Gaysinsky, S.; Davidson, P. M.; McClements, D. J.; Weiss, J. Food Biophys. 2008, 3(1), 54–65. (22) Gaysinsky, S.; Taylor, T. T.; Davidson, P. M.; Bruce, B. D.; Weiss, J. J. Food Prot. 2007, 70, 2631–2637. (23) Nagarajan, R. J. Chem. Phys. 1989, 90(3), 1980–1994. (24) Brackman, J. C.; Engberts, J. Chem. Soc. ReV. 1993, 22(2), 85–92. (25) Smitter, L. M.; Guedez, J. F.; Muller, A. J.; Saez, A. E. J. Colloid Interface Sci. 2001, 236(2), 343–353. (26) Tam, K. C.; Wyn-Jones, E. Chem. Soc. ReV. 2006, 35(8), 693–709. (27) Lin, T.; Wang, H. X.; Wang, H. M.; Wang, X. G. Nanotechnology 2004, 15(9), 1375–1381. (28) Yao, L.; Haas, T. W.; Guiseppi-Elie, A.; Bowlin, G. L.; Simpson, D. G.; Wnek, G. E. Chem. Mater. 2003, 15(9), 1860–1864. (29) Kriegel, C.; Kit, K. M.; McClements, D. J.; Weiss, J. Polymer, 2008, doi: 10.1016/j.polymer.2008.09.041 (30) Raina, V. K.; Srivastava, S. K.; Aggarwal, K. K.; Syamasundar, K. V.; Kumar, S. FlaVour Fragrance J. 2001, 16(5), 334–336. (31) Huang, X. J.; Ge, D.; Xu, Z. K. Eur. Polym. J. 2007, 43(9), 3710–3718.
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part of the study, we evaluated the release characteristics of the lipophilic compounds incorporated in electrospun fibers as a function of microemulsion composition and concentration and determined their antimicrobial activity.
Materials and Methods Materials. All solutions were prepared with distilled and deionized water. Poly(vinyl alcohol) (PVA) with a molecular weight of 130 kDa and a degree of hydrolysis of 86.7-88.7 mol %, sodium acetate trihydrate (#71188), and eugenol 99% (4-allyl-2-methoxyphenol) (E51791) were obtained from Sigma Aldrich (St. Louis, MO). Eugenol has a molecular weight of 164.21 Da, a specific gravity of 1.065, a vapor pressure of 4 Pa at 20 °C, and a melting point of -7.5 °C and a boiling point of 253.2 °C at atmospheric pressure. Glacial acetic acid (CAS #64197, UN 2789) was purchased from Acros Organics (Morris Plains, NJ). Tris HCl (#BP1756-500, pH 7.0) was obtained from Fisher Scientific (Pittsburgh, PA). The nonionic surfactant Surfynol 465 was provided by Air Products and Chemical, Inc. (Allentown, PA). The reported critical micellar concentration was 0.65 wt % for Surfynol 465. Surfynol 465 is an ethoxylated oligomer of 2,4,7,9-tetramethyl-5-decyne-4,7-diol with an average molecular weight of approximately 519 Da (total number of ethoxyl groups m + n ≈ 12). Surfynol surfactant was selected because of its characteristically low dynamic surface tension and high stability in high ionic strength environments.32 The surfactant is approved for applications in food contact surfaces (FDA 21 CFR 175-177).33 Reagents and polymers were used as received from the manufacturer without further purification. Preparation of Micellar Solutions. Surfactant solutions were prepared with distilled and deionized water and reagent grade glacial acetic acid. Bulk surfactant solutions were obtained by dispersing Surfynol 465 in water at room temperature to yield concentrations ranging from 5 to 10% (w/w). Eugenol was added to the surfactant solutions at 0.75-1.5% (w/w). These concentrations were chosen based on studies previously conducted in our research laboratory that identified the range of surfactant and eugenol concentrations where microemulsions could be formed.19-21 Solutions were stirred for approximately 15 min at room temperature until the absorbance decreased to zero which indicated completed solubilization and formation of microemulsions. After sterile filtration (0.22 µm) with a syringe filter (Corning, NY) to remove any impurities, solutions were stored up to 2 weeks at 25 ( 2 °C. Polymer and Polymer-Microemulsion Solution Preparation. To find a suitable polymer concentration for the microemulsionincorporation experiments, pure PVA solutions of various concentrations were initially electrospun. To this purpose, PVA solutions were prepared by dissolving 5-15% (w/w) PVA in 1 wt % aqueous acetic acid at 80 °C for 3 h to ensure complete dissolution of the polymer. This type of solvent was chosen because of its ability to facilitate the electrospinning of PVA compared to pure water systems. After cooling to room temperature, solutions were immediately electrospun. Uniform, thin fibers were formed at a polymer concentration of 7.5 wt % that increased in diameter with increasing polymer concentration. Based these initial experiments, a polymer concentration of 7.5% (w/w) was chosen for all subsequent studies. For preparation of microemulsion-containing PVA solutions, PVA solutions were prepared as stated above. After cooling to room temperature, the polymer solution was blended with a specific amount of eugenol-containing microemulsions to yield PVAS solutions with microemulsions at surfactant and eugenol concentrations ranging from 5 to 10% and 0 to 1.5% (w/w), respectively. After blending for 2 h to ensure a homogeneous distribution, the polymer-micellar solutions were immediately electrospun. Determination of Critical Micellar Concentration (cmc) by Plate Tensiometry. The cmc of surfactants in the absence and presence of polymer was determined by measuring the surface tension as a function of surfactant concentration using a digital tensiometer (32) Parees, D. M.; Hanton, S. D.; Clark, P. A. C.; Willcox, D. A. J. Am. Soc. Mass Spectrom. 1998, 9, 282–291. (33) Menger, F. M.; Keiper, J. S. Angew. Chem., Int. Ed. 2000, 39(11), 1907– 1920.
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(model K10ST, Kruss, Germany) utilizing the Wilhelmy plate method. An amount of 40 g of test solution was poured into a 70 mm diameter glass beaker which had been rinsed with absolute ethanol and deionized and double distilled water and then dried at 70 °C overnight to remove any surface-active material. The solutions were equilibrated at 25 °C, and the platinum plate was lowered into the solution to form a meniscus. The surface tension σ was calculated in mN/m from the measured force F acting on the platinum plate, where L is the length of the total meniscus (2 × the length + thickness of the plate) and θ is the contact angle.
σ)
F L cos θ
(1)
The force F was read after equilibration for 5 min when values had stabilized and did not change with time. No correction calculations were required with this method, and results shown are means of triplicate measurements. Solution Viscosity. Solution viscosity was measured using an oscillatory rheometer with a double coaxial cup and bob measurement system of the Couette type: bob length ) 32.05 mm, diameter ) 25 mm, and cup diameter ) 26.97 mm (ARES LS, Patel Scientific, NJ). The shear stress σ (Pa) of solutions was recorded as a function of shear rate (s-1) at shear rates ranging from 10-2 to 103 s-1. Solutions were equilibrated to 25 °C prior to all measurements using a Peltier system. Reported results are means of triplicate measurements. Measurements were fitted to the power law model:34
σ ) Kγ˙ n
(2)
where K is the consistency coefficient and n is the flow behavior index. If the flow behavior index n equals 1, the solution behaves as a Newtonian fluid; if the index is smaller than 1, the solution exhibits shear thinning, and if the index is larger than 1, the solution is a shear thickening fluid. Solution Conductivity. Electrical conductivity of polymersurfactant or polymer-microemulsion solutions was determined using a microelectrophoresis instrument (NanoZS, Malvern Instruments, Worcestershire, U.K.). The temperature was adjusted to 25 °C prior to the measurements. Reported results are means of triplicate measurements. Solution Surface Tension. The surface tension of each polymer-micellar or polymer-microemulsion solution was determined using a digital tensiometer as described above. Electrospinning. An electrospinning setup described previously was used to electrospin solutions.29 Briefly, a 20 ml glass syringe (Micro-Mate, Popper & Sons, New Hyde Park, NY) with a 0.69 mm diameter stainless steel capillary (Hamilton, NE, no. 91019) bluntend tip was filled with polymer-microemulsion solutions. The syringe was placed in a syringe pump (Harvard apparatus; 11plus, Holliston, MA) which permitted adjustment and control of solution flow rates. The metal capillary of the syringe was connected to the positive lead of a high voltage power supply (Gamma High Voltage; ES 30P-5W, Ormond Beach, FL) operated in positive DC mode. A grounded copper plate wrapped in aluminum foil and mounted onto two polypropylene blocks was used as the target collector plate for collection of fibers and/or beads. The target was placed 10 cm from the capillary tip. The syringe pump delivered polymer solution at a controlled flow rate of 0.02 mL/min, while the voltage was maintained at 20 kV and the temperature at 25 °C. These conditions were kept constant throughout all experiments. Scanning Electron Microscopy (SEM). The morphology of electrospun nanofibers was observed with a field emission scanning electron microscope (FESEM 6320 FXV, JEOL, MA) operated at an accelerating voltage of 5 kV. Nanofibers were electrospun directly onto aluminum SEM stubs which were mounted on the grounded collector plate. After collection of the fibers, samples were sputter coated with Au in a sputter coater (Cressington 108, Cressington, Watford, U.K.) for 60 s to reduce electron charging effects. The (34) Wongsasulak, S.; Kit, K. M.; McClements, D. J.; Yoovidhya, T.; Weiss, J. Polymer 2007, 48(2), 448–457.
Figure 1. Apparent viscosities of solutions at a shear rate of 100 s-1.
fiber diameter distribution was determined from the scanning electron images using image analysis software (ImageJ, NIH) from >60 randomly selected fibers from SEM micrographs for each sample. For transmission electron microscopy (TEM) examination, nanofibers were directly electrospun onto copper grits followed by staining with ruthenium tetroxide vapor for 15 min for visualization of the phenol ring structure in eugenol.
Results and Discussion Apparent Viscosity of Polymer Solutions in the Presence and Absence of Surfactants and Microemulsions. The apparent viscosity of polymer solutions with or without loaded and unloaded surfactant micelles at a shear rate of 100 s-1 (ηa,100) was measured using constant shear rheology (Figure 1). In pure polymer solutions, the apparent viscosity increased as the polymer concentration was raised. For example, the apparent viscosity increased from 32.1 ( 0.7 mPa · s at a polymer concentration of 5 wt % to 308 ( 7 mPa · s at a solution concentration of 10 wt %, equaling an approximate 10-fold increase in the apparent viscosity at double the polymer concentration. At 7.5 wt %, the PVA solution had an apparent viscosity of 126 ( 3 mPa · s which is about half that of the 10 wt % solution. The solvent had an apparent viscosity of 1.6 ( 0.5 mPa · s. Generally, viscosity is directly affected by concentration, molecular weight, and structure of a polymer as well as solvent type, properties that have a profound influence on electrospinning and the resulting morphology of the fibers.38 Viscosity is a key parameter that is also related to polymer-polymer interactions such as polymer chain entanglements. The loss of electrospinnability in solutions of low viscosity is often due to reduced or insufficient polymer chain entanglement required for the formation of a stable and continuous polymer jet that has to be ejected from the tip of the capillary.39 Insufficient entanglement can lead to a break up of the jet into small droplets, yielding beads instead of fibers. As (35) Rosen, M. J. Surfactants and Interfacial Phenomena, 3rd ed.; Wiley Interscience: New York, 2004. (36) Farn, R. J. Chemistry and technology of surfactants; Blackwell Publishing: Oxford, 2006. (37) Almgren, M.; Hansson, P.; Mukhtar, E.; Vanstam, J. Langmuir 1992, 8(10), 2405–2412. (38) Ramakrishna, S. An Introduction to Electrospinning and Nanofibers; World Scientific: Singapore, 2005. (39) Buchko, C. J.; Chen, L. C.; Shen, Y.; Martin, D. C. Polymer 1999, 40(26), 7397–7407.
Eugenol Microemusions in Electrospun PVA Nanofibers
evidenced by the scanning electron micrograph shown later (Figure 5A), electrospinning of 5 wt % PVA solution with lower viscosity led to formation of beaded fibers with an occurrence of spindlelike beads. Conversely, higher concentrated solutions favored the formation of smooth regularly shaped nanofibrous structures (Figure 5C). At higher solution viscosities, viscoelastic forces counteract Coulombic forces, preventing a stretching of the polymer jet which favors the formation of nanofibers with a larger diameter.1,40-42 However, while a certain optimum viscosity will favor the formation of fine fibers by electrospinning, increasing the viscosity above a certain limit will cause difficulty in pumping the polymer solution through the syringe needle and a gel may be formed at the tip of the syringe due to drying, which prevents electrospinning.43 On the basis of these results, all subsequent experiments were performed at a constant polymer concentration of 7.5 wt %. Addition of surfactant micelles at a concentration of 5, 7.5, and 10 wt % Surfynol 465 to the 7.5 wt % PVA solutions increased the apparent viscosity from 186 ( 7 to 223 ( 7, and 272 ( 9 mPa · s, respectively. At the highest surfactant concentration, the apparent viscosity was approximately 150 mPa · s higher than that of the pure PVA solution, while a 10 wt % Surfynol solution only had an apparent viscosity of 1.68 ( 0.01 mPa · s. Thus, the viscosity of the composite solution was somewhat larger than that of the individual solutions, which could suggest a weak interaction between surfactant micelles and PVA. In nonionic polymers-nonionic surfactants, interactions have been reported to be generally weak compared to the interaction in ionic polymers and nonionic surfactants, nonionic polymers and ionic surfactants, and ionic surfactants and ionic polymers. Addition of microemulsion to the 7.5 wt % PVA solution increased the apparent viscosity to 288 ( 7, 329 ( 2, and 432 ( 4 mPa · s for 0.75, 1.125, and 1.5 wt % eugenol in 10 wt % Surfynol 465, respectively. Keeping the loading ratio of eugenol to Surfynol 465 in the microemulsion constant (10% Surfynol/1.5% eugenol) but increasing the overall concentration of microemulsions in the PVA solution also resulted in a steady increase in apparent viscosity. For example, at the lowest concentration of both eugenol and Surfynol, 0.75 and 5 wt %, respectively, the apparent viscosity was 230 ( 10 mPa · s, which represents an increase of about 100 mPa · s compared to the pure polymer solution, while the system containing 1.125 wt % eugenol and 7.5 wt % Surfynol 465 had an apparent viscosity of 289 ( 7 mPa · s. Flow Behavior of Polymer Solutions in the Presence and Absence of Surfactants and Microemulsions. Flow curves of solutions were measured over a shear rate range of 10-3-103 s-1 and fitted to the power law (eq 1) to calculate the consistency coefficient (K) and power law flow behavior index (n) for a more in-depth understanding of the rheological behavior of solutions. The consistency coefficient provides a measure of the apparent viscosity of the solution at low shear rates: the higher K, the more viscous the solution. The flow behavior index indicates whether the solution behaves as a pseudoplastic (n < 1), dilatant (n > 1), or Newtonian liquid (n ) 1). In the absence of surfactant and eugenol, the consistency coefficient increased with increasing PVA concentration as would be expected for polymer solutions (Table 1). The power law index was close to unity for the 5 wt % PVA solutions, indicating that they behaved like Newtonian (40) Geng, X. Y.; Kwon, O. H.; Jang, J. H. Biomaterials 2005, 26(27), 5427– 5432. (41) Han, S. O.; Son, W. K.; Youk, J. H.; Park, W. H. J. Appl. Polym. Sci. 2008, 107(3), 1954–1959. (42) Helander, I. M.; Nurmiaho-Lassila, E. L.; Ahvenainen, R.; Rhoades, J.; Roller, S. Int. J. Food Microbiol. 2001, 71(2-3), 235–244. (43) Zhong, X. H.; Kim, K.; Fang, D. F.; Ran, S. F.; Hsiao, B. S.; Chu, B. Polymer 2002, 43(16), 4403–4412.
Langmuir, Vol. 25, No. 2, 2009 1157 Table 1. Power Law Indices K and n of PVA-Surfactant and PVA-Microemulsion Dispersions samplea
power law consistency power law flow coefficient (K) behavior index (n)
5%P 7.5%P 10%P
0.0306 ( 0.001 0.1343 ( 0.006 0.3762 ( 0.000
1.002 ( 0.001 0.982 ( 0.006 0.966 ( 0.001
7.5%P 5%S 7.5%P 7.5%S 7.5%P 10%S
0.210 ( 0.014 0.259 ( 0.012 0.3320 ( 0.017
0.979 ( 0.005 0.973 ( 0.002 0.964 ( 0.003
7.5%P 7.5%P 7.5%P 7.5%P 7.5%P
0.2655 ( 0.016 0.3435 ( 0.006 0.3525 ( 0.016 0.4000 ( 0.023 0.9625 ( 0.008
0.974 ( 0.003 0.966 ( 0.001 0.962 ( 0.004 0.941 ( 0.008 0.764 ( 0.001
5%S 0.75%E 10%S 0.75%E 7.5%S 1.125%E 10%S 1.125%E 10%S 1.5%E
a The letters P, S, and E denote poly(vinyl alcohol), Surfynol 465, and eugenol, respectively.
liquids, but decreased at higher PVA concentrations, indicating that these solutions became shear thinning (Table 1). A reduced n is generally an indication for polymer entanglement or interactions, which is a fundamental prerequisite for deposition of nanofibers during the electrospinning process as mentioned above. For 7.5 wt % PVA solutions in the absence of eugenol, there was an appreciable increase in the consistency coefficient and decrease in the power law index as the surfactant concentration was increased (Table 1). These results suggest that the surfactant altered either the conformation and/or the interactions of the PVA molecules in the polymer solutions. This increase in viscosity and shear thinning behavior can be attributed to a number of physicochemical phenomena: (i) the binding of surfactant molecules to the PVA molecules may have caused the polymers to expand and occupy a greater volume; and (ii) surfactant micelles may have acted as bridges between PVA molecules, causing the polymer molecules to be weakly attracted to each other. Electrical Conductivity of Polymer Solutions in the Presence and Absence of Surfactant. In electrospinning, charged ions are expelled from the meniscus formed at the tip of a capillary and accelerated toward a grounded target. During this process, the polymer jet is stretched and orientation occurs.44 The electrical conductivity of the polymer solutions is critically important in this process. Figure 2 shows the electrical conductivity of polymer-surfactant systems. Pure PVA solution (7.5 wt %) had the highest conductivity of 0.58 ( 0.02 mS/cm, while pure surfactant solution (10 wt %) had a conductivity of 0.52 ( 0.01 mS/cm. Blending of 7.5 wt % PVA with 10% Surfynol yielded a solution conductivity of 0.42 ( 0.01 mS/cm. When less surfactant was added to the PVA solution, conductivities increased to 0.44 ( 0.02 and 0.50 ( 0.01 mS/cm for 7.5 and 5 wt % surfactant, respectively. Loading of micelles with eugenol and addition to PVA solution had no significant influence on the conductivity of solutions. Results may be attributed to the fact micelles were composed of nonionic surfactants loaded with an uncharged lipophilic phytophenol and increases in either concentration thus should not noticeably influence the overall conductivity of the solution. It should be noted though that addition of charged microemulsions could yield substantially different results. We previously added micellar solutions of ionic surfactants (SDS, DTAB) to polymer solutions which resulted in significant increases in conductivity.29 In electrospinning, if the conductivity of a solution is increased due to addition of ionic salts, (44) Son, W. K.; Youk, J. H.; Park, W. H. Biomacromolecules 2004, 5(1), 197–201.
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Figure 4. Surface tension in mN/m of polymer-surfactant and polymer-microemulsion solutions dispersed in 1% acetic acid. Figure 2. Electrical conductivity in mS/cm of polymer-surfactant and polymer-microemulsion solutions dispersed in 1% acetic acid. Surface tension in mN/m.
Figure 3. Surfynol 465 solutions of varying surfactant concentration with and without addition of 7.5 wt % poly(vinyl alcohol).
polyelectrolytes, or a solvent that dissociates in water, more charges can be carried in the polymer jet, allowing for greater elongation forces to act on the jet. As a result, bead formation as well as resulting fiber diameter tend to be reduced.43 Further, with an increase in conductivity, a lower acceleration voltage is required for jet initiation.44 Polymer-Surfactant Interactions and Solution Surface Tension. The surface tension of the aqueous acetic acid solution used in this study was 69.00 ( 0.01 mN/m. As the surfactant concentration in the aqueous acidic acid solution was increased, a gradual decrease of surface tension was observed, indicative of a solution containing surfactant monomers (Figure 3).35 At a certain concentration (0.75 wt % surfactant), the surface tension reached a value of 29.70 ( 0.02 mN/m and remained relatively constant even when more surfactant was added to the solution. At this concentration commonly known as the critical micelle concentration (cmc), surfactant monomers associated with each other to form micellar aggregates.36 To study the interaction between the PVA and Surfynol 465, the surfactant was titrated
into a 7.5 wt % PVA solution (the concentration used in subsequent electrospinning experiments). In the absence of surfactant, the surface tension of the polymer solution was 46.65 ( 0.14 mN/m (Figures 3 and 4). The surface tension gradually decreased with increasing surfactant concentration from 0 to 0.02 wt %, fell more rapidly from 0.02 to 1 wt %, and then attained a relatively constant value (∼30 mN/m) at higher surfactant concentrations (Figure 3). From 0 to 0.02 wt % surfactant, the surface tension was considerably lower in the presence of PVA than in its absence. From 0.02 to 10 wt % surfactant, the surface tension versus surfactant concentration profile was fairly similar in the presence and absence of PVA, which suggests that absorption of surfactant to the air-water interface dominated the behavior of the system. Nevertheless, the surface tension was slightly higher in the presence of PVA from 0.02 to 1 wt % surfactant, which may have been because surfactant molecules formed a complex with the polymer that reduced the amount of surfactant monomers available to adsorb to the air-water surface and/or that was less surface active than the surfactant monomers.25,37 For example, surfactant monomers may have associated with polymer chains via hydrogen bonding and/or hydrophobic interactions at nonpolar patches on the PVA chains. Even so, the presence of fairly high concentrations of polymer (7.5 wt % PVA) did not have a major impact on the surface tension versus surfactant concentration profile (Figure 3), compared to other surfactant-polymer systems.25,37 However, these phenomena were observed in systems containing nonionic polymers and anionic surfactants or in the case of oppositely charged polymer and surfactants rather than nonionic surfactants and uncharged polymers as used in our studies. The fact that the PVA-surfactant interaction appeared to be relatively weak for the system used in this study may be an advantage for subsequent release and antimicrobial activity of the encapsulated eugenol, since liberation of microemulsions containing the active ingredient occurs freely and is primarily diffusion controlled. Strong interactions between the polymer and surfactant aggregates might instead impede a sufficient release, thus lowering the biofunctional activity. Furthermore, surface tension has a direct influence on the ability to electrospin a polymer solution as well as on the nature
Eugenol Microemusions in Electrospun PVA Nanofibers
Langmuir, Vol. 25, No. 2, 2009 1159 Table 2. Morphological Characteristics of Fiber Deposits Shown in Figures 5 and 6a sampleb
fiber diameter range (nm)
average fiber diameter (nm)
standard deviation (nm)
5 wt % PVA 7.5 wt % PVA 10 wt % PVA
50-240 80-140 140-340
83.40 111.43 218.92
31.84 11.94 28.87
30-160 50-280 60-240 20-120 10-190 20-120
75.84 125.87 125.70 64.58 82.39 56.71
27.47 56.52 34.41 22.57 32.03 21.24
A B C D E F
a Denoted are minimum, maximum, and average observed diameters as determined by image analysis using scanning transmission electron microscopy images. b Samples A-F correspond to samples A-F in Figure 6, respectively.
Figure 5. Scanning electron micrographs of electrospun poly(vinyl alcohol) at a concentration of (A) 5 wt %, (B) 7.5 wt %, and (C) 10 wt %.
of the structures formed, for example, fibers versus beads.45 The surface tension must be overcome by the electrical forces acting upon the polymer solution before a fine polymer jet is expelled from the injector. In addition, the surface tension has a tendency to cause the fine polymer jet to deform into spheres during its transit between the injector and collector plate, which should be avoided if fibers rather than beads are to be produced.46 For this reason the surface tension of the polymer solutions was measured in the presence of surfactant and eugenol (Figure 4). Both the polymer and surfactant were surface active as indicated by their reduced surface tensions of 46.65 ( 0.14 and 30.8 ( 0.1 mN/m, respectively, when compared to the pure solvent. The addition of different levels of surfactant (5-10 wt %) or eugenol (0.75-1.5 wt %) to the polymer solutions (7.5 wt % PVA) caused little change in the measured surface activity of the solutions compared to that of the 10 wt % surfactant solution (Figure 4), which suggests that their behavior was dominated by the surfactant rather than by the polymer or eugenol. We propose that the air-water interface was saturated with surfactant molecules (which were the most surface-active component in the system) (45) Fong, H.; Chun, I.; Reneker, D. H. Polymer 1999, 40(16), 4585–4592. (46) Gupta, P.; Elkins, C.; Long, T. E.; Wilkes, G. L. Polymer 2005, 46(13), 4799–4810.
in all of the systems and that any polymer-surfactant-eugenol complexes remained in the bulk of the solution. Fiber Morphology, Average Fiber Diameter, and Localization of Eugenol in the Nanofibers. The functional performance of nanofibers is usually governed by their microstructure, and so we characterized the structure of the materials produced by electrospinning the polymer solutions using electron microscopy. SEM micrographs of pure PVA nanofibers are shown in Figure 5, while the morphological characteristics of the nanofibers determined from these micrographs are tabulated in Table 2. At lower PVA concentrations (5 wt %, Figure 5A), many fiber defects such as spindlelike elongated beads were observed together with fibers of a broader size distribution ranging between 50 and 240 nm, however with a relatively smaller mean fiber diameter of 83 ( 32 nm compared to the other pure polymer nanofibers. In this case, the viscoelastic force was not large enough to counteract the surface tension and favored a partial breakup of the charged polymer jet into droplets. At 7.5 wt % (Figure 5B), fibers had smooth surfaces and were of regular round shape and almost defect free with a relatively small average fiber diameter of 111 ( 12 nm and a fiber size distribution ranging from 80 to 140 nm. At the highest tested polymer concentration of 10 wt % PVA, the fibers had very smooth surfaces with a larger average fiber diameter of 219 ( 29 nm and a distribution ranging from 140 to 340 nm. In this case, the solution viscosity was higher, increasing the viscoelastic forces which counteracted the Coulombic force and prevented a stretching of the polymer jet, favoring the formation of nanofibers with a larger diameter1,40-42,47 in accordance with results of the rheological measurements presented above. The obtained sizes are in good agreement with values obtained by other researchers, who reported similar fiber sizes when electrospinning poly(vinyl alcohol).48,49 Addition of Surfynol 465 to the polymer dispersion yielded thinner nanofibers with an average diameter of 76 ( 27 nm (Figure 6A) and a broad size distribution range of 30-160 nm. Many fibers of smaller diameter are intersecting larger ones, and their surfaces are somewhat rough and uneven unlike pure PVA fibers. This indicates potential occurrence of surfactant micelles just beneath the nanofiber surface. Addition of loaded micelles or microemulsions resulted in irregular shaped nanofibrous structures with rough and patchy surfaces regardless of microemulsion composition. Furthermore, fiber size distributions tended to be broader while a fiber size reduction was observed in almost all cases. Nanofiber diameters obtained from polymer solutions (47) Yang, D. Z.; Li, Y. N.; Nie, J. Carbohydr. Polym. 2007, 69(3), 538–543. (48) Jun, Z.; Hou, H. Q.; Wendorff, J. H.; Greiner, A. e-Polym. 2005. (49) Supaphol, P.; Chuangchote, S. J. Appl. Polym. Sci. 2008, 108(2), 969– 978.
1160 Langmuir, Vol. 25, No. 2, 2009
Kriegel et al.
Figure 6. Scanning electron micrographs of electrospun 7.5 wt % poly(vinyl alcohol) solution containing (A) 10 wt % Surfynol 465, (B) 10 wt % Surfynol 465 loaded with 1.5 wt % eugenol, (C) 7.5 wt % Surfynol 465 loaded with 1.125 wt % eugenol, (D) 10 wt % Surfynol 465 loaded with 1.125 wt % eugenol, (E) 5 wt % Surfynol 465 loaded with 0.75 wt % eugenol, and (F) 10 wt % Surfynol 465 loaded with 0.75 wt % eugenol. Images are shown at a magnification of 10 000×.
that also contained 10 wt % Surfynol 465 loaded with 1.5 wt % eugenol ranged from 50 to 280 nm with an average fiber size of 126 ( 57 nm (Figure 6B). A decrease in loading ratio at the same surfactant concentration led to a reduction in size to 65 ( 23 and 57 ( 21 nm at levels of 1.125 and 0.75 wt % eugenol, respectively, with the latter being the smallest size obtained in this set of experiments (Figure 6D, F). However, while fiber size remained small, the bead defects were numerous. The fiber size distribution ranged from 20 to 120 nm in both cases. Electrospinning of polymer solution with microemulsions composed of 7.5 wt % Surfynol 465 and 1.125 wt % eugenol resulted in fibers of 126 ( 34 nm with a relatively broad distribution of 60-240 nm (Figure 6C), whereas fibers with an average diameter of 82 ( 32 nm and a distribution of 10-190 nm were obtained from solutions containing 5 wt % Surfynol 465 and 0.75 wt % eugenol (Figure 6F). Micelles were not observed on the surfaces of nanofibers; however, fibers containing surfactant micelles or microemulsions had somewhat rugged surfaces. It is known from previous studies that nonionic surfactant micelles which may associate with a noncharged polymer (i.e., poly(vinyl alcohol)) form polymer-bound micellar structures of a much smaller size than those observed in polymer free micelles,23 which likely made it impossible to visualize them using this type of imaging technique.
Figure 7A, B shows transmission electron micrographs of pure PVA nanofibers, while Figure 7C-H shows nanofibers at varying magnifications obtained from electrospinning of PVAmicroemulsion solutions. Nanofibers of pure PVA have a smooth surface with no visible inclusions inside the fibers and are of regular shape (see also SEM images mentioned above). However, in nanofibers containing microemulsions, patches of microemulsion are visible which are homogeneously dispersed throughout the nanofibers. Furthermore, nanofiber surfaces have a rough and uneven appearance indicative of micelles located beneath the surface. For these images, the phenol ring of the essential oil component eugenol was stained with ruthenium tetroxide and microemulsions thus correspond to the black stained areas within fibers. Thus, addition of surfactant micelles and microemulsions may influence the morphology of the nanofibers in a variety of ways. First, surfactant monomers may adsorb to the surfaces of nanofibers, facilitating the fabrication of thinner fibers due to a reduction in surface tension. This may also lead to a decrease in the number of bead defects. Second, the dispersion of intact microemulsion droplets throughout the fiber may lead to increased surface roughness due to the presence of subcutaneous microemulsion droplets.
Eugenol Microemusions in Electrospun PVA Nanofibers
Langmuir, Vol. 25, No. 2, 2009 1161
Figure 7. Transmission electron micrographs of 7.5 wt % pure PVA nanofiber at a magnification of (A) 33 000× and (B) 20 000×. (C-H) Various magnifications of nanofibers electrospun from 7.5 wt % PVA solutions blended with 10 wt % Surfynol 465 and 1.5 wt % eugenol: (C) 66 000×, (D,E) 50 000×, (F) 33 000×, and (G,H) 20 000×.
Conclusions Functional nanofibers were produced by electrospinning antimicrobial microemulsion carrying poly(vinyl alcohol) blend solutions at varying loading ratios. While viscosity and conductivity both showed a slight increase upon addition of increasing amounts of eugenol or Surfynol 465, surface tension lowering effects of the phytophenol and surfactant were minor. However, solution properties were in the processing window to facilitate electrospinning. Fiber morphology and fiber size was affected by the composition of the microemulsion. For example, increasing amounts of eugenol led to an increase in fiber diameter and a decrease in the number and occurrence of bead defects, while fibers decreased in diameter at higher concentrations of Surfynol 465. Transmission electron microscopy images suggested the presence of micellar-dispersed eugenol in the fibers. In part two of this study, we will present results that illustrate the enhanced functionality of the fibers (i.e., release of the active compound eugenol from fibers and microbial
growth inhibition in the presence of fibers). Based on the results presented in this study, one could envision use of microemulsioncarrying nanofibers in a variety of applications. For example, such fibers could serve as the inner coating of an active packaging material. Fibers could also carry pharmaceutical compounds for use in tissue engineering. In general, the ability to incorporate lipophilic compounds in a solid system that provides mechanical strength may lead to unique applications that cannot possibly be designed by inclusion in liquid systems such as emulsions. Acknowledgment. This work was supported by the Environmental Protection Agency Star Grant Program (Grant No. GR832372) and the Massachusetts Experiment Station supported by the Cooperative State Research, Extension, Education Service, United States Department of Agriculture, Massachusetts Agricultural Experiment Station (Projects No. 831 and 911). LA803058C
