Raman microscopic imaging of electrospun fibers made from a polycaprolactone and polyethylene oxide blend

Vibrational Spectroscopy 92 (2017) 27–34

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Vibrational Spectroscopy journal homepage: www.elsevier.com/locate/vibspec

Raman microscopic imaging of electrospun fibers made from a polycaprolactone and polyethylene oxide blend Geoffrey P.S. Smitha , Andrew W. McLaughlinb , Andrew N. Clarksonc , Keith C. Gordona,* , Greg F. Walkerb a b c

Dodd-Walls Centre for Photonic and Quantum Technologies, Department of Chemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand School of Pharmacy, University of Otago, PO Box 56, Dunedin 9054, New Zealand Department of Anatomy, University of Otago, PO Box 56, Dunedin 9054, New Zealand

A R T I C L E I N F O

Article history: Received 7 September 2016 Received in revised form 26 April 2017 Accepted 9 May 2017 Available online 15 May 2017 Keywords: Raman imaging Scanning electron microscopy Electrospun nanofibers Polycaprolactone Polyethylene oxide

A B S T R A C T

Raman spectroscopy is a useful technique for providing compositional information about samples. By combining this technique with microscopy, Raman data can be collected from domain sizes smaller than one micron in diameter. This research aims to utilize this technique to examine the submicron component distribution in electrospun nanofibers. Nanofibers containing a 50:50 blend of polyethylene oxide (PEO) and polycaprolactone (PCL) were analyzed using Raman microscopy and scanning electron microscopy (SEM). This was performed before and after the samples were incubated in Milli-Q water to observe the effects of PEO dissolution on the nanofiber structure. Raman results indicate that both polymers are distributed evenly throughout individual nanofibers, but that some nanofibers may contain more of one component than the other, and the technique provided direct evidence for PEO dissolution after submersion in water. The dissolution of PEO observed by Raman microscopy correlated with results of mass loss analysis. SEM results also provided evidence for component dissolution and suggest that electrospinning may result in the formation nanofibers made up of PEO and PCL nanofibrils. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Nanofibers have several characteristics which make them useful in a variety of applications. These characteristics include a large surface area to volume ratio, they can be used as porous materials with variable pore shapes and sizes, and they can be used to create strong, rigid structures [1]. Electrospinning is a technique whereby nanofiber mats can be made relatively simply by passing a polymer solution through a thin capillary tube, applying an electric field, and collecting the resulting nanofibers on a charged collection plate [2]. In order to make the technique more useful on an industrial scale, this technique has been modified in various ways in the literature [3]. This has been accomplished through use of a larger number of capillary tubes [4], using electrostatic and rotational forces to generate nanofibers [5,6], or other methods which forgo the use of a capillary tube for generating the electrostatically charged jets of polymer solution [7–9]. Nanofibers are now being investigated for their uses in the pharmaceutical industry as it is possible to produce drug/polymer

* Corresponding author. E-mail address: [email protected] (K.C. Gordon). http://dx.doi.org/10.1016/j.vibspec.2017.05.002 0924-2031/© 2017 Elsevier B.V. All rights reserved.

and polymer/polymer nanofiber composites through the process of electrospinning [5,10–17]. There are many potential benefits to using such composites for medical treatments, especially for use in drug delivery [13]. These benefits include: lack of potentially harmful solvent; improved dissolution rate due to increased surface area; improved dissolution rate due to drug amorphization; improved drug solubility due to reduced particle size; improved stability of amorphous drug; and due to the improved stability of a supersaturated solution [13]. Many of these benefits are also useful when constructing nanofiber mats used for wound dressings or tissue scaffolds [18–20]. In such systems, the choice of which polymer or polymer blend can be very important. Polycaprolactone (PCL) is a synthetic polyester which has been found to have several medical applications due to the low cost of the material, its biocompatibility, its strength and due to its low degradation rate in the body [21–26]. However, this material has two disadvantages with respect to being used as a scaffold for guiding tissue regeneration. Firstly, the hydrophobicity of PCL hinders cell adhesion and proliferation; and secondly, PCL membranes take too long to be resorbed in vivo [21]. Polymer blends are created in order to mitigate these disadvantages, and create a material with moderated properties. In general, naturally

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occurring polymers tend to be better suited to incorporation into biological systems but lack mechanical strength and often degrade too quickly, whereas synthetic polymers often have the strength necessary, but degrade too slowly and are less compatible with biological systems [21,23]. In the literature this has been achieved using blends of PCL with gelatin [18], or chitosan [20,27]. Despite being a synthetic polymer, polyethylene oxides (PEOs) have also been blended with PCL as it has some properties which are similar to natural polymers, namely biocompatibility, hydrophilicity, and fast degradation in the body [28–32]. These properties have allowed these polymers to be used in relatively short-term drug delivery systems such as nanoparticles, nanofibers, hydrogels, and liposomes [29,30,33]. Raman spectroscopy has been extensively used in the analysis of electrospun fibers. Indeed Stephens et al. [34] showed that it is possible to characterize spun fibers virtually in real time as a way of providing the bulk (not single fibers) properties. Since then Raman spectroscopy studies of electrospun fibers may be categorized as follows: (i) Studies which examine bulk molecular properties of fibers, such as polymer conformation. In a study on electrospun silk nanofibers it has been shown that the index of crystallinity for the fibers may be determined using the band intensity ratio amide I/ amide III [35]. It may also be used to indicate the presence of two materials in a fiber mix and the removal of one of these via processes such as heating. Wang et al. [36] have shown that graphene oxide/polyvinyl alcohol (PVA) nanofibers show Raman signatures from each component with the PVA signal disappearing when the material is heated above 150
C. In combination with SEM and other results they conclude that the graphene oxide is a nanofiller within the PVA matrix; (ii) Studies in which time dependent changes of the bulk molecular properties are observed. In a study of silk fibroins (some with acrylate grating) and their interaction with aqueous methanol, Raman spectroscopy showed that the b-sheet conformation of the silk was altered by grafting of the 2-hydroxyethyl methacrylate (HEMA) and 4-hydroxybutyl acrylate (HBA) [37]. Treatment with methanolic solution did increase crystallization to the b-sheet conformation as shown by the changes in the bandwidth of the amide I and band ratio changes  this recrystallization was inhibited by grafting. The dynamic behavior of isotactic poly(1butene) electrospun membranes has been studied in which Raman spectroscopy was used to show that initially spun fibers were form I and II polymorphs but with aging a conversion to the thermodynamically stable form I occurred [38]; (iii) Studies in which mapping is used to determine compositional makeup. Nanofibers made from hyaluronic acid (HA) and either PCL or PEO have been investigated using Raman spectroscopic mapping [39]. Single value decomposition (SVD) was used to give an image of composition. By calculating the SVD coefficient for each spectrum and plotting it against the “spectrum number”, the relative concentration of drug was able to be compared for each mapped point. This analytical method showed that the nanofibers made from PEO and hyaluronic acid were homogeneous with regards to their component distribution, but nanofibers made from PCL and hyaluronic acid were not. This was attributed to PEO and hyaluronic acid having high water solubilities, but PCL having relatively low water solubility [39]. Therefore it is evident that a homogeneous distribution of components within electrospun nanofibers cannot be assumed. Nagy et al. [13] used Raman microscopy to image a nanofiber matrix of spironolactone and Soluplus1 in a 1200  1200 mm area using a 10  objective [13]. Results showed that spironolactone distribution could be imaged effectively and that electrospinning appears to be the more effective technique for attaining homogeneous drug/polymer mixtures than extrusion or physical mixing

processes. Sóti et al. [11] performed similar comparative experiments using Raman microscopy to image caffeine distribution on spray dried and electrospun nanofiber drug delivery systems. The domains imaged in these experiments were approximately 50  50 mm and contained 41  41 spectra, resulting in a step size of approximately 1 mm. The results of this study indicated that electrospinning provided better drug homogeneity than spray drying. Raman imaging may also be used to visualize the efficiacy of interaction between a polymer (polycaprolactone) and molecule (cyclodextrin or amine functionalized cyclodextrin). The analysis of the Raman maps with hierarchical cluster analysis suggests that amine functionalized cyclodextrin coats the fiber more effectively than the non-functionalised; One study has combined mapping with a chemical change [40]. In a study of molecularly imprinted polymer microspheres within a polymer membrane, Raman microscopy (with lateral resolution of 270 nm) was used to distinguish between the microspheres (made up of methacrylic acid and divinylbenzene) within a polyacrylonitrile nanofiber matrix and the same sample loaded with a target drug (–)-cinchonidine [40]. Nanofibers may be characterized using scanning electron microscopy (SEM) [14–16,27,41–44]. SEM provides excellent spatial resolution and is therefore informative with respect to the fiber structure and morphology. High spatial resolution Raman microscopy could potentially be an effective complementary technique for imaging the distribution of components within a nanofiber as the technique is non-destructive and has the ability to provide detailed structural and compositional information. Because Raman spectroscopy is an optical technique, a spatial resolution of 300 nm may be achieved but higher spatial resolutions would require the use of other spectroscopic techniques such as tip enhanced Raman spectroscopy (TERS) [45]. This research aimed to determine whether Raman microscopy could provide structural and compositional information about electrospun nanofibers on smaller length scales between 300 and 400 nm and the changes in these fibres with process effects, such as dissolution. While it may not be possible to resolve exact component distributions in smaller nanofibers, the signals from smaller nanofibers will still be detectable, and will therefore still be useful for providing information on the general fiber contents. Fibers in this investigation were made from synthetic polymers, PCL and PEO, and were analyzed before and after dissolution in water. This system was studied because the dissolution properties of PEO are well known for the nanofibers [46–49]. Following this analysis, scanning electron microscopy was performed on the same samples to provide the higher resolution morphological information. A further goal of this study was to determine whether any structural information can be gathered about individual nanofibers. Ultimately, this high resolution data may prove useful for understanding the mechanisms through which electrospun nanofibers made from polymer blends function in drug delivery and tissue scaffolds. 2. Experimental 2.1. Preparation of electrospinning solutions PCL (MW, 70–90 kDa), PEO (200 kDa) and formic acid were obtained from Sigma Aldrich Co. (St. Louis, Mo, USA). Glacial acetic acid was purchased from Merck (Darmstadt, Germany). Acetic acid and formic acid (AA/FA) was the solvent system used for all electrospun solutions at a ratio of 90:10 v/v. PCL was prepared by dissolving 0.2 g in 2 mL of AA/FA to give a 10% (w/v) solution. PEO was prepared by dissolving 0.2 g in 1.33 mL of milli-Q water to give a 15% (w/v) solution. PCL/PEO solutions were prepared by dissolving 0.1 g of PCL and 0.1 g PEO in 1 mL of AA/FA to give a

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20% (w/v). The polymer solution was placed on a magnetic stirrer to dissolve overnight. 2.2. Electrospinning Polymer solutions were electrospun in a fume hood at ambient temperature. A syringe with a blunted 20 gauge needle was connected to a Fusion 200 syringe pump (Chemyx Inc. Stafford, TX, USA), which delivered the solution at a flow rate of 0.3 mL/h. A voltage range from 18 to 20 kV was applied to the needle using a Bertan HVPS Series 230 power supply (Spellmann, Hauppauge, NY, USA). Fibers were collected on a mandrel rotating at 6 rpm, the distance between the needle and rotating mandrel was 15 cm. Fiber mats were typically 100 mm thick. 2.3. Mass loss A hole-punch was used to create 14 mm diameter nanofiber discs of pure PEO, pure PCL, and a PEO/PCL mixture. Each dry disc was weighed and then placed in a glass vial containing 2 mL of Milli-Q water and placed in an incubator at 37
C without stirring. At time points 1, 3, 5 and 7 days (n = 3) the disc was removed and dried in a vacuum oven at 25
C for at least 24 h. The dried disc was reweighed and the mass loss percentage was calculated by the following formula: Mt/Mi  100%. Where Mi is the initial mass of the dry disc and Mt is the mass of the dry disc after incubation in Milli-Q water. 2.4. Raman microscopy A WITec confocal Raman microscope was used for the high resolution imaging of nanofiber samples. The instrument was an Alpha 300RA+ (WITec, Ulm, Germany). A 100  objective with a numerical aperture of 0.9 was used to view the sample. Raman scattering was generated using a 532 nm laser wavelength with a power of 50.6 mW. The combination of the 100  objective and 532 nm laser wavelength produced a theoretical laser spot size of 0.366 mm; however, due to a variety of factors including sample inhomogeneity, reflection, and refraction, it is more logical to assume that the practical spot size is actually between 0.4-0.7 mm. Two different sizes of Raman maps were produced for each sample: 10  10 mm, and 50  50 mm. These maps contained 28  28, and 125  125 Raman spectra, respectively. At least three maps were collected for each of the different map sizes, and this was done for each of the samples analyzed. The integration time used to obtain Raman spectra varied between 0.1–0.5 s. The collected Raman spectra were not processed in any way before images were produced. The presence of each component was monitored by calculating the Raman band integrals of characteristic bands for each component. The larger the band integral, the higher the relative concentration of the component in that region. Circular nanofiber mats were presented to the microscope on glass slides without the use of a coverslip.

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3. Results and discussion Images of the nanofibers were obtained using optical microscopy, Raman microscopy, and scanning electron microscopy (Fig. 1). These techniques were applied to electrospun nanofibers made from pure PEO, pure PCL, and a 50:50 blend of PEO with PCL. Only images obtained from the blend will be presented here; however, Raman data were collected from the pure nanofibers and used as a reference to discern the distribution of components within the PEO/PCL blend nanofibers. Reference Raman spectra of the pure components are presented in Fig. 2. Histograms of typical nanofiber diameters are provided in the Supplementary material. 3.1. Single polymer nanofibers Reference Raman spectra obtained from PCL nanofibers contained several distinct bands (Fig. 2). The band at 913 cm1 can be attributed to CCOO stretching, the bands in the region from 1030 to 1110 cm1 are due to skeletal stretching, the bands around 1304 cm1 can be attributed to CH2 wagging, bands around 1443 cm1 are caused by CH2 scissoring, and the band at 1726 cm1 is caused by C¼O stretching [39,50]. Reference Raman spectra from PEO also contain several distinct Raman bands (Fig. 2). The low frequency Raman band at 278 cm1 is indicative of disordered longitudinal acoustic modes (D-LAM) which indicates that the PEO is amorphous within the nanofibers [51,52]. The bands at 366, 538, and 582 cm1 can be attributed to C O C bending, the 846 cm1 band is likely due to CH2 wagging, the 1064 cm1 band is likely due to C O C stretching, the 1142 cm1 band is attributable to CC COC stretching, bands at 1234 and 1280 cm1 can be attributed to C O C antisymmetric

2.5. Field emission scanning electron microscopy The morphology of nanofiber scaffolds was characterized by field emission scanning electron microscopy (FESEM). Samples were mounted on 10 mm aluminium stubs using double sided carbon tape. Samples were coated with 10 nm carbon in an Emitech K575X Peltier-cooled high resolution sputter coater with carbon coater attachment (EM Technologies Ltd, Kent, England). Samples were viewed in a JEOL JSM-6700F field emission scanning electron microscope (JEOL Ltd, Tokyo, Japan) at an accelerating voltage of 5 kV, using the SEI (secondary electron imaging) detector, at a working distance of 6 mm.

Fig. 1. Comparison of images of PEO/PCL blend nanofibers obtained using simple optical microscopy (top left), Raman microscopy (top right) and scanning electron microscopy (bottom).

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Fig. 2. Averaged Reference Raman spectra collected from PCL, PEO, and PEO/PCL nanofibers (left column). Raman images collected from mixed PEO/PCL nanofibers are presented in the column on the right. A Raman image mapping the distribution of PCL using the 1726 cm1 band integral has been presented in red (top image, right column); a Raman image mapping the distribution of PEO using the 366 cm1 band integral is presented in blue (middle image, right column); and a combined Raman image of both PEO and PCL distributions is also presented where purple regions indicate component overlap (bottom image, right column). Raman images contain 28  28 Raman spectra in a 10  10 mm area, using an integration time of 0.5 s (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

stretching, and the band at 1481 cm1 can be attributed to CH2 scissoring [39,50]. 3.2. PEO/PCL mixed nanofibers Electrospinning the 50:50 PEO/PCL mixture produced nanofibers which typically ranged in size from approximately 50– 1000 nm in diameter (Fig. 1). However, analysis of SEM images revealed that the average size of PEO/PCL blend nanofibers were between 200 and 300 nm in size. While SEM is able to easily resolve fibers with diameters less than 200 nm, the fibers which are more readily visible using optical microscopy are typically more than 300 nm in diameter. This indicates that the nanofibers visible using simple optical microscopy and Raman microscopy are typically larger than the average nanofiber. Therefore it is important to note that this study assumes that the composition of the larger nanofibers is similar to that of the smaller nanofibers.

Raman images obtained from different sized areas were collected from nanofiber samples. The Raman spectra collected to create the 10  10 mm image were found to be consistent throughout the nanofibers with no noticeable domains which contained pure PCL or pure PEO. Raman spectra were found to all contain features which were attributable to the presence of both components. An averaged spectrum of PEO/PCL nanofibers is presented in Fig. 2, which shows the presence of PEO features between 200 and 600 cm1 and PCL features at 1443 and 1726 cm1, as well as other bands indicative of each of these components. This suggests that: (1) chemical interactions between components are not likely to be occurring as the PEO/PCL blend Raman spectrum resembles a spectrum which would be produced through combining pure PCL and pure PEO spectra  with exception to the disappearance of the PEO band at 582 cm1, the reason for which is not known to the author at this time; (2) the two components within these nanofibers are highly mixed and

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that the individual nanofibers are homogeneous at this scale; and (3) that the domain sizes of PEO and PCL are smaller than the laser spot size. In order to determine whether all nanofibers within the sample are homogeneous, Raman images were obtained over a larger area, but small enough that individual fibers were still distinguishable. The larger images were taken from an area of 50  50 mm (presented in Fig. 3). Results from these images indicate that the majority of nanofibers consist of a mixture of PEO and PCL. However, some regions of the nanofibers appear to consist of primarily one component. The most prominent example is the large blue region of a nanofiber in the PEO/PCL combined image in Fig. 3 which indicates that this domain consists primarily of PEO. The combined image also contains some red regions which indicate that the area is primarily made up of PCL. Therefore, the apparent presence of single component domains reinforces the notion that mixing the initial PEO/PCL solution effectively is crucial to the formation of homogeneous component distribution in nanofiber mats. Raman microscopy has effectively demonstrated its ability to image these nanofibers and its ability to show whether individual nanofibers contain the desired mixture of components. While these images do not provide a value for the PEO and/or PCL content within the individual nanofibers, it may be possible to calculate relative component concentrations in future experiments using partial least squares analysis. 3.3. PEO dissolution Mass loss experiments involved electrospinning pure PCL (the control) and mixed PEO/PCL nanofiber samples, measuring their initial dry mass (Mi), placing the samples in Milli-Q water, and measuring their dry mass (Mt) periodically thereafter (Fig. 4). The purpose of this experiment was to observe how quickly these nanofibers lost mass over the seven day period and what proportion of the nanofibers remained undissolved during this period. Results indicated that pure PCL nanofibers lost very little mass while incubated in water over the seven day period, losing less than 2% of its mass over this timescale. The relatively small loss of mass of the pure PCL nanofibers was expected as the polymer is known to be relatively insoluble both in water and in the body [21– 26]. By comparison, the mass loss of the mixed PEO/PCL nanofibers was considerably larger. These samples lost approximately half of their mass over the seven day period. The majority of the nanofiber mass was lost by then end of the first day (46.7%) and only a further 2.3% lost over the following six days. Considering that PCL loses minimal mass when incubated in water, these results indicate that

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the vast majority of the lost mass can be attributed to the dissolution of PEO. Furthermore, considering the fact that almost half of the mass of the PEO/PCL nanofibers was lost indicates that almost all of the PEO present is likely to have dissolved. This suggests that there are minimal areas within the nanofiber where the solvent does not have access to PEO. This is because a scenario where a large number of PEO domains were encapsulated by PCL would result in reduced losses of nanofiber mass, as the PEO would not be accessible to the solvent. Therefore, the process of electrospinning appears to distribute the two components evenly and readily available to solvents at the surface of the fiber. To further investigate whether the majority of PEO has been removed from these nanofibers, Raman microscopic imaging was performed on the samples (Fig. 4). These images also indicate that PEO has dissoluted out of the mixed PEO/PCL nanofibers. The Raman image created using the PCL band integral at 1726 cm1 shows distinctive nanofiber structures in the area. However, the integral of the PEO band at 366 cm1 now produces an image with no discernible uniform distribution of the PEO polymer as the 366 cm1 band is no longer present. Therefore, the absence of this band suggests that there is very little PEO remaining within the fibers. This is also supported by taking the average Raman spectrum of the fibers in this area (Fig. 5), which shows that the average Raman spectrum taken from PEO/PCL blend nanofibers now resembles a spectrum of pure PCL. Overall, assessment of the mass loss and Raman imaging data indicates that the majority of PEO is no longer within the nanofibers. Examining the PEO/PCL blend nanofibers before and after the ‘mass-loss’ experiment using SEM showed that significant changes in the appearance of the fibers had occurred after only one day incubated in Milli-Q water (Fig. 6). Fibers appeared to have consistently smooth surfaces before submersion; however, afterwards they appeared to have small cavities and striations along the nanofiber. This supports the results of the mass loss and Raman imaging experiments, as it suggests that one of the components has dissoluted out of the nanofiber structure. Furthermore, the authors speculate that the appearance of striations along the length of the nanofibers suggests that the components are possibly present in the nanofiber as intertwined nanofibril-like structures. However, the formation of nanofibril structures does not appear to be uniform, as the striations (or grooves) seem to follow irregular paths along the nanofiber surface and appear to have formed at irregular intervals. The presence of intertwined nanofibrils would also explain the fast and near complete dissolution of PEO. Arguably, this fast and effective dissolution of PEO could have occurred if PCL nanofibers had been coated in PEO. However, the

Fig. 3. Three Raman images collected from the PEO/PCL mixed nanofiber sample. This figure includes a Raman image of PCL distribution created using the 1726 cm1 band integral (left); a Raman image of PEO distribution created using the 366 cm1 band integral (middle); and a combined Raman image of PCL and PEO distributions (right). These images contain 125  125 Raman spectra in a 50  50 mm area, using an integration time of 0.1 s.

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Fig. 4. Plot of the average percentage of remaining mass of the nanofiber samples versus the amount of time the sample spent submerged in Milli-Q water (left). At day zero, each sample had not been exposed to water and therefore maintains 100% of its original mass. Raman images on the right were collected from the PEO/PCL blend at day 1 (after the sample was incubated for one day). The red image (top) maps the distribution of PCL and the blue image (bottom) attempts to map the distribution of PEO. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Averaged Raman spectrum collected from the PEO/PCL blend nanofibers before and after the sample had been incubated in Milli-Q water for one day.

electrospinning equipment was not set up for this purpose (this would require the use of a hollow tip injector), and if the nanofibers had lost an outer coating of PEO, the observed diameter of nanofibers would have decreased after PEO dissolved. This was not observed, so it is not likely that the nanofibers consisted of a PCL nanofiber core with a PEO coating. Furthermore, nanofibrils may also explain the much more ‘stringy’ appearance of the submerged sample as it is possible that the PCL fibrils began to unwind and become exposed when PEO dissolved. While the structure of these nanofibers is purely theoretical, future experiments should explore this possibility further.

4. Conclusions These investigations showed that Raman microscopy can provide complementary compositional information about nanofibers when utilized with scanning electron microscopy. Despite the assumption that composition of larger nanofibers is similar to that of smaller nanofibers, this assumption could potentially be tested in future work using TERS techniques. Raman microscopy was effectively able to map the distribution of PEO and PCL in nanofibers made from a PEO/PCL blend. Both PEO and PCL appeared to be homogeneously distributed within individual

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Fig. 6. SEM images of PEO/PCL blend nanofibers before (left) and after (right) incubation in Milli-Q water for one day.

nanofibers with very few notable regions exclusively one component or the other. This indicated that the domain sizes were smaller than the 0.366 mm spot size used. However, larger scale analysis of nanofibers indicated that not all nanofibers contained even mixtures of the two components. Use of Raman microscopy and SEM showed that incubating samples in water resulted in rapid dissolution of almost half the nanofiber mass in less than a day. Raman microscopy was used to conclude that PEO was dissoluting out of the nanofiber system, and SEM showed that the components are potentially present within the nanofibers as intertwined nanofibrils. To the authors’ knowledge, this is the first time the dissolution behavior of PEO/PCL blend nanofibers has been observed on such a small scale using Raman microscopy and results encourage future investigations into the potential presence of nanofibril structures.

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