Production of essential oil-based composite nanofibers by emulsion electrospinning

Öz This study aimed to produce polyvinylpyrrolidone (PVP)/gelatin (GEL)/lavender essential oil (LEO)-based nanofibers by means of oilin-water emulsion electrospinning. Firstly, the polymer solution properties were measured, and then optimization of nanofiber production and characterization of the nanofibrous web were carried out. As gelatin was added to the PVP solution, viscosity was found to increase while surface tension and conductivity decreased. PVP/GEL (50/50) was determined to be the optimum sample in terms of nanoweb quality, fiber diameter, diameter uniformity, and gelatin content. Nanofiber production proceeded with PVP/GEL (50/50) and various concentrations of LEO. FT-IR results confirmed that LEO, PVP, and gelatin were incorporated in the chemical structure of the nanofibers. Generally, ultra-fine and uniform nanofibers were obtained, except when using pure PVP or PVP/GEL (50/50) including 8 wt % LEO. The finest fibers were obtained from PVP/GEL (100/0) (183 nm), and the most uniform fibers were obtained from PVP/GEL (50/50) (fiber diameter uniformity coefficient of 1.04). All nanofiber samples displayed unimodal distribution curves of histograms. While the addition of gelatin affected solution properties and average fiber diameter, the addition of LEO did not affect fiber properties. Bu çalışmada, su içinde yağ emülsiyon elektro lif çekimi ile polivinilpirolidon (PVP)/jelatin (GEL)/lavanta uçucu yağ (LEO) esaslı nanolif üretilmesi amaçlanmıştır. Öncelikle; polimer çözelti özelliklerinin ölçümü ve daha sonra nanolif üretim optimizasyonu ve nanolifli ağ yapının karakterizasyonu gerçekleştirilmiştir. PVP çözeltisine jelatin ilavesi ile viskozite artar iken, iletkenlik ve yüzey gerilimi azalmaktadır. Nano ağ kalitesi, lif çapı, çap üniformitesi ve jelatin içeriği bakımından PVP/GEL (50/50) optimum numune olarak belirlenmiştir. Çeşitli konsantrasyonlarda LEO içeren PVP/GEL (50/50)’den nanolif üretimine devam edilmiştir. FT-IR sonuçları, nanoliflerin kimyasal yapısında LEO, PVP ve jelatin varlığını doğrulamıştır. Saf PVP ve % 8 LEO içeren PVP/GEL (50/50) hariç, genellikle oldukça ince ve üniform nanolifler elde edilmiştir. En ince lifler PVP/GEL (100/0) (183 nm) çözeltisinden ve en üniform lifler PVP/GEL (50/50) (1.04 lif çapı üniformite katsayısı) çözeltisinden elde edilmiştir. Tüm nanolif numunelerinin histogramında tek tepeli dağılım eğrileri elde edilmiştir. Jelatin ilavesi, çözelti özelliklerini ve ortalama lif çapını istatistiksel olarak etkilemiştir fakat LEO ilavesi lif özelliklerini etkilememiştir.


Introduction
Emulsion electrospinning is a new, green approach for the production of nanofibers. It enables the generation of nanofiber from immiscible liquids such as essential oils and hydrophobic drugs and proteins; indeed, it is the most suitable method for combining nanofibers with essential oils [1][2][3][4]. Normally, it is very difficult but not impossible to produce nanofibers from an aqueous polymer solution and an essential oil. Emulsion electrospinning enables the preparation of stable and homogenous emulsion solutions, making it far easier to produce nanofibers. Another advantage of this method is that it does not require extra apparatus over the conventional electrospinning system. Furthermore, emulsion electrospinning represents a big step towards green electrospinning. The main aim of green electrospinning is the use of green chemicals, which are very important in terms of environmental impact and end-product properties [5][6]. In recent years, nanofibers have become very attractive for medical and cosmetic applications, for which a green electrospinning approach has vital importance. For these reasons, this study explored the production of nanofibrous composite material including PVP/gelatin/lavender essential oil (LEO) by oil-in-water emulsion electrospinning.
Essential oils are odorant oils that can be produced from different parts of medicinal and aromatic plants [30][31][32]. The essential oil from lavender (Lavandula hybrida L.), which was used as an additive in this study, has antiseptic, antibacterial, sedative, tranquillizer, antioxidant, and relaxing properties; and therefore can be used in the application areas of perfumery, pharmacology, medicine, and especially aromatherapy [33][34][35]. Also, LEO is a commercial product of Isparta province, Turkey, and it is thought that with this study, this commercial product can enter into new application areas such as nanoscale cosmetics and biomedical materials.
Limited studies exist concerning nanofibers produced with essential oils or the major constituents of essential oils. [36] Kayaci et al. (2013) investigated the thermal stability and release profile of eugenol in polyvinyl alcohol (PVA) nanofibers containing eugenol (EG)/cyclodextrin (CD) inclusion complexes. Three type of cyclodextrin (α-CD, β-CD, and γ-CD) were used in the nanofiber structures. They found that the PVA/EG/γ-CD inclusion complex demonstrated higher thermal stability and slower release of eugenol, and suggested that this nanofibrous surface can be used in the food industry to leverage properties of eugenol such as its antibacterial, antifungal, and antioxidant efficacies. investigated the production of nanofibers from polylactic acid (PLA) and candeia (Eremanthus erythropappus), and investigated the fiber morphology and structure. They determined that the nanofibers had homogeneous structures incorporating the candeia essential oil, and that increased proportions of candeia essential oil increased the nanofiber diameter and decreased the glass transition and melting temperatures. However, it could not be found any literature on emulsion electrospun nanofibers incorporating LEO. To explore the properties and potential of such nanofibers, PVP and gelatin polymers and the additive LEO were chosen as raw materials for this study of a nanofibrous composite material that might be useful for cosmetic and medical applications.

Materials
PVP (mw 360.000 g/mol) and gelatin (type A) were used as polymers, a surfactant (PEG-40 hydrogenated castor oil) was used as an emulsifier and lavender essential oil (Lavandula hybrida L.) was used as an additive for the produced nanofibrous composite material. Green solvents were selected from solvent selection guides [39], and consisted of distilled water (DW) and acetic acid (AA). PVP, gelatin, and acetic acid were purchased from Sigma-Aldrich; the surfactant was supplied by Ersa Chemistry, İzmir, Turkey; and the lavender essential oil was acquired from Botalife, Isparta, Turkey. All chemicals were analytical grade and used without further purification. Polymer solution optimization consisted of two stages. In the first stage, solutions were prepared with different proportions of PVP (12 wt %) in distilled water and gelatin (6 wt %) in acetic acid (Table 1).  (Table 2). Also, the surfactant concentration was set at 3 wt %, as determined from our preliminary studies. All polymer solutions were prepared under the same conditions such as; stirring time, stirring rate and temperature (Figure 1).

Method
Solution properties such as; conductivity, viscosity (under a shear rate of 5 s -1 ) and surface tension (by the Wilhelmy plate method) were determined. Next, nanofiber production was carried out with the electrospinning method. Optimum process parameters are given in Table 3, and all nanofibers were produced for the same duration (30 minutes). Figure 2 shows a representation of the emulsion electrospinning method used in this study.
(a) (b)  (1) and (2) given below [40]. Lastly, nanofiber diameter histograms were overlaid with a normal distribution curve, and nanofiber diameters were analysed by one-way ANOVA with statistical significance set at p<0.05. Figure 3 shows the obtained conductivity, surface tension, and viscosity values for PVP/GEL solutions. As illustrated in Figure  3( Figure 3(b) shows that surface tension values increase with less gelatin content. Also, it is possible to say that there is a strong relationship between spinnability, fiber morphology, and surface tension. During the experiment, spinnability was observed to decrease from sample PVP25 to sample PVP100. Therefore, gelatin addition improves spinnability excitingly. The conductivity, surface tension, and viscosity of PVP/GEL (50/50) (sample PVP50) solutions also including LEO are given in Figure 4. As is clearly seen in Figure 4(a), conductivity decreased with increasing LEO concentration. This result is expected because the conductivity value of pure LEO is 0.13 µS/cm, which is quite low. In addition, increasing LEO concentration caused viscosity to increase and surface tension to decrease.

Fiber Morphology
SEM images and fiber diameter histograms of various mixtures of PVP/GEL nanofibers are given in Figure 5. Mostly, the nanofibers produced were quite fine and had a homogeneous distribution; the exception is sample PVP100 (PVP/DW), which had some beads in its fiber structure. In addition, it was not possible to spin nanofibers from sample PVP0, because of solution gelation. All samples displayed unimodal histogram curves (PVP25, PVP50, PVP75 and PVP100).  From analyses of fiber diameter and diameter uniformity coefficient (Figure 6), it was determined that gelatin addition decreases average fiber fineness. This finding is compatible with the viscosity results (Figure 3(a)) and existing literature. It is possible to say that the addition of gelatin had a statistically significant effect on the average fiber diameter of PVP nanofibers. The finest fibers (183 nm) were obtained from sample PVP100, (PVP/DW) but it also had the worst fiber morphology, with beads observed. The most uniform nanofibers (at FDUC = 1.04) were obtained from sample PVP50 (PVP/GEL 50/50). Sample PVP50 was chosen as the most suitable sample for the second stage of the study in terms of average fiber diameter, fiber diameter uniformity coefficient, fiber morphology, and quantity of gelatin content. The results showed that LEO concentration did not significantly affect nanofiber diameter and FDUC (Figure 8). Some beads were present in sample PVP50-L8, and it is thought that morphological deformation starts at this concentration; therefore, it is possible to say that 8 wt % LEO is not suitable for use with PVP/GEL (50/50). Generally, quite fine and uniform nanofibers were produced in the second stage of this study. The finest fibers (202.57 nm) were obtained from sample PVP50-L2, and the most uniform fibers (FDUC = 1.04) were obtained from sample PVP50-L6. All samples again displayed unimodal histogram curves ( Figure  7).
During the experimental studies and analyses, it became apparent that solution conductivity has an important effect on spinnability and the fiber morphology. A graph of the relationship between solution conductivity and average fiber diameter for PVP/GEL solutions is given in Figure 9. In short, there is an inverse correlation between solution conductivity and average fiber diameter. Therefore, finer nanofibers, which are important for nanoweb quality, can be produced with high-conductivity polymer solutions. These results are compatible with reports in the literature [43][44].
In the second stage of this study, neither solution conductivity nor average fiber diameter were changed significantly by the addition of LEO ( Figure 10). Figure 10. Relationship between solution conductivity and average fiber diameter for PVP/GEL/LEO solutions It is also possible to say there is a direct relationship between solution conductivity and FDUC ( Figure 11); that is, as the gelatin content decreases, conductivity increases and fiber diameter uniformity decreases, which results in greater fineness of fibers. As was mentioned in relation to Figure 9, average fiber diameter decreases with increasing solution conductivity, and this result is expected from literature [43][44]. Figure 11. Relationship between solution conductivity and FDUC for PVP/GEL solutions Analysis showed that FDUC is affected slightly by LEO addition, and that solution conductivity decreases with increasing LEO concentration ( Figure 12).  Table 4.

Conclusions
Firstly, this study produced composite nanofibers using various mixture ratios of PVP/GEL polymers (0/100, 25/75, 50/50, 75/25 and 100/0). After the determination of the optimum mixture, PVP/GEL (50/50), the addition of various concentrations of LEO was applied (0, 2, 4, 6, and 8 wt %). The results demonstrated that gelatin concentration has important effects on the solution conductivity, surface tension, viscosity, and fiber morphology. It was determined that as the gelatin concentration increases, solution viscosity also increases while conductivity and surface tension decrease. Another result of added gelatin was enhanced fiber morphology (without beads) and increased average fiber diameter. In contrast, LEO concentration did not have any significant effect on the average fiber diameter, but did impact solution properties (conductivity, surface tension, and viscosity). Solution viscosity increased with LEO concentration while conductivity decreased, but surface tension did not change. Generally, very fine and uniform nanofibers were produced from PVP/GEL/LEO solutions. Lastly, FT-IR results confirmed that LEO, PVP, and gelatin were present in the structures of all nanofibers samples. These aromatic composite materials have potential use in cosmetic and biomedical applications and the advantages of incorporating biocompatible polymers, green solvents, a non-toxic surfactant, and a natural additive.  Nanoparticles and Nanofibres Based on Tree Gums for