# Electrospinning of Essential Oils
## 3 Electrospun Fibres Containing EOs and Their Applications
### Clove Essential Oil  
Clove EO is extracted from the buds, leaves or stems of _Syzygium aromaticum_ (synonym: _Eugenia carophyllata_), a plant of the _Mytraceae_ family that is cultivated in Tanzania, Indonesia, Sri Lanka, and Madagascar [88; 89]. This essential oil and its main component, eugenol, are traditionally used as analgesics and antiseptics to treat dental caries and periodontal diseases, being active against cariogenic bacteria, such as _S. mutans_ and _S. sobrinus_, and periodontal pathogens, such as _P. gingivalis_ and _P. intermedia_[90; 91]. They detrimtally impact on the metabolic pathways of other strains of Gram-positive (such as _S. aureus_, _B. cereus_, _B. subtilis_) and Gram-negative bacteria (such as _E. coli_, _P. aeruginosa_) by disrupting cell membrane integrity, enhancing membrane wall permeability and therefore leading to bacteria death [92; 93].  
Clove EO has been encapsulated in electrospun fibres of PCL [53], gelatine [54], PCL/gelatine [55], polyacrylamideonitrile [56], alginate/PVA [41] and polyvinylpyrrolidone [57], and its efficacy has been investigated against _S. aureus_, _E. coli_, _B. subtilis_, _Klebsiella pneumoniae_, _Candidia tropicalis_, and _Candidia albicans_. PCL/gelatine fibres (with a 7:3 PCL:gelatine ratio) containing different concentrations of clove EO (1.5%, 3.0% and 6.0% v/v) have been produced for wound care applications [55]. The fibres were characterised by an average diameter of 250-300 nm, obtained by electrospinning at a voltage of 20 kV, flow rate of 0.6 mL/hour and working distance of 12 cm. The composite fibres inhibited the growth of _S. aureus_ after 6 h of in vitro antibacterial tests, with a reduction of 30-40% of cell viability. However, they were less effective after 24 h, when the bacteria viability returned to values higher than 90%. A similar trend was observed for _E. coli_ colonies exposed to electrospun mats containing the lowest concentration of EO: a decrease in cell viability at 6 h, followed by an increase at 24 h. Instead, fibres with 3% and 6% v/v of clove EO induced a gradual decrease of _E. coli_ viability, reaching values of 35-40% after 24 h of exposure. Cytotoxicity tests on human dermal fibroblasts demonstrated that PCL-gelatine fibres containing clove EO were biocompatible and did not induce changes in the morphology of the cells, after 48 h. In vitro wound healing (scratch) assays revealed that migration and proliferation of fibroblasts were inhibited in a dose-dependent way, when the cells were exposed to the composite fibres. After 24 h, wound closure rates of -80% and -65% were measured for cells exposed to PCL-gelatine fibres with the lowest and highest concentration of EO, respectively. Complete wound closure was instead observed for the control samples (24-well plate) at the same time point.  
Previous studies have reported on the dose-dependent cytotoxicity of clove EO [94; 95]. When endothelial cells and fibroblasts were incubated in growth media containing different concentrations of clove EO (from 0.002 to 0.250% v/v), it was observed that a concentration as low as 0.03% v/v reduced cell viability to levels lower than 20% after 1 h of exposure [94]. The toxicity was attributed to eugenol, which is expected to induce cell apoptosis (programmed cell death) [95]. It was also demonstrated that eugenol diluted in the cell culture media at concentrations lower than 600 uM caused strong genotoxic effects (DNA damages) on human fibroblasts after 24 h of exposure, because these cells lack metabolic enzymes involved in activation/detoxification processes. The encapsulation of clove EO in electrospun fibres can be a valid strategy to limit the harmful effects of eugenol on cells [96]. By controlling the diffusion rate of eugenol out from the polymeric fibres, it is possible to ensure that the dose released stays within safe limits.