The largest organ of the human, with about one-sixth of the total body weight, is skin. It involved in many functions such as sensation, thermoregulation, and immune functions . Due to the large surface of the skin, it is likely to be injured. Skin wounds are introducing as any interruption in the continuity of the skin. They are caused by many types of stress, including physical, chemical, and mechanical trauma, or triggered by a medical condition (diabetes). Nevertheless, all open skin wounds are colonized with bacteria; this can lead to an extension of the wound healing time [2, 3].
Human skin is covered with microflora; it inhibits the growth of pathogenic bacteria through bacterial interference, nutrient competition, as well as producing metabolites that inhibit pathogen growth [4, 5]. Besides, phagocytosis cells such as neutrophils and macrophages in chronic or open skin wounds have a crucial role in the control of bacterial infections [6, 7].
On the other hand, many opportunistic bacteria from gram-positive or -negative such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and others, can enter the wounds . It caused a complicated infection, especially in humans, with a weak immune system [9, 10]. The emergence of microbial resistance and high load of pathogens in the environment has led to many challenges in the treatment of skin wounds; unfortunately, sometimes treatment with systemic antibiotics are advised [11, 12].
Nanofibers (NF)s containing green antibacterial agents, such as plant-derived essential oils, are introduces as a new generation of dressings that able to cure bacterial infections and facilitate wound healing [13, 14]. For example, in two studies, polycaprolactone (PCL) and PCL-gelatin NFs containing Peppermint and clove essential oils were prepared, respectively; they can be reduced the growth of S. aureus, E. coli [15, 16]. PCL is a biodegradable and hydrophobic polymer from Ɛ-caprolactone, as well as has been approved by the FDA (Food and Drug Administration of the USA) . It has many usages in medical applications, e.g., drug delivery, surgical suture, and wound dressing .
Citrus sinensis belong to family Rutaceae . The essential oil of C. sinensis (CSEO) is a well‐established natural antimicrobial activity against different types of pathogens, such as bacteria, fungi, or even viruses [20-22]. Therefore, it has been used in food chemistry and pharmaceutics .
In this study, the ingredients of CSEO were identified using GC-MS analysis. Its nanogel was then developed to facilitate topical application. Then, electrospun NFs of PCL were prepared, as dressing. Besides, the nanogel was impregnated on PCLNFs, NGelNFs. Antibacterial activity of NGelNFs was investigated against four human pathogens using ATCC100 standard method.
MATERIALS AND METHODS
Zardband Pharmaceuticals Co (Iran) provided CSEO. Tweens (20, 80), NaOH, Mueller-hinton broth, and Mueller-hinton agar were bought from Merck Chemical Co. (Germany). PCL with a mean molecular weight of 80.000 was purchased from Sigma–Aldrich (USA). Hexafluoroisopropanol (HFIP) and Carbomer 940 were supplied by two Indian companies, SDFCL and Suvchem, respectively.
For identification components of CSEO, analysis of GC-MS was performed, as described in our previous study .
Investigation of antibacterial activity of CSEO
Antibacterial activity of CSEO was investigated against Klebsiella pneumonia (ATCC: 13883), Pseudomonas aeruginosa (ATCC: 27853), Escherichia coli (ATCC: 25922), and Staphylococcus aureus (ATCC: 25923) using 96-well plate broth microdilution . For this purpose, CSEO was dissolved in normal saline (35°C) for the preparation of serial dilution in a concentration range of 16000 – 62.5 µg.mL-1.
Briefly, the mentioned bacteria colonies were dissolved in Mueller-hinton broth; 1.5×108 CFU/mL with a turbidity of 0.5 McFarland. Eighty and twenty µL of the Mueller-hinton broth and the bacterial suspension was added to each well using an 8-channel pipette, respectively. After the addition 100 µL/well of the prepared serial dilutions, the concentration of CSEO finally fixed at 8000,4000, 2000, 1000, 500, 250, 125, 62.5, and 31.25 µg.mL-1, respectively. The treated plates were then incubated for 24 h at 37ºC.
Followed by, the absorption (A) of wells was read at 630 nm using a plate reader (Synergy HTX Multi-Mode Reader, USA). Growth (%) of bacteria at each concentration was determined by equation 1. Finally, inhibitory concentration 50% (IC50) CSEO against each of the bacteria strains with their lower and upper confidence limits were calculated by CalcuSyn free version (Biosoft Co. UK)
This test was performed in triplicates; in each replicate, control and blank groups were also considered. In the control groups, 80, 20, and 100 µL of the Mueller-hinton broth, bacteria suspension, and normal saline, respectively, were used. Blank wells were filled by the Mueller-hinton broth and normal saline (100: 100 µL).
Growth (%)= (1)
Preparation and characterization of nanoemulsion-based nanogel
Fixed amounts of CSEO (125 µL) and ethanol (20 µL) and different concentrations of tween 80, tween 20, and span 80 were stirred (single or mixed) to form a homogenous solution; 500 rpm, 10 min. Followed by, distilled water was added dropwise to the homogenous solution reach to 5000 µL. The prepared mixture was stirred for another time (2000 rpm, 30 min).
The particle size and particle size distribution (SPAN) of prepared samples were carried out using the DLS type apparatus (K-One Nano, Ltd, Korea). SPAN was calculated as . Where, D10, 50, and 90 are percentile of particles that have a diameter lower than these values. Optimum nanoemulsion with appropriate particle size (around 200 nm) and SPAN (< 1) was selected for the preparation of nanogel.
Nanogel dosage form
Nanogel was prepared by the addition of carbomer 940 (1.5% w/v) to the optimum nanoemulsion. First, carbomer was hydrated in a mild condition; 120 rpm, ambient temperature, overnight. After that, the pH was adjusted to 6-7 by the addition of NaOH 25% w/v for completing the gelling process.
The viscosity of the nanogel was investigated under atmospheric pressure at 25ºC, by a rheometer apparatus (Anton Paar, model MCR-302, Austria). Furthermore, a blank gel was also prepared using a similar process, and the same constituents in comparison to the nanogel; only no CSEO was used.
Preparation and characterization of electrospun PCLNFs
Electrospun NFs of PCL were prepared using a previously described method with slight modifications . By dissolving granules of PCL in HFIP at room temperature (overnight), the PCL solution (14% w/v) was prepared. The solution was loaded into a 10 mL syringe connected to a stainless steel blunt needle (gauge 22) in an electrospinning machine (Fnm. Co. Iran). The PCL solution was injected (0.5 mL/h) using a syringe pump. The distance of the needle and rotating collector (100 rpm) was fixed at 140 mm as well as a voltage of 15 kV was applied between them. For the facilitation of the separation of formed NFs, the surface of the collector was wrapped with aluminum foil.
The morphology, diameter, and distribution diameter of the NFs were investigated using SEM analysis (TESCAN Vega3, Czech Republic). Before the observations, the samples were cut in squares pieces (1 × 1cm) for coating with gold vapors (Quorum Technologies, Q150R- ES). The diameter and size distribution of NFs was determined by Digimizer analysis software, a free version (MedCalc Software Ltd, Belgium).
ATR-FTIR (Bruker Company, Model Tensor II) was applied to characterize functional groups of the NFs. Spectra were recorded in the range of 400–4000 cm−1.
Contact angle measurement
The wettability of the surface of the NFs was evaluated by the contact angle measurement machine (Sharif Solar, Tehran, Iran). A 7 µL of deionized water was dropped onto the surface of the NFs. After 5 s, a photograph was gotten for investigating the hydrophobic behavior of the NFs .
Impregnation of the nanogel on the surface of PCLNFs
As shown in Fig.1, circular pieces of PCLNFs with a diameter of 50 mm (50 ± 5 mg) were cut from the prepared mat of PCLNFs. Both sides of them were sterilized using a UV light for 20 min. After that, 3000 mg of nanogel of CSEO was impregnated on the surface of the pieces, named NGelNFs. Furthermore, by impregnating the blank gel on PCLNFs, another sample with the name of Gel(-oil)NFs was prepared.
Antibacterial effect of NGelNFs
The antibacterial action of NGelNFs was investigated by a standard test method for texture, AATCC100, with slight modification. The new colonies of each bacteria strains were added to Mueller Hinton broth to reached 2 × 105 CFU/mL turbidity. After that, NGelNFs were immerged in 6 cm plates containing 5 mL of each bacteria suspension and then incubated, 24 h at 37°C. The concentration of the CSEO at each plate eventually fixed at 15000 ppm.
After that, 10 μL of the supernatant of each plate was cultured on Mueller Hinton agar, separately and incubated for another 24 h at 37°C. Finally, the number of grown colonies on plates was counted using a colony counter. The growth reduction of each sample was calculated by equation 1. This test was performed in triplicate, in each replicates negative and control groups were considered. In negative groups, antibacterial activities of PCLNFs containing blank nanogel was examined (Gel(-oil)NFs). In the control group, no treatment was applied.
RESULTS AND DISCUSSIONS
Ingredients of CSEO
Thirty-two compounds were determined in CSEO using GC–MS analysis (see Table 1). Limonene (61.83%), trans-p-2,8-Menthadien-1-ol (4.95%), Limonene oxide, Trans- (2.29 %), Limonene oxide, Cis- (2.58 %), and trans-Carveol (2.90%) defined as five major ingredients.
Antibacterial activity of CSEO.
Observed IC50s with lower and upper confidence limits of CSEO against target bacteria strains are summarized in Table 2. The effectiveness (IC50) of CSEO against S. aureus significantly better than other (one-way ANOVA, p < 0.05).
Prepared nanoemulsion-based nanogel
The constituents of the prepared emulsions (26 samples) and their size analyses are listed in Table 3. For the formation of a nanoemulsion, the balance between the constituents, as well as the coordination between them, is necessary [25, 28]. For finding proper surfactants as well as the right amount, three types of typical surfactants in a single form or mixture were examined. Among the prepared samples, only No. 21 shows acceptable particle size and SPAN value; 125 ± 4 nm and 0.95 ± 0.01 (see Fig. 1A).
By the addition of carbomer 940 (1.5% w/v) to the chosen nanoemulsion (No. 21), nanogel was prepared. The effect of different shear rates on its viscosity is illustrated in Fig. 2B. The viscosity decreased by increasing of shear rate; this relation was fitted with Carreau–Yasuda model. This model is a well-known equation for non-newtonian fluids . Furthermore, a blank gel was also prepared with the same process and ingredients; only no CSEO was used.
Nanogels have 3d-dimensional structures like to the biomacromolecules; thus have a wide range of biomedical applications . Their polymeric network can be both hydrophilic and hydrophobic so they can carry all of the ionic, nonionic, and nonpolar nanoparticles . Carbomers are derivatives of polyacrylic acid that used as thickening agents for the preparation of nanogels . For example, topical delivery of amphotericin B was improved by preparing its nanoemulsion-based nanogel, using carbomer 980. The percutaneous permeation flux rate of nanogel (18.09±0.6 µg/cm2/h) was better than drug solution (4.59±0.01 µg/cm2/h) or even nanoemulsion (15.74±0.4 µg/cm2/h) .
Characteristics of PCLNFs
SEM image of smooth and non-branched PCLNFs with an average size of 186 ± 36 is depicted in Fig. 3A. The contact angle of de-ionized water with the surface of PCLNFs is given in Fig. 3(B and C). The contact angle (θ) was 145 ± 1°, concluded that the surface showed superhydrophobic behavior. Noted, if the contact angle of water with any solid surface exhibits larger than 90ο, it is showed hydrophobic behavior and lower values called hydrophilic .
The ATR-FTIR spectra of PCLNFs is displayed in Fig. 4. FTIR spectroscopy is an important identification tool for determining functional groups . The absorption bands of 2943 and 2865 cm-1 are attributed to the C-H stretching vibration of the hydrocarbon of PCL. A strong band at 1721 cm−1 is related to the stretching vibration of carbonyl groups (C=O stretching of ester) of PCL. The characteristic absorption band in 1239cm−1 belongs to the stretching vibration of (C–O).
Antibacterial activity of NGelNFs
The antibacterial activities of PCLNFs impregnated with CSEO nanogel and blank gel (i.e., NGelNFs and Gel(-oil)NFs) are illustrated in Fig. 5. There is no significant difference seen between the effectiveness of Gel(-oil)NFs and the control group against all examined bacteria strains (Independent sample t-test, p > 0.05). Interestingly, the growth of all bacteria after treatment with NGelNFs reduced to ~ 0%, concluding excellent effectiveness.
Reviewing the literature, no report was found on using impregnated NFs with essential oil-based nanogel as an antibacterial agent. However, one study tried to preparing photosensitive antibacterial agents using PCLNFs and gel of silver nanoparticles. The nanoparticles were released from the nanogel after irradiation by UV light 405 nm. The released nanoparticles act as antibacterial agents against S. aureus and E. coli. However, the antibacterial test was carried out by disc diffusion; therefore, the results are not comparable with this study . ATCC100 is the standard method for the investigation antibacterial effect of textures.
In another study, antibacterial properties of PCLNFs mat containing Peppermint EO against S. aureus and E. coli have reported. However, the growth of examined bacteria strain reduced to 50% . In addition, a nanopad of cellulose acetate containing a mixture of orange and rosemary essential oil was able to partially inhibit the growth of E. coli and S. aureus 
The observed results in this study are promising, completely inhibition of the growth of several important bacteria. Moreover, the prepared prototype can be used as a potent antibacterial material. However, the introduced model to make this type of antibacterial agent or antibiotic-free wound dressing is much more valuable.
In this system, by preparing nanoemulsion-based nanogel, the volatility and effectiveness of the essential oil were improved. Besides, its topical usage was facilitated. Many achievements were also obtained by impregnating the nanogels onto the surface of the NFs. For instance, easier topical application, the possibility of packing with a certain amount of nanogels as well as preventing the entry of environmental pathogens into the wound site.
Ingredients of CSEO were identified by GC-MS analysis, and its nanoemulsion-based nanogel of CSEO was prepared. Besides, electrospun NFs of PCL were successfully prepared and impregnated with the nanogel, NGelNFs. Interestingly, the NGelNFs completely inhibited the growth of all examined bacteria strains, including K. pneumonia, P. aeruginosa, E. coli, and S. aureus.
The prepared prototype can be used as a potent antibacterial agent as well as the antibiotic-free type of wound dressing. Moreover, this study introduced a new approach for the preparation of nano-dressing using green constituents.
This study was supported by Fasa university of medical sciences, grant No. 97152. Besides, it has been ethically approved, IR.FUMS.REC.1397.142.
CONFLICT OF INTEREST
No researchers have a conflict of interest in this study.