1. Introduction
Skin is one of the mechanical defense systems in the human body that acts as a barrier against pathogen invasion [
1]. However, it can be exposed to microbial infections that require certain treatments via topical application. The strategy of applying drugs over the skin and providing their effectiveness directly to the target site is termed a topical drug delivery system [
2]. Topical delivery is a more desirable and convenient strategy than other routes of administration owing to its great advantages [
3], since it can overcome the first pass mechanism and the problems associated with swallowing [
4]. Topical drug delivery systems can be used for treating a wide variety of disorders where they are available as analgesic [
5], antifungal [
6], anti-inflammatory [
7], anticancer [
8], antioxidant [
9] and antibacterial formulations [
10]. Ointments and creams are different conventional dosage forms that are broadly used topically; however, certain problems could limit their formulation. Mostly, inadequate drug loading capacity, poor spreadability, and certain stability problems are the most challenging complications. Therefore, it was necessary to overcome these weaknesses to reach a more comfortable and reproducible activity [
11].
In light of that, an advanced approach termed nanotechnology has critically attracted attention. Nanotechnology is the science of developing different nanosystems with a nanoscale range of size, hiding the unrequired properties of active constituents and maximizing their therapeutic actions [
12]. Nanosystems are formed by nanocarriers carrying the active moiety of the drug. These nanocarriers are numerous, such as liposome, ethosome, niosome, nanoparticles, and nanoemulsion [
13].
Nanoemulsion (NE) is one of the currently settled nanocarriers that are colloidal systems of tiny globular size and, consequently, large surface area, which can enhance drug absorption and bioavailability as well [
14]. Additionally, NE can offer controlled drug release and protect the formulation against degradation [
15]. NE can be used for delivering drugs via different ways of drug administration, oral [
16], parenteral [
17], transdermal [
18], and topical routes [
19]. Though, for topical medication delivery, it is more appropriate for the drug to be incorporated into a more viscous preparation such as hydrogel base providing the NE-hydrogel base formulation.
From the extensive spread of skin disorders, the prevalence of bacterial infections has appeared widely and should be handled wisely using antibacterial agents named antibiotics [
20]. Neomycin sulfate (NEO) is one of the antibiotics that has revealed a broad-spectrum activity against Gram-positive and Gram-negative bacterial strains [
21]. It is a 2-deoxystreptamine-containing aminoglycoside antibiotic exhibiting nephrotoxicity and ototoxicity problems upon long-term treatment, which explains its limited therapeutic range [
22]. Despite that, its use was approved by the United States Food and Drug Administration [
23]. Meanwhile, it was reported that the toxicity of the antibacterial agents is greatly minimized upon topical application [
24,
25]. Therefore, it was recommended to develop a topical formulation incorporating NEO rather than other routes. However, NEO was discovered long ago; thus, it could exhibit some kind of bacterial resistance [
26]. Several investigations were explored in order to overcome bacterial resistance against NEO by producing a new derivative and applying structural modification [
27,
28]. Another strategy focused on combining NEO with other antibacterial agents that provided a superior influence than each one alone and helped in reducing the burden of bacteria, which is renowned as a combination therapy [
29]. Given that there is a lack in the development of new antibiotics to face antimicrobial resistance, a combination therapy was an alternative way of choice for managing such complications that threaten the medicinal field [
30].
The best category to be combined with these antibiotics is natural products owing to their excellent efficacy and safety [
7]. Tea tree oil (TTO) is an essential oil of the Australian native plant
Melaleuca alternifolia, a well-known genus derived from the Myrtaceae family [
31]. With regard to its origin, it is well-known as melaleuca oil. TTO showed a broad-spectrum activity, namely, antiprotozoal, antifungal, and antiviral effects [
32]. In addition to its antibacterial influence, which can be attributed to its cyclic monoterpenes structure from which terpoen-4-ol is responsible for such behavior [
33], it also exhibits antioxidant and anticancer activity, which has formerly been proven, in addition to its established antiseptic and disinfectant influence [
34].
In light of the previous facts, our target in the study has been raised. It is an attempt to formulate NE using TTO containing NEO. As far as we know, this is the first combination of TTO and NEO into a nanoemulsion formulation intended for topical application. CCD strategy as a tool for quality by design approach was run employing a 22 full factorial design to obtain a high-quality product and selected the optimized NE formulation. The optimized formula was characterized, incorporated into a hydrogel base, and inspected for its antibacterial activity.
2. Materials and Methods
2.1. Material
Neomycin was obtained from (Sisco Research Laboratories Pvt. Ltd., Taloja, Maharashtra, India). Tea tree oil was acquired from NOW® Essential Oils (NOW Foods, Bloomingdale, IL, USA). Diethylene Glycol Monoethyl Ether (Transcutol® P) was bought from Gattefosse SAS (Saint-priest Cedex-France). Tween 80 and Carboxymethylcellulose Sodium (NaCMC) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Distearoyl phosphatidylethanolamine-N-[methoxy poly (ethylene glycol)-2000] (PEG-DSPE) was bought from Lipoid LLC., (Newark, NJ, USA). All other chemicals were of the finest grade available.
2.2. Designing the Experiment
QbD approach was exploited to maximize the desirable characteristics of the formulations via implementing CCD in which two factors, 2 level (2
2) factorial design was constructed. In that design, two independent factors were selected, amount of TTO and tween 80 with symbols, A and B, respectively. They were inspected at two levels to show their influence on two different dependent responses, namely, globule size (Y
1) and in vitro release (Y
2). The strategy was implemented using Design-Expert version 12.0 software (Stat-Ease, Minneapolis, MN, USA) since it helps in data interpretation via analyzing the results using the Analysis of variance (ANOVA) test. The data were illustrated further by constructing certain modeling graphs such as a 3D response surface plot, one-factor graph, and perturbation plot. The linearity between the actual and observed response could be demonstrated by predicted versus actual plot. Moreover, mathematical polynomial equations provided by the design could as well establish the influence of the nominated independent factors on the studied response [
35].
2.3. Development of NEO-NE
Different NE formulations were prepared using TTO and including NEO; a method lately reported by Shehata et al. was followed and data were displayed in
Table 1 [
7]. Fundamentally, the aqueous phase was prepared by dissolving 50 mg of NEO in distilled water containing a specified amount of tween 80. On the other side, 0.5 g of transcutol
® P and 50 mg of PEG-DSPE were added to a quantified amount of TTO to provide an oily phase. Together, the two phases were mixed for 10 min at 15,000 rpm using a high shear homogenizer (T 25 digital Ultra-Turrax, IKA, Staufen, Germany) after adjusting the volume to 10 mL with distilled water. The formed NE was exposed to sonication for 30 s in order to obtain a suitable globule size using probe sonicator (XL-2000, Qsonica, Newtown, CT, USA).
2.4. Characterization of Developed NE
Globule Size and Polydispersity Index (PDI) Determination
One of the essential parameters to be evaluated in NE preparation is their globule size along with the relative size distribution. For that, Zetasizer apparatus (Malvern Instruments Ltd., Worcestershire, UK) was used for determining the globule size and the corresponding PDI of the preparation. Briefly, about 5 µL of each NE was added to 3 mL distilled water in a disposable cuvette and measured at 25 °C [
36].
2.5. In Vitro Study
To detect the percentage of NEO released from the preparation, in vitro release study was conducted using the ERWEKA dissolution system (ERWEKA, GmbH, Heusenstamm, Germany) as mentioned previously by Almostafa et al. [
10]. Briefly, 1 mL of NEO-NE sample was added into a glass tube hung into the apparatus and closed from one side with a cellophane membrane (MWCO 2000–15,000). The tubes were suspended into the acceptor vehicle composed of 500 mL phosphate buffer pH 5.5 and kept at 32 °C to mimic the skin condition. The system was operated and tubes were allowed, rotating at 50 rpm. Samples of 3 mL were withdrawn from the media at specified time up to 3 h and replaced with the same volume of fresh media. The withdrawn sample was analyzed at ƛ
max 277 nm using UV. Spectrophotometer (JENWAY 6305, Bibby Scientific Ltd., Staffs, UK). Experiment was performed three times for each sample.
2.6. Zeta Potential
The optimized NEO-NE was evaluated for its surface charge by conducting zeta potential measurements using Zetasizer apparatus (Malvern Instruments Ltd., Worcestershire, UK). A special electrophoretic cuvette was used at which 5 µL of the sample was diluted with distilled water and checked for their electrophoretic mobility at 25 °C [
37].
2.7. Development of NEO-NE-Based Hydrogel
Following validation of the optimization, the optimized NEO-NE formulation was loaded into a pre-formulated hydrogel base in order to facilitate the formulation topical application over the skin. Then, 4% NaCMC hydrogel was prepared simply by dispersing the gelling agent over 10 mL distilled water and keep stirring using magnetic stirrer (Jeio Tech TM-14SB, Medline Scientific, Oxfordshire, UK) until the homogenous NaCMC hydrogel base was obtained. The optimized NEO-NE formulation was added to the hydrogel base and mixed continuously for 5 min using a mixer (Heidolph RZR 1, Heidolph Instruments, Schwabach, Germany) in receipt of consistent NEO-NE-based hydrogel formulation [
38].
2.8. Characterizing the Developed NEO-NE-Based Hydrogel
2.8.1. Visual Examination
Visual inspection of the developed formula is very important, to follow up on the state of the preparation. Therefore, the developed NEO-NE-based hydrogel formulation was visually observed for its physical characteristics such as appearance, color, and homogeneity.
2.8.2. pH Measurement
In order to avoid any probable irritation that could happen due to variation in pH value between the skin and the applied formula, this measurement was performed. pH value was determined using a standardized pH meter (MW802, Milwaukee Instruments, Szeged, Hungary) [
39].
2.8.3. Viscosity
Viscosity of the topical formulation is essential in evaluating the preparation. Therefore, appropriate viscosity evaluates the run-off of the formulation as it is better to stay adhered to the affected area for a longer time [
40]. The viscosity of the examined NEO-NE-based hydrogel formulation was measured utilizing Brookfield viscometer (DV-II+ Pro, USA) using spindle 63 and worked at 25 °C [
41].
2.8.4. Spreadability
Spreadability is a critical parameter that has to be validated in any topical formulations as it greatly affects their viscosity. Proper spreadability gives an assumption about the formulation that would spread easily and evenly over the skin. It was conducted by holding a certain amount of the examined formulation in between two slides made of glass and of about (25 cm × 25 cm). Specific load, usually 500 g was added over the slides for 1 min. The spreadability is calculated by measuring the diameter of the spreading formulation [
42].
2.9. Scanning Electron Microscopy (SEM)
Morphology of the fabricated NEO-NE-hydrogel formula could be estimated by a microscopic technique using scanning electron microscopy (SEM), (JSM-6390LA, JEOL, Tokyo, Japan). Typically, a sample of the formulation was added on slabs, shielded with gold using a sputter coater, and scanned. Then, the morphology was identified under a lower vacuum at an acceleration voltage of 10 kV using different magnifications [
43].
2.10. In Vitro Release of NEO from Different Developed Formulations
The same method mentioned in
Section 2.5 was followed to detect the in vitro release of NEO from the developed NEO-NE based hydrogel compared to the optimized NEO-NE formula.
2.11. Kinetic Study
Kinetic studies explain the mechanism by which the drug could be released from the developed formulation. It could take place by one of the kinetic modeling systems, namely, zero-order reaction, first-order, Higuchi, and Korsmeyer–Peppas modeling. Each mechanism illustrates a relation between drug concentration and the time, in a special way to provide the most fitted model with the highest correlation value R
2. Meanwhile, zero-order kinetics demonstrates a relationship between the drug concentrations against time, while first-order kinetics clarifies the relation between Log concentrations against the time. Regarding the Higuchi equation, it illustrated the relationship between drug concentrations against the square root of time (t
0.5). However, if the relation was among Log concentration against Log time, the model seemed to obey the Korsmeyer–Peppas equation [
44].
2.12. Stability Test
The developed NEO-NE-based hydrogel preparation was checked for its capability to stay unchanged upon storage in different conditions and for a definite period of time. The study was performed according to the guidelines of the International Conference on Harmonization (ICH) to evaluate different criteria of the formulation such as pH, viscosity, spreadability, and in vitro drug release. The study was conducted after storing the formulation at 4 ± 1 °C and at 25 ± 1 °C for a period of 1 and 3 months [
45].
2.13. Animal
2.13.1. Animals
Male Wister rats were required for the present investigation from the Experimental Animal Research Center at King Saud University, Riyadh, KSA; with an average weight of 220–250 g. The rats were housed in an appropriate environmental condition with a 12 h dark/light cycle and free access to water and food.
2.13.2. Statement of Animal Ethics
Handling of animals and the entire in vivo experiments implemented were performed in accordance with the regulations of ethical conduct for animal use at King Faisal University. The protocol of the experiment was issued by the Research Ethics Committee (REC) of King Faisal University approval number (KFU-REC/2022-May–ETHICS17).
2.13.3. Skin Irritation Test
The study provides an indication about the safety of the formulation. Primarily, one day before proceeding with the test, the hair from the dorsal part of the animal was shaved using clippers. The inspected formulation was uniformly distributed over the shaved area. Rats were kept under observation for 7 days following topical application of the formulation. Rats were checked for any abnormal signs such as irritation, edema, or erythema (redness). The reactions were determined by applying a sensitivity scale ranging as 0, 1, 2, or 3, which represents no reaction, minor, moderate, and severe erythema with or without edema, respectively [
46].
2.14. Microbiological Study
A microbiological examination was conducted to determine the antibacterial activity of the NEO-NE-based hydrogel using the disk diffusion method. The study was carried out using different bacterial strains provided by the American Type Culture Collection (ATCC). Consequently, Bacillus subtilus (ATCC 10400), Staphylococcus aureus (ATCC 29213), Klebsiella pneumoniae (ATCC 10013), and Escherichia coli (E. coli) (ATCC 25922) were used in the study as representative microorganisms. Simply, a disk of about 12 mm diameter was made by a sterile cork borer in a Petri dish containing Moller–Hinton Agar, which is a media for bacterial culturing. Small amounts of the preparation were added in each disk in order to evaluate the inhibition zone made by NEO-NE-based hydrogel and blank NE, compared with NEO solution as a control. The experiments were carried out in triplicate for each bacterium with a mean value ± SD and the plates were incubated for 24 h at 37 °C. Afterward, the zone of inhibition was measured in each plate and recorded.
2.15. Statistics
Results were regarded as significant when p value being < 0.05. All studies were performed in triplicate. To compare results between two groups, Student’s t-test was followed. Analysis of variance (ANOVA) followed by the least significant difference (LSD) as a post hoc test was conducted when comparing between groups. The analysis was carried out using SPSS statistics software, version 9 (IBM Corporation, Armonk, NY, USA).