Methods and materials
Preparation and characterization of the nanoemulsion and nanoemulsion-based nanogel
Bark-extracted A. dracunculus EO was purchased from Zardband Pharmaceuticals Company (Iran). The nanoemulsion was prepared as follows; the EO (100 μL) and tween 20 (300 μL) was first mixed at 2000 rpm for 3 min at ambient temperature to form a homogeneous solution. Distilled water was then added to the mixture up to desired volume (5000 μL) and was stirred for another 40 min at 2000 rpm. Finally, the prepared nanoemulsion's droplet sizes and droplet size distribution were investigated utilizing a Dynamic Light Scattering (DLS) apparatus (K-One NANO- Ltd. Korea). Droplet size distribution was computed as d90-d10/d50, where d10, 50, and 90 are percentiles of droplets with diameters less than these values.
The nanoemulsion was gelified by adding 3.5% w/v carboxymethylcellulose; the mixture was stirred at a mild speed (180 rpm) for 4 h. Moreover, nanoemulsion (-oil) and nanogel (-oil) were prepared using the same process, only without the EO.
The viscosity of the prepared nanogel was assayed at different shear rates at 25° C under atmospheric pressure (Rheometer machine model MCR-302, Anton Paar, Austria). Besides, ATR-FTIR analysis was used to investigate the successful loading of the EO in the nanoemulsion and nanogel. Spectra of the EO, nanoemulsion (-oil), nanoemulsion, nanogel (-oil), and nanogels were recorded in a wavenumber range of 400–3900 cm−1 using a spectrometer (Tensor II model, Bruker Co, Germany).
Evaluation of larvicidal activity
In the current study, A. stephensi late-3rd or young-4th instar larvae were used; they were reared and maintained at 29 ± 2 °C with 70 ± 5% humidity at Urmia University of Medical Sciences (Iran). Mosquitoes are not exposed to any insecticides for more than 10 years. According to the WHO guideline, the larvicidal activity was done with a slight modification [17]. Briefly, beakers containing 200 mL of no-chlorine water and 25 larvae were first prepared. The larvicidal effects of nanoemulsion and nanogel were then investigated at 6.3, 12.5, 25, 50, and 100 µg/mL. Larval mortality after 24 h exposure was counted, while the larvae were not fed during the test. The larvae were exposed to 1.5 mL ethanol and nanoemulsion (-oil) and nanogel (-oil) in the control and negative control group.
Evaluation of antibacterial activity
The antibacterial activity of nanoemulsion and nanogel against S aureus (ATCC 25,923) and P aeruginosa (ATCC 27,853) were investigated using ATCC100 standard method [18]. Briefly, 4 mL of each bacterial suspension (2 × 105 CFU/mL) was first poured into 5 cm plates separately. Antibacterial effects of nanoemulsion and nanogel were then investigated at 1250, 2500, and 5000 µg/mL. The treated plates were incubated at 37 °C for 24 h, and 10 μL-microbial suspensions were cultured on agar plates and incubated for 24 h. The number of grown colonies on the plates was counted and compared to the control sample. The control groups were not treated, and the negative control group was treated with nanoemulsion (-oil) and nanogel (-oil). Growth (%) of bacteria in each plate was calculated as (CFU sample /CFU control) × 100.
Statistical analyses
Three replicates were carried out for all tests, and final values were given as mean ± standard deviations. The samples were compared with SPSS software using one-way ANOVA with at least a confidence interval of 95%.
Results
Prepared nanoemulsion and nanogel
DLS profile of the nanoemulsion with a droplet size of 152 ± 6 nm is shown in Fig. 1A. The nanoemulsion had narrow particle size distribution as its droplet size distribution was 0.98; its single sharp peak also confirmed its uniform droplet size [19]. The viscosity of nanogel at different shear rates (1/s) is fully fitted with the Carreau-Yasuda models (Fig. 1B). This well-known empirical equation has been used for non-Newtonian fluids; viscosity is decreased with increasing shear rates [20].
Besides, successful loading of the EO in nanoemulsion and nanogel was confirmed using ATR-FTIR analysis (Fig. 1C). The spectrum of A. dracunculus EO showed the bands at 3061 and 3028 cm−1 related to = C-H. The bands at 3076, 3001, 2933, 2096, and 2834 cm−1 displayed –CH stretching vibration in SP3 and SP2. Besides, the bands at 1727 and 1638 cm−1 can be related to the stretching vibration carbonyl C = O group. The peak at 1243 cm−1 corresponds to the stretching vibrations of C-O. The peak at 1035 cm−1 is assigned to C-H bending absorption, and the peak at 808 cm−1 is attributed to benzene rings C-H vibration absorption.
The spectrum of nanoemulsion (-oil) showed broadband between 3300 to 3600 cm−1 can be attributed to the presence of hydroxyl group due to hydrogen bonding. Besides, the peak at 2923 cm−1 is attributed to C-H stretching in tween. Moreover, the peak at 1732 cm−1 corresponds to C = O stretching exhibiting ester groups in tween 20. The sharp band at 1088 cm−1 is assigned to C-O stretching vibration.
In the spectrum of nanoemulsion, the broadbands at about 3200 to 3600 cm−1 are related to the hydroxyl group due to hydrogen bonding. The absorbance band at 2923 cm−1 showed CH stretching vibration in tween 20 and EO. Besides, the band at 1734 cm−1 can be related to the carbonyl group in the EO and tween 20. The band at 1457 cm−1 is related to CH2 bending in the EO and tween 20. All the other characteristic bands appear in the spectra of the EO and nanoemulsion (-oil).
The spectrum of nanogel (-oil) showed the broadband at about 3200 to 3600 cm−1 is attributed to the OH group due to hydrogen bonding. The band at 1738 cm−1 showed C = O stretching that represents the carbonyl group in CMC and tween 20. The characteristic band at 1579 cm−1 is attributed to the carboxyl group in CMC.
In the spectrum of nanogel, the broadband at around 3200 to 3600 cm−1 is attributed to the OH group due to hydrogen bonding. The interaction between CMC and the EO during gel formation is related to the preparation of hydrogen bonding. The formation of hydrogen bonds increases the degree of polarization of chemical bonds. Besides, the peak at 1733 cm−1 exhibited carbonyl stretching that confirmed the carbonyl group in CMC, tween 20, and the EO. The peak at 1579 cm−1 corresponded to the carboxyl group in CMC. All the other characteristic peaks appear in the EO and nanogel (-oil) spectra at the same wavenumber.
Larvicidal effect of the nanoemulsion and nanogel
Larvicidal effects of nanoemulsion and nanogel against A. stephensi are given in Fig. 2. Dose-dependent responses are observed in their efficacy; however, the nanogel with LC50 6.6 (2–19) µg/mL was more potent than the nanoemulsion with LC50 13.5 (7–25) µg/mL. Besides, nanogel was significantly more potent than nanoemulsion at 6.3 µg/mL (P < 0.001), 12.5 µg/mL (P < 0.001), and 50 µg/mL (P < 0.028). Interestingly, perfect efficacy (100% larval mortality) was observed at 25, 50, and 100 µg/mL nanogel. Moreover, nanoemulsion (-oil) and nanogel (-oil) with 0 and 3% larval mortality did not affect larvae.
Antibacterial effects of the nanoemulsion and nanogel
The antibacterial effect of nanoemulsion and nanogel against P. aeruginosa and S. aureus are shown in Fig. 3(A and B). The efficacy of nanogel was more potent than nanoemulsion; however, this difference was not significant at all examined concentrations (P > 0.05). The best efficacy (~ 20% growth inhibitory) against P. aeruginosa was observed at 5000 μg/mL nanogel and nanoemulsion. However, 70% growth inhibitory was observed at this point against S. aureus. Moreover, nanoemulsion (-oil) and nanogel (-oil) did not affect bacterial growth.