2. Introduction
• Essential oils (Eos) exert their antibacterial activity through
destruction of the bacterial cell membrane.
• Eos is suitable delivery system such as nanoemulsions, is an
effective technique to boost the bioavailability and physical stability
of active lipophilic substances.
• Nanoemulsions with mean particle sizes less than 100 nm, that
preserve lipophilic bioactive chemicals from chemical degradation.
• Many studies evaluated nanoemulsification of EOs is an efficient
delivery technique to increase the antimicrobial activities.
• Nanoemulsions can be produced from both high-energy and low-
energy methods.
3. Objectives
• Nanoemulsions were prepared using trans-cinnamaldehyde as the
active compound and 1,8-cineol as a co-additive with
antibacterial properties and the effect of sonication time,
emulsifier type, and component concentration on nanoemulsion
stability was investigated.
• In addition, the antibacterial activity of the produced
nanoemulsions was studied, as well as the mechanism of
antibacterial action, against a variety of harmful microorganisms.
• Furthermore, lipid membrane analysis was used to track the
effect of nanoemulsion on bacterial membrane permeability.
• Finally, IR analysis was used to assess the influence of
nanoemulsion on bacterial membrane breakdown.
4. Material and Methods
2.1. Chemicals
• Trans-Cinnamaldehyde (P98%)
• 1,8-cineol (P98%)
• Tween 80
• Tween 20
(Obtained from Merck Millipore (Darmstadt, Germany).
5. 2.2. Fabrication of nanoemulsions using probe sonication
• Tween 80 or Tween 20 were used as emulsifiers, with trans-cinnamaldehyde as
the oil phase and 1,8-cineol as a ripening inhibitor (1:4).
• Surfactant to oil ratios (SOR 2:1, 1:1, 1:2, 1:3) and emulsification times (5, 10,
and 15 minutes) for fabrication of stable NEs with the lowest particle size.
• A 20.5 kHz probe sonicator power output of 400W was used for emulsions.
• With a maximum probe diameter of 8 mm, a piezoelectric crystal sonotrode
conveyed input energy into the mixture.
• The combination was 3 mL in volume, and the sonotrode was immersed in the
reaction vessel's center, with an ice bath used to keep the temperature constant
throughout sonication.
6. 2.3. Nanoemulsions stability
• The accelerated stability test for nanoemulsion was performed by centrifugation
at 3500 rpm for 30 min.
• The particle size and size distribution for samples stored at room temperature for
period of six months.
2.4. Nanoemulsions characterization
• Morphology and structure of optimum nanoemulsion were evaluated using
transmission electron microscopy (TEM).
• TEM micrographs were obtained using Zeiss-EM10C (Oberkochen, Germany)
at an accelerating voltage of 80 keV.
• Also, droplet size measurements were carried out using a Dynamic Light
Scattering (DLS) instrument (Nanophox Sympatec GmbH, Claushtal,
Germany).
7. 2.5. Antibacterial activity
Bacterial strains
• Antibacterial activity of the nanoemulsions and trans-
cinnamaldehyde oil were analyzed against
1) Escherichia coli
2) Pseudomonas aeruginosa
3) Staphylococcus aureus
9. Fig. 1. Effect of sonication time and Emulsifier type for Tween 80 (A) and Tween 20 (B) on CIN:CIN
nanoemulsions particle size, difference between preparation time and 10 days for Tween 20 were significant
(C) * (p < 0.009), **(p < 0.01), ***(p < 0.004), ****(p < 0.002).
3.1.1. Effect of sonication time, surfactant to oil ratio and emulsifier type
on the particle size and stability of nanoemulsions
10. Fig. 2. TEM micrograph of optimum formulation (T804) at tow magnification: 40,000 (A), 63,000 (B)
3.1.2. Transmission electron microscopy
11. 3.3. Antibacterial activity of nanoemulsions
3.1.3. The influence of particle size and emulsifier type on antibacterial
activity
Table 2: Antibacterial activity of CIN:CIN nanoemulsions with different
formulations against selected bacteria
12. 3.4. Kinetic kill curve
Fig. 3. Time killing curve of T804 nanoemulsion and CIN:CIN oil in 1 h.
13. 3.5. IR analysis
Fig. 4. IR analysis of E. coli after 10 min treatment with T804 nanoemulsion and CIN:CIN oil.
14. 3.6. Effect of the nanoemulsion on the fatty acid
composition of E. coli membrane
Table 3: Alternation in FAMEs of E. coli after 10 min treatment with CIN:CIN oil and T804
nanoemulsion
15. • Finally, trans-cinnamaldehyde oil was ultrasonically emulsified
utilizing two non-ionic surfactants (Tween80 and Tween 20), as well
as 1,8-cineol as a co-additive and OR inhibitor.
• Concluded 1,8-cineol is a good carrier oil for forming transparent
and stable nanoemulsions by a controlled OR destabilizing process.
• The results also showed that probe sonication is a good way to make
a stable nanoemulsion from CIN:CIN oil.
• The particle size may be changed from 27.76 (T 804) to 113.57
(T201) nm (after 15 minutes of sonication) without affecting the
antibacterial activity.
Conclusion
16. • Changing the kind of emulsifier from Tween 80 to Tween 20
increased particle size and disrupted the nanoemulsion stability
profile, according to the findings.
• Because of the small particle size (27.7 nm), high stability and
appearance, and potent antibacterial activity, optimization of the
ultrasound-assisted formulation revealed that nanoemulsion with
Tween 80 as a surfactant, surfactant to oil ratio of 2:1 (w/w), and 15
min sonication time is the best formulation condition.
• Mechanistic investigations on the permeability and fluidity of the E.
coli membrane found that cell contents are released into the
environment, resulting in bacterium cell death.
Conclusion
To evaluate the influence of emulsifier type on the particle size, the nanoemulsions with Tween 20 as emulsifier were compared to those prepared using Tween 80. As can be seen in Fig. 1, comparing T801 with T201 particle size show increasing droplet size from 120.03 nm to 136.27 nm and 113.57 nm to 84.95 nm after 5 and 15 min sonication time, respectively. This. investigation showed that changing the emulsifier type from Tween 80 to Tween 20 increased the particle size significantly.
Interestingly, following centrifugation at 3500 rpm, the Tween 20 formulations (T201, T202, T203 and T204) showed phase separation and also the particle size increased over 10 days (Fig. 1C). Moreover, phase separation was observed after one month of storage. However, all prepared formulations using Tween 80 showed no phase separation after centrifugation and were stable over six month period
This phenomenon could show that Tween 80 is more appropriate than Tween 20 for emulsification of CIN:CIN nanoemulsions.
TEM images were obtained from optimum nanoemulsion (T804) for analyzing the size, size distribution and morphology.
The TEM images of T804 nanoemulsion droplets shows spherical nanoemulsion droplets with mean particle size and size distribution within the size ranges of 28 nm (Fig. 2A, B).
type of emulsifier (Tween 80 or Tween 20) could not influence the antibacterial activity. These results may propose that nanoemulsification of CIN:CIN oil improves the bioavailability of the lipophilic oils followed by the release of active compounds from the oil droplet near the bacterial cell surface. On the other hand, the study of particle size impact on the antibacterial activity showed that all formulations with different particle size had the same antibacterial activity. Indeed, variation of particle size from 27.76 nm (T804) to 113.57 nm (T201) had no effect on the antibacterial activity.
Exposure of E. coli to the nanoemulsion in 5 min caused the viable counts to decrease from 1 1011 CFU/mL to 500 CFU/mL which equates to an 8 log reduction in viable counts. This experimental data shows complete loss of viability following 10 min of incubating with the MIC concentration of T804 nanoemulsion. Whereas incubating the bacteria within equivalent amount of CIN:CIN oil for 10 min Resulted in a loss of the viable cell count from 1 1011 CFU/mL to 1 109.9 CFU/mL
Upon treatment with T804 nanoemulsion, the intensity of shoulder at about 1460 cm1 -attributed to the lipid CH2 stretching vibrations – was increased.
nanoemulsion treatment increasing the band intensity at about 1160 cm1 (attributed to CAO stretching vibrations in polysaccharides). may be described by the increase in the polysaccharides content of E. coli cell membrane.
Moreover, enhancement of the peak intensity of fatty acid region between 2800 and 3000 cm1 was observed in the nanoemulsion treated samples, which is attributed to the increase of fatty acid content. This enhancement because of cell membrane destruction.
The dominant bands at amide I region (1600–1700 cm1 ) were attributed to the protein content of the E. coli cells [33]. This region has greater magnitude in the nanoemulsion treated bacteria.
These results can be attributed to the cellular membrane damage or conformational and compositional changes in some components of the cell membrane.
There are several studies that investigated the alternation of membrane fluidity in stress condition [35–38]. In some cases the membrane fluidity was increased in response to the environmental stress such as EOs. Active lipophilic compounds including EOs could penetrate through the double lipid layer of the membrane, modifying the membrane fluidity of the microorganism may be altered by change of fatty acids synthesis [
