1 

Chapter 2 
 
Bio-based Nanoemulsion Formulations Applicable in Agriculture, Medicine 
and Food Industry 
 
Josef Jampílek1*, Katarína Kráľová2, Estefânia V. R. Campos3 and Leonardo F. Fraceto3 
 
1Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 27, 783 
71 Olomouc, Czech Republic; E-mail: josef.jampilek@gmail.com 
2Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Ilkovičova 6, 842 15 
Bratislava, Slovakia 
3São Paulo State University (UNESP), Laboratory of Environmental Nanotechnology, Institute 
of Science and Technology of Sorocaba, Sorocaba, São Paulo State, Brazil 
 
Abstract  
 
Nanotechnology providing “a new dimension” accompanied with new properties conferred to 
many current materials is widely used for production of a new generation of agrochemicals, in 
medicine it enables improved drug bioavailability, reducing undesirable side effects, minimizing 
non-specific uptake and specific targeting to certain target cells, while in food industry it has 
great importance in food protection and biofortification of food with valuable ingredients. In bio-
based nanoemulsions belonging to lipid nanocarriers plant oils used for oil phase, emulsifiers, 
biosurfactants, cosurfactants, targeting ligands on the surface of nanoemulsion (e.g., folate) or 
encapsulated active ingredients are of natural origin. The biocomponents of such nanoemulsions 
show low toxicity to living organisms, could protect encapsulated compounds for degradation, 
ensure their sustainable release and reduce the amount of active ingredient necessary for required 
effect. This chapter presents a comprehensive current overview of recent findings in the field of 
nanoemulsions and their utilization in agriculture and food industry, with the main emphasis on 
formulations encapsulating essential oils or plant extracts suitable as effective pesticide 
preparations as well as medicinal applications of bio-based nanoemulsions, where attention is 
paid to transdermal nanoemulsion formulations, the use of nanoemulsions in cancer therapy and 
for pulmonary and ocular drug delivery. Nanoemulsions formulated with natural emulsifiers, 
biosurfactants and biopolymers are presented and bio-based nanoemulsions of essential oils and 
their constituents as well as nanoemulsions with encapsulated vitamins, fatty acids and some 
bioactive compounds are discussed. Applications of nanoemulsions in edible coatings are 
outlined as well. 
 
Key words: nanoemulsions, nanoformulations, nanocomposites, encapsulation, drugs, bioactive 
agents, foodstuffs, essential oils, packaging materials 
 
 
2.1 Introduction 
 
Nanotechnology is a fast growing field that provides for the development of materials that have 
new dimensions, novel properties, and a broader array of applications (e.g., (Achari and Kowshik 
2018; Agarwal et al. 2018;  Jampílek and Kráľová 2015a, 2017a,b; 2018a,b; Prasad et al. 
2017a,b; Sekhon 2014; Ventola 2017). It is regarded as one of the key technologies of the 21st 
century. U.S. National Nanotechnology Initiative defines nanoparticles (NPs) in the range 1–100 
nm (National Nanotechnology Initiative 2008). NPs and nanoformulations can be prepared from 
both inorganic and organic materials (e.g., Bhushan et al. 2014; Singh et al. 2015; Jampílek and 
Kráľová 2018a; Pisarčík et al. 2018). As mentioned above, nanoscale materials change properties 
and behavior of all materials and thus a variety of industrial, agricultural, pharmaceutical and 



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medical products have been improved and innovated in such a way (e.g., Dolez 2015; Patra et al. 
2018; Sekhon 2014). 
In modern agriculture, the main form of control of diseases and agricultural pests is performed 
using agrochemicals. However, extensive and intensive use of these compounds has resulted in 
environmental contamination, development of resistance in some species, decreased food safety, 
and side effects in non-target organisms (Aktar et al. 2009; Fountain and Wratten 2013; Prasad et 
al. 2014; 2017a). To address these issues, the development of nano-based pesticides and also the 
utilization of bio-based pesticides has become an important research tool (Campos et al. 2016, 
2018; Hayles et al. 2017; Hemraj 2017; Kah et al. 2018; Jampílek and Kráľová 2015a, 2017a, 
2018c, 2019a) and the application of nanofertilizers results in increased nutrient use efficiency in 
crop production (Achari and Kowshik 2018; Jampílek and Kráľová 2017c; Raliya et al. 2018). 
Nanoemulsions (NEs) showing desirable physico-chemical characteristics have been extensively 
studied as carriers for pesticide delivery (Wang et al. 2007; Knowles 2008; Hayles et al. 2017; 
Hazra et al. 2017). 
However, nanotechnology is increasingly being applied also in medicine, in theranostic and drug 
delivery (Prasad et al. 2016; 2017b). By encapsulation into nanoformulations sustainable release 
of drugs as well as reduction of the required drug amount could be obtained and nanomaterials 
represent also an alternative approach to treating and mitigating infections caused by resistant 
bacteria (e.g., Jampílek et al. 2015b; Jampílek and Kráľová 2017b, 2018a, 2019b,c; Pentak et al. 
2016; Patra et al. 2018; Prasad M et al. 2018). 
In the food sector the nanotechnologies are used for food protection, including nanocomposites 
for protection of fruits, vegetables, cheese, dairy products, meat or fish, in smart active 
packaging, responsive packaging or edible coatings and like that significantly contribute to 
enhanced food quality. Moreover, they found wide application also as nanosensors (e.g., 
Chellaram et al. 2014; Jampílek and Kráľová 2015a, 2018b; Malhotra et al. 2014; 
Mihindukulasuriya and Lim 2014; Singh et al. 2017a). 
For preparation of bio-based NEs many compounds of natural origin, including encapsulated 
active ingredients (e.g., essential oils; EOs), but also plant oils for NE oil phase (e.g., palm oil, 
rapeseed oil, sunflower oil etc.) (e.g., Raviadaran et al. 2018; Kaci et al. 2018; Abdou et al. 
2018), emulsifiers, biosurfactants or cosurfactants (e.g., Quillaja saponin, phospholipids, lecithin, 
gum arabic, pectin, whey protein, lactoferrin, lactoferrin/alginate) (Bai et al. 2016; McClements 
and Gumus 2016; Ozturk et al. 2014, 2015; Liu et al. 2017; Verma et al. 2016; Artiga-Artigas et 
al. 2018; Zhao et al. 2018a; Pinheiro et al. 2016), or targeting ligands on the surface of NE such 
as folate (Liu et al. 2017; Ganta et al. 2016; Afzal et al. 2016a) increasing drug bioavailability, 
reducing undesirable side effects, minimizing non-specific uptake and thus allowing specific 
targeting to certain target cells are used. Targeting technology usually utilizes the nanocarrier 
functionalization, which can be surface modification (e.g., Attia et al. 2017; Liu et al. 2017) 
and/or ligand grafting (e.g., Geng et al. 2016). Chitosan (CS)-based NE coatings are used to 
enhance mucoadhesive properties (Fachel et al. 2018; Mendes et al. 2017; Kumar et al. 2009) as 
well as antimicrobial activity of these nanoformulations (Marei et al. 2018; Severino et al. 2014, 
2015), and also pectin, sodium caseinate or carrageenan (Abdou et al. 2018; Qian and 
McClements 2011; Alarcon-Alarcon et al. 2018) are used as NE coatings. 
This chapter is focused on the use of NEs in agriculture, with the main emphasis on formulations 
encapsulating EOs or plant extracts suitable as effective pesticide preparations as well as 
medicinal applications of bio-based NEs, where attention is paid to transdermal NE formulations, 
the use of NEs in cancer therapy and for pulmonary and ocular drug delivery. NEs formulated 
with natural emulsifiers, biosurfactants and biopolymers are presented and bio-based NEs of EOs 
and their constituents as well as NEs with encapsulated vitamins, fatty acids and some bioactive 
compounds are discussed. Applications of NEs in edible coatings are outlined as well. 
 
2.2 Nanoemulsions and methods of their preparations 
 



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Nanoemulsions (NEs) are biphasic dispersions of two immiscible liquids: either water in oil 
(W/O) or oil in water (O/W) droplets stabilized by an amphiphilic surfactant, whereby very small 
emulsion droplets (generally oil droplets in water) with sizes in the order of 100 nm (usually 
>500 nm) and occurring from a thermodynamic point of view in a non-equilibrium state, are 
characterized by slow kinetics of destabilization and therefore they could be considered as 
kinetically stable. Small size of NEs results in convenient features such as high surface area per 
unit volume, robust stability, optically transparent appearance, and tunable rheology (Anton and 
Vandamme 2011; Gupta et al. 2016; Singh et al. 2017b; Sabry and Ragaei 2018). Very small size 
of NEs pronouncedly contributes to the prevention of droplet flocculation and coalescence and 
thus, the destabilizing process is governed alone by the Ostwald ripening (Anton and Vandamme 
2011). As the destabilization of NE occurs due to the change in the droplet size by Ostwald 
ripening, surface functionalization of NEs could be used to stabilize the O/W interface during 
emulsification (Quadir et al. 2016). NEs prepared using biocompatible and biodegradable 
constituents could be used in medicine and food industry for drug/active ingredient encapsulation 
and sustained and controlled release of encapsulated compounds also belong to their advantages. 
On the other hand, according to the IUPAC definition, the microemulsion (ME) is “dispersion 
made of water, oil, and surfactant(s) that is an isotropic and thermodynamically stable system 
with dispersed domain diameter varying approximately from 1 to 100 nm, usually from 10 to 50 
nm, whereby the term “oil” refers to any water-insoluble liquid (Slomkowski et al. 2011). In 
general, NEs could be formulated using so-called “high-energy” methods that utilize specific 
devices (e.g., ultrasound generators or high pressure homogenizers) supplying enough energy to 
increase the W/O interfacial area for generating nanoscale droplets or by „low-energy” methods, 
in which spontaneous emulsification without requiring any device or energy generate nanoscale 
droplets (e.g., Jaiswal et al. 2015; Håkansson and Rayner 2018; Hadžiabdic et al. 2017). 
The high pressure homogenization, microfluidization, sonication or ultrasonic homogenization, 
jet disperser high-amplitude ultrasonic method or membrane emulsification belong to the high-
energy methods used for preparation of NEs. The high-energy methods are designed to supply 
the energy required for emulsification by subjecting it to a disruptive hydrodynamic stress, that 
is, laminar or turbulent shear or cavitations (e.g., Håkansson and Rayner 2018; Hadžiabdic et al. 
2017). For example, a dual-channel microfluidization is a suitable method to fabricate fine NEs 
with high oil loading levels, which may be advantageous for many commercial applications, 
while the single-channel method was found to be effective only at producing NEs at relatively 
low oil concentrations (10%) (Bai and Clements 2016). In β-carotene NEs prepared by 
microfluidization technique droplet size decreased from 416.0 to 97.2 nm with increasing 
microfluidization pressure, number of cycles, and emulsifier concentration and as the optimum 
conditions for fabricating such NEs homogenization pressure of 120 MPa and 3 cycles were 
estimated (Jo and Kwon 2014). Abbas et al. (2013) outlined the principles and production 
technology of high-intensity ultrasound, analyzed the role of acoustic cavitation in the 
preparation of food-grade O/W NEs discussed technical hurdles, issues and future prospects of 
this technology. 
Low-energy transitional emulsification methods to prepare NEs based on phase transitions of 
nonionic surfactants (PEGylated surfactants) related to a sudden change in their relative 
solubility in the oily phase and aqueous phases was overviewed by Anton et al. (2018). 
Spontaneous emulsification is a low-energy method that simply involves addition of an organic 
phase (oil + surfactant) into an aqueous phase and the produced droplet size is affected by 
surfactant-to-oil ratio, surfactant type, surfactant location, and oil type (Komaiko and 
McClements 2016). The catastrophic phase inversion method for fabrication of NE is based on 
gradually diluting, under mild flow conditions, one liquid (such as water) with another 
immiscible liquid (such as oil) until phase inversion occurs and a NE is formed (e.g., O/W) that 
could be used to encapsulate many active compounds (Perazzo and Preziosi 2018). 
 
2.3 Nanoemulsions applicable in agriculture 
 



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The primary object of preparing chemical pesticide colloids is significantly increase the apparent 
solubility of these molecules in water and consequently the delivery of the active compound 
homogeneously to the pests and/or plants. However, recent research has shown that the 
production of these colloids can also improve other properties of pesticides, such as 
bioavailability, increased physico-chemical stability and promote sustained release 
(Bhattacharyya et al. 2016). 
Pesticides formulated with NEs use less organic solvents when compared with conventional 
formulations (emulsifiable concentrate) (Chin et al. 2012) and also, have lower surfactant 
concentration (between 3−10%) than microemulsions (20% or higher) (McClements 2012). In 
comparison to the emulsions the advantages of the NEs due to the small droplet size are the 
greater spreadability, wettability and superior mechanical stability (McClements 2012). 
Consequently, NEs incorporating pesticides settle evenly on the leaves of plants. Some studies 
have shown that NE improves the solubility of poorly water-soluble pesticides, resulting in the 
increasing of bioactivity of the pesticides in comparison with conventional pesticides (Leng et al. 
2014; Wang et al. 2017). 
Using a Quillaja saponin as a natural surfactant Kumari et al. (2018) prepared and characterized 
thymol NE. The authors tested the antibacterial activity of the NE against Xanthomonas 
axonopodis pv.glycine, which is the organism responsible for bacterial pustule disease in 
soybean. The most stable NE was produced with fifty minutes of sonication and resulted in a NE 
with mean diameter 274±2 nm, polydispersity index (PDI) 0.1 and zeta potential -31 mV. 
Thymol NE showed strong antibacterial activity, and no bacterial colony was observed in the 
concentration range of 0.02−0.06 (v/v) of NE as compared to control and thymol and saponin. 
The incidence of the disease was evaluated in vivo and the results showed that the control plants 
and plants treated with thymol and saponin alone presented a higher disease incidence (64−78%), 
while lower disease incidence (3.3−29%) was observed in plants treated with thymol NE. 
Treatment with NE also resulted in enhancements of plant growth. 
In the study of Lim et al. (2013) the authors prepared and characterized a water-soluble herbicide 
formulation based on NEs of glyphosate isopropylamine. The herbicidal activity was evaluated 
against three weeds: Asystasia gangetica, Diodia ocimifolia and Paspalum conjugatum. The 
authors found that the glyphosate NEs showed a lower spray deposition in all species when 
compared to a commercial formulation (Roundup®). However, after 14 days of the treatment the 
injury rates of the weeds treated with NE were similar to those observed with plants that were 
treated with commercial formulation. The similar herbicidal activities of NE and the commercial 
formulation, even with the lower NE deposition, could be attributed to potentiation of the 
biological activity when glyphosate was formulated as NE. 
In another paper, Du et al. (2016) described the preparation and characterization of O/W NEs 
using laurate as oily phase and a mixture of two surfactants, alkyl polyglycoside (AL) and 
polyoxyethylene 3-lauryl ether (PLE). In order to evaluate the potential application of the NEs a 
model insoluble molecule, β-cypermethrin, was incorporated into the NEs. Two NEs were 
analyzed, the first one containing laurate/AL:PLE(6:4)/water in a ratio 10:5:85 and in the second 
one  this ratio was 20:6:74. NE prepared with the lowest laurate concentration showed lower 
mean diameter (∼110 nm) in comparison with NE prepared with higher concentration of laurate 
(∼200 nm). However, in both cases there were no significant changes in droplet size between the 
NEs prepared with and without β-cypermethrin. Even after dilution the β-cypermethrin the NE 
remained as single-phase and homogeneous. After dilution an increase in droplet size was 
observed. As consequence, diluted NE showed excellent wetting and spreading properties on the 
hydrophobic surface. 
Badawy et al. (2017) prepared O/W NE with an insecticide (diazinon) using Tween or lecithin as 
surfactants. In the prepared optimized NE the dependent variables were droplet size, PDI, 
dynamic viscosity and pH. NE prepared using synthetic surfactant (Tween) showed a size droplet 
varying from 30 to 138 nm and PDI values from 0.084 to 0.256. The NEs prepared using natural 
surfactant (lecithin) showed a broader range of droplet size varying from 56.2 to 920.2 nm and 
also broader range of PDI values (0.122−0.946). Diazinon NE prepared using Tween showed low 



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viscosity ranging from 2.33 to 21.33 cP, whereas viscosity of lecithin NE incorporating diazinon 
were higher (55−1039.33 cP). Both NEs prepared with natural or synthetic surfactant showed an 
acid pH value. In another study aiming to NEs applications in agriculture, Zhao et al. (2017) 
developed and characterized a positive charged O/W NE as a carrier system for lambda-
cyhalothrin and evaluated the influence of addition of externally ionic liquids on Z-average, size 
distribution, zeta potential, viscosity and stability of the NEs. The measurement of contact angles 
of NEs on wheat leaves (Triticum aestivum) was evaluated. The zeta potential of the NE was 
affected by the addition of ionic liquids with different alkyl chain length. Increases in alkyl chain 
length resulted in a change of droplet charge from negative to positive. The droplet size showed 
no increases in diameter after 90 days of storage and NE exhibited a non-Newtonian fluid 
behavior. NE showed a tough adsorption on negative surface decreasing the contact angle on 
wheat leaves. The addition of ionic liquids provided an increase in the stability due to 
electrostatic repulsion and the NE could be considered as a potential carrier system for 
agrochemicals. 
Choupanian et al. (2017) studied the preparation and characterization of neem oil NE and 
evaluated the increase in the NE stability by addition of two non-ionic surfactants (naturally 
based polysorbate and alkylpolyglucoside). The biological activity of neem oil NE was evaluated 
against two important pests of stored products: Sitophilus oryzae (L.) and Tribolium 
castaneum (Herbst). All NEs showed a droplet size ranging from 200 to 600 nm and the 
formulation, that showed higher ratio of non-ionic surfactants, had smaller droplet size in 
comparison to the formulations showing the same composition of surfactants, however with 
lower concentration. The addition of non-ionic surfactants resulted in increases in viscosity of the 
NE. Both pests treated with 1% of NE showed 100% of mortality after two days of treatment, 
except for the NE with higher droplet size (507 nm) causing 85 and 74% mortality of S. 
oryzae and T. castaneum, respectively. However, all NEs showed more toxic effects against both 
pests compared with free neem oil and Neemix®. 
Díaz-Blancas et al. (2016) prepared O/W NEs entrapping tebuconazole using non-ionic 
surfactant (Tween 80) or AG54, which is a surfactant composed by a mixture of non-ionic and 
anionic amphiphilic components. According to a pseudo-ternary diagram, the phase equilibrium 
area was achieved in the range of 0.49–0.90, 0.01–0.23, and 0.07–0.49 of organic phase, aqueous 
phase, and surfactant, respectively. The viscosity of both NEs depended on aqueous phase 
percentage and showed the same behavior. When the concentration of the aqueous phase 
increased from 4 to 30 wt%, a 4-fold decrease in the viscosity was observed, whereas increase in 
aqueous phase concentration from 30 to 50 wt% resulted in viscosity increase, however, NE 
prepared with Tween 80 showed higher viscosity than NE fabricated with AG54. The droplet 
size of the NE containing Tween 80 was 9±1 nm and the size remained constant with the increase 
of concentration in aqueous phase from 1 to 50 wt%. In contrast, NE produced with AG54 
showed a strong dependence on the aqueous phase concentration up to 20 wt%. However, further 
increases in the percentage of the aqueous phase did not influence the size droplet that remained 
around 250 nm. 
Hazrati et al. (2017) studied O/W NE containing Satureja hortensis essential oil (EO) and 
evaluated the herbicidal activity of the NE against worldwide weeds Amaranthus retroflexus and 
Chenopodium album. The NE showed a droplet size of 92.7±2.6 nm and PDI 0.29±0.01 and after 
30 days of storage an increase of mean diameter and a decrease in PDI was observed. Under 
laboratory conditions the germination percentage showed a dose-dependent response and the best 
germination inhibition was observed for the concentration 800 µL/L NE. In this assay, the root 
elongation was more affected in comparison with shoot elongation. In greenhouse assays a 
decrease in growth of the plants in a dose-dependent manner was observed as well. In addition, a 
decrease in chlorophyll content and increase in relative in electrolyte leakage 5 days after the 
treatment was estimated, which could be attributed to cell membrane disruption and increased 
membrane permeability. This NE presented herbicidal activity against weeds due to the presence 
of carvacrol that is a phenolic monoterpene showing strong phytotoxic effects. 



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Feng et al. (2016) evaluated the effect of adding the aqueous phase to the organic phase and vice 
versa, as well as the initial location of the surfactant (organic phase or aqueous phase) on the 
stability of NE using β-cypermethrin as pesticide model. According to the authors the NE 
stability depended strongly on the emulsification process. NEs, in which the emulsifier agent was 
diluted in organic phase showed higher stability than NEs, in which it was diluted in aqueous 
phase. In addition, the most stable NE was prepared by adding the organic phase containing the 
emulsifier in the aqueous phase. This NE showed a droplet size of 166 nm and PDI of 0.16. 
Mossa et al. (2017) fabricated NEs of camphor EO and evaluated their insecticide activity against 
Sitophilus granaries. NEs were produced with fixed concentration of camphor oil (5%), different 
ratios of Tween 20 (w/w) and different sonication time. Optimized NEs were achieved at a ratio 
1:1.5 of Tween 20 : camphor EO and 40 minutes of sonication, showing a droplet size of 
99.0±0.605 nm and remained unchanged during three months of storage. Both camphor EO and 
NE showed insecticidal activity against S. granaries in a dose-dependent manner. After 72 h of 
exposure to the highest concentrations of the NE tested (250 and 300 µg/g) 100% of insect 
mortality and reduction in the progeny (98 and 100%) was observed and higher concentration of 
the free EO was necessary to reach the same effect. The estimated LC50 for EO and NE were 
282.01 µg/g and 181.49 µg/g, respectively. The insect mortality caused by EO was increased by 
36.5% when this oil was incorporated into the NE. 
In addition, Fernandes et al. (2014) developed a NE with an extract consisting of the apolar 
fraction from fruit extract of Manilkara subsericea and evaluated its insecticidal activity against 
Dysdercus peruvianus. The best oil phase was found to be octyldodecyl myristate that was able 
to solubilize equal amount of apolar extract (1:1 w/w). Optimized emulsion was composed of the 
apolar extract (5%), surfactants (5%), octyldodecyl myristate (5%) and water phase (85%). The 
NE without extract showed a droplet size of 57±0.3 nm and zeta potential of -59.6 ±4.1 mV, 
while the NE containing the extract showed an increase in mean diameter (155.2±3.8 nm) and a 
decreased zeta potential value (-47.4±3.2 mV). The mortality of the insects treated with NE 
containing the extract, that started in the first day reached 12.23±0.58%, after 30 days of 
exposure was enhanced to 44.43±6.66%. It could be mentioned that NE containing extract did 
not induce effect against acetylcholinesterase or mortality in mice. 
In order to improve the permeability and efficiency of delivery of antibacterial (ampicillin) into 
the citrus phloem by foliar spray Yang et al. (2015) designed a NE using eight adjuvants. Among 
the adjuvants tested, Brij 35 showed the highest increases (3.33-fold) in the cuticular 
permeability when compared with control (water). Two O/W NEs were prepared with different 
physicochemical properties; however, both formulations showed good thermodynamic stability. 
One NE showed droplet size of 5.26±0.04 nm and pH value 7.76±0.03 (NE-1) and another one 
had higher droplet size (94±1.48 nm) and higher pH value (8.31±0.05) (NE-2). After addition of 
the adjuvant (Brij 35) to both NEs a laboratory assay with Bacillus subtilis was performed in 
order to evaluate the antibacterial activity. The NEs showed higher inhibitory zone diameters of 
5.75 and 6.6 mm for NE-1 and NE-2, respectively in comparison with Brij 35 alone (4.34 mm) 
and free ampicillin (2.83 mm). In addition, in vivo assays also showed that the NEs were more 
efficient at suppressing or eliminating Las bacterium when compared with free ampicillin and 
Brij 35 alone. 
Ali et al. (2017) developed NEs containing neem and cintronella oil using a method of low 
energy spontaneous emulsification. Antifungal activity of NEs was evaluated against two 
phytopathogenic fungi Rhizoctonia solani and Scletotium rolfsii. Optimized primary emulsion for 
both oils was achieved using the ratio of oil, surfactant and water of 0.50:1:8.50. This primary 
emulsion was employed to make neem NE with diverse amounts of citronella oil (0.5−5%) or 
citronella oil NE with diverse amounts of neem oil (0.5-5%). Neem NEs with different 
percentage of citronella oil showed a droplet size varying between 11.23±3.86 nm to 
17.80±4.52 nm, whereas citronella oil with diverse amounts of neem oil showed a droplet size 
varying between 8.12±2.80 nm to 12.04±3.74 nm. In vitro antifungal activity of the different NEs 
against R. solani and S. rolfsii was screened by poisoned food method. Neem NE with the highest 
percentage of citronella oil and citronella NE with the highest percentage of neem oil showed the 



7 

best antifungal activity against the both fungi tested. The estimated LC50 related to R. solani were 
13.67 and 25.64 mg/L and those related to S. rolfsii were 14.71 and 20.88 mg/L respectively for 
neem NE and citronella NE, respectively. 
Liu et al. (2011) studied O/W NEs composed of different mixture of non-ionic surfactant 
polyoxyethyene 3-lauryl ether (C12E3) and anionic surfactant dipotassium 
monododecylphosphate (MAPK) containing the insecticide bifenthrin. According to the authors 
the most stable emulsion was achieved with a mixed ratio of 6:4 (MAPK:C12E3) and 10 wt% 
surfactant mixture. The NE showed a droplet size of 200.7 nm that increased after 180 days of 
storage to 218.6 nm but phase separation was not observed. Pant et al. (2014) prepared NEs of 
eucalyptus oil and evaluated the potentiation of their insecticidal activity by addition of the 
aqueous filtrate of Pongamia glabra and Jatropha curcas. Insecticidal activity was estimated 
against Tribolium castaneum that is an important pest of stored grains. Four formulations, all 
containing the same composition and the same concentration of eucalyptus oil (10% w/w) were 
prepared, whereby the only component that varied was the aqueous filtrate concentration of 
karanja and jatropha (0, 20, 49 and 60 %). The droplet size of the NEs decreased with increasing 
concentration of the aqueous filtrate in the formulation. NE produced only with water as 
continuous phase showed the highest PDI (0.8), while the NE with 20% of aqueous filtrate 
showed PDI of 0.113, which increased to 0.278 at the highest concentration of aqueous filtrate 
utilized. The percentage of insecticidal activity of T. castaneum did not show significant 
difference among all formulations tested; however, it was observed a significant decrease in the 
value of LC50 from 5.49 mg/L estimated for the NE without plant aqueous filtrate to 0.1646 mg/L 
for the NE with 60% of aqueous filtrate. In addition, the volatilization of the eucalyptus oil was 
stabilized in the NE with aqueous filtrate. 
Sharma et al. (2018) evaluated NEs prepared using non-ionic surfactants and containing a 
mixture of two EOs, clove oil and lemongrass oil, for antifungal activity against Fusarium 
oxysporum f.sp. lycopersici (FOL). Optimized formulation contained 5% of a blend of clove oil 
and lemongrass oil (1:1), 10% of surfactant mixture (Tween 20: CoE-40, 7:3), 5% propylene 
glycol and 80% water. This NE showed droplet size 76.73±4.8 nm, PDI 0.207±0.02 and viscosity 
26.9±1.9 cP. Such NE was more efficient (48.5% more fungistatic activity) in inhibiting the 
mycelial growth (MIC 4000 mg/L) in comparison with free oil mixture (MIC 7000 mg/L). In 
addition, NE showed more pronounced fungicidal activity (5000 mg/L) than free oil mixture 
(9000 mg/L) against FOL, whereby the nanoemulsion disrupted the membrane integrity of FOL. 
Hashem et al. (2018) developed a NE containing Pimpinella anisum L. EO and evaluated its 
insecticidal activities against Tribollium castaneum adults and progeny. The NE showed a 
droplet size of 198.9 nm, PDI 0.303, zeta potential of -25.4 mV and low viscosity 0.8872 cP. 
Adult mortality increased proportionally with increasing NE concentrations as well as time of 
exposure, while the number of progeny and grain weight showed gradual decrease. After 12 
hours there was 81.33 ±0.08% of adult mortality and reduction of 70.85% if the progeny when 
exposed to 10% NE and the LC50 value after 72 hours of exposure was 9.84%. The NE adhered 
to different parts of the insect body, such as, head, thorax, abdomen, elytra, mouth and legs. In 
addition, the NE caused a distinct kind of alteration in the midgut cells of the insects. Abd-
Elsalam and Khokhlov (2015) prepared eugenol NE using Tween 20, a non-ionic surfactant, and 
water as continuous water phase and evaluated its antifungal activities in vitro and in vivo against 
Fusarium oxysporum f. sp. Vasinfectum (FOV) isolates. Eugenol NE showed a droplet size of 
80 nm, spherical shape and no increases in droplet size after 30 days of storage at room 
temperature (25 °C). In vitro antifungal activity (zone inhibition) showed that among the four 
FOV tested the NE was more efficient against DQO86833 (5 cm) and AY264267 (4.5 cm) at 
application of 2% NE, whereas treatment with 5%, NE inhibited the mycelial growth of all FOV 
isolates. Fungal morphology was also affected by the NE, and reduction in the size and number 
of conidiospores as well as reduction in hyphae pigmentation were observed. 
As a summary, in literature there are many examples of systems with potential to be used in 
agriculture, however, a lot of work to become all initiatives in products to be used in crop 
protection. 



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2.4 Nanoemulsions applicable in medicine 
 
NEs as stabilized heterogeneous systems of two immiscible liquids have a great potential in 
various biomedical applications; especially they are very attractive for drug delivery. 
Encapsulation in particular lipophilic drugs leads to the formation of therapeutic 
nanoformulations providing modification of bioavailability, alternative administration routes, 
release of drugs and, thus, a reduction of the required drug amount (e.g., Jampílek et al. 2015b; 
Jampílek and Kráľová 2017b, 2018a, 2019b,c; Pentak et al. 2016; Patra et al. 2018; Prasad et al. 
2017b; Prasad M et al. 2018; Tayeb and Sainsbury 2018). 
  
2.4.1 Transdermal nanoemulsion formulations 
 
Drugs with low oral bioavailability due to the first pass metabolism are good candidates for 
transdermal delivery. Transdermal permeation of majority drugs is hindered by the upper layer of 
the epidermis (stratum corneum), therefore for topical delivery of drugs chemical permeation 
enhancers that are able to make the stratum corneum more permeable for drugs and reduce the 
primary skin barrier by different mechanisms are used. In addition, physical methods (e.g., 
sonophoresis, iontophoresis, electroporation, microneedles, etc.) can be used to increase 
permeability of drugs (Jampílek and Brychtová, 2012). For enhancing epidermal and dermal drug 
deposition also nanoscale drug delivery systems, including NEs, are widely applied (Iqbal, et al. 
2018). Although the skin represents a natural physical barrier against particle penetration, the 
therapeutic NPs could be delivered especially in diseased skin and to the openings of hair 
follicles (Prow et al. 2011) and good candidates for transdermal delivery are drugs with low oral 
bioavailability due to the first pass metabolism. It is advantageous that NEs are vehicles acting 
also as transdermal permeation enhancers without utilizing additional permeation enhancers 
(Shakeel et al. 2010). 
The progressive advancement in the delivery of drugs via NE with special reference to the 
dermal and transdermal administration and the most suitable semisolid dosage forms for the 
particular type of NEs (O/W, W/O and others), including effects of particle size and zeta 
potential on the delivery of drugs through dermal or transdermal route was overviewed by Rai et 
al. (2018). Low viscosity of NEs that might be unsuitable for topical application could be 
overcome by hydrogel-thickened NEs using thickening polymer, e.g. CS (Barradas et al. 2017). 
Challenges and future prospects of NEs as a drug delivery system were presented by Yukuyama 
et al. (2017). Applications of NEs in the field of dermatology highlighting the advantages over 
the other dermatological therapies connected with increased contact surface area by the particle 
size, which leads to increased drug efficacy were overviewed by de Souza et al. (2018). For 
example, Salim et al. (2016) discussed the potential of drug-loaded NEs for the treatment of 
psoriasis to achieve better efficacy and eliminate side effects and noted that the delivery and 
penetration of a drug through the psoriasis skin layer could be enhanced by a small droplet size. 
Nastiti et al. (2018) in their review paper focused their attention on the composition and 
characterization of MEs and NEs for topical and transdermal delivery and the mechanism of skin 
delivery across stratum corneum and via hair follicles. 
Nanoemulgels that are basically O/W NEs gelled with the use of some gelling agent in it, in 
which the gel phase in the formulation is nongreasy and stabilizes the formulation through 
reduction in surface as well as interfacial tension could be considered as a novel transdermal 
delivery system that is able to overcome poor oral bioavailability of drugs, more specifically 
target to the site of action, can avoid first-pass metabolism and relieve the user from 
gastric/systemic incompatibilities (Choudhury et al. 2017). In nanoemulgels the NE containing 
drug is incorporated into a gel base. Lipophilic drugs could be easily incorporated and the skin 
permeability of the incorporated drugs can be enhanced in several folds due to the finely 
distributed droplets of NE phase resulting in notably improved pharmacokinetic and 
pharmacodynamic profiles of the lipophilic drugs. Consequently, nanoemulgel formulations 



9 

could be considered as potential and promising candidates for topical delivery of lipophilic drugs 
in the future (Sengupta and Chatterjee 2017). A review paper of Pawar and Babu (2014) is 
devoted to various lipid materials (vegetable oils, fatty acids, fatty alcohols, medium chain 
glycerides, and fatty acid esters) used in the preparation of NEs for topical and transdermal drug 
delivery. 
Integral NEs with particle size of 80 nm can diffuse into but not penetrate the viable epidermis, 
however, they can efficiently fill the whole hair follicle canals and reach as deep as 588 µm 
underneath the dermal surfaces and the “cargos” released from the NEs diffuse into the 
surrounding dermal tissues. On the other hand, NEs with mean particle size of 500 nm, cannot 
penetrate the stratum corneum and can only migrate along the hair follicle canals, while NEs 
with median size, e.g. 200 nm show moderate transdermal permeation effect (Su et al. 2017). 
Capsaicin O/W NEs with droplet sizes 20−62 nm were reported to permeate all skin layers from 
the stratum corneum to the dermis (Kim et al. 2014a). 
Permeation flux of antifungal drug fluconazole from optimized drug-loaded olive oil NEs 
through artificial skin was approximately three fold higher than the control (Ansaril et al. 2017). 
A testosterone transdermal delivery system developed using a palm oil base (HAMIN) with 
particle sizes 97−774.0 nm was tested using in vivo skin permeability test and it was found that 
testosterone was well absorbed with a mean Cmax and Tmax of 60.94 ng/mL and 2.29 h after in 
vivo application on rabbit skin indicating that such nanoformulation could have great potential 
for topical delivery of testosterone (Haron et al. 2015). The optimized clove oil based olmesartan 
NE (droplet size of 53.11±3.13 nm, PDI 0.335±0.008) showing a 1.23-fold increase in the 
bioavailability compared with oral formulation of drug due to better permeation through rat skin 
could be used as an antihypertensive dosage form for effective transdermal delivery of 
olmesartan (Aqil et al. 2016). O/W NEs containing the skin penetration enhancer oleic acid or 
eucalyptol as oil pronouncedly enhanced the skin penetration of encapsulated caffeine and 
naproxen compared to their aqueous control solutions. Caffeine maximum flux enhancement was 
connected with a synergistic increase in both caffeine stratum corneum solubility and skin 
diffusivity, while increased solubility in the stratum corneum was the dominant determinant for 
higher naproxen fluxes (Abd et al. 2016). Transdermal NE formed of 2% Foeniculum vulgare 
Mill. EO, 5.6% oleic acid, 68% Smix (1:1) and distilled water showed a high potential of reducing 
plasma glucose levels in rats that continued for 7-days after a single topical application of a dose 
of 120 mg/kg of fennel EO, bringing glucose to normal levels in diabetic rats (Mostafa, et al. 
2015a). O/W NEs containing minoxidil, an antihypertensive vasodilator, and the skin penetration 
enhancer oleic acid or eucalyptol as oil phases pronouncedly enhanced drug permeation through 
skin compared with control solutions. Minoxidil retention in the stratum corneum and deeper 
skin layers was promoted to higher extent with eucalyptol NEs, while oleic acid formulations 
gave the greatest hair follicle penetration. The increases in both minoxidil stratum corneum 
solubility and skin diffusivity in both nanoemulsion systems were reflected in drug maximum 
flux enhancement connected with enhanced fluidity and disruption of stratum corneum lipids 
(Abd et al. 2018). NEs containing isoflavone-rich soybean extracts that are considered as 
promising skin antiaging products due to their antioxidant activity could be applied as suitable 
topical formulations to protect skin from UVA/UVB oxidative damage (Back et al. 2018). 
Antioxidant hydrogels containing an Achyrocline satureioides extract-loaded NEs aimed at 
topical application were found to be suitable to protect the porcine ear skin against oxidative 
stress generated by UVA/UVB light (Balestrin et al. 2016). Brownlow et al. (2015) developed 
vitamin E-enriched NE vehicles loaded with genistein for chemoprevention against UVB-
induced skin damage showing enhanced dermal delivery of the drug. Also Nam et al. (2018) 
developed W/O NE (diameter of <5 nm) that contained nitric oxide (NO) by mixing surfactant 
with vitamin E antioxidant body oil (Product No. 04800, Cococare, Dover, NJ) and NaNO2 
solutions. After spreading of this NEs with NO on penis skin of the middle aged dogs, blood 
NOx concentration in the penis increased, resulting in penile erection without any notable topical 
and systemic side effects, suggesting that such W/O NEs could be used in non-invasive 



10 

medication for patients suffering in erectile dysfunction with low response to phophodiesterase 
type 5 (PDE5) inhibitors such as Viagra or Cialis.  
Formulations of hydrogels containing negatively or positively charged Copaiba oil NEs exhibited 
anti-inflammatory effects, which was reflected in mouse ear edema (69 and 67%) and rat paw 
edema inhibition (32 and 72%) and decrease of inflammatory factors, such as dermis and 
epidermis hyperplasia and inflammatory cells infiltration in histological cuts were estimated as 
well (Lucca et al. 2018). Skin permeation with the positively charged Copaiba oil NEs increased 
threefold the retention of the major component in copaiba oil, β-caryophyllene, in the epidermis, 
and also in the receptor fluid compared to the negatively charged NEs (Lucca et al. 2017). Coffee 
oil−algae oil-based NEs with a particle size of 30 nm, zeta potential -72.72 mV, and 100% 
encapsulation efficiency (EE) of docosahexaenoic acid (an important component of algae oil) 
applied at dose of 0.1% efficiently mitigated trans-epidermal water loss, skin erythema, melanin 
formation, and subcutaneous blood flow in animal experiments and were found to inhibit the 
growth of melanoma cells B16-F10 (IC50: 26.5 µg/mL) and arrest the cell cycle G2/M phase, 
whereby the apoptosis pathway of melanoma cells may involve both mitochondria and death 
receptor (Yang et al. 2017). 
Transdermal administration of nanogel based on optimized catechin NEs showed sustained 
release profile of catechin and enhanced photoprotection potential due to its improved 
permeability as well as bioavailability compared to the conventional gel and it could represent an 
effective strategy for decreasing UV-induced oxidative damage in the skin tissues (Harwansh et 
al. 2016). Natural pentacyclic triterpenes-loaded NEs showed greater ability to inhibit 
inflammation than NEs loaded with the synthetic mixture of triterpenes (Alvarado et al. 2015). 
NEs of CS oleate encapsulating α-tocopherol with particle size of 220 nm were reported to be 
suitable for topical application in wound healing (Bonferoni et al. 2018). Astaxanthin (ASX)-
loaded carboxymethyl CS functionalized NE formulation with spherical droplets showing mean 
diameter >100 nm and a small negative surface charge exhibited higher ASX chemical stability 
and skin permeability than ASX NEs and ASX solution and were characterized with low 
cytotoxicity (Hong et al. 2017).  
Genistein-loaded cationic NEs (mean droplet size of ca. 200−300 nm) prepared by spontaneous 
emulsification and using hydroxyethyl cellulose as a thickening agent (at 3%) showed 
considerable increase of drug retention in mucosa compared to the genistein propylene glycol 
solution, and exhibited antiherpetic activity in vitro against herpes simplex virus 1 (HSV-1, strain 
29R) (Argenta et al. 2016). Factorial design applied to the optimization of lipid composition of 
topical antiherpetic NEs containing isoflavone genistein was presented by Argenta et al. (2014). 
The 10,11-methylenedioxycamptothecin loaded hyaluronic acid (HA) NEs were reported to 
perform desirable skin permeable capacity across human keloid skin, whereby the drug was 
transferred directly to keloid lesion area. The growth-inhibitory effect was further clarified upon 
cell cycle regulation, which arrested cells at G1/S and prevented them entry into mitosis (Gao et 
al. 2014). 
NEs with co-encapsulated C6 ceramide (0.35%) and paclitaxel (0.50%) containing tributyrin 
delivered 2- and 2.4-fold more paclitaxel into viable skin layers of porcine skin in vitro at 4 and 8 
h post-application than the MEs, and 1.9-fold more C6 ceramide at 8 h, whereby the drugs were 
co-localized mainly in the epidermis. The EC50 values related to melanoma cells viability 
estimated for individually encapsulated paclitaxel and ceramide in NEs were 4- and 13-fold 
lower (ceramide) than for unencapsulated formulation and at co-encapsulation of both active 
ingredients synergic effect was observed, because further decrease of EC50 by 2.5−4.5-fold was 
estimated, and calculated combination index also indicated a synergistic effect. Topical 
application of NEs on 3D bioengineered melanoma models for 48 h stimulated marked epidermis 
destruction and only few cells remained in this layer (Carvalho et al. 2017). 
The olein-based β-D-glucan-loaded NE prepared using ultrasound exhibited higher antioxidant 
activity as compared to free antioxidant β-D-glucan (Alzorqi et al. 2016). In sulpiride ME 
formulations containing glycerylmonooleate, Labrafil and Avocado as oily phases drug solubility 
increased to 43.35 mg/mL with drug content >97% and based on pharmacodynamic performance 



11 

and antipsychotic activity of sulpiride this formulations could be used as antipsychotic nasal drug 
delivery to overcome sulpiride low oral bioavailability (Ayoub et al. 2016). Optimized eugenol-
NE prepared using Tween 80 and Labrasol as surfactant and cosurfactant, respectively showing 
hydrodynamic diameter 89.98±6.48nm, PDI of 0.238±0.021 and zeta potential of -10.05±0.11 
mV were found to enhance the transdermal delivery of eugenol without causing skin irritation 
(erythema and edema) in vivo suggesting their potential to be used in wounds healing and anti-
inflammatory treatments (Ahmad et al. 2018a). The NE containing cumin EO/oleic acid and 
Tween 20/ethanol (2:1) showed high phenolic encapsulation efficiency and remarkable 
cumulative phenols permeation through rat skin as well as high in vitro and in vivo antioxidant 
efficiency and provided high hepatoprotective potential and reserved rats' body weight after a 
period of seven days of a single transdermal application (Mostafa et al. 2015b). 
Staphylococcus aureus treated with optimized NE containing eucalyptus oil as organic phase, 
water as continuous phase, and non ionic surfactant, Tween 80, as emulsifier prepared by 30 min 
sonication and showing the mean droplet diameter of 3.8 nm resulted in complete loss of 
viability within 15 min of interaction and the membrane of treated bacterial cells was damaged. 
This NE was not irritant and exhibited higher wound contraction rate in Wistar rats with respect 
to control and neomycin treated rats (Sugumar et al. 2014). 
The optimized W/O NE of thiocolchicoside (TCC), an effective therapeutic agent against the 
orthopedic, traumatic and rheumatologic disorders, containing in ratio 1:1 linseed and sefsol 
(propylene glycol caprylate) as the oil phase, showed mean globule diameter of 117 nm, PDI of 
0.285 and the steady-state flux (Jss) and permeability coefficient (Kp) of 30.63±4.18 µg/cm2/h and 
15.21×10-3±2.81 cm2/h, respectively in in vitro permeation experiment using porcine skin 
suggesting that W/O NEs, which are compatible with the lipophilic sebum environment of the 
hair follicle, facilitate the transport of TCC, which might be predominantly transfollicular in 
nature (Kumar et al. 2016).  
Curcumin (CUR) NE for transdermal application with mean droplet diameter, PDI and zeta 
potential of optimized NE 85.0±1.5 nm, 0.18±0.0 and -5.9±0.3 mV, respectively notably 
improved the permeation flux of CUR from the hydrophilic matrix gel Viscolam AT 100P 
(sodium polyacryloyldimethyl taurate, hydrogenated polydecene, polyoxyethylene), whereby NE 
formulation not only improved CUR permeability but also protected the drug from chemical 
degradation (Rachmawati et al. 2015). CUR-loaded NE (droplet size of 41.13±3.34 nm and zeta 
potential of -33.1±1.45 mV) that was incorporated into gel using carbopol-980 (1% w/v) and 
tested on Freund's complete adjuvant induced arthritic rat model after topical application of 
CUR-NE gel in Wistar rats showed substantial reversal of arthritic symptoms suggesting that the 
nanoformulation could exhibit therapeutic effects locally in inflammatory arthritic disorders with 
improved topical bioavailability (Naz and Ahmad 2015). Study of CUR distribution in neonate 
pig skin using CUR-loaded myristic acid microemulsions showed dermal CUR accumulation 
(326 µg/g skin) and transdermal CUR penetration (87 µg/cm2/d). CUR encapsulated in ME 
inhibited bacterial growth of Staphylococcus epidermidis (EC50 of 0.86 µg/mL) and was found to 
be 12-fold more effective than CUR dissolved in dimethyl sulfoxide suggesting that such MEs 
could be used as alternative treatment for S. epidermidis-associated diseases and acne vulgaris 
(Liu et al. 2012). 
NEs composed of propylenglycol, Transcutol®, water, Labrasol®, Plurol Oleique®, isostearyl 
isostearate, oleic acid, and D-limonene with incorporated imipramine or doxepin in the NE 
system (3% w/w) were tested for an analgesic and anti-allodynic activity at transdermal delivery 
of drugs and it was found that in vivo analgesic and anti-allodynic activity in rats was stronger for 
the doxepin loaded NE suggesting that such nanoformulation could be an alternative analgesic 
therapy with a potential clinical application (Sandig et al. 2013). 
NEs consisting of mono ammonium glycyrrhizinate, Span 80, Brij 35, isopropyl alcohol, 
soyabean oil and distilled water were reported as appropriate vehicles for transdermal delivery of 
glycyrrhizin through human cadaver skin and while excipients of NEs acted as permeation 
enhancers themselves, the use of additional permeation enhancers was not necessary (Harwansh 
et al. 2011). 



12 

 
2.4.2 Nanoemulsions for cancer therapy 
 
Surface modification of nanocarriers could contribute to their enhanced functions in imaging, 
targeting and delivery, increase drug bioavailability, reduce undesirable side effects, and 
minimize non-specific uptake, thus allowing specific cancer targeting to certain target (Yu and 
Zhang 2009). NEs in the translational research and their role in targeted cancer therapy were 
summarized by Ganta et al. (2014). The current status of NEs in the cancer therapeutics and 
commercial field on the basis of morphology, formulation, characteristics and characterization 
parameters was overviewed by Sahu et al. (2017). Sasikumar and Kamalasanan (2017) analyzed 
the possibilities of exploring NE platform for targeted drug delivery to prostate cancer. Recent 
advances in lipid nanocarriers, including NEs, applicable in the fight against cancer were 
summarized by Jampílek and Kráľová (2019b). 
Zhao et al. (2018b) reported that woody oil-based emulsive nanosystems could efficiently deliver 
poorly soluble natural alkaloids resulting in increases in the sensitivity of lung cancer cells. O/W 
cinnamon oil NE (40.52 nm) and vitamin D encapsulated cinnamon oil NE (48.96 nm) showed 
anti-cancerous activity in human alveolar carcinoma cells, they induced DNA damage along with 
corresponding increase in micronucleus frequency, arrested the cell cycle progression in G0/G1 
phase, showed increased expression of Bax, capase-3 and caspase-9 and decreased expression of 
BcL2 proteins along with considerable increase in apoptotic cell population and loss of 
mitochondrial membrane potential (Meghani et al. 2018). The antitumor potential of both O/W 
NE with encapsulated vitamin K2 (VK2) and NE incorporating VK2 with a ligand conjugate sialic 
acid-cholesterol (showing enhanced affinity towards the membrane receptors overexpressed in 
tumors) anchored on the surface was evaluated in S180 murine sarcoma tumor cells. It was found 
that i.v. or intragastric administration of VK2 NE to syngeneic mice with subcutaneously 
established S180 tumors resulted in considerable tumor growth suppression, higher effect being 
observed with surface-modified NE and both NEs were non-toxic (Shi et al. 2018). 
Ahmad et al. (2018b) reported that the optimized silymarin NE with mean particle size of 21.24 
nm reduced the cancer cell viability, increased intensity of reactive oxygen species (ROS) and 
chromatin condensation and could be considered as an efficient carrier for oral delivery of 
silymarin against human hepatocellular carcinoma without damaging normal cells. Methyl 
jasmonate loaded NE with mean droplet size of 75.06 nm and PDI 0.017 was found to be more 
effective in killing cancer cells, it induced a stronger sub-G1 arrest than methyl jasmonate 
solution and showed a considerable absence of toxicity in human umbilical vein endothelial cells 
(Habibi et al. 2017). The EC50 values related to cytotoxic activity against A549 tumor cell line 
(human lung carcinoma) estimated with pure Casearia sylvestris Sw. extract and its NE were 4.0 
µg/ mL and 1.0 µg/mL, respectively suggesting 4-fold higher efficiency of the nanoformulation 
(Pereira et al. 2017). 
The cytotoxic effect of exopolysaccharides extracted from brown seaweed (Sargassum 
longifolium) encapsulated in orange oil NE with particle size 178 nm and zeta potential -43.9 mV 
estimated by MTT method in colon (HCT 116) cell lines was lower (70%) than that of seaweed 
polysaccharide encapsulated with nanostructured lipid carrier (Shofia et a;. 2018). Weekly i.v. 
administration of O/W NE encapsulating DHA-SBT-1214, a novel ω-3 fatty acid conjugated 
taxoid prodrug against prostate cancer stem cells, to NOD/SCID mice bearing subcutaneous 
PPT2 tumor xenografts resulted in strong suppression of tumor growth compared to Abraxane 
and placebo NE and viable cells that survived from this in vivo treatment regimen were no longer 
able to induce floating spheroids and holoclones (Ahmad et al. (2017). Migotto et al. (2018) 
developed a cationic bioadhesive NE surface modified with CS and particle sizes of 46.3 nm for 
intraductal administration of C6 ceramide showing 4.5-fold lower EC50 value of C6 ceramide 
related to the reduction of MCF-7 cells viability and this NE prolonged drug localization for 
more than 120 h in the mammary tissue following intraductal administration compared to its 
solution. NEs prepared using tanshinone extract of Salvia miltiorrhiza with the mean particle size 
of 14.2 nm inhibited human lung carcinoma cell (A549) proliferation more effectively than the 



13 

extract alone, they penetrated into cytoplasm through endocytosis and caused up-regulation of p-
JNK, p53 and p21 and down-regulation of CDK2, cyclin D1 and cyclin E1 expressions in a dose-
dependent manner and caused cell cycle arrest at G0/G1 phase (Lee et al. 2016). NE formulation 
of Nigella sativa L. EO with droplet diameter 20−50 nm notably reduced the viability of MCF-7 
breast cancer cells, whereby the treated cells included cell membrane blebbing, cytoplasmic 
vacuolation, marginalization of chromatin, and fragmentation of the nucleus suggesting induction 
of apoptosis in MCF-7 cells (Periasamy et al. 2016). 
O/W NE coated with a thiol modified CS designed for co-delivery of piperine (weight ratio 100: 
1) was fabricated and high degree of CS modification with particle sizes of 110 nm did not show 
any cytotoxic effect on normal fibroblasts and promoted death in colon cancer cells (Vecchione 
et al. 2016). Formulation of diallyl disulfide and α-linolenic acid prepared as protein NEs 
showing antioxidant and radical scavenging property and acting also as optimal H2S slow-release 
donors exhibited considerable anti-proliferative effect on MCF-7 breast cancer cell lines and 
HuT 78 T-cell lymphoma cells, induced apoptosis and cell cycle arrest at the G0/G1 phase and 
improved the Lin– Sca1+ human cardiac progenitor cells proliferation suggesting that they could 
be used in selective cancer therapy and for promoting the muscle tissue repair (Ciocci et al. 
2016). 
Evaluation of anticancer activities of ginger EO (GEO) and frankincense EO (FEO) NEs 
prepared by a high-pressure homogenization technique with incorporated antineoplastic agent, 
mitomycin C (MMC) showed that NE-based EOs ameliorated the apoptotic effects of MMC on 
the cancer cells (IC50 values of GEO-MMC and FEO-MCC NEs estimated for HeLa cells were 
reduced by 44.12 and 29.42 folds, respectively, while those for MCF-7 cells were decreased by 
29.29 and 55.3 folds when compared to MMC solution). FEO-MMC NE caused also the greatest 
change on the HeLa cellular morphology, while MCF-7 cells were most damaged and their 
nuclei were segmented when subjected into high GEO-MMC NE. Consequently, mixing of drug 
with GEO NE and FEO NE considerably improved its cytotoxicity on the MCF-7 and HeLa cells 
(Al-Otaibi et al. 2018). MMC formulated into NEs based on EOs of chamomile (ChEO) and 
garlic (GarEO) reduced the cell viabilities of HeLa cervical cancer cells by 42- and 20-fold 
compared to free MMC, whereby treatment with GarEO NE or GarEO-MMC NEs resulted in 
stronger alteration of the cell membrane of the HeLa cells than treatment with ChEO or ChEO-
MMC NEs. Using staining with 4′,6-diamidino-2-phenylindole it was found that NEs of GarEO 
and GarEO-MMC have got attached to the cell membrane causing damage to the cell, while 
those of ChEO or ChEO-MMC passed the cell membrane and affected the nucleus directly 
(Alkhatib et al. 2018a). 
In vitro cytotoxicity against MCF-7 and HeLa cells of an albumin anchored docetaxel (DTX) 
lipid NE was higher than that of the plain NE, and in in vivo experiment it caused 80.01±2.74% 
inhibition of solid tumours induced in C57BL/6 mice compared to 55.62±5.41% inhibition 
caused by administration of the plain NE, whereby also its tumour targeting activity was 3-fold 
higher than that of plain NE (Afzal et al. 2016b). Transferrin coupled DTX lipid NE (200−393 
nm) prepared by homogenization and ultra-sonication process caused 84.66±4.29% tumor 
inhibition in tumor induced C57BL/6 mice and was found to have also 3.54-fold higher tumor 
targeting activity compared to the plain lipid NE (Afzal et al. 2016c). 
Carvacrol NE showing a negative surface charge of -29.89 mV and 99.1 nm mean droplet size 
powerfully induced apoptosis in doxorubicin (DOX)-resistant A549 lung carcinoma cells 
(A549DR), displayed cell senescence leading to cell cycle arrest and inhibited the autophagy 
suggesting that it could be used as a potential candidate for lung cancer therapy (Khan et al. 
2018).  
Paclitaxel (PTX) NEs, in which α-tocopherol oil core of Tocosol was substituted with γ-
tocotrienol, and D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) with 
PEGylated γ-tocotrienol showing droplet size <300 nm were tested against Bx-PC-3 and PANC-
1 pancreatic tumor cells. Fastest release was observed with NEs loaded with free PTX, when γ-
tocotrienol was used as the core and anticancer activity was improved also by substituting α-
tocopherol with γ-tocotrienol and pronounced increase in activity was estimated when PTX lipid 



14 

conjugates were used (Abu-Fayyad et al. 2018). Entrapment of gemcitabine-γ-tocotrienol 
conjugates into NEs pronouncedly improved their anticancer activity against Bx-PC-3 and 
PNAC-1 pancreatic cancer cells when compared to the free drug.  Moreover, it was found that 
gemcitabine-γ-tocotrienol conjugates were least affected by deamination deactivation reaction in 
vitro when compared with the free and conjugated gemcitabine in solution (Abu-Fayyad and 
Nazzal 2017). CUR/δ-T3 tocotrienol NEs with average particle size of 261 nm, PDI 0.27 and 
zeta potential of 35 mV significantly suppressed constitutive NF-κ B activation, and significantly 
induced apoptosis in breast and ovarian cancer cells, MCF-7 and OVCAR-8 (Steuber et al. 
2016). Hyaluronic acid-complexed PTX NEs containing DL-α-tocopheryl acetate and soybean oil 
with a diameter of 85.2±7.55 nm and a zeta potential of -35.7±0.25 mV administered at a dose of 
25 mg/kg to nude mice transplanted with CD44-overexpressing non–small cell lung carcinoma 
cells (NCI-H460) xenografts suppressed cancer cell growth more than the Taxol and strongly 
inhibited tumor growth, which could be connected with the specific tumor-targeting affinity of 
HA for CD44-overexpressed cancer cells (Kim and Park 2017a). HA-coated NEs composed of 
DL-α-tocopheryl acetate, soybean oil, polysorbate 80, and ferric chloride incorporating PTX with 
particle diameter and zeta potential of 65±15 nm and -39.5±0.33 mV, respectively showed superb 
targeting of ovarian SK-OV-3 tumor cells overexpressing CD44 (Kim and Park 2017b). 
The study of the antitumor activity and cardiotoxicity of the garlic oil (GarO) NE and GarO NE 
with incorporated DTX with particle sizes 63.19±1.85 nm and 110.±14.37 nm, respectively in 
Ehrlich ascites carcinoma (EAC)-bearing mice showed that the administration of GarO NE 
enhanced the lactate dehydrogenase activity in the ascetic fluid and ameliorated the heart 
enzymes, while treatment with DTX-Gar NE improved the mean survival time of the mice 
(27.7±11.63 days) that at treatment with aqueous DTX solution reached 23.1±1.52 days 
(Alkhatib et al. 2017a). It is important to note that incorporating the DTX into NEs based on 
orange oil also improved its antitumor efficacy and reduced its cardiotoxicity in female Swiss 
Albino mice bearing Ehrlich tumor in their ascetic fluid (Alkhatib et al. 2017b). Efficient 
accumulation of NEs incorporating fraxinellone (Frax), an anti-fibrotic drug, prepared by an 
ultrasonic emulsification method with a particle size approx. 145 nm in the tumor site after 
systemic administration was observed and NE was taken up by tumor-associated fibroblasts 
(TAFs) and tumor cells. Following i.v. administration of Frax NE a notable decrease in TAFs 
and stroma deposition was estimated and an increase of natural-killer cells, cytotoxic T cells as 
well as a decrease of regulatory B cells, and myeloid-derived suppressor cells in the tumor 
microenvironment suggested that this treatment remolded the tumor immune microenvironment 
as well. Enhanced tumor-specific T-cell infiltration, activated death receptors on the tumor cell 
surface and increased apoptotic tumor cell death was achieved using combination of a tumor-
specific peptide vaccine with Frax NE suggesting that such approach could be an effective and 
safe strategy to remodel fibrotic tumor microenvironment resulting in enhanced immune 
response activation and thus, in a prolonged efficiency for advanced desmoplastic melanoma 
(Hou et al. 2018). Improved in vivo antitumor activity in female Swiss Albino mice inoculated 
with Ehrlich ascietes and reduced hepatotoxicity was observed also with sorafenib incorporating 
NE formulated with flaxseed oil with particle diameter and zeta potential of 77.46±8.28 nm and -
3.4±1.2 mV, respectively (Alkhatib et al. 2017c). Similarly, incorporation of sorafenib into carrot 
seed oil NE improved the antitumor efficacy of drug and reduced its hematoxicity and 
hepatotoxicity (Alkhatib et al. 2018b). 
Acid-sensitive lipidated DOX prodrug (C16-DOX) entrapped in lipid NE improved 
chemotherapeutic index and tumor-control efficacy in an in vivo murine 4T1 breast cancer model 
compared to free drug and caused considerable reduction in lung metastasis due to possibility 
using higher dose of DOX (Camara et al. 2017). Folate-functionalized soy lecithin lipid NEs of 
DOX and ytrium 90 (90Y) were found to inhibit growth of folate receptor rich nasopharyngeal 
carcinoma (NPC) cells CNE1 in vitro and pronouncedly decrease tumor volume in NPC-induced 
nude mice compared to DOX + 90Y-lipid NE, causing massive (89.9%) necrosis and hemorrhage 
of CNE1 cells but lower growth inhibition (21%) of folate deficient nasal epithelial cells (RPMI 



15 

2650) than DOX + 90Y-lipid NE (43.65%) (Liu et al. 2017). DTX NE containing as 
biocomponents soybean oil and lecithin with droplet size, PDI and zeta potential 233.23±4.3 nm, 
0.240±0.010 and -43.66±1.9 mV, respectively exhibited strong cytotoxic activity against MCF-7 
cancer cells (IC50: 13.55±0.21 µg/mL at 72 h) as well as 2.83-fold higher cell uptake than 
control, whereby at a dose of 20 mg/kg no toxicity or necrosis was observed with liver and 
kidney tissues of mice (Verma et al. 2016). DTX NEs surface-functionalized with folate with 
particle sizes <150 nm showed a 270-fold decrease in IC50 value in chemoresistant ovarian cancer 
cells SKOV3TR as compared to DTX alone, while in SKOV3 tumour-bearing mice these NEs 
delivered DTX by folate receptor mediated endocytosis resulting in cytotoxicity capable of 
overcoming ABC transporter mediated taxane resistance (Ganta et al. 2016). Folate-PEG-
decorated DTX lipid NE was superior in tumor targeting by 4.81- and 2.08-fold over controls and 
in tumor regression as well providing better results as PEGylation, albumin and transferrin 
strategies (Afzal et al. 2016a). 
Theranostic NEs incorporating PTX and contrast agents prepared using linear polyglycerol-
poly(ε-caprolactone) diblock copolymers and ethiodized poppyseed oil, lipiodol, as a core oil 
exhibited superb anticancer activities against HeLa ovarian cancer cells as well as the ability to 
be used as a contrast agent (Le Kim 2017). NE with co-encapsulated PTX and baicalein (5,6,7-
trihydroxyflavone) showed better antitumor efficacy in MCF-7/Tax cells exhibiting progressive 
resistance to PTX in vitro as well as much higher antitumor efficacy in vivo than other PTX 
formulations suggesting that this NE formulation could be used to overcome multidrug resistance 
via oxidative stress augmentation and P-glycoprotein inhibition (Meng et al. 2016). 
Multifunctional smart CUR-loaded CS/perfluorohexane nanodroplets of 101.2 nm and 77.8% 
CUR entrapment designed for contrast-ultrasound imaging and on-demand drug delivery showed 
notably higher inhibition of 4T1 human breast cancer cells in vitro by ultrasound exposure, 
suggesting that they could have a great potential for image-guided cancer therapy (Baghbani et 
al. 2017). Ultrasound-responsive multifunctional smart alginate/perfluorohexane nanodroplets 
with the mean particle size of 55.1 nm designed for co-delivery of DOX and CUR and showing 
92.3% entrapment efficiency of DOX exhibited enhanced cytotoxicity in adriamycin-resistant 
A2780 ovarian cancer cells compared to DOX nanodroplets due to synergistic effects of DOX 
and CUR and their combined application with ultrasound irradiation leads to strong tumor 
regression (Baghbani and Mortarzadeh 2017). MEs of CUR with docosahexaenoic acid (DHA)-
rich oil showed strong cytotoxicity on human glioblastoma U-87MG cell line (IC50: 3.755±0.24 
ng/mL), which could be a attributed to the synergistic effect of CUR and DHA in the ME and 
following administration of this ME to Sprague-Dawley rats the CUR concentration at 24 h 
achieved 466-fold (for intranasal) and 421-fold (for intravenous) of the IC50 value estimated in 
the U-87MG cell line suggesting that designed ME could be used for therapy of brain cancer by 
both routes of administration (Shinde and Devarajan 2017). 
Photodynamic therapy (PDT) is a clinically approved cancer therapy utilizing photochemical 
reaction between a light activable molecule or photosensitizer, light, and molecular oxygen 
resulting in formation of reactive oxygen species that directly damage cells and/or vasculature 
leading to tumor destruction and induce inflammatory and immune responses (van Straten et al. 
2017). For both hydro-alcoholic extract from Tectona grandis L.f. leaves (TGE) alone as well as 
extract incorporated into O/W NE with particles sizes approx. 20 nm and tested against 
melanoma B16 F10 cells photodynamic effect was estimated due to increasing toxicity under 
illumination with red light, however NE formulation was much less toxic towards normal cells in 
the dark compared to free TGE, which exhibited notable dark toxicity towards both B16 F10 and 
murine fibroblast NIH3T3 cells (Furtado et al. 2017). Acai oil in NE, a novel photosensitizer for 
PDT, applied at PDT treatment of NIH/3T3 normal cells and B16F10 melanoma cell lines caused 
85% cell death for melanoma cells, while maintaining high viability in normal cells and tumor 
volume reduction of 82% in tumor bearing C57BL/6 mice was observed following 5-fold 
treatment with PDT using acai oil in NE (Monge-Fuentes et al. 2017). Magneto low-density NE 
(MLDNE) that can carry maghemite NPs and Chlorin e6 (Phytochlorin) as an active 
photosensitizer drug was designed as a potential vehicle for combined hyperthermia and PDT to 



16 

treat cancer, because it is selectively taken up by MCF-7 cancer cell surfaces with receptor 
recognition based on the overexpression of low density lipoprotein receptor. MLDNE showing 
particle size <200 nm and high drug encapsulation efficiency was found to enhance tumor 
damage after minor heat dissipation and/or minimum visible light photosensitization doses by 
classical magnetic hyperthermia and PDT resulting in notable synergic action on MCF-7 cells 
reflected in reduced cell viability (Pellosi et al. 2018). de Matos et al. (2018) studied the effects 
of PDT on cellular viability using CUR NE as a photosensitizing drug in cervical carcinoma cell 
lines and found that NEs were internalized inside cells and were observed in the intracellular 
environment for up to 36 hours after incubation with cell lines, whereby after the PDT high 
phototoxic effect of CUR NE (<5% of viable cells after irradiation) was estimated suggesting 
that CUR NEs have potential as an alternative treatment to cervical lesions using an endoscopic 
diode fiber laser setup for in situ activation or cavity activation using a diffuse fiber delivery 
system. 
 
2.4.3 Nanoemulsions for pulmonary drug delivery 
 
Optimized quercetin loaded O/W NE prepared using palm oil ester/ricinoleic acid as oil phase 
(droplet size of 131.4 nm, PDI 0.257, zeta potential 51.1 mV) showing good stability against 
phase separation and storage at 4°C for 3 months induced cytotoxicity towards A549 lung cancer 
cells without affecting the normal cells suggesting that NE could be used as a potential carrier 
system for pulmonary delivery of molecules with low water solubility (Arbain et al. 2018). 
Minz and Pandey (2018) designed recombinant hepatitis B surface antigen loaded solid fat NEs 
as carrier system and monophosphoryl lipid A as an adjuvant-carrier system and evaluated it as 
multiadjuvanted vaccine system for deep pulmonary vaccination. The observed humoral (sIgA 
and IgG) and cellular (IL-2 and IF-γ) immune responses considerably exceeded those estimated 
with naive antigen (recombinant surface antigen without any excipient) solution. 
Nasr et al. (2014) used commercially available lipid NE, the Intralipid or Clinoleic, to 
prepare amphotericin B lipid NE aerosols for targeting peripheral respiratory airways via 
nebulization. 
Tea tree oil NEs prepared using Cremophor EL (average size of 12.5 nm) exhibited excellent 
antimicrobial activities on Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, S. 
aureus and Candida albicans and after inhalation to the lung showed higher anti-fungal effect 
than fluconazole on the fungal pneumonia rat models, causing reduced lung injury, highly 
microbial clearance, blocking of leukocyte recruitment, and decrease of pro-inflammatory 
mediators. Such inhalable formulation could be used in local therapies of fungal and bacterial 
pneumonia with no obvious adverse events (Li et al. 2016a). 
Shah et al. (2017) tested rifampicin-oleic acid first-generation NE and its respective CS- and CS-
folate conjugate-decorated second and third-generation NEs showing mean droplet sizes of 
40−60 nm related to aerosolization, pulmonary inhalation, intracellular trafficking potential in 
macrophages and pharmacokinetics profiles. Chitosan oligosaccharide lactate was employed as 
mucoadhesive, bioadhesive and targeting moiety for macrophages, while folic acid was used as 
targeting ligand of macrophage. It was found that tested NEs exhibited >95% aerosol output and 
inhalation efficiency >75% and the size and surface tension of NEs affected  the aerosol output, 
aerosolized and inhaled fine particle fractions in an inverse relationship. Higher cell 
internalization potential, reduced plasma drug concentration, and higher lung drug content 
showed third-generation NEs. NE containing highly-refined soybean oil together with cetyl 
pyridinium chloride, Tween 80 and ethanol in water produced by high-speed emulsification 
showing droplets with mean diameter of 450 nm and safe mucosal adjuvants, when delivered 
intranasally along with Mycobacterium tuberculosis specific immunodominant antigens, induced 
potent mucosal IL-17 T-cell responses and conferred protection upon M. tuberculosis challenge 
in mice. Moreover, when such NE tuberculosis vaccine is delivered along with Mycobacterium 
bovis bacillus Calmette-Guerin licensed vaccine, decreased disease severity could be observed 
(Ahmed et al. 2017). 



17 

 
2.4.4 Nanoemulsions for ocular drug delivery 
Nanotechnology-based systems for treating and managing various ocular diseases include more 
accessible formulations for deeper segments of the eyes, which enable the availability of drugs at 
required site in a required amount without inversely affecting the eye tissues (Lalu et al. 2017). 
Findings related to topical application of lipid-based systems (MEs, NEs, liposomes and solid 
lipid NPs), polymeric systems (hydrogels, contact lenses, polymeric NPs and dendrimers) and 
used physical methods (iontophoresis and sonophoresis) was reviewed by Souza et al. (2014). 
The use of nanocarriers, including NEs for gene therapy of retinal diseases, was overviewed by 
Al-Halafi (2014). 
Coumarin-rich extract from Pterocaulon balansae incorporated into NEs composed of medium 
chain triglycerides (MCT) and egg-lecithin with droplet sizes >300 nm caused a 95% reduction 
of trophozoite viability after 24 h of incubation with a NE containing 1.25 mg/mL of coumarins, 
whereby the effect was comparable with that of chlorhexidine suggesting potential of such NEs 
to be used for the local treatment of ocular keratitis caused by Acanthamoeba (Panatieri et al. 
2017). 
Castor oil and mineral oil NEs with mean particle size of 234 nm, PDI >0.16, zeta potential of -
8.562±3.49 mV, slightly acid pH and viscosity of about 1.2 cP, which were instilled on the 
surface of a commercial contact lens, were found to be situated inside and on the surface of the 
lenses and their transparency remained near 100% suggesting their suitability for ocular 
application, because the contact lens remained transparent and ion-permeable after association 
with this formulation (Katzer et al. 2014). 
The introduction of a positive charged mucoadhesive polymer, CS, into NEs designed for ocular 
administration of timolol containing the drug as maleate or as ion-pair with bis(2-ethylhexyl) 
sulfosuccinate increased drug permeation probably due to the interaction of CS with corneal 
epithelial cells (Gallarate et al. 2013). 
A combination of NE with 2% hydroxyethyl β-cyclodextrin resulted in 9.2-fold higher lutein 
accumulation (119±6 µg/g/h) compared to lutein suspension alone and the scleral accumulation 
of lutein increased by increasing the cyclodextrin content, whereby the nanoformulation showed 
low cytotoxicity in retinal cells (Liu et al. 2015a). Lutein-loaded NE consisting of isopropyl 
myristate, triacetin, Tween 80, and ethyl alcohol with particle sizes approx. 10−12 nm showed 
sustained and pronouncedly increased lutein release. Such NE could be considered as a potential 
alternative delivery system for lutein, an effective drug used in therapy of macular degeneration 
(Lim et al. 2016).  
Dexamethasone acetate (DEX) and polymyxin B sulfate were formulated as a cationic NE, in 
which soy phosphatidylcholine represented 30% of the lipid phase containing DEX, while 
polymyxin B sulfate was dissolved in the water phase and this NE could be considered as a 
viable alternative to the commercial ophthalmic suspensions designed for the treatment of 
ophthalmic infections (Li et al. 2016b). Triamcinolone acetonide-loaded NE consisting of MCT 
oil, soybean lecithin and Poloxamer 188 released the drug in a fast rate and could provide an 
adequate drug release rate on the eye surface to be used in topical ophthalmic administration 
(Silva and Lemos-Senna 2016). 
Alternative controlled-release ocular terbinafine hydrochloride-loaded NE in situ gels prepared 
by NE dispersion in gellan gum solution (0.2%, w/w) were transparent, pseudoplastic, 
mucoadhesive, showed more retarded zero-order drug release rates and increased bioavailability 
(Tayel et al. 2013). 
Acetazolamide loaded NEs fabricated using peanut oil, Tween 80 and/or cremophor EL as 
surfactant and transcutol P or propylene glycol as cosurfactant were incorporated into ion 
induced in situ gelling systems composed of gellan gum alone and in combination with xanthan 
gum, hydroxypropyl methylcellulose (HPMC) or carbopol. The gels showed a considerably 
sustained drug release in comparison to the NEs, whereby gellan/xanthan and gellan/HPMC 
exhibited improved therapeutic efficacy and more prolonged intraocular pressure lowering effect 
relative to that of commercial eye drops and oral tablet (Morsi et al. 2017). 



18 

Cyclosporine A (Cy-A) NEs containing oleic acid, Tween 20 and Transcutol P and coated with 
CS were evaluated as non-irritant and non-toxic, being well tolerated by rabbit eye much more 
sensitive than human. In corneal penetration studies for low molecular weight CS coatings 59.2 
and 57.5% of drug penetration was estimated, while for medium high molecular weight CS 
coatings it was 55.5% and 52.3%, respectively (Shen et al. 2018). Optimized Cy-A loaded 
polymeric mucoadhesive NE with higher Cy-A payload, in which the concentration of CS was 
adjusted according to the blinking force of eyelids, was able to maintain the therapeutic 
concentrations (≥50−300 ng/g) of drug in the rabbit cornea and conjunctiva over the period of 24 
h (Akhter et al. 2016). 
Celecoxib NE containing 5% oleic acid-Transcutol P (10:1) in oil phase with droplet sizes 
ranging from 6.96 to 26.65 nm and pH 6.5−6.9, respectively showed 82.6% of the drug released 
in the 24 h of the experiment. NE containing 85% of surfactant (Tween 80, Span 20) and 
cosurfactant (propylene glycol) in a ratio 2:1 showed that 15.73% drug permeated through rabbit 
cornea and the flux (Jss) of Celecoxib from this NE (0.65 mg cm-2/h) was 22-fold higher than this 
of control (Moghimipour et al. 2017). 
 
 
2.5 Nanoemulsions applicable in food industry 
 
There are various nanocomposites suitable for the protection of different foodstuffs, smart active 
or responsive packaging materials, edible coatings, as well as diverse nanosensors applicable for 
monitoring of food quality, safety, and integrity and the application of nanocomposites in food 
packaging systems could diminish the harshness of food processing and the amount of additives 
and chemical preservatives in food industries (e.g., Jampílek and Kráľová 2018b). Nanoemulsion 
technology is particularly appropriate for the preparation of encapsulating systems for functional 
compounds, because it prevents their degradation and improves their bioavailability. An 
overview of food-grade NEs, their recent applications in the food systems and patent review of 
emulsions was presented by Yalcinoz and Ercelebi (2018). Promising advantages reached with 
the use of NEs as delivery systems of flavoring and preservative agents in the food industry were 
summarized by Salvia-Trujillo et al. (2015). Adjonu et al. (2014) overviewed 
emulsifiers/surfactant (ionic, non-ionic, phospholipid, polysaccharide, and protein) used in NEs 
designed for food applications as well as suitability of proteins and protein hydrolysates as 
nanoemulsifiers and analyzed the potential of whey protein derived peptides as both emulsifiers 
and bioactive compounds in NE delivery systems. The influence of particle characteristics such 
as size and interfacial properties on potential biological fate (digestion and absorption) of 
ingested NEs was described by McClements and Xiao (2012). The influence of droplet 
characteristics on the physicochemical and sensory properties of beverage emulsions, with 
special focus on their influence on product stability as well as developments in the soft drinks 
area, including fortification with vitamins, reduced calorie beverages, and "all-natural" products 
were overviewed by Piorkowski and, McClements (2014). Although to the potential advantages 
of food-grade NEs over conventional emulsions for applications in the food industry belong 
higher stability to particle aggregation and gravitational separation, higher optical transparency 
and increased bioavailability of encapsulated components, potential risks associated with 
consumption of lipid nanoparticles that are able to alter the fate of bioactive components within 
the gastrointestinal tract and possible toxicity of surfactants and organic solvents could not be 
marginalized (McClements 2013). 
 
2.5.1 Nanoemulsions formulated with natural emulsifiers, biosurfactants and biopolymers 
 
Mungure et al. (2018) overviewed the potential application of pectin, an anionic polysaccharide, 
for the stabilization of NE systems. Synthesis and applications of pectin-based nanomaterials that 
combine the advantages of both pectin and the nanoscale particles was overviewed by Zhao and 
Zhou (2016). Bai et al. (2016) compared the effectiveness of a number natural emulsifiers (whey 



19 

protein, gum arabic, Quillaja saponin and soy lecithin) at fabricating corn O/W NEs using dual-
channel microfluidization and found that whey protein isolate and Quillaja saponin more 
effectively formed NEs with fine droplets than gum arabic and soy lecithin (lower amount of 
emulsifier needed, fabrication of smaller droplets), which could be connected with faster 
emulsifier adsorption and a greater reduction in interfacial tension resulting in more effective 
droplet disruption within the homogenizer for saponins and whey proteins. McClements and 
Gumus (2016) analyzed the possibility of the replacement of synthetic surfactants with natural 
emulsifiers, such as amphiphilic proteins, polysaccharides, biosurfactants, phospholipids, and 
bioparticles, discussed the physicochemical basis of emulsion formation and stabilization by 
natural emulsifiers and compared the benefits and limitations of different natural emulsifiers. 
Konjac glucomannan, a renewable natural polysaccharide occurring in the tuber Amorphophallus 
konjac K. Koch, composed of β-1,4-linked D-mannose and D-glucose in either a 1.6:1 or 1.4:1 
molar ratio, exhibit good swelling, gelling, or emulsifying properties. Its derivative, konjac 
glucomannan octenyl succinate (KGOS), was found to be a good potential emulsifier and 
stabilizer for encapsulation of lipophilic bioactive compounds such as β-carotene. Emulsification 
yield of the KGOSC nanoemulsion containing 0.03% β-carotene, 0.3% KGOS and 10% MCT 
exceeded 95% and after 30 days of storage, the particle size and PDI of the KGOSC NE did not 
reach 5 nm and 0.5, respectively, whereby the sensitivity of KGOSC NEs to storage conditions 
decreased in following order: temperature > oxygen > light (Li et al. 2018). 
W/O NEs of Sacha Inchi oil (SIO), containing a very high content of the ω-3 fatty acid, α-
linolenic acid (approx. 50%) and tocopherols (176–226 mg/100 g) prepared using olive leaf 
phenolics (OLP) showed droplet size 2.15±0.13 nm and were stable without phase separation 
during 30-day storage, whereby the release of OLP controlled the oxidative progress of SIO by 
prolonging the induction time, preventing the production of primary and secondary oxidative 
products and also the deterioration of polyunsaturated fatty acids and tocopherols (Liu et al. 
2018). Among O/W NEs prepared with 10% avocado oil using natural (lecithin) and synthetic 
(Tween 80) emulsifiers at different concentrations (2.5−10%) with zeta potential ranging from  -
26 to -59 mV and lipid droplets of 103−249 nm, Tween 80 was found to be more effective than 
lecithin (Arancibia et al. 2017). Multilayer emulsions produced by a high-pressure 
homogenization method using an electrostatic layer-by-layer deposition process of lecithin-CS 
membranes encapsulating linseed oil and α-lipoic acid simultaneously were found to be an 
effective delivery system to incorporate them into functional foods and beverages. CS 
encapsulation inhibited the degradation of α-lipoic acid and improved the oxidation stability of 
linseed oil in multilayer emulsions that showed good centrifugal, dilution and storage stabilities 
(Huang et al. 2018).  
Using MCT oil the rhamnolipids biosurfactants can effectively form small droplets (d32 
<0.15 µm) at low surfactant-to-oil ratios (<1:10). Rhamnolipid-coated droplets were found to be 
stable to aggregation at pH values 5−9, salt concentrations <100 mM NaCl and 20−90 °C. 
Droplet aggregation at pH 2−4 and high ionic strength 200−500 mM NaCl could be connected 
with a reduction in electrostatic repulsion at low pH and high salt levels. Rhamnolipids could be 
considered as effective natural surfactants suitable to replace synthetic surfactants in certain 
commercial applications (Bai and McClements 2016). 
Quillaja saponin (Q-Naturale), a natural food-grade surfactant isolated from the bark of the 
Quillaja saponaria Molina tree, containing saponin-based amphiphilic molecules, was reported 
to be able to replace synthetic surfactants in food and beverage products (Yang et al. 2013). A 
promising NE delivery system for oregano as an essential oil model with long-term stability 
using a sugar-based biosurfactant (Quillaja saponin) produced by microfluidization was found to 
be suitable to be used in antimicrobial as well as flavoring and potential antioxidant applications 
in food and beverages or in pharmaceutical and cosmetic products (Doost et al. 2018). 
The study of high pressure homogenized whey protein emulsions prepared by mixing whey 
protein concentrate (10%), soybean oil (0 or 5%) and soy lecithin (0 or 5%) showed that 
increasing homogenization pressures significantly decreased particle sizes of all samples but 



20 

increased electrical conductivity of samples. After 25 MPa treatment emulsions with lecithin had 
smaller particle sizes than those made without lecithin, but at 50 MPa further reduction of 
particle sizes in the presence of lecithin was not observed (Yan et al. 2017). Yerramilli et al. 
(2017) studied partially replacement of sodium caseinate (SCas) in the formation and long-term 
stabilization of 5 wt% O/W NEs prepared using a high-pressure homogenization by pea protein 
isolate (PPI) and found that NEs stabilized by 1:1 mixture of SCas and PPI did not display any 
creaming or aggregation and remained stable for more than 6 months (droplets <200 nm), which 
could be connected with the interaction of pea proteins disrupted by high-pressure 
homogenization with SCas in the continuous phase of the NEs. Consequently, the plant proteins 
have potential to be applicable in the long-term stabilization of NEs in the food and beverage 
industry. O/W NEs stabilized by high-pressure homogenized lentil proteins isolate (LPI) showed 
pronouncedly higher in vitro lipid digestibility than unmodified LPI NEs, which could be 
connected with the higher interfacial area of smaller droplets and weaker interfacial moduli of 
modified LPI-stabilized interfaces compared to those with unmodified LPI. It could be noted that 
high-pressure homogenization notably decreased LPI particle size distribution, surface 
hydrophobicity and the interfacial storage moduli relative to the unmodified LPI (Primozic et al. 
2018). 
As an attractive biosurfactant suitable for preparation of NE formulations applicable in food and 
beverage products saponin extracted of Brazilian ginseng roots was reported (Rosa et al. 2016). 
 
2.5.2 Nanoemulsions of essential oils and their constituents 
 
Excellent antimicrobial efficacy of plant EOs predestines them to be used as an alternative of 
health hazardous synthetic preservatives in food products and encapsulation of EOs in NEs, MEs, 
solid-lipid NPs and liposomes improve their shelf-life already at low doses (Prakash et al. 
2018a). Donsi and Ferrari (2016) in a review paper focused their attention on EO NEs as 
antimicrobial agents in food, critically analyzed the reported antimicrobial activity data, both in 
vitro and in products and discussed the regulatory issues associated with their use in food 
systems. An overview related to the different systems for the encapsulation of bioactive oils and 
the currently applied elaboration methods was presented by Rodriguez et al. (2016). 
Although EOs in the food industry are mainly used as flavoring agents, O/W NE of betel (Piper 
betle L.) leaf EO showed antimicrobial activity against five strains of Gram-positive and Gram-
negative bacteria with MIC of 0.5−1.25 µL/mL and MBC of 1−2.5 µL/mL suggesting that it can 
serve as natural antimicrobial agent for food system (Roy and Guha 2018). Mexican oregano 
(Lippia graveolens Kunth) EO containing thymol and γ-terpinene, which was incorporated into 
active coatings and spread on fresh pork meat as free, nanoemulsified, and microencapsulated 
EO at a dose of 2.85 mg EO/cm2 caused growth inhibition of Lactobacillus plantarum (5 log 
population reduction) and Pseudomonas fragi (4 log reduction), while ≤1.5 log population 
reduction was observed for Brochothrix thermosphacta and Salmonella Infantis, whereby meat 
microbiota was most efficiently controlled by microencapsulated EO resulting in delayed lipid 
and oxymyoglobin oxidation of fresh pork meat (Hernandez-Hernandez et al. 2017). Anise oil 
NE more effectively reduced the population of E. coli O157:H7 and Listeria monocytogenes 
(count by 2.51 and 1.64 log cfu/mL, respectively) than bulk anise oil (by 1.48 and 0.47 log 
cfu/mL) after 6 h of contact time (Topuz et al. 2016). The EO-based NEs could improve the 
microbial quality of minimally processed fruits and vegetables (Prakash et al. 2018b). Basil oil 
(Ocimum basilicum) NE fabricated by ultrasonic emulsification with droplet diameter 29.3 nm 
showed antibacterial activity against E. coli causing alteration in permeability and surface 
features of bacterial cell membrane (Ghosh et al. 2013). Long-term stability over 21 days of 
storage was estimated with Zataria multiflora Boiss EO NE (droplet size of 200 nm and PDI 0.2) 
fabricated by emulsion phase inversion that showed MIC values of 2500 and 5000 µg/mL against 
L. monocytogenes and Salmonella typhimurium, respectively and this NE was more effective in 
inhibiting the growth of bacteria in milk than in culture media (Shahabi et al. 2017). 



21 

Optimized clove oil NEs prepared using SCas (5%) and pectin (0.1%) as coating material by 
high speed homogenization with spherical NPs of 172.1±4.39 nm, zeta potential of -37±1.93 mV 
and EE of 88% showing stability at all food processing conditions except pH 3.0−5.0 could be 
recommended as delivery system for antimicrobial bioactive substances in food preservation 
(Sharma et al. 2017). Optimized ultrasound-mediated nettle oil (1.25 wt%) NEs stabilized by 
purified jujube polysaccharide with 86.75 nm droplet size inactivated the Gram-positive 
bacterium more effectively than the Gram-negative one (Gharibzahedi 2017). Stable NEs 
prepared by mixing eugenol with SCas using shear homogenization observed up to 38.5 mg/mL 
eugenol showed droplet diameters <125 nm at pH 5−9 after ambient storage for up to 30 days 
and more effective inhibition of E. coli O157:H7 than free eugenol during incubation at 37 °C for 
48 h. A greater reduction of intracellular ATP and a greater increase of extracellular ATP was 
observed in bacteria treated with encapsulated eugenol (20 min interaction at 21 °C) compared to 
free eugenol suggesting enhanced permeation of eugenol due to its nanoencapsulation, although 
the possible membrane adaptation could not be excluded (Zhang et al. 2018). Salvia-Trujillo et 
al. (2014) reported that lemongrass oil−alginate NEs prepared by microfluidization exhibited 
enhanced antimicrobial activity against E. coli, while ultrasound processing of NEs led to loss of 
their bactericidal action. 
Thyme O/W NEs (pH 3.5) were found to be highly unstable to droplet growth and phase 
separation, which could be connected with Ostwald ripening due to the relatively high water 
solubility of thyme oil. Inhibition of Ostwald ripening could be achieved by mixing thyme oil 
with a water-insoluble ripening inhibitor (≥60 wt% corn oil or ≥50 wt% MCT in the lipid phase) 
before homogenization, yielding NEs with good physical stability. However, ripening inhibitor 
type and concentration had a notable impact on the antimicrobial activity of EO and increasing 
the ripening inhibitor levels in the lipid phase resulted in the reduction of the antimicrobial 
efficacy of NEs (Chang et al. 2012). 
Comparison of the in vivo oral bioavailability study of conventional emulsion (droplet diameter 
1.285 µm) and NEs of vitamin E prepared using sunflower oil and saponin (droplet diameter 
0.277 nm) showed that the in vivo oral bioavailability of vitamin E in male Wistar rats at NE 
application reached a 3-fold higher AUC compared to the conventional emulsion (Parthasarathi 
et al. 2016). Bovi et al. (2017) reported that NEs of Buriti (Mauritia flexuosa L.) oil, one of the 
richest vegetal sources of carotenoids, could be successfully incorporated in isotonic sports drink 
and replace the artificial coloring by natural dyes. 
In O/W NEs stabilized by soy lecithin and encapsulating carvacrol the zwitterionic lecithin 
molecules adsorbed to the O/W interface for 24 h formed a notably viscoelastic layer, at pH 7 the 
NEs were highly stable, yielding monodispersed droplet size distributions and high resistance to 
increases in droplet size over 30 days, although the initial size of oil droplets slightly depended 
on pH (smaller droplets at pH 7 and larger droplets at pH 3) (Nash and Erk 2017). 
Eugenol NE prepared by using ultrasonication as emulsification techniques, CS NPs as carrier, 
and Tween 20 surfactant as emulsifier, with regularly spherical shape and sizes ranging from 80 
to 100 nm were characterized with great storage and thermal stability and exhibited superb 
antioxidant capacity and antimicrobial activity suggesting their great potential to be used in food 
formulations for extending the shelf life (Shao et al. 2018). 
O/W chia seed oil NE systems prepared by spontaneous emulsification and microfluidization and 
stabilized with Tween 80 and Span 80, as well as SCas- and sucrose monopalmitate-stabilized 
NEs fabricated by microfluidization were characterized by storage stability at 4 °C during 2 
weeks. The NEs prepared with sucrose monopalmitate showed best transparency with droplet 
diameter  ca. 47 nm (Teng et al. 2018). Blended cloves/cinnamon EO NEs fabricated using 
Tween 80 surfactant and ethanol (cosurfactant) with oil to the mixed surfactant/cosurfactant ratio 
of 1:9 showed higher antimicrobial activity against E. coli, B. subtilis, S. typhimurium, and S. 
aureus, even at far lower concentrations than free EOs and this NE could be applied as a natural 
antimicrobial agent in food industry (Zhang et al. 2017). 
Digested Hibiscus cannabinus L. seed O/W NEs stabilized by SCas, Tween 20 and β-
cyclodextrin complexes produced using high pressure homogenization showed good lipid 



22 

digestion (85.25%), good bioaccessibility of antioxidants (tocopherols and total phenolic 
contents) and lower degradation rate of phytosterols compared to digested bulk oil suggesting 
potential use of such formulation in food and nutraceutical preparations (Cheong et al. 2016a). 
Addition of β-cyclodextrin to primary emulsion containing SCas and Tween 20 was found to 
improve the physical stability of kenaf (Hibiscus cannabinus L.) seed O/W NEs (Cheong and 
Nyam 2016). 
Negatively charged NEs of black cumin EO fabricated using different ratios of EO with canola 
and flax seed oils (ripening inhibitors) that were stabilized with octenyl succinic anhydride 
(OSA) modified waxy maize starch (mean droplet diameter >200 nm and zeta potential above -
30 mV) showed prolonged bactericidal activities against Bacillus cereus and L. monocytogenes 
than the pure black cumin EO, which could be explained with its better stability, controlled 
release and self-assembly with the cell membrane of Gram-positive bacteria resulting finally in 
destruction of cellular constituents (Sharif et al. 2017a). 
Alexandre et al. (2016) reported about activated films with improved physical properties 
prepared by incorporation of montmorillonite and nanoemulsioned ginger EO into gelatin-based 
films with notably better elongation at break, puncture force and puncture deformation and 
showing antioxidant activity, which could be used in food packaging applications. 
 
2.5.3 Vitamin nanoemulsions 
 
The use of natural surfactants, Quillaja saponin and lecithin, on the formation and stabilization of 
NE-based vitamin E delivery systems was described by Ozturk et al. (2014). At simulated small 
intestine conditions, the rate of lipid digestion and tocotrienol bioaccessibility in bulk oil and 
within O/W conventional emulsions (>10 µm) and NEs (<350 nm) fabricated using MCT as an 
oil phase (5 to 40% wt) and Quillaja saponins as a natural surfactant decreased as follows: NEs > 
emulsions > bulk oil (Xu et al. 2018). The increase of the bioavailability of α-tocopherol as a 
food supplement could be secured through edible (coconut) oil NE showing a 9.5 mg/mL of 
encapsulation capacity and almost 100% release of the loaded active ingredient within 24 h, 
whereby the contribution of kinetic-controlled release was found approx. 70% and that of 
diffusion-controlled release was found approx. 30%. Beside of good stability of NE a reasonable 
cell viability (biocompatibility) with apposite antimicrobial activity was estimated suggesting 
potential application of such edible oil NE in food, beverages, and health care industries (Saxena 
et al. 2018). The α-tocopherol based O/W NE fabricated using sodium stearoyl lactate and 
Tween 80 surfactants by high energy ultrasonication method with encapsulated 
benzylisothiocyanate acted as better antioxidant compared to pure and CUR encapsulated NE; 
the prepared emulsions exhibited good stability up to 90 days in salt solution (50−200 mM) and 
different pH conditions and the degradation of CUR by UV light was successfully controlled by 
trapping in NE (Kaur et al. 2017). 
The impact of antioxidants on the thermal stability of β-carotene encapsulated in diluted O/W) 
emulsions prepared using gum arabic decreased as follows: α-tocopherol > tertiary butyl 
hydroquinone > ascorbyl palmitate, the most effective antioxidant being α-tocopherol at the 
concentration of 0.10 wt% under light exposure (Liu et al. 2015b). Digested Ulva fasciata 
polysaccharide-stabilized emulsions encapsulating β-carotene (mean particle size of 0.82 µm) 
showed higher lipid digestion rate and increased β-carotene bioaccessibility than gum arabic- or 
beet pectin-stabilized emulsions suggesting t