nanomaterials

Article
Preparation and Characterization of
Water-Based Nano-Perfumes
Małgorzata Miastkowska 1, *, Elwira Lasoń 1 , Elżbieta Sikora 1 and
Katarzyna Wolińska-Kennard 2
1 Institute of Organic Chemistry and Technology, Cracow University of Technology, Warszawska 24,
31-155 Krakow, Poland; [email protected] (E.L.); [email protected] (E.S.)
2 Finea Sp. Z o.o., Sternicza 129/55, 01-350 Warsaw, Poland; [email protected]
* Correspondence: [email protected]




Received: 25 October 2018; Accepted: 23 November 2018; Published: 27 November 2018


Abstract: The application of nanoemulsions as a novel delivery system for lipophilic materials,
such as essential oils, flavors, and fragrances is one of the growing technologies used in cosmetic,
pharmaceutical, and food industries. Their characteristic properties, like small droplet size with
high interfacial area, transparent or semi-transparent appearance, low viscosity, and high kinetic
stability, make them a perfect vehicle for fragrances, in the perfume industry. They could be a great
alternative to water-based perfumes, without alcohol, and solve problems related to the oxidation and
low bioavailability of fragrances with other non-alcoholic vehicles of perfumes like pomades or gels.
The aim of our study was to develop stable Oil-in-Water (O/W) nanoemulsions that are compatible
with selected fragrance compositions, without ethanol, polyols, and ionic surfactants, and to study
their physicochemical, microbiological, and dermatological properties. The nano-perfume systems
were obtained with a low-energy (Phase Inversion Composition; PIC) and with a high-energy
(ultrasound, US) method, taking into account the possibility of moving from the laboratory scale to
an industrial scale. The optimized nano-perfume formulations, prepared with different methods,
yielded the same physicochemical properties (stability, medium droplet size of the inner phase,
polydispersity, viscosity, surface tension, pH, density). Stable systems were obtained with a fragrance
composition concentration within 6–15% range. These formulations had a low viscosity and a pH
suitable for the skin. Moreover, the obtained results confirmed the protective role of nanoemulsions.
The peroxide number measurement (POV) showed that the tested fragrance compositions had a high
chemical stability. The results of the microbiological tests confirmed that the obtained products were
free of microbiological contamination and were appropriately preserved. The dermatological test
results confirmed the safety of the developed preparations.

Keywords: fragrances; nano-perfumes; nanoemulsions

1. Introduction
Ethanol forms the base for traditional perfumes available on the market, since it is the
solvent of hydrophobic aromatic substances. Depending on the contents of a fragrance composition,
we distinguish between eau de parfum which contains 10–15% of a fragrance composition, eau de
toilette which contains 5–10% of a fragrance composition, and eau de cologne which contains 3–5% of
a fragrance composition [1,2]. Unfortunately, people prone to allergies or with sensitive skin, who use
ethanol-based perfumes, may suffer from skin irritation and inflammation because ethanol is a solvent
with a defined irritating potential [3].
As an alternative to alcohol compositions, there are perfumes in a solid state (solid emulsions,
gels, and pomades) and perfumed oils. However, their main disadvantage is that they leave greasy

Nanomaterials 2018, 8, 981; doi:10.3390/nano8120981 www.mdpi.com/journal/nanomaterials

Nanomaterials 2018, 8, 981 2 of 15

and slippery spots on the perfumed surfaces. Some examples of perfumed products are presented
in the literature and they are based on a safe solvent like water, the so-called “alcohol-free perfumes”
(emulsions, microemulsions, liposomes, and micelles) [4–9]. Yet from a technological perspective,
the introduction of lipophilic systems to water without a cosurfactant (ethanol, among others) is very
difficult, with respect to thermodynamic stability. It requires the use of solubilizers of essential oils
which can be polyols (glycols and glycerin) or surfactants.
An interesting solution for the water-based perfumes without alcohol, seem to be
nanoemulsions—liquid colloidal systems characterized by a high degree of dispersion (20–500 nm),
consisting of aqueous and oil phases and a surfactant, sometimes with the addition of a cosurfactant.
Similar to microemulsions, nanoemulsions increase the expiry date of many products, due to their
resistance to sedimentation and creaming. An additional advantage over microemulsions is that they
have a much lower amount of a surfactant (approx. 5–10%), which allows maintaining adequate
stability of the system and it makes them safe for human body [10–14]. Clear appearance, liquidity,
and low viscosity, make nanoemulsions even more popular with the cosmetic industry. The examples
of nanoemulsion cosmetics found on the market are anti-UV hair spray by Korres, Nanocream by
Sinerga, Nanogel by Kemira, Vital Nanoemulsion A-VC serum by Marie Louise, Bepanthol Ultra face
cream by Bayer, or face cleanser NanoVital by Vitacos Cosmetics [15,16]. Moreover, the international
cosmetics company L’Oreal patented a number of formulas for nanoemulsion cosmetics [15,16]. In the
case of perfume products, nanoemulsions can be not only a medium for a fragrance composition,
but they also increase chemical stability of the compounds in the composition (protection against
oxidation) [12,13,17,18].
Alcohol-free perfumes based on micro- and nanoemulsions are already present in the patent
literature [19–22], however, apart from additional solubilizers, such as polyols or paraffin hydrocarbons,
the compositions of the perfumes in the above-mentioned patents have cationic or anionic surfactants,
which can be an irritant to the skin. Nanoemulsions that serve as a matrix for fragrance substances
used in cosmetics are also described in the literature [18,23–28] these are primarily solutions
for emulsification of a single lipophilic component that forms the oil phase of a nanoemulsion
(e.g., D-limonene) [18,23,24]. On the other hand, the fragrance compositions contain up to twenty
compounds with various chemical structures (alcohols, phenols, aldehydes, esters, saturated,
unsaturated, cyclic, and branched hydrocarbons), and as a result they are a difficult base for obtaining
stable nanoemulsion systems.
Nanoemulsions turned out to be a great solution for solving problems related to the oxidation and
low-bioavailability of fragrances and they could be applied as nano-encapsulated fragrance systems,
in the perfume industry. Therefore, the most important aspect for further research would be to make
the preparation of fragrance nanoemulsions practicable, at a pilot scale, so as to make them possible to
be adopted in the industrial full-scale production.
The aim of the research was to develop stable oil-in-water (O/W) nanoemulsions that are
compatible with selected fragrance compositions, without ethanol, polyols, and ionic surfactants, and to
study the physicochemical, microbiological, and dermatological properties, as well as permanence
of the fragrance of the obtained nano-perfumes. The nano-perfume systems were obtained with
a low-energy method (Phase Inversion Composition; PIC), as well as with an ultrasound (US)
high-energy method, taking into account the possibility of moving from the laboratory scale to
an industrial scale.

2. Materials and Methods

2.1. The Properties of Raw Materials

In order to obtain dispersive systems with the droplet size of 20–500 nm of the inner phase,
the research concerned the influence of the type and concentration of the oil phase (of a given
fragrance composition) and of the type and concentration of the surfactant on the stability of
Nanomaterials 2018, 8, 981 3 of 15

nanoemulsion systems. The emulsifiers used in the study were supplied by the Croda company
(Krakow, Poland) (Table 1). Fragrance compositions were made by European Flavours & Fragrances
PLC (Hertfordshire, UK). All tested fragrances are of GRAS (Generally Recognised As Safe) status,
allergen free. Milli-Q®filtered water (Merck, Warsaw, Poland) was used as the aqueous phase of the
nanoemulsions. The preservative used was Dermosoft 1388 (up to 1%), kindly supplied by Evonik Dr.
Straetmans GmbH (Germany).

Table 1. Emulsifiers used in the research.

No. Polish Name/Trade Name INCI Name HLB

1. Cithrol 10GTIS PEG-20 Glyceryl Triisostearate 10.3
2. Decyl Glucoside Decyl Glucoside 13.0–14.0
Polyglyceryl-4 Laurate/Sebacate
3. Natragem S140 (and) Polyglyceryl-6 14.0
Caprylate/Caprate (and) Water
4. Etocas 35 PEG-35 Castor Oil 13.0

Nonionic surface active agents were used in the research. Those surfactants belong to the group
of polyoxyethylated esters of glycerin and fatty acids, polyoxyethylated castor oil, polyglycerol and
fatty acids esters, and alkyl polyglucosides. Surfactants of this type are known for their very good
performance and dermatological properties. They show biocompatibility with the skin and they
are used in cosmetics, such as solubilizers, humectants, dispersing agents, and emulsifiers, for the
stabilization of O/W emulsions. In comparison to the ionic surfactants, both cationic and anionic,
they are not susceptible to pH change and the addition of electrolytes. They can be used with other
emulsifiers to increase the system stability [13,29–31].

2.2. The Method of Obtaining Nanoemulsions

2.2.1. Phase Inversion Composition Method (PIC)

To obtain perfumes in the form of a nanoemulsion, the low-energy method was used
(Phase Inversion Composition—PIC). The nanoemulsions were obtained by a gradual addition of
water to the mixture of a surfactant with the oil phase (the fragrance composition), at room temperature
(25 ◦ C), with constant stirring (IKA Vortex Genius 3 shaker).

2.2.2. Ultrasonic Homogenization Method (US)

The nano-perfumes in the nanoemulsion form were obtained with a high-energy method that
requires ultrasonic homogenization with initial pre-emulsification. A specified amount of the oil phase
(fragrance composition), surfactant, and demineralized water was dispersed at room temperature
(25 ◦ C), with a mechanical stirrer (IKA◦ RW 20 digital), 500 rpm, for 10 min. The obtained pre-emulsion
underwent ultrasonic homogenization (probe-type sonicator UP200Ht, Hielscher Company, Teltow,
Germany) with 15 W for at least 60 s.

2.3. Tests of the Physicochemical Properties of the Fragrance Compositions

In order to establish the properties of the fragrance compositions used in the research, their
surface tension was measured with a tensiometer STA-1 by Sinterface (Berlin, Germany) with a du
Noüy ring. Viscosity was determined with an R/S (Cone/Plate) rotational rheometer with cone/plate
measuring elements (by Brookfield). Moreover, the value of logP was calculated (i.e., lipophilicity of
the composition) on the basis of the qualitative and quantitative constituents of the used fragrance
compositions provided in material data sheets. This was determined by calculating the lipophilicity
(logP, P—Partition coefficient) of individual relevant constituents and the percentage composition of
the fragrance [32].
Nanomaterials 2018, 8, 981 4 of 15

2.4. Tests of the Physicochemical Properties of the Nanoemulsion

In order to determine the physicochemical properties of the obtained systems, the following
analytical methods and techniques were used. The size of the droplets of the inner phase was analyzed
with a Zetasizer Nano ZS droplet analyzer (Malvern Instruments, Malvern, UK). The kinetic stability
of the emulsion systems was monitored with the analysis of the droplet size of the inner phase of the
nanoemulsion and the polydispersity index (PDI) over time. To determine the rheological properties
of the obtained formulations, just like in the case of pure compositions, an R/S rotational rheometer
was used with cone/plate measuring elements (cone C25-1), at room temperature (25 ◦ C). Viscosity
tests were conducted with a variable cutting rate, within the range of 1–500 rps. The surface tension
of the emulsion was also measured with a tensiometer and a du Noüy ring, in the same way as
in the case of the fragrance compositions. The pH value of the emulsion system was determined
with a multi-functional measuring tool (Seven Multi by Mettler Toledo, Warsaw, Poland) which was
equipped with an electrode for measuring pH. Density was established with a hydrostatic method,
using an analytical balance (AS310X0, Radwag, Krakow, Poland), with a density measuring device.

2.5. The Test of the Microbiological Properties of the Nanoemulsion

The test of microbiological purity of the obtained stable products was conducted according to the
following polish standards: PN-EN ISO 21149:2009, PN-EN ISO 162012:2011, PN-EN ISO 22718:2010,
PN-EN ISO 21150:2010, and PN-EN ISO 18416:2009. The preservation tests were carried out according
to the standard PN-EN ISO 11930:2012. The following sample strains were used: Pseudomonas aeruginosa
ATCC 9027, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 11229, Candida albicans ATCC
10231, and Aspergillus brasilensis ATCC. With regard to every checked microorganism, the test involved
the treatment of a cosmetic preparation with a standardized inoculum and then the measurement of
the number change of the microorganisms, at given intervals, during a specified period and under
a given temperature.

2.6. The Test of the Dermatological Properties of the Nanoemulsion

The dermatological test of the obtained products was conducted with a patch test on a group
of twenty-five testers (15 women and 10 men, age 19–55), under a dermatologist’s supervision,
in accordance with the applicable legal provisions and guidelines for dermatological research on
humans. The tests were conducted in a specialist dermatological medical room. Patch tests were
applied on the inner side of the forearm. The tested preparation was rubbed onto a 1 × 1 cm skin
patch, the area with an applied cosmetic was covered with a 2 × 2 cm filter paper and foil attached
with an adhesive. The dressing was taken off after 24 h and the result was assessed organoleptically.
Further dermatological check was carried out after 3, 4, and 5 days, after application. The assessment
was conducted according to the generally accepted scale for dermatological tests [33].

2.7. The Test of Fragrance Permanence of the Nanoemulsion

The test of fragrance permanence was carried out in accordance with the standard BN-84/6148-02.
Two strips of filter paper, 150 mm long and 15–20 mm broad, were dipped 20 mm in the samples;
the first one was dipped in a tested sample, and the second one in a reference sample, and left at the
temperature of 25 ◦ C. The intensity of the fragrance was assessed organoleptically after 1, 24, and 48 h
after dipping. The samples were assessed by comparing the intensity of the fragrance of the tested
sample and the reference sample.

2.8. The Test of Chemical Stability of the Fragrance Composition (Oxidative Stress Test)
The oxidative stress test was carried out to demonstrate the chemical stability of the fragrances and
protecting effect of nanoemulsions against peroxidation of the fragrance composition. The oxidation
process induced by the oxygen from air and by UV, was determined by measuring the peroxide value
Nanomaterials 2018, 8, 981 5 of 15

of the fragrances, according to Wheeler DGF standard method C-VI 6a [34]. The value was measured
as the amount of iodine which was formed by the reaction of peroxides (formed in oil) with iodide ion.
The sample was added to a mixture of glacial acetic acid and isooctane (60/40 v/v) and then allowed
to react with potassium iodide (0.5 cm3 of saturated solution). The iodine released was determined by
titration, using 0.01 N sodium thiosulfate solution. The titration end point was specified iodometrically.
The peroxide value (POV) was calculated according to Equation (1):

( a − b) × M × 1000
POV = (1)
2×Q

where a is the consumed volume of sodium thiosulfate solution, b is consumed volume of sodium
thiosulfate solution in the blank test, M is the molarity of the sodium thiosulfate solution, and Q is the
quantity of the tested sample with accuracy ±0.1 mg. Each sample was assessed in triplicates (n = 3).

2.9. Statistical Analysis

All data presented in the plots were presented as a mean of three different experiments ± SD.
Differences between the calculated means of each individual group were determined by one-way
ANOVA tests, using the statistical software Statistica Version 12 StatSoft Company., Cracow, Poland.
A value of p < 0.05 was considered statistically significant.

3. Results and Discussion

3.1. The Properties of the Tested Fragrance Compositions

In order to establish the properties of a fragrance composition, their logP was calculated and the
surface tension (τ) and viscosity coefficient (η) were tested.
As can be seen from Table 2, composition B had the highest logP, surface tension, and viscosity
values. In the case of other fragrances, the values were comparable.

Table 2. Physicochemical properties of fragrance compositions.

Name of the Fragrance Composition logP τ (mN/m) η (mPa·s) for γ = 1/100 s

A 2.92 23.3 123
B 4.35 30.7 150
C 3.12 25.1 108
D 3.48 24.8 102
E 3.75 26.1 140
F 3.25 26.4 129

3.2. The Physicochemical Properties of the Obtained Nanoemulsions

So far, a few methods of obtaining nanoemulsions with a low-energy method have been proposed,
including spontaneous emulsification, phase inversion method, and solvent evaporation method,
which perform well in the laboratory but not on an industrial scale [35]. Among the different literature
reports, ultrasound homogenization is one of the most attractive methods for direct production of
nanoemulsions on a larger scale [36]. However, comparative studies between scalable and non-scalable
methods have not been documented enough. Even if numerous studies report the properties
of nanoemulsions obtained with different methods [37], still there is no substantial knowledge
about the possibility of producing a preparation optimized with a laboratory method, on a small
scale, by applying a large-scale production method. This paper presents the comparison of the
physicochemical properties of the optimized nanoemulsions as the media of the selected fragrance
compositions obtained with a non-scalable PIC method, with systems obtained with a scalable
US method.
Nanomaterials 2018, 8, 981 6 of 15

3.2.1. The Influence of a Surfactant on the Nanoemulsion Stability

In the course of the study, four hydrophilic emulsifiers were selected with an HLB
(Hydrophilic-Lipophilic Balance) of 10.3–14.0, due to their very good dermatological and application
properties. In order to select an optimal emulsifier, two tests were carried out—for a 6% fragrance
composition concentration (corresponding to concentrations of eau de toilette, i.e., 5–15%), and for an
emulsifier concentration up to 10% (not more than the traditional emulsifier systems that are safe for
the skin). Table 3 presents the composition and stability of preparations obtained with the PIC and the
US methods.

Table 3. The influence of a fragrance composition (O) and emulsifiers (S) on the physicochemical
properties of nanoemulsions (ratio S:O = 0.625:0.375) obtained with both methods.

Physicochemical Properties
Emulsifier Fragrance Composition Appearance
Z-Ave (nm) PDI Stability
(n = ±S.D.) (n = ±S.D.) After 24 h
A Milky - - -
B Milky - - -
C Milky - - -
Cithrol 10GTIS
D Milky - - -
E Milky - - -
F Milky - - -
A Milky - - -
B Milky - - -
C Milky - - -
Decyl Glucoside
D Transparent 21.0 ± 0.2 0.269 ± 0.013 +
E Milky - - -
F Milky - - -
A Semi-transparent, bluish 123 ± 15 0.750 ± 0.008 +
B Milky - - -
C Semi-transparent, bluish 106 ± 20 0.692 ± 0.066 +
Natragem S140
D Semi-transparent, bluish 151 ± 44 0.633 ± 0.002 +
E Semi-transparent, bluish 120 ± 10 0.592 ± 0.066 -
F Semi-transparent, bluish 105 ± 14 0.433 ± 0.002 +
A Transparent, bluish 27.2 ± 0.9 0.509 ± 0.061 +
B Transparent, bluish 23.5 ± 0.4 0.308 ± 0.007 +
C Transparent, bluish 19.8 ± 0.2 0.214 ± 0.002 +
Etocas 35
D Transparent, bluish 22.0 ± 0.3 0.346 ± 0.007 +
E Transparent, bluish 30 ± 2 0.467 ± 0.060 +
F Transparent, bluish 25 ± 1 0.537 ± 0.012 +
- unstable sample (separation); + stable sample (homogeneous).

The data in Table 3 show that stable nanoemulsion systems were obtained with both methods,
for all six fragrance compositions, with Etocas 35 (PEG-35 castor oil) only among four of the tested
emulsifiers. Nanoemulsions obtained by the US method had properties similar to formulations
obtained with a PIC method. Z-Ave diameter and PDI did not show any significant differences
(p > 0.05).
According to the literature, the physicochemical properties of the nonionic emulsifiers, used for
obtaining nanoemulsions, play a crucial role during emulsification. The most popular group of
nonionic emulsifiers used for obtaining nanoemulsions are Tweens (ethoxylated esters of sorbitan and
fatty acids) and Cremophors (ethoxylated derivatives of castor oil) [38].
Zheng et al. [38] worked on obtaining O/W nanoemulsions with a spontaneous emulsification
method. On the basis of their studies, they concluded that Cremophors had better emulsification
properties than Tweens. Out of the tested emulsifiers (Cremophor RH 40, Cremophor EL, Tween 20,
Tween 80), nanoemulsions with the smallest droplet size and lowest PDI were obtained by stabilizing
systems with Cremophor RH 40 (INCI: PEG-40 hydrogenated castor oil) and Cremophor EL
(INCI: PEG-35 castor oil). The authors of the study found no correlation between the droplet size of
the nanoemulsion and the HLB of the applied emulsifier. Tween 20 and Tween 80 have a similar HLB
and the nanoemulsions stabilized with them had different particle sizes—179 and 122 nm, respectively.
They explained the results with the impact of the molecular structure of the surfactant and, therefore,
Nanomaterials 2018, 8, 981 7 of 15

the packing parameter on emulsification. The differences between packing parameters of a surfactant
affect the curvature of its monolayer on the inter-phase surface [39]. The changes of this curvature,
during nanoemulsion emulsification, are crucial parameters in the process of obtaining nanoemulsions
with low-energy methods (Phase Inversion Temperature (PIT); Phase Inversion Composition (PIC);
Self-Emulsification (SE); Catastrophic Phase Inversion (CPI); Emulsion Phase Inversion (EPI)).
On the other hand, Tang et al. [31] obtained nanoemulsions that were stabilized with
Cremophor EL with the US method. The oil phase was formed by propylene glycol monolaurate.
Nanoformulations were characterized with kinetic stability and droplet size under 200 nm. It was
observed that the optimal surfactant concentration for the greatest dispersion rate of the systems was
in the range of 5–6%. The authors of the research supposed that a significant decrease in the droplet
size could be attributed to the presence of the polar covalent parts in the Cremophor EL chemical
structure (polyethylene glycols and ethoxylated glycerol). These two components have good properties
to solubilize various essential oils and hydrophobic drugs. Due to these two highly hydrophilic groups
in the aqueous phase, the viscosity difference between the two immiscible phases is reduced, leading to
a decrease in the critical Weber number, followed by an increased droplet break-up efficiency. The good
stability of nanoemulsion droplets can be attributed to the steric stabilizing effect of nonionic emulsifier
(Cremophore EL), in which it prevents flocculation and coalescence, by forming a thick steric barrier
against droplet merging.
Moreover, many other studies showed that Cremophor EL was perfect to obtain nanoemulsion
systems [10,40–43].

3.2.2. The Effect of the Surfactant:Oil (S:O) Mass Ratio on Nanoemulsion Properties
In the next stage of the study, the emulsifier concentration was reduced to 6%, while
maintaining the same fragrance composition concentration (6%) (S:O ratio = 0.5:0.5). Attempts
were also made to increase the fragrance composition concentration to 15% (corresponding to the
composition concentrations in eau de parfum), while maintaining the surfactant concentration at 10%
(S:O ratio = 0.4:0.6). Table 4 shows the results of the analysis of particle size and PDI for three different
surfactant:oil (S:O) mass ratios on the day of obtaining the preparation.

Table 4. The effect of the mass ratio of the surfactant—Etocas 35 (S)—in relation to
fragrance composition (O) on the particle size (Z-Ave) and polydispersity index (PDI) of the
obtained nanoemulsions.

S:O
Composition (1)—0.625:0.375 (2)—0.5:0.5 (3)—0.4:0.6
Z-Ave (nm)/PDI (n = ±S.D.)
A 27.2 ± 0.9/0.509 ± 0.061 29.1 ± 0.1/0.241 ± 0.021 96 ± 20/0.536 ± 0.059
B 23.5 ± 0.4/0.308 ± 0.007 84 ± 14/0.557 ± 0.019 133 ± 5/0.471 ±0.013
C 19.8 ± 0.2/0.214 ± 0.002 30.2 ± 0.4/0.217 ± 0.016 88 ± 5/0.497 ± 0.085
D 22.0 ± 0.3/0.346 ± 0.007 28.0 ± 0.4/0.179 ± 0.009 62 ± 1/0.416 ± 0.004
E 30 ± 2/0.467 ± 0.060 32 ± 2/0.292 ± 0.012 40.3 ± 0.6/0.286 ± 0.006
F 25 ± 1/0.537 ± 0.012 23.0 ± 0.3/0.093 ± 0.009 41.2 ± 1.1/0.345 ± 0.029
Legend: (1)—surfactant:oil (S:O) mass ratio = 0.625:0.375; (2)—surfactant:oil (S:O) mass ratio =
0.5:0.5; (3)—surfactant:oil (S:O) mass ratio = 0.4:0.6.

As can be seen from the data in Table 4, all fragrances (A–F) achieved satisfactory values
with reduced emulsifier concentration (S:O = 0.5:0.5) and with increased fragrance composition
concentration (S:O = 0.4:0.6) The droplet size did not exceed 150 nm and the PDI was not greater
than 0.5, in most cases. However, increasing the fragrance composition concentration (S:O = 0.4:0.6)
resulted in increasing the average droplet size of the nanoemulsion and PDI, which is consistent
with the literature data, according to which the droplet size increases with the concentration of the
 As can be seen from the data in Table 4, all fragrances (A–F) achieved satisfactory values with
reduced emulsifier concentration (S:O = 0.5:0.5) and with increased fragrance composition
concentration (S:O = 0.4:0.6) The droplet size did not exceed 150 nm and the PDI was not greater than
0.5, in most cases. However, increasing the fragrance composition concentration (S:O = 0.4:0.6)
Nanomaterials 2018, 8, 981 8 of 15
resulted in increasing the average droplet size of the nanoemulsion and PDI, which is consistent with
the literature data, according to which the droplet size increases with the concentration of the inner
inner
phase phase [10,40,44–46].
[10,40,44–46]. On theOn thehand,
other other the
hand, the systems
systems in whichinthe
which the surfactant
surfactant concentration
concentration was the
was the (S:O
highest highest (S:O = 0.625:0.375),
= 0.625:0.375), showed significantly
showed significantly larger dispersion
larger dispersion rate. Asin
rate. As reported reported in the
the literature,
literature, high surfactant
high surfactant concentration
concentration can cause can cause dissolution
complete complete dissolution of the in
of the oil phase oilwater,
phase atinthe
water, at the
emulsion
emulsion
inversioninversion
point, butpoint, but low surfactant
low surfactant concentration
concentration resultedresulted in the formation
in the formation ofdroplets.
of larger larger droplets.
When
When surfactant:oil
surfactant:oil ratio (S:O)
ratio (S:O) was was
highhigh enough,
enough, the the surfactant
surfactant formed
formed thelayer
the layerstructure
structure at at phase
phase
inversion
inversion point,
point, and
and then the minimal interfacial tension was formed and promoted promoted the formation of
small droplets
droplets [18].
[18].

3.2.3.
3.2.3. The
The effect
effect of
of the
the Fragrance
Fragrance Compositions
Compositions on
on Nanoemulsion
Nanoemulsion Properties
Properties
With
With regards
regards to
to the
the effect
effect of
of the
the type
type of
of composition
composition on on the
the physicochemical
physicochemical properties
properties ofof the
the
nanoformulation (Figure 1, Table 4), Composition B with the highest lipophilicity (logP
nanoformulation (Figure 1, Table 4), Composition B with the highest lipophilicity (logP = 4.35, Table = 4.35, Table 2),
surface tension
2), surface (τ =(τ30.7
tension mN/m,
= 30.7 mN/m, Table
Table2),2),
and viscosity
and viscositycoefficient
coefficient(η (η
= 150 mPa
= 150 ·s, Table
mPa·s, 2) 2)
Table yielded
yieldeda
nanoemulsion with the largest droplet size and the highest PDI (for S:O = 0.5:0.5 and
a nanoemulsion with the largest droplet size and the highest PDI (for S:O = 0.5:0.5 and 0.4:0.6). In the 0.4:0.6). In the
case
case of
of other
other Compositions
Compositions A, A, C,
C, D,
D, E,
E, and
and FF (Table
(Table 4,
4, Figure
Figure 1),
1), no
no major
major differences
differences were
were observed
observed
between
between thethe droplets
droplets ofof the
the inner
inner phase.
phase.

Figure 1.
Figure 1. Effect
Effect of
of aa fragrance
fragrance composition
composition on the
the particle
particle size
size and
and polydispersity
polydispersity index (PDI) of the
the
nanoemulsion (S:O
nanoemulsion (S:O = = 0.5:0.5) (SD, n = 3).

According to the literature,

literature, there
there is
is no
no clear
clear relationship
relationship between
between the
the size
size of
of the
the nanoemulsion
nanoemulsion
droplets
droplets and physicochemical properties
properties of the oil phase (refractive
(refractive index,
index, density,
density, interfacial tension,
and viscosity). Instead, the
viscosity). Instead, the phase
phase behavior
behavior of the surfactant-oil-water system is likely to be more
surfactant-oil-water system
important
important [44,47].
[44,47].
The effect
effect ofofthe
theoiloil phase
phase on nanoemulsion
on the the nanoemulsion droplet
droplet size obtained
size obtained with a low-energy
with a low-energy method
method (EPI), was studied by Ostertag et al. [47]. The systems were stabilized
(EPI), was studied by Ostertag et al. [47]. The systems were stabilized with emulsifiers from with emulsifiers
from oxyethylated
oxyethylated estersesters of sorbitan
of sorbitan and acids
and fatty fatty (Tween
acids (Tween 20, Tween
20, Tween 80, Tween
80, Tween 85).
85). On theOn theof
basis basis
the
of the conducted experiments, the researchers found out that the smallest nanoemulsion droplet
sizes were obtained with medium-chain triglycerides, then with essential oils (orange and limonene),
and the largest droplets when the oil phase consisted of natural oils with long-chain triglycerides
(olive, grape, sesame, peanut, and canola oils). Out of the natural oils, the nanoemulsions with the
smallest droplet size of the inner phase were obtained with olive, and the largest ones with canola oil.
Similar studies were carried out by Kmaiko and McClements [44], with the same types
of oil phases and surfactants. The difference lay in that in order to receive the nanoemulsion,
a different low-energy method—spontaneous emulsification (SE)—was used. Additionally, in this case,
the droplet size followed the order—medium chain triglycerides < flavor oils < long-chain triglycerides.
However, in contrast to the observations made by Ostertaga et al. [47], the nanoemulsions made on
the basis of olive oil had the lowest dispersion index.
As was already mentioned, the literature reports on nanoemulsion, as a matrix for fragrance
composition, concern mostly the emulsification of a single lipophillic component, mainly
phases and surfactants. The difference lay in that in order to receive the nanoemulsion, a different
low-energy method—spontaneous emulsification (SE)—was used. Additionally, in this case, the
droplet size followed the order—medium chain triglycerides < flavor oils < long-chain triglycerides.
However, in contrast to the observations made by Ostertaga et al. [47], the nanoemulsions made on
the basis of olive oil had the lowest dispersion index.
Nanomaterials 2018, 8, 981 9 of 15
As was already mentioned, the literature reports on nanoemulsion, as a matrix for fragrance
composition, concern mostly the emulsification of a single lipophillic component, mainly D-
limonene. [18,23,24].
D-limonene. [18,23,24].Li Li et etal.al.[18]
[18]obtained
obtainednanoemulsions
nanoemulsions with with thethe CPI
CPI (Catastrophic Phase Phase
Inversion) method,
method, on onthe
thebasis
basisofofolive oiloil
olive as as
a carrier of D-limonene,
a carrier of D-limonene, stabilized withwith
stabilized Tween 80. The
Tween 80.
nanoemulsions were obtained with a maximum 15% concentration of
The nanoemulsions were obtained with a maximum 15% concentration of olive oil. Similar resultsolive oil. Similar results were
achieved
were whenwhen
achieved corn corn
oil, sunflower
oil, sunfloweroil, and
oil, soybean oil were
and soybean usedused
oil were instead of olive
instead oil. However,
of olive oil. However, the
obtained
the systems
obtained differed
systems in droplet
differed sizes (the
in droplet smallest
sizes (the in the caseinofthe
smallest olive oil with
case 15% oil
of olive concentration
with 15%
of oil phase). Moreover,
concentration of oil phase).the nanoemulsions containing only containing
Moreover, the nanoemulsions olive oil hadonlylarger sizes
olive oilthan
had those
largerwhich
sizes
also had
than those D-limonene
which alsoinhad the D-limonene
oil phase. The in authors explained
the oil phase. Thethis phenomenon
authors explained with
thisthe differences
phenomenon
in thethe
with viscosity of the
differences inoil
thephase.
viscosity D-limonene
of the oil was characterized
phase. D-limonenebywas a much lower viscosity
characterized by a much(approx.
lower2
mPa·s) than
viscosity the vegetable
(approx. 2 mPa·s)oils than(above 30 mPa·s).oils
the vegetable The(above
viscosity of the
30 mPa ·s).oilThe
phase could affect
viscosity of thethe
oil rate
phase of
mass affect
could transport of surfactant
the rate molecules
of mass transport through the
of surfactant oil and through
molecules into thethe aqueous
oil and phase
into the oraqueous
droplet
disruption,
phase within
or droplet a flow field
disruption, [47]. a flow field [47].
within
The nanoemulsion
The nanoemulsion in in which
which the the oil
oil phase
phase was
was formed
formed only
only with
with D-limonene
D-limonene was was obtained
obtained by by LiLi
and Chiang
and Chiang [23],
[23], with
with the
the US
US method.
method. The The system
system was
was stabilized
stabilized with
with aa surfactant
surfactant mixture—sorbitane
mixture—sorbitane
trioleate and
trioleate and polyoxyethylene
polyoxyethylene (20) (20) oleyl
oleyl ether.
ether. The
The ultrasound
ultrasound power
power was was 18 W, sonification
18 W, sonification timetime
100–140 s.
100–140 s. Nanoemulsions
Nanoemulsionshad hada droplet
a dropletsize under
size under100100
nm nm
andand
maintained
maintained kinetic stability
kinetic for eight
stability for
weeks.
eight weeks.

3.2.4. The Kinetic Stability of the Obtained Nanoemulsions

Nanoemulsions
The
The kinetic
kineticstability of the
stability of obtained fragrance
the obtained composition
fragrance nanocarriers
composition was studied
nanocarriers wasbystudied
measuringby
droplet sizes
measuring and PDI
droplet forand
sizes nanoemulsions stored for one
PDI for nanoemulsions year,
stored foratone
ambient
year, attemperature. The resultsThe
ambient temperature. of
the measurements
results are presented
of the measurements in the Figure
are presented 2. Figure 2.
in the

Figure 2. Particle
Figure 2. Particle size
size distribution
distribution over time for
for nanoemulsions
nanoemulsions based on fragrance A with with different
different
mass
mass ratios
ratios of
of the
the surfactant:oil
surfactant:oil (NA(1)
(NA(1) == 0.625:0.374; NA(2) = 0.5:0.5; NA(3) = 0.4:0.6) (SD, n =3).
=3).

In Figure 2, it can be clearly seen that the nanoemulsions on the basis of the fragrance Composition
A, in which the surfactant:oil ratio (S:O) was 0.625:0375 and 0.5:0.5, maintained kinetic stability for
12 months. However, in the case of preparations with higher content of oil phase, the droplet size
increased significantly, most likely due to coalescence and Ostwald ripening.
It should be noted that nanoemulsions with oil phase and surfactant in mass ratio surfactant:oil
(S:O) of 0.625:0375, maintained kinetic stability in all used fragrance compositions for 12 months
of storage (Figures 3 and 4). The droplet sizes did not exceed 30 nm and virtually did not change,
over 365 days.
 In Figure
stability for 122, months.
it can beHowever,
clearly seen thatcase
in the the ofnanoemulsions on the
preparations with basis content
higher of the fragrance
of oil phase, the
Composition A, in which the surfactant:oil ratio (S:O) was 0.625:0375 and 0.5:0.5, maintained
droplet size increased significantly, most likely due to coalescence and Ostwald ripening. kinetic
stability for 12 months. However, in the case of preparations with higher content of oil phase, the
It should be noted that nanoemulsions with oil phase and surfactant in mass ratio surfactant:oil
droplet size increased significantly, most likely due to coalescence and Ostwald ripening.
(S:O) of 0.625:0375, maintained kinetic stability in all used fragrance compositions for 12 months of
It should be noted that nanoemulsions with oil phase and surfactant in mass ratio surfactant:oil
storage (Figures
Nanomaterials
3 and 4). The droplet sizes did not exceed 30 nm and virtually did not change,
2018, 8, 981
over
(S:O) of 0.625:0375, maintained kinetic stability in all used fragrance compositions for 12 months of 10 of 15
365 days.
storage (Figures 3 and 4). The droplet sizes did not exceed 30 nm and virtually did not change, over
365 days.

Figure
Figure
Figure 3. Droplet
3. 3.
Droplet sizes
sizes
Droplet ofofthe
of the
sizes thenanoemulsions
nanoemulsions on theon
nanoemulsions thethe
basis
on basis
of A–F ofcompositions
basis A–F compositions
of A–F as a timeasfunction
compositions aas
time function (S:O
(S:Ofunction
a time
= 0.625:
= (S:O
0.625: 0.375)
0.375) (SD,
(SD, n = n =
3). 3).
= 0.625: 0.375) (SD, n = 3).

Figure 4. Droplet size distribution of the nanoemulsions on the basis of A–F fragrance composition,
over time, for S:O = 0.625:0.375 (t = 0 and t = 365 days).

These results are consistent with the literature data. Sadurni et al. [40] obtained nanoemulsions
also stabilized with Cremophor EL (PEG-35 castor oil), on the basis of caprylic and capric
triglycerides (Miglyol 812), with a low-energy method. For a water/Cremophor EL/Miglyol 812
system, the nanoemulsion was obtained with an oil:surfactant ratio of 10:90 to 40:60. They had a
droplet size between 14 and 39 nm and maintained kinetic stability for 7 months.
Nanomaterials 2018, 8, 981 11 of 15

3.2.5. The Physicochemical Properties of the Obtained Nanoemulsions

In the next stage of the research, the physicochemical properties were established for the stable
nanoformulations containing Etocas 35 emulsifier. Viscosity, pH, surface tension, and density were
measured. The results are shown in Table 5.

Table 5. Physicochemical properties of nanoemulsions containing different fragrance compositions

with an S:O ratio of 0.625:0.375 (SD, n = 3).

Name of Viscosity Coefficient Surface Tension Density

Z-Ave (nm) pH
the Sample (mPa·s), γ = 100 s−1 (mN/m) (g/cm3 )
NA(1) 27.2 ± 0.9 4.95 ± 0.2 78.2 ± 5.0 32.1 ± 1.5 1.005 ± 0.001
NB(1) 23.5 ± 0.4 5.28 ± 0.3 70.4 ± 2.5 35.6 ± 2.0 1.007 ± 0.003
NC(1) 19.8 ± 0.2 5.05 ± 0.15 69.5 ± 9.0 33.6 ± 1.4 1.008 ± 0.002
ND(1) 22.0 ± 0.3 5.08 ± 0.10 70 ± 4 33.2 ± 2.0 1.006 ± 0.003
NE(1) 30 ± 2 4.87 ± 0.25 71 ± 7 33.5 ± 0.9 1.007 ± 0.003
NF(1) 25 ± 1 4.47 ± 0.18 69.8 ± 9.6 33.5 ± 1.0 1.007 ± 0.005

Table 5 shows that the obtained stable preparations had pH values, appropriate for the skin [48,49],
low viscosity, density, and surface tension, which would facilitate the application of nano-perfumes as
aerosol. The method used to obtain the composition with the above-mentioned content, did not have
any effect on the properties of the preparations. Nanoemulsion obtained by US method had properties
similar to those of formulations obtained with a low-energy method. Z-Ave diameter, PDI, viscosity,
surface tension, pH, and density values did not show any significant differences (p > 0.05).

3.2.6. Oxidative Stress Test

In order to confirm the effect of the nanoemulsions on the chemical stability of fragrance
compositions, an oxidative stress test was carried out. The peroxide number measurement (POV)
helped to determine the chemical stability of the applied fragrances. The same amount of the oils
(fragrance compositions) was used in all of the formulations. The calculated values of the peroxide
number (POV) indicated the level of the oil oxidation process. The lower were the peroxide number
differences between the fresh fragrances and those protected by nanoemulsions during storage,
the higher was the protective ability of the nanoemulsion and chemical stability of the oils. Figure 5
shows the protection effect of nanoemulsion against oxidation of the oils. The obtained formulations
showed a high protection against the oxidation of fragrance compositions, compared to the reference
samples of pure oils. This protective ability is connected with the nanoemulsion structure, which
hinders the oxygen from reaching the oils, and oxidizing them.

3.2.7. The Results of the Test of Fragrance Permanence in the Nanoemulsions

The test of fragrance permanence was carried out according to the standard BN-84/6148-02 by
an organoleptical assessment of the intensity of the fragrance after 1, 24, and 48 h, after dipping the
filter paper in the tested formulation. The samples were assessed by comparing the intensity of the
fragrance of the tested sample and the reference sample. In all cases, the intensity was the same.

3.2.8. The Results of the Test of Microbiological Properties of the Nanoemulsions

The results of the microbiological tests confirmed that the obtained products were free of
microbiological contamination. The total number of aerobic mesophilic microorganisms, as well
as the number of yeast and mold, was below 10 cfu. The tested samples were free of pathogens, such as
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and the Candida albicans yeast. The tests
of the preservation method showed that it fulfilled the standard requirements, acceptance criteria lgRx
(logarithm of the reduction of the number of viable microorganisms); in all cases it had the value of
<2. It should be noted that the microbiological stabilization of the preparations did not involve any of
Nanomaterials 2018, 8, 981 12 of 15

the compounds from the list of preservatives allowed in cosmetic products presented in the Annex
V of the Regulation (EC) No. 1223/2009. The raw materials used were sodium salt of levulinic acid
and sodium salt of anisic acid, which are substances of natural origin that are used in cosmetics as
ingredients of fragrance
Nanomaterials 2018, compositions
8, x FOR PEER REVIEW and alternative preservatives. 12 of 15

40
35
POV [mmol O2/kg]

30 Fragrances protected
25 by nanoemulsions,
after 12 months
20 storage
15 Fresh fragrances
10
5
0
A B C D E F
Fragrance compositions
Figure 5. The peroxide values of the fragrances protected by nanoemulsions, after 12 months storage,
and the fresh fragrance
fragrance compositions (SD, nn == 3).
compositions (SD,

3.2.9. The Results of the Test of the Dermatological Properties of the Nanoemulsions
3.2.7. The Results of the Test of Fragrance Permanence in the Nanoemulsions
The dermatological test for the obtained products was conducted with a patch test on a group
The test of fragrance permanence was carried out according to the standard BN-84/6148-02 by
of twenty-five testers (15 women and 10 men, age 19–55), under a dermatologist’s supervision.
an organoleptical assessment of the intensity of the fragrance after 1, 24, and 48 h, after dipping the
The assessment was carried out in accordance with a generally-accepted scale for determination
filter paper in the tested formulation. The samples were assessed by comparing the intensity of the
of various of
fragrance dermatological changes
the tested sample and (Table 6).
the reference sample. In all cases, the intensity was the same.
Table 6. Skin assessment by a dermatologist.
3.2.8. The Results of the Test of Microbiological Properties of the Nanoemulsions
Designation Type of Change
The results of the microbiological tests confirmed that the obtained products were free of
(-) No reaction
microbiological contamination. The total number of aerobic mesophilic microorganisms, as well as
(+/-) Weak short-term itching
the number of yeast and mold, was below 10 cfu. The tested samples were free of pathogens, such as
(+) Weak itching and local weak erythema
Staphylococcus aureus, Pseudomonas(++) aeruginosa, Escherichia
Itching andcoli,
localand the Candida albicans yeast. The tests
erythema
of the preservation method (+++) showed that itItching,
fulfilled the
large standard
erythema andrequirements,
papules acceptance criteria
lgRx (logarithm of the reduction of the number of viable microorganisms); in all cases it had the value
of < 2.
OnItthe
should
basisbeof noted that theresults,
the obtained microbiological
it was found stabilization
out that the oftested
the preparations
products did did
notnot involve
cause any
side effect symptoms, irritation, or sensitization in the test group. The patch tests for all subjectsingave
any of the compounds from the list of preservatives allowed in cosmetic products presented the
Annex V of the Regulation (EC) No. 1223/2009. The raw materials used were sodium salt
a negative result (no reaction), therefore, neither skin irritation nor sensitization was observed as a of levulinic
acid and
result sodium
of the salt of anisic
application of theacid, which areItsubstances
preparation. should be of natural
noted thatorigin that are does
this opinion used not
in cosmetics
apply to
as ingredients of fragrance compositions and alternative preservatives.
people who have an allergy to any of the components of the tested cosmetic.

3.2.9.
4. The Results of the Test of the Dermatological Properties of the Nanoemulsions
Conclusions
Theadermatological
As test for the
result of the performed obtained
studies, products
stable was conducted
oil-in-water with a patch test
(O/W) nanoemulsions onobtained,
were a group
of twenty-five
which testers (15
were compatible women
with the sixand 10 men,
selected age 19–55),
fragrance under a dermatologist’s
compositions, and they did notsupervision. The
contain ethanol
assessment was carried out in accordance with a generally-accepted scale for determination
or any other solvent. These systems can be successfully applied as modern carriers of selected fragrance of
various dermatological changes (Table 6).
compositions, both with a low-energy method (PIC), at a laboratory scale, and with a high-energy
method (US), at a production scale. The optimized nano-perfume recipes that were obtained with
different methods, yielded the Table
same 6. Skin assessment by
physicochemical a dermatologist.
properties (stability, medium droplet size of the
Designation Type of Change
(-) No reaction
(+/-) Weak short-term itching
(+) Weak itching and local weak erythema
(++) Itching and local erythema
Nanomaterials 2018, 8, 981 13 of 15

inner phase, polydispersity, viscosity, surface tension, pH, and density). The simple composition of the
formulation is worth noting. The use of a nonionic surfactant that was gentle to the skin as an emulsifier,
i.e., castor oil (Etocas 35) ethoxylated with 35 moles of ethylene oxide, allowed to obtain stable systems,
without the need to use additional solubilizers, such as polyol, e.g. glycerin, an oil phase component,
or an oil with low polarity (e.g., isohexadecane). Stable, transparent nano-perfumes were obtained
with a fragrance composition concentration within 6–15% range [50,51]. These formulations have
low viscosity and pH suitable for the skin. Moreover, the obtained results confirmed the protective
role of nanoemulsions. The peroxide number measurement (POV) showed that the tested fragrance
compositions had a high chemical stability. The results of the microbiological tests confirmed that
the obtained products were free of microbiological contamination and were appropriately preserved.
The dermatological test results confirmed the safety of the developed preparations.

5. Patents
• P.426105: patent application, Sikora, E.; Miastkowska, M.; Lason, E.; Gut, K.; “Method of making
non-alcoholic perfumes”, application for granting a patent adopted in the Patent office of the
Republic of Poland on 28.06.2018, unpublished yet.
• P.426104, patent application, Sikora, E.; Miastkowska, M.; Lason, E.; Gut, K.; “Non-alcoholic
perfumes and the method of making non-alcoholic perfumes”, application for a patent granted in
the Patent office of the Republic of Poland on 28.06.2018, unpublished yet.

Author Contributions: Conceptualization, M.M., E.L. and E.S., Methodology, M.M. and E.L.; Software, M.M. and
E.L., Validation, M.M. and E.L. Formal Analysis, M.M. and E.L.; Investigation, M.M. and E.L., Resources, K.W.-K.,
Data Curation, M.M. and E.L., Writing-Original Draft Preparation, M.M. and E.L.; Writing-Review & Editing,
M.M.; Visualization, E.L.; Supervision, E.S.; Project Administration, E.S., Funding Acquisition, K.W.-K.
Funding: Work financed from the funds of the “Regional Operational Program for Mazowieckie Voivodeship
2014-2020, project no. RPMA.01.02.00-14-5729/16”, entitled: Project aimed at creating ecological, safe-to-use,
water-based perfumes, with the application of allergen free fragrance compositions.
Conflicts of Interest: The authors declare no conflict of interest.

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