Article

Targeted Hybrid Nanocarriers as a System Enhancing the Skin

Structure
Agnieszka Lewińska 1,*, Marta Domżał-Kędzia 2, Kinga Kierul 3, Michał Bochynek 2, Dominika Pannert 3,
Piotr Nowaczyk 4,5 and Marcin Łukaszewicz 2,*

1 Faculty of Chemistry, University of Wroclaw, Joliot-Curie 14, 50-383 Wroclaw, Poland

2 Department of Biotransformation, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14,
50-383 Wroclaw, Poland; [email protected] (M.D.-K.); [email protected] (M.B.)
3 InventionBio Sp. z o.o., Wojska Polskiego 65 st., 85-825 Bydgoszcz, Poland;

[email protected] (K.K.); [email protected] (D.P.)

4 Faculty of Health Science, University of Opole, ul. Katowicka 68, 45-060 Opole, Poland;

[email protected]
5 Dr. Nowaczyk Research and Innovation Center Sp. z o.o. Sp. K., ul. Żmigrodzka 81-83 lok. 205,

51-130 Wrocław, Poland

* Correspondence: [email protected] (A.L.); [email protected] (M.Ł.)

Abstract: The skin is constantly exposed to external and internal factors that disturb its function. In
this work, two nanosystems-levan nanoparticles and a surfactin-stabilized nanoemulsion were pre-
served (tested for microbial growth) and characterized (size, polydispersity, Zeta potential, and sta-
bility). The nanosystems were introduced in the model formulations-cream, tonic, and gel, and con-
Citation: Lewińska, A.; Domżał- firmed by TEM. The analysis showed that nanoemulsion has a spherical morphology and size 220–
Kędzia, M.; Kierul, K.; Bochynek, M.; 300 nm, while levan nanoparticles had irregular shapes independently of the use of matrix and with
Pannert, D.; Nowaczyk, P.; Łukasze- particle size (130–260 nm). Additionally, we examined the antiradical effect of levan nanoparticles
wic, M. Targeted Hybrid Nanocarri- and nanoemulsion in the prototype of formulations by scavenging DPPH (2,2-diphenyl-1-picrylhy-
ers as a System Enhancing the Skin drazyl; EPR spectroscopy). The model cream with both nanosystems and the whole range of prod-
Structure. Molecules 2021, 26, 1063. ucts with nanosystems were evaluated in vivo for hydration, elasticity, smoothness, wrinkles and
https://doi.org/10.3390/molecules
vascular lesions, discoloration, respectively. The cream improved skin condition in all tested pa-
26041063
rameters in at least 50% of volunteers. The use of more comprehensive care, additionally consisting
of a tonic and gel, reduced the previously existing skin discoloration to 10.42 ± 0.58%. The presented
Academic Editors: María De La Luz
prototype formulations are promising in improving skin conditions.
Cádiz-Gurrea, Antonio Segura-Car-
retero and David Arráez-Román
Keywords: surfactin; levan; nanoemulsion; nanoparticles; skin; formulation; anti-aging
Received: 3 February 2021
Accepted: 17 February 2021
Published: 18 February 2021

Publisher’s Note: MDPI stays neu- 1. Introduction

tral with regard to jurisdictional The topical route of administration has many advantages for the treatment of various
claims in published maps and insti-
skin disorders as well as cosmeceutical purposes [1]. The demand for ever more effective
tutional affiliations.
treatments inspires scientists as well as industry to develop interdisciplinary solutions.
The boundaries between pharmacy and cosmetics are blurring. Newly developed carriers
of active substances are frequently examined in cosmetic formulations since it is easier
and less bureaucratic than conducting detailed clinical trials. Consequently, obtained re-
Copyright: © 2021 by the authors. Li-
sults can further be developed in pharmaceutical research. This concept is represented by
censee MDPI, Basel, Switzerland.
This article is an open access article
the gradually increasing number of published articles in pharmaceutical journals. Stand-
distributed under the terms and con-
ard cosmetic compounds are described as newly discovered for medicine in the treatment
ditions of the Creative Commons At- of various diseases, e.g., nanoparticles of hyaluronan are used in the treatment of skin
tribution (CC BY) license (http://crea- inflammatory diseases [2], encapsulated curcumin tested for treatment of psoriasis and
tivecommons.org/licenses/by/4.0/). melanoma [3], coenzyme Q-10 encapsulated in microemulsion used to improve regener-

Molecules 2021, 26, 1063. https://doi.org/10.3390/molecules26041063 www.mdpi.com/journal/molecules

Molecules 2021, 26, 1063 2 of 23

ation of skin [4]. The constant urge for better products forces development of sophisticated
formulations, aiming to improve performance, appearance, and sensorial benefit and
safety.
In the past few years, applied nanotechnology has received increasing attention in
pharmaceutical, food, cosmetic, textile, personal care, agricultural, chemical, biotechnol-
ogy, biomedical, and sensor industries. Colloidal carrier systems are advantageous in sol-
ubilizing poorly soluble active substances [5]. Biologically active substances’ encapsula-
tion increases their stability and protects them from the harsh environment and thus saves
them from degradation. The preparation and application of nanostructured systems have
become an integral part of the development of cosmetology, providing significant ad-
vances in delivering active substances. Colloidal systems from various raw materials cre-
ate polymer nanoparticles, micelles, solid lipid nanostructures, liposomes, nano-, and mi-
croemulsion, etc. [6,7]. By using distinct formulations and preparation methods, nanosys-
tems can be incorporated into diverse types and formats of cosmetics.
Most cosmetics are applied topically. Therefore, an important aspect is the penetra-
tion ability through the skin barrier. The external layer stratum corneum is an obstacle to
the penetration of active substances, especially hydrophobic. Due to encapsulation, hy-
drophobic compounds are easier to deliver to the deeper skin layers [8]. Encapsulation
also has an economic dimension. Encapsulation enables significantly lower amounts of
active substances, reducing the potential skin irritation effect, but still obtaining the de-
sired effect. Therefore, cosmetic products containing nanocarrier can significantly im-
prove their effectiveness, enabling overall lower concentration than “traditional” prod-
ucts, delivering a higher amount of active ingredients to the skin’s deeper layers.
One of the more desirable effects is skin hydration. For this purpose, nano-systems
composed of moisturizing substances are used. One of the most commonly used moistur-
izing substances in cosmetics is hyaluronic acid (HA) [9]. However, conventional HA
preparations are characterized by difficult penetration into the skin due to the large diam-
eter of its particles. Another natural polymer is levan. It is not naturally present in the
skin, but due to its properties, it can be a good candidate for HA replacement. The tran-
sepidermal water loss (TEWL) test, showed that the moisturizing effect of levan is com-
parable to that after using hyaluronic acid [10]. Levan is a fructose polymer synthesized
by Bacillus subtilis and is one of the novel components which might improve skin condi-
tion. Levan provides deep and long-lasting skin hydration due to its porous structure. It
is non-toxic to human fibroblasts and keratinocytes and has no hemolytic effect on eryth-
rocytes. Our previous research has also shown that it has antioxidant properties [11]. De-
spite its high molecular weight, levan can be incorporated into a variety of active sub-
stance delivery systems [12]. Both of these compounds are polymers with large particle
diameters, which makes it difficult for their penetration through the skin. Hence, their use
in polymeric nano-systems can significantly affect their skin penetration possibilities. Pol-
ymeric nanoparticles (PNs) with their specific ability have attracted enormous attention.
PNs are composed of amphipathic copolymers that tend to self-assemble into particles
with unique architecture [13].
It is also very important to provide nutrients, the nanoemulsions are perfect as
nanocarriers. Composed of a suitable surfactant, they ensure the formation of small vehi-
cles with a bioactive substance inside [14]. The use of nanoemulsions is a very good solu-
tion, especially for hydro and lipophilic active substances. Their enclosure in a nanoemul-
sion protects them against the influence of the external environment and increases their
stability, so they can be delivered deep into the skin while maintaining their activity. Sur-
factants are an essential component of the dispersion system and their presence improves
the penetration of nanoemulsions through the stratum corneum. However, synthetic sur-
factants often cause allergies and irritate the skin. Surfactin (SF), synthesized by B. subtilis,
is a naturally derived surfactant (biosurfactant). It is a cyclic lipopeptide with high surface
activity, and at the same time, it is biodegradable. Surfactin is non-toxic to healthy cells,
including the keratinocytes HaCaT cell line and has anticancer properties [15]. The ideal
Molecules 2021, 26, 1063 3 of 23

solution is to use two simultaneous nanosystems-allowing to deliver the active substances

into the deeper layers of the skin and increasing skin hydration.
This research aimed to incorporate two types of nanosystems–levan nanoparticles
(NP) and nanoemulsion (NE) stabilized by surfactin–in the three topical formulations.
Model formulations of tonic, gel, and cream were chosen for incorporation during the
production process in matrix single and hybrid double dispersion. Firstly, preservatives
have been tested, and their effect on the stability of the systems has been examined. Ad-
ditionally, the results have been confirmed by the DLS method. Change in the size of
drops, the zeta potential of the dispersion, and polydispersity were measured. All formu-
lations were compared to each other in antiradical properties by radical scavenging EPR
technique, analyzing the synergistic effect of nanosystems with matrices. The incorpora-
tion of NP and NE has been confirmed by imagining by TEM. In the end, the skin condi-
tion was assessed using the analyzer: structure, hydration, pore size, depth of wrinkles,
degree of discoloration, and the properties were confirmed by ten women, aged 42– 54
years, using the preparation.

2. Results and Discussion

2.1. Preservation, Evaluation of Microbial Protection, and Stability of Nanosystems
The main task of the preservative is to keep the product-during storage and use-in
the same microbiological purity in which it was produced. The addition of preservatives
also prevents the formation of microbial metabolic products, which can cause skin irrita-
tion. Preparations of products, especially those that contain a large amount of water, are
prone to the growth of microorganisms[16]. As a result of microbiological contamination,
they lose their physicochemical properties and utility values. Thanks to preservatives, it
is possible to maintain microbiological purity during use and storage. A preservative in-
troduced into a system should meet several criteria: it should be active at a low concen-
tration and in a wide pH range and be effective against a wide range of microorganisms
[17]. It mustn’t be toxic, irritating, or allergenic. It must be chemically inert, resistant to
light, oxygen, and temperature changes. Therefore, the selection of a preservative for a
particular system is a challenging task.
Our work aims to incorporate nanoemulsion systems and nanoparticles of levan into
cosmetic matrices. These systems, dispersed in water, are introduced into more complex
matrices in which the main component is water. Therefore, the first step was to select an
appropriate preservative for nanosystems. In the case of preservation of nanocarriers, an-
timicrobial agents should not affect their stability as well. Preservatives are known for
causing physical instability of disperse systems such as aggregation, coalescence [18]. The
first stage in this part of the work was to select an appropriate preservative for the devel-
oped nanoemulsion. Nanoemulsions are more complex and are less stable systems than
nanoparticles. Hence, the preservative must not disturb the stability of the nanoemulsion.
Pivotal role during selection was driven by their chemical performance: hydrophilic
(e.g., propylene glycol, sodium levulinate, sodium benzoate), and hydrophobic (e.g., glyc-
eryl caprylate, glyceryl undecylenate) (Table 1). Additionally, an emerging class of multi-
functional-ingredients that besides antimicrobial properties, possess skin-caring proper-
ties, thus do not have to be declared as preservatives such as pentylene glycol, which was
already used in nanostructured carriers [19]. The concentration of applied preservatives
was at the highest dose allowed since, at this time point, it is difficult to predict for which
products each nanocarrier will be used, or the kind of microbial exposure during the shelf
life of the final products. Adequate pH is an important issue, as it can destabilize the car-
rier, in particular nanoemulsions. For this reason, a pH range of preservative had to
strongly correlate with the actual pH of the nanocarrier. The influence of 26 preservatives,
in three different temperature ranges, was tested on the stability of nanoemulsion. Since
nanodispersions are vulnerable to chemical destruction, it was important to conduct pos-
sibly the widest range of inspections. A group of preservatives containing benzyl alcohol
Molecules 2021, 26, 1063 4 of 23

(C, J, M, P), caused changes in the visual appearance of the samples, due to sedimentation
or phase separation. Similar results were obtained when preservation systems for
nanostructured lipid carriers (NLC) were evaluated [18]. Other preservatives such as B,
D, E, H, I, O, S, AC caused destabilization of the systems by sedimentation or phase sep-
aration. Preservatives showing signs of aggregation, precipitation, or sedimentation were
rejected when visually tested.
After selecting the appropriate preservatives for the nanoemulsions, the next step
was to check whether any of the typed preservatives would also be stable and have anti-
microbial effect on the solution of levan nanoparticles. Since levan nanoparticles are con-
sidered to be stable systems, stability tests were narrowed regarding temperature, as well
as the number of tested preservation systems (Table 1). Among 12 selected preservatives,
four did not influence the stability of levan nanoparticles while inspected visually (Z, AA,
AB, AD). When those systems were inspected regarding the presence of microorganisms,
the only preservative consisting of glycerin and propylene glycol was shown to be effec-
tive (AA). The pH value of the unpreserved levan nanoparticles dropped significantly
during observation time, as well as in the case of preservatives that were not suppressing
growth of microorganisms.
Molecules 2021, 26, 1063 5 of 23

Table 1. Nanoemulsion and nanoparticles of levan-stability test.

D0 D30 D90
Nanoemulsion System
Sample T [°C] T [°C] T [°C]
4 22 37 4 22 37 4 22 37
pH O pH O pH O pH O pH O pH O pH O pH O pH O
control 6.04 A 6.28 A 6.38 A 6.3 A 4.48 A 6.9 D 6.50 A 4.60 A 7.05 E
A* 3.77 A 3.74 A 3,52 A 3.54 A 3.43 A 3.4 A 3.54 A 3.43 A 3.4 A
B 6.01 C 6.06 C 6.04 C F F
C 6.11 C 6.22 C 6.21 C F F
D 5.98 C 5.94 C 6.19 C F F
E 5.93 C 5.93 C 6.06 C F F
F* 6.04 A 6.01 E 6.01 E 5.64 E 5.94 C 5.90 A 5.64 E 5.94 B 5.9 A
G* 7.0 A 7.05 A 7.14 A 4.67 A 4.46 A 4.68 A 4.78 A 4.56 A 4.77 A
H 6.13 C 6.08 C 6.11 C F F
I 5.19 B 5.24 B 5.08 B F F
J 5.46 B 5.26 B 5.54 B F F
K* 7.06 A 7.17 A 7.1 A 6.95 A 6.86 A 4.78 A 7.10 A 6.99 A 4.80 A
L* 7.39 A 7.32 A 7.39 A 6.99 A 7.28 A 6.99 A 7.08 A 7.40 A 7.08 A
M 3.8 C 3.39 C 3.6 C F F
N* 4.26 A 4.31 A 4.27 A 4.5 A 4.23 A 4.25 A 4.65 A 4.29 A 4.36 A
O 6.19 C 6.17 C 6.28 C F F
P 4.49 C 4.48 C 4.4 C F F
R* 5.98 A 6.18 A 6.22 A 4.65 A 5.0 A 4.73 A 4.72 A 5.12 A 4.83 A
S 5.98 B 5.8 B 5.93 B F F
T 5.57 B 5.55 B 5.55 B F F
W* 5.95 A 4.87 A 5.99 A 5.97 A 4.82 A 5.96 A 6.09 A 4.92 A 6.08 A
Z* 5.77 A 5.79 A 5.83 A 5.79 A 5.82 A 5.86 A 5.91 A 5.94 A 5.98 A
AA* 5.94 A 5.43 A 6.09 A 5.96 A 5.48 A 6.04 A 6.08 A 5.59 A 6.16 A
AB* 5.92 A 4.93 A 5.97 A 5.96 A 4.97 A 5.91 A 6.08 A 5.07 A 6.03 A
AC 5.97 B 5.96 B 5.94 B F F
AD* 5.95 A 5.17 A 6.23 A 5.97 A 5.23 A 6.27 A 6.09 A 5.33 A 6.40 A
AE* 5.98 A 5.35 A 6.1 A 5.99 A 5.37 A 6.12 A 6.12 A 6.11 A 5.48 A
Nanoparticles of Levan System
control - - 7.18 C - - - - 4.51 C - - - - 4.27 C - -
A - - 6.44 C - - - - 6.27 C - - - - 5.20 C - -
F - - 7.03 A - - - - 6.30 A - - - - 4.94 E - -
G - - 7.05 A - - - - 6.43 E - - - - 6.40 C - -
R - - 7.00 A - - - - 4.58 E - - - - 4.27 E - -
W - - 7.04 A - - - - 5.47 E - - - - 4.73 E - -
Z - - 6.96 A - - - - 6.99 A - - - - 6.83 A - -
Molecules 2021, 26, 1063 6 of 23

AA - - 7.03 A - - - - 7.06 A - - - - 7.00 A - -

AB - - 7.00 A - - - - 7.01 A - - - - 6.11 A - -
AD - - 7.04 A - - - - 7.07 A - - - - 6.40 A - -
AE - - 7.04 A - - - - 5.91 C - - - - 5.08 E - -
(O) observation, (A) no changes (pass test), (B) phase separation, (C) sedimentation, (D) precipitation, (E) milky, (F) no further observations; * antimicrobials used in
preservation efficacy test (Challenge test).
Molecules 2021, 26, 1063 7 of 23

In nanosystems, it is not always possible to see the symptoms of aging, instability, or

aggregation. Hence, an important element is the DLS analysis. It allows determination of
not only the size of a given carrier but also their polydispersity and the Zeta potential.
Analysis of those parameters may indicate their stability. The most desirable PdI value is
around 0.2. However, in the case of the Zeta potential high values indicate high stability
of the carrier, which is important during the manufacturing processes.
Samples of nanoemulsions after preservation with previously selected antimicrobial
agents for further tests were tested by means of DLS analysis (Table 2). Shortly after
preservation, most of the samples had a small particle size below 220 nm. In the case of
glycerin, when added to the nanoemulsion, the size of the carrier increased (preservative
Z, AA). The same observation was made for preservative N. Reduction of carrier size was
observed when antimicrobials composed of levulinate salts were added (K, L). The de-
crease in the carrier size was obtained when 1.2 hexanediol was added to the system (R,
AE), propylene glycol (W) as well as phenylpropanol combined with pentylene glycol (F).
Very little changes in the particle size were observed when pentylene glycol on its own
(AB) or combined with citrus extracts (A), butylene glycol (AD), or salts of benzoic acids
and sorbic acid were added (G).
There were also changes in the polydispersity of these samples, but they are still very
optimal and close to 0.200. Changes resulting from the stabilization of the systems were
observed with regard to the Zeta potential.

Table 2. DLS analysis of nanocarriers with the preservatives.

Day 0 Day 90
DH [nm] PdI ξ [mV] DH [nm] PdI ξ [mV]
Sample Nanoemulsion System
Control 110.5 ± 1.9 0.220 ± 0.01 −27.5 ± 2.83 120.2 ± 1.2 0.212 ± 0.010 −12.80 ± 1.18
A 165.3 ± 0.5 0.190 ± 0.003 40.34 ± 0.84 169.0 ± 3.0 0.195 ± 0.004 38.27 ± 1.26
F 385.2 ± 26.6 0.430 ± 0.01 −18.04 ± 0.42 195.7 ± 7.5 0.650 ± 0.120 −12.84 ± 0.40
G 146.6 ± 5.0 0.220 ± 0.01 −47.27 ±3.67 162.4 ± 1.1 0.094 ± 0.010 −22.20 ± 3.34
K 155.3 ± 0.9 0.190 ± 0.001 −31.60 ± 0.37 55.4 ± 10.9 0.591 ± 0.050 −14.07 ± 1.36
L 143.9 ± 7.4 0.210 ± 0.01 −39.00 ± 4.29 91.6 ± 5.5 0.636 ± 0.040 −26.10 ± 1.93
N 163.5 ± 3.3 0.220 ± 0.01 −4.92 ± 0.37 212.1 ± 1.7 0.280 ± 0.020 −7.41 ± 0.10
R 194.1 ± 0.8 0.240 ± 0.001 −20.07 ± 0.17 165.2 ± 1.4 0.130 ± 0.010 −16.13 ± 0.46
W 173.9 ± 1.1 0.160 ± 0.02 −29.34 ± 1.67 153.8 ± 1.2 0.190 ± 0.003 −15.97 ± 0.42
Z 212.8 ± 5.3 0.190 ± 0.02 −25.10 + 0.71 741.1 ± 19.5 0.199 ± 0.020 −9.88 ± 0.28
AA 184.2 ± 3.1 0.18 ± 0.20 −24.53 ± 1.72 256. 7 ± 3.6 0.116 ± 0.003 −12.10 ± 0.35
AB 165.9 ± 7.9 0.171 ± 0.02 −31.07 ± 1.89 160.8 ± 2.9 0.193 ± 0.005 −16.53 ± 0.40
AD 175.1 ± 1.0 0.180 ± 0.01 −28.93 ± 0.19 177.2 ± 2.6 0.181 ± 0.020 −15.23 ± 0.50
AE 487.3 ± 12.8 0.230 ± 0.01 −6.46 ± 0.15 169.0 ± 0.8 0.243 ± 0.004 −16.80 ± 0.20
Nanoparticles of Levan System
control 203.1 ± 5.3 0.280 ± 0.001 −11.37 ± 2.17 311.1 ± 3.6 0.441 ± 0.01 −8.53 ± 0.95
AA 236.6 ± 3.9 0.115 ± 0.01 −4.68 ± 0.19 224.3 ± 4.6 0.136 ± 0.02 −1.07 ± 0.35

Preservation systems that gave satisfactory results after DLS measurements were
subjected to preservation efficacy test, so-called challenge test. There were 12 antimicro-
bial agents tested for their protection efficiency (Table 1). Nanoemulsions preserved with
promising antimicrobials were challenged with two microbial strains E. coli, S. aureus, and
yeast C. albicans. The initial number of microbial cells in the inoculum and 1g of the tested
formulation is given in Table S1. Prepared microbial suspensions meet criteria set in nor-
mative document ISO 11930. Validation of neutralizer efficiency was performed, and ac-
cording to the collected data diluent used (D/E broth), neutralized preservatives without
inhibition of microbial growth (Table S2).
Pentylene glycol, by itself or combined either with phenylpropanol or with citrus
extracts meet the criteria given in Table S3 (preservative symbol A, AB, and F). Nanosys-
tems preserved with 1.2-hexanediol gave a satisfactory reduction of microbial growth (R).
A synergistic effect was observed when 1.2-hexanediol was added together with butylene
Molecules 2021, 26, 1063 8 of 23

glycol (AE), the former did not present antimicrobial properties when added alone. Satis-
factory results were obtained for a mixture of levulinic acid and its sodium salt (N). How-
ever, these preservatives are considered to be mild, and may not withstand harsh produc-
tion conditions (Tables S4 and S5).
Levan nanocarriers are prone to microbial deterioration since they are of bacterial
origin. Preliminary tests, consisting of a simple microbial purity check, showed, that out
of 13 tested preservatives, only one was effective to suppress bacterial growth, and further
was examined for its preservation efficacy. Antimicrobial agent composed of glycerin and
propylene glycol (AA) turned out to be effective during a challenge test (Tables S4 and
S5) and the next part of the research.

2.2. Incorporation of Nanosystems

2.2.1. Into the Model Matrix
The ingredients used in cosmetic products depend greatly on the type of formulation
(cream, gel, emulsion, lotion, etc). However, the principal raw materials used to manufac-
ture cosmetics are water, oily materials, fats, and wax, surfactants, humectants, thickening
agents, antioxidants, preservatives, coloring agents, along with vitamins, as well as active
agents. Cosmetic emulsions based on vegetable oils are the subject of many studies re-
ported in the literature. They are preferred instead of mineral oils, due to their character-
istics: biocompatibility and biodegradability, effectiveness in protecting the skin against
solar radiation, inflammation, insect attack, microorganisms, and viruses [20]. Moreover,
in comparison to mineral oils, vegetable oils exhibit low viscosity and molecular weight,
which makes them less occlusive [21]. Therefore, research cream formulation (emulsion-
EM) was created based on vegetable oils. An emulsion is a mixture of two immiscible
liquids with different polarities where one liquid is dispersed as often deformed droplets
into the other phase. The emulsions used in cosmetic formulations are usually based on
water (W)-oils mixtures (O). There are two main types of W–O systems: one of them con-
sists of water droplets dispersed in an oil phase (called water-in-oil (W/O) emulsion); the
other one is represented by dispersed droplets of oil in the aqueous phase (called oil-in-
water (O/W) emulsion). As a general rule, the type of emulsion depends primarily on the
emulsifier type (i.e., its HLB number) used in the formulation. O/W emulsions are com-
monly used as water-washable drug bases and for general cosmetic purposes while W/O
emulsions are widely used as emollients and for dry skin, treatment [22,23]. Important to
note that when applied topically, constituents of the formulation may act synergistically
by several mechanisms: promoting skin barrier homeostasis; antioxidative activities; anti-
inflammatory properties; anti-microbial properties; promoting wound healing; and anti-
carcinogenic properties [24]. This is the reason for using in our study mixtures of all the
above components. The developed base formulation is stabilized by nonionic emulsifiers
that create hydrogen bonds and spatial obstacles that prevent dispersed droplets from
approaching one another. The introduction of an ionic emulsifier in nanoemulsion creates
an electrically charged film on the surfaces of the dispersed phase droplets; this effect re-
pels the droplets from one another, therefore the product is stable.
The cream’s composition has been developed exclusively based on natural ingredi-
ents. The cream has been specially designed in such a way as to take into account all as-
pects, both technological, application, and safety-related. The prepared recipe is an O/W
emulsion. When developing a recipe, the ingredient necessary to create an emulsion is an
emulsifier. The emulsifier polyglyceryl-3 picitrate/stearate (TEGO® Care PSC 3-Evonik)
was used. It is a non-ionic emulsifier, obtained in the process of esterification of polyglyc-
erol-3 with stearic acid and citric acid. This emulsifier has a good emulsifying effect and
good skin tolerance. Polyglyceryl-3 dicitrate/stearate in combination with consistency-
forming ingredients, such as glyceryl stearate and cetearyl alcohol, creates emulsions
building lamellar structures. This emulsifier is adapted to emulsions, in which it works
synergistically with the natural organic acids used.
Molecules 2021, 26, 1063 9 of 23

Emollients are an essential element of the cream. They show regenerative, and mois-
turizing properties. Emollients help replenish epidermal lipids. They include, among
other ceramides, triglycerides, linoleic and linic acid, and cholesterol. Additionally, they
contain natural vegetable oils. Such a set of ingredients allow the rebuilding of the dam-
aged lipid layer of the epidermis. Occlusive substances are another component of emol-
lients. Producers play this role, among other petroleum jellies, paraffin, fatty acids, and
phospholipids. These substances allow you to create a thin protective layer on the epider-
mis, which will prevent water loss from the deeper parts of the skin.
To provide the emulsion with appropriate sensory properties and better distribution,
the following emollients were used, such as Butyrospermum parkii (Shea) butter, squalane,
sesame oil, oleic/linoleic/linolenic polyglyceride, caprylic/capric triglyceride, oc-
tyldodecanol emollient, triheptanoin. They are all characterized by great skincare proper-
ties.
As for substances that increase and control the condition of skin hydration, propylene
glycol, and glycerin were used. Thanks to the ability to penetrate the stratum corneum,
glycerin acts as a promoter, making it easier for active substances to penetrate deep into
the skin. It homogenizes cosmetic ingredients and is also a humectant with gentle preser-
vation properties. Propylene glycol, vegetable glycol, is an excellent carrier of active sub-
stances and a solvent for acids and plant extracts. Thanks to a small molecule, it easily
penetrates deep into the epidermis, improving its hydration level.
The cream also contains two ingredients from the group of active substances-tocoph-
erol and Punica Granatum fruit extract. Tocopherol is an antioxidant. It protects cosmetic
products, including oils, against oxidation. The Punica Granatum fruit extract helps protect
the skin from damage by free UV radicals. It contains large amounts of polyphenols, in-
cluding anthocyanins, gallotanin, and ellagitanin with strong antioxidant and astringent
properties. In addition, substances increasing the viscosity of the entire formulation were
used, such as microcrystalline cellulose and xanthan gum, as well as the preservatives
benzyl alcohol, benzoic acid, and dehydroacetic acid.
The serum components have been selected in a way to facilitate the penetration of
active substances. Permeation promoters are a mixture of natural glycols (propylene gly-
col and pentylene glycol). These substances also improve the condition of the skin.
One of the ingredients of the tonic is betaine. It has a protective effect on cell mem-
branes and has a soothing and moisturizing effect. Another ingredient is sodium L-py-
roglutamate-it is present in the skin as a component of NMF (Natural Moisturizing Fac-
tor), which allows water to be retained in the stratum corneum. It is one of the most effec-
tive moisturizing factors. It permanently moisturizes the epidermis and deeper layers of
the skin softening it and toning. Another active ingredient is aloe vera juice, which has a
strong moisturizing, softening, and antioxidant effect. It also has anti-inflammatory prop-
erties and speeds up wound healing.

2.2.2. Compatibility of Nanosystems with Matrix

The IR analysis shows that there are no spectral differences between empty formula-
tions and formulations enriched with levan nanoparticles or nanoemulsion (Figure 1).
This observation proves that there are not any side processes after adding ingredients to
the matrix and the matrix remains stable [25]. For all three types of formulation, the most
intensive signals are connected with water molecule oscillation (~3000–3700 cm−1) and the
presence of alcohols like glycerol, propylene glycol (hydroxyl group oscillations ~1500
cm−1). In the case of cream, there are also characteristic bands associated with aliphatic
groups from fatty acids (~2900 cm−1) [26].
Molecules 2021, 26, 1063 10 of 23

Figure 1. IR spectra of (A) cream, (B) tonic and (C) serum. In red-empty formulation, in blue-with
levan nanoparticles, in green-with nanoemulsion.

2.2.3. Imaging of Implementation

Transmission electron microscopy (TEM) is the rapid, powerful, and relatively non-
invasive visual technique to obtain information about the mean size and the actual surface
and morphology characteristics of the prepared and incorporated nanosystems in the ma-
trix. Imaging was performed for all three prototype formulations with the incorporation
of two types of systems (Figure 2).
Molecules 2021, 26, 1063 11 of 23

Figure 2. Implementation of nanosystems in matrix: (A) nanoemulsion; (B) levan nanoparticles;

(C) hybrid double dispersion-in cream; (D) nanoemulsion; (E) levan nanoparticles; (F) hybrid dou-
ble dispersion-in serum; (G) nanoemulsion; (H) levan nanoparticles; (I) hybrid double dispersion-
in tonic.

The TEM photographs revealed that nanoemulsion was of spherical morphology

having diameter range from 220 nm to 300 nm, and levan nanoparticles show a defor-
mation from a spherical form, appearing in irregular shapes independently of the matrix
used and with particle size 130–260 nm. The diameter of NE and NP-levan obtained using
DLS (Table 2) and TEM were in reasonable agreement with each other and the difference
came from the different sample preparation processes and different principles.

2.3. Antiradical Properties of Incorporated Nanosystems in the Matrix

Due to the capability to oxidize lipids, proteins, and nucleic acids, free radicals are
one of the skin aging factors. Hence, radical scavenging is an essential property of cos-
metic formulations [27]. Moreover, it was investigated that some antioxidant compounds
can exhibit also antimicrobial agents [28]. Therefore, the antiradical effect of levan nano-
particles and nanoemulsion in the prototype of cosmetic formulations as cream, tonic, and
serum was examined. The most accessible and most common method to evaluate the an-
tiradical properties is scavenging of DPPH by UV-Vis spectroscopy [29] but can only be
used for clear samples. In the case of cosmetic preparations, with a consistency of creams,
the turbidity of the system was difficult, so it was decided to use EPR technique [30].
DPPH radical is used as a radical molecule to evaluate the free radical scavenging ability
of the compounds. The DPPH radical is characterized by a strong EPR signal intensity,
and it can accept an electron or hydrogen atom to become a stable diamagnetic molecule.
The loss of the DPPH EPR signal intensity in the presence of antioxidants is directly pro-
portional to the concentration (or number) of electrons (protons) accepted. The EPR signal
Molecules 2021, 26, 1063 12 of 23

intensity was stable but it was rapidly reduced after the addition of the test compound in
a concentration-dependent manner.
At first, the antiradical potential of levan nanoparticles, nanoemulsion, and their link-
age in three different formulations (cream, tonic, serum) were investigated. The samples
were measured 30 min after adding the DPPH solution. These results showed us that in
all formulations free radical scavenging effect is observed (Figure 3). Empty formulations
were investigated simultaneously with enriched ones to examine if there is any antiradical
effect associated with the matrix. Our results showed that there is no change in radical
signals’ intensities-differences between samples measured after 5 and 30 min were smaller
than their standard deviations. Therefore, we concluded that in the case of empty cream,
serum and tonic there is no antiradical effect.

A B C
100%
Scavenged DPPH

80%

60%

40%

20%

0%

Figure 3. Antiradical effect of ingredients in three different types of formulations: A-levan nanoparticles, B-nanoemulsion,
C– the linkage of levan nanoparticles and nanoemulsion. -cream; -tonic; -serum.

In the case of tonic and serum, the scavenging of free radicals was moderate, but the
cream showed much greater properties in removing them. Moreover, in all three formu-
lations, a synergy of nanoemulsion and levan nanoparticles linkage was observed. Such
an effect was described also in other systems [31]. Due to the satisfying effect of the cream,
radical scavenging in time was determined (measuring EPR signal after 5, 15, 30, and 60
min after adding DPPH). Figure 4 shows the differences in the percentage of scavenged
DPPH in time. According to the results, a cream containing both levan nanoparticles and
nanoemulsion is capable of terminating 88% of DPPH after 5 min and 100% after 30 min.
It means that 100 µL of our cream formulation diluted 5 times in water can neutralize
0.152 µmol of radical in half an hour. Similar research was done with a formulation con-
taining Sterculia populifolia extract as an antioxidant [32]. Its authors showed that a con-
centration of 100 µg/mL of this extract can neutralize 50% of radicals in 3 mL of 0.004%
(w/w) solution of DPPH, which gives 0.152 µmol. Moreover, 100 µL of 0.015 mM solution
of ascorbic acid can terminate 0.2 µmol of DPPH [33], hence it can be concluded that the
tested formulations have comparable antioxidant properties.
Molecules 2021, 26, 1063 13 of 23

Figure 4. Time-dependent radical scavenging in cream. - levan nanoparticles; - nanoemulsion; - linkage

2.4. Application Study and Skin Conditioning-In Vivo

Cosmetic preparations for application on the skin (such as creams, serums, tonics,
etc.) are designed to protect the skin, remove its small defects, such as by smoothing wrin-
kles or whitening discoloration. However, for these processes to take place, the active sub-
stances contained in the cosmetic must penetrate the deeper layers of the skin. The use of
nanosystems enables the transport of active substances and may support skin repair pro-
cesses. Application tests carried out on volunteers using non-invasive methods are one of
the possibilities of testing preparations before their commercialization [34]. In this study,
the influence of prepared base cream and cream with nanosystems were evaluated. The
effectiveness of the product is confirmed with positive results in at least 50% of the vol-
unteers participating in the study. The moisturizing, elasticity, smoothing, depth, and vol-
ume of wrinkles were measured in a non-invasive way. The cream containing nanosys-
tems increased skin hydration by 14.9%, while the base cream alone increased the hydra-
tion by 6.6% (Figure 5A).

Figure 5. In vivo application tests: (A) moisturizing, (B) elasticity, (C) smoothing. -before test-
ing, -after series of applications of the base cream, -after series of applications of the cream
with nanosystems.

In the case of elasticity, both creams had a similar effect, but the one containing levan
and nanoemulsion was 1.1% more effective (for the base cream, an increase of 5%) (Figure
5B). The respondents paid attention to the smoothing of the skin, which was also shown
by apparatus tests-the cream with nano-systems smoothed the skin by 1.7% better than
the base cream itself (Figure 5C).
After using the cream with levan and the formulation, the volume and area of wrin-
kles decreased by 2.3 and 3.2%, respectively (where for the base cream the parameter im-
proved by 2.1 and 2.8%) (Figure 6). In the case of wrinkle depth for the base cream, there
was a 6.8% decrease (Figure 7A), while for the cream with nanosystems this decrease was
Molecules 2021, 26, 1063 14 of 23

smaller and amounted to 5.2% (Figure 7B). All results are given in Tables in SM (Tables
S6–S10).

Figure 6. Differences in the volume of the wrinkles (red) and its surface (blue) (A) after a base cream and (B) cream with
the nanosystems.

Figure 7. The depth of the wrinkles before application (blue) and after application (red) (A) of base cream, (B) of cream
with nanosystems.

Based on skin analyzes carried out with the use of NatiV3, the effect of the cream,
serum, and tonic with two nanosystems on vascular lesions and discoloration was deter-
mined. Comparing the base cream with the range of products with nano-systems, their
effect on the vascular lesions was negligible. The values for both tested versions of skin-
care are similar to each other, and additionally, they are burdened with quite a large
standard deviation. For the base cream, the size of the vials was 1.87 ± 1.28 mm2, and after
28 days of use, 1.41 ± 1.28 mm2. For the products with nanosystems, this value was 2.27 ±
0.95 mm2 before application, and after 28 days of daily applications, 1.37 ± 1.16 mm2. In
the case of discoloration, it was found that the base cream did not reduce the discoloration
on the skin after 28 days of use, unlike the cosmetics with nano-systems. Initially, the dis-
coloration was 10.91 ± 3.20% and 10.88 ± 0.72%, and after 28 days of use, it was 11.29 ± 0.79
and 10.42 ± 0.58%, respectively for the base cream and cosmetics with nano-systems. Sam-
ple of changes before applying the formulation and after 28 days of use in Supplementary
Materials (Figure S1).
Molecules 2021, 26, 1063 15 of 23

The analysis of a given preparation is difficult to interpret. The skin is not a homoge-
neous tissue and it is characterized by high variability in parameters, even within one
examined person. The skin’s reaction to a given cosmetic also depends on the state of
health, environmental conditions, etc. The developed preparation with a double nanosys-
tem is only at the initial stage of research. Further work is underway to improve it. The
studies conducted so far have shown that their use in cosmetic preparations has a positive
effect on the condition of the skin. The use of polymers in the production of cosmetics is
very common. Their addition has a great influence on the consistency of the preparations,
increasing their viscosity and ensuring greater stability of the emulsion. The addition of
more than one polymer can lead to faster coalescence or isolation of the emulsion droplets.
The use of polymers in cosmetics also affects their care properties. They can retain water
in their structures, thus increasing skin hydration. The polymers can also form a scaffold
for other essential ingredients, e.g., ions, or form a coating around sensitive active sub-
stances. The use of bacterial cellulose in cosmetics include facial masks, facial scrub, per-
sonal cleansing formulations, and contact lenses has been reported so far [35]. Another
used polymer is chitosan and its derivatives. The encapsulation of, for example, essential
oils or polyphenols with strong antioxidant properties in chitosan protects their action, as
they are usually very sensitive to external factors [36]. However, the use of at least two
polymers in a cosmetic preparation may result in the creation of new materials based on
the molecular interaction between polymer molecules [37]. One of the greatest advantages
of using nanoemulsions in cosmetics is dissolving large quantities of hydrophobic com-
pounds and the ability to protect drugs from degradation [38]. In addition to the interest
in nanoemulsions due to their unique consistency, other benefits of nanoemulsions have
been appreciated in many cosmetic applications, especially skincare for solubilizing and
delivering active ingredients [39]. Enclosing the active substances in the nanoemulsion
enables their transport through the stratum corneum to the deeper layers of the skin [7].
So far, various nanoemulsions have been tested for their cosmetic use. Their use is to mois-
turize the skin [40,41], lighten discoloration [42], or reduce vascular lesions [7]. The for-
mulation presented in this study has the potential to improve the condition of the skin
and slow down the aging processes.

3. Materials and Methods

3.1. Levan Nanoparticle Preparation
Nanoparticles of levan were prepared with a precipitated and lyophilized polymer.
An appropriate amount of levan was taken to prepare a 5% solution in distilled water. It
was stirred till it dissolved. Then the solution of levan nanoparticles was incorporated into
the cream. Levan was obtained from microbial fermentation with Bacillus subtilis natto
KB1, described below. Levan standard was purchased in Sigma-Aldrich (Poznan, Poland).
All medium components were purchased from BioShop LabEmpire (Rzeszow, Poland).
Ethanol and minerals were purchased in Chempur (Chempur, Poland). All other reagents
were of analytical grade and used as provided. Water used for all experiments was doubly
distilled and purified through a Milli-Q water purification system (Millipore, Bedford,
MA, USA).
The culture of Bacillus subtilis natto KB1 was grown at 37 °C for 24 h with continuous
shaking in a 5 L bioreactor. After 24 h, the culture was stopped and centrifuged to separate
the bacterial biomass. Levan was separated from the supernatant as described before [11].

3.2. Nanoemulsion Preparation

Nanoemulsion concentrate was prepared as follows: 50% of sodium surfactin pow-
der, 30% of 2-(2 ethoxyethoxy) ethanol (trade name Transcutol HP; Gattefossé SAS, Saint-
Priest, France) and 20% of ascorbyl tetraisopalmitate (trade name Nikkol VC-IP, Nicco
Chemicals Co. Ltd., Tokio, Japan) were subjected to ultrasound for 20 min, at 50 °C, as
previously described [7]. Concentrates were organoleptically inspected for the absence of
Molecules 2021, 26, 1063 16 of 23

any thickenings. In case there were none, the obtained pre-concentrate was diluted in a
glass beaker, 50 mg to 10 mL of water at temperature 37 °C, and further stirred on a mag-
netic stirring plate.
Surfactin was obtained from microbial fermentation with B. subtilis natto KB1, as de-
scribed before [7,43]. All medium components were purchased from BioShop LabEmpire
(Rzeszow, Poland). All solvents for chromatographic separations were of HPLC and/ or
MS grade for HPLC and MS analyses, respectively. Trifluoracetic acid (TFA) and surfactin
standards were purchased from Sigma Aldrich.

3.3. Other Components

Following ingredients were obtained as a gift: Dermosoft PEA, Dermasoft GMCY,
Dermosoft 1388 Eco (Evonik, Essen, Germany), Activonol BG (Activon, Suwon City, Ko-
rea), Stabil Zero, Kem Nat, Kem Nat Beta, Kem DH (Akema,Coriano, Italy), Liqupar ME
(Ashland, Wilmington, NC, United States), Cosphagard Elbe, Cosphagard Pol, Cospha-
derm LA-T, Cosphaderm TOM, Cosphaderm Hexiol, Cosphaderm Octiol, Cosphaderm
Sodium LAAS (Cosphatec, Hamburg, Germany), InBioLev, InBioSur (InventionBio, Byd-
goszcz, Poland), Geogard ECT, Geogard Ultra (Lonza, Basel, Switzerland), E-Leen Green
OR, MinaSolve Green A, A-Leen Aroma-3 (MinaSolve, Mont-Saint-Guibert, Belgium),
Euxyl PE 9010, Euxyl K340, Euxyl K320 (Schülke, Warsaw, Poland), Sharomix 703 (Do-
nauchem, Rokietnica, Poland). Other raw materials were purchased, Ajidew NL-50
(Ajinomoto, Tokyo, Japan), Viamerine 2500 (Aldivia, Saint-Genis-Laval, France), Aloe
Vera SDP 200X (Ashland, Wilmington, United States), Myritol 312, Eutanol G, Cetiol SB
45, Lanette O, Cutine GMSV, Plantacare 810 (BASF, Ludwigshafen, Germany), Propylene
Glycol (Brenntag, Kędzierzyn-Koźle, Poland), Cosphaderm 1,3 -Propanediol, Cospha-
derm X34 (Cosphatec, Hamburg, Germany), Glycerin (CQM Masso, Warsaw, Poland),
Fruitliquid Pomegranate (Crodarom, Chanac, France), Dermofeel PA 12, Tego Natural Be-
taine, Tego Care PSC-3, Dermofeel Toco 70 non, Dermofeel PA-12 (Evonik, Essen, Ger-
many), Transcutol HP (Gattefossé SAS, Saint-Priest, France), Sesame Oil (Henry Lamotte,
Bremen, Germany), Miglyol T-C7 (IOI Oleochemical, Putrajaya, Malaysia.), A-Leen 5 (Mi-
nasolve, Mont-Saint-Guibert, Belgium), Nikkol VC-IP (Nicco Chemicals, Tokyo, Japan),
Vivapur CS 032 XV (Rettenmaier, Rosenberg, Germany), Natural Parfume (Robertet,
Grasse, France), Euxyl K903, Euxyl K903 (Schülke, Norderstedt, Germany), Sharomix 713
(Donauchem Polska, Rokietnica, Poland), Phytosqualan (Sophim, Peyruis, France).

3.4. Influence of Conservation Systems on Stability

3.4.1. Preservation Efficiency
Obtained nanoemulsions were subjected to various preservation strategies. Homog-
enization of nanoemulsions was followed by the addition of antimicrobial agents accord-
ing to Table 3, and tubes were kept in three different conditions: refrigerator at 4 °C, an
incubator at 37 °C, and climate chamber set at 50 °C, 75% humidity, light 7500 lux/h, 1.1
W/m2 (UVA). After every month of incubation, microbiological tests for the presence or
absence of mesophilic microorganisms as well as for yeast and molds were conducted as
described in SM1. The obtained results were assessed according to criteria given in the
normative document ISO 17516 (Table S11).

Table 3. List of used preservatives.

Max. Content
Preservative Symbol INCI pH Range
[%]

Pentylene Glycol, Glycerin, Citrus Aurantium Amara Fruit Extract, Citrus Reticulata Fruit
3–6.5 3
Extract, Citrus Aurantium Dulcis Peel Extract, Ascorbic Acid, Citric Acid, Lactic Acid

AA Glycerin + Propylene Glycol unlimited 10 + 5

AB Pentylene Glycol 2–12 5
Molecules 2021, 26, 1063 17 of 23

AC Phenethyl Alcohol unlimited 1

AD Butylene Glycol unlimited 10
AE Butylene Glycol + 1.2 Hexanediol unlimited 1.5
B Phenoxyethanol, Ethylhexylglicerin < 12 1
C Benzyl Alcohol, Benzoic Acid, Dehydroacetic Acid <6 1.2
D Phenoxyethanol, Methylparaben, Ethylparaben, Propylparaben, Butylparaben <8 1.2
E Phenoxyethanol, Methylparaben, Ethylparaben, Propylene Glycol to 8 1.4
F Pentylene Glycol, Phenylpropanol 3–10 3
G Potassium Sorbate, Sodium Benzoate to 5.5 1.5
H Phenylpropanol 3–10 1
I Pentylene Glycol, Caprylyl Glycol, Ethylhexylglycerin 3–9 2
J Benzyl Alcohol, Glyceryl Caprylate, Glyceryl Undecylenate 4–8 2
K Sodium Levulinate, Sodium Benzoate <6 2.5
L Sodium Levulinate, Potassium Sorbate <6 2
M Benzyl Alcohol, Glyceryl Caprylate, Benzoic Acid, Propylene Glycol 4–6 1.2
N Levulinic Acid, Glycerin, Sodium Levulinate 6 1
O Pentylene Glycol, Glyceryl Caprylate, Glyceryl Undecylenate 3.5–7.5 2.5
P Benzyl Alcohol, Dehydroacetic Acid 3–7 1
R 1.2-Hexanediol unlimited 2.5
S Caprylyl Glycol unlimited 0.5
W Propylene Glycol unlimited 8
Z Glycerin unlimited 20

3.4.2. Evaluation of Microbial Protection

The preservation efficiency test was performed according to the method described in
the normative document ISO 11930 (SM2). Nanoemulsions have been subjected to stress
tests to assess the effectiveness of preservation [44]. Microorganisms used in the test were:
Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus
(ATCC 6538). The obtained results were assessed according to the criteria given in Table
S3. The composition of the media used in the experiment are given in Table S12, incuba-
tion conditions are given in SM2. Bacterial suspensions were prepared as mentioned in
SM4. Enumeration of microbial cells in the inoculum was performed using the pour plate
method (SM3). The initial number of viable microorganisms in the inoculum and 1g of the
tested formulation is listed in Table S1.

3.4.3. Stability of Nanosystems

The integrity of nanocarriers with preservatives was inspected organoleptically right
after preparation (D0), after 30 (D30), and 90 (D90) days of incubation at three temperature
conditions. pH was measured after preparation (D0) of all prepared nanocarriers and was
continued in case of visual integrity. DLS measurements were taken in case of organolep-
tic acceptance to overcome the problem of unstable formulations, nanoemulsions were
subjected to time-dependent size (DH) and Z-potential measurements after preparation
(D0) and 90 days (D90) of storage as well as dispersion stability test [7].

3.5. Incorporation of Nanosystems into the Model Matrix

The formulation prototypes (new design) have been developed for this work. All the
ingredients were carefully selected and used for the formulations according to the rules
provided by The European Cosmetics Legislation [45]. The main raw materials used to
obtain cosmetics are water, oily materials (oils, fats), humectants, antioxidants, preserva-
tives, active agents, fragrances. The compositions of the prepared cosmetic emulsions are
shown in Table 4,5,6. The classic type emulsion oil in water (O/W), was chosen as the
cosmetic matrix model, where the external phase is water and the internal (dispersed
phase) is oil (Table 4). Whereas to test the compatibility of nanosystems with water for-
mulations, recipes for serum (Table 5) and face tonic (Table 6) were prepared.
Molecules 2021, 26, 1063 18 of 23

Table 4. Ingredients of the emulsion (face cream- C1) formulation.

Content [%]
Phase INCI Function
C C1 C2 C3
Polyglyceryl-3 Dicitrate/Stearate Emulsifier 2 2 2 2
Caprylic/ Capric Triglyceride Emollient 3.2 3.2 3.2 3.2
Octyldodecanol Emollient 2 2 2 2
Triheptanoin Emollient 2 2 2 2
Butyrospermum Parkii (Shea) Butter Emollient 3.6 3.6 3.6 3.6
A Cetearyl Alcohol Viscosity controlling, Emollient 2.5 2.5 2.5 2.5
Glyceryl Stearate Viscosity controlling, Emollient 1.8 1.8 1.8 1.8
Oleic/Linoleic/Linolenic Polyglyceride Emollient, co-emulsifier 1 1 1 1
Squalane Emollient 2 2 2 2
Tocopherol, Helianthus Annuus (Sunflower) Seed Oil Antioxidant 0.1 0.1 0.1 0.1
Sesamum Indicum Seed Oil Emollient 1.5 1.5 1.5 1.5
Aqua Solvent 40 40 40 40
B
Sodium Phytate Chelating agent 0.1 0.1 0.1 0.1
Microcrystalline Cellulose, Xanthan Gum Viscosity controlling agent 0.9 0.9 0.9 0.9
C Glycerin Humectant 0.5 0.5 0.5 0.5
Aqua Solvent 30 30 30 30
Propylene Glycol Solvent 1.5 1.5 1.5 1.5
Perfume Parfum 0.1 0.1 0.1 0.1
D Water, Glycerin, Punica Granatum Fruit Extract, Potassium Sorb-
Active ingredient 0.8 0.8 0.8 0.8
ate, Sorbic Acid
Benzyl Alcohol, Benzoic Acid, Dehydroacetic Acid, Tocopherol Preservative 0.8 0.8 0.8 0.8
Levan, Glucose, Fructose, Sucrose Active ingredient - 0.08 - 0.08
Sodium Surfactin Surfactant - 0.375 0.375 -
E
Diethylene Glycol Monoethyl Ether Solvent - 0.225 0.225 -
Ascorbyl Tetraisopalmitate Active ingredient - 0.15 0.15 -
Aqua Solvent to 100 to 100 to 100 to 100

Preparation:
Phases A and B were heated separately to a temperature of 70–75 °C, which was fol-
lowed by homogenization. Raw materials from phase C were added into the water at 40–
45 °C and mechanically stirred for about 1 h. Phases A and B were poured into one vessel
and subsequently phase C was added. Such a prepared mixture was homogenized. After-
ward, the emulsion was cooled down by a mechanical stirrer at low speed. Phase D was
added and pH was set to the value of 5.5 with citric acid. Previously prepared nanosys-
tems (phase E) were mixed into the emulsion at the final step (Table 4).

Table 5. Ingredients of the face serum (S) formulation.

Content [%]
INCI Function
S S1 S2 S3
Propylene Glycol Solvent 4.5 4.5 4.5 4.5
Pentylene Glycol Solvent, Skin conditioning 3.5 3.5 3.5 3.5
Caprylyl/Capryl Glucoside Surfactant 0.3 0.3 0.3 0.3
Glycerin Humectant 0.8 0.8 0.8 0.8
Xanthan Gum Viscosity controlling 0.95 0.95 0.95 0.95
Benzyl Alcohol, Benzoic Acid, Dehydroacetic Acid, Tocopherol, Preservative 0.95 0.95 0.95 0.95
Perfume Parfum 0.08 0.08 0.08 0.08
Levan, Glucose, Fructose, Sucrose Active ingredient - 0.08 - 0.08
Sodium Surfactin Surfactant - 0.375 0.375 -
Diethylene Glycol Monoethyl Ether Solvent - 0.225 0.225 -
Ascorbyl Tetraisopalmitate Active ingredient - 0.15 0.15 -
Aqua Solvent to 100 to 100 to 100 to 100
Molecules 2021, 26, 1063 19 of 23

Table 6. Ingredients of the face tonic (T) formulation.

Content [%]
INCI Function
T T1 T2 T3
Betaine Active Ingredient 1 1 1 1
Aloe Barbadensis Leaf Juice Active ingredient 0.05 0.05 0.05 0.05
Sodium Phytate Chelating agent 0.1 0.1 0.1 0.1
Perfume Parfum 0.05 0.05 0.05 0.05
Propylene Glycol Solvent 2 2 2 2
Sodium PCA Active ingredient 1.5 1.5 1.5 1.5
Potassium Sorbate; Sodium Benzoate Preservative 1 1 1 1
Levan, Glucose, Fructose, Sucrose Active ingredient - 0.08 - 0.08
Sodium Surfactin Surfactant - 0.375 0.375 -
Diethylene Glycol Monoethyl Ether Solvent - 0.225 0.225 -
Ascorbyl Tetraisopalmitate Active ingredient - 0.15 0.15 -
Aqua Solvent to 100 to 100 to 100 to 100

3.5.1. Images of Incorporated Nanosystems in Different Matrix

The transmission electron microscopy (TEM) measurements were performed to
measure the morphology and size distribution of lipid nanocarriers. Images were taken
using an FEI Tecnai G2 20 XTWIN electron microscope (FEI, Hillsboro, OR, USA). The
size distribution of the nanocarriers for each sample was determined by counting the size
of approximately 250 nanocarriers from several TEM images obtained from different parts
of the TEM grids. A few drops of the diluted suspension were placed on the grid and
stained with 2% uranyl acetate, and then the image was captured. The size distribution
plots were fitted by using Gauss curve approximation.

3.5.2. IR analysis of Nanosystems in the Cosmetic Matrix

Functional groups and chemical bonds of the obtained cosmetic products were de-
termined using Fourier-transform infrared (FT-IR) spectroscopy on Spectrometer Bruker
Vertex 70 FT-IR (Bruker Optics, Ettlingen, Germany). The sample was prepared as a thin
film. All of the samples were scanned over a wavelength range of 4000–400 cm−1.

3.6. Antiradical Properties of Incorporated Nanosystems in the Matrix

Antiradical properties of self-assembly nanosystems incorporation in the different
matrix were studied by radical scavenging activity using DPPH by electron paramagnetic
resonance. A stable radical solution in ethanol was stirred in dark for 2 h to obtain a ho-
mogeneous solution. Next, the tested systems were prepared as follows: 75 µL of radical
and 100 µL of the sample (5x diluted in water) were mixed. The capillary tube with the
sample was sealed and placed inside a standard EPR quartz tube, then placed in the res-
onant cavity. Measurements were carried out at room temperature. Data were reported
as the average of three measurements. The EPR spectra were recorded using a Bruker
Elexsys 500 spectrometer operating at the X-band frequency (~9.7 GHz). A microwave
power of 1 mW, modulation amplitude of 1 G, a sweep width of 200 G were adopted. An
analysis of the EPR spectra was carried out using the WinEPR software package, version
1.26b (Bruker WinEPR GmbH, Rheinstetten, Germany). The double integral of the signal
was evaluated as representative of the free radical concentration. The field under the ab-
sorption curve is proportional to the number of stable radicals remaining in the sample.
The percent value of scavenged DPPH was calculated with the following formula:
𝐼 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 − 𝐼 𝑠𝑎𝑚𝑝𝑙𝑒
𝑆𝑐𝑎𝑣𝑒𝑛𝑔𝑒𝑑 𝐷𝑃𝑃𝐻 = ∗ 100%
𝐼 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
Isample–integrity of signal measured in the sample
Istandard–integrity of signal measured in the sample
Molecules 2021, 26, 1063 20 of 23

3.7. Conditioning Skin In Vivo

For in vivo evaluation of vascular lesions, discoloration, NatiV3 Analyzer (Beauty of
Science, Poland) was used [7]. The measurements were analyzed based on the manufac-
turer’s database (more than 2,500 studies of people between 18 and 90 years old, both
women and men) [46]. Prepared cosmetic formulations C1, C2, C3, C4 of cream separately.
In the next research, the effect of using the product group consisting of tonic, serum, and
cream was tested. T4, S4, and C4 were applied to the skin and compared with part of the
face with C4 only. The measurements were carried out before the application of the for-
mulations and at 28 days after application.

3.8. Application Tests In Vivo

The test was performed on 10 women, age 42–54 years. Measurements were taken on
healthy skin, without irritation signs. Volunteers were advised not to use any antihista-
mines or pharmacological drugs, which could interfere with the results. Both formulations
were applied daily, one without the addition of NP levan, and NE was applied on the left
part of the face, neck, and neckline, while the formula with NP levan and NE on the right
part of the body. The measurements were taken before the application of formulations,
and after four weeks of application. All measurements were carried out in a room with a
temperature of 20 °C and relative humidity of 50 ± 10%.
Performed methods were designed in accordance with Regulation (EC) No 1223/2009
of the European Parliament and of the Council of 30 November 2009 on cosmetic products;
Cosmetics Europe-The Personal Care Association Guidelines Product Test Guidelines for
the Assessment of Human Skin Compatibility 1997; Cosmetics Europe-The Personal Care
Association Guidelines for the Evaluation of the Efficacy of Cosmetic Products 2008 and
the laboratory procedure No. PB-19 ed. I from November 10, 2017 “Cosmetic products.
Apparatus application research”.
For the assessment of skin hydration, a Multi Probe Adapter System was used (Cour-
age+Khazaka Electronics, Cologne, Germany), which is connected to a probe used for the
determination of the skin surface hydration-Corneometer® CM 825, for firmness/elastic-
ity–Cutometer MPA580, smoothening effect–Visioscan VC98, reduction of wrinkles–Vi-
sioline VL650. All the measurements were performed in triplicate (mean ± SD, n = 3). The
obtained results are presented as absolute numbers.

3.9. Statistical Analysis

All data are expressed as the mean value ± standard deviation of three measure-
ments. Statistical analyses were accomplished using an online one-way analysis of vari-
ance with the post hoc Tukey honest significant difference calculator. A value of p < 0.05
was considered statistically significant.

4. Conclusions
Antimicrobial agents were tested on the nanoemulsion and levan nanocarrier stabil-
ity. For nanoemulsions pentylene glycol, 1.2-hexanediol and butylene glycol proved to
have a preservation effect. In the case of levan nanocarriers, only glycerin combined with
propylene glycol presented this effect. Systems with preservatives showed stability in 90
days and had a slight influence on the particle size. The incorporation of nanosystems into
three prototypes of matrices was proved by infrared spectroscopy and microscopy imag-
ing. Antiradical properties were evaluated by radical scavenging-EPR spectroscopy. The
strongest synergy NP, NE, and matrix were observed in the emulsion. The effect was ob-
served and confirmed by an application study. Cream tested on volunteers increased skin
hydration, elasticity, and had a smoothening effect. In regard to wrinkles, it reduced their
area and depth.
Molecules 2021, 26, 1063 21 of 23

Supplementary Materials: Figure S1. Images of vascular lesions and discoloration for the tested
skin. Table S1. The initial number of microbial cells in the inoculum (N) and the initial number of
cells found in 1g of the product tested (N0). Table S2. Demonstration of neutralizer efficacy. Table
S3. Criteria for the evaluation of the preservation of cosmetic products. Table S4. The number of
viable cells of test microorganisms after a given contact time with nanoemulsion and levan nanocar-
riers. Table S5. Log reduction values (Rx = lgN0−lgNx). Table S6. Skin moisturization effect. Table
S7. Influence of skin firmness/elasticity. Table S8. Skin smoothening effect. Table S9. Reduction of
wrinkles. Base cream. Table S10. Reduction of wrinkles. Formulations with nanosystems. Table S11.
Microbial limits for cosmetic products according to EN ISO 17516: 2014. Table S12. Media used to
assess the effectiveness of product preservation. SM 1. Microbiological analysis, SM 2. Evaluation of
microbial protection: criteria and experimental conditions. SM. 3 Method used for inoculum enu-
meration. SM. 4 Preparation of bacterial suspensions and their incorporation into the tested formu-
lations. SM. 5. Determination of the effectiveness of a preservative.
Author Contributions: Conceptualization, A.L; methodology, A.L., M.D.-K., K.K., M.B., P.N., M.Ł.;
software, A.L, M.D.-K., M.B.; validation, A.L., M.D.-K. and M.Ł.; formal analysis, A.L, M.D.-K., K.K.
M.B. M.Ł; investigation, A.L., M.D.-K., K.K., M.B., D.P., P.N.; resources, A.L. M.Ł.; data curation
A.L., M.Ł.; writing—original draft preparation, A.L., M.D.-K., K.K., M.B.; writing—review and ed-
iting, all authors.; visualization, A.L.; supervision, A.L, M.Ł.; project administration, A.L. M.Ł.;
funding acquisition, A.L., M.Ł. All authors have read and agreed to the published version of the
manuscript.
Funding: This project was partially supported by The National Center for Research and Develop-
ment (NCBR) by POIR.02.01.00-00-0159/15-00 and the Wroclaw Center for Biotechnology (WCB)
and the Leading National Research Centre (KNOW) for years 2014–2018.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Bioethics Commission at the Lower Silesian Chamber
of Physicians and Dentists (1/PNHAB/2020).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study.
Data Availability Statement: The data presented in this study are available in supplementary ma-
terial.
Acknowledgments: Financial support from the statutory activity of subsidy from the Polish Minis-
try of Science and Higher Education for the Faculty of Chemistry and Faculty of Biotechnology of
the University of Wroclaw is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Ghasemiyeh, P.; Mohammadi-Samani, S. Potential of nanoparticles as permeation enhancers and targeted delivery options for
skin: Advantages and disadvantages. Drug Des. Devel. Ther. 2020, 14, 3271–3289, doi:10.2147/DDDT.S264648.
2. Rao, N.V.; Rho, J.G.; Um, W.; Ek, P.K.; Nguyen, V.Q.; Oh, B.H.; Kim, W.; Park, J.H. Hyaluronic acid nanoparticles as
nanomedicine for treatment of inflammatory diseases. Pharmaceutics 2020, 12, 1–18, doi:10.3390/pharmaceutics12100931.
3. Yousef, S.A.; Mohammed, Y.H.; Namjoshi, S.; Grice, J.E.; Benson, H.A.E.; Sakran, W.; Roberts, M.S. Mechanistic Evaluation of
Enhanced Curcumin Delivery through Human Skin In Vitro from Optimised Nanoemulsion Formulations Fabricated with
Different Penetration Enhancers. Pharmaceutics 2019, 11, doi:10.3390/pharmaceutics11120639.
4. Ryu, K.A.; Park, P.J.; Kim, S.B.; Bin, B.H.; Jang, D.J.; Kim, S.T. Topical delivery of coenzyme Q10-loaded microemulsion for skin
regeneration. Pharmaceutics 2020, 12, doi:10.3390/pharmaceutics12040332.
5. Molska, A.; Nyman, A.K.G.; Sofias, A.M.; Kristiansen, K.A.; Hak, S.; Widerøe, M. In vitro and in vivo evaluation of organic
solvent-free injectable melatonin nanoformulations. Eur. J. Pharm. Biopharm. 2020, 152, 248–256, doi:10.1016/j.ejpb.2020.05.003.
6. Bazylińska, U.; Lewińska, A.; Lamch, Ł.; Wilk, K.A. Polymeric nanocapsules and nanospheres for encapsulation and long
sustained release of hydrophobic cyanine-type photosensitizer. Colloids Surfaces A Physicochem. Eng. Asp. 2014, 442, 42–49,
doi:10.1016/j.colsurfa.2013.02.023.
7. Lewińska, A.; Domżał-Kędzia, M.; Jaromin, A.; Łukaszewicz, M. Nanoemulsion stabilized by safe surfactin from Bacillus
subtilis as a multifunctional, custom-designed smart delivery system. Pharmaceutics 2020, 12, 1–21,
doi:10.3390/pharmaceutics12100953.
Molecules 2021, 26, 1063 22 of 23

8. Abd, E.; Namjoshi, S.; Mohammed, Y.H.; Roberts, M.S.; Grice, J.E. Synergistic Skin Penetration Enhancer and Nanoemulsion
Formulations Promote the Human Epidermal Permeation of Caffeine and Naproxen. J. Pharm. Sci. 2016, 105, 212–220,
doi:https://doi.org/10.1002/jps.24699.
9. Sato, Y.; Joumura, T.; Takekuma, Y.; Sugawara, M. Transfer of orally administered hyaluronan to the lymph. Eur. J. Pharm.
Biopharm. 2020, 154, 210–213, doi:10.1016/j.ejpb.2020.07.007.
10. Kim, K.S.H.; Chung, C.B.; Kim, Y.H.; Kim, K.S.H.; Han, C.S.; Kim, C.H. Cosmeceutical properties of levan produced by
Zymomonas mobilis. Int. J. Cosmet. Sci. 2006, 28, 231, doi:ICS314_2 [pii]\r10.1111/j.1467-2494.2006.00314_2.x.
11. Domżał-Kędzia, M.; Lewińska, A.; Jaromin, A.; Weselski, M.; Pluskota, R.; Łukaszewicz, M. Fermentation parameters and
conditions affecting levan production and its potential applications in cosmetics. Bioorg. Chem. 2019, 1–8,
doi:10.1016/j.bioorg.2019.02.012.
12. Pantelić, I.; Lukić, M.; Gojgić-Cvijović, G.; Jakovljević, D.; Nikolić, I.; Lunter, D.J.; Daniels, R.; Savić, S. Bacillus licheniformis
levan as a functional biopolymer in topical drug dosage forms: From basic colloidal considerations to actual pharmaceutical
application. Eur. J. Pharm. Sci. 2020, 142, doi:10.1016/j.ejps.2019.105109.
13. Abolmaali, S.S.; Tamaddon, A.M.; Salmanpour, M.; Mohammadi, S.; Dinarvand, R. Block ionomer micellar nanoparticles from
double hydrophilic copolymers, classifications and promises for delivery of cancer chemotherapeutics. Eur. J. Pharm. Sci. 2017,
104, 393–405, doi:10.1016/j.ejps.2017.04.009.
14. Desai, J.D.; Banat, I.M. Microbial production of surfactants and their commercial potential. Microbiol. Mol. Biol. Rev. 1997, 61,
47–64, doi:10.1128/.61.1.47-64.1997.
15. Santos, V.S.V.; Silveira, E.; Pereira, B.B. Toxicity and applications of surfactin for health and environmental biotechnology. J.
Toxicol. Environ. Heal. Part B Crit. Rev. 2018, 21, 382–399, doi:10.1080/10937404.2018.1564712.
16. Alvarez-Rivera, G.; Llompart, M.; Lores, M.; Garcia-Jares, C. Preservatives in Cosmetics: Regulatory Aspects and Analytical Methods,
2nd ed; Elsevier B.V.: Amsterdam, The Netherlands, 2018; ISBN 9780444635167.
17. Lundov, M.D.; Moesby, L.; Zachariae, C.; Johansen, J.D. Contamination versus preservation of cosmetics: A review on
legislation, usage, infections, and contact allergy. Contact Dermatitis 2009, 60, 70–78, doi:10.1111/j.1600-0536.2008.01501.x.
18. Obeidat, W.M.; Schwabe, K.; Müller, R.H.; Keck, C.M. Preservation of nanostructured lipid carriers (NLC). Eur. J. Pharm.
Biopharm. 2010, 76, 56–67, doi:10.1016/j.ejpb.2010.05.001.
19. Hommoss, A. Preservative system development for argan oil-loaded nanostructured lipid carriers. Pharmazie 2011, 66, 187–191,
doi:10.1691/ph.2011.0272.
20. Chanchal, D.; Swarnlata, S. Novel approaches in herbal cosmetics. J. Cosmet. Dermatol. 2008, 7, 89–95, doi:10.1111/j.1473-
2165.2008.00369.x.
21. Bajerski, L.; Michels, L.R.; Colomé, L.M.; Bender, E.A.; Freddo, R.J.; Bruxel, F.; Haas, S.E. The use of Brazilian vegetable oils in
nanoemulsions: An update on preparation and biological applications. Brazilian J. Pharm. Sci. 2016, 52, 347–363,
doi:10.1590/s1984-82502016000300001.
22. Zhao, Y.; Khalid, N.; Shu, G.; Neves, M.A.; Kobayashi, I.; Nakajima, M. Formulation and characterization of oil-in-water
emulsions stabilized by gelatinized kudzu starch. Int. J. Food Prop. 2017, 20, 1329–1341, doi:10.1080/10942912.2017.1347675.
23. Smaoui, S.; Hlima, H.B.; Jarraya, R.; Kamoun, N.G.; Ellouze, R.; Damak, M. Cosmetic emulsion from virgin olive oil: Formulation
and bio-physical evaluation. African J. Biotechnol. 2012, 11, 9664–9671, doi:10.5897/ajb12.163.
24. Lin, T.K.; Zhong, L.; Santiago, J.L. Anti-inflammatory and skin barrier repair effects of topical application of some plant oils.
Int. J. Mol. Sci. 2018, 19, doi:10.3390/ijms19010070.
25. Dinache, A.; Tozar, T.; Smarandache, A.; Andrei, I.R.; Nistorescu, S.; Nastasa, V.; Staicu, A.; Pascu, M.L.; Romanitan, M.O.
Spectroscopic characterization of emulsions generated with a new laser-assisted device. Molecules 2020, 25,
doi:10.3390/molecules25071729.
26. Pasieczna-Patkowska, S.; Olejnik, T. Analysis of Cosmetic Products Using Different IR Spectroscopy Techniques. Ann. UMCS,
Chem. 2014, 68, 1–2, doi:10.2478/umcschem-2013-0008.
27. Bouftira, I.; Abdelly, C.; Sfar, S. Characterization of cosmetic cream with Mesembryanthemum crystallinum plant extract:
Influence of formulation composition on physical stability and anti-oxidant activity. Int. J. Cosmet. Sci. 2008, 30, 443–452,
doi:10.1111/j.1468-2494.2008.00469.x.
28. Joshi, V.K.; Kumar, A.; Kumar, V. Antimicrobial, antioxidant and phyto-chemicals from fruit and vegetable waste. Intl. J. Food.
Ferment. Technol. 2012, 2, 123–136.
29. Ali, A.; Akhtar, N.; Khan, H.M.S. Assessment of physical stability and antioxidant activity of polysiloxane polyalkyl polyether
copolymer-based creams. J. Chem. 2013, 2013, doi:10.1155/2013/938042.
30. Makarova, K.; Zawada, K.; Wagner, D.; Skowyra, J. Optimization of antioxidant properties of creams with berry extracts by
artificial neural networks. Acta Phys. Pol. A 2017, 132, 44–51, doi:10.12693/APhysPolA.132.44.
31. Ilyasov, I.; Beloborodov, V.; Antonov, D.; Dubrovskaya, A.; Terekhov, R.; Zhevlakova, A.; Saydasheva, A.; Evteev, V.;
Selivanova, I. Flavonoids with glutathione antioxidant synergy: Influence of free radicals inflow. Antioxidants 2020, 9, 1–20,
doi:10.3390/antiox9080695.
32. Khairi, N.; As’ad, S.; Djawad, K.; Alam, G. The determination of antioxidants activity and sunblock Sterculia populifolia extract-
based cream. Pharm. Biomed. Res. 2018, 4, 20–26, doi:10.18502/pbr.v4i1.142.
33. Lo Scalzo, R. Organic acids influence on DPPH{radical dot} scavenging by ascorbic acid. Food Chem. 2008, 107, 40–43,
doi:10.1016/j.foodchem.2007.07.070.
Molecules 2021, 26, 1063 23 of 23

34. Danby, S.G.; Andrew, P.V.; Brown, K.; Chittock, J.; Kay, L.J.; Cork, M.J. An Investigation of the Skin Barrier Restoring Effects of
a Cream and Lotion Containing Ceramides in a Multi-vesicular Emulsion in People with Dry, Eczema-Prone, Skin: The
RESTORE Study Phase 1. Dermatol. Ther. (Heidelb). 2020, 10, 1031–1041, doi:10.1007/s13555-020-00426-3.
35. Ullah, H.; Santos, H.A.; Khan, T. Applications of bacterial cellulose in food, cosmetics and drug delivery. Cellulose 2016, 23,
2291–2314, doi:10.1007/s10570-016-0986-y.
36. Aranaz, I.; Acosta, N.; Civera, C.; Elorza, B.; Mingo, J.; Castro, C.; de los Gandía, M.L.; Caballero, A.H. Cosmetics and
cosmeceutical applications of chitin, chitosan and their derivatives. Polymers (Basel). 2018, 10, doi:10.3390/polym10020213.
37. Sionkowska, A.; Kaczmarek, B.; Michalska, M.; Lewandowska, K.; Grabska, S. Preparation and characterization of
collagen/chitosan/hyaluronic acid thin films for application in hair care cosmetics. Pure Appl. Chem. 2017, 89, 1829–1839,
doi:10.1515/pac-2017-0314.
38. Lewińska, A.; Jaromin, A.; Jezierska, J. Role of architecture of N-oxide surfactants in the design of nanoemulsions for Candida
skin infection. Colloids Surfaces B Biointerfaces 2019, 187, doi:10.1016/j.colsurfb.2019.110639.
39. Sonneville-Aubrun, O.; Yukuyama, M.N.; Pizzino, A. Application of Nanoemulsions in Cosmetics; Elsevier Inc.: Amsterdam, The
Netherlands, 2018; ISBN 9780128118399.
40. Rocha-Filho, P.A.; Ferrari, M.; Maruno, M.; Souza, O.; Gumiero, V. In vitro and in vivo evaluation of nanoemulsion containing
vegetable extracts. Cosmetics 2017, 4, doi:10.3390/cosmetics4030032.
41. Barreto, S.M.A.G.; Maia, M.S.; Benicá, A.M.; de Assis, H.R.B.S.; Leite-Silva, V.R.; da Rocha-Filho, P.A.; de Negreiros, M.M.F.; de
Oliveira Rocha, H.A.; Ostrosky, E.A.; Lopes, P.S.; et al. Evaluation of in vitro and in vivo safety of the by-product of Agave
sisalana as a new cosmetic raw material: Development and clinical evaluation of a nanoemulsion to improve skin moisturizing.
Ind. Crops Prod. 2017, 108, 470–479, doi:10.1016/j.indcrop.2017.06.064.
42. Boonme, P.; Junyaprasert, V.B.; Suksawad, N.; Songkro, S. Microemulsions and nanoemulsions: Novel vehicles for whitening
cosmeceuticals. J. Biomed. Nanotechnol. 2009, 5, 373–383, doi:10.1166/jbn.2009.1046.
43. Jajor, P.; Piłakowska-Pietras, D.; Krasowska, A.; Łukaszewicz, M. Surfactin analogues produced by Bacillus subtilis strains
grown on rapeseed cake. J. Mol. Struct. 2016, 1126, 141–146, doi:10.1016/j.molstruc.2016.02.014.
44. ISO 11930. International Organization for Standardization ISO 11930:2012 Cosmetics—Microbiology—Evaluation of the Antimicrobial
Protection of a Cosmetic Product; ISO: Geneva, Switzerland, 2012.
45. Couteau, C.; Coiffard, L. Regulation (EC) no 1223/2009 of the European Parliament and of the Council of 30 November 2009 on
Cosmetic Products. Nouv. Dermatologiq. 2010, 29, 59.
46. Nowicka, D. Diagnostyka kosmetologiczna—studium przypadków. LNE Spa 2012, 2012, 38–42.