International Journal of Nanomedicine

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A Cosmeceutical Topical Water-in-Oil

Nanoemulsion of Natural Bioactives: Design of
Experiment, in vitro Characterization, and in
vivo Skin Performance Against UVB Irradiation-
Induced Skin Damages

Carol Yousry, Mona M Saber & Wessam H Abd-Elsalam

To cite this article: Carol Yousry, Mona M Saber & Wessam H Abd-Elsalam (2022) A
Cosmeceutical Topical Water-in-Oil Nanoemulsion of Natural Bioactives: Design of Experiment,
in vitro Characterization, and in vivo Skin Performance Against UVB Irradiation-Induced Skin
Damages, International Journal of Nanomedicine, , 2995-3012, DOI: 10.2147/IJN.S363779

To link to this article: https://doi.org/10.2147/IJN.S363779

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ORIGINAL RESEARCH

A Cosmeceutical Topical Water-in-Oil

Nanoemulsion of Natural Bioactives: Design of
Experiment, in vitro Characterization, and in vivo
Skin Performance Against UVB Irradiation-Induced
Skin Damages
1
Carol Yousry , Mona M Saber 2 , Wessam H Abd-Elsalam 1

1
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt; 2Department of Pharmacology and
Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt

Correspondence: Wessam H Abd-Elsalam, Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt,
Email [email protected]

Introduction: Damage to human skin occurs either chronologically or through repetitive exposure to ultraviolet (UV) radiation,
where collagen photodegradation leads to the formation of wrinkles and skin imperfections. Consequently, cosmeceutical products
containing natural bioactives to restore or regenerate collagen have gained a remarkable attention as an ameliorative remedy.
Methods: This study aimed to develop and optimize collagen-loaded water-in-oil nanoemulsion (W/O NE) through a D-optimal
mixture design to achieve an ideal multifunctional nanosystem containing active constituents. Vit E was included as a constituent of
the formulation for its antioxidant properties to minimize the destructive impact of UV radiation. The formulated systems were
characterized in terms of their globule size, optical clarity, and viscosity. An optimized system was selected and evaluated for its
physical stability, in vitro wound healing properties, and in vivo permeation and protection against UV radiation. In addition, the effect
of collagen-loaded NE was compared to Vit C-loaded NE and collagen-/Vit C-loaded NEs mixture as Vit C is known to enhance
collagen production within the skin.
Results: The optimized NE was formulated with 25% oils (Vit E: safflower oil, 1:3), 54.635% surfactant/cosurfactant (Span 80:
Kolliphor EL: Arlasolve, 1:1:1), and 20.365% water. The optimized NE loaded with either collagen or Vit C exhibited a skin-friendly
appearance with boosted permeability, and improved cell viability and wound healing properties on fibroblast cell lines. Moreover, the
in vivo study and histopathological investigations confirmed the efficacy of the developed system to protect the skin against UV
damage. The results revealed that the effect of collagen-/Vit C-loaded NEs mixture was more pronounced, as both drugs reduced the
skin damage to an extent that it was free from any detectable alterations.
Conclusion: NE formulated using Vit E and containing collagen and/or Vit C could be a promising ameliorative remedy for skin
protection against UVB irradiation.
Keywords: water-in-oil nanoemulsion, collagen, vitamin C, vitamin E, fibroblast, UVB irradiation

Introduction
Collagen is a major structural protein that forms most of the connective tissue, and thus functions as a support network to
the cellular structures. collagen is available in the dermis layer of the skin, where the dermal fibroblasts produce the
distinct polypeptide chains of types I and III collagen as precursor molecules (Procollagen).1 It was found that the dermis
comprises mainly type I collagen (85–90%) and lesser quantities of type III collagen (10–15%).2 In general, both the
abundance and strength of collagen are directly related to the healthy appearance of the skin, including hydration,
smoothness, and luminosity.3

International Journal of Nanomedicine 2022:17 2995–3012 2995

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Damage to human skin occurs either over time (chronologic aging) or through repetitive exposure to solar ultraviolet
(UV) radiation (photoaging). Chronologic aging or skin aging is a normal consequence of the damage of the collagen
fibers over time, where the skin loses its thickness and strength.4 On the other hand, photoaging occurs as a result of
exposure to solar UV radiation, which is composed of UVA (320–400 nm), UVB (280–320 nm), and UVC (200–280
nm).5 UVB was found to be responsible for the majority of the injury to the skin, as it is absorbed by the epidermis and
brings about damage to the DNA by producing harmful photoproducts. Besides, UVB reacts with the water content of the
skin generating reactive oxygen species (ROS) which interact with cellular lipids and proteins, resulting in apoptosis and
genetic mutations.6 Moreover, ROS were found to trigger proteases in human skin that upregulate the production of
matrix metalloproteinases (MMPs), which by their role destroy collagen.7 Photoaged skin is characterized by coarse
wrinkles, dyspigmentation, and telangiectasia.2 As illustrated in Figure 1, the healthy skin of a rat (Figure 1A) showed
some defects upon exposure to UVB radiation (Figure 1B), where skin alterations such as erythema and wrinkles, as well
as, hyperpigmentation in certain areas were observed.
In context with these facts, vitamin C (Vit C), in its most biologically active form; L-ascorbic acid, has shown to
possess anti-aging effects by enhancing collagen production and stabilizing its fibers, thus lessening collagen
degradation.8 In addition, Vit C has antioxidant properties via reducing the formation of ROS such as hydroxyl radicals,
superoxide radicals, and singlet oxygen.6 Also, Vit C was proved to minimize melanin formation, therefore dropping skin
pigmentation.9 However, Vit C is photochemically unstable, where it gets oxidized to dehydro ascorbic acid, which
imparts a yellow color upon exposure to light.10 To circumvent the stability issues of vitamin C, previous formulation
approaches were to lower the pH of the medium or limit the water content.11
In a parallel line, the use of vitamin E (Vit E) in the protection against oxidative damage caused by UV radiation is
well established as per its antioxidant properties, and it has been incorporated in many antioxidant cocktails, along with
other ingredients to minimize the destructive impact of UV radiation by heightening the antioxidative capacity of the
epidermis.12 Furthermore, fibroblast attachment, proliferation, and collagen secretion were enhanced in the presence of
Vit E.13
Nanoemulsions (NEs) are colloidal systems usually employed in the nutraceutical, pharmaceutical, and cosmetic
industries.14 NEs are isotropic thermodynamically stable nanoformulations that are characterized by a fast onset of action
with improved drug efficacy, effortless preparation and scale-up, and act as a cargo shield against hydrolysis and
oxidation.15–17 In addition, NEs are well-tolerable by the skin, with a skin-friendly appearance, in addition to their
suitability for accommodating hydrophilic and lipophilic drug substances.18 Consequently, NEs have become famous for
dermal drug delivery, adding to the existence of surfactants in their composition, which in turn surges the membrane

Figure 1 Rats’ dorsal skin photographs of (A) normal control group (unexposed to UVB-irradiation) and (B) positive control group (exposed to UVB-irradiation, where
skin alterations were visible, as the skin was erythematous and wrinkled, as well as, being hyperpigmented in certain areas).

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permeability and circumvents the skin barrier, thus facilitating drug transport through the skin.19 NEs are usually
prepared using a random formulation approach, which is based on trial and error and lacks a cause–effect relationship
between independent variables and dependent response, leading to an erratic and a non-optimized formulation. On the
contrary, the formulation of NEs on basis of a validated statistical experimental design results in reliable outcomes and
realistic evaluations of the impact of different variables on the formulation process. Numerous studies including different
statistical experimental designs for formulating NEs have been established and among them, the D-optimal mixture
design. This type of design allows the formulator to apply a limitation that the sum of all components is constant (ie,
100% w/w), which is a typical feature related to the pseudo-ternary phase diagram plotted to contour NEs regions.20
Based on all the above-stated pieces of evidence, collagen has been introduced as a counteracting therapy in different
cosmetic formulations owing to its high biodegradability, biocompatibility, and non-toxicity,21 and to boost the valuable
effect of collagen on the skin, rethinking a multifunctional nanosystem that co-deliver collagen along with multi-
antioxidant vitamins such as Vit C and E would be beneficial. Taking into consideration the aforementioned properties
of NEs, employing them as a water-in-oil (W/O) NE for hydrophilic actives such as collagen or Vit C seems reasonable
to improve their stability and enhance their permeation and thus boosting their efficacy. Thus, the primary objective of
this study was to develop and optimize the formulation of collagen-loaded W/O NE through a D-optimal mixture design
and select an optimized formulation. The oily phase was suggested to be a mixture of the natural oils; safflower oil and
Vit E, while Span 80 and Kolliphor EL were incorporated as a surfactant, and Arlasolve was used for the first time as
a cosurfactant in the formulation of NE. Safflower oil is an edible oil that is obtained from safflower seeds, and it
comprises various bioactive compounds such as linoleic acid, tocopherols, sterols, squalene hydrocarbons, and
polyphenols.22 The safflower seed oil has been recommended to be used as a promoter of skin barrier homeostasis, in
addition, safflower has shown different physiological functions, including antioxidation and melanin production
inhibition.23 The inclusion of dermally safe surfactants is crucial in formulating topical skin formulations, where
surface-active agents with structure blocks of natural origin are frequently considered to be more biocompatible and
biodegradable compared to chemically created counterparts. An add-on property that minimizes the skin sensitization
potential and favors their usage for dermal application is being uncharged or nonionic.24 Span 80 is a non-ionic lipophilic
surfactant with an HLB value of 4.3, which is composed of a natural fatty acid (oleic acid) and sugar alcohol sorbitol,24
while Kolliphor EL, polyoxyl 35 castor oil, is a nonionic surface-active agent (HLB value = 12–14).25 Arlasolve is
3,6-dimethoxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan, that acts as a skin adjuvant by boosting the performance and
enhancing the delivery of actives within formulations for many personal care applications. To test the boosted permeation
character of the developed system, the permeation of the optimized NE was investigated through a confocal laser
scanning microscopy study. The secondary objective was to load the optimized NE with Vit C and to compare its effect
with that of collagen or a mixture of both collagen- and Vit C-loaded NEs on fibroblast cell lines, namely normal human
fibroblast (HFB4) and Baby Hamster Kidney fibroblasts (BHK). In addition, an in vivo study and histopathological
investigations were conducted to verify the hypothesis of the skin protection outcome of the optimized formulations
against UVB irradiation in Wistar rats.

Materials and Methods

Materials
Porcine type-I collagen (Cell matrix Type I-A), safflower seed oil (Carthamus tinctorius), Vitamin E (α-tocopherol),
Span 80 (Sorbitan monooleate), Kolliphor EL, vitamin C (L-ascorbic acid), and Rhodamine B dye were acquired from
Sigma Aldrich (Missouri, USA). Arlasolve was obtained from Croda International Plc (East Yorkshire, UK). Any other
used chemicals were of high purity and used as received.

Construction of Pseudo-Ternary Phase Diagrams

According to the outcomes of the preliminary trials (data not shown), vitamin E and safflower oil (1:3) represented the
oily phase, Span 80, and Kolliphor EL (1:1) were selected as surfactants, while Arlasolve was used as a cosurfactant. The
selected surfactants were mixed with the cosurfactant at a ratio of 2:1 to form S/Cos mixture, which was further blended

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with the oily phase at weight ratios of 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, and 10:90. Nanoemulsions
(NEs) were prepared using the aqueous titration method, where water was added dropwise to the formed blends under
vortexing (Stuart variable speed vortex mixer/SA8; UK) till the last point the system was transparent and clear. The
concentrations of the different ingredients (as percentages) were plotted on a pseudo-ternary phase diagram, and the
borders of the clear zone were determined.

Design of Experimentation (DOE)

Design Expert® software (version 7.0.0, Stat-Ease Inc., Minneapolis, USA) was employed to create a D-optimal mixture
design aiming to provide maximized prediction power in the designated cluster of 16 experimental runs, thus lessening
any possible variance correlated to the estimations of the coefficients in the model. The independent variables were set so
that the percentage of the oils (A) ranges from 20% to 45%, the percentage of the surfactant/cosurfactant (S/Cos) (B) lies
between 45% and 70%, and the percentage of water (C) fall within range 10–25%.
The prepared runs were characterized according to the dependent variables, which were selected to include the
globule size (GS) (Y1) in nm, polydispersity index (PDI) (Y2), the percent transmittance (%T) (Y3), and viscosity (Y4) in
cp. The resultant linear, quadratic, cubic, and special cubic mathematical models were analyzed by the software. The
value of standard deviation (SD), R2, adjusted R2, predicted R2, and the predicted residual error sum of squares (PRESS),
as statistical parameters, were compared to test the significance of the model, where the one with the higher values of R2,
adjusted R2, and predicted R2, and with a difference of less than 0.2 (between the adjusted R2 and the predicted R2) was
chosen. On the contrary, the least SD and PRESS values were targeted. The composition and the responses of the
suggested experimental runs, loaded with collagen (1% w/w), are represented in Table 1. The optimized NE was elected
by the software, in which the GS (Y1) and PDI (Y2) were minimized, while %T (Y3) and viscosity (Y4) were maximized.
The optimized NE was prepared and loaded with either collagen or Vit C (both at a concentration of 1% w/w) to compare
the effectiveness of the carrier to deliver both actives, as Vit C is known to be a viable supplement to enhance collagen
synthesis and accelerate soft tissue healing.

Table 1 The Composition of the Experimental Runs According to the D-Optimal Statistical Design and Their
Responses
Runs A Oils (%) B S/Cos (%) C Water (%) Y1* GS (nm) Y2* PDI Y3* %T Y4* Viscosity (cp)

1 28.49 53.20 18.31 26.42±0.37 0.42±0.08 98.76±0.08 342.83±3.55

2 24.36 51.23 24.41 29.52±0.10 0.41±0.02 86.12±0.72 495.83±5.78
3 23.20 62.03 14.77 25.87±1.31 0.43±0.07 98.36±0.57 285.50±2.04
4 31.38 58.62 10.00 34.12±1.42 0.35±0.05 92.56±1.54 320.20±3.44
5 40.55 49.45 10.00 73.01±3.03 0.59±0.07 75.25±0.51 336.30±2.46
6 45.00 45.00 10.00 68.64±2.18 0.29±0.05 61.93±4.28 324.17±5.92
7 38.46 45.00 16.54 58.22±3.58 0.42±0.06 79.05±2.32 354.77±6.23
8 45.00 45.00 10.00 63.96±3.10 0.25±0.02 65.21±3.23 333.03±2.59
9 20.00 57.26 22.74 18.75±1.86 0.68±0.02 99.11±0.30 433.90±1.47
10 20.00 70.00 10.00 151.00±28.82 0.58±0.01 72.03±0.65 312.40±5.20
11 20.00 70.00 10.00 147.00±33.93 0.56±0.01 68.85±0.06 340.87±2.95
12 29.44 45.56 25.00 29.89±1.78 0.84±0.01 85.29±2.10 418.97±2.50
13 29.44 45.56 25.00 28.37±2.26 0.80±0.01 80.86±1.82 456.63±3.43
14 20.00 57.26 22.74 15.20±1.18 0.65±0.00 99.63±0.60 470.23±19.20
15 38.46 45.00 16.54 46.15±3.02 0.47±0.04 87.39±2.56 321.77±4.46
16 35.32 51.81 12.87 59.49±3.37 0.31±0.03 77.85±1.35 382.80±3.46
Notes: *All values are represented as mean ± standard deviation of three measurements for three different batches (n = 3). Collagen (10 mg) was
incorporated in all formulations with a final concentration of 1% w/w.
Abbreviations: S/Cos, surfactant/cosurfactant; GS, globule size; PDI, polydispersity index; %T, percent transmittance.

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In vitro Characterization of Collagen-Loaded NEs

Globule Size (GS) and Polydispersity Index (PDI) Determinations
The mean globule size (GS) and polydispersity index of the size distribution (PDI) for each NE were measured by photon
correlation spectroscopy using the Malvern zeta-sizer (Ver.6.20, Malvern Instruments Ltd., Worcestershire, UK).26
Analysis was performed at 25±2 °C and each reported value is the average of three measurements for three different
batches.27

Optical Clarity
The optical clarity or the percent transmittance (%T) of NEs was measured via a UV-vis spectrophotometer (UV-1800;
Shimadzu, Kyoto, Japan) at a wavelength of 600 nm.

Rheological Measurements
The rheological properties of NEs were determined via a cone-plate Brookfield Digital Rheometer Model DV-III
(Brookfield Engineering LABS, Stoughton, MA) equipped with a flat spindle, type CP 40. The determination of the
apparent viscosity measurements of NEs samples (1.5 g) was conducted at a variable spindle speed (0, 10, 15, 20, 30, 50,
70, 90, 100, and 120 rpm) within 90 seconds, and torque between 15.0% and 85.0%, at 25±2 °C. To compare the
viscosity values of NEs between the dissimilar stresses, a 100 rpm as a spindle speed (after 30 seconds) was selected. The
average ± standard deviation of three runs for three different batches was recorded.

Transmission Electron Microscopy (TEM) and pH Measurements

The inherent structure and topography of the optimized NE, containing either collagen or Vit C, were elucidated by Joel
JEM 1230 transmission electron microscope (Tokyo, Japan), while their pH value was measured with a pH meter
(Jenway, UK).28

Stability Studies
The thermodynamic stability of the optimized NE containing either collagen or Vit C was assessed by the protocol
previously reported by Shafiq et al.29,30 The optimized NEs were subjected to six heating-cooling cycles between the
temperature of (4°C) and (45°C) for 48 h at each temperature and then centrifuged at 3500 rpm for 30 min. Finally, the
optimized NEs were exposed to three freeze–thaw cycles between (−20°C) and (+25°C) for 48 h at each temperature.
Moreover and according to ICH guidelines, the optimized NEs were stored in amber glass vials at 25±2 °C/60±5% RH
for 6 months. At the end of the storage period, GS, PDI, %T, and viscosity were determined thrice and the results were
compared to that attained from the freshly prepared NE, where the statistical significance was determined using
GraphPad Prism Version 9 (San Diego, CA, USA), at p < 0.05.

In vitro Cellular Studies

Cell Culture
All in vitro cellular studies were performed at the Egyptian National Cancer Institute (Cairo, Egypt). Normal human
fibroblast (HFB4) (PCS-201-010™) and Baby Hamster Kidney fibroblasts (BHK) (CCL-10™) cell lines were obtained
commercially from the American Type Culture Collection (ATCC) (Manassas, USA). The cell lines were cultured and
maintained in Roswell Park Memorial Institute-1640 (RPMI-1640) medium, which was charged with 10% fetal bovine
serum, 1.5 g/L sodium bicarbonate, 2 mM L-glutamine, and 1% penicillin/streptomycin. The cells were kept at 37 °C in
the prescence of 5% CO2.

Cell Viability Assay

Sulforhodamine B (SRB) assay was used to investigate the cytotoxicity of the investigated treatments. In 96-well plates,
exponentially growing cells were seeded at an initial density of 5×103/well and left in the incubator for 24 h. The
considered treatment groups were Vit E, Vit C, collagen, plain NE lacking Vit E, plain NE, Vit C-loaded NE, collagen-
loaded NE, and a mixture of collagen- and Vit C-loaded NEs (1:1). Different treatments with various concentrations
ranging from 0 to 50 µL/mL were added to human HFB4 and BHK cell lines and incubated for 48 h at 37 °C to assess
their effect on cell growth and survival. Trichloroacetic acid (10%) was used for cell fixation and left for 1 h at 4 °C,

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while SRB (0.4%) was added to stain the cells. After 30 min, the cells were rinsed with acetic acid (1%) four times, then
left to dry. Tris base (10 mM, pH 10.5) was added to dissolve the dye, then the optical density (O.D.) was measured
spectrophotometrically at 570 nm via the microplate reader (Tecan SunriseTM, Switzerland). GraphPad InStat Software,
version 7.01 (GraphPad, USA) was used to create sigmoidal dose–response curve-fitting models and to carry out the
analysis of the data by applying a one-way analysis of variance (ANOVA) then Tukey multiple comparison post-hoc test.
Results were considered statistically significant at P < 0.05.

Scratch Wound Healing Assay

HFB4 and BHK cells were cultured in six-well plates and then left for 24 h to form an adherent monolayer. This layer
was then gently injured in a unidirectional way across the center of the well using a sterile pipette tip (1 mL). A gap
distance equivalent to the external diameter of the pipette tip (0.5 mm) was produced and the elimination of the detached
cells was completed by washing the wells with fresh medium twice. Different treatments (Vit E, Vit C, collagen, plain
NE lacking Vit E, plain NE, Vit C-loaded NE, collagen-loaded NE, and a mixture of collagen and Vit C-loaded NEs
(1:1)) were added at 50 µL/mL and the plates were incubated for 48 h. After washing the cells with phosphate buffer
saline (PBS), they were fixed for 30 min with 3.7% paraformaldehyde. The cells were stained with crystal violet (1%) in
2% ethanol for 30 min. Micrographs of the stained monolayer were captured on a microscope and the gap distance was
measured (when possible) by drawing lines through the edge of repopulating fibroblast cells and quantifying the cell-free
distance remaining via the Leica Qwin-Plus software (Leica Microsystems, UK).

In vivo Studies
The procedures of the in vivo studies performed using male Wistar rats were pre-approved by the Research Ethics
Committee in the Faculty of Pharmacy (REC-FOPCU), Cairo University (PI 2971) and were in compliance with the
Guide for Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 2011). The animals (200–250 g)
were maintained in a regulated environment of controlled temperature (23 ± 2 °C) and humidity (55±10%), and
alternating light–dark cycles (12 h). The rats were nourished with a commercial chow and water (ad libitum).

Confocal Laser Scanning Microscopy (CLSM) Study

CLSM affords data about the shuttling of drugs by delivery systems through biological tissues. Rhodamine B dye (a
hydrophilic fluorescent agent) was selected to mimic the behavior of water-soluble actives. The penetration and
distribution of Rhodamine B dye in the skin layers after its topical application as an aqueous solution or loaded into
the optimized NE were compared using CLSM. Before the experiment, the dorsal skin of six male Wistar rats was shaved
and circumscribed, then the animals were allocated randomly into two groups (n=3). Group (A) received topical
application of Rhodamine B dye aqueous solution, while group (B) was treated with Rhodamine B dye-loaded NE.
After 8 h, the rats were decapitated and the skin samples were excised and washed with phosphate buffer saline (pH 7.4),
then directly sandwiched between a glass slide and a coverslip, and finally inspected via CLSM.

In vivo Evaluation of the Optimized NE as a Protective Agent Against UVB Irradiation-Induced Skin Damage
Before the study, the dorsal hair was shaved by an electric clipper and specified areas were circumscribed. In total, 25
rats were separated randomly into 5 groups (n=5), where the calculation of the sample size was based on Mead’s test.31
Group (A): Control group; the rats did not receive any treatment and were not irradiated, group (B): Positive control
group; the rats were UVB-irradiated and did not receive any treatment, group (C): Vit C-loaded NE-treated group;
group (D): collagen-loaded NE-treated group, and group (E); Mixture of collagen and Vit C-loaded NEs (1:1)-treated
group. The rats in groups (C), (D), and (E) received prophylactic topical treatment on the circumscribed dorsal skin
with 0.5 g of Vit C-loaded NE, collagen-loaded NE, as well as collagen and Vit C-loaded NEs mixture (all at
a concentration of 1% w/w), respectively, once daily for 1 week before starting the irradiation protocol, and 30 min
before irradiation. For UV exposure, the rats were irradiated with a UVB lamp (290–320 nm), at a dose of 100 mJ/cm2/
day for 5 days, receiving a total dose of 500 mJ/cm2. Three hours after the last exposure to UVB, the rats were
euthanized by decapitation.32 Specimens of dorsal skin were excised and fixed by immersion into 10% formalin. The
dehydration of the specimens was performed with gradient series of alcohol then treated with xylene and finally

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embedded in paraffin blocks. Five-micrometer thick sections were microtomed and stained with hematoxylin and eosin
(H&E). Skin sections were inspected by light microscopy and photographed with a full HD microscopic camera
operated by the Leica application module (Leica Microsystems GmbH, Wetzlar, Germany).

Results
Pseudo-Ternary Phase Diagram
According to the outcomes of the preliminary trials (data not shown), vitamin E and safflower oil (1:3) represented the
oily phase, Span 80, and Kolliphor EL (1:1) were set as surfactants, while Arlasolve was used as a cosurfactant. As
depicted in Figure 2, the pseudo-ternary phase diagram allows for distinguishing nanoemulsion (contoured) areas and
emulsion regions. Consequently, and according to the NE area, defined percentages of the oils (20–45%), the surfactant/
cosurfactant (S/Cos) (45–70%), and water (10–25%) were selected (represented by the black triangle), which were
further included as independent variables in a D-optimal mixture design.

Fitting of the Models

The Design Expert® software proposed 16 runs considering three independent variables: the percentage of oils (A), the
percentage of S/Cos (B), and the percentage of water (C) with four experimental responses, namely (GS) (Y1) in nm,
(PDI) (Y2), (%T) (Y3), and viscosity (Y4) in cp, as displayed in Table 1. The relationship between the dependent and
independent variables was constructed as per the polynomial equations generated by the statistical analysis, where the
cubic model was selected for the analysis of GS and PDI, whereas the quadratic model was selected for %T and the linear
model for the viscosity, Table 2. The P values were found to be less than 0.05 which confirmed the significance of the
model-fitting following the ANOVA, and this was accompanied by a non-significant lack of fit (P > 0.05). Remarkably,
a model with an R2 of more than 0.90 signifies a high correlation between the data.33 A difference of less than 0.2
between the predicted and adjusted R2 demonstrates the fitness of the nominated model to the data, which was detected in
all the measured responses except PDI. The adequacy of precision that determines the ratio of signal to noise was quite
higher than four which supported the adequacy of the signal in the selected model,34 in addition, the chosen models
showed the lowest PRESS values. The GS and PDI varied widely in the design space, with values of 15.20±1.18 to
151.00±28.82 nm, and 0.25±0.02 to 0.84±0.01, respectively, which infers the influence of the concentration of different
ingredients on the GS and PDI. The %T ranged from 61.93±4.28 to 99.63±0.60%, while the viscosities of the developed
systems were in the range of 285.50±2.04–495.83±5.78 cp suggesting suitability for topical application. The final
equations in terms of actual components were as follows:

Figure 2 Pseudo-ternary diagram of vitamin E and safflower oil (1:3), Span 80/kolliphor EL/ Arlasolve (1:1:1), and water. The defined percentages of the oils (20–45%), the
surfactant/ cosurfactant (S/Cos) (45–70%), and water (10–25%) are represented by the black triangle.

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Table 2 Summary of Model Statistics for the Measured Responses

Response Model SD R2 Adjusted R2 Predicted R2 PRESS

Y1: GS Linear 29.67 0.5488 0.4794 0.2536 18,933.41

Quadratic 16.80 0.8887 0.8331 0.7157 7212.14
Cubic 4.07 0.9961 0.9902 0.9823 449.19
Y2: PDI Linear 0.14 0.4569 0.3734 0.1654 0.39
Quadratic 0.13 0.6364 0.4546 0.2286 0.36
Cubic 0.026 0.9915 0.9787 0.7273 0.13
Y3: %T Linear 10.02 0.4405 0.3544 0.1000 2100.27
Quadratic 5.39 0.8755 0.8133 0.6779 751.64
Cubic 3.26 0.9727 0.9318 0.3152 3069.33
Y4: Viscosity Linear 38.29 0.6911 0.6436 0.5711 26,470.12
Quadratic 32.74 0.8264 0.7395 0.6138 23,830.24
Cubic 21.63 0.9545 0.8863 1.6081 160,900
Note: The statistic values of the selected model for each response are underlined.
Abbreviations: SD, Standard deviation; GS, globule size; PDI, Polydispersity index; %T, Percent transmittance, PRESS, Predicted residual error sum of squares.

GS ¼ 79:57 � A þ 37:48 � B þ 150:18 � C þ 0:81 � A � B 3:31 � A � C 3:06 � B � C þ 0:07

3
(1)
�A � B � C þ 0:02 � A � B�ðA BÞþ0:02 � A � C�ðA CÞ 2:59x10 �B � C�ðB CÞ

PDI ¼ 0:92 � A þ 0:07 � B 4:71 � C þ 0:02 � A � B þ 0:12 � A � C þ 0:08 � B � C 1:42x10 3 �A � B

(2)
�C þ 1:83x10 4 �A � B�ðA BÞ 6:08x10 4 �A � C�ðA CÞ 2:43x10 4 �B � C�ðB CÞ

%T¼ 3:86 � A 1:37 � B 13:24 � C þ 0:11 � A � B þ 0:19 � A � C þ 0:26 � B � C (3)

Viscosit ¼ 2:16 � A þ 2:37 � B þ 11:00 � C (4)

It should be noted that the main variables ([A], [B], and [C]) signify the middling fallouts of fluctuating one variable at
a time between its low to high value, and the interactions [A][B], [B][C], and [A][C] express how the responses alter
when two or three factors vary simultaneously. Moreover, a positive sign preceding each term points to a synergistic
consequence; however, a negative sign entitles an antagonistic outcome on the measured responses. Figure 3 represents
the 3D surface plots demonstrating the effect of the independent variables, the percentage of oils (A), the percentage of S/
Cos (B), and the percentage of water (C) on (I) globule size in nm (GS), (II) polydispersity index (PDI), (III) percent
transmittance (%), and (IV) viscosity in cp.

Optimization of the Design

To establish the criteria for selecting the optimized formulation, minimum GS and PDI, and maximum %T and viscosity
were set, and the software was allowed to predict the optimized NE. The predicted NE (with a desirability value of 0.808)
was prepared with 25% oils, 54.635% S/Cos, and 20.365% water in triplicate, and the responses were measured. The
optimized collagen-loaded NE showed GS of 19.41±4.17 nm with a PDI value of 0.50 ± 0.11, in addition, % T was 99.98
±0.01%, and the measured viscosity was 402.00±5.02 cp. The practically obtained responses showed insignificant
different results (P < 0.05) compared to the predicted values, Table 3. Moreover, the optimized NE was prepared and
loaded with Vit C, which exhibited GS of 26.15±1.02 nm, PDI of 0.49±0.04, % T of 98.34±0.21%, and viscosity of
357.00±11.31 cp.

TEM and pH Measurements

The TEM micrographs of the optimized collagen-loaded NE and Vit C-loaded NE are exhibited in Figure 4. The
micrographs revealed dispersed spherical water globules with a size comparable to those obtained from the zeta-sizer.

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Figure 3 3D surface plots demonstrating the effect of the independent variables; the percentage of oils (A), the percentage of S/Cos (B), and the percentage of water (C) on
(I) globule size in nm (GS), (II) Polydispersity index (PDI), (III) Percent transmittance (%), and (IV) Viscosity in cp.

The measured pH value of collagen-loaded NE was found to be 5.68, and the pH value of Vit C-loaded NE was found to
be 4.30.

Stability Studies
After storage at 25±2 °C/60±5% RH for 6 months, the optimized collagen-loaded NE and Vit C-loaded NE continued to
be transparent and clear lacking any precipitation or phase separation. The values of GS, PDI, %T, and viscosity were not
altered significantly (P > 0.05), suggesting that the optimized NEs were stable under the above conditions (Table 4).
Additionally, the physical stability of the optimized NEs was maintained without any phase separation or drug
precipitation upon centrifugation at 3500 rpm for 30 min or exposure to repetitive extreme conditions of temperature
through heating-cooling and freeze–thaw cycles.

Table 3 The Observed and the Predicted Values of the Optimized

Collagen-Loaded NE
Factor Optimized Level (D=0.808)

A: Oils (%) 25
B: S/Cos (%) 54.635
C: Water (%) 20.365

Response Expected Observed# Residual*

Y1: GS (nm) 15.20 19.41±4.17 −4.21

Y2: PDI 0.51 0.50 ± 0.11 −0.01
Y3: %T 99.63 99.98±0.01 −0.35
Y4: Viscosity (cp) 407.89 402.00±5.02 4.11
Notes: *Residual = expected value – observed value. #Mean ± Standard deviation (n = 3).
Abbreviations: D, desirability value; S/Cos, surfactant/cosurfactant; GS, globule size; PDI,
polydispersity index; %T, percent transmittance.

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Figure 4 TEM micrographs of (A) collagen-loaded NE, and (B) Vit C-loaded NE.

In vitro Cellular Studies

Cell Viability and Proliferation
In this study, the influence of the different treatments on cell viability was evaluated on the HFB4 and BHK cell lines as shown
in Figure 5A and Figure 5B, respectively. After 48 hours of treatment, NE loaded with Vit C, collagen, or a mixture of both
increased the cell proliferation and enhanced the surviving fraction of both cell lines significantly (P < 0.05) compared to the
untreated cells or cells treated with either collagen or Vit C solutions or Vit E. The mixture of collagen and Vit C-loaded NEs
produced the highest increase in cell survival at all concentrations that ranged from 0 to 50 µL/mL. As displayed in Figure 5C
and D, at 50 µL/mL as the highest concentration, collagen and Vit C-loaded NEs mixture resulted in a 1.18- and 1.21-fold
increase in cell survival in HFB4 and BHK cell lines, respectively. This increase was statistically significant (P < 0.05)
compared to those observed with NEs containing the solo drugs. Vit C- or collagen-loaded NEs also produced a statistically
significant elevation (P < 0.05) in cell survival compared to their solutions.

Wound Healing Study

This experiment aimed to evaluate the effect of the different treatments on wound healing and cell migration in the HFB4
and BHK cells as shown in Figure 6. As shown in Figure 6A, the gap distance (0.5 mm) produced by the pipette tip was
obvious at zero time and decreased to approximately 0.15–0.2 mm after 48 h without treatment (Figure 6B). This gap
was filled to a certain extent with proliferated cells in the wells treated with Vit E, Vit C, collagen, plain NE lacking Vit
E, and plain NE (Figure 6C–G, respectively). Treatment with Vit C (Figure 6H) or collagen-loaded NE (Figure 6I)
resulted in healing to a greater extent compared to their drug solutions, which could be demonstrated by the presence of
more regenerated cells in the gap. The gap distance in the HFB4 and BHK cell lines reached a complete closure with
collagen- and Vit-C-loaded NE mixture (Figure 6J), which indicates a synergistic effect.

In vivo Studies
Confocal Microscopy
To envision the influence of the formulation (NE) on drug shuttling through the skin following topical application, the
penetration and diffusion of Rhodamine B dye from the optimized NE and its solution (as a control) in the skin layers

Table 4 Characteristics of the Optimized Collagen-Loaded NE and Vit C-Loaded NE Before and After
Storage at (25±2 °C/60±5% RH) for 6 Months
Formulations GS (nm) PDI T (%) Viscosity (cp)

Collagen-loaded NE before storage 19.41±4.17 0.50 ± 0.01 99.98±0.01 402.00±5.02

Collagen-loaded NE after storage 19.95 ± 3.37 0.51± 0.1 99.98± 0.12 411.00±9.31
Vit C-loaded NE before storage 26.15 ± 1.02 0.49±0.04 98.34±0.21 357.00±11.31
Vit C-loaded NE after storage 25.82 ± 2.35 0.52±0.03 98.57±0.11 365.00±8.78
Abbreviations: GS, globule size; PDI, polydispersity index; %T, percent transmittance.

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Figure 5 Surviving fraction after applying the different treatments (Vit E, Vit C, collagen, plain NE lacking Vit E, Plain NE, Vit C-loaded NE, collagen-loaded NE, and a mixture
of collagen and Vit C-loaded NEs) for 48 h on (A) HFB4 cells, (B) BHK cells, (C) HFB4 cells after applying the treatments at the highest concentration (50 µL/mL), and (D)
BHK cells after applying the treatments at the highest concentration (50 µL/mL). The actual data represent the mean ± SD of three separate experiments performed in
duplicates. Results were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by Tukey multiple comparison post-hoc test. aSignificant
difference from Vit E, bSignificant difference from Vit C, cSignificant difference from collagen, dSignificant difference from Plain NE lacking Vit E, eSignificant difference from
Plain NE, fSignificant difference from Vit C-loaded NE, and gSignificant difference from collagen-loaded NE (p<0.05).

were observed via CLSM. Figure 7 illustrates rhodamine B retention in the stratum corneum layer when introduced to the
skin in its aqueous solution (Figure 7A), whereas the dye was permeated through the skin layers at the depth of up to 260
µm after the topical application of the optimized NE (Figure 7B). Moreover, the dye was represented with a weak
fluorescence in the specimens treated with the dye aqueous solution, while higher fluorescence intensities were observed
in the case of NE.

In vivo Protection Against UVB Irradiation-Induced Skin Damage

Figure 8 demonstrates the histopathological observations of untreated denuded rat skin, UVB-irradiated rat skin, and UVB-
irradiated rat skin pretreated with Vit C-loaded NE, collagen-loaded NE, and a mixture of collagen and Vit C-loaded NEs. The
histological examination of the control group (1) revealed normal epidermal and dermal layers along with normal structure of
different skin appendages, including hair follicles, sebaceous glands, and sweat glands (Figure 8A). The shaved rat skin
exposed to UVB irradiation displayed alterations mainly in the epidermis, where hyperkeratosis was frequently observed in
many sections, and acanthosis was manifested by thickening in the epidermal layer. The epidermis contained numerous
shrunken cells known as sunburn cells near the basal cell layer, as demonstrated by arrows in Figure 8B. Topical treatment
with Vit C-loaded NE and collagen-loaded NE showed better skin shape and less roughness owing to their healing effects
(Figure 8C and D, respectively). However, the effect of collagen and Vit C-loaded NEs mixture was more pronounced
compared to Vit C-loaded NE or collagen-loaded NE, where NE containing both drugs reduced the skin damage to an extent
that the skin was free from any detectable alterations (Figure 8E).

Discussion
Nanoemulsions consist of water, oil, and surfactant, so these systems can be represented by ternary diagrams; and since
a co-surfactant is incorporated in the systems, pseudo-ternary diagrams are used. A co-emulsifier is usually added to

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Figure 6 Micrographs of the stained healed monolayer of HFB4 and BHK cells. (A) Control at 0 h, (B) control after 48 h, (C) Vit E, (D) Vit C, (E) collagen, (F) plain NE
lacking Vit E, (G) plain NE, (H) Vit C-loaded NE, (I) collagen-loaded NE, and (J) A mixture of collagen- and Vit C-loaded NEs.

advance the system’s stability.35 As for the oily phase, an optimum composition is required to form stable NEs. The
inclusion of Vit E in such formulation was optimum as per its structural features, where the presence of the hydroxyl
group allowed the system to incorporate more amounts of water compared to those formulated using safflower oil solely.
Also, it can donate a hydrogen atom to reduce free radicals and thus enhancing the system’s stability. Moreover, the
hydrophobic side chain of Vit E permits boosted penetration into biological membranes.36 The choice of the surfactants is
also critical and this selection was based on their solubility and miscibility with the oils, taking into consideration that the
used non-ionic surfactants are considered to be less toxic than their ionic counterparts. The surfactant mixture of Span 80
and Kolliphor EL was reported earlier to work synergistically to reduce the water–oil interfacial tension to an extent more
than that produced by each of them. Previously during the preparation of NE, it was stated that the adsorption and
arrangement of Span 80 molecules on the oil-water interfacial boundary are easier in the presence of Kolliphor EL as the
mixture boosts the dispersion and the solubilization of the internal phase into sub-micrometer droplet size.14 The
selection of Arlasolve as a co-surfactant was based on its miscibility with the selected oils and surfactants, besides its
ability to enhance the delivery of actives through being incorporated in many cosmeceutical preparations.
As mentioned earlier and as per the results of model fitting, the cubic model was selected for the analysis of GS and
PDI, whereas the quadratic model was selected for %T and the linear model for the viscosity. The GS of NE ought to be
as small as possible so that their penetration into the deeper layers of the skin is facilitated to deposit collagen. As per the
3D surface plot of GS (Figure 3I) and ANOVA results, the oil and S/Cos proportions interaction (AB) and the oil, S/Cos,

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Figure 7 CLSM micrographs of (A) Rhodamine B dye aqueous solution and (B) Rhodamine B dye-loaded NE.

Figure 8 Microscopic examination of skin samples from (A) control group revealing normal histology of skin, (B) positive control showing histopathological alterations
mainly in the epidermis. Hyperkeratosis and acanthosis (black arrows) are manifested by thickening in the epidermal layer (red arrow). The epidermis contained numerous
shrunken cells known as sunburn cells near the basal cell layer (3 black arrows), (C) Vit C-loaded NE showing apparently normal skin with mild inflammatory cells
infiltration, (D) collagen-loaded NE showing apparently normal skin, and (E) a mixture of collagen and Vit C-loaded NEs revealing skin free from any detectable alterations.

and water proportions interaction (ABC) showed a significant impact on GS (P < 0.05). According to equation (1), the oil
proportion showed an antagonistic effect on the GS of NE, which could be explained in light of the fact that the higher
percentages of oil, as an external phase of the W/O NE, minimized the probability of water droplets aggregations owing

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to the dilution effect. In addition, more oil proportions increased the viscosity of the medium, and thus restricted the
migration of water droplets.37,38 Taking into consideration the significant oil and S/Cos proportions interaction (AB) and
the oil, S/Cos proportions, and water interaction (ABC), higher proportions of oils when accompanied by small amounts
of S/Cos yielded large GS, and this could be due to the adsorption of an insufficient number of S/Cos molecules at the
interface. On the contrary, increasing the proportion of S/Cos and water, as an inner phase of the W/O NE, resulted in
a decrease in GS, which is attributed to the lower possibility of water droplets coalescence due to the sufficiency of S/Cos
molecules adsorption at the interface, and probably because of certain surface activity of collagen which was comfortably
soluble in an increased proportion of inner water phase.39 The largest GS (Run 10 and 11) was observed with the highest
amount of S/Cos, and a possible explanation is that a highly viscous liquid crystalline phase could be formed which
interfered with the spontaneous collapse of the water–oil interface in the NE.40 Shakeel and Ramadan reported earlier
that upon increasing the ratio of S/Cos to water, the droplet size significantly increases.41 PDI is a dimensionless
parameter that measures the breadth of size distribution considered from the cumulant analysis, with values from zero to
one. According to the 3D surface plot of PDI (Figure 3II) and ANOVA results, the oil and water proportions interaction
(AC), S/Cos and water proportions interaction (BC), and the oil, S/Cos, and water proportions interaction (ABC) showed
a substantial influence on PDI (P < 0.05). As per equation (2), and regarding the oil and water proportions interaction
(AC), PDI decreased with increasing oil proportion and decreasing water content as per the dilution effect of the external
oily phase. Vis-à-vis, S/Cos and water proportions interaction (BC), where decreasing both S/Cos and water proportions
decreased PDI values while increasing one of them or both resulted in the formation of systems with higher PDI values.
This could be due to possible formulation instability caused by higher S/Cos proportion and hence give a chance for
globule coalescence; known as Ostwald ripening.42,43 Higher water content with lower S/Cos proportions led to the
formulation of NE with a high PDI value, and this could be accredited to the inability of S/Cos to emulsify the inner
aqueous phase in the outer oily one, where coalescence of the inner phase occurred. The 3D surface plot of %T
(Figure 3III) and ANOVA results showed that the oil and S/Cos proportions interaction (AB), the oil and water
proportions interaction (AC), and S/Cos and water proportions interaction (BC) significantly affected %T (P < 0.05).
According to equation (3), decreasing the proportions of all components led to an increase in %T, while different
combinations of the components showed a positive effect on %T. This could be explained by the fact that the formulation
of NE requires an optimum combination of all components, and varying the proportion of any of them would alter the
overall equilibrium of the system, which is mandatory to keep the drug at its ideal solubility level.44 This fact is highly
related to the physical properties of the surfactant-oil-water mixtures, which are dynamic and variable over time and
space upon mingling of the organic phase with the aqueous phase.45 It should be noted that the results of %T are in good
harmony with that of GS, where NEs with smaller GS displayed a high %T. All NEs showed acceptable viscosity values
as a topically applied dosage form; whereupon their application on the skin, this viscosity helps in retaining the actives
for a suitable period of time, which guarantees better drug permeation and absorption. The viscosity of the NE is known
to be a function of its components and their concentrations.46 Moreover, viscosity analysis is crucial in explaining the
interactions between the dispersed phase droplets and the continuous phase.47 According to equation (4) and the 3D
surface plot of the viscosity displayed in Figure 3IV, the viscosity of NEs was found to be directly related to the
concentrations of all of its components. Higher viscosity values were observed with the formulated systems due to the
inherent high viscosities of the oils, surfactants and cosurfactant, in addition to the oily nature of the external phase of W/
O NE.
Based on the design analysis, the optimum formulation was elected and furtherly characterized for morphology, pH,
and stability. The distinct spherical droplets observed with the TEM revealed the dispersion of water globules, which is
attributed to the presence of a monolayer of S/Cos around each globule that acts as a barrier against globules coalescence
leading to the formulation of a stable system.48 In addition, no precipitation of the drug was observed which would affect
the system stability.49 As per the results of the stability study, the high stability observed with the formulated NEs is
attributed to the plasticity of the interfacial film, which was maintained throughout the shelf life of NE and hindered
phase separation.50 The measured pH values of the formulated NEs were found to be appropriate for skin topical
application.24 In addition, the slightly acidic pH value observed with Vit C-loaded NE is preferable to enhance the
stability of Vit C.11

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Relating to the results of the in vitro cellular studies, it could be observed that the formulation played a profound role
in enhancing the permeation of the actives, which in turn led to a significantly improved cell proliferation and an
enhanced surviving fraction of HFB4 and BHK cell lines, in addition to the wound healing effect, especially when
collagen and Vit C were applied together. This could be attributed to the process of tissue repair which involves a series
of cellular events that include cell migration and the secretion of several mediators.51 Previous studies highlighted the
importance of collagen in tissue regeneration, where applying collagen or chemicals that increase its endogenous release
to cutaneous skin lesions has shown an enhancement in cell migration and wound healing.52,53 Based on this fact, several
collagen-based formulations and matrices were proved to be very effective in halting the skin aging process due to
collagen deficiency in fibroblast cultures.54–56 Similarly, different studies have reported that Vit C plays a pivotal role in
decelerating the aging process either through its direct effects or indirectly through increasing collagen synthesis,57–60 as
well as, acting by itself as a promoter of tissue repair.61,62 Shibuya et al demonstrated an additive effect on aging-related
skin atrophy when collagen and Vit C were administered together.63 Also, Vit E was found to have cell protectant and
antioxidant properties, which sustain the structural and functional integrity of the cellular membrane, besides being a free
radical scavenger so averting the peroxidation of membrane fatty acids.64 The results of the CLSM study came to prove
the boosted permeability and retention of the formulated NE. This could be accredited to the nanoemulsion penetration-
enhancing potentiality and its ability to advance the transport of drugs throughout the skin, via disturbing the lipid barrier
of the stratum corneum, enriching skin hydration, and empowering the drive of drugs through the skin.65
As per the results of the in vivo study, it was pragmatic that the UVB irradiation caused some alterations in the skin
principally in the epidermis. It is widely recognized that excessive contact with pollutants or long exposure to UV
radiation results in skin aging with wrinkle formation due to loss of collagen.66 Moreover, some studies revealed the skin
changes implicated with UVB exposure, where hyperkeratosis and acanthosis are among the common alterations in skin
morphology after irradiation.67,68 Another important factor that causes skin damage is the oxidative stress that depletes
vitamin C levels in the skin,39,40 which is known to stimulate collagen synthesis and protect against UV-induced
photodamage. According to the results, the group that was treated with collagen- and Vit C-loaded NEs mixture showed
the most healthy skin features, as collagen was found to elevate the degree of hydration at the inner layers of the skin
through the topical treatment with cosmetic formulations containing collagen peptides,69 and boost the smoothness and
the luminosity of the skin surface at the microtextural level.3 Besides, Vit E (as a component of NE) and Vit C are famous
for their skin protectant and topical antioxidants characteristics,70,71 where applying the three ingredients led to the
superlative results.

Conclusion
Skin delivery of collagen and prophylactic antioxidant therapy (including Vit C and Vit E) through a W/O nanoemulsion
has been introduced to lessen UV-induced skin injury. In this study, a novel system was formulated using a mixture of
safflower oil and Vit E as the oily phase, while Span 80 and Kolliphor EL were incorporated as a surfactant, and
Arlasolve was employed as a cosurfactant for the first time. The optimized nanoemulsion, either loaded with collagen or
Vit C, displayed a small globule size with spherical morphology, a high optical clarity, a topically accepted viscosity, and
pH, as well as a good physical stability. The confocal laser scanning microscopy study proved the boosted permeability
and retention of the formulated W/O nanoemulsion. The optimized NE loaded with collagen or Vit C, or the mixture of
collagen and Vit C-loaded NEs revealed improved cell viability and enhanced wound healing properties on fibroblast cell
lines (HFB4) and (BHK) compared to the actives’ solutions. In addition, an in vivo study and histopathological
investigations proved the efficacy of the developed system in shielding the skin against UV damage. In conclusion,
the formulated NE containing skin protectant actives, namely collagen, Vit C, and Vit E, could be a promising
ameliorative cosmeceutical preparation for skin protection against harmful UVB irradiation. Further investigations to
compare the efficacy and long-term safety of the formulated NEs to commercial products, as well as clinical studies are
considered.

Disclosure
The authors report no conflicts of interest in relation to this work.

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