gels

Article
Preparation and Characterization of Curcumin Nanoemulgel
Utilizing Ultrasonication Technique for Wound Healing:
In Vitro, Ex Vivo, and In Vivo Evaluation
Mohammed S. Algahtani 1 , Mohammad Zaki Ahmad 1 , Ihab Hamed Nourein 2 , Hassan A. Albarqi 1 ,
Hamad S. Alyami 1 , Mohammad H. Alyami 1 , Abdulsalam A. Alqahtani 1 , Ali Alasiri 1 , Thamer S. Algahtani 1 ,
Abdul Aleem Mohammed 1 and Javed Ahmad 1, *

1 Department of Pharmaceutics, College of Pharmacy, Najran University, Najran 11001, Saudi Arabia;
[email protected] (M.S.A.); [email protected] (M.Z.A.); [email protected] (H.A.A.);
[email protected] (H.S.A.); [email protected] (M.H.A.); [email protected] (A.A.A.);
[email protected] (A.A.); [email protected] (T.S.A.); [email protected] (A.A.M.)
2 Department of Clinical Laboratory (Histopathology and Cytology), College of Applied Medical Sciences,
Najran University, Najran 11001, Saudi Arabia; [email protected]
* Correspondence: [email protected]; Tel.: +966-17542-8744

Abstract: Hydrogels being a drug delivery system has great significance particularly for topical appli-





 cation in cutaneous open wound. Its specific physicochemical properties such as non-adhesiveness,
moisture retention, exudate absorption, and gas permeability make them ideal as a drug delivery
Citation: Algahtani, M.S.; Ahmad,
vehicle for wound healing application. Further, curcumin (a natural bioactive) was selected as a
M.Z.; Nourein, I.H.; Albarqi, H.A.;
therapeutic agent to incorporate into the hydrogel system to design and develop nanogel pharma-
Alyami, H.S.; Alyami, M.H.;
Alqahtani, A.A.; Alasiri, A.;
ceutical products for wound healing. Although, curcumin possesses remarkable anti-inflammatory,
Algahtani, T.S.; Mohammed, A.A.; antioxidant, and anti-infective activity along with hastening the healing process by acting over the
et al. Preparation and different stages of the wound healing process, but its poor biopharmaceutical (low aqueous solubility
Characterization of Curcumin and skin penetrability) attributes hamper their therapeutic efficacy for skin applications. The current
Nanoemulgel Utilizing investigation aimed to develop the curcumin-loaded nanogel system and evaluated to check the
Ultrasonication Technique for Wound improvement in the therapeutic efficacy of curcumin through a nanomedicine-based approach for
Healing: In Vitro, Ex Vivo, and In wound healing activity in Wistar rats. The curcumin was enclosed inside the nanoemulsion system
Vivo Evaluation. Gels 2021, 7, 213. prepared through a high-energy ultrasonic emulsification technique at a minimum concentration of
https://doi.org/10.3390/gels7040213
surfactant required to nanoemulsify the curcumin-loaded oil system (Labrafac PG) having droplet
size 56.25 ± 0.69 nm with polydispersity index 0.05 ± 0.01 and negatively surface charge with
Academic Editor: David Díaz Díaz
zeta potential −20.26 ± 0.65 mV. It was observed that the impact of Smix (surfactant/co-surfactant
Received: 15 October 2021
mixture) ratio on droplet size of generated nanoemulsion is more pronounced at lower Smix concen-
Accepted: 10 November 2021 tration (25%) compared to the higher Smix concentration (30%). The optimized curcumin-loaded
Published: 14 November 2021 nanoemulsion was incorporated into a 0.5% Carbopol® 940 hydrogel system for topical application.
The developed curcumin nanoemulgel exhibited thixotropic rheological behavior and a significant
Publisher’s Note: MDPI stays neutral (p < 0.05) increase in skin penetrability characteristics compared to curcumin dispersed in conven-
with regard to jurisdictional claims in tional hydrogel system. The in vivo wound healing efficacy study and histological examination of
published maps and institutional affil- healed tissue specimen further signify the role of the nanomedicine-based approach to improve the
iations. biopharmaceutical attributes of curcumin.

Keywords: curcumin; nanoemulsion; ultrasonic emulsification; nanoemulgel; thixotropy; wound

healing
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and 1. Introduction
conditions of the Creative Commons
Wounds are physical injuries, leading to an opening or break of the skin [1]. The
Attribution (CC BY) license (https://
appropriate healing of wounds is vital for the repair of disrupted anatomical and functional
creativecommons.org/licenses/by/
status of the skin. Restoring wounds is one of the most complicated physiological processes
4.0/).

Gels 2021, 7, 213. https://doi.org/10.3390/gels7040213 https://www.mdpi.com/journal/gels

Gels 2021, 7, 213 2 of 17

that starts from the response to an injury to restoring the function and integrity of damaged
tissues [2]. Wound healing involves numerous physiological events as clotting, coagulation,
inflammation, and the generation of fresh tissues, which may follow varied timescale from
minutes to numerous months or years [3]. Any deviations or delays to the multistage
healing process might be the reason for the failure of wound healing and the development
of an incomplete healing process [4]. Long-lasting wounds can seriously affect the quality
of life and their treatment requires a very high quality of care [5,6]. It may lead to an
increase in the risk of morbidity and mortality. This is especially true for people who suffer
from vascular diseases and diabetes mellitus [4]. Therefore, it is essential to optimize a
wound healing method that can minimize tissue damage and accelerate the wound healing
time. Wound healing agents are available in different routes of administration including
oral and parenteral; however, the systemic drug administration of these agents can cause
undesirable systemic side effects. Hence, a topical drug delivery system is an attractive
approach that can improve the wound healing process and minimize systemic side effects.
Curcumin (o-methoxy phenol derivative compound) is a hydrophobic polyphenol
derived from the rhizome of the herb Curcuma longa, belonging to the family Zingiberaceae.
Curcumin has been used traditionally for several diseases due to its wide spectrum of
biological and pharmacological activities [7]. It has been reported to exhibit multifunctional
properties, including antioxidant, anti-inflammatory, anti-microbial, and anti-carcinogenic
activities [8,9]. Furthermore, topical application of curcumin has been shown to improve
the wound healing process and prevent oxidative damage to tissues [8,9]. Additionally, it
has been reported that curcumin enhances the production of granulation in body tissue,
including a greater amount of cellular content and new vascularization, along with increas-
ing the process of re-epithelialization of wounds [9]. Despite the significant pharmaceutical
value of curcumin, the therapeutic applications of curcumin are limited due to its poor
aqueous solubility, poor absorption, rapid metabolism, rapid systemic elimination, and low
stability [5]. Moreover, the poor aqueous solubility of curcumin limits its skin permeation
through the stratum corneum (SC), and poses a major limitation on its topical application
in wound healing.
To solve this, there is a need for a topical delivery formulation that enhances the water
solubility of curcumin, which promotes its permeation through the skin. Recent studies
reported in which drugs are loaded inside the core of nanoemulsion (NE) have contributed
to increasing the drug permeation through the skin due to high penetrability through the
subcutaneous barrier [10,11]. For example, Algahtani et al. designed a NE formulation
to deliver curcumin utilizing the low energy technique for psoriasis treatment [12]. The
formulation then was loaded into Carbopol®gel to form a curcumin nanoemulgel (NEG).
This formulation has shown faster healing for the psoriatic symptoms when compared
to the curcumin suspension and betamethasone-17-valerate, which is commonly used for
the treatment of psoriasis. The NE-based formulation for topical delivery of curcumin
increases drug concentrations at the area treated, drug-loading capacity, improves skin
penetrability, and prolongs the amount of drug release and those properties helpful for
successful wound healing [13,14]. The poorly water-soluble curcumin is encapsulated into a
lipophilic environment of an oil droplet of nano dimension, which offers high drug-loading,
and is stabilized through an optimized amount of surfactant/co-surfactant mixture (Smix)
along with an aqueous phase [14].
The low-energy technique used for the production of NE consumes a high amount of
surfactants and co-surfactants that might irritate when applied to an open wound. Here,
the curcumin NE formulated utilizing the high energy technique through ultrasonication
of the formulation that reduces the needed amount of surfactants [15]. Different NE
compositions were evaluated to select the optimum formulation. Due to low retention
of NE system at the site of application because of low viscosity of NE system [14] and
because of the inconvenient application of such formulation, the optimized NE system was
dispersed into a hydrogel system to make NEG. Hydrogels being a drug delivery system
has great significance particularly for topical application in cutaneous open wound. Its
Gels 2021, 7, 213 3 of 17

specific physicochemical properties such as non-adhesiveness, moisture retention, exudate

absorption, and gas permeability make them ideal as a drug delivery vehicle for wound
healing application [16]. In vitro evaluation has been applied to the developed NE system
including the measurement of the droplet size, size distribution, zeta potential, and drug
release. Ex vivo drug permeation and deposition through rat skin were also evaluated in a
developed NEG system. The in vivo wound healing property of the developed formulation
was evaluated on rat model and the speed of healing was compared to silver sulfadiazine
cream, a common wound healing formulation available in the market.

2. Results and Discussion

2.1. Screening of NE Components
The Labrafac PG was selected as the oil phase of the NE system based on maximum
curcumin solubility in it as previously investigated in our lab [12]. The Tween 80 and PEG
400 are selected as favorable Smix (surfactant and co-surfactant combination) phases based
on the desirability of curcumin solubility. Further, it is a widely accepted Smix combination
utilized in pharmaceutical products and compatible with the skin. Although the solubility
of curcumin in Labrafac PG, Tween 80, and PEG 400 is desirable to achieve sufficient drug
loading (10 mg/mL) in the NE system, indeed these components should be helpful to
sufficiently nanoemulsify at a low concentration of Smix for the better suitability in wound
healing application. The results of emulsification efficiency of Smix phase at different ratios
(1:1, 2:1, 3:1, and 4:1) and corresponding %T values were determined and illustrated in
supplementary Figure S1.
Therefore, to design NE system of <100 nm (size dimension desirable for topical
application) utilizing Labrafac PG, Tween 80, and PEG 400 as formulation components
at a minimum concentration of surfactant and maximum concentration of oil phase, a
high-energy ultrasonication technique was adopted. The low-energy self-emulsification
technique is a commonly preferred method to prepare an NE system of <100 nm for topical
application at a maximum concentration of surfactant and minimum concentration of
oil phase.

2.2. Preparation of NE through Ultrasonication

Curcumin-loaded NE system was prepared by high-energy ultrasonication tech-
nique [17,18] utilizing Labrafac PG as oil phase, Tween 80 as a surfactant, and PEG 400 as
co-surfactant at variable % composition shown in Table 1. The % composition of different
formulations shown in Table 1 has been selected based on the curcumin loading in each
formulation being 10 mg/mL. Therefore, the maximum possible concentration of the oil
phase (20%) was selected and it remains constant in each formulation to achieve uniform
drug loading (10 mg/mL) in each NE system.
In the beginning, the uniform dispersion system was obtained by mixing the 1% w/w
(10 mg/g) of curcumin in the mixture of the oil phase and Smix phase (at a ratio of 1:1 and
2:1 with variable concentration) through vortex mixture. After that, the aqueous phase was
added with continuous vortexing for 1 min [15]. The uniform dispersion system obtained
was further ultrasonicated (Ultrasonic Homogenizer, FS-300N, Zhejiang, China) in a water
bath for varying time duration (3, and 5 min) at a constant ultrasonication amplitude
of 40% [17,19]. The impact of the ultrasonication process and composition variables on
droplet size and PDI of the generated NE system loaded with curcumin was evaluated as
shown in Table 1.
 Table 1. Formulation composition of curcumin loaded NE system prepared through ultrasonication
technique.
Gels 2021, 7, 213 4 of 17
Ultrasoni- Polydis-
Formula- Smix Ra- Mean Drop-
Oil% Smix% Water% cation Time persity In-
tion Code tio let Size (Nm)
(Min) dex
Table 1. Formulation composition of curcumin loaded NE system prepared through ultrasonication technique.
NE1 20.0 25.0 55.0 1:1 3.0 232.80 ± 11.04 0.23 ± 0.16
Formulation Code Oil% NE2 Smix
Smix% Water% 20.0
Ratio 25.0 55.0 Time (Min)
Ultrasonication 1:1 5.0 Size 133.76
Mean Droplet Polydispersity
(Nm) ± 2.84 0.29 ± 0.11
Index
NE1 20.0 25.0
NE3
55.0
20.0
1:1
30.0 50.03.0
1:1 3.0
232.80 ± 11.04
152.70 ± 3.08 0.20 ± 0.04
0.23 ± 0.16
NE2 20.0 25.0 NE4
55.0 20.0
1:1 30.0 50.05.0 1:1 5.0 ± 2.84 116.63 ± 2.22
133.76 0.290.14 ± 0.12
± 0.11
NE3 20.0 30.0 50.0 1:1 3.0 152.70 ± 3.08 0.20 ± 0.04
NE4 20.0 30.0 NE5
50.0 20.0
1:1 25.0 55.05.0 2:1 3.0
116.63 ± 2.22 84.23 ± 1.33 0.23 ± 0.05
0.14 ± 0.12
NE5 20.0 25.0 55.0 2:1 3.0 84.23 ± 1.33 0.23 ± 0.05
NE6 20.0 25.0 NE6
55.0 20.0
2:1 25.0 55.05.0 2:1 5.0 ± 0.69 56.25 ± 0.69
56.25 0.050.05 ± 0.01
± 0.01
NE7 20.0 30.0 NE7
50.0 20.0
2:1 30.0 50.03.0 2:1 3.0 ± 0.71 51.47 ± 0.71
51.47 ± 0.04
0.130.13 ± 0.04
NE8 20.0 30.0 50.0 2:1 5.0 49.61 ± 0.53 0.10 ± 0.01
NE8 20.0 30.0 50.0 2:1 5.0 49.61 ± 0.53 0.10 ± 0.01

2.2.1.
2.2.1. Influence
Influence of of Smix
Smix Ratio
Ratio and
and Smix
Smix Concentration
Concentration on on Droplet
DropletSize
Size
The droplet size of the curcumin-loaded NE system obtained
The droplet size of the curcumin-loaded NE system obtained through through the high-energy
the high-en-
ultrasonication technique was greatly influenced by the ratio of
ergy ultrasonication technique was greatly influenced by the ratio of Smix phase Smix phase present as
present
formulation
as formulation components
components of the
of NE
the system. It was
NE system. observed
It was that the
observed thatNEthesystem composed
NE system com-
of Smixofratio
posed Smix2:1 (at constant
ratio Smix concentration
2:1 (at constant and ultrasonication
Smix concentration time) significantly
and ultrasonication time) signifi-
(p < 0.05)
cantly (p <reduced the droplet
0.05) reduced size compared
the droplet to the to
size compared NEthesystem composed
NE system of Smix
composed of ratio
Smix
1:1 (at constant Smix concentration and ultrasonication time). This influence
ratio 1:1 (at constant Smix concentration and ultrasonication time). This influence is shown is shown in
Figure 1 at Smix concentration of 25 and 30% respectively. This influence
in Figure 1 at Smix concentration of 25 and 30% respectively. This influence of Smix ratio of Smix ratio on
droplet
on droplet sizesize
is more
is morepronounced
pronounced at at
lower
lowerconcentration
concentration (25%)
(25%)ofofSmix
Smixcompared
comparedto tothe
the
higher Smix concentration
higher Smix concentration (30%). (30%).

Figure
Figure 1.1. Impact
Impactofofthe
theprocess
processvariable
variable(ultrasonication
(ultrasonicationtime
time3 and 5 min)
3 and andand
5 min) composition variables
composition varia-
(Smix ratioratio
bles (Smix and and
% concentration) on on
% concentration) droplet size
droplet of of
size NE NE system
systemgenerated
generatedthrough
throughhigh-energy
high-energy
ultrasonication technique
ultrasonication technique at Smix
Smix concentration
concentration 25% (Smix
(Smix ratio
ratio 1:1
1:1 and
and 2:1)
2:1) and
and Smix
Smix 30%
30%(Smix
(Smix
ratio 1:1
ratio 1:1 and
and 2:1).
2:1). Colors
Colors and
and shaded
shaded bars
bars represent
represent variability
variability ininformulation
formulation compositions
compositions and
and
process conditions.
process conditions.

2.2.2. Influence of Ultrasonication Time on Droplet Size

The droplet size of the curcumin-loaded NE system obtained through the high-energy
ultrasonication technique was also greatly influenced by the ultrasonication time. It was
observed that 5 min ultrasonication significantly (p < 0.05) reduces the droplet size of
the NE system of constant concentration and the ratio of Smix compared to the 3 min
Gels 2021, 7, 213 5 of 17

ultrasonication. This influence is shown in Figure 1 at Smix concentration 25 and 30%,

respectively. This influence of ultrasonication time on droplet size is more pronounced at
25% concentration of Smix compared to the 30% of Smix concentration. The NE system
of comparatively higher Smix concentration is sufficient to reduce the droplet size up to
a certain extent compared to the NE system of low Smix concentration. Therefore, the
influence of ultrasonication time on droplet size is more pronounced in the NE system
having low Smix concentration compared to the NE system of higher Smix concentration.
The Smix ratio, the concentration of Smix phase, and ultrasonication time have no
remarkable effect on PDI. It is might be correlated to the HLB value of Labrafac PG (HLB
value 2) compared to the natural origin fixed oils (such as black seed oil, sesame oil, and
castor oil, etc.). It was observed in the previous investigation of our lab that the impact of
Smix ratio, the concentration of Smix phase, and ultrasonication time on PDI of generated
NE system through high-energy ultrasonication technique was significantly evident in the
case of the NE system of black seed oil [17].
Out of the eight formulations developed, four formulations (NE5, NE6, NE7, and
NE8) show droplet size <100 nm (Table 1). The mean droplet size of these four selected
NE systems varies between 84.23 ± 1.33 to 49.61 ± 0.53 nm, and the PDI varies between
0.23 ± 0.05 to 0.10 ± 0.01. It is very interesting to observe that curcumin NE prepared
through high energy ultrasonic emulsification technique can achieve the droplet size
<100 nm at a significantly (p < 0.05) lower concentration of Smix compared to the curcumin
NE prepared through low energy spontaneous emulsification technique. It is widely seen
that NE prepared through low energy spontaneous emulsification technique commonly
required two or more than two times of Smix concentration relative to oil concentration
in the NE composition to achieve the droplet size <100 nm [12,20–23], while NE prepared
through high energy ultrasonic emulsification technique able to achieve the same droplet
size even at Smix concentration less than 1.5 times relative to oil concentration [15]. Indeed,
droplet size of NE can also be influenced by the type of oil, surfactant, and co-surfactant
used in its preparation in addition to the type of emulsification technique (high-energy/low-
energy) utilized for the formation of NE system.
Those four curcumin-loaded NE systems having droplet size <100 nm were selected
for further characterization such as thermodynamic stability, viscosity, and % drug content.

2.3. Characterization of NE
2.3.1. Analysis of Thermodynamic Stability and Zeta Potential
Thermodynamic stress testing was performed to find out the presence of any metastable
NE in the screened formulations. It is determined by the exchange of free energy between
the system’s milieu and the system itself [24]. Table 2 shows the results of the thermo-
dynamic stability investigation of the selected formulations (NE5, NE6, NE7, and NE8).
The four NE formulations tested were stable under the heat-cooling cycle, freeze-thaw
cycle, and centrifugation study. This stability may be related to the high zeta poten-
tial of the screened NE formulations (NE5, NE6, NE7, and NE8), which ranges between
−15.96 ± 0.55 mV and −20.26 ± 0.65 mV (Table 2). The magnitude of the surface charges
has a direct relationship to the stability of the NE system. The high repulsive force between
NE droplets minimizes any chances of coalescence and prevents the possibility of physical
instability of generated NE system [25].

2.3.2. Viscosity
The viscosity of four screened NE system (NE5, NE6, NE7, and NE8) were tested at
room temperature through rotational viscometer. The NE5 has the lowest viscosity value
with 83.74 ± 1.92 mPas, while NE8 has the highest viscosity value with 89.82 ± 1.27 mPas
(Table 2). This variation in viscosity value is related to the higher concentration of surfactant
(Tween 80) in the NE8 formulation system compared to the NE5 formulation system.
Gels 2021, 7, 213 6 of 17

2.3.3. Analysis of Drug Content

The UV-visible spectrophotometric analysis was carried out to determine the curcumin
content in NE systems [26,27] and it was used here to quantify the % content of the curcumin
in the selected four NE formulations (NE5, NE6, NE7, and NE8). The % drug content in
curcumin NE formulations are between 98.86 ± 0.58 to 99.23 ± 0.28 % (Table 2), which
indicates that the curcumin is almost completely encapsulated into oil droplets of Labrafac
PG-based NE system.
All of the selected formulations have passed the thermodynamic stability tests and
show complete drug encapsulation. The droplet size of the NE system around 50 nm
and PDI less than one has great significance, particularly for topical application. The
same droplet size distribution has been widely reported for improved skin penetrability of
loaded therapeutics after topical application [28,29]. NE6, NE7, and NE8 were selected for
further investigation as they have droplet size around 50 nm with PDI less than one and
negative zeta potential (as shown in Supplementary Figures S2–S4), which is the optimum
droplet size and desirable surface charge reported for the topical application as it results in
improved skin permeability and deeper penetration.

Table 2. Characterization of selected CUR-loaded NE for thermodynamic stability, droplet size distribution, zeta potential,
% drug content, and viscosity.

Thermodynamic Stability Mean Droplet Zeta Potential Viscosity

Formulation Heating Centrifugation Freeze-Thaw Polydis-persity Drug Content
Code Size (nm) Index (mV) (%) (mPas)
Cooling Cycle Study Cycle
√ √ √
NE5 √ √ √ 84.23 ± 1.33 0.23 ± 0.05 −15.96 ± 0.55 98.86 ± 0.58 89.82 ± 1.27
NE6 √ √ √ 56.25 ± 0.69 0.05 ± 0.01 −20.26 ± 0.65 99.23 ± 0.28 83.74 ± 1.92
NE7 √ √ √ 51.47 ± 0.71 0.13 ± 0.04 −19.26 ± 0.20 99.16 ± 0.15 85.05 ± 2.30
NE8 49.61 ± 0.53 0.10 ± 0.01 −19.6 ± 0.41 99.03 ± 0.41 87.37 ± 0.66

2.4. In Vitro Drug Release

Figure 2 shows the in vitro release behavior of curcumin from the screened formulation
(NE6, NE7, and NE8) and curcumin aqueous suspension for 24 h. All investigated NE
formulations were able to release 85% of curcumin within the first 12 h, while nearly 10% of
curcumin was released from the curcumin aqueous suspension within the same time. This
indicates that the droplet size of the NE system is an important determinant for in vitro
drug release irrespective of the % composition of the NE system. This is in agreement
with the previous investigation of Algahtani et al., which reported that curcumin NE
(prepared by low energy emulsification technique with a higher concentration of Smix)
of droplet size 70 nm with PDI 0.5 was able to release nearly 60% of curcumin within the
first 12 h [12]. All the curcumin-loaded NE formulations showed a significantly (p < 0.05)
higher release of curcumin compared to the in vitro release of curcumin from the aqueous
suspension. Since the three tested formulations have similar release profiles, the selection
of the formulation to be converted to NEG was based on the Smix concentration. Less
Smix concentration in the topical formulation is more preferable when applied to an open
wound. NE6 formulation was selected to be converted to NEG as it has the minimum
concentration of Smix phase with maximum drug release rate (Figure 2).
Gels 2021, 7, 213 7 of 17
Gels 2021, 7, x FOR PEER REVIEW 7 of 17

Figure2.2.In
Figure Invitro
vitrorelease
releaseofofcurcumin
curcuminfrom
fromcurcumin-loaded
curcumin-loadedNE
NE(nanoemulsion)
(nanoemulsion)system.
system.

2.5.
2.5.Preparation
PreparationandandCharacterization
CharacterizationofofNanoemulgel
Nanoemulgel
NE
NEformulations
formulationsin ingeneral
generalhave
havelowlowviscosity
viscosityandandliquid
liquidininnature
natureandandspecifically
specifically
the
the curcumin NE has the addition of staining ability, which makes it difficult to beapplied
curcumin NE has the addition of staining ability, which makes it difficult to be applied
topically. ®
topically. Therefore,
Therefore, thetheNE6NE6waswasevenly
evenlydispersed
dispersedininthetheCarbopol
Carbopol® 940 940gelgelmatrix
matrixtoto
achieve
achievethe
thefinal
finalconcentration
concentrationofof0.5% 0.5%(w/w)
(w/w)of ofcurcumin
curcumininto intoaadeveloped
developedNEG NEGsystem
system
with
withthe
thedesired
desiredconsistency
consistencyfor forpatient-friendly
patient-friendlytopical
topicalapplication.
application.The Thegelgelstrength
strengthofof
curcumin
curcuminNEG NEG system (46.33 ±±1.154
system (46.33 1.154s) s)
andand placebo
placebo gelgel system
system (44.66
(44.66 ± 1.154
± 1.154 s) was s)meas-
was
measured and observed to be comparable to each other. The spreadability
ured and observed to be comparable to each other. The spreadability coefficient of curcu- coefficient
of
mincurcumin NEG and
NEG system system and gel
placebo placebo
system gelwere
system were determined
determined and comparatively
and comparatively illustrated
illustrated in supplementary Figure S5. The pH range
in supplementary Figure S5. The pH range of the prepared curcumin NEG of the prepared curcumin
system NEG
was
system was within the acceptable range of skin application and found close
within the acceptable range of skin application and found close to the pH skin acid mantle to the pH skin
acid
(5.53mantle This±suggests
± 0.03).(5.53 0.03). Thisthatsuggests that theformulation
the developed developed formulation
is safe to useisfor safe
skinto applica-
use for
skin
tion,application,
particularlyparticularly
for woundfor wound
healing healing
[20,23]. The[20,23].
% drugThe % drug
content content uniformity
uniformity of
of the curcu-
the curcumin NEG system was calculated. The formulation of curcumin
min NEG system was calculated. The formulation of curcumin NEG exhibited a uniform NEG exhibited a
uniform dispersion of curcumin in the hydrogel system with a uniformity of 98.93 ± 0.11%.
dispersion of curcumin in the hydrogel system with a uniformity of 98.93 ± 0.11%. The
® 940 of
The rheological profile of developed curcumin NEG and placebo gel of Carbopol
rheological profile of developed curcumin NEG and placebo gel of Carbopol® 940 of the
the same concentration (0.5% w/v) are graphically shown in Figure 3a,b. The prepared
same concentration (0.5% w/v) are graphically shown in Figure 3a,b. The prepared curcu-
curcumin NEG demonstrated a similar rheological profile as compared to placebo gel, and
min NEG demonstrated a similar rheological profile as compared to placebo gel, and the
the incorporated curcumin NEG did not affect its rheology behavior. It is observed that the
incorporated curcumin NEG did not affect its rheology behavior. It is observed that the
viscosity of placebo gel and curcumin NEG decreased upon an increase in applied shear
viscosity of placebo gel and curcumin NEG decreased upon an increase in applied shear
rate and vice-versa (illustrated through downward and upward curves in Figure 3a,b).
rate and vice-versa (illustrated through downward and upward curves in Figure 3a,b).
This property indicates the thixotropic behavior of the developed curcumin NEG system
This property indicates the thixotropic behavior of the developed curcumin NEG system
which is a desirable attribute for pharmaceutical dosage forms for topical application [30].
which is a desirable attribute for pharmaceutical dosage forms for topical application [30].
Gels 2021, 7, 213 8 of 17
Gels 2021, 7, x FOR PEER REVIEW 8 of 17

Figure
Figure 3. 3. Rheology
Rheology profile
profile ofof
(a)(a) placebo
placebo gelgel
andand
(b)(b) Nanoemulgel
Nanoemulgel ofof curcumin.
curcumin.

2.6.
2.6.Ex-Vivo
Ex-VivoSkin
SkinPermeability
PermeabilityStudy
Study
For
Fordrug
drugpermeability
permeabilitythrough
throughthe
theskin
skinand
anditsits
skin deposition,
skin deposition,analysis
analysisfrom
from NEG
NEG
and
andconventional
conventionalgelgelofofcurcumin
curcumin were
werecarried
carriedwith
with the help
the ofof
help Franz-diffusion
Franz-diffusion cell. The
cell. The
results
resultsobtained
obtainedfrom
fromthetheex-vivo
ex-vivo skin permeability
permeability are areshown
shownininTable
Table3.3.The
ThepHpHforfor
cur-
curcumin-loaded
cumin-loaded NEG NEG (5.53
(5.53 ± 0.03)
± 0.03) andand conventional
conventional gel gel of curcumin
of curcumin (5.56
(5.56 ± 0.02)
± 0.02) was was
main-
maintained with drug content uniformity of 98.93 ± 0.11 and 98.60 ± 0.52,
tained with drug content uniformity of 98.93 ± 0.11 and 98.60 ± 0.52, respectively. respectively.
For drug permeation, the cumulative amount of curcumin permeated through the
skin was 773.82 ± 1.08 from the curcumin NEG and 156.90 ± 0.95 µ g/cm2 from the curcumin
gel preparation. Whereas, the amount of curcumin deposited on the skin was 1161.54 ±
2.78 from the curcumin NEG and 179.47 ± 1.56 µ g/cm2 from the curcumin gel preparation.
It is noteworthy to mention that there was an approximately six-fold increase of the
percutaneous drug flux of curcumin from CUR-NEG (13.74 ± 1.08), compared to the drug
flux of curcumin from the curcumin gel (2.19 ± 0.10). Likewise, the permeability coefficient
Gels 2021, 7, 213 9 of 17

Table 3. Characterization of curcumin-loaded nanoemulgel to determine skin penetrability profile compared to conventional
curcumin gel. “ER—enhancement ratio”.

Cumulative Amount Drug Deposited Permeability

Formulation of Drug Permeated Lag Time (h) Flux Coefficient ER Local Accumulation
in Skin (µg/cm2 ) (µg/cm2 .h) (K × 10−3 ) Efficiency (LAE)
(µg/cm2 )
CUR-NEG 773.82 ± 1.08 1161.54 ± 2.78 0.75 ± 0.03 13.74 ± 1.08 5.49 ± 0.67 6.27 ± 0.77 1.50 ± 0.01
CUR-gel 156.90 ± 0.95 179.47 ± 1.56 2.37 ± 0.09 2.19 ± 0.10 0.876 ± 0.01 - 1.14 ± 0.01

For drug permeation, the cumulative amount of curcumin permeated through the skin
was 773.82 ± 1.08 from the curcumin NEG and 156.90 ± 0.95 µg/cm2 from the curcumin gel
preparation. Whereas, the amount of curcumin deposited on the skin was 1161.54 ± 2.78
from the curcumin NEG and 179.47 ± 1.56 µg/cm2 from the curcumin gel preparation.
It is noteworthy to mention that there was an approximately six-fold increase of
the percutaneous drug flux of curcumin from CUR-NEG (13.74 ± 1.08), compared to the
drug flux of curcumin from the curcumin gel (2.19 ± 0.10). Likewise, the permeability
coefficient (K × 10−3 ) of curcumin increased approximately six-fold from the curcumin
NEG formulation (5.49 ± 0.67) when compared to the curcumin gel (0.876 ± 0.01). The
permeation enhancement ratio (ER) of curcumin from the curcumin NEG was 6.27 ± 0.77,
whereas the local accumulation efficiency (LAE) for curcumin was 1.50 ± 0.01 from the
curcumin NEG and 1.14 ± 0.01 from the curcumin gel preparation.
The ex-vivo studies were performed to evaluate and compare the permeation and skin-
deposition profile for the curcumin loaded in the two types of gel formulations. The high
skin permeability of curcumin from the curcumin NEG system might be correlated to the
encapsulation of curcumin inside the NE system. The encapsulation of loaded therapeutics
inside nano oil droplets enhances its skin permeability profile and decreases the lag time
for permeation [31]. The lag time (h) for curcumin released from the curcumin NEG
was decreased to (0.75 ± 0.03) compared to the released curcumin from the conventional
curcumin gel (2.37 ± 0.09). The enhanced skin-deposition of curcumin from curcumin
NEG is imparted due to the use of Tween 80 in the Smix formulation as surfactant and it is
known to enhance the dermal delivery of drugs [32]. Tween 80 modifies the skin barriers
via readily entering the stratum corneum of the skin and showing a strong interaction with
water within the cells, which affects the skin’s lipid and protein permeability, whereby the
skin-permeation and deposition for the drugs are increased [33]. In this study, the high
LAE value of curcumin released from the curcumin NEG is suggested to be related to the
negatively charged surfaces of the drug-loaded oil droplets (Table 2) [34].

2.7. In-Vivo Wound Healing Activity

The wound healing activity of curcumin from the curcumin NEG and the conven-
tional gel of curcumin were evaluated and compared to the commonly used and marketed
formulation silver sulfadiazine (Figure 4). The topical applications of the studied formula-
tions in Wistar rats were monitored for 20 days. Figure 5a, shows the appearance and the
contraction of the wound at the days 0, 4, 8, 12, 16, and 20. Figure 5b shows the wound
contraction percentage as the wound size at day zero was considered 100%.
 Gels 2021, 7, x FOR PEER REVIEW 10 of 17

Gels 2021, 7, 213 10 of 17

formulation of curcumin in the form of NEG system further enhanced the wound healing
activity of curcumin compared to the conventional gel formulation of curcumin.

4. (a) In-vivo
Figure Figure wound
4. (a) In-vivo healing
wound activity
healing in Wistar
activity in Wistarrats (b)(b)
rats percentage
percentagecontraction
contraction of woundarea
of wound areaasasananevaluation
evaluation
parameter for wound
parameter healinghealing
for wound activity. Four groups
activity. were were
Four groups studied, Group
studied, I is the
Group I is untreated group,
the untreated Group
group, II was
Group treated
II was with
treated
with silver sulfadiazine
silver sulfadiazine cream,
cream, Group Group
III was III was
treated treated
with with the conventional
the conventional curcumin curcumin gel (CUR-gel),
gel (CUR-gel), and Group
and Group IV was IVtreated
was
with thetreated with NEG
curcumin the curcumin NEG (CUR-NEG).
(CUR-NEG). Shaded barsShaded barsthe
represent represent the day number.
day number.
Group I animals the
Furthermore, revealed a swellingevaluation
histopathological with exudates on day four
was performed of post-wound
to record the inflam-ob-
servations
mation,(Figure
collagen4a). The three
formation, and treated
growth ofgroups showed
the epithelial a soft thrombus
membrane. with a lack
For this purpose, the of
discharge and reduced inflammation with a descending order of wound healing activity as
Group IV > Group II > Group III. Moreover, the formation of brown-reddish tissues in the
wound of G-I and III was observed on day eight; however, the formation of this structure
was observed on day six for Group II and IV (not shown).
All the treated groups (Group II, III, IV) exhibited a remarkable wound healing activity
compared to the untreated group (Group I), particularly the group treated with curcumin
NEG (Group IV) and the group treated with silver sulfadiazine marketed cream (Group II)
with almost complete wound contraction at the end of study i.e., day 20 (Figure 5a,b).
For a proper epithelization of the observed wound, the untreated group required 16 days,
Gels 2021, 7, 213 11 of 17

whereas the time for epithelization observed in the three treated groups was as following:
14 days for the group treated with the curcumin gel (Group III), 11 days for the group
treated with the silver sulfadiazine cream (Group II), and 10 days for the group treated
with the curcumin NEG (Group IV).
Figure 4a,b denotes an almost equivalent wound healing activity for the animals
treated with silver sulfadiazine marketed cream (standard drug) and with the curcumin
NEG system. Curcumin is well-known for its wound healing activity [35,36], whereas, the
formulation of curcumin in the form of NEG system further enhanced the wound healing
Gels 2021, 7, x FOR PEER REVIEW 11 of 17
activity of curcumin compared to the conventional gel formulation of curcumin.
Furthermore, the histopathological evaluation was performed to record the inflam-
mation, collagen formation, and growth of the epithelial membrane. For this purpose,
Wistar
the rats’rats’
Wistar skinskin
from the the
from tested groups
tested (day(day
groups 20) was
20) wassubjected to histopathological
subjected to histopathological pro-
cedures (Figure 5).
procedures (Figure 5).
The comparative
The comparative analysis
analysis revealed
revealed aa high
high amount
amount of of granulomatous
granulomatous mass, mass, fewer in-
flammatory cells, and extensive collagen fibers for the animals
flammatory cells, and extensive collagen fibers for the animals treated with curcumin treated with curcuminNEG
NEG (Group
(Group IV) followed
IV) followed by the by the treated
treated group
group with withsulfadiazine
silver silver sulfadiazine
marketed marketed cream
cream (Group
(Group
II) II) and
and the the treated
group group treated
with thewith the conventional
conventional gel ofgel of curcumin
curcumin (Group(GroupIII) III) (Figure
(Figure 5).
5). Additionally,
Additionally, the histopathological
the histopathological studies
studies foranimal
for the the animal treated
treated with with curcumin
curcumin NEGNEG and
and silver
silver sulfadiazine
sulfadiazine revealed
revealed the presence
the presence of papillary
of papillary dermis dermis
with awiththicka epidermal
thick epidermallayer,
layer, regeneration
regeneration of sebaceous
of sebaceous glands, glands,
as wellas as
well as follicles
hair hair follicles
withwith
no signno sign of inflamma-
of inflammation
tion (Figure
(Figure 5). formation
5). The The formation of granulation
of granulation tissues,
tissues, woundwound contraction,
contraction, tissue
tissue remodel-
remodeling,
and
ing, collagen
and collagendeposition properties
deposition for curcumin
properties for curcumin are already widely
are already reported
widely in the
reported lit-
in the
erature [8,9].
literature Indeed,
[8,9]. Indeed,the thewound
woundhealing
healingproperties
propertiesofofloaded
loadedtherapeutics
therapeuticsalong along with
with
incorporation
incorporation of it into NEG NEG as asaadelivery
deliveryvehicle
vehiclefurther
furtherimparts
impartsananauxiliary
auxiliary property
property to
to curcumin
curcumin through
through deeper
deeper skin
skin penetration,
penetration, local
local deposition
deposition of of
drugdrugin in
thethe skin,
skin, hence
hence an
an augmented
augmented woundwound healing
healing activity
activity was was observed
observed for curcumin.
for curcumin. The outcomes
The outcomes for
for in vivo
in vivo healing process and histopathological examinations are in line
healing process and histopathological examinations are in line with the previous investi- with the previous
investigation
gation [17,24].[17,24].

Figure 5. Histopathology analysis of newly healed tissue at day 20. (a) Stained with hematoxylin-eosin; (b) stained with
Figure 5. Histopathology
vangeison analysis
to observe collagen of newly
formation healed
(at 10 tissue at dayA—stratum
× magnification). 20. (a) Stained with hematoxylin-eosin;
corneum; (b) C—collagen
B—papillary dermis; stained with
vangeison to observe collagen formation (at
fibers; D—sebaceous gland; E—hair follicles. 10× magnification). A—stratum corneum; B—papillary dermis; C—collagen
fibers; D—sebaceous gland; E—hair follicles.

3. Conclusions
A nanoemulgel system with a uniform dispersion of curcumin was successfully de-
signed and evaluated for ex vivo skin penetrability attributes along with in vivo wound
healing efficacy in Wistar rats. The encapsulation of curcumin in O/W nanoemulsion of
Gels 2021, 7, 213 12 of 17

3. Conclusions
A nanoemulgel system with a uniform dispersion of curcumin was successfully
designed and evaluated for ex vivo skin penetrability attributes along with in vivo wound
healing efficacy in Wistar rats. The encapsulation of curcumin in O/W nanoemulsion of
droplet size around 50 nm using a minimum concentration of surfactant is desirable for
wound healing application and achieved effectively exploiting ultrasonic emulsification
technique. This simple step product development process is of great significance for
the industrial scalability of a nanoemulsion-based pharmaceutical product containing a
therapeutic agent of poor biopharmaceutical attributes.

4. Materials and Methodology

4.1. Materials
Labrafac PG (Propylene glycol dicaprylocaprate) was obtained from Gattefose, France.
Tween 80 (Polyoxyethylene sorbitan monooleate) was purchased from Sigma Aldrich,
Germany. PEG 400 (Polyethylene glycol 400) was purchased from Merck, Schuchardh,
Hokenbrunn, Germany. Water was obtained from the Milli-Q-water purification system
(Millipore, Billerica, MA, USA). All the other excipients and chemicals were of pharmaceu-
tical grade and/or analytical grade.

4.2. Screening of NE Components

The principle formulation components utilized to design an NE system for topical
application prepared through low-energy/high-energy emulsification technique are oil,
surfactant, and co-surfactant [20]. The oil phase of the NE system was screened based
on the maximum amount of curcumin solubilize in it to achieve loading of therapeutic
concentration. The solubility of the drug was also assessed in the surfactant/co-surfactant
phase, which will also be helpful to predict the maximum loading of therapeutic concentra-
tion in the designed NE system. Briefly, excess amount of the drug in 2 mL of the selected
vehicle in 5 mL capacity stoppered vials and mixed using a vortex mixer. These vials were
then kept at 25 ± 2 ◦ C in an isothermal water bath shaker for 48 h to equilibrate. The
equilibrated samples were centrifuged at 5000× rpm for 15 min and supernatants were
filtered through a syringe membrane filter (Whatman® Puradisc, 0.22 µm) and the amount
of solubilized drug was measured by UV-visible spectrophotometer at 425 nm [12].
Further, the emulsification efficiency of the surfactant and co-surfactant combination
(Smix) was evaluated for screened oil phase. The emulsification efficiency of the Smix phase
for screened oil phase was determined according to the previously reported method with
slight modification [37]. Briefly, the oil-in-water (O/W) dispersion system was prepared
through shearing with the addition of a known amount of oil phase (5 µL) to the 5%
aqueous dispersion system of Smix, through a vortex mixture after each addition until
the appearance of turbidity just developed. The percentage transmittance (%T) of the
developed oil-in-water (O/W) dispersion system was determined through a UV-visible
spectrophotometer at 683.2 nm [17]. The value of %T should be more than 80% to consider
the endpoint of incorporation of oil phase into the 5% aqueous dispersion system of Smix
phase. The aqueous dispersion system of Smix phase, having the ability to emulsify the
desired amount of the screened oil phase, was allowed to equilibrate and visually observe
to remain as a homogenous dispersion system.
The results obtained from the solubility of the drug in formulation components as
well as emulsification efficiency of Smix phase for screened oil phase formed the basis to
select the formulation components of the designed NE system.

4.3. Preparation of Curcumin NE through High-Energy Emulsification

The high-energy ultrasonication technique was utilized to prepare the curcumin-
loaded NE system [18]. Briefly, the O/W aqueous dispersion system consists of 1% w/w
(10 mg/g) of curcumin in the mixture of the oil and Smix phase through vortex mixture
followed by addition of the aqueous phase with continuous vortexing for 1 min [15]. The
Gels 2021, 7, 213 13 of 17

generated O/W aqueous dispersion system was ultrasonicated (Ultrasonic Homogenizer,

FS-300N, Zhejiang, China; 300 W with continuous adjustable ultrasonication amplitude
0–100%) further in a water bath for different periods (3 and 5 min) at a constant ultra-
sonication amplitude of 40% (power 120 W) [17,19]. Eight NE formulations of different
compositions were designed and developed for further investigation for droplet size and
polydispersity index (PDI) to select the desirable formulation of the curcumin-loaded
NE system.

4.4. Characterization of Curcumin NE

Initially, curcumin-loaded NE formulations were prepared in triplicate by high-energy
ultrasonication technique and formulation of desirable attributes were evaluated for ther-
modynamic stability, droplet size distribution, PDI, zeta potential, viscosity, and analysis
of % drug content. These experiments were carried out in triplicate.

4.4.1. Thermodynamic Stability

The prepared curcumin loaded-NE system was subjected to different stress conditions
tests such as heating–cooling cycles (4 and 40 ◦ C) and freeze–thaw cycles (−21 and +25 ◦ C)
with storage at specified temperature for 48 h. For centrifugation stress study, 1 mL of the
curcumin-loaded NE system was diluted to 100 mL with distilled water and centrifuged at
5000× rpm for 30 min, and visually observed for any phase separation [21].

4.4.2. Droplet Size, Polydispersity Index, and Zeta Potential

The average droplet size and PDI of different compositions of curcumin-loaded NE
system were analyzed at 25 ◦ C by Dynamic Light Scattering (DLS) technique using a
Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK) [22]. The zeta potential of the
curcumin-loaded NE system was also determined using the same instrument.

4.4.3. Determination of Curcumin NE Viscosity

The viscosity of the optimized curcumin-loaded NE system was analyzed, without
dilution, using a Bohlin rotational viscometer at ambient temperature (25 ◦ C) [19,21].

4.4.4. Analysis of the Drug Content

The % content of curcumin in the screened NE formulations was determined by a
UV-visible spectrophotometer at λmax 425 nm [12]. A 100 µL sample of curcumin-loaded
NE was diluted to 1000 times with methanol.

4.5. In-Vitro Drug Release

The curcumin-loaded NE system, which passes the thermodynamic stability and has
a droplet size near 50 nm (NE6, NE7, and NE8), was chosen for the in vitro drug release
investigation through the dialysis bag technique [38]. Dialysis bags (Merck dialysis sacks,
12–14 kDa) were filled with 1 mL of curcumin-loaded NE formulation and suspended in
phosphate buffer release medium of pH 7.4 and maintained at 37 ± 0.5 ◦ C. At a defined
interval of time, 1 mL aliquots were taken out and replaced by the same volume of release
medium. The amount of curcumin in the aliquots was quantified by UV-spectroscopy at
λmax 425 nm. These experiments were carried out in triplicate.

4.6. Preparation and Characterization of Curcumin Nanoemulgel

Curcumin NEG system was prepared by dispersing the optimized formulation (NE6)
in Carbopol® 940 (0.5% w/w) gel [12]. Glycerin (5% w/w) was added as a humectant to the
dispersion system to provide a smooth and soothing effect [20]. Triethanolamine was added
into the dispersion system drop-by-drop to neutralize the pH to 5.5, resulting in instant
conversion to a hydrogel system [17]. The pH, rheology, and drug content uniformity of
the curcumin in the NEG system were evaluated by the method previously reported by
our group [17,20].
Gels 2021, 7, 213 14 of 17

4.7. Ex-Vivo Skin Permeability

The ex-vivo skin permeability profile of the developed curcumin-loaded NEG and con-
ventional gel of curcumin were performed as per the method described previously [17,20].
The excised skin from the Wistar rat was utilized for the ex vivo skin permeation experi-
ment. Local accumulation efficiency (LAE) of the curcumin in NEG and conventional gel
were obtained as a ratio of curcumin accumulated in the skin to that permeated through
the skin [38,39].

4.8. In-Vivo Study

4.8.1. Experimental Protocol
The animal protocol to carry out the in-vivo wound healing activity and histopatholog-
ical examination were approved by the institutional ethical committee (Najran University,
KSA) and followed their guidelines to perform the studies (Ref. No: 25-01-02-20-EC).
Albino rats (weight 200–250 g) were used for the study. The animals were kept under
standard laboratory conditions (temperature: 25 ± 2 ◦ C; relative humidity: 55 ± 5%). The
animals were housed in polypropylene cages, with free access to a standard laboratory
diet and water ad libitum. Animals were anesthetized under aseptic conditions, using
50 mg/kg Ketamine HCl intraperitoneally [40]. Animals were placed on a level surface,
their back was shaved, and a deep wound area was created using a sterile 8 mm biopsy
punch (Acu punch, Acuderma Inc, Louderale, FL, USA).
All animals were divided into four groups with four rats in each group. Group I is the
negative control group with no treatment, group II is the treated group with the marketed
preparation of 1% w/w silver sulfadiazine cream twice a day, group III is the group treated
with 0.5% curcumin-gel twice a day, and group IV is the group treated with 0.5% curcumin
NEG twice a day. All the treated groups (Group II, III, and IV) received the therapy for 20
consecutive days.

4.8.2. Evaluation of Wound Healing Area

Evaluation of wound healing area was performed in terms of wound contraction
percentage, wound closure time, and epithelialization period [41,42]. Percentage of wound
contraction was calculated taking the initial size of the wound as 100% using the following
formula.
( Initial wound area − Speci f ic day wound area)
%wound contraction = × 100 (1)
Initial day wound area

4.8.3. Histopathology
On the last day of the wound healing experiment, the animals were anesthetized
using Ketamine HCl (50 mg/kg, i.p.), euthanized, and specimens of wound tissue with the
adjacent healthy tissue were collected. The collected samples were fixed in 10% formalin
and were subjected to routine histopathological tissue examination [17]. The wound
tissue specimen was sectioned with a microtome (Leica RM 2245) and then stained with
hematoxylin-eosin. The prepared tissue slide was examined under a light microscope.
Further, to evaluate the collagen content, the wound tissue specimen was sectioned using
a microtome, stained with Van Gieson stain for collagen fiber, and examined under a
microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany).

4.9. Statistical Analysis

The statistical analysis was performed using SPSS (version 23 SPSS Inc., Chicago, IL,
USA). The obtained data were analyzed utilizing one-way ANOVA followed by Tukey’s
multiple comparisons tests, where p < 0.05 was considered statistically significant.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/gels7040213/s1, Figure S1: Volume of optimized oil phase (Labrafac PG) emulsified in Smix
phase (Tween 80 and PEG 400) at different ratios (1:1, 2:1, 3:1, and 4:1) to measure its emulsification
Gels 2021, 7, 213 15 of 17

efficiency and corresponding value of the percentage transmittance., Figure S2: Droplet size (56.64
nm with PDI 0.054) and zeta potential (−20.3 mV) of NE6., Figure S3: Droplet size (51.10 nm with
PDI 0.181) and zeta potential (−18.8 mV) of NE7, Figure S4: Droplet size (49.39 nm with PDI 0.113)
and zeta potential (−18.1 mV) of NE8., Figure S5: Spreadability behavior of curcumin NEG system
and placebo gel system.
Author Contributions: Conceptualization, M.S.A. and J.A.; Formal analysis, M.Z.A., I.H.N., H.A.A.,
H.S.A., M.H.A., A.A.A., A.A., T.S.A., A.A.M. and J.A.; Funding acquisition, M.S.A.; Investigation,
M.S.A., H.A.A., H.S.A., M.H.A., A.A.A., A.A. and J.A.; Methodology, M.Z.A., T.S.A., A.A.M. and J.A.;
Project administration, M.S.A.; Software, M.S.A. and M.Z.A.; Supervision, J.A.; Validation, I.H.N.
and J.A.; Visualization, M.Z.A. and I.H.N.; Writing—original draft, M.Z.A., H.A.A., H.S.A., M.H.A.,
A.A.A., A.A., T.S.A. and A.A.M.; Writing—review & editing, M.S.A. and J.A. All authors have read
and agreed to the published version of the manuscript.
Funding: The authors extend their appreciation to the Deputyship for Research & Innovation,
Ministry of Education in Saudi Arabia for funding this research work through the project number
(NU/IFC/ENT/01/005).
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Ethics Committee of NAJRAN UNIVERSITY (protocol
code 25-01-02-20-EC; 25.01.2020).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in article or Supplemen-
tary Materials.
Acknowledgments: The authors extend their appreciation to the Deputyship for Research & Inno-
vation, Ministry of Education in Saudi Arabia for funding this research work through the project
number (NU/IFC/ENT/01/005).
Conflicts of Interest: The authors declare no conflict of interest.

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