International Journal of Biological Macromolecules 101 (2017) 569–579

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Development of Curcumin loaded chitosan polymer based

nanoemulsion gel: In vitro, ex vivo evaluation and in vivo wound
healing studies
Lydia Thomas a , Foziyah Zakir a , Mohd. Aamir Mirza b , Md. Khalid Anwer c ,
Farhan Jalees Ahmad a , Zeenat Iqbal a,∗
a
Nanomedicine Lab, Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India
b
New Zealand Fulvic Limited, Mount Maunganui, Tauranga 3116, New Zealand
c
Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-kharj 11942, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: In the present study, various nanoemulsions were prepared using Labrafac PG + Triacetin as oil, Tween
Received 11 December 2016 80 as a surfactant and polyethylene glycol (PEG 400) as a co-surfactant. The developed nanoemul-
Received in revised form 10 March 2017 sions (NE1-NE5) were evaluated for physicochemical characterizations and ex-vivo for skin permeation
Accepted 13 March 2017
and deposition studies. The highest skin deposition was observed for NE2 with 46.07% deposition
Available online 16 March 2017
amongst all developed nanoemulsions (NE1-NE5). Optimized nanoemulsion (NE2) had vesicle size of
84.032 ± 0.023 nm, viscosity 78.23 ± 22.2 cps, refractive index 1.404. Nanoemulsion gel were developed
Keywords:
by incorporation of optimized nanoemulsion (NE2) into 1–3% chitosan and characterized by physical
Curcumin
Nanoemulsion
evaluation and rheological studies. Chitosan gel (2%) was found to be suitable for gelation of nanoemul-
Gel sion based on its consistency, feel and ease of spreadability. The flux of nanoemulsion gel was found
68.88 ␮g/cm2 /h as compared to NE2 (76.05 ␮g/cm2 /h) is significantly lower suggesting limited skin per-
meation of curcumin form gel. However, the retained amount of curcumin on skin by gel formulation
(980.75 ± 88 ␮g) is significantly higher than NE2 (771.25 ± 67 ␮g). Enhanced skin permeation of NE2
(46.07%) was observed when compared to nanoemulsion gel (31.25%) and plain gel (11.47%). The out-
come of this study evidently points out the potential of curcumin entrapped nanoemulsion gel in wound
healing.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction of serious side effects or the disadvantage of early inactivation of

Healing wound is of perennial interest and fundamental impor-
tance to the surgeon. Wound is typically a tissue injury which
involves inflammation followed by oxidative stress mediated by the
formation of free radicals thus leading to DNA breakage, lipid per-
oxidation and enzymes inactivation. Various factors such as such as
diabetes, infection or metabolic deficiencies are susceptible to fail-
ure in healing of wound [1]. A variety of therapeutic regimes have
been investigated experimental and clinical settings for accelera-
tion of wound healing [2]. However, treatment of wounds by oral
and parenteral application of drugs can be accompanied by the risk
∗ Corresponding author at: Dept. of Pharmaceutics, Jamia Hamdard, New Delhi
110062, India.
E-mail address: ziqbaljh@yahoo.co.in (Z. Iqbal).
the drug.
Studies on curcumin revealed its antioxidant activity that may
have play an excellent role in the management of wound healing
[3]. Curcumin, a natural product obtained from the rhizomes of
Curcuma longa has been reported to be effective in wound repair
used both orally and topically. Curcumin is a o-methoxyphenol
derivative which possesses antioxidant activity and stimulates
detoxification enzymes. It has been reported that when curcumin is
applied topically it significantly improves wound healing and pre-
vents oxidative damage [4]. Further curcumin treatment result in
an increased formation of granulation in tissue, including greater
cellular content and neo vascularisation and a faster re epithelial-
isation of the wound [5]. However being a poorly water soluble
drug it is preferably localized in the superficial stratum corneum
(SC) after topical administration. Nevertheless using an optimal
vehicle, even for lipophilic substances like curcumin, it is possi-
ble to minimize this accumulation. In addition, direct application

http://dx.doi.org/10.1016/j.ijbiomac.2017.03.066
0141-8130/© 2017 Elsevier B.V. All rights reserved.
570 L. Thomas et al. / International Journal of Biological Macromolecules 101 (2017) 569–579
of curcumin to the skin is a matter of concern because of toxic
effects of high concentration of polyphenols. Therefore a formu-
lation which releases the drug slowly in a controlled manner will
be acceptable for efficient delivery of curcumin. Hence a water
based controlled released formulation such as curcumin loaded
nanoemulsion (NE) can be a suitable approach for cutaneous wound
healing [6]. Recently, a number of investigations have been car-
ried out which demonstrated that drugs incorporated into NEs
penetrate efficiently into the skin and through the subcutaneous
barrier [7].So it is imperative to design such a formulation which
enhances the solubilisation of curcumin and improves its perme-
ation through the skin. Many literatures are available regarding
enhancement of solubility and dissolution by different techniques
such as commplexation, nanoemulsion, polymeric nanoparticles
etc [8,9].
The encapsulation of active ingredients into nanoemulsions as
carrier systems is a novel approach for drug delivery. NEs are
defined as clear, thermodynamically stable, isotropic mixture of
oil, water and surfactant/cosurfactant combination [10]. Lipophilic
drugs usually get localised in the superficial skin layers after topical
application. Recent investigations show lipophilic drugs incorpo-
rated into NEs penetrate efficiently into the skin. As topical vehicles,
NEs can increase the local or systemic delivery of a drug by differ-
ent mechanisms [11]. First their composition and structure enable
them to incorporate a greater amount of drug than other conven-
tional topical formulations like ointments, creams, gels and lotions.
NEs can enhance the solubility of poorly water-soluble drugs via
the finely dispersed oil droplet phase. Second the diffusional bar-
rier of the skin may be modified depending on the composition
of the NE [12]. Third an increased thermodynamic activity of the
drug may favour its partitioning into the skin [13]. Thus the formu-
lation will enable the drug to reach the underlying skin layers of
the wound, specifically to the stratum germinativum and the der-
mis where wound repair and angiogenesis take place and act as a
microreservoir with a slow release of the entrapped agent.
2. Materials and methods
2.1. Materials
Curcumin was obtained from Sigma Aldrich, (France). Triacetin
was obtained from Central Drug House, (Mumbai). Labrafac PG was
a gift sample from Gattefosse (Saint Priest, France). Tween 80 and
Polyethylene glycol (PEG 400) were purchased from (Schuchardh,
Germany). All others chemicals were of analytical grade.
2.2. Determination of the solubility of curcumin in oils and
surfactants
The solubility studies were carried out as described by Yuan
et al., 2008 [14], with slight modification. Briefly, 2 ml of different
oils were taken in small vials and excess amount of the curcumin
was added. The vials were tightly stoppered and continuously
stirred for 72 h in mechanical shaker at 37 ± 5 ◦ C and the samples
were centrifuged at 10,000 rpm for 10 min. The supernatant was
separated, filtered and appropriately diluted with methanol and
the amount of drug content was quantified by spectrophotometric
method. The same method was followed for determination of solu-
bility of curcumin in surfactants and cosurfactants. The selected oils
and surfactants were further used for the construction of pseudo-
ternary phase digrams as discussed below.
2.3. Construction of pseudo-ternary phase diagrams
For the determination of existence zone of NE, pseudoternary
phase diagrams were constructed using aqueous titration method
[15]. To construct pseudoternary phase diagrams, the selected
oil phase (Labrafac PG: Triacetin, 1:1) was mixed with surfactant:
cosurfactant mixture (Tween 80 and PEG 400, Smix ). The ratios of
Smix used for titration are 1:0, 1:1, 1:2, 1:3, 1:4, 2:1, 3:1 and 4:1
and the mixture was titrated with distilled water until it turned
turbid. The volume of water used was recorded. Water titration
was continued until a clear, isotropic and thermodynamically sta-
ble dispersion with low viscosity was obtained. The pseudo ternary
phase diagrams were constructed by plotting water phase, oil
phase and surfactant: cosurfactant phase (Smix ) used in the exper-
iment. Different formulations were selected from pseudoternary
phase diagrams for thermodynamic evaluation. The best ratio was
selected for the development of curcumin loaded NEs [16].
2.4. Thermodynamic stability of nanoemulsions (NEs)
In order to select the stable NEs and to discard the unstable
or metastable ones, the drug loaded NEs were subjected to ther-
modynamic stability testing, which comprises of heating cooling
cycles. The formulations were kept between 37 ◦ C and 4 ◦ C for
24 h (3 cycles) at each temperature and observed for any physi-
cal changes. Various aspects like phase separation, turbidity etc. at
room temperature were observed. The selected stable formulations
were subjected to centrifugation for 30 min at 5000 rpm. The stable
formulations which did not show any phase separation or turbidity
were treated for freeze thaw cycles at −20 ◦ C for 24 h and −20 ◦ C for
2–3 min (3 cycles). The NEs which returned to their original form
within 2–3 min were selected for further studies [17].
2.5. Nanoemulsion characterisation
2.5.1. Transmission electron microscopy
Morphology of the NE was studied using TEM (Morgagni 268D
SEI, USA) operating at 200 kV and a 0.18 nm capable of point to
point resolution. Combination of bright field imaging at increasing
magnification and diffraction modes was used to reveal the form
and size of the NE. In order to perform the TEM observations, the
diluted NE was deposited on the holey film grid and observed after
drying [18].
2.5.2. Measurement of droplet size
Droplet size was determined by photon correlation spec-
troscopy that analyzes the fluctuations in light scattering due to
brownian motion of the particles [19] using a Zetasizer (1000 HS,
Malvern Instruments, UK). The formulation (0.1 mL) was dispersed
in 10 ml of water in a volumetric flask, mixed thoroughly with vig-
orous shaking and light scattering was monitored at 25 ◦ C at a 90◦
angle.
2.5.3. Viscosity
The viscosity of the formulations was determined using Brook-
field DV III ultra V6.0 RV cone and plate rheometer (Brookfield
Engineering Laboratories, Inc., Middleboro, MA) using spindle #
CPE40 at 25 ± 0.5 ◦ C [18].
2.5.4. Refractive index and pH
The refractive index of the NEs were determined using an Abbes
type refractometer (Nirmal International, New Delhi) at 25 ± 5 ◦ C.
The apparent pH of the formulation was measured by pH meter
(Accument AB15, Fischer Scientific, USA) in triplicate at 25 ± 1 ◦ C
[18].
2.6. Formulation of optimized nanoemulsion gel
The optimised NE was incorporated into gel by using chitosan
2% as gelling agent. Chitosan was mixed with 1% aqueous solution
 L. Thomas et al. / International Journal of Biological Macromolecules 101 (2017) 569–579
of acetic acid manually in a mortar. After that it was hydrated for 1 h
at room temperature to obtain a gel of desirable viscosity and com-
patibility with the nanoemulsions [20]. 0.025% cetyl pyridinium
chloride was incorporated into the chitosan gel as an antiseptic.
The optimized NE was then mixed with the gel in 1:1 ratio with
gentle stirring.
2.7. Evaluation of nanoemulsion gel formulation
2.7.1. Drug content
For determination of drug content, about 1 g of nanoemulsion
gel (NEG) formulation was weighed in a 100 ml volumetric flask and
dissolved in methanol. It was diluted appropriately and analyzed
at 420 nm by UV spectrophotometry.
2.7.2. Viscosity and pH
The viscosity and pH of the NEG formulation was determined
using methods described previously [18].
2.7.3. Spreadability
Spreadbility of the NEG is an important parameter from patient
compliance point of view. Briefly, 0.5 g test formulation was placed
within a circle of 1 cm diameter pre-marked on a glass plate over
which a second glass plate was added [21]. A weight of 500 g was
allowed to rest on the upper glass plate for 5 min. The increase in
the diameter due to spreading of the test formulation was noted
[22].
2.8. Preparation of rat skin
Albino rats (200–250 g) obtained from central animal facility at
Jamia Hamdard (protocol number 526) were sacrificed with pro-
longed ether anesthesia. Full thickness rat skin was excised from
the abdomen and hair on the skin was removed with depilatory
cream. Subcutaneous tissue was surgically removed and dermis
side was wiped with isopropyl alcohol to remove residual adhering
fat. The skin was washed with distilled water and stored in deep
freezer at −20 ◦ C till further use [23].
2.9. Skin permeation study
Ex-vivo permeation studies through rat skin was performed
using an automated diffusion cell sampling system (SFDC6, LOGAN
Inst, NJ, USA). Following pre-treatment, the skin was cut and
trimmed to appropriate size and mounted on a vertical diffusion
sampling cell. Epidermal membrane samples were mounted into
the diffusion cells (area 0.653 cm2 ) equilibrated at 37 ± 0.5 ◦ C for
8–10 h. The stratum corneum of the skin faced the drug donor
cell whereas dermal side was in contact with the receptor phase.
The donor and receptor compartment was filled to capacity with
acetate buffer pH 5.5 [24]. The receiver fluid was stirred by a Teflon-
coated mini magnetic bar at a speed of 600 rpm and equilibrated at
37 ± 0.5◦ C for 8–10 h. After application of the test formulation on
the donor side, 0.5 ml of aliquot was collected from the receiver
cell at designated time intervals (viz. 0, 1, 2, 3,4, 5, 6, 8, 10, 12
and 24 h) for 24 h period and replaced immediately with the same
volume of fresh media maintained at 37± 0.5◦ C. After appropriate
dilutions, the samples were filtered using 0.45 ␮m membrane filter
and the amount of drug in the receptor media was analyzed using
UV spectrophotometer at 420 nm. Receiver volume was immedi-
ately replenished with the same amount of fresh acetate buffer pH
5.5. Ethanol was added to the buffer to increase the solubility of
curcumin for maintaining sink conditions [25].
571
2.10. Permeation data analysis
The cumulative amount of curcumin permeated through the
skin (Q, ␮g/cm2 ) was plotted as a function of time (hr). The drug
flux (permeation rate) at steady state (Js, ␮g/cm2 /h) was calculated
from the slope of linear portion of the curve [26].
Cumulative amount of drug permeated
Concentration (g/ml)x Volume of diffusion cell
=
Area (cm2 )
Permeability coefficient (Pb )
Flux
=
Drug concentration in donor compartment (g/ml)
2.11. Skin deposition study
For optimum wound healing it is necessary that a reasonable
amount of the drug be retained in the skin. Skin deposition stud-
ies were conducted in order to quantify the amount of curcumin
deposited in the skin. After 24 h of ex-vivo permeation study, the
skin surface was washed five times with ethanol:water (1:1), fol-
lowed by water to remove excess drug from the surface. The skin
was then cut in to small pieces and homogenized with methanol
(5 ml) by Teflon homogenizer for 6 h at room temperature. After
shaking for 5 min, it was centrifuged for 5 min at 5000 rpm and
supernatant was collected, filtered using 0.45 ␮m membrane fil-
ter and analyzed for curcumin content by UV spectrophotometer
at ␭max 420 nm [27]. The skin permeation and deposition studies
were carried out for optimised nanoemulsion gel formulation in a
similar manner.
2.12. Skin permeation and deposition studies
Skin permeation and deposition studies for optimized NEG were
carried out according to the procedure as described previously.
The NEG formulation permeation parameters were also compared
with plain curcumin gel (control) in order to elucidate the effect of
nanoemulsion on skin permeation of curcumin.
2.13. Skin irritation test
The animal protocol (number 526) to carry out skin irritation
study was reviewed and approved by Institutional Animal Ethics
Committee, Jamia Hamdard and their guidelines were followed
for the studies. The skin irritation test of the optimised NEG for-
mulation was carried out on Swiss albino mice weighing 20–25 g.
The animals were kept under standard laboratory conditions, with
temperature of 25 ◦ C ± 1◦ C and relative humidity of 55% ± 5%. The
animals were housed in polypropylene cages, 6 per cage, with free
access to a standard laboratory diet and water ad libitum. The ani-
mals were divided into 3 groups as follows:
Group I Control (Plain curcumin gel)
Group II Blank Nanoemulsion gel (Nanoemulsion gel without
curcumin)
Group III Curcumin loaded nanoemulsion gel (NEG)
A single dose of the NEG containing 2.5 mg curcumin was
applied to the left ear of the mouse, with the right ear as control.
The development of erythema was monitored for 6 days using a
method established by Van- Abbe et al. [28].
572 L. Thomas et al. / International Journal of Biological Macromolecules 101 (2017) 569–579
70
60
Solubility (mg/ml)
50
40
30
20
10
0
OILS
Fig. 1. Solubility of curcumin in various oils, surfactants and co-surfactants (n = 3).
2.14. Stability studies
The optimised NEG was stored at different temperatures for 3
months. As per ICH guidelines, it was stored at 25 ± 2 ◦ C and 75 ± 5%
RH. Then the consistency and concentration of curcumin was inves-
tigated at the end of 0, 30, 60, 90 days. The samples were withdrawn,
diluted with methanol and analysed using UV spectrophotometer.
The amount of drug degraded and the shelf-life of the formulation
was determined from Arrhenius plot.
2.15. In-vivo wound healing study
The animal protocol (number 526) to carry out in vivo study was
reviewed and approved by Institutional Animal Ethics Committee,
Jamia Hamdard and their guidelines were followed for the stud-
ies. Albino rats, 7–9 weeks old and weighing 200–250 g were used
for the study. Screening for wound-healing activity was performed
by incision wound model. The hair on the skin of back surface of
animals was removed by using a suitable depilatory cream (Anne
French hair removing cream). The animals were starved for 12 h
prior to wounding. After shaving the backs of the animals, a lin-
ear 3 cm incision was made over the skin of the back. Eighteen
animals were randomly divided into three groups namely, experi-
mental control, placebo and treatment groups. The vehicle control
group did not receive any treatment. Placebo group received the
NEG devoid of curcumin and the treatment group received NEG
with curcumin. Application was carried out once in a day. For com-
putation of the percentage of wound healing, a transparent paper
was placed on the location of wound and its shape was drawn on the
paper. Then the wound area was measured by matching the shape
of wound with a graph paper. The percentage of wound healing was
calculated by walker formula after measurement of the wound area.
Percentage of wound healing was computed every three days upto
the 18th day [29].
Wound area on day X
Percentage of wound area =
Wound area on day 1
Percentage of wound healing = 100 − Percentage of wound area
SURFACTANTS CO-SURFACTANTS
3. Result and discussion
3.1. Solubility of curcumin
The solubility studies of curcumin were carried out in selected
oils, surfactants and co-surfactants and their results are depicted in
Fig. 1. Higher solubilities were found in synthetic oils as compared
to edible natural oils. The results are in accordance to previous
reports that edible oils are incapable of dissolving large amounts
of lipophillic drugs. The maximum solubility of curcumin was
found in Sefsol, Labrafac PG and Triacetin with solubility values of
14 ± 2.1 mg/ml, 17.5 ± 0.9 mg/ml and 20 ± 1.1 mg/ml respectively.
However because of insignificant differences in curcumin solu-
bility in these three oils, their combination was also evaluated
for solubility. Among the various combinations, namely Sefsol:
Labrafac (1:1), Sefsol: Triacetin (1:1), Labrafac PG: Triacetin (1:1)
and Labrafac PG: Triacetin (1:2), the drug exhibited highest solu-
bility (25 ± 1.2 mg/ml) in Labrafac PG, Triacetin mixture in a ratio
of 1:1. Since curcumin is hydrophobic, we aim to formulate o/w
nanoemulsion. Hence the amount of drug incorporated in oil phase
is an important criterion as poor soluble drugs needs more amounts
of oil for maximum drug loading this will increase the cost of
the formulation. Further, such a formulation will demand higher
amounts of surfactant blends to produce a stable NE [30].
The choice of surfactant is another important factor as the right
blend with sufficient HLB value can form stable nanoemulsion. In
order to formulate o/w NE, the HLB value of surfactant mixture
should be more than 10. To achieve this purpose, we tried vari-
ous non-ionic surfactants such as Tween 80, Tween 20, Labrafil and
Labrasol due its least toxicity as compared to their counterparts.
It is imperative that we conduct solubility study of curcumin in
surfactant and co-surfactant so that the surfactant with highest sol-
ubility can be selected. Due to toxic nature of surfactants, they can
only be used in limited amounts thus necessitating their judicious
selection. Curcumin was found to be highly soluble in Tween 80
(60 ± 3.1 mg/ml) when compared with other surfactants. Therefore,
Tween 80 was selected for our study. Generally short to medium
chain alcohols are used as co-surfactants. The solubility of cur-
cumin in various co-surfactants was also carried out and maximum
solubility was found in PEG 400 (45 ± 3.1 mg/ml). An important cri-
teria which cannot be overlooked is the safety profile of surfactants
and co-surfactants [16]. All the surfactants selected are pharma-
ceutically acceptable and categorized as generally regarded as safe
 L. Thomas et al. / International Journal of Biological Macromolecules 101 (2017) 569–579 573

WATER WATER WATER

A Smix: 1:0 B C
Smix 1:1 Smix 1:2

OIL Smix OIL Smix OIL Smix

WATER WATER
WATER

D E F
Smix 1:3 Smix 1:4 Smix 2:1

OIL Smix OIL Smix OIL Smix

WATER WATER

G H
Smix 3:1 Smix 4:1

OIL Smix OIL Smix

Fig. 2. Pseudoternary phase diagrams indicating oil-in-water nanoemulsion (shaded area) region of Labrafac PG and Triacetin (oil), Tween 80 (surfactant), and PEG 400
(cosurfactant) at different Smix ratios; A (Smix 1:0), B (Smix1:1), C (Smix1:2), D (Smix 1:3), E (Smix 1:4), F (Smix 2:1), G (Smix 3:1), H(Smix 4:1).
Yellow dots – nanoemulsion zone; Purpule dots – microemulsion zone.

“GRAS” but only within a limited quantity. Therefore using a blend
of surfactant and co-surfactant allows the use of low concentrations
of individual surfactant, which is a suitable approach to reduce the
toxicity of the formulation (development of o/w). Additionally, HLB
value of the selected surfactant blend i.e. Tween 80 and PEG 400 was
found to be 13.4, which fulfills the criteria of minimum HLB value
required for the formulation of a stable o/w NE.
3.2. Pseudo-ternary phase diagram
The pseudo-ternary phase diagrams were constructed to elu-
cidate any changes in the area of NE region with change in
excipients. The Figures show phase diagrams constructed using
Labrafac PG:Triacetin (1:1) as oil phase and different Smix ratios
of Tween 80 and PEG 400; 1:0, 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, 4:1.
574 L. Thomas et al. / International Journal of Biological Macromolecules 101 (2017) 569–579

The relationship between the phase behaviour of a mixture and its Table 1
Compositions oil, Smix and water for selected nanoemulsion formulations.
composition can be captured with the aid of a phase diagram. Pseu-
doternary phase diagrams were constructed separately for each Nanoemulsions % v/v components S/Cos ratio
Smix ratio (Fig. 2), so that o/w NE regions could be identified and
Oil Smix Distilled water
NE formulations could be optimized. The translucent nanoemulsion
NE1 12 40 48 1:1
region is presented in phase diagrams. The rest of the region on the
NE2 15 40 45 2:1
phase diagram represents the turbid and conventional emulsions NE3 18 40 42 2:1
based on visual observation. In Fig. 2A, B and H), Two color could NE4 15 35 50 3:1
be seen (Yellow and Purpule). During phase diagram construction NE5 18 36 46 4:1
two zone were selected in software. Yellow and purpule color in
phase diagram describes nanoemulsion and microemulsion zone
respectively. It is clearly evident from the graph (Fig. 2A), NE pre-
pared without any co-surfactant exhibit a low NE region. However
when the co-surfactant is added this region has increased signif- drug can be solubilised per ml of oil. Therefore 12% was selected as
icantly. Further when the Smix ratio was increased from 1:1 to the least oil concentration to be taken for 1 ml of formulation from
1:3, the nanoemulsion area also increased, though insignificantly the phase diagram. Curcumin when applied topically in a concen-
(Fig. 2B–D). From these observations, it can be inferred that the tration of 0.1% has been found to be effective for wound healing [5].
use of a single surfactants cannot appreciably reduce the interfa- Keeping this in mind the drug stock solutions were prepared in such
cial energy required for the formation of NE. Therefore a second a way that 2.5 mg dose is present in each formulation complying the
surfactant known as co-surfactant is generally added, which will oil percentage for each formulation. For the amount of oil selected,
give sufficient flexibility to interfacial film such that it can change the minimum possible concentration of Smix should be used. Smix
its curvature to allow formation of nanoemulsion over a wide range ratios 1:0 and 1:4 were not used for making drug loaded formula-
of composition. In addition, co-surfactants also enhance the mobil- tions because of small NE area which implies decreased ability for
ity of hydrocarbon tails of oil thus allowing better penetration. On emulsification.
further raising the co-surfactant concentration i.e. Smix 1:4, the NE
area was again reduced most probably indicating formation of less
number of micelles thus it was not able to sufficiently reduce the
3.4. Thermodynamic stability of nanoemulsions (NE)
interfacial energy (Fig. 2E).
In order to prepare a stable NE, the effect of change in surfac-
NEs are thermodynamically stable systems and are formed
tant concentration (Tween 80) has been observed with respect to
at a particular concentration of oil, surfactant, and water, mak-
changes in NE area. When the Smix ratio was enhanced from 2:1 to
ing them stable and not subject to phase separation, creaming,
4:1, the NE area concomittantly increased (Fig. 2F–H). These results
or cracking. It is the thermostability that differentiates nano- or
suggest surfactants have the ability to reduce the interfacial energy
microemulsions from emulsions that have kinetic stability and
but only upto to a certain concentration (CMC). When the concen-
eventually phase-separate [18]. Thus the formulations were tested
tration exceeds the CMC, the capability is hindered without any
for their thermodynamic stability by checking their stability at low
effect on interfacial energy.
temperature and high shear. The formulations which passed the
thermodynamic protocol were namely 12:40:48 (1:1), 15:40:45
3.3. Selection of stable nanoemulsion (NE) (2:1), 18:40:42 (2:1), 15:35:50 (3:1) and 18:36:46 (4:1); these were
then coded as NE1, NE2, NE3, NE4 and NE5 respectively (Table 1).
From the pseudo-ternary phase diagram studies, placebo formu- These were also the ones that contained the minimum amount
lation was found sufficiently stable. Therefore our next step was to of surfactant which is a major criterion for selection of such for-
prepare drug loaded NE keeping the oil and Smix ratios same. The mulations. The rest of the formulations showed phase separation,
amount of oil should be sufficient enough to solubilise the dose turbidity, change in color or drug precipitation suggest instabil-
of curcumin in 1 ml of the NE. As per saturation solubility studies ity and were rejected. The composition of selected nanoemulsions
of curcumin in the oil phase (Labrafac PG: Triacetin, 1:1), 25 mg of (NE1-NE5) were given in Table 1.

Fig. 3. In-vitro skin permeation profile of curcumin from 5 different nanoemulsion formulations (NE1-NE5).
 L. Thomas et al. / International Journal of Biological Macromolecules 101 (2017) 569–579 575

Table 2 Table 4
Permeability Parameters of Different nanoemulsions (NE1-NE5). Skin irritation studies of optimized nanoemulsion (NE2).

Formulation code Flux (␮g/cm2 /h) (n = 3) Permeability coefficient Rat Group Score after (days) Mean Score

NE1 75.306 ± 5.4 0.030 1 2 3 4 5 6 7

NE2 77.175 ± 7.2 0.031
NE3 107.1 ± 5.9 0.043 I 0 1 0 0 1 0 1 0.43
NE4 95.106 ± 9.2 0.038 II 1 0 0 0 0 1 0 0.29
NE5 92.733 ± 8.6 0.037 III 0 1 1 0 0 0 0 0.29

Table 5
Optimization of chitosan concentration for development of nanoemulsion gel.
3.5. Ex-vivo skin permeation study and skin deposition study
S.No. Percentage of chitosan Gel formation Spreadability
Ex-vivo skin permeation studies were performed to compare the 1. 1% Not formed Good
release of drug from five different NE formulations (NE1 –NE5 ), all 2. 2% Formed Good
having the same quantity of curcumin. Fig. 3 clearly demonstrates 3. 4% Formed Poor

the skin permeation of curcumin from various NEs. The flux values
abruptly increased from 75.3 ␮g/cm2 /h (NE1) to 107.1 ␮g/cm2 /h
in smaller in size as compared to the droplet size measured using
(NE3) followed by gradual decrease to 92.7 ␮g/cm2 /h (NE5), sug-
photon correlation spectroscopy.
gesting enhanced skin permeation shown by NE3 formulation
Viscosity measurements were carried out and average viscos-
(Table 2). Upon investigation, It was observed that NE2 and NE3
ity of NE2 was found to be 78.23 cps. It is noted that the viscosity
had same content of Tween 80 (2:1) but flux values found less in
of the formulations was low due to low amount of Tween 80 and
NE2 as compared to NE3, which may be due to difference in ratio
oil content, which is expected for nanoemulsion suggesting ease in
of oil and water composition used in formulation.
handling. Refractive index of the NE was determined using Abbes
Higher content of oil present in formulation NE3 was respon-
type refractometer. Thr mean value of refractive index for the for-
sible for higher flux values as lipophilic drugs are preferably
mulation was 1.404, suggesting isotropic nature of formulation. The
solubilized in o/w nanoemulsion. Since Tween 80 is a polysor-
pH values of the prepared formulations ranged from 6.2 to 6.3,
bate surfactant by nature, therefore it possesses the capability
which is considered acceptable as the skin pH is reported to be
to enhance drug permeation by penetrating into the intracellular
5.5-6.5 Therefore the formulation avoids the risk of irritation upon
regions of stratum corneum and eventually solubilise lipid compo-
application to the skin.
nents. However, this effect was observed only upto a certain extent.
As the amount of Tween 80 is increased further (NE4- 3:1, NE5- 4:1),
3.7. Skin irritation test
the rate of skin permeation fell gradually. This can be possibly due
to overall reduced Smix content, 35% (NE4) and 36% (NE5) vs 40%
Curcumin is a known irritant and in addition the formulation
in NE3. These findings have been supported by permeability coeffi-
contains varying amounts of surfactants making it necessary to
cient data for NE1 (0.03), NE2 (0.031), NE3 (0.043), NE4 (0.038) and
conduct skin irritation study. The test revealed a mean irritation
NE5 (0.037) which shows a similar pattern. Since the formulation is
score value of 0.34 for the optimized NE formulation (NE2). It has
designed for use in the treatment of wounds, more skin retention is
been previously reported that a value between 0 and 9 suggest
required rather than permeation. Therefore, skin deposition of the
non-irritant potential (Table 4) hence our formulation is safe and
curcumin in the five formulations (NE1-NE5) were investigated and
compatible for topical delivery to human skin [28].
were found in the range of 17.46–46.07%. Since highest skin depo-
sition was observed for formulation NE2 with 46.07% deposition
(Table 3), therefore it was selected for further studies. 3.8. Formulation and evaluation of nanoemulsion gel

On the basis of permeation and characterization studies, the

3.6. Characterization of nanoemulsion (NE)
Since our aim is to formulate NE therefore observing droplet size
is of immense significance. By definition, a typical nanoemulsion
should have droplet sizes less than 100 nm and the results of our
study prove that nanoemulsions were formed. The average droplet
size of nanoemulsion NE2 was found to be 84.032 nm with PDI
0.232. Further TEM studies were conducted to determine the sur-
face morphology of nanoemulsion. In the TEM positive image, the
nanoemulsions droplets appear circular. The light surroundings is
due to opaque nature of oil phase whereas the darker area signifies
the embedded curcumin (Fig. 4). TEM image showed the presence of
spherical non-aggregated droplets within nanorange, which were
optimized formulation NE2 was selected to be formulated into gel.
Chitosan was selected for the preparation of gel due to its biocom-
patible and non-toxic nature. It posesses an ability to form gels at
acidic pH values, because it is hydrophilic and can retain water in
its structure. Gels containing 1%, 2% and 4% chitosan were prepared
and the best concentration was selected on the basis of visual obser-
vations. 2% gel was found to be suitable for gelling the NE because
of its desirable consistency, feel and ease of spreadability (Table 5).
Additionally, 0.025% of cetyl pyridinium chloride was incorporated
into chitosan gel to incur anti-infectious properties.
The NEG was subjected to the study of various parameters for its
characterization (Table 6). The spreadability of the gel was found
to be 7.1 cm and viscosity was measured to be 504 ± 21.4 cps.

Table 3
Skin deposition studies of developed nanoemulsions (NE1-NE5).

Nanoemulsions Concentration (␮g/ml) Amount of drug in skin (␮g) (mean ± SD)a (n = 3) % of drug deposition

NE1 132.5 662.5 ± 22 26.5%

NE2 194.25 1811 ± 97 46.07%
NE3 112.8 564.0 ± 27 22.56%
NE4 87.3 436.5 ± 35 17.46%
NE5 94.45 472.25 ± 41 18.89%
576 L. Thomas et al. / International Journal of Biological Macromolecules 101 (2017) 569–579

Fig. 4. Transmission electron microscopic image of curcumin nanoemulsion.

Table 6
Comparative Evaluation.

Formulation Spreadability Drug content (mg) Droplet size (nm) Viscosity (cps) pH Cumulative Amount of drug
amount of drug retained in skin
permeated (␮g) (␮g)

NEG 7.1 ± 0.115 2.45 ± 0.008 504 ± 21.4 6.4 ± 0.121 1356.46 980.75 ± 88
NE2 – – 84.032 ± 0.023 78.23 ± 22.2 6.2 ± 0.224 1811.35 771.25 ± 67
Plain gel – – – – – 392.14 154.32 ± 15

(n = 3).

80 of chitosan which enmeshed the drug and precluded the release.

70 The thickening effect of chitosan gel has lent the formulation a bet-
% Cumulative drug permeated

ter spreadability. It also ensured that the drug deposition is higher,

60
50
40
30
20
10
0 This significant difference can be attributed to small droplet size
0 5 10 15
Time (hr)
Fig. 5. Ex-vivo skin permeation through rat skin at different time intervals on appli-
cation of Contro, NE2 and NEG (n = 3).
These parameters signify sufficient viscosity that facilitates ease
in application and spreading. Further the pH of the formulation
was 6.4 ± 0.121, suggesting that the formulation is compatible to
human skin. The drug content was also evaluated since it is an
important factor which determines the amount of drug reaching
the skin. 98% (2.45 mg) drug content signifies good drug loading
capacity. The flux of nanoemulsion gel was 68.88 ␮g/cm2 /h, when
compared to that of nanoemulsion NE2 (76.05 ␮g/cm2 /h) is signifi-
cantly lower suggesting limited skin permeation of curcumin from
gel formulation (Fig. 5). Further the amount of curcumin retained in
skin by gel formulation (980.75 ± 88 ␮g) is significantly higher than
NE2 formulation (771.25 ± 67 ␮g). The reduction in permeation is
actually attributable to the gel formulation and thickening action
nearing approximately 40%. This retention of curcumin in the skin
will ensure the hastening of re-epithelialisation process. Moreover
NE2
the presence of chitosan load with cetyl pyridinium chloride as
a bed within the midst of the wound would probably ensure an
NEG
infectious moist environment for fast healing of the wound. In addi-
control tion skin permeation of curcumin from NEG was compared with
plain curcumin gel. Enhanced skin permeation was observed on
using NEG (31.25%)/NE2 (46.07%) in contrast to plain gel (11.47%).
20 25 30 of nanoemulsion which can permeate through skin barriers effi-
ciently. Numerous surfactant/co-surfactants have been evaluated
as penetration enhancer for the development of nanoemulsions
such as Tween 80 and PEG 400 which facilitate improved perme-
ation of curcumin in topically applied dosage forms. [18]. Since
curcumin is hydrophobic in nature therefore limited penetration is
observed from plain gel. Therefore, it was found that NE have suc-
cessfully enhanced skin permeation and deposition of curcumin by
upto 6 fold.
3.9. Physical stability studies
Stability testing was carried out to investigate the effect of envi-
ronmental factors such as variations in temperature, humidity and
light on the formulation. A NEG should undergo physical and chem-
ical estimations to determine their stability. The gel formulation is
considered stable if there is no phase separation, turbidity or coa-
lescence. For chemical stability, we have tested the drug content in
the formulation at different time periods. The log% drug remaining
 L. Thomas et al. / International Journal of Biological Macromolecules 101 (2017) 569–579 577

2.002 120
2
100
Log % drug remaining

1.998

% Wound Healing
1.996 80
1.994 30°C Control
60
1.992 40°C Placebo

1.99 50°C Treatment

40
1.988
20
1.986
0 20 40 60 80 100
0
Time (days)
Day3 Day6 Day9 Day12 Day15 Day18
Fig. 6. Stability studies of nanoemulsion gel (NEG), Log percentage remaining vs
Fig. 8. Incisional wound healing study in of nanoemulsion gel on albino rat model
time.
(n = 3).

1/T*1000
-3.56
dragged behind significantly. This suggests that treatment with
the cucumin NEG enhanced wound healing as compared with the
-3.58 3.05 3.1 3.15 3.2 3.25 3.3 3.35
groups not receiving it. It can be observed that placebo formulation
-3.6
was successful in healing wound in 15 days as compared to con-
-3.62 trol group (18 days). The wound healing properties of curcumin
-3.64 is mainly due its ability to enhance granulation tissue formation,
log K

-3.66 collagen deposition, tissue remodeling and wound contraction as

-3.68
-3.7
-3.72
-3.74
Fig. 7. Arrhenius plot for the calculation of shelf-life.
for each temperature (30 ◦ C, 40 ◦ C and 50 ◦ C) was calculated and
plotted against time (Fig. 6). The degradation rate constant was
calculated by the following equation (Table 7):
Slope = −K/2.303
Arrhenius plot was drawn by plotting log K vs 1/T (Fig. 7) and
value of K at 25 ◦ C (K25 ) was obtained by extrapolation. The shelf-
life (t0.9 ) i.e. time required for 10% degradation of the drug was
calculated from the following equation: t0.9 = 0.1054/ K25. The shelf-
life of the formulation was found to be 1.58 years. The results shows
that the formulation is chemically and physically stable for 1.58
years.
3.10 In-vivo wound healing study
The suitability of any new formulation is best tested through its
in-vivo performance. Often the success of a formulation in in-vitro
conditions is not reproduced in the in-vivo situation and hence its
testing in a suitable animal model before progressing for commer-
cialization is needed. The effect of the application of the formulation
on the healing of incisional wounds in wistar rats was studied. Per-
cent reduction in the wound area in control, placebo and treatment
groups is presented in Figs. 8 and 9. All wounds showed a reduction
in size over time, regardless of the group to which they belonged.
But the group treated with the NEG showed full wound closure by
the 12th day of the study whereas the control and placebo groups
reported in literature. Hence,this wound healing potential of NEG
was probably due to definite wound contraction [31]. It has been
reported that chitosan acts a wound healing accelerator. The effi-
cacy of chitosan for wound healing is mainly due to its acceleration
of the infiltration of polymononuclear cells into the wound area,
stimulation of the migration of microphages, proliferation of fibrob-
lasts and the production of collagen [32]. For detailed mechanistic
insights on the wound healing activity of NEG, further studies are
needed. A primary requirement for wound healing is to include a
moist and bacteria free environment. Availability of the antisep-
tic (cetyl pyridinium chloride) in the aqueous phase will prevent
chances of infection. On the contrary, enhanced wound healing
effect observed with NEG formulation can be attributed to the com-
bined effect of chitosan and curcumin.
Additionally, the NE was able to facilitate permeation of cur-
cumin to the underlying skin layers of the wound because of its
tendency to dissolve the superficial layers of skin lipids. They
also provide better reservoir for a poorly soluble drug through
their capacity for enhanced solubilisation. An added advantage of
nanoemulsion, is the depot effect of curcumin in the skin which
helps in residing the drug for longer periods of time [7]. These
outcomes have been justified and explained in detail in skin per-
meation studies. Since curcumin used alone can be highly irritant
to the animal because of high amounts of plyphenol, therefore it
was not included in the study protocol [4].
4. Conclusion
Our studies have clearly shown that curcumin nanoemulsion
was formulated which was proved by pseudo-ternary phase dia-
grams and TEM studies. Further the in-vitro studies proved the
formulation was sufficiently thermodynamically and physically

Table 7
Degradation rate constant for curcumin in nanoemulsion gel at different temperatures.

Temperature ◦ C Slope*105 K*104 (days−1 ) Log K Absolute temperature 1/T 1/T *103

30 −8.73 2.010 −3.6968 0.003300 3.3003

40 −9.96 2.293 −3.6395 0.003189 3.189
50 −11.70 2.694 −3.5696 0.003091 3.0951
25 0.003350 3.3500
578 L. Thomas et al. / International Journal of Biological Macromolecules 101 (2017) 569–579
Fig. 9. Photographical representation of wound contraction rate at different time intervals for A- control, B- placebo, C- treatment groups.
stable. The optimised formulation containing 15% Labrafac PG: Tri-
acetin (1:1) as the oil phase and 40% Tween 80 and PEG 400 as
surfactants and co-surfactants, respectively in a ratio of 2:1, were
subjected to ex-vivo and in-vivo studies. These studies have proved
that nanoemulsion can be used for loading curcumin for the treat-
ment of wound healing.
4.1. Statistical analysis
All the results obtained were expressed as mean ± S.D using
Graph Pad Software (Instat 3.06, USA) with more than three repli-
cates for each experiment.
Conflict of interest statement
Authors report no conflict of interest of this work. Author is
responsible for content and the writing of the paper.
Acknowledgement
Authors are thankful to Department of Pharmaceutics, Faculty
of Pharmacy, Jamia Hamdard University, New Delhi, India, for pro-
viding essential facility in this research.
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