After 7 days burned, all three treated groups were found to be not different by the external
morphology. From day 7 to day 12, the morphology of 3 treatment groups was changed. The
area of the wound had significant changes, especially in group treatment by nCur-CP that
decreased from 82.2 ± 17.35 mm2to 12.2 ± 4.9 mm2 (p < 0.05). After 20 days of Silvirin and
nCur-CP treatment, wounds were healed completely. The new blood vessels were appeared in
the nCur-CP treatment group while less in the group treated with Silvirin. Epithelial formation
process in treatment group with nCur-CP was faster than that treated with Silvirin (p < 0.05). In
addition, the appearance of the collagen fibers in the nCur-CP treatment group was earlier than
treatment group with Silvirin. Growth of hair follicle in the nCur-CP-treated wound was more
than that of Silviri treatment. Besides, the epidermis layer of the nCur-CP and Silvirin-treated
models were thinner than the non-treated group, and the skin became normal (Figure 6A) as for
both morphology and histology. From the above results it can be concluded that the burn
wounds treated with nCur-CP had better efficiency than the other groups.
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Vietnam Journal of Science and Technology 56 (5) (2018) 594-603
DOI: 10.15625/2525-2518/56/5/11075
NANOCURCUMIN AND CHITOSAN-PLURONIC F127-BASED
HYDROGEL FOR 3
RD
DEGREE BURN TREATMENT
Dinh Van Tuan
1, *
, Pham Thi Ly Na
2
, Dang Le Hang
3
, Cao Van Du
1
, Mach Thi Anh
3
,
Doan Nguyen Vu
3
, Tran Le Bao Ha
3
, Nguyen Dinh Trung
4
, Tran Ngoc Quyen
4, 5
1
Lac Hong University, 10 Huynh Van Nghe Street, Dong Nai Province
2
University of Science, VNU-HCM, 227 Nguyen Van Cu Street, District 5, HCMC
3
International University, VNU-HCM, Quarter 6, Linh Trung Ward, Thu Duc District, HCMC
4
Graduate School of Science and Technology Viet Nam, Vietnam Academy of Science and
Technology (VAST),01A TL29 Street, Thanh Loc Ward, District 12, HCMC
5
Department of Material and Pharmaceutical Chemistry, Institute of Applied Materials Science,
(VAST), 01A TL29 Street, Thanh Loc Ward, District 12, HCMC
*
Email: tuandinhvan15101995@gmail.com
Received: 16 January 2018; Accepted for publication: 3 June 2018
Abstract. Burn is one of the popular accidents today and usually leaves serious physical and
mental damage. Previously, burn was considered acute wounds, but now it is evolution into
chronic wounds if inadequately managed. Up to now, there have been abundant of natural and
synthetic products for burn healing. In the study, we fabricated a thermosensitive nanocomposite
hydrogel, which incorporated dual active curcumin and chitosan. Beside of a well-known
characteristic of chitosan for wound healing, curcumin has been a lot of interest in burn wound
healing application due to ability in depleting the action of oxidative radicals and stimulation of
fibroblast cells. In order to enhance the therapeutic efficacy of curcumin, we introduced a new
method to synthesize nanocurcumin in the thermosensitive chitosan-g-Pluronic F127 copolymer
solution under ultrasonication. The rheology of aqueous solutions of this material was studied as
a function of temperature. The solutions of this material undergo a transition to a gel at higher
temperature, above which a complex rheological behavior is observed. In addition, a minimum
inhibitory concentration of this material was determined for a variety of bacterial and was
compared to that of curcumin. It was found that the aqueous dispersion of this material was
much more effective than curcumin against both positive and negative gram bacterial. In the
third degree burn models, the nCur-CP hydrogel performed a higher burn healing rate as
compared to Silvirin-treated burn. These data suggest that the nanocomposite hydrogel may be a
great potential for burn treatment.
Keywords: burn healing, curcumin, nanocomposite hydrogel, chitosan, pluronic F127.
Classification numbers: 1.2.6; 2.9.3.
Nanocurcumin and chitosan-pluronic F127-based hydrogel for 3
rd
degree burn treatment
595
1. INTRODUCTION
The desire to restore skin after being damaged remains a major target of tissue regeneration
technology [1, 2]. Nowadays, the substances derived from nature that used to treat and improve
human health are increasingly being concerned. In some serious damage cases such as third
degree burns it is difficult to recover as original. One of the factors affecting the healing process
is the appearance of oxidative free radicals [3, 4]. The reduction of free radicals increases the
ability to heal wounds [5, 6]. Curcumin (Cur; 1.7-bis (4-hydroxy-3-methoxyphenyl)-1.6-
hepadiene-3.5-dione) is a natural substance found in turmeric (Curcuma longa). It is known as
an antioxidant and anti-inflammatory stimulating the growth of fibroblasts [7, 8]. Curcumin has
great potential in medical applications, however it is of poor solubility so its application is
limited [9, 10]. To enhance the therapeutic effect of curcumin, we developed the formula of
injectable nanocomposite hydrogel, which has brought much hope regarding the use of curcumin
in the medical field [11, 12, 13].
Pluronic F-127 is a thermosensitive hydrogel capable of dissolving in water and has low
toxic properties. It is one of the pluronics belongs to Food and Drug Administration (FDA) for
clinical [14]. For clinical application the Pluronic F127 is capable to be dissolved well,
biologically compatible (Pluronic F127 as a Cell Encapsulation Material: Utilization of
Membrane-Stabilizing Agents) and sensitive to heat [15, 16]. It is considered as a material for
loading drug. However, it is possible to combine F127 with chitosan, to increase bio-
compatibility and increase antibacterial activity. Chitosan is a natural polymer widely used in the
medical field by bio-compatibility, biodegradable, hemostatic activity, and their antibacterial
properties [17, 18, 19]. Therefore, chitosan could be used as a factor against bacterial infiltration
[20]. From the results of the previous researches, we have conducted the further evaluation of
the copolymer-based hydrogel encapsulated nanocurcumin in the treatment of third degree burn
[11]. Regarding to synergic effects of nanocurcumin and chitosan-based hydrogel, the extensive
study could be expected to pave a way for exploiting the injectable nanocomposite hydrogel in
clinical application.
2. MATERIALS AND METHODOLOGY
2.1. Material
The hydrogel nCur-CP was synthesized following the procedures in our previous study
[11]. The chemicals and solvents in this study were purchased from Sigma (USA) or Schalaurs
(Spain). Male white mice (scientific name: Musmusculus var. Albino), of weight of about 35 g
was offered from the Pasteur Institute in Ho Chi Minh City.
2.2. Storage study
The curcumin in absolute ethanol was added drop-wise into CP solution under
ultrasonication process as shown in Figure 1. Then ethanol was evaporated by the rotary
evaporator to obtain an nCur-loaded CP nanocomposite material which could be dissolved in
cold distilling water and then form a nanocomposite hydrogel by increasing the solution
temperature. The sol-gel transition temperature of the sample was tested. The stability of
nanocurcumin-loaded nCur-CP samples was evaluated at time interval of 0, 1, 4 and 24 weeks.
These samples were stored in two different temperature conditions, at cold temperatures of 4-8
Dinh Van Tuan et al.
596
o
C and at room temperature (RT). In the specified period, the product was taken out to evaluate
stability of the nanocurcumin in hydrogel.
Figure 1. Prepared nanocurcumin loaded chitosan-g-pluronic F127.
2.3. In vitro evaluation of the injectable nCur-CP materials
2.3.1. Antibacterial activity
The types of bacteria used to evaluate the antibacterial activity of curcumin were
Escherichia coli (ATCC8739), Salmonella typhimurium (ATCC 14028), Pseudomonas
aeruginosa (ATCC27853) and Staphylococcus aureus (ATCC 6538). The Kierby-Bauer disk
diffusion method used in this experiment was obtained from Department of Biochemistry,
Faculty of Biology and Biotechnology, University of Science, Vietnam National University, Ho
Chi Minh city. These bacteria are cultured in nutrient agar before carrying out the tests. The
bacteria after culture were diluted in sterile LB solution and adjusted to a number of bacteria in
the colony forming (unit: cfu/mL) by a UV spectrophotometer at 660 nm. In this experiment, we
have used 4 different variables including hydrogel, nCur-CP, curcumin solutions and antibiotics
chloramphenicol as the control. The concentration of curcumin used in this testing was
equivalent to the concentration of curcumin in nCur-CP composite.
Raw curcumin solution was prepared by dissolving in DMSO, the remaining variables were
dissolved in water. For each individual agar plate were added with varying concentrations of
curcumin (0.08-4 mg/ml), hydrogel (0.75-3.7 mg/mL) and nanocomposite solutions. A plate of
agar added DMSO was used as the control plates. Minimum Inhibitory Concentration (MICs)
was the lowest concentration that completely inhibits the growth of bacteria.
2.3.2. Cell culture
In this study, the Human Foreskin fibroblasts (HFF-1; SCRC-1041TM; USA) were used to
evaluate the toxicity and proliferation of living cells. The HFF1 cells were cultured in standard
culture medium (Dulbecco's 10 % bovine fetal modified Eagle medium serum, 100 U/ml
penicillin G, and 100 μg/ml streptomycin). The culture medium was replaced twice a day. Cell
growth (HFF-1) was evaluated on three groups: nCur-CP, hydrogel and 2D tissue culture
Nanocurcumin and chitosan-pluronic F127-based hydrogel for 3
rd
degree burn treatment
597
control. HFF1- cell proliferation was measured with Alamar Blue (Sigma Aldrich, St. Louis,
MO, USA) and fluorescence microscope (TE2000, Nikon, Seoul, Korea).
2.4. In vivo evaluation of burn wound healing in animal model
The 3
rd
-degree burn wounds on mice were conducted at the Laboratory of the Department
of Physiology and Animal Biotechnology, University of Science, Vietnam National University-
Ho Chi Minh City. Mice were kept stable for 4 days before carrying out experiments. Mice were
weighed and anesthetized with ketamine intraperitoneal (100 mg/ml) and xylazine (20 mg/ml)
with dosage of 0.2 ml / 100 g body weight before shaving the dorsal skin of mice. Sterilize the
shaved skin with 1 % povidine before proceeding to burn. To create the 3
rd
-degree burn, a
cylindrical shaped stainless steel cup with a diameter of 1 cm, weight 114 g was heated in a hot
water (100 degrees). Then steel bar was placed over the cleaned skin and held at different
intervals (5s, 10s and 20s). After 2 or 3 hours, these skins were collected and kept in
formaldehyde 10 % about 24 hours for H&E dyeing.
In this study, animals were divided into three groups, each group consisted of three mice:
Group I: Control (non-treatment),
Group II: Using commercial drugs (Silvirin),
Group III: Using nCur-CP.
The 3
rd
-degree burn was studied within 22 days, the wound was measured twice a day.
After 7, 14, 22 days treatment, skin samples were collected and stored in formaldehyde 10% to
dyeing H & E and using ImageJver 1.41 to evaluate the effect of treatment.
2.5. Statistical analysis
The data collected from the experiments were analyzed statistically by using one-way
ANOVA for particle size, the test of antibiotics or curcumin content, etc. The data related to the
comparison between the two independent variables were evaluated by using two-way ANOVA
for cell viability and closed wound surface. They are considered statistically significant with the
value p < 0.05.
3. RESULTS
3.1. The sol-gel transition temperature
Rheological measurement was used to determine the sol - gel transition temperature of the
nanocomposite hydrogel. The point was observed on the change of storage modulus G' and loss
modulus G'' at temperature ranging from 4 °C to 45 °C with frequency and amplitude fixed. The
operation defined here as the temperature at which the storage modulus G' and loss modulus G''
are equal. As shown in Figure 2, at low temperature G">>G' it means that the hydrogel is in sol
state. At 30
oC both G’ and G’’ rise together, when temperature reach 35 oC the two values of G’
and G’’ are nearly equal. This indicates that the sol - gel transition temperature (Tgel) of the
hydrogel is about 35 °C. This Tgel is closer to human body temperature (about 37 °C).
Therefore, injectable nanocomposite hydrogel has great potential to develop into a drug delivery
system applicable for wound healing [11].
Dinh Van Tuan et al.
598
Figure 2. Rheology of nCur-loaded CP hydrogel (CS: F127 = 1:15) as dependent on temperature.
After 4 and 24 weeks of storage, the storage samples were collected to test stability of the
nanocurcumin. The results show that there is no change in color of all nanocomposite hydrogel
samples as well as size distribution of the nanoparticles as stored at cool or room temperature for
4 weeks as shown in Figure 3. Significant increase in the particle size in RT condition and
increase slightly in cool condition after 24 weeks were observed.
Figure 3. Size of curcumin nanoparticles in the CP copolymer phase at low temperature and
ambient conditions (RT).
3.2. In vitro evaluation of nCur-CP nanocomposite hydrogel
3.2.1. Anti-biotic ability
The zones of growth inhibition were measured after 18 to 24 h incubation at 37 °C for bacteria.
The antibacteria activity of materials was determined by the measured diameter of growth
inhibitory zones as indicated in Figure 4, and if the value is equal or less than 6 mm which
means that no antibacteria activity occurred. The antibacterial activity of the antibiotics
chloramphenicol was the same for all four bacterial groups. nCur-CP and hydrogel were more
effective against P. aeruginosa, E. coli and S. aureus than against S. typhi. Raw curcumin
Nanocurcumin and chitosan-pluronic F127-based hydrogel for 3
rd
degree burn treatment
599
samples showed the most effective against S. aureus while less effective against E. coli, P.
aeruginosa and S. typhi. In addition, by using ANOVA one way for each bacterial group, the
nCur- CP had maximum antibacterial efficiency in all the tests (p < 0.05).
Figure 4. Zone of inhibition of hydrogel, nCur-CP and raw curcumin solutions against 4 bacteria strains:
Escherichia coli; Pseudomonas aeruginosa; Salmonella typhimurium and Staphylococcus aureus.
Table 1 has shown that the minimum inhibitory concentration (MIC) of raw curcumin for
E. coli, S. typhimurium, P. aeruginosawas and S. aureus were 1.6, 4, 2 and 0.96 (ppm),
respectively; and the MIC of hydrogel in turn is 18.75, 37.5, 9 and 15 (mg/ml). From the results
it is found that the raw curcumin had antibacteial activity better than that of hydrogel. However,
MIC of nCur-CP was lower than both MIC of raw curcumin and hydrogel. The effect could be
contributed by a synergically active combination of both chitosan hydrogel and curcumin.
Table 1. MIC of raw curcumin, hydrogel and nCur-CP against different microbes.
Organisms MIC
Raw curcumin (ppm) Hydrogel (mg/ml) nCur-CP (mg/ml)
E. coli 1.6 18.75 3.0 (0.32 ppm curcumin)
S. typhimurium 4 37.5 4.5 ( 0.48 ppm curcumin)
P. aeruginosa 2 9.0 0.75 (0.08ppm curcumin)
S. aureus 0.96 15 1.5 ( 0.16 ppm curcumin)
3.2.2. Biocompatibility test
In this study, the fibroblast cells were used for evaluation because of their involvement in
the wound healing [21]. The number of fibroblasts in different environments was shown in
Figure 5A. The number of fibroblasts on the nCur-CP sample was greater than all other
environments. This could be caused by the presence of small amount of curcumin released into
media culture, which could contribute to increase proliferation of fibroblasts [22]. In addition,
Figure 5B also confirms that the chitosan hydrogel is well-biocompatible as shown that most of
Dinh Van Tuan et al.
600
cells are alive. For the fluorescent staining, living cells are stained with green color and dead
cells with red color.
Figure 5. Effect of culture environment on cell proliferation (A) and live/dead staining of cell incubated in
the chitosan hydrogel (B).
3.3. In vivo evaluation of third burn wound healing in mice model
Third degree burn model was performed following the protocol at the University of
Science. Examination of healing rate and wound contraction were based on the rate of closed
wound and histological staining (H&E), respectively. The calculation of closed wound was
applied following the formula of Lyman et al, as below:
S =
In that: S_ is area of the wound; R _ width of the wound and D _length of the wound.
Figure 6. Digital photographical images of morphological wounds (A), and wound contraction of
treated and non-treated models (B).
After 7 days burned, all three treated groups were found to be not different by the external
morphology. From day 7 to day 12, the morphology of 3 treatment groups was changed. The
Nanocurcumin and chitosan-pluronic F127-based hydrogel for 3
rd
degree burn treatment
601
area of the wound had significant changes, especially in group treatment by nCur-CP that
decreased from 82.2 ± 17.35 mm
2
to 12.2 ± 4.9 mm
2
(p < 0.05). After 20 days of Silvirin and
nCur-CP treatment, wounds were healed completely. The new blood vessels were appeared in
the nCur-CP treatment group while less in the group treated with Silvirin. Epithelial formation
process in treatment group with nCur-CP was faster than that treated with Silvirin (p < 0.05). In
addition, the appearance of the collagen fibers in the nCur-CP treatment group was earlier than
treatment group with Silvirin. Growth of hair follicle in the nCur-CP-treated wound was more
than that of Silviri treatment. Besides, the epidermis layer of the nCur-CP and Silvirin-treated
models were thinner than the non-treated group, and the skin became normal (Figure 6A) as for
both morphology and histology. From the above results it can be concluded that the burn
wounds treated with nCur-CP had better efficiency than the other groups.
Figure 7. Histological examination of the healing process at different treatment groups (7, 14 and 22 days
post wounding). All the images were observed at 10x magnification and scale bar = 500. Black arrows and
red arrows used to point out the crush and re-epithelialized layer, respectively.
4. CONCLUSION
Injectable nanocurcumin formulated chitosan-g-pluronic hydrogel based on curcumin and
thermosensitive pluronic F127-grafted chitosan copolymers were prepared and efficiency-
evaluated on the 3
rd
-degree burn model. In addition, assessments of biocompatibility,
antibacterial activity and treatment efficacy on 3
rd
-degree burn model were performed. The
results have shown the positive efficiency of the nCur-CP nanocomposite in healing 3rd burn
injury. The material has great potential and is useful in human health care.
Acknowledgement. This work was financially supported by Vietnam Academy of Science and
Technology (VAST) under Grant Number VAST03.08/17-18.
Dinh Van Tuan et al.
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