TYPE
Original Research
27 December 2023
10.3389/fpubh.2023.1238961
PUBLISHED
DOI
OPEN ACCESS
EDITED BY
Pradeep Kumar,
University of the Witwatersrand, South Africa
REVIEWED BY
Deni Rahmat,
Pancasila University, Indonesia
Surapaneni Krishna Mohan,
Panimalar Medical College Hospital and
Research Institute, India
Pranay Punj Pankaj,
Nagaland University, India
*CORRESPONDENCE
Md Habban Akhter
habban.akhter@dituniversity.edu.in
Mariusz Jaremko
mariusz.jaremko@kaust.edu.sa
RECEIVED 12
June 2023
November 2023
PUBLISHED 27 December 2023
ACCEPTED 15
CITATION
Akhter MH, Al-Keridis LA, Saeed M,
Khalilullah H, Rab SO, Aljadaan AM,
Rahman MA, Jaremko M, Emwas A-H,
Ahmad S, Alam N, Ali MS, Khan G and
Afzal O (2023) Enhanced drug delivery and
wound healing potential of berberine-loaded
chitosan–alginate nanocomposite gel:
characterization and in vivo assessment.
Front. Public Health 11:1238961.
doi: 10.3389/fpubh.2023.1238961
COPYRIGHT
© 2023 Akhter, Al-Keridis, Saeed, Khalilullah,
Rab, Aljadaan, Rahman, Jaremko, Emwas,
Ahmad, Alam, Ali, Khan and Afzal. This is an
open-access article distributed under the terms
of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction
in other forums is permitted, provided the
original author(s) and the copyright owner(s)
are credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted which
does not comply with these terms.
Frontiers in Public Health
Enhanced drug delivery and
wound healing potential of
berberine-loaded chitosan–
alginate nanocomposite gel:
characterization and in vivo
assessment
Md Habban Akhter 1*, Lamya Ahmad Al-Keridis 2, Mohd Saeed 3,
Habibullah Khalilullah 4, Safia Obaidur Rab 5, Adel M. Aljadaan 6,7,
Mohammad Akhlaquer Rahman 8, Mariusz Jaremko 9*,
Abdul-Hamid Emwas 10, Sarfaraz Ahmad 11, Nawazish Alam 11,
Md Sajid Ali 12, Gyas Khan 13 and Obaid Afzal 14
1
School of Pharmaceutical and Population Health Informatics (SoPPHI), DIT University, Dehradun, India,
Department of Biology, Faculty of Science, Princess Nourah Bint Abdulrahman University, Riyadh,
Saudi Arabia, 3 Department of Biology, College of Science, University of Hail, Hail, Saudi Arabia,
4
Department of Pharmaceutical Chemistry and Pharmacognosy, Unaizah College of Pharmacy Qassim
University, Unaizah, Saudi Arabia, 5 Department of Clinical Laboratory Sciences, College of Applied
Medical Sciences, King Khalid University, Abha, Saudi Arabia, 6 Department of Pharmacology, College of
Pharmacy, Najran University, Najran, Saudi Arabia, 7 University of Nottingham Graduate Entry Medicine,
Royal Derby Hospital, Nottingham, United Kingdom, 8 Department of Pharmaceutics and Industrial
Pharmacy, College of Pharmacy, Taif University, Taif, Saudi Arabia, 9 Division of Biological and
Environmental Sciences and Engineering (BESE), King Abdullah University of Science and Technology
(KAUST), Thuwal, Saudi Arabia, 10 Core Labs, King Abdullah University of Science and Technology
(KAUST), Thuwal, Saudi Arabia, 11 Department of Clinical Pharmacy Practice, College of Pharmacy, Jazan
University, Jazan, Saudi Arabia, 12 Department of Pharmaceutics, College of Pharmacy, Jazan University,
Jazan, Saudi Arabia, 13 Department of Pharmacology and Toxicology, College of Pharmacy, Jazan
University, Jazan, Saudi Arabia, 14 Department of Pharmaceutical Chemistry, College of Pharmacy,
Prince Sattam Bin Abdulaziz University, Al-Kharj, Saudi Arabia
2
Berberine–encapsulated polyelectrolyte nanocomposite (BR–PolyET–NC) gel
was developed as a long-acting improved wound healing therapy. BR–PolyET–
NC was developed using an ionic gelation/complexation method and thereafter
loaded into Carbopol gel. Formulation was optimized using Design-Expert®
software implementing a three-level, three-factor Box Behnken design (BBD).
The concentrations of polymers, namely, chitosan and alginate, and calcium
chloride were investigated based on particle size and %EE. Moreover, formulation
characterized in vitro for biopharmaceutical performances and their wound healing
potency was evaluated in vivo in adult BALB/c mice. The particle distribution
analysis showed a nanocomposite size of 71 ± 3.5 nm, polydispersity index (PDI)
of 0.45, ζ–potential of +22 mV, BR entrapment of 91 ± 1.6%, and loading efficiency
of 12.5 ± 0.91%. Percentage drug release was recorded as 89.50 ± 6.9% with pH
6.8, thereby simulating the wound microenvironment. The in vitro investigation
of the nanocomposite gel revealed uniform consistency, well spreadability, and
extrudability, which are ideal for topical wound use. The analytical estimation
executed using FT-IR, DSC, and X-ray diffraction (XRD) indicated successful
formulation with no drug excipients and without the amorphous state. The colony
count of microbes was greatly reduced in the BR–PolyET–NC treated group on
the 15th day from up to 6 CFU compared to 20 CFU observed in the BR gel treated
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10.3389/fpubh.2023.1238961
group. The numbers of monocytes and lymphocytes counts were significantly
reduced following healing progression, which reached to a peak level and vanished
on the 15th day. The observed experimental characterization and in vivo study
indicated the effectiveness of the developed BR–PolyET–NC gel toward wound
closure and healing process, and it was found that >99% of the wound closed by
15th day, stimulated via various anti-inflammatory and angiogenic factors.
KEYWORDS
wound healing, polymer, nanomedicine, nanotechnology, chitosan, alginate,
nanocomposite, berberine
1 Introduction
efficacy (7, 8). Furthermore, passively targeted nanocarriers enable the
increase in drug concentration for a longer duration, thereby
maintaining drug concentration in the blood for the therapeutic
window to obtain efficient recovery of the wound surface (9–11).
Berberine (BR) is an isoquinoline alkaloid extract obtained from
the rhizomes of plants such as Phellodendron amurense, Berberis
aristata, and species of Coptis (12). A considerable increase in interest
in exploring the medicinal alkaloid has been observed worldwide due
to their potential biological activity. Previous literature reported that
berberine may exhibit anti-inflammatory (13), anti-oxidant (14),
anticancer (15), anti-microbial (16), anti-diabetic (17), antihypertension (18), antiviral (19) properties and may be used for the
treatment of mood disorders (20). The naturally existing hydrophobic
feature and limited aqueous solubility raise questions about BR’s
effectiveness (20). However, several attempts have been made in the
past to enhance their dissolution and oral bioavailability, such as
polysaccharide-based nanoparticles for enhanced oral bioavailability
(21), berberine liposomes for oral delivery (22) and cardiac therapy
(23), chitosan-layered nanoliposome for oral administration (24), and
pharmacokinetic evaluation and hypoglycemic effect of berberine
loaded with solid lipid nanoparticles (25). The anti-inflammatory and
anti-oxidative properties of benzylisoquinoline alkaloide berberine
(BR) is regulated by many molecular pathways, including the
inhibition of the mitogen-activated protein kinase (MAPK) signaling
pathways (26). Berberine has shown anti-inflammatory properties
both in vitro and in vivo. It suppresses the gene transcriptions
associated with interleukin-1, interleukin-6, and tumor necrosis
factor-α and reduces the concentration of inflammatory proteins.
Berberine also inhibits cyclooxygenases, thereby inhibiting
prostaglandin production (27).
The alginic acid forms a complex with chitosan via electrostatic
attraction of the chitosan molecules via its γ-carboxyl moiety. The
chitosan–alginate complex protects encapsulated drug molecules from
biodegradation in the biological system (28). The chitosan–alginate
complex enhanced the rate of BR dissolution and the permeability of
BR. The process of ionic polymerization accelerated when brought in
contact with calcium ions through binding with alginate guluronic
residues, resulting in the formation of polyanionic nanoparticles (29).
Several studies reported the use of chitosan as a natural polymer in
wound healing application (30–33). Nanoparticles have been used in
nano therapy as a carrier for the delivery of a large number of
therapeutic regimens for several diseases over the past decade, and
their substantial use in the modern therapeutic system owing to
overwhelming responses based on tissue selectivity and specificity,
Approximately 4.5 million individuals in the US alone undergo
medical care for chronic wound and an estimated cost of nearly USD$
25 billion is spent every year in the management of chronic wound
therapy. However, the impediment due to chronic wounds is growing
day by day with every increase in the incidence of diabetes and
obesity (1).
Skin is the largest organ of the body, and it acts as a protective
barrier for internal organs against threats of environmental hazards.
The excise area or injuries over the skin surface can be healed after it
goes through several physiological and biological upshots.
Physicochemical features of the wound environment, such as, pH and
temperature, may vacillate with the level of inflammation, the level of
microbial infection, and aeration to the wound area (2). The normal
temperature of wound area varies from 32°C to 34°C or higher
depending on the level of inflammation. Post-excising of the skin,
temporary pH was reported to be 7, and this pH may decrease to
acidic levels of 4 throughout the stages of healing. The decrease in pH
also depends on the level of microbial infection (3). Wound healing
generally restores the damaged tissues through a plethora of changes
involving, intially, the phase of hemostasis that occurs post-injury,
followed by the phases of inflammation, proliferation, and remodeling
of tissues. Following hemostasis, fibrin triggers clot formation in the
wound matrix and platelets start releasing several growth factors to
initiate the healing process. The inflammation phase starts within 24 h
and may last upto 2 weeks in normal injury or longer in case of chronic
wound. Macrophages, neutrophils, and monocytes are key cells in the
inflammation phase. These cells act as phagocytes to clean the cell
debris and further release pro-inflammatory mediators that involve
fibroblast formation and re-epithelization of the wounds (4).
Subsequent to this stage, a proliferative phase involving the synthesis
of fibroblast cells, collagens, and the extracellular matrix and, finally,
a remodeling phase triggering the basic alteration in collagen
organization, replacement of scar tissues with an organized normal
extracellular matrix occur. Besides these two phases, other factors
affect wound healing such as oxygen supply to the growing tissue, age,
degree of infection, cytokines, nutrition, hormones, production of
ECM, and enzyme proteases (5, 6). Due to the dynamic characteristics
of the wound environment, the controlled drug release via an active
targeted drug delivery system is an efficient treatment option to
be explored over the traditional hypodermic injection approach,
which enable an increase in drug concentration outside the above
therapeutic indications and tends to cause side effects and reduce
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improving drug concentration to the target, and minimizing off-target
delivery is notable (34).
reduced particle size and PDI. The ANOVA test was performed to
validate polynomial equations generated by the Design-Expert
software, which was validated using the ANOVA test. All collected
responses were provided from the use of the Design-Expert software.
Various possibilities in the experimental run were explored to obtain
the optimum composition of the formulation BR-PolyET-NC. The
Design-Expert software generated 3D surface morphology, contour
plots, and the predicted-to-observe response of selected dependent
variables to demonstrate the significant impact of excipients under use
on dependent variables of the formulation.
2 Materials and methods
2.1 Materials
Berberine (BR) was purchased from Sisco Research Laboratories,
which also (Mumbai, India) provided sodium alginate and chitosan
(MW–50–190 KDa, 85% deacetylation). Various analytical grade
reagents such as HPLC water, solvents, and other chemicals were used
in the analysis. The components of the solution used for buffer
preparation/phosphate saline buffer (PBS) were of analytical grades.
The Central Drug House Pvt. Ltd. (CDH, New Delhi) provided
sodium dihydrogen phosphate, potassium dihydrogen phosphate,
sodium hydroxide, disodium hydrogen phosphate, glacial acetic acid,
dichloromethane, acetonitrile, and ethanol.
2.3 Preparation of
BR-loaded-nanocomposite
A nanocomposite of BR-loaded alginate–chitosan nanoparticles
(BR–PolyET–NC) was prepared using the ionic gelation technique
modifying the preceding study (39). A solution of sodium alginate
(10 mL) was first prepared in deionized water (30 mg/mL) and a pH of
5.2 ± 0.02 was maintained. The BR (10 mg) vortex was dissolved in 96%
ethanol and then incorporated dropwise into the alginate solution,
ensuing the preparation of a homogeneous pregel solution. Then, 5 mL
of an aqueous calcium chloride solution was supplied dropwise to the
alginate solution and stirred continuously using a magnetic plate at
1,000 rpm for 30 min, leading to the formation of the alginate complex
gel. Chitosan (50 mg/mL) was separately thawed in 1% v/v acetic acid;
then, 10 mL of this solution was imparted in the alginate solution
dropwise and stirred using a magnetic plate thereafter, following which
the plate was sonicated for 10 min. Then, the chitosan-coated NPs were
harvested after centrifugation at 15,000 rpm for 40 min and finally
obtained the alginate–chitosan complex (Alg–Ch complex), which was
lyophilized for characterization (39).
2.2 Optimization using Design-Expert
®
Using the BBD in Design-Expert software (Version 10; Stat-Ease
Inc., Minneapolis, Minnesota), we generated 17 formulations for
experiments implementing three levels and three factors for
optimizing the formulations (35–37). The levels of independent
factors were decided considering the outcomes of the preliminary test
in the laboratory, which was classified into minimum level (−1),
intermediate level (0), and higher level (+1), as summarized in the
Table 1. Chitosan (X1), sodium alginate (X2), and calcium chloride
(X3) as independent factors were 40 mg, 60 mg, and 80 mg; 20 mg, 30
mg, and 40 mg; and 20 mg, 30 mg, and 60 mg, respectively
corresponding to the levels (−1), (0), and (+1). Using the BBD in
Design-Expert software, 17 formulations were generated, which
underwent an extensive characterization process for the responses PS
(y1) and entrapment efficiency (y2). The details of the obtained
response analysis of individual factors are shown in Table 2. The
details of the individual and interaction effects of the input attributes
on the response were sorted out. This design was often helpful in
sorting the problem of selecting three or more independent variables
as the analysis requires an elite group of experimental runs than what
is required for the central composite design (38). The primary aim of
using the Design-Expert software was to optimize the formulation and
make it stable and robust by enchancing improved entrapment
efficiency, drug loading, and higher drug release concomitantly with
®
2.4 Nanocomposite characterization
2.4.1 Particle size, polydispersity index, and
surface charge
The particle size of synthesized BR–PolyET–NC was measured
using Malvern Zetasizer instrument (Nano ZSP, Malvern Instruments,
Worcestershire, United Kingdom). The working of the instrument was
based on the dynamic light scattering (DLS) technique. When a beam
of light passed through the particle of the samples, it scattered due to
the Brownian movement or the zigzag motion of sample particles. The
scattering phenomenon was observed at a scattering angle of 90° at
room temperature. The NPs were extracted from the gel formulation
by diluting in Milli-Q grade water at a ratio of 1:100, vortexed, and
sonicated to reach homogeneous consistency. The particle size was
measured in triplicate (n = 3). The surface charge on the BR–Ach–NC
was also measured.
TABLE 1 The formulation variables and the levels employed as low (0),
medium (−1), and high (+1) in optimizing BR–PolyET–NC.
Level employed
Independent variables
X1: Chitosan (mg)
Low (−1)
Medium (0)
High (+1)
40
60
80
X2: Sodium alginate (mg)
20
30
40
X3: Calcium chloride (mg)
30
45
60
2.4.2 Percentage BR entrapment and loading
Dependent variables
Y1: Particle size (nm)
Maximize
Y2: Entrapment efficiency
Maximize
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The BR-loaded nanocomposite, BR–PolyET–NC, was centrifuged
at 12,000 rpm for 20 min. The aliquot of suspension was pulled out
and analyzed at λmax of 422 nm in the UV spectrophotometer for
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TABLE 2 The different experimental run (1–17) predicted by in the BBD for responses Y1 and Y2 for the optimization of BR–PolyET–NC.
Run
Factor 1 = X1
Factor 2 = X2
Factor 3 = X3
Response = Y 1
Response = Y2
A:Chitosan
B:Sodium alginate
C:Calcium chloride
Particle size
Entrapment efficiency
mg
mg
Mg
nm
%
1
80
30
60
120
60
2
60
20
30
70
69
3
40
20
45
90
67
4
40
30
60
85
60
5
60
20
60
60
68
6
80
30
30
120
60
7
60
30
45
65
88
8
80
40
45
140
60
9
80
20
45
90
69
10
60
40
28
90
60
11
40
40
45
80
65
12
60
30
45
62
87
13
40
30
30
90
60
14
60
30
45
59
89
15
60
40
60
85
60
16
60
30
45
70
87
17
60
30
45
62
89
A, chitosan (mg); B, sodium alginate (mg); X3, calcium chloride (mg); Y1, particle size (nm); Y2, drug encapsulation.
free BR concentration. The amount of BR entrapped and BR loading
in the nanocomposite was determined using equations given below.
%EE =
(Total BR added − Total BR in aliquot ) × 100
(3)
%DL =
(Total BR added − Total BR in aliquot ) ×100
(4)
Total BR added
Total weight of nanocomposites
the grid was coated with phosphotungstic acid. Finally, the developed
sample was examined under the microscope.
2.4.5 Percentage drug release and kinetic study
BR release from the BR–PolyET–NC nanocmoposite was carried
out in simulated intestinal fluid (SIF) along with PBS with pH of 6.8
and then compared with BR dispersion. Then, the study
nanocomposite was passed through the activated dialysis membrane
in PBS. The measured quantity of BR–PolyET–NC was 10 mg, and the
BR gel was enwrapped in a dialysis bag with a PBS volume of 95 mL
(MW; 8–12 kDa; Repligen, Waltham, MA, United States) with its ends
closed and transferred into dissolution medium. The dissolution
medium was maintained at a physiological temperature of 37 ± 0.5°C
with uninterrupted stirring (40, 41). The requisite sample volume of
1 mL in various time frames, that is, 0, 8, 16, 24, 32, 40, and 48 h, were
withdrawn, and the exact volume of fresh buffer was put back in
dissolution medium. The collected samples were suitably treated (i.e.,
filtered and diluted) and quantified using UV-spectrophotometry. BR
release from the carrier was interpreted through plotting data between
%drug release versus time. Furthermore, the incurred drug release
data fitted into various mathematical models, and a good model fit for
BR release from the nanocomposite was predicted. The mathematical
equations expressing different models are shown below.
2.4.3 Nanocomposite FT-IR and x-ray
diffractometer
A total of 5–10 mg of the BR–PolyET–NC sample was used for
FTIR spectral analysis (BRUKER Corporation, Billerica, MA,
United States). The sample was kept in contact with a beam of light
that emerged from the FTIR instrument, and the sample was scanned
over the wave number range of 4,000–500 cm−1. XRD was performed
for BR, alginate, chitosan, and physical mixture and optimized gel
applying diffractometer (PANalytical X’pert PRO, Netherland). The
measures of electric current from the x-ray tube of the XRD were
40 kV and 100 mA during the process of Cu Kb reduction using a
nickel-filter. The sample scanned at 2θ angle ranged from 10 to 50°, at
a speed limit of 10°/min.
Mt / Mo = K × t → Zero order
(5)
M
or , ln
= K × t → Zero order
Mo
(6)
2.4.4 Transmission electron microscopy
Transmission electron microscope further evaluated the particle
size with a TEM instrument (Techni TEM 200 Kv, Fei, Electron
optics). For electron microsope examination, a drop of the diluted
sample was disseminated onto the copper grid (carbon-coated) and
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1
Mt
= K × t → Higuchi model
2
Mo
(7)
Mt
= K × tn → Korsemeyer − Peppas
Mo
(8)
2.4.7.3 Surface morphology
Nanocomposite gel was spread on an aluminum stub, air-dried,
then gold coated, and finally observed under a JSM 6100-Digital
Scanning Electron Microscope (JEOL, Ltd. Tokyo, Japan).
2.5 In vivo studies
where, K represents a kinetic rate constant, Mt./Mo indicates the
fraction of drug released at time t, and n is the diffusion exponent. The
n-value expresses the mechanism of drug release, herein, n ≤ 0.5
(Fickian diffusion), 0.5 < n < 1.0 (Anomalous, non-Fickian transport),
and n = 1.0 (relaxation Case-II).
For this study, BALB/c rats weighing between 200 and 230 g were
procured. The studies were performed in ethical compliance to the
research guidelines approved by the Institutional Animal Ethics
Committee (IAEC) of DIT University (Dehradun, Uttrakhand, India
under ref. no. DITU/IAEC/22/04/28) dated 26 September 2022. The
animals were placed inside polypropylene cages that were kept under
12-h light/dark cycles at ambient temperature and relative humidity
was maintained. Animals were categorized into four groups (n = 4).
Surgical incisions were made on the rat skin. On the day following
surgical incision, treatment protocol was followed with the BR–
PolyET–NC gel, and BR dispersion occurred topically twice a day
until the 15th day, which may be compared either to untreated
control or negative control. On completion of the treatment protocol,
animals were euthanized, and sections of skin tissues were stored in
10% formalin at −20°C for histopathological investigation.
2.4.6 Fabrication of nanocomposite gel
The BR-loaded nanocomposite, i.e., BR–PolyET–NC gel, was
developed by compounding and mixing using a propeller-type mixer
1% BR–PolyET–NC in previously dissolved 1% Carbopol 940 in
distilled water, which is followed by the addition of 1% propylene
glycol and preservative, 0.01% methyl paraben, and, thereafter, few
drops of triethanolamine to the mixture. The mixing process was
continued until a clear, transparent gel has not been formed, and its
pH was adjusted to make it compatible with the physiological skin
pH. The development of the chitosan–alginate nanocomposite gel
using polymer Carbopol 940 grades was carried out in accordance
with our previously reported study with some modifications (36).
2.5.1 Macroscopic wound area closure
measurement
2.4.7 Gel characterization
Wound contraction percentage (%wound contraction ) was
determined after measuring the diameter with a scale and, thereafter,
using the following formula:
2.4.7.1 Organoleptic features, drug content, viscosity, and pH
The BR–PolyET–NC gel was examined visually for colour, odor,
and taste. A digital meter was employed for measuring the pH of the
gel (Thermo Scientific, Waltham, MA, United States). Gel pH was
measured in triplicate (n = 3). The drug content can be expressed as
the percentage of drug showcased in gel preparation. It is the ratio of
the drug in the nanocomposite to the true value of the drug loaded
on the gel. The viscosity profile of the developed gel was determined
by applying the shear rate (1/s).
Wound diameter on 5th day
− Wound diameter on 15th day
%Wound contraction =
× 100 (10)
Wound diameter on 5th day
2.5.2 Microbial assessment
The swabs excluded from the wound part of the skin on every 5th,
10th, and 15th days of wound dressing change. Using the serial dilution
technique for quantitative investigation of bacterial colon count, swabs
were diluted by 10-fold using normal saline. A total of 500 μL of each
diluted specimen was kept on 2% agar medium spread on petri plates
and incubated at physiological temperature for 24 h. After incubation
time, the colony count of bacterial generation was investigated.
2.4.7.2 Gel homogeneity, spreadability and extrudability
The homogeneity of the developed gel was investigated via visual
inspection by placing the gel in a clear glass beaker kept in a fixed
position. The spreadability of the gel was tested by sandwiching 0.5 g
of the gel in between two glass plates, and the gel spread to a diameter
of 1 cm. A weight box of 0.5 kg was then set above the plate for 5 min,
and, thereafter, spreading of the gel under the influence of weight was
ascertained. The spreading area of the gel was determined using a
Vernier caliper (n = 3). The limit of the gel’s spreadability was
considered to be 2 cm2 for topical application (42). An aluminum
collapsible tube was completely filled with 15 g of the gel from the
bottom with care to prevent the entrapment of air. After filling, the
bottom side was folded in triplicate and then compressed under a
crimping machine, and then a plastic cap was wrapped on the top side
of the tube and sealed. The process of extrusion was commenced after
removing the sealed cap from the tube followed by a gentle pressing
of the tube. Gel extrudability was determined using following formula.
Extrudability =
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Weight applied to the tube
Area ( cm 2 )
2.5.3 Preparation of wound tissue specimen
On 5th, 10th, and 15th days, mice from different groups were
captured after removing the dead tissues from the wound site, and
these tissues were taken for microscopic analysis to investigate the
pattern of re-epithelization, fibrogenesis, and collagen formation.
2.5.4 Histopathological analysis
For this analysis, after euthanizing the animals, the wounded area
was carefully removed and rinsed with saline water, and the skin tissue
was dissected and preserved in formalin (10%). Thereafter, sections of
approximately 5 μm thickness were paraffinized using a microtome
with prior treatment with xylene and ethanol. Then, the specimens
were treated to remove a paraffin layer, hydrated, and stained using
hematoxylin and eosin (43). The prepared slides were examined by a
(9)
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pathologist who was blindled to the origin of the specimens using a
CX31 microscope (Olympus Corporation, Tokyo, Japan) connected
with a NanoZoomer-SQ Digital slide scanner C13140-01 (Hamamatsu
Photonics, Shizuoka, Japan).
variable and factors, namely, particle size and % EE, are
displayed below.
Particle size = +63.57 + 15.63 × X 1 + 10.33 × X 2 − 2.23 × X 3
+ 15.00 × X 1 × X 2 + 1.25 × X 1 × X 3 + 1.79 ×
X 2 × X 3 + 32.24 × X 12 + 4.15 × X 22 + 7.99 × X 32
(1)
2.6 Stability studies
%EE = +87.99 − 0.375 × X1 − 3.75 × X 2 − 0.606 × X3
− 1.75 × X1 × X 2 − 0.711 × X 2 × X3 − 13.98
× X12 − 8.78 × X 22 − 14.00 × X32
The developed optimized gel preparation in a glass container well
covered with aluminum foil was subjected to stability tests under
refrigerated condition (5 ± 3°C) and at elevated temperature
(40 ± 2°C/75 ± 5% RH) in stability chamber as per International
Conference on Harmonization (ICH) guidelines. The formulation
was inspected at regular intervals on days 0, 30, and 60 for specific
physico-chemical changes such as particle size, PDI, pH, viscosity,
particle size, PDI, spreadability, and extrudability. In addition,
physical appearence features such as clarity, turbidity, and phase
separation were also inspected visually.
(2)
The quadratic equation (1) shows a positive impact of chitosan and
sodium alginate on particle size. The particle size ranges between 58 to
140 nm, indicating a uniform and narrow distribution. The smallest
particle size obtained was 58 nm at a chitosan concentration of 60 mg
and the highest particle size obtained was 140 nm at a chitosan
concentration of 80 mg. Increasing chitosan concentration may increase
the bulk of the polymer in the formulation, resulting in bigger particles
(44). The high viscosity of the formulation may lead to binding to the
alginate gel matrix that result in increased particle size (45).
Furthermore, sodium alginate had a positive effect on the particle size
due to the formation of a complex with positive charges bearing
chitosan. In an aqueous sodium alginate solution, calcium ion replaces
sodium ion to form calcium alginate, thereby forming a calcium alginate
complex. Cross-linker Ca2+ ions provide elasticity and, thus, stabilize the
nanocarrier (46). Ahdyani et al. pointed out that raising Ca2+ ion
concentration in chitosan-alginate formulation led to their reduction in
chitosan-alginate NPs (39). In addition to the individual effect, the
combined effect of chitosan and alginate was positive on particle size.
3D response surface curve and 2D contour plots well explicated the
impact of independent variables on response particle size (Figure 1).
In terms of particle size, %EE is an important parameter under
consideration in the development of a successful nanoplatform. The
impact of an independent variable on %EE is comprehensively
explained in equation (2), with 3D response surface curve and
contour plot. The encapsulation of BR in various developed
formulation ranges from 60 to 89%. The formulation with the highest
chitosan concentration showed a slightly reduced entrapment of
BR. A high chitosan concentration might increase the viscosity of the
preparation solution, causing the barrier to deliver the BR inside the
matrix core of the polymer. Increasing the concentration of sodium
alginate in the preparation solution led to a slightly reduced %
EE. Zimet et al. prepared Nisaplin -entrapped alginate NPs through
the ionic gelation complexation technique. They observed reduced
%EE aside from an increase in particle size from 86 to 204 nm and a
zeta potential from −33.2 to −38.7 mV (47). The cross-linker Ca2+ ion
marginally reduced the %EE of the chitosan–alginate NPs, probably
due to being in contact with porous hydrophilic sodium alginate in
the aqueous medium (48).
2.7 Statistical analysis
The samples were analyzed using a one-way ANOVA plus the
Tukey–Kramer test using GraphPad Prism (version 7). The data were
represented in the form of mean ± SD (n = 3). The significance level
was considered at a value of p of <0.05.
3 Results and discussion
3.1 Optimization
The present study aimed to develop, characterize, and evaluate the
effectiveness of a BR-loaded chitosan–alginate polyanionic complex
gel in treating self-induced wound in rat model. Employing a threefactor, three-level Box Behnken design (BBD), the formulation was
optimized. The various levels of independent variables are shown in
Table 1. Response surface morphology and contour plots
demonstrating the influence of excipients on particle size and %EE are
shown in Figures 1A−E and Figures 1F−J. Contour plots and response
surface morphology demonstrating the influence of excipients on
particle size and %EE are shown in Figure 1. The quadratic model was
determined as the best-fitted model with a high coefficient of
correlation (R2) of approximately 1 among the other 2FI and linear
models while considering the significant influence of independent
variables on responses. A polynomial equation established according
to the best fit model guidelines well explained the different components
on independent variables combined with the quadratic effect on
dependent variables. Seventeen formulations were developed in the
BBD, accommodating five center points to advertently check for any
replicas (05) of these formulations (Table 2) (35).
®
3.3 Checkpoint analysis
3.2 Impact of X1, X2, and X3 on particle size
(Y1) and % EE (Y2)
The numerical optimization technique revealed the optimized
composition of BR formulation, restraining minimum particle size
(Y1) and maximum % EE. The desirability value close to 1, i.e., 0.987,
elaborates the stable and coherent nature of the formulation. The
The optimum relationship between chitosan concentration (X1),
sodium alginate (X2), and calcium chloride (X3) as independent
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FIGURE 1
Response surface curve (A–E) exploring the impact of independent variables on dependent variables in the preparation of NPs. Two dimensional
contour curve (F–J) exhibiting the influence independent variables have on dependent variables of NPs.
smaller the particle size, the large will be the surface area, thereby
contributing to better solubility and dissolution of the drug and,
hence, improving overall drug availability from the nanosystem in
the biological medium (36). The optimized formulation, BR–PolyET–
NC, comprised of a chitosan concentration of 58.8 mg, a sodium
alginate concentration of 27 mg, and a calcium chloride concentration
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of 45.27 mg. The model anticipated value for the responses particle
size and %EE were noted as 65 nm and 88%, respectively. The
experimental estimated value of the responses particle size and % EE
were 71 ± 3.5 nm and 91 ± 1.6%, respectively. Less variation in the
model anticipated value determine by software and experimental
value was observed, which was not statistically significantly different
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(p > 0.05), as expressed in Table 3. Furthermore, the surface charge on
the surface of the NPs was estimated to be +22 mV, indicating a stable
optimized formulation. Moreover, the low polydispersity index (PI)
shows a homogeneous and uniform and narrow distribution of
nanosized particles throughout the developed formulation (40).
consistent and unimodal. The PI value of <0.5 could be considered as
the monodispered system. The polydispersity index of formulation
was low, that is, 0.45, which suggested homogeneity and monodispered
nanosize system (Figure 2A). The surface charge on BR–PolyET–NC
was reported to be +22 mV using Malvern Zetasizer Nano ZSP. The
positive surface charge on the chitosan NPs was due to the presence
of the cationic functional group, and it provides the colloidal stability
to the NPs. The surface charges help to integrate the cell membrane in
vivo (49). The high percentage of BR entrapment (91 ± 1.6%) and
loading (12.5 ± 0.91%) was estimated.
3.4 Characterization of optimized
formulation
3.4.1 Nanoparticle characterization, BR
entrapment, and BR loading
3.4.2 Percentage drug release and kinetic model
The particle size of BR–PolyET–NC observed to be 71 ± 3.5 nm,
and the particle population in the formulation was uniform and
Percentage release of BR from BR–PolyET–NC and BR-dispersion
at predefined time points has been investigated, as shown in Figure 3.
TABLE 3 Independent variables, estimated value, and model predicted value of optimized the BR–PolyET–NC gel.
Independent
variables
Response
variables
Optimized
formula
Estimated value
of responses
Predicted
response
Anticipated error†
X1: Chitosan (mg)
Particle size (nm)
58.5 mg
71 ± 3.5 nm
65 nm
8.4
X2: Sodium alginate (mg)
EE (%)
27.0 mg
91 ± 1.6%
88%
3.29
X3: Calcium chloride (mg)
-
45.27 mg
-
-
-
†Calculated as (estimated value – predicted value/predicted value)/estimated value × 100.
A
105
90
Intensity (%)
75
60
45
30
15
0
0
25
50
75
Particle size, nm
B
100
125
C
FIGURE 2
Histogram showing particle size distribution curve (A); TEM image of BR–PolyET–NC (B); SEM image of BR–PolyET–NC gel (C).
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nanocomposite complex was enhanced by aqueous penetration into
the polyelectrolyte complex, causing gel swelling, matrix erosion, and,
therefore, drug diffusion into the circumferential medium.
Additionally, drug release from the nanocomposite also relies on pH
of the dissolution medium, physicochemical properties of the drug
and nanocarriers, and nanocarrier interaction with the biological
system at the wound surface (41).
The BR release of the nanocomposite was fitted to kinetic models
of first order, and Higuchi, Korsmeyer–Peppas, and Hixson–Crowell
were screened out as the best models of good fit. The coefficient of
correlation (R2) of the selected kinetic model was estimated, and it
indicatded that the model of good fit for BR release from the
nanocomposite was Korsmeyer–Peppas, with highest value of R2
being 0.9632. Furthermore, the n exponent value was determined to
be 0.398 (0.5 < n < 1), revealing Fickian diffusion of BR from the
polymeric composite, BR–PolyET–NC (52).
FIGURE 3
Percentage of BR release from BR–PolyET–NC and BR-dispersion at
pH 6.8.
3.4.3 Nanocomposite XRD and FTIR spectral
analysis
The drug release characteristics follows the biphasic pattern, where it
appears that an abrupt release of the drug ahead of time abide by the
sustained release model from the nanocomposite mentioned at the
end of the study. In the initial 2 h of injection, a fast drug release was
noticed, followed by a slow and sustained release in a controlled way
for a period of 72 h. The burst release was due to adsorbed or weakly
held drug particle on to theNP surface, and BR-entrapment and
dispersion were estimated to be 38 ± 5.19% and 13 ± 3% in the initial
2 h at room temperature in pH 6.8. The poor release of BR from BR
dispersion may be ascribed to the hydrophobicity of the drug, resulting
in low aqueous solubility and dissolution. The maximum BR release
from the nanocomposite after the completion of 72 h was observed to
be 89.50 ± 6.9% at pH 6.8 compared to 46 ± 10% the BR release from
BR dispersion. Overall, % cumulative release from BR–PolyET–NC
was apparently high over drug dispersion. A higher release from the
nanocomposite may be attributed to the straightaway dissolution and
then drug diffusion at pH 6.8. The dissolution study reported herein is
in concurrence with previous studies in the literature (50, 51).
The polysaccharide nanocomposite of alginate–chitosan, bearing
Contrary surface charge or opposite surface charge, has been widely
explored in drug delivery and biomedical application (52). Owing to
the swelling property of polysaccharides, chitosan is more repulsive in
an environment of positive charge, which thereby initiates the
movement of positive ions from chitosan and negative ions from
alginate in their combined complexes with calcium, with Ca2+ ion
triggering improved drug release. The PolyNC formed primarily due
to the ionic interaction of chitosan with alginate in the presence of
divalent cations encapsulate protection of the surface by reducing
pores in the complex and thus enhancing the controlled release of
BR. The prompt solubility of chitosan at slightly acidic pH and
contrarily high solubility of alginate at neutral pH in the
nanocomposite concomitantly led to the sustained release of BR,
which is an ideal feature for improved therapy at the wound site.
Despite the good sustainability of BR through the gel system, the
bioadhesive nature of chitosan prolonged the abidance of BR at the
wound site, minimized rapid clearance of the drug, and thereby
improved the efficacy of BR at the target (44, 53). The alginate fraction
of the complexes showed sustained release and improved longeracting BR release. Furthermore, BR release from the alginate–chitosan
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XRD is an important analytical parameter to illustrate the molecular
state of drug encapsulated in the nanoparticles. The physical state of the
drug indicates the extent of solubility, dissolution, absorption and thus
reflects the bioavailability of the drug from the formulation. The XRD
peaks of BR-loaded nanocomposite clarified that some of the berberine
peaks could be noticed in XRD of the nanocomposite (Figure 4A).
These peaks were observed in reduced intensity at 2Θ of 14.81, 21.26°,
23.54° and 29.72°. Based on the diffraction pattern of the compound,
BR has been greatly reduced in the nanocomposite, stating the
amorphization or conversion into molecular BR in the formulation (37,
54, 55). All the characteristics peaks of berberine appeared in the
formulation, although their intensity slashed to zero level or reduced,
which affirmed the encapsulation of the alcohol and carboxylate groups
or other groups of the compound berberine in the chitosan–alginate
nanocomposite. Although some reduced characteristic peaks appeared
at 2,293.36 cm−1 due to the C=C bond stretching, at 1,639.49 cm−1 due
to C=O stretching, at 1,346.31 cm−1 due to C–C stretching, and at
1,166.93 cm−1 due to N-H stretching. The peak shown at 1,037.70 cm−1
is due to C–O stretching (alcoholic and carboxylic) (Figure 4B). Thus,
the FTIR spectrum of nanocomposite substantiated the drug excipients’
stability in the formulation.
3.4.4 Measurements of particle size distribution
and electron microscopy
Particle size and their distribution in the bulk of nanosize
preparation are shown in Figure 2A. The TEM image shows that
particle size is uniform, and the spherical, scattered, and agreeable
size distribution was uniform and monodisperse with prior
investigation with Malvern Zetasizer. A TEM study revealed the
particle distribution was 71 ± 3.5 nm (Figure 2B). NP in this range is
assorted with antimicrobial activity at the wound site. The SEM image
revealed a spherical morphology of uniform consistency and was
non-porous (Figure 2C).
3.4.5 Characterization of BR–PolyET–NC gel
3.4.5.1 Organoleptic features, drug content and pH
The color of the gel appeared pale yellow, and the odor was
acceptable. Drug content in the gel was reported to be 99 ± 0.50%.
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FIGURE 4
(A) XRD, and (B) FTIR of berberine nanocomposite.
The high drug content in the gel indicated the reliable method of
high accuracy in gel preparation and that is desirable for
pharmaceutical gel. A digital pH meter measured the pH of the
developed gel to be 6.3. The ideal pH range of topical gel
preparation was 4.5–6.5. A higher pH value of the gel may set off
scaly skin, and low pH may set off dermatitis. The deviation in pH
of the gel may cause modification in the skin pH preceding the
adverse impact on the cutaneous barrier layer, dermal microflora,
and also the normal healing mechanism of skin (56). A
nanocomposite gel comprised of the gelling agent is Carbopol 940.
The current preparation used a Carbopol (an acrylic polymer)
concentration of 1%, which produced a transparent, consistent, and
stable preparation and showed better permeation or drug release.
Polyethylene glycol was used as a humectant and solubilizer.
Methylparaben was used as a preservative to prevent microbial
growth. Trithanolamine neutralized the caboxylic group from
carbomer and maintained the desired pH and it also acts as an
emulsifier and thickening agent.
FIGURE 5
Viscosity (Pa.s) versus shear rate (1/s) profile of BR-PolyET-NC gel.
3.5 In vivo wound healing activity
3.5.1 Macroscopic wound area closure
measurement
3.4.5.2 Viscosity of BR–PolyET–NC gel
BR–PolyET–NC gel homogeneity was inspected visually by
placing it in a settled position in a container. It was of uniform
consistency and homogeneous with no aggregates. The viscosity of
the BR–PolyET–NC gel was determined to be 9.23 Pa.s. Applying
the shear rate (1/s) indicating that the shear-thinning system means
the application of shear force, which led to decreased viscosity of
the gel system, which helped in easy application to the target site
(Figure 5).
The macroscopic features of various treated groups of animals
with BR–PolyET–NC gel, BR gel, blank gel, and untreated/positive
control for the wound healing progress and closure are shown in
Figure 6A. As shown in the Figure 6B, among the entire treated group
of the wound, the BR–PolyET–NC gel treated group had their wound
closed at approximately the 15th day. On 5th, 10th, and 15th days, the
wound closure percentage was reported to be 42 ± 5%; 85.55 ± 3.5%;
and 98.22 ± 1%, respectively, in the animals in the BR–PolyET–NC gel
treated group. Similarly, in the BR gel and blank gel treated groups,
wound closure percentage was determined to be 68 ± 9% and 55 ± 9%
on the final day of treatment. In the case of the control group
(untreated), only 35 ± 8% wound closure was measured on the 15th
day. The BR–PolyET–NC gel reduced the wound gap significantly
compared to the BR, blank, and control gels on every 5th, 10th and
15th days (Figure 6B).
It is believed that berberine exert the anti-inflammatory action in
wound healing process, which primarily relies on the signalling
pathway of NF-кB, that is, silent information regulator 1 (Sirt1). The
wound healing mechanism of berberine involves upregulation of the
NF-κB protein Sirt1 and thus lead to decreased expression of NF-κB,
inhibiting TNF-a and IL-6 expression and decreasing inflammatory
protein expression. The reduced inflammatory response altered the
wound microenvironement by increasing the expression of VEGF and
3.4.5.3 Gel homogeneity, spreadability and extrudability
The developed gel was visually observed to be homogeneous in
consistency when settled in a container. It was reported to be free of
any particle aggregates. The spreadability test was performed to
estimate the nature of flow of the gel, and it indicated good flowability
in the range of 5–7 cm. Gel spreadability was determined to
be 6.2 ± 0.12 cm, indicating better dissemination and suitability in
topical application. Good spreadability of the gel after application at
the wound or disease site ensures easy spread over an area and cover
of the whole wound in less time (~2 s) and thus helps in effective and
fast healing. Furthermore, the extrudability of the gel was measured
to be >90%, indicating excellent extrudability. Gel retention was
excellent without draining at the wound site after application and
thus ensures better adherence (57).
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****
**
P o s itiv e c o n tro l
**
80
*
60
***
ns
***
B la n k g e l
B R -g e l
(B R -P o ly -N C ) g e l
ns
*
40
ns
20
15
10
5
0
80
N e g a tiv e c o n tro l
70
B la n k g e l
60
B R -g e l
50
*
40
(B R -P o ly -N C ) g e l
*
30
*
20
10
0
15
% W o u n d c lo s u r e
100
**
10
C
****
5
B
C o l o n y f o r m in g u n i t s ( c f u ) /d i s h
A
Days
Days
FIGURE 6
(A) Time dependent (0, 5, 10, and 15 days) healing of the wound in Balb C mice following treatment with BR–PolyET–NC, BR gel, blank gel, and positive
control (untreated). (B) Wound closure measured on days 5, 10, and 15 after incision made on skin following treatment with BR–NP gel, BR gel, and
blank and positive control. (C) Microbial colony count at the wound site. Data were expressed as mean ± SD (mean ± SD = 3). Observations among the
group were statistically compared using a one-way ANOVA and Tukey’s multiple comparison test. The level of significance; *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001 when compared to different groups.
CD31 and eventually wound healing progressed. Sirt1 also directly
involves reversal of the inflammatory cells and anti-oxidant stress (58).
Berberine as a wound healing agent has been recommended by
various researchers. A few illustrative examples have been indicated
in this study. Panda et al. recently developed BR–loaded lecithin–
chitosan nanosystem for wound healing in diabetic rats. The findings
of these authors suggested that combining chitosan and berberine
together in a carrier gave synergistic effect toward wound healing.
These authors further observed that optimum formulation worked by
eliminating inflammatory cells and increasing mature collagen
fibers (44).
Yin et al. developed a berberine-decorated zinc oxide colloid
nanohydrogel (ZnO-Ber/H) for the wound healing activity in diabetic
rats. The developed gel had an excellent wound closure rate of 92.9%
by the end of the 15th day. The developed preparation ZnO-Ber/H
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downregulated inflammatory factors, upregulated vascular factors,
and improved the re-epithelization in the wound (59).
Zhang et al. developed BR nanohydrogel of alginate. The
molecular mechanism of healing relied on BR-mediated activation
of Sirt1, which, in turn, promotes wound healing by reducing
inflammatory factors and progressing angiogenesis (58). In addition,
Samadian et al. developed a berberine-loaded electrospun cellulose
acetate/gelatin mat in treating diabetic foot ulcers (60). The
nanofiber produced a mean diameter of 502 ± 150 nm. In vivo studies
disclosed that CA/Gel bandage dressing exhibited their antibacterial
potential and improved collagen density by 8.8 ± 6.7% and received
an angiogenesis score of 19.8 ± 3.8. Thus, an electrospun mat bearing
BR proved to be helpful in wound healing activity in animals.
Moreover, Amato et al. developed a nanogel consisted of BR,
hyaluronan, and poly-L-lysine (61). The results expressed that a
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nanogel encapsulating BR reduced the fibroblast gap after 42 h. Zhou
et al. proved that TrxR1 plays an important role in governing redox
homeostasis in different pathologic conditions. The BBR works on
TrxR1 was elucidated. BBR outstandingly promotes the synthesis of
the extracellular matrix and remarkably damages HaCaT cells in the
wound healing process. Furthermore, BBR activated TrxR1, leading
to the suppression of downstream JNK signal and preventing
oxidative stress and apoptosis, and thus raised cell proliferation,
growth factor, amd tissue inhibitors and later lowered matrix
metalloproteinase, thus promoting wound healing in diabetic rats
(62). Remarkably, we observed that, compared to the BR gel, the
BR–PolyET–NC gel enabled the replacement of the damaged tissues
at the wound site and complete reversal of the wound area with
normal tissues on the 15th day of treatment.
the number of neutrophils and macrophages significantly increased
on 4th day and then their number significantly decreased on 7th day
onwards for the entire group, but the decline was observed to higher
in the BR–PolyET–NC treated group. The inflammatory infiltrate
greatly reduced in the early phase in the BR–PolyET–NC treated
animals compared to the BR gel treated group and other groups.
Furthermore, an increased number of macrophages was observed
until 7th day, and thereafter, it decreased on 14th day in all the groups,
and the decrease was more pronounced in the BR–PolyET–NC treated
group than other groups. Moreover, a steep increase in fibroblasts and
fibrocytes was observed on 7th day, indicating re-epithelization, and
then a sharp decline on 14th day of treatment in the BR–PolyET–NC
treated group compared to other groups (63).
The epidermal layer of the control group shows evidence of
stern acanthosis and follicular hyperkeratosis, as depicted in
Figure 7. On the other hand, the dermal layer bearing an ample
number of fibers from the extracellular matrix such as collagen
fibers, disperse blood vessels, and large chronic inflammatory
infiltrates such as macrophages, lymphocytes, and B and T cells.
These characteristics of hispathological alteration skin tissues are a
clear indicative of chronic wound. In the BR–PolyET–NC gel treated
group, as shown in Figure 7, the epidermal, dermal, and hypodermal
tissues showed normal characteristics after treatment compared to
the control group.
Acanthosis and follicular hyperkeratosis were absent in the
epidermis layer. The dermal tissue bears bundle fibers of collagen,
muscle fibers, and skin appendages. Fatty tissues and blood vessels are
shown in the hypodermal layer. In the BR gel and blank gel treatment
groups (Figure 7), the epidermis layer indicated mild acanthosis as
well as follicular hyperkeratosis. The dermal layer showed muscle
fibers, bundle fibers of collagen, skin appendages, less inflammatory
infiltrates, and lymphocytes. The hypodermis layer depicted
subcutaneous fatty tissues and blood vessels (42).
In the early inflammatory phase of the healing process after 5th
day, polymorphonuclear cells such as neutrophil infiltration trigger
the recruitment of monocytes on the wound site, which later leads to
the formation of macrophages. Following this, the proliferative phase
begins with the formation of the granulation tissues, fibroblasts, and
angiogenesis cells. The appearance of different cells such as
lymphocytes, monocytes, angiogenesis, and fibroblast cells at the
wound site supports the healing progress with the formation of new
blood vessels and capillaries.
The numbers of monocytes and lymphocytes were largely
reduced once the healing progression reached a peak level and
disappeared completely at the end of the 15th day. The fibroblast
cell number significantly reduced after the 10th day and the
percentage of re-epithelization increased after the 7th day, leading
to the formulation of epidermal cells around wound edges. As
shown in the macroscopic structure of the wound covered almost
completely on the 15th day. Collagen deposition in the later stage
of wound healing is an important stage toward the normalization
of cells, and the deposition was more pronounced in the advanced
phase of healing. Overall, the results suggested that animals in the
BR–PolyET–NC treated groups showed rapid recovery in the
healing process involving different transition phases such as
inflammatory and proliferative ones, and re-epithelization of,
collagen deposition at, neovascularization of, and normalization of
the wound site indicates wound closure (64, 65).
3.5.2 Bacterial colony counts at wound site
The antibacterial effect of BR–PolyET–NC at the wound site was
compared to BR gel, blank gel, and control (untreated) is shown in
Figure 6C. The bacterial colony was measured lowest for the BR–
PolyET–NC gel, followed by BR gel and blank gel. The colony counts
for a group of animals treated with BR–PolyET–NC gel, BR gel, and
blank gel and negative control were noted. The highest and lowest
numbers of colony counts on the 15th day were found to be for
negative control and BR–PolyET–NC, respectively. The bacterial
colony counts of the BR–PolyET–NC gel on 5th, 10th, and 15th days
were recorded to be 15, 17, and 6 CFU, respectively compared to 26,
35, and 20 CFU for the BR gel. The bacterial count on these days at
the wound site in the case of the BR–PolyET–NC gel was significantly
lower than the BR gel (p < 0.05), indicating the potential of therapeutic
use in wound healing therapy (61).
3.5.3 Histopathogical investigation of the wound
Histopathology is an important parameter for investigating the
wound microenvironment and the healing process.
Histolopathogical images of the wound site are expressed in
Figure 7. The microscopy assessment of wound tissue calls for the
measurement of macrophagic cells, neutrophil cells, fibroblast,
fibrocytes, and collagen cells in the animal groups included for
wound study. The microscopic evaluation of the histopathology in
various treated groups are shown in Figure 6. The observations
revealed that the BR–PolyET–NC treated group of animals had the
best re-epithelization in the wound healing process and achieved
the highest percentage of healing compared to the BR gel treated
group in the entire treatment schedule of 15 days in BALB/c mice.
The BR–gel treated group of animal showed better result in healing
process compared to placebo gel and untreated group. The process
of re-epithelization was observed to begin on 5th day and,
consequently, wound closure completed on 15th day. The BR–
PolyET–NC treated group showed statistically significant difference
in the wound closure rate, i.e., >99% of the wound closed on the
15th day, followed by the BR gel and blank gel treated groups and
the untreated group.
Microscopic studies demonstrated that the treatment outcome of
the BR–PolyET–NC gel treated group was excellent and statistically
significantly different compared to other groups, including the BR gel
treated group. The haematoxylin and eosin (H and E) stained slides of
various treated groups illustrate the re-epithelization and
normalization of the wound treated skin. It was further disclosed that
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FIGURE 7
Histopathological examination of various treatment groups showing the changes from the 5th day to the 15th day of therapy in BALB/c mice: BR–
PolyET–NC gel treated group; BR gel treated; blank gel treatment group; and untreated or positive control and negative or normal at the end of the
15th day. The BR–PolyET–NC gel treated group stated the great influence in wound healing capability as demarcated by keratinization, formation of
hair follicles, or skin appendages. The blank gel and BR gel groups indicated acanthosis and follicular hyperkeratosis and inflammatory infiltrates in
dermal tissues. The connective fibrous tissue marks the existence of inflammatory infiltrates. Scale bar: 400 μm.
Generally, wound healing is a complex physiological process
mediated through various cells such asgrowth factors, cytokines,
and chemokines (1). These cells may directly or indirectly
be involved in the healing process, which is triggered by various
imbricating phases, i.e., hemostasis, inflammation, proliferation,
and remodeling of tissues to achieve proper healing of the intended
site (38). The first response to the injury site starts with hemostasis,
which leads to the arresting of bleeding and hemorrhage. Thereafter,
an aggregation of platelets and inflammatory cells accumulates at
this site and binds with newly formed collagen tissues in the
extracellular matrix (ECM). Platelets secrete proteins such as
vitronectins, fibronectins, and Sphingosine-1-Phosphate which help
in fibrin clots, accelerates vasocontriction, and stops bleeding. They
further assist in supplying inflammatory cells, which harbor cell
scaffolds that produce chemokines and cytokines that orchestrate
the early phase of wound repair (66, 67).
conditon. The external feature showed no turbidity or phase
separation. The stability experiment unveils that optimized BR–
nanocomposite gel was physico-chemically stable for 2 months. This
could be due to the complexing agent/gelling agent at elevated
temperatures, which raised the free energy of the gel system, resulting
in collision and thin particle aggregation. The stability study
confirmed that the stable nanocomposite gel can safely preserve for
2 months at this temeperature (68–70).
4 Conclusion
The BR-loaded BR–PolyET–NC nanocomposite was developed
for improved BR delivery and long-acting efficacy of the involved
drug in the wound healing process. The nanocomposite of BR was
prepared by applying the ionic gelation/complexation technique
and then loaded into the gel. A robust and stable formulation was
obtained using a three-level, three-factor experimental design.
The optimum composition of the developed formulation
comprising chitosan (X1 = 58.5 mg), sodium alginate (X2 = 27 mg),
and calcium chloride (X3 = 45.27 mg) had the mean particle size
and %EE of 71 ± 3.5 nm and 91 ± 1.6%, respectively. The drug
release behavior was controlled over a period of 72 h to maximize
the therapeutic effect and fasten wound healing. The electron
microscopy of the nanocomposite revealed distinct and
de-aggregated particles of uniform shape and size, indicating the
stability of the nanosystem. The in vivo model proved that the
developed formulation act upon by declining inflammation,
3.6 Stability studies
The different assessed parameters of BR–PolyET–NC gel such as
particle size, PDI, pH, viscosity, spreadability and extrudability under
stability studies of 2 months at elevated and at refrigerated
temeperatures are listed in Table 4. After inspecting the gel, changes
in pH and viscosity of the gel were not observed; however, less
marked changes in particle size and PDI were observed under the
stated condition in either of the temeperature. Furthermore, the
spreadability was not changed and extrudability was in good
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TABLE 4 Stability appraisal characteristics of the BR–PolyET–NC gel at elevated temeprature (40 ± 2°C/75 ± 5% RH) and at refrigerated temperature
(5 ± 3°C).
Time
(Days)
pH
Paticle
size, nm
PDI
Viscosity,
Pa.s
Spreadability
0
6.3 ± 0.01
71 ± 3.5
0.45
9.23
6.20 ± 0.12
Good
30
6.3 ± 0.03
73 ± 6.5
0.46
9.32
6.25 ± 0.15
Good
60
6.4 ± 0.02
75 ± 5.2
0.47
9.26
6.23 ± 0.13
Good
At refrigerated
0
6.3 ± 0.01
71 ± 3.5
0.45
9.23
6.20 ± 0.12
Good
temperature
30
6.3 ± 0.01
72 ± 2.3
0.44
9.24
6.23 ± 0.20
Good
60
6.4 ± 0.03
73 ± 4.2
0.46
9.27
6.24 ± 0.18
Good
At elevated
temperature
Extrudability
Acknowledgments
depositioning collagen fibers, enriching blood supply to the
wound, and replacing damaged tissues and cell debris. Overall,
our study highlights the important use of berberine in the nanoplatform in wound healing application, and we believe that it
could be translated clinically.
LA-K who was supported by a research program at Princess
Nourah bint Abdulrahman University, Riyadh, Saudia Arabia
(Reference number: PNURSP2023R82). The authors expressed their
gratitude to the Deanship of Scientific Research at King Khalid
University for funding this research through the Large Research Group
Project under grant numbers (RGP.02/535/44) and (RGP.02/557/44).
Data availability statement
The original contributions presented in the study are included in
the article/supplementary material, further inquiries can be directed
to the corresponding authors.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Author contributions
MHA, SR, and LA-K: conceptualization. MHA and MS:
methodology. MHA and HK: software. SA, NA, and MSA: validation.
MHA, GK, and SR: formal analysis. MHA, HK, and AA:
investigation. MR and HK: resources. HK and SR: data curation.
MHA and LA-K: writing–original draft preparation. A-HE, GK, and
MJ: writing–review and editing. MS and MR: visualization. LA-K
and MHA: supervision. MHA: project administration. SR, A-HE,
and MJ: funding acquisition. All authors contributed to the article
and approved the submitted version.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
References
9. Bawa P, Pillay V, Choonara YE, Du Toit LC. Stimuli-responsive polymers and
their applications in drug delivery. Biomed Mater. (2009) 4:022001. doi:
10.1088/1748-6041/4/2/022001
1. Frykberg RG, Banks J. Challenges in the treatment of chronic wounds. Adv Wound
Care. (2015) 4:560–82. doi: 10.1089/wound.2015.0635
2. Takeo M, Lee W, Ito M. Wound healing and skin regeneration. Cold Spring Harb
Perspect Med. (2015) 5:a023267. doi: 10.1101/cshperspect.a023267
10. Akhter MH, Rizwanullah M, Ahmad J, Ahsan MJ, Mujtaba A, Amin S.
Nanocarriers in advanced drug targeting: setting novel paradigm in cancer therapeutics.
Artif Cells Nanomed Biotechnol. (2018) 46:873–84. doi: 10.1080/21691401.2017.1366333
3. Tamayol A, Akbari M, Zilberman Y, Comotto M, Lesha E, Serex L, et al. Flexible
pH-sensing hydrogel fibers for epidermal applications. Adv Healthcare Mat. (2016)
5:711–9. doi: 10.1002/adhm.201500553
11. Akhter MH, Ahsan MJ, Rahman M, Anwar S, Rizwanullah M. Advancement in
Nanotheranostics for effective skin Cancer therapy: state of the art. Curr Nanomed.
(2020) 10:90–104. doi: 10.2174/2468187308666181116130949
4. Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: mechanisms,
signaling, and translation. Sci Transl Med. (2014) 6:265sr6. doi: 10.1126/scitranslmed.3009337
12. Mirhadi E, Rezaee M, Malaekeh-Nikouei B. Nano strategies for berberine delivery,
a natural alkaloid of Berberis. Biomed Pharmacother. (2018) 104:465–73. doi: 10.1016/j.
biopha.2018.05.067
5. Schultz GS, Chin GA, Moldawer L, Diegelmann RF. Principles of wound healing
In: R Fitridge and M Thompson, editors. Mechanisms of vascular disease: A reference
book for vascular specialists. Adelaide, AU: University of Adelaide Press (2011)
6. Afshar M, Taheri MH, Zardast M, Honarmand M. Efficacy of topical application of
coumarin on incisional wound healing in BALB/c mice. Iran J Dermatol. (2020)
23:56–63. doi: 10.22034/ijd.2020.110925
13. Tsang MSM, Jiao D, Chan BCL, Hon K-L, Leung PC, Lau CBS, et al. Antiinflammatory activities of Pentaherbs formula, Berberine, Gallic acid and Chlorogenic
acid in atopic dermatitis-like skin inflammation. Molecule. (2016) 21:519. doi: 10.3390/
molecules21040519
7. Ahmad J, Ahmad MZ, Akhter H. Surface-engineered Cancer nanomedicine:
rational design and recent Progress. Curr Pharm Des. (2020) 26:1181–90. doi: 10.217
4/1381612826666200214110645
14. Khalki LE, Tilaoui M, Jaafari A, Mouse HA, Zyad A. Studies on the dual cytotoxicity
and antioxidant properties of Berberis vulgaris extracts and its Main constituent
Berberine. Adv Pharm Pharm Sci. (2018) 2018:1–11. doi: 10.1155/2018/3018498
8. Akhter MH, Beg S, Tarique M, Malik A, Afaq S, Choudhry H, et al. Receptor-based
targeting of engineered nanocarrier against solid tumors: recent progress and challenges
ahead. Biochim Biophys Acta. (2021) 1865:129777. doi: 10.1016/j.bbagen.2020.129777
15. Tong L, Xie C, Wei Y, Qu Y, Liang H, Zhang Y, et al. Antitumor effects of Berberine
on gliomas via inactivation of Caspase-1-mediated IL-1β and IL-18 release. Front Oncol.
(2019) 9:364. doi: 10.3389/fonc.2019.00364
Frontiers in Public Health
14
frontiersin.org
Akhter et al.
10.3389/fpubh.2023.1238961
16. Wojtyczka RD, Dziedzic A, Kępa M, Kubina R, Kabała-Dzik A, Mularz T, et al.
Berberine enhances the antibacterial activity of selected antibiotics against coagulasenegative Staphylococcus strains in vitro. Molecules. (2014) 19:6583–96. doi: 10.3390/
molecules19056583
40. Md S, Alhakamy NA, Neamatallah T, Alshehri S, Mujtaba MA, Riadi Y, et al.
Development, characterization, and evaluation of α-Mangostin-loaded polymeric
nanoparticle gel for topical therapy in skin Cancer. Gels. (2021) 7:230. doi: 10.3390/
gels7040230
17. Yin J, Xing H, Ye J. Efficacy of berberine in patients with type 2 diabetes mellitus.
Metabolism. (2008) 57:712–7. doi: 10.1016/j.metabol.2008.01.013
41. Mahya S, Ai J, Shojae S, Khonakdar HA, Darbemamieh G, Shirian S. Berberine
loaded chitosan nanoparticles encapsulated in polysaccharide-based hydrogel for the
repair of spinal cord. Int J Biol Macromol. (2021) 182:82–90. doi: 10.1016/j.
ijbiomac.2021.03.106
18. Affuso F, Mercurio V, Fazio V, Fazio S. Cardiovascular and metabolic effects of
Berberine. World J Cardiol. (2010) 2:71–7. doi: 10.4330/wjc.v2.i4.71
42. Fatima F, Aleemuddin M, Ahmed MM, Anwer MK, Aldawsari MF, Soliman GA,
et al. Design and evaluation of solid lipid nanoparticles loaded topical gels: repurpose
of fluoxetine in diabetic wound healing. Gels. (2023) 9:21. doi: 10.3390/gels9010021
19. Varghese FS, van Woudenbergh E, Overheul GJ, Eleveld MJ, Kurver L, van
Heerbeek N, et al. Berberine and Obatoclax inhibit SARS-Cov-2 replication in primary
human nasal epithelial cells in vitro. Viruses. (2021) 13:282. doi: 10.3390/v13020282
20. Fan J, Zhang K, Jin Y, Li B, Gao S, Zhu J, et al. Pharmacological effects of berberine
on mood disorders. J Cell Mol Med. (2019) 23:21–8. doi: 10.1111/jcmm.13930
43. Iqubal A, Syed MA, Haque MM, Najmi AK, Ali J, Haque SE. Effect of nerolidol on
cyclophosphamide-induced bonemarrow and hematologic toxicity in swiss albino mice.
Exp Hematol. (2020) 82:24–32. doi: 10.1016/j.exphem.2020.01.007
21. Kohli K, Mujtaba A, Malik R, Amin S, Alam MS, Ali A, et al. Development of
natural polysaccharide–based nanoparticles of Berberine to enhance Oral bioavailability:
formulation, optimization, ex vivo, and in vivo assessment. Polymers. (2021) 13:3833.
doi: 10.3390/polym13213833
44. Panda DS, Eid HM, Elkomy MH, Khames A, Hassan RM, Abo El-Ela FI, et al.
Berberine encapsulated lecithin-chitosan nanoparticles as innovative wound healing
agent in type II diabetes. Pharmaceutics. (2021) 13:1197. doi: 10.3390/
pharmaceutics13081197
22. Duong TT, Isomaki A, Paaver U, Laidmae I, Tõnisoo A, Yen TTH, et al.
Nanoformulation and evaluation of Oral Berberine-loaded liposomes. Molecules. (2021)
26:2591. doi: 10.3390/molecules26092591
45. Cattelan G, Guerrero Gerbolés A, Foresti R, Pramstaller PP, Rossini A, Miragoli
M, et al. Alginate formulations: current developments in the race for hydrogel-based
cardiac regeneration. Front Bioeng Biotechnol. (2020) 8:414. doi: 10.3389/
fbioe.2020.00414
23. Allijn IE, Czarny BMS, Wang X, Chong SY, Weiler M, da Silva AE, et al. Liposome
encapsulated berberine treatment attenuates cardiac dysfunction after myocardial
infarction. J Control Release. (2017) 247:127–33. doi: 10.1016/j.jconrel.2016.12.042
46. Li S, Wang X, Chen J, Guo J, Yuan M, Wan G, et al. Calcium ion cross-linked
sodium alginate hydrogels containing deferoxamine and copper nanoparticles for
diabetic wound healing. Int J Biol Macromol. (2022) 202:657–70. doi: 10.1016/j.
ijbiomac.2022.01.080
24. Nguyen TX, Huang L, Liu L, Elamin Abdalla AM, Gauthier M, Yang G. Chitosancoated nano-liposomes for the oral delivery of berberine hydrochloride. J Mater Chem
B. (2014) 2:7149–59. doi: 10.1039/C4TB00876F
47. Zimet P, Mombrú ÁW, Faccio R, Brugnini G, Miraballes I, Rufo C, et al.
Optimization and characterization of Nisin-loaded alginate-chitosan nanoparticles with
antimicrobial activity in lean beef. LWT Food Sci Technol. (2018) 91:107–16. doi:
10.1016/j.lwt.2018.01.015
25. Xue M, Yang MX, Zhang W, Li XM, Gao DH, Ou ZM, et al. Characterization,
pharmacokinetics, and hypoglycemic effect of berberine loaded solid lipid nanoparticles.
Int J Nanomedicine. (2013) 8:4677–87. doi: 10.2147/IJN.S51262
48. Hasnain MS, Nayak AK. Alginates: Versatile polymers in biomedical applications
and therapeutics. Cambridge, MA, USA: Apple Academic Press (2020).
26. Li Z, Geng YN, Jiang JD, Kong WJ. Antioxidant and anti-inflammatory activities
of berberine in the treatment of diabetes mellitus. Evid Based Complement Altern Med.
(2014) 2014:289264. doi: 10.1155/2014/289264
49. Pavinatto FJ, Pavinatto A, Caseli L, Santos DS, Nobre TM, Zaniquelli ME, et al.
Interaction of chitosan with cell membrane models at the air-water interface.
Biomacromolecules. (2007) 8:1633–40. doi: 10.1021/bm0701550
27. Ayati SH, Fazeli B, Momtazi-borojeni AA, Cicero AFG, Pirro M, Sahebkar A.
Regulatory effects of berberine on microRNome in Cancer and other conditions. Crit
Rev Oncol Hematol. (2017) 116:147–58. doi: 10.1016/j.critrevonc.2017.05.008
28. Martău GA, Mihai M, Vodnar DC. The use of chitosan, alginate, and pectin in the
biomedical and food sector-biocompatibility, bioadhesiveness, and biodegradability.
Polymers (Basel). (2019) 11:1837. doi: 10.3390/polym11111837
50. Sahibzada MUK, Sadiq A, Faidah HS, Khurram M, Amin MU, Haseeb A, et al.
Berberine nanoparticles with enhanced in vitro bioavailability: characterization and
antimicrobial activity. Drug Design Dev Ther. (2018) 12:303–12. doi: 10.2147/DDDT.
S156123
29. Abasalizadeh F, Moghaddam SV, Alizadeh E, Akbari E, Kashani E, Fazljou SMB,
et al. Alginate-based hydrogels as drug delivery vehicles in cancer treatment and their
applications in wound dressing and 3D bioprinting. J Biol Eng. (2020) 14:8. doi: 10.1186/
s13036-020-0227-7
51. Choudhury D, Jala A, Murty US, Borkar RM, Banerjee S. In vitro and in vivo
evaluations of Berberine-loaded microparticles filled in-house 3D printed hollow
capsular device for improved Oral bioavailability. AAPS PharmSciTech. (2022) 23:89.
doi: 10.1208/s12249-022-02241-9
30. Basit HM, Ali M, Shah MM, Shah SU, Wahab A, Albarqi HA, et al. Microwave
enabled physically cross linked sodium alginate and pectin film and their application in
combination with modified chitosan-curcumin nanoparticles. A novel strategy for 2nd
degree burns wound healing in animals. Polymers. (2021) 13:2716. doi: 10.3390/
polym13162716
52. Thai H, Nguyen CT, Thach LT, Tran MT, Mai HD, Nguyen TTT, et al.
Characterization of chitosan/alginate/lovastatin nanoparticles and investigation of their
toxic effects in vitro and in vivo. Sci Rep. (2020) 10:909–15. doi: 10.1038/
s41598-020-57666-8
53. Biranje S, Madiwale PV, Patankar KC, Chhabra R, Dandekar-Jain P, Adivarekar
RV. Hemostasis and anti-necrotic activity of wound-healing dressing containing
chitosan nanoparticles. Int J Biol Macromol. (2019) 121:936–46. doi: 10.1016/j.
ijbiomac.2018.10.125
31. Li F, Shi Y, Liang J, Zhao L. Curcumin-loaded chitosan nanoparticles promote
diabetic wound healing via attenuating inflammation in a diabetic rat model. J Biomater
Appl. (2019) 34:476–86. doi: 10.1177/0885328219860929
54. Battu SK, Repka MA, Maddineni S, Chittiboyina AG, Avery MA, Majumdar S.
Physicochemical characterization of berberine chloride: a perspective in the
development of a solution dosage form for oral delivery. AAPS PharmSciTech. (2010)
11:1466–75. doi: 10.1208/s12249-010-9520-y
32. Hou B, Qi M, Sun J, Ai M, Ma X, Cai W, et al. Preparation, characterization and
wound healing effect of vaccarin-chitosan nanoparticles. Int J Biol Macromol. (2020)
165:3169–79. doi: 10.1016/j.ijbiomac.2020.10.182
33. Choudhary A, Kant V, Jangir BL, Joshi VG. Quercetin loaded chitosan
tripolyphosphate nanoparticles accelerated cutaneous wound healing in Wistar rats. Eur
J Pharmacol. (2020) 880:173172. doi: 10.1016/j.ejphar.2020.173172
55. Niu J, Yuan M, Chen C, Wang L, Tang Z, Fan Y, et al. Berberine-loaded Thiolated
Pluronic F127 polymeric micelles for improving skin permeation and retention. Int J
Nanomedicine. (2020) 15:9987–10005. doi: 10.2147/IJN.S270336
34. Su S, Kang PM. Recent advances in Nanocarrier-assisted therapeutics delivery
systems. Pharmaceutics. (2020) 12:837. doi: 10.3390/pharmaceutics12090837
56. Khan MFA, Ur Rehman A, Howari H, Alhodaib A, Ullah F, Mustafa ZU, et al.
Hydrogel containing solid lipid nanoparticles loaded with Argan oil and simvastatin:
preparation, in vitro and ex vivo assessment. Gels. (2022) 8:277. doi: 10.3390/
gels8050277
35. Soni K, Mujtaba A, Akhter H, Zafar A, Kohli K. Optimisation of ethosomal
nanogel for topical nano-CUR and sulphoraphane delivery in effective skin
cancer therapy. J Microencapsul. (2019) 37:91–108. doi: 10.1080/02652048.
2019.1701114
57. Mahdi WA, Bukhari SI, Imam SS, Alshehri S, Zafar A, Yasir M. Formulation and
optimization of Butenafine-loaded topical Nano lipid carrier-based gel: characterization,
irritation study, and anti-fungal activity. Pharmaceutics. (2021) 13:1087. doi: 10.3390/
pharmaceutics13071087
36. Kausar H, Mujeeb M, Ahad A, Moolakkadath T, Aqil M, Ahmad A, et al.
Optimization of ethosomes for topical thymoquinone delivery for the treatment of skin
acne. J Drug Deliv Sci Technol. (2019) 49:177–87. doi: 10.1016/j.jddst.2018.11.016
37. Akhter MH, Kumar S, Nomani S. Sonication tailored enhance cytotoxicity of
naringenin nanoparticle in pancreatic cancer: design, optimization, and in vitro studies.
Drug Dev Ind Pharm. (2020) 46:659–72. doi: 10.1080/03639045.2020.1747485
58. Zhang P, He L, Zhang J, Mei X, Zhang Y, Tian H, et al. Preparation of novel
berberine nano-colloids for improving wound healing of diabetic rats by acting Sirt1/
NF-B pathway. Colloids Surf B Biointerfaces. (2020) 187:110647. doi: 10.1016/j.
colsurfb.2019.110647
38. Greaves NS, Ashcroft KJ, Baguneid M, Bayat A. Current understanding of
molecular and cellular mechanisms in fibroplasia and angiogenesis during acute wound
healing. J Dermatol Sci. (2013) 72:206–17. doi: 10.1016/j.jdermsci.2013.07.008
59. Yin X, Fan X, Zhou Z, Li Q. Encapsulation of berberine decorated ZnO nanocolloids into injectable hydrogel using for diabetic wound healing. Front Chem. (2022)
10:964662. doi: 10.3389/fchem.2022.964662
39. Afzal O, Akhter MH, Ahmad I, Muzammil K, Dawria A, Zeyaullah M, et al. A
β–Sitosterol encapsulated biocompatible alginate/chitosan polymer nanocomposite for
the treatment of breast Cancer. Pharmaceutics. (2022) 14:1711. doi: 10.3390/
pharmaceutics14081711
60. Samadian H, Zamiri S, Ehterami A, Farzamfar S, Vaez A, Khastar H, et al.
Electrospun cellulose acetate/gelatin nanofibrous wound dressing containing berberine
for diabetic foot ulcer healing: in vitro and in vivo studies. Sci Rep. (2020) 10:8312. doi:
10.1038/s41598-020-65268-7
Frontiers in Public Health
15
frontiersin.org
Akhter et al.
10.3389/fpubh.2023.1238961
61. Amato G, Grimaudo MA, Alvarez-Lorenzo C, Concheiro A, Carbone C,
Bonaccorso A, et al. Hyaluronan/poly-L-lysine/Berberine Nanogels for impaired wound
healing. Pharmaceutics. (2021) 13:34. doi: 10.3390/pharmaceutics13010034
66. Saghazadeh S, Rinoldi C, Schot M, Kashaf SS, Sharifi F, Jalilian E, et al. Drug
delivery systems and materials for wound healing applications. Adv Drug Deliv Rev.
(2018) 127:138–66. doi: 10.1016/j.addr.2018.04.008
62. Zhou R, Xiang C, Cao G, Xu H, Zhang Y, Yang H, et al. Berberine accelerated
wound healing by restoring TrxR1/JNK in diabetes. Clin Sci. (2021) 135:613–27. doi:
10.1042/CS20201145
67. Locatelli L, Colciago A, Castiglioni S, Maier JA. Platelets in wound healing: what
happens in space? Front Bioeng Biotechnol. (2021) 9:716184. doi: 10.3389/fbioe.2021.716184
68. Alam Shah MK, Azad AK, Nawaz A, Ullah S, Latif MS, Rahman H, et al.
Formulation development, characterization and antifungal evaluation of chitosan NPs
for topical delivery of Voriconazole in vitro and ex vivo. Polymers. (2021) 14:135. doi:
10.3390/polym14010135
63. Wulandari PAC, Ilmi ZN, Husen SA, Winarni D, Alamsjah MA, Awang K, et al.
Wound healing and antioxidant evaluations of alginate from Sargassum ilicifolium and
Mangosteen rind combination extracts on diabetic mice model. Appl Sci. (2021) 11:4651.
doi: 10.3390/app11104651
69. Khan MK, Khan BA, Uzair B, Niaz SI, Khan H, Hosny KM, et al. Development of
chitosan-based Nanoemulsion gel containing microbial secondary metabolite with
effective antifungal activity: in vitro and in vivo characterizations. Int J Nanomedicine.
(2021) 16:8203–19. doi: 10.2147/IJN.S338064
64. Rodrigues M, Kosaric N, Bonham CA, Gurtner GC. Wound healing: a cellular
perspective. Physiol Rev. (2019) 99:665–706. doi: 10.1152/physrev.00067.2017
65. Bhubhanil S, Talodthaisong C, Khongkow M, Namdee K, Wongchitrat P,
Yingmema W, et al. Enhanced wound healing properties of guar gum/curcuminstabilized silver nanoparticle hydrogels. Sci Rep. (2021) 11:21836. doi: 10.1038/
s41598-021-01262-x
Frontiers in Public Health
70. Akhter MH, Ahmad A, Ali J, Mohan G. Formulation and development of CoQ10loaded s-SNEDDS for enhancement of Oral bioavailability. J Pharm Innov. (2014)
9:121–31. doi: 10.1007/s12247-014-9179-0
16
frontiersin.org