Accepted Manuscript

Title: Chitosan–Hyaluronic acid composite sponge scaffoldenriched with Andrographolide-loaded lipid nanoparticles forenhanced wound healing

Authors: Rania Abdel-Basset Sanad, Hend MohamedAbdel-Bar

PII: S0144-8617(17)30623-9DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2017.05.098Reference: CARP 12384

To appear in:

Received date: 2-3-2017Revised date: 25-4-2017Accepted date: 31-5-2017

Please cite this article as: Sanad, Rania Abdel-Basset., & Abdel-Bar, Hend Mohamed.,Chitosan–Hyaluronic acid composite sponge scaffold enriched with Andrographolide-loaded lipid nanoparticles for enhanced wound healing.Carbohydrate Polymershttp://dx.doi.org/10.1016/j.carbpol.2017.05.098

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Chitosan–Hyaluronic acid composite sponge scaffold enriched with

Andrographolide-loaded lipid nanoparticles for enhanced wound

healing.

Rania Abdel-Basset Sanad a,*, Hend Mohamed Abdel-Barb

aDepartment of Pharmaceutics, National Organization for Drug Control and Research (NODCAR),

Giza, Egypt. (E-mail: sanadrania3@yahoo.com)

b Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, University of Sadat

city, Egypt. (E-mail: hend.abdelbar@fop.usc.edu.eg)

* Corresponding author at: Department of Pharmaceutics, National Organization for Drug Control

and Research (NODCAR), Cairo, Egypt, 6 Abou Hazem Street, Pyramids,Giza, Egypt; Tel: +002

01224980700; e-mail: sanadrania3@yahoo.com

Highlights

Andrographolide lipid nanoparticles were prepared and statistically optimized.

Chitosan-hyaluronan/Andrographolide nanocomposite scaffold was successfully prepared.

Addition of Andrographolide nanocarrier showed proper porosity and swelling ratio.

Chitosan-hyaluronan/Andrographolide scaffold showed enhanced wound healing capacity.

Abstract

In this work chitosan–hyaluronic acid composite sponge scaffold enriched with

andrographolide (AND) lipid nanocarriers was developed. Nanocarriers were prepared using

solvent diffusion method by applying 23 factorial design. NLC4 had the highest desirability value

(0.882) and therefore, it was chosen as an optimal nanocarrier. Itexhibited spherical shape with

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253 nm, 83.04% entrapment efficiency and a prolonged release of AND up to 48-h. Consecutively,

NLC4 was incorporated in a chitosan-hyaluronic acid gel and lyophilized for 24 h to obtain

chitosan-hyaluronic acid/NLC4 nanocomposite sponge to enhance AND delivery to wound sites.

The morphology of sponge was characterized by scanning electron microscopy. Nanocomposites

showed a porosity of 56.22%with enhanced swelling. The in vivo evaluation in rats reveals that

the chitosan-hyaluronic acid/NLC4 sponge enhances the wound healing with no scar and improved

tissue quality. These results strongly support the possibility of using this novel chitosan-HA/NLC4

sponge for wound care application.

Keywords: Chitosan; hyaluronic acid;andrographolide; lipid nanoparticles; sponge

scaffold; in vivo wound healing.

1. Introduction

Chitosan is a unique biodegradable, biocompatible polymer possessing hemostatic

properties which make it a prospective candidate in the wound healing substrates (Deng, He, Zhao,

Yang, & Liu, 2007). It could be applied in wound healing sponge scaffolds (Lu et al., 2017) which

should be non-toxic, non-allergic and allow proper nutrient and gas exchange (Jayakumar,

Prabaharan, Sudheesh Kumar, Nair, & Tamura, 2011). Due to their well-interconnected

microporous structure, chitosan sponges have good cell interaction, fluid absorption capability,

and hydrophilicity (Anisha et al., 2013b). Unfortunately, the high brittleness of chitosan is the

most pronounced inadequacy in formulating wound healing materials, which could be overcome

by blending them with other polymers (Han et al., 2014) such as Hyaluronic acid (HA) (Mohandas,

Anisha, Chennazhi, & Jayakumar, 2015a). HA is the main constituent of the skin extracellular

matrix with good hydrophilic property. It provides a moist environment and protects the wounded

tissue surface from dryness and encourages healing. Besides, it enhances collagen secretion at the

3

wound site by fibroblast proliferation and have an optimistic effect in scarless wound healing

(Anisha, Biswas, Chennazhi & Jayakumar, 2013a).Utilizing these polymers to produce improved

wound management products especially those based on natural medicines represents an essential

research area (Boateng and Catanzano, 2015). Andrographolide (AND) is a natural diterpene

lactone separated from Andrographis paniculata leaves with improved wound healing activity in

rats (Al-Bayaty, Abdulla, Abu Hassan& Ali, 2012). This could be due to the reported anti-

inflammatory and antioxidant activities of AND (Li, Hu, Sun, Luo, Lu, & Tian, 2016). These could

play a significant role in protecting the tissues from oxidative damage and developing the wound-

healing process (Al-Bayaty, Abdulla, Abu Hassan& Ali, 2012). However, formulation

development of AND has been limited by its poor aqueous solubility (Jiang et al., 2014).

Therefore, encapsulation of AND in a nanoparticle platform can overcome the aforementioned

shortcoming to an extent. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers

(NLCs) applied in the treatment of wounds could selectively permeabilize the stratum corneum,

and also afford sufficient drug localization and deposition (Pachuau, 2015). However, as SLN and

NLC suspensions do not acquire sufficient viscosity to encourage its use for topical application.

Therefore, the inclusion of these active nanoparticles in secondary vehicles such as scaffolds to

elaborate their application and dosage, enhance the beneficial effects of the formulations,

microenvironment protection, enhance the consistency of final formulations, co-delivery of active

molecules at target sites and promote the long-term stability of the incorporated nanoparticles

(Oyarzun-Ampuero et al., 2015). Yet, there is no published work used AND in a wound healing

nanocomposite sponge scaffold.

The novelty of this work is to exploit the lucid biocompatibility and biodegradability of

chitosan-HA scaffold loaded with the antioxidant and anti-inflammatory AND lipid nanocarrier as

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an innovative wound healing agent. To the best of our knowledge, although chitosan-HA sponges

enriched with nanoparticles proved their high efficiency in wound healing, but yet there is no

published work deal with enrichment of these chitosan sponge scaffolds with NLCs. Moreover,

AND has not been formulated in NLCs before. Consequently, this in vitro and in vivo study aimed

to evaluate the wound healing efficiency of chitosan- HA sponge scaffold enriched with AND-

NLCs.

2. Materials and methods

2.1. Materials

Chitosan (MW 100–150 kDa, degree of deacetylation-85%) was purchased from Koyo

Chemical Co., Ltd. (Japan). Hyaluronic acid (HA) (MW 1500-1800 KDa) was purchased from

Qingdao Haitao Biochemical Co., Ltd. (China). AND (Xi'an Sonwu Biotech Co.,Ltd., China).

Solid lipids: Cetyl palmitate and Geleol, liquid lipid: Labrafac lipophile WL1349 (medium chain

triglyceride) (Gattefossè, France). Tego care 450 (polyglyceryl-3 methylglucose distearate) was

obtained from Goldschmidt, Germany). Brij 58 (Polyoxyethylene 20 cetyl ether) (Acros Organic,

Morris Plains, NJ). All other reagents were of analytical grade.

2.2. Chemical compounds studied in this article

Chitosan (CID: 71853); Hyaluronic acid (CID: 24728612); Andrographolide (CID:

5318517); Cetyl palmitate (CID: 10889); Labrafac (CID: 198429); Brij 58 (CID: 2724259).

2.3. Methods

2.3.1. Preparation of SLNs and NLCs

AND-loaded SLNs and NLCs were prepared by solvent diffusion method (Sanad,

Abdelmalak, Elbayoomy, & Badawi, 2010). Briefly, the lipid phase (7% w/w of the formulation)

composed of cetyl palmitate or Geleol with or without Labrafac lipophile WL1349 and AND (2%

5

w/w of the formulation) were completely dissolved in a mixture of acetone and ethanol 1:1 (v/v)

at 50°C. The resultant lipid solution was poured into 120 mL of an aqueous phase containing 2%

surfactant (tego care 450 or Brij 58) under agitation by mechanical stirrer (Falc Instruments, Italy)

at 400 rpm at 70°C for 5 min. The obtained dispersion was cooled to room temperature to obtain

AND-nanoparticles. The drug-free nanoparticles were also prepared. The composition of the

prepared nanoparticles is given in Table 1.

*Percent with respect to total lipid concentration. 2.3.2. Optimization of nanoparticle formulations

23 full-factorial design was used to optimize the variables for the preparation of AND lipid

carriers. Dependent, independent variables and the desirability constraints are shown in Table 2.

The statistical analysis of responses was made by Design-Expert®7 Soft-ware (Stat-Ease Inc.,

Minneapolis, MN, USA).

2.4. In-vitro characterization of SLNs and NLCs

2.4.1. Particle Size (PS) analysis

PS and the polydispersity index (PDI) of the nanoparticles were determined by using laser

scattering particle size analyzer (LA-920, Horiba, Japan)

2.4.2. Entrapment efficiency (EE %) and drug loading (DL)

AND nanoparticle suspensions were diluted 1:9 (v/v) in ethanol and put into a Pall

Nanosep® MF centrifugal device with Bio-Inert membrane pore size 0.2 μm (Sigma Aldrich,

India), then centrifuged (Hermle Z 326 K, Hermle Labortechnik GmbH, Germany) at 8,000 rpm

for 60 min at 10ºC. The supernatants were then analyzed for AND content by measuring at 225

nm using spectrophotometer (Shimadzu Co., Kyoto, Japan). EE and DL were then calculated

(Moreno-Sastre et al., 2016):

EE%= (Di– Dn)/Di×100 (1)

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DL = (Di - Dn)/L X 100 (2)

Di: initial drug amount

Dn: unentrapped drug.

L: total lipid.

2.4.3. In vitro drug release

AND-nanoparticles or drug suspension equivalent to 4mg drug were put into the dialysis

bags (molecular weight cutoff 12–14 kDa; Serva Electrophoresis GmbH, Germany) which placed

in 500 mL PBS, pH 7.4 (Yang et al., 2013), at 37ºC and stirred at 100 rpm for 48 h. Samples were

withdrawn at 1, 2, 3, 4, 5, 6, 7, 8, 24 and 48 h and then AND was quantified by UPLC system

(Waters Corporation, USA). The chromatographic analysis was performed using a BEH300 C18

(1.7 μm 2.1 × 50 mm) column. The mobile phase was: water and methanol (50:50, v/v). The flow

rate was 0.1 mL/min; the injection volume was 10 μl and UV detection at 227 nm was used (Du

et al., 2012).The coefficient of determination (R2) of the AND calibration curve in PBS in the

concentration range of 0.2–50 µg/mL, was 0.9994 and the respective limits of detection (LOD)

and quantification (LOQ) were 0.08 and 0.2 µg/mL. The CV% ranged from 0.85 to 9.78% and the

accuracy for AND determination was within acceptable range (not more than 1%) with mean%

drug recovery of 99.68%. In vitro drug release data was fitted to various kinetic models and the

regression analysis was performed.

2.5. Selection of the optimized formulation

Design Expert software was utilized for the selection of optimized formulation with small

particle size, higher in vitro cumulative drug release after 48 h, and higher EE%. (Tiwari & Pathak,

2011)

2.6. Transmission Electron Microscope (TEM)

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The morphology of the optimized AND nanoparticles was inspected by TEM (JEOL, JEM-

HR-2100, Japan). Nanoparticles were dried on a carbon coated copper 300-mesh grid and stained

with 2% phosphotungstic acid and visualized at 200 kV.

2.7. Preparation of chitosan–HA/AND-nanoparticle scaffold

Chitosan–HA scaffold enriched with AND-nanoparticle was prepared in two steps. First,

chitosan-HA blend was prepared by mixing solution 1 (2% w/v chitosan dissolved in 1% acetic

acid) with solution 2 (1% w/v HA dissolved in double distilled water) in a ratio 2:1 (Anisha,

Biswas, Chennazhi & Jayakumar, 2013). Subsequently, the optimized lipid nanoparticle dispersion

was added to the chitosan–HA blend in a ratio 3:7, and stirred for 4 h to obtain a homogenous

mixture which was poured into a mold, frozen and lyophilized for 24 h.

2.8. Characterization of chitosan–HA/AND-nanoparticle scaffold.

2.8.1. Fourier- Transformed Infrared spectroscopy (FT-IR)

FTIR spectra of Chitosan, HA, and chitosan-HA scaffold, were recorded on a FT/IR-4100 FT-IR

(JASCO, Tokyo, Japan). IR spectra were obtained over the range 450–4000 cm−1.

2.8.2. Morpholgy

The morphology of chitosan-HA scaffold and chitosan-HA/AND-nanoparticle scaffold

was observed by scanning electron microscopy (Quanta 250 FEG, FEI Co) under low vacuum at

400x magnification. TEM was used for examining the integrity of the optimized AND-

nanoparticle in sponge by dispersing it in water, sonicated for 15 minutes, and then treated

similarly as described under section (2.6) (Hazzah, Farid, Nasra, EL-Massik & Abdallah, 2015).

2.8.3. Porosity

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Samples of identified volume (V) and weight (Wi) were dipped in a graduated cylinder

containing ethanol and soaked for 24 h (Loh and Choong, 2013). The final weight of the wet

scaffold was noted as Wf. Porosity % was determined:

Porosity%= ((Wf – Wi / ρ ethanol)/V)X 100 (3)

ρ ethanol: Density of ethanol.

2.8.4. Swelling ratio

Scaffolds were immersed in PBS (pH 7.4) for different time points and incubated at 37°C.

Swelling ratio was calculated:

Swelling ratio= Ww-Wi/Wi (4)

Where Wi is the initial weight of the scaffolds and Ww is the wet weight of the samples

after taking them out and removing the excess buffer from them.

2.8.5. In vitro release of AND sponge:

A sponge equivalent to 4 mg AND was transferred to a dialysis bag (cutoff 12-14 KDa)

and immersed into 500 mL PBS pH 7.4 and the release study was conducted as previously

discussed (section 2.4.3.) up to 72 h.

2.8.6. Antioxidant Activity

The antioxidant activity of nonmedicated and medicated scaffolds was determined by using

DPPH as free radical (Rossi et al., 2007). Briefly, 20 mg of each scaffold was added to 975 μL of

a 6 ×10−5 mol-1 methanol/KH2PO4/NaOH buffer (50:50 v/v) DPPH solution, shaken and allowed

to stand at room temperature in the dark for 100 min. The absorbance of each sample was

determined at 517 nm using a UV VIS spectrophotometer (UV-2450; Shimadzu Co., Kyoto,

Japan). The control was prepared by mixing methanol/ KH2PO4/ NaOH buffer and DPPH radical

solution. The DPPH scavenged activity percentage (AA%) was determined:

9

DPPH scavenged %= A con - A test / A con X 100 (5)

A con : absorbance of the control.

A test : is the absorbance in the presence of the sample of examined sponge.

2.9. In-vivo wound healing study.

2.9.1. Animals

The Ethics Committee at the National Organization for Drug Control and Research, Giza,

Egypt approved all experiments. Six male albino rats (220 - 240 g) were kept in separate cages

with free access to food and water at a 12 h light/12 h dark cycle in a temperature controlled room

at 25±1°C. Rats were anesthetized by intraperitoneal thiopental injection (100 mg/kg) immediately

before inflicting the burn. The hair of each rat dorsum was shaved and cleaned with 70% ethanol.

2.9.2. Burn-wound model

The back of each rat was exposed for 5 s to stainless steel stamp heated for 5 min to form

a second degree burn wound (Morsi, Abdelbary, & Ahmed, 2014). Each rat was subjected to three

burns, the first was treated with chitosan-HA scaffold, the second treated with the chitosan-

HA/AND-nanoparticle scaffold and the third wound was kept without treatment as control. After

applying each scaffold on the burn-wounds, the wounds were covered by Tegaderm® (3 M Health

Care, Germany). Scaffolds were applied every other day to the burned areas for 21 days. On 1st,

4th, 12th and 21st post wounding days, the appearance of the wound was photographed. Tracing the

margin of the wound area onto the wound-photograph was performed by using 10.0 Adobe

Photoshop CS3 Extended software. The relative wound size reduction was determined (Anjum,

Arora, Alam & Gupta, 2016):

The relative wound size reduction (%) = (Ao-At)/ Ao X 100 (6)

A0 and At: the wound areas one day after operation and the specified day, respectively.

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2.9.3. Histopathological examination

At the 1st and 21st day after wound treatment, collection of full thickness skin biopsies of

wounds was done. Samples were fixed in 10% formol saline for 24h, washed by tap water and

dehydrated by serial dilutions of alcohol. Specimens were cleared in xylene and inserted in paraffin

for 24h in hot air oven at 56°, cut into 4 µm thickness pieces, stained by hematoxylin & eosin

(H&E) and subsequently visualized under a light electric microscope.

2.10. Statistical analysis

All presented in vitro data are the mean of three replicates ±SD and in vivo data are the

mean of six replicates ±SD. For comparing two variables, student’s t-test was applied. To compare

difference between groups, one-way analysis of variance (ANOVA) was applied followed by

Tukey HSD test (SPSS 18, Chicago, USA.), differences were considered statistically significant

at p≤0.05.

3. Results and discussion

3.1. Nanoparticle characterization

3.1.1. PS

The mean size of the prepared nanoparticles was shown in Table 3. Fig. 1a shows the effect

of the three independent variables on the PS. Geleol formed significantly (P < 0.0001) smaller

nanoparticles as it contains monoglycerides that possesses surfactant properties (Jensen, L. B.,

Magnussson, Gunnarsson, Vermehren, Nielsen, & Petersson, 2010). In the existence of water,

Geleol will rearrange into the aqueous–organic interface and facilitates emulsification and

formation of smaller particles (Abdelbary and Fahmy, 2009). Furthermore, the differences in

hydrophilicity of both surfactants (Sznitowska, Wolska, Baranska, Cal, & Pietkiewicz, 2017)

causes brij58 nanoparticles (HLB =15.7) to be significantly smaller (p < 0.0001) when compared

11

to tego care 450 (HLB= 12) particles. The incorporation of 40% Labrafac lipophileWL1349

significantly decreased PS (p < 0.0001). This might indicate better emulsification due to decreasing

the viscosity of the melted droplets (Elnaggar, El-Massik, & Abdallah, 2011).

3.1.2. EE% and DL

The EE% and DL of all nanoparticles are shown in Table 3. The DL was found directly

proportional to EE%. The ANOVA results illustrated in Fig. 1b shows that cetyl palmitate

significantly (p<0.05) increased EE% when compared to Geleol due to its higher lipophilicity

(Shah and Agrawal, 2013) which resulted in increased accommodation of AND which is a

lipophilic drug (log P=2.9) (Baek, Shin, & Cho, 2012, Levita, Nawawi, Abdul Mutalib & Ibrahim,

2010). Also, Brij 58 gave significantly (p<0.05) higher EE% than tego care 450. This is in

agreement with Tavano, De Cindio, Picci, Ioele, & Muzzalupo (2014) who stated that EE% is

directly proportional to the HLB value of the surfactant.

In addition, the incorporation of 40% liquid lipid significantly (p<0.05) increased the EE%

due to the decrease of the perfect structure of the solid lipid with a subsequent increase of

imperfections, hence leading to the formation of a not crystalline lipid blend and consequently the

presence of free spaces existing for the incorporation of higher amount of drug molecules (Shah

et al., 2016).

3.1.3. In vitro release

12

(A)

(B)

13

(C)

Fig. 2: In vitro release profile of AND from AND-nanoparticle formulations and AND suspension in

PBS (pH 7.4) (A), Correlation between mean particle size and cumulative % AND released after 48

h (Q48) for nanoparticle formulations (B) and Transmission electron micrographs of the optimized

AND-loaded nanostructured lipid carrier (C).

The prepared nanoparticles improved the release of AND over drug suspension in a

controlled manner up to 48 h (Fig. 2 a). This is probably correlated to the solubilizing effect of the

surfactants (Brij 58 and tego care 450) and the smaller PS (larger surface area) which enhanced

the dissolution rate of AND lipid nanoparticles (Shamma and Aburahma, 2014). In vitro AND

release from all nanoparticles exhibited biphasic release pattern with a low initial burst release up

to 22.46% within 1h, due to minor AND density at the surface of the formulated nanoparticles

(Jiang et al., 2014). Later, a controlled release manner was observed due to the slow liberation of

AND imbedded in the lipid matrix. This profile enables the use of AND-naoparticles as a

promising vector in wound healing (Mohandas, Anisha, Chennazhi, & Jayakumar, 2015a). An

initial release is sufficient to allow the existence of drug at the wound site in a short period of time,

whereas further steady release offers the drug over an extended period of time (Chereddy,

Vandermeulen, & Préat, 2016).

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Furthermore, there is a negative correlation (r2= -0.892) between PS and Q48 (Fig. 2b). As

larger nanoparticles make more total inner space for AND to be accommodated (Elsayed,

Abdelbary, & Elshafeey, 2014).

Moreover, release kinetic studies revealed that the release of AND from nanoparticles fitted

a sustained-release first-order model, the mechanism for which might be explained by the fact that

the drug incorporated into the solid-liquid lipid matrix was released in a sustained manner (Zhang

et al., 2013).

3.2. Optimization of AND-lipid nanoparticles

The optimized NLC4 composed of Geleol, 2% w/w Brij 58 and 40% w/w Labrafac-

lipophile WL1349 had the highest desirability value (0.882) and therefore, it was chosen as an

optimal formulation. As depicted in Table 3, NLC4 showed a particle size of approximately 253

nm, 83.04% EE and 100 % AND was released after 48h.

3.3. Morphology of the optimized AND-nanoparticles

The optimized formula NLC4 appears as deformed hexagonal-shaped particles (Fig.2c) it

is viewed from a top view (Esposito, Drechsler, & Cortesi, 2013). The mean size of NLC4

appeared in TEM was clearly smaller than that measured by Dynamic light scattering. As during

TEM sample preparation, nanoparticles become dehydrated; therefore their morphological size in

the solid state was revealed. Whereas particle size analyzer measures hydrodynamic diameter

(apparent size) of NLC4, including hydrodynamic layers around these nanoparticles leading to

overestimation of the nanoparticles size (Ghorab, Gardouh, &Gad, 2015).

3.4. Characterization of the prepared scaffold

15

(A)

B (i)

B (ii)

16

(C)

Fig. 3: (A) FT-IR of Chitosan, HA and chitosan-HA scaffold, (B) SEM micrographs of (i)

chitosan–HA scaffold (ii) chitosan–HA/NLC4 composite scaffold at magnification 400x and

(C) TEM micrograph of chitosan–HA/NLC4 composite scaffold.

3.4.1. FT-IR spectra

By inspecting the FT-IR spectrum of the prepared chitosan-HA scaffold sponge and

comparing it with those of chitosan and HA (Fig. 3a ), a new peak at 1572 cm-1 could be observed

due to the formation of NH3+ group after the complexation of chitosan with HA (Kim, Shin, Lee,

Park, & Kim, 2004).

3.4.2. Morphology

The surface morphology of chitosan–HA and chitosan–HA/NLC4 scaffolds was shown by

SEM images (Fig. 3b (i) and 3b (ii) respectively). A highly porous interconnected structure can be

seen with extra sheets like structures were shown in chitosan–HA scaffolds and irregular porous

morphology while a highly porous structure with a uniform distribution of pores in the case of

chitosan–HA/NLC4 scaffold. The removal of water from the NLC4 suspension incorporated into

the scaffold by lyophilization generated the vacant spaces which contributed to the recognized

17

large number of uniform pores (Florczyk et al., 2013). Fig. (3c) depicted the distribution of NLC4

in the prepared scaffold and its integrity which indicated that the preparation process had no

significant effect on the incorporated nanoparticle (Hazzah, Farid, Nasra, EL-Massik, & Abdallah,

2015).

3.4.3. Porosity

(A)

(B)

18

(C)

(D)

Fig. 4: (A) Porosity% of chitosan–HA composite scaffold and Chitosan–HA/NLC4 composite

scaffold and (B) swelling ratios in PBS (pH 7.4) of composite scaffolds and (C) In vitro release

of AND from chitosan–HA/NLC4 composite scaffold and NLC4 in PBS (pH 7.4). (D)

Antioxidant activity of chitosan–HA and chitosan–HA/NLC4 composite scaffolds.

Porosity of a scaffold determines how well the material can transport nutrients and oxygen

or absorb wound exudates. The fluid absorption would be cooperative in controlling infection at

the wound site (Mohandas, Sudheesh Kumar, Raja, Lakshmanan, & Jayakumar, 2015b). These

properties cause an improvement in cell respiration rate, skin regeneration, exudates removal and

hemostasis (Rath, Hussain, Chauhan, Garg, & Goyal, 2016). Chitosan–HA/NLC4 scaffold showed

improved porosity with respect to chitosan–HA scaffold (Fig. 4a) due to the incorporation of

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nanoparticle in the suspension form, consequently, lyophilization would yield a highly porous

structure (Anisha et al., 2013b).

3.4.4. Swelling behavior

Fig. 4b showed that the degree of swelling of chitosan–HA scaffold is higher than that of

chitosan–HA/NLC4 scaffold. This observation can be explained based on the effect of NLCs

components. In this respect, the system might reveal the following possible interactions: hydrogen

bonding between the hydroxyl and carbonyl groups present in Brij 58 (Polyoxyethylene 20 cetyl

ether) and the ammonium ions and hydroxyl groups of the chitosan, in addition to hydrophobic

interactions between Brij 58 tail and chitosan hydrophobic sites (Grant, Lee, Liu, & Allen, 2008).).

Therefore, the presence of NLC is hypothesized to interfere with polymer swelling ability.

Furthermore, Studies showed an increase in swelling ratio with time till 4th day which

decreased on the 7th day. Similar results were obtained by Anisha, Biswas, Chennazhi, &

Jayakumar (2013a) and Anisha et al. (2013b).

3.4.5. In vitro release of AND from scaffold

In vitro release results (Fig 4c) showed that incorporation of NLC4 into a chitosan-HA scaffold

decreased the amount of AND release compared to that from NLC4 dispersion. Upon scaffold

hydration, the polymers regain their gel structure leading to increase in diffusional path length of

the drug release. Furthermore, the thick gel layer formed on the swollen scaffold surface is capable

of preventing matrix disintegration and controlling additional water penetration hence retarding

the drug release (Hazzah, Farid, Nasra, EL-Massik, & Abdallah, 2015).

3.4.6. Antioxidant activity measurements

DPPH scavenging activity of the non-medicated scaffold was significantly lower than that

of the medicated scaffold (p< 0.05) (Fig. 4d). These results indicates that chitosan-HA possess

antioxidant activity which subsequently improved by the addition of AND. Various experimental

models previously demonstrated the antioxidant activity of chitosan (López-Mata et al., 2015).),

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HA (Sato et al., 1988) and AND (Karmegam, Nagaraj, Karuppusamy, & Prakash, 2015). The

antioxidant properties of chitosan-HA/NLC4 which play a significant role in protecting the tissues

from oxidative damage could contribute to the healing process (Al-Bayaty, Abdulla, Abu Hassan,

& Ali, 2012).

4.4.7. In vivo wound healing

(A)

(B)

21

(C)

Fig. 5: (A) Photographs of treated wounds at different time intervals (B) the wounds closure

rate (a+: significant difference from control group (untreated) at the same time interval at

p< 0.05, b+: significant difference from chitosan-HA scaffold group at the same time interval

at p< 0.05. (C) Photomicrographs (H&E stain) of normal skin (i), burned skin at day 1 post-

wounding (ii), untreated control burned skin at day 21 post-wounding (iii), burned skin

treated with chitosan-HA scaffold at day 21 post-wounding (iv) and burned skin treated with

chitosan-HA/NLC4 scaffold at day 21 post-wounding (v)

The wound healing results on day 1, 4, 12 and 21 are presented in Fig. 5 a. All the wounds

showed progressive healing up to 21 days. The wounds treated with chitosan–HA/NLC4 scaffold

healed faster than chitosan–HA scaffold treated groups and control. Fig. 5b shows the wound

closure rate of the chitosan–HA/NLC4, chitosan–HA scaffolds and control groups. On the 4th day,

reductions in wound area began in all wounds. On the 4th and 12th days, the wound closure rates

in the group treated with chitosan–HA/NLC4 scaffold was faster than those of chitosan–HA

scaffold treated and control groups. On day 21, the wounds treated with the chitosan–HA/NLC4

scaffold had healed completely with no scar and improved tissue quality, whereas in chitosan–HA

scaffold and control groups the wound closure rates are 95.3 % and 85.3 % respectively. This

indicates that chitosan–HA/NLC4 scaffolds were effective and beneficial for accelerated and

healthy wound healing.This positive healing effect might be due to the combined anti-

22

inflammatory and antioxidant effect of AND (Li et al., 2016) that is slowly released from the

optimized NLC4 together with the effect of chitosan and HA. AND as one of the main components

of Andrographis paniculata extract that has been reported to accelerate the wound healing process

(Al-Bayaty, Abdulla, Abu Hassan& Ali, 2012). In addition, it was stated (Archana et al., 2013)

that throughout the process of wound healing, depolymerization of chitosan gradually took place

to release N-acetylglucosamine, which helps in the deposition of ordered collagen and initiates

fibroblast proliferation and stimulates the synthesis of a high level of natural HA at the wound site.

Besides, HA is known to have high potential for scar reduction. Also, the incorporation of NLC4

in such scaffold increases the probability of its dermal localization and subsequently improving its

efficacy (El-Refaie, Elnaggar, El-Massik,& Abdallah, 2015).

3.4.8. Histological observation

At the 21st day study, the control showed a complete loss of keratin layer as well as details

of the prickle cells layer in the epidermis, fibrosis and few newly formed granulation tissue in the

dermis. The subcutaneous tissue showed edema with few inflammatory cells infiltration while the

musculature was intact (Fig.5c (iii)). On the other hand, wounds treated with chitosan-HA scaffold

showed fine keratin layer with underlying ill-defined prickle cell layer of the epidermis as well as

wide granulation tissue formation in the dermis. The subcutaneous tissue and musculature were

intact (Fig.5c (iv)). The wound treated with chitosan-HA/NLC4 scaffold indicate intact keratin

layer with underlying defined prickle cell layer of the epidermis as well as granulation tissue

formation in the dermis. The subcutaneous and musculature tissues were intact as shown in Fig.5c

(iv). The presence of hair follicles and matured fibrous tissues confirmed efficient healing. This

can easily be compiled with a statement that it provides the moist environment at the wound

site,which speeds up various stages of wound healing especially epithelialization (Jin et al., 2015).

23

These results are in line with what detected by Al-Bayaty, Abdulla, Abu Hassan, & Ali, (2012)

who observed that histologically, wounds dressed with Andrographis paniculata extract (its main

bioactive component is AND) contained high amounts of fibroblast proliferation.

4. Conclusion

In this study, AND-nanoparticles were developed and optimized. The optimized lipid

nanoparticle formulation was then successfully incorporated into a chitosan-HA scaffold. The

developed chitosan–HA/NLC4 scaffold showed appropriate porosity, swelling ratio controlled

drug release up to 72 has well as superior wound healing, reduced scar formation and improved

histological progress when compared to control. This was attributed to the combined anti-

inflammatory and antioxidant effect of chitosan and HA together with the effect of AND. In

conclusion this chitosan–HA/NLC4 scaffold would be a potential candidate for wound healing

applications.

Conflict of interest

The authors report no financial or personal conflict of interest.

Abbreviation list:

AND: Andrographolide.

HA: Hyaluronic acid

SLNs: Solid lipid nanoparticles.

NLCs: Nanostructured lipid carriers.

PS: Particle size.

PDI: Polydispersity index.

EE%: Entrapment efficiency percent.

DL: Drug loading capacity.

Q48: % andrographolide released after 48 h.

UPLC: Ultra Performance Liquid Chromatography.

PBS: Phosphate buffered saline.

TEM: Transmission electron microscope.

SEM: Scanning electron microscope.

DPPH: 2, 2-diphenyl- 1-picrylhydrazyl.

AA%: The DPPH scavenged activity percentage.

Log P: Octanol-water partition coefficient.

24

HLB: Hydrophilic lipophilic balance.

25

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Fig. 1: 3D response plots for the effect of solid lipid type (X1), surfactant type (X2) and liquid

lipid concentration (X3) on the PS (A), EE% (B) and cumulative release after 48 h (Q48) (C)

of AND-nanoparticle formulations.

31

Table 1 Experimental runs and independent variables of full factorial design for

nanoparticle formulations

Formulation

code

X1: Solid lipid

type.

X2: Surfactant type. X3: Labrafac conc*.

(%)

SLN1 Cetyl palmitate Tego care 450 0

SLN2 Cetyl palmitate Brij 58 0

SLN3 Geleol Tego care 450 0

SLN4 Geleol Brij 58 0

NLC1 Cetyl palmitate Tego care 450 40

NLC2 Cetyl palmitate Brij 58 40

NLC3 Geleol Tego care 450 40

NLC4 Geleol Brij 58 40

Table 2: Full factorial design used for optimization of nanoparticle formulations Factors (independent variables) Levels

X1: solid lipid type Cetyl palmitate Geleol

X2: surfactant type Tegocare 450 Brij 58

X3: Labrafac lipophileWL1349 concentration 0% 40%

Responses (dependent variables) Desirability constraints

Y1: PS (nm) Minimize

Y2: EE% Maximize

Y3: Q 48 h (%) Maximize

Table 3: The measured responses of full factorial design for nanoparticle formulations:

Formulation

code

Y1: PS (nm) Y2: %EE Y3: %AND released

after 48 h

PDI* Drug

loading

capacity*

SLN1 465±4.20 75.75±6.42 50.56±4.29 0.86 21.65

SLN2 348.5±7.70 75.66±6.41 71.00±6.02 0.91 21.64

32

SLN3 322±2.80 58.18±4.93 92.84±7.87 0.55 16.61

SLN4 343±8.48 69.49±4.20 100.00±9.89 0.73 19.85

NLC1 438±11.3 80.15±6.80 68.31±6.76 0.84 22.91

NLC2 297±11.3 87.36±7.41 98.64±11.16 0.90 24.96

NLC3 288±9.89 70.50±3.43 98.83±12.57 0.48 20.14

NLC4 253±16.97 83.04±7.04 100.00±7.07 0.53 23.72