Accepted Manuscript

Curcumin-Releasing Chitosan/Aloe Membrane for Skin Regeneration

Xiaocui Liu, Lijun You, Solaiman Tarafder, Lin Zou, Zhexiang Fang, Jingdi
Chen, Chang Hun Lee, Qiqing Zhang

PII: S1385-8947(18)32305-2
DOI: https://doi.org/10.1016/j.cej.2018.11.073
Reference: CEJ 20375

To appear in: Chemical Engineering Journal

Received Date: 7 August 2018

Revised Date: 8 November 2018
Accepted Date: 10 November 2018

Please cite this article as: X. Liu, L. You, S. Tarafder, L. Zou, Z. Fang, J. Chen, C.H. Lee, Q. Zhang, Curcumin-
Releasing Chitosan/Aloe Membrane for Skin Regeneration, Chemical Engineering Journal (2018), doi: https://
doi.org/10.1016/j.cej.2018.11.073

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 Curcumin-Releasing Chitosan/Aloe Membrane for Skin Regeneration

Xiaocui Liua#, Lijun Youa#, Solaiman Tarafderb, Lin Zoua, Zhexiang Fanga, Jingdi

Chena,b*, Chang Hun Leeb, Qiqing Zhanga,c**

aInstitute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou

350002, China
bRegenerative Engineering Laboratory, Columbia University, New York 10032,USA
cInstitute of Biomedical Engineering, Chinese Academy of Medical Science &

Peking Union Medical College, Tianjin 300192, China

#These authors contributed equally to this work

* Corresponding author: Jingdi Chen

Phone: +86-591-83725260; fax: +86-591-83725260

E-mail address: ibptcjd@fzu.edu.cn

** Corresponding author: Qiqing Zhang

E-mail address: zhangqq@126.com

1
Abstract:

Wound dressing, primarily used to protect skin lesions, has recently evolved into

advanced biomaterial-based membranes that support or induce skin regeneration.

Here, we fabricated a chitosan/aloe film that provides a sustaining release of curcumin

for promoting wound healing and skin regeneration. Uniform embedding of

curcumin-encapsulated poly(lactic-co-glycolic acid) microspheres in the chitosan/aloe

membrane were achieved with a high-power ultrasonic emulsification and a tape-

casting process. The micro-structural, physicochemical and mechanical properties of

the composite film were examined by SEM, XRD, ATR-FTIR and tensile tests. The

biocompatibility of the composite film was tested with NIH-3T3 cells culture in vitro.

Then the composite film is implanted in a full-thickness skin wound model in Wistar

rats. The implantation of curcumin-releasing film led to the accelerated would healing

and promoted skin tissue regeneration as compared to the untreated control and the

film without curcumin. In addition, sustain-released curcumin likely shows effect on

restraining inflammation and cicatrices. Our findings demonstrate that the

chitosan/aloe film with controlled delivery of curcumin promotes the intrinsic

regeneration of skin tissues.

Keywords: Chitosan, Aloe, Curcumin, film, microspheres, wound healing

1. Introduction

Skin injuries can be caused by physical and chemical damages. Although vertebrates’

skin can respond to the damages through the routine maintenance process by the
2
underlying regenerative machinery,[1] the intrinsic regenerative capacity is limited

upon an excessive tissue damage.[2] Moreover, the skin wound healing frequently

ends up a fibrotic scar with limited functional restoration.[3] To promote scar-less

wound healing, various types of skin grafts have been applied as a gold standard

treatment. However, the skin grafts suffer from several limitations including a

shortage of donor sites, donor sites morbidity, pain and a risk of infection.[4-7] To

overcome these limitations, tissue engineering approaches have been investigated to

regenerate skins by stimulating endogenous or exogenous cells with a support of

biomaterials (e.g. film or hydrogel), growth factors (GFs) and antibacterial drugs.[8-

11]

The effective skin graft materials should be able to recruit host cells and promote

their proliferation and differentiation,[12, 13] which highly depend on the surrounding

microenvironment created by materials through biochemical interactions with the host

cells and tissues.[14-16] Natural polysaccharides such as aloe vera (Aloe) and

chitosan (CS) have received attentions as efficient materials for skin wound dressing

and repair,[17-20] given their promising moisturizing properties and biocompatibility.

In addition, aloe contains dozens of nutrients including vitamins B2, B6, and B12,

eight kinds of essential amino acids and minerals, which is associated with its

myogenic repair capability. Aloe also has antibacterial and anti-inflammatory

properties,[21, 22] associated with its role in promotion of healing of cutaneous

wounds.[19] Similarly, CS is a film-forming non-toxic biocompatible and

biodegradable natural polymer with antimicrobial properties.[10, 23] Given its

3
cationic nature, CS can have ionic interactions with other compounds like aloe which

contains negatively charged bioactive polysaccharides (e.g. glucomannans and

acetylated mannan).[24, 25]

Besides the biomaterials, various GFs have been applied with wound dressing to

promote cell proliferation and skin repair.[26-28] In our study, we delivered curcumin

(Cur) via our film, which plays roles in regeneration and functional reconstruction of

skin tissue by stimulating secretion of transforming growth factor (TGF-β), followed

by promoting collage synthesis and reviving of epithelial cells.[29] Moreover, Cur

can also act as an anti-inflammatory agent by suppressing inflammatory mediators

such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)

induced by lipopolysaccharide (LPS).[30, 31] In order to take the advantages of Cur

and simultaneously control its release in wound healing and skin regeneration, self-

assembled microsphere (µS) have been prepared to provide controlled delivery.[32,

33] Previous works demonstrated a composite film combined with GF-encapsulated

µS that provides a sustained release of drug at controlled rates.[11]

In the present study, we developed and tested organic substrate-based films

embedded with Cur-loaded PLGA µS for skin regeneration. Cur-loaded PLGA µS

were introduced into the chitosan/Aloe composite films to enhance anti-inflammatory

characteristics and repair capability. A series of in vitro and in vivo studies evaluated

the potential of the chitosan/aloe composite film with Cur-loaded PLGA µS for

wound healing and skin regeneration.

2. Materials and methods

4
2.1. Materials

Chitosan (CS, 90% deacetylated), aloe vera (Aloe), Cell counting kit-8 (cck-8),

and Polyvinyl alcohol (PVA) were obtained from Sigma-Aldrich (St. Louis, MO,

USA). Curcumin (Cur) was purchased from Aungshing biomedical technology Co.,

Ltd. (Shanxi, China). The carboxyl-terminated poly (lactic-co-glycolic acids) (PLGA)

(Mw = 30 kDa, LA: GA = 75:25) was purchased from the Medical Research Institute

(Shandong, China). PVA was dissolved in ultrapure water at 2.0% (w/v). AO/EB

staining reagents were obtained from Nanjing Keygen Biotech. Co., Ltd. Fetal bovine

serum (FBS), Dulbecco’s minimum Eagle’s medium (DMEM),

penicillin/streptomycin, trypsin and other materials for cell culture were purchased

from HyClone Co. (USA). All chemicals and solvents were of analytical grade, and

ultrapure water was used in preparation of solutions.

2.2. Preparation of Chitosan-Aloe composite film

Cur-loaded PLGA µS embedded chitosan/Aloe composite film was prepared by

an ultrasound emulsification technology and a tape-casting processing. Briefly, total

1.0 g of chitosan (CS) was evenly dispersed in 30 mL PVA aqueous solution (2% w/v)

and then mixed with 0.5 mL acetate solution by magnetic stirring. Followed by

adding 300 mg aloe, Cur-loaded PLGA µS were prepared by dissolving 500 mg

PLGA in Cur acetone/dichloromethane solution (0.25 mg/mL) by ultrasonification for

2 minutes. PLGA/Cur solution was then added dropwise to the chitosan/aloe solution,

followed by emulsifying at 3000 rpm by a high-speed homogenizer (XHF-D, Ningbo

Science Biotechnology Co. Ltd., China) at 4 °C for 5 minutes. The organic solvent in

5
the water/oil (w/o) emulsion was removed by stirring and then poured into the pre-

designed molds (10×5×1 cm polyfluortetraethylene plate). The polymer film,

CS/Aloe-PLGA@Cur (Film-3), was prepared by storing the molded emulsion

solution at -20 °C for 24 h, -80 °C for another 24 h, and freeze-dried for 3 days. As a

separate group, air-dried films were prepared by placing the emulsion in the cupboard

and letting it naturally dry at room temperature. The freeze-dried and air-dried

polymer films were sterilized using gamma ray for cell culture and animal experiment.

The control group of CS/Aloe (Film-1) and CS/Aloe-PLGA µS (Film-2) were

fabricated following the same method described above.

2.3. Characterization of composite films

2.3.1. Characterization

In order to examine the crystal structure of films, the samples were scanned by

X-ray diffraction (X’Pert MPD Pro, PANalytical, Netherlands) in the range of 10-70°

at 30 kV and 20 mA (λ=0.154178 nm). Infrared (IR) reflectance spectra of the

constituents of the films were measured by an Attenuated Total Reflection Fourier

Transformed Infrared Spectroscopy (ATR-FTIR) (PerkinElmer Life and Analytical

Sciences, USA), which were collected in the range of 4,000-500 cm-1. The thermal

stability and dynamic characteristics of the composite films were investigated using a

thermal analyzer (TGA 449C, TA Instruments-Waters LLC, USA), with heating from

75 to 660 °C at a heating rate of 10 °C/min under constant nitrogen purging. Scanning

electron microscopy (SEM, 30XLFEG, Philips) was used to evaluate the surface

microtopography of the films. The macrostructure of films was observed by a

6
stereoscopic microscope.

2.3.2. Water retention and absorption capacity

Water retention capacity was measured to determine the porosity of the films

using the liquid displacement method.[34] Briefly, films were immersed in ethanol for

24 h, followed by measuring the saturated wet weight. The porosity of the films was

calculated using the following equation:

𝐷 2
Porosity (%) = (Wt－Wi)/(ρ × 𝜋 × ( ) )
2

Where Wt is the saturated wet weight, Wi is the initial dry weights. ρ is the

density of ethanol (0.789 g/cm3) , D is the diameter, and H is the thickness of the film.

To measure the water absorption capacity, the films with equal initial dry

weights were kept immersed in 6 mL PBS (pH=7.4) at 37 °C until they reached

equilibriums. pH varies from 1 to 8 in human organs with tight regulation in blood

and epithelia of barrier organs.[35, 36] At predetermined intervals, the films were

taken out, the excess PBS was removed with filter paper, and then the weight of the

films were measured. The swelling ratio was calculated using the flowing formula:

SR (%) = (Wt－Wi) × 100%

Where SR is the percent swelling capacity (%), Wt and Wi denotes swollen weights

and initial dry weights of the films, respectively.

2.3.3. In vitro enzymatic degradation

The initial dry weight of the composite films were measured and placed into 5

mL of PBS (0.01 mol/L, pH=7.4) with 500 μg/mL lysozyme.[37, 38] The specimens

were then incubated at 80 rpm in a reciprocating shaking bath. After 1, 4, 7, 14 and 28

7
days, the samples were removed from the degradation medium. The excess lysozymes

were washed away from the sample using distilled water, and the specimens were

lyophilized through a vacuum freeze dryer for 3 days. The weight of the specimens

was determined to evaluate the composite film degradation percentage.

2.3.4. Cur encapsulation rate, loading efficiency and drug release profiles

To determine Cur encapsulation rate (ER) and drug content (DC) of Cur within

PLGA µS, Cur-loaded Film-3 was put into 20 ml ethyl alcohol, followed by

measuring the Cur concentration by ultraviolet spectroscopy (Perkin Elmer

Instrument Lambda 35 UV-vis spectrometer) at 426 nm, where an intense

characteristic peak of Cur was displayed. All measurements were carried out in

triplicate (n = 3). The ER and DC were calculated,[39] according to the following

equations:
𝐴
ER (%) = ( ) × 100%
𝐵
𝐴
DC (%) = ( ) × 100%
𝐶
Where the A (mg) is the weight of loaded drug, B (mg) is the dosage of the drug, C

(mg) is the weight of the film with loaded drug.

In vitro release profile of the drug from the films and the influencing factors on

drug release were studied by a similarity factor method.[40] Briefly, the films were

immersed in 10 mL of PBS (pH 7.4) containing 10% ethanol and incubated in a

constant temperature shaker (120 rpm) at 37 °C for 360 hours. At different time points,

2 mL supernatant was collected and equal amount of fresh medium was added to each

specimen. The amount of Cur in the media was determined by measuring absorption

8
at 426 nm.

2.3.5. Tensile properties

The wet composite films were used for tensile tests with a universal mechanical

testing system.[41] The polymer films were cut into 20×10.5 mm rectangular-shaped

specimens. The thickness of Film-1, Film-2, and Film-3 were 3.51 ± 0.03, 4.09 ± 0.02

and 3.92 ± 0.02 mm, respectively. Samples were tested at a constant displacement rate

of 2 mm/min. Total 3 samples were used per group for the tensile tests.

2.3.6. Antibacterial activity assays

Antibacterial activities were investigated against Escherichia coli (as Gram-

negative bacteria) and Staphylococcus aureus (as Gram-positive bacteria) using agar

diffusion method.[42] A bacterium suspension (8×107 bacteria per mL) was seeded

onto an agar plate. The diameter of the inhibition zone (in mm) was measured to

determine the antibacterial activity of the films. The films with and without PLGA µS

(3×3 cm2) were placed into the plate, including Cur-loaded Film-3 and Film-1 (as

control). After incubating at 37 °C for 24 h, an inhibition zone around the samples

was recorded as an indication of antimicrobial activity. Each zone was then measured

and recorded three times by caliper to get an average value as an inhibition zone

diameter.

2.4. Cell culture

The NIH-3T3 cells were cultured with DMEM containing 10% FBS and 1%

antibiotics (100 μg/mL penicillin, 100 units/mL streptomycin) in an incubator at

37 °C and in 5% CO2. Cell adhesion and proliferation were evaluated after seeding

9
the NIH-3T3 cells (5000 cells per mL) on composite films in the 96-well plates for 24,

48 and 72 h. After removing culture medium at pre-defined time points, the samples

were washed twice with PBS, followed by adding 10 μL cck-8 in 100 μL PBS

solution in each well. After 4 h, a microplate reader was used to quantify the

absorbance at 490 nm. The cell viability index was calculated by the following

equation:

𝑂𝐷𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡 ‒ 𝑂𝐷𝑏𝑙𝑎𝑛𝑘
Relative cell viability (%) = ( ) × 100%
𝑂𝐷𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ‒ 𝑂𝐷𝑏𝑙𝑎𝑛𝑘

Five replicate measurements were obtained for each sample.

Cell adhesion and morphology of NIH-3T3 cells on the composite films were

observed by SEM.[43] Before SEM measurement, the cell-seeded films were washed

twice with PBS buffer (pH 7.4) and fixed with 4 wt% paraformaldehyde for 30 min,

then rinsed twice in PBS buffer. The films were then dehydrated through a graded

series of ethanol solution (50%, 70%, 85%, 95%, and 100%) for 20 min each,

followed by freeze-dried for 3 days. Finally, the samples were sputter-coated with

gold for 60 s and observed by FE-SEM (JSM-6700F, Japan) with an accelerating

potential of 5.0 kV.

AO/EB staining was performed to evaluate the cell growth behavior and

cytocompatibility of the composite films.[37] NIH-3T3 cells (9 × 103 cells per mL)

were cultured on composite films in 24-well plates at 37 °C for 24 h, 48 h and 72 h.

Culture medium was removed at the end of the pre-determined time points and the

samples were washed twice with PBS, followed by adding 100 μL of the dual

fluorescence reagents (100 μL AO and 100 μL EB were dissolved in 1 mL PBS) into

10
each well. The fluorescent reagents were removed after 20 min incubation and 300 μL

of PBS was added to each well. The behavior of the cells was examined with an

inverted fluorescence microscope (Leica, DMIL LED model, Germany).

2.5. Animals study

All animals were taken care for according to the policies and principles

established by the National Institutes of Health Guide for the Care and Use of

Laboratory Animals in this research. Male Wistar rats with an average weight of 300

g were purchased from the Fuzhou Laboratory Animal Research Center. The rats

were randomly divided into 4 groups (n = 5 animals in each group). A Full-thickness

skin injury was created by removing a round 20 mm diameter section of dorsal flank

skin after anesthetization by intraperitoneal injection of 2% pentobarbital sodium (1

mL/kg). Rats were treated with conventional gauze and composite films, and then

kept in a single cage for observation. Gross photographs of injury region were taken

to visualize changes in the wound size after established postoperative days. The

percentage of wound closure (wound healing rate) was calculated using the following

formula: [43]

𝐴𝑒 ‒ 𝐴𝑖
Wound closure (%) = ( ) × 100%
𝐴𝑖

Where Ae is the wound area and Ai is the initial wound area.

The animals were sacrificed at 7 and 14 days after surgery and wound skin

tissues were harvested for histopathological analysis. Tissue samples were sectioned

after being embedded in paraffin and stained with H&E. An inverted microscope was

11
used to record histological images.

2.6. Statistical analysis

The values presented are expressed as mean ± standard deviation (SD). The

SPSS 16.0 software and Origin 8.5 software were used for statistical analysis via

unpaired t-test and the graph plotting. P* < 0.05 and p** < 0.01 were significant

differences and greatly remarkable differences between the two groups, respectively.

Each experiment included at least three replicates.

3. Results and discussion

3.1. Characterization of the composite film

Figure 1. The XRD diffraction patterns (A) and TGA curves (B) of composite films.

(C) The ATR-FTIR spectra of CS, Aloe, CS+PLGA and films with different

compositions were tested. Abbreviations: CS/Aloe films (Film-1); CS/Aloe-PLGA

films (Film-2); CS/Aloe-PLGA@Cur films (Film-3).

12
 The phase structures of the composite films were characterized by XRD patterns,

as shown in Figure 1A. Although the crystallinity was different with respect to

different phase composition, all the samples showed a semi-crystal structure of CS,

indicating that composite film did not change with respect to chitosan. Film-1

exhibited the characteristic diffraction peak centered at 2θ value of 22º, which related

to the reflection of (110) crystalline plane, indicating its low crystallinity.[10] Film-2

and Film-3 showed a single broad peak that confirms its predominantly amorphous

structure. As compared to Film-2, Film-3 also showed the diffraction peak centered at

2θ = 43.5º, and its peak has enhanced, which confirmed that PLGA µS with Cur in

Film-3 had been successfully loaded.

Thermal stability of biomedical materials is a matter of paramount significance

for evaluating their commercial application.[41] The TGA results for the composite

films were displayed on Figure 1B. All samples were subjected to three obvious

weight losses. The first weight loss for Film-1, Film-2 and Film-3 was about 12%, 9%,

and 10%, respectively, which occurred in the temperature ranging from 75 °C to

165 °C. This is likely attributed to the loss of water by evaporation or to a reduction in

moisture content. The initial weight loss for films could be due to the water sorption

of CS from the environment and the excellent moisturizing efficacy of Aloe,

indicating that Film-2 and Film-3 still reserve the water sorption with the addition

PLGA microspheres. Furthermore, these outcomes demonstrated that Film-2 and

Film-3 composites may also absorb excess exudates when they were used as wound

dressing. All the samples had a second obvious weight loss starting around 340 °C,

13
which is likely related to the degradation of CS, Aloe or PLGA components. The

weight loss for the Film-2 and Film-3 composites were higher than for Film-1 below

340 °C, and the identical weight loss of Film-2 and Film-3 amount was ~48%. These

results appeared to show the equal addition of PLGA microspheres within the

CS/Aloe network structure. The weight loss of Film-2 and Film-3 occurring in the

temperature range from 340 °C to 500 °C was 13% and 21%, respectively. This 8%

difference in mass loss was possibly affected by the electrostatic interaction between

the positively charged chitosan and the negatively charged Cur that was not loaded in

Film-2.

Significant changes in the chemistry and the fine structure of CS, Aloe, PLGA,

Film-1, Film-2 and Film-3 were found in the ATR-FTIR spectra in Figure 1C. The

ATR-FTIR spectrum of CS showed characteristic absorption bands corresponding to

saccharide structure of pure CS at about 1067-831 cm-1. Particularly, the absorbance

peak at 3277 cm−1 was indicative of hydrogen bonded O-H stretching or N-H

vibration. The spectrum corresponding to chitosan presented C-H stretching

vibrations at 2939-2909 cm-1. The bands located at ~1637 cm-1 was associated with

C=O for amide I.[10] The characteristic vibrational absorption band located at ~1559

cm-1 corresponds to N-H bending vibrations for amide II. The peak at ~1327 cm-1

stands for C-N stretching vibrations of amide III.[18] The merged IR peaks of non-

cross-linked CS/Aloe kept its stability, which confirmed that the CS and Aloe formed

organic substrate through electrostatic interactions. In comparison with Film-1, the

Film-2 and Film-3 also showed characteristic absorption bands corresponding to

14
saccharide structure at about 1080 and 820 cm−1, suggesting that they hold biological

functions of CS. Furthermore, the spectra of the Film-2 and Film-3 still showed

typical absorptions bands of PLGA at ~1750 cm-1 correspond to C=O stretching

vibrations peaks of carboxylate groups and at ~1265 cm-1 relates to C-O stretching

vibrations. The band at 1072-1024 cm-1 represented C-O-C stretching vibrations for

glycosidic bonds.[32] It is suggested that the self-assembled PLGA microspheres

successfully immobilized within Film-3 by bond.

Figure 2. (A) FE-SEM micrographs of the upper surface and inner morphology of the

composite films, inset: SEM image of high magnification. AFM images (B), the

porosity (C) and swelling properties (D) of composite films. * and ** indicates

significantly different compared to Film-1 groups.

As shown in Figure 2A, Film-1 displayed a continuous, smooth and uniform

surface morphology, showing no phase separation. In contrast to Film-1, Film-2 and

Film-3 images showed the particles that are uniformly dispersed throughout the film
15
surface, suggesting the successful, even incorporation of PLGA µS. In addition,

electrostatic interaction and hydrogen bonding in the CS/Aloe formed uniform

emulsion of organic matrix, which can assure homogeneous distribution of the

microspheres. The surface tomography also revealed the notable roughness on Film-2

and Film-3, which may be considered favorable for cell attachment.[41] Moreover,

the addition of Cur led to the decrease in roughness (Figure 2A) likely due to special

laminar structure of unloaded-Cur.

The 3D morphologies of the composite films obtained from atomic force

microscopy (AFM) images show the different surface roughness in between these

composite films.[44] As shown in Figure 2B, Film-1 possessed root mean square

height (Sq parameter) of approximately 8.82 nm. However, the Sq parameter of Film-

2 and Film-3 were significantly higher than Film-1, which was contributed to the

anchoring PLGA microspheres on surface. The Sq parameter of films after PLGA

loading tend to increase with values of about 17.1 and 15.3 nm, respectively

according to ISO 25178. The height increased markedly about 9 nm in Film-2 and

Film-3 compared to Film-1 according to their values of Sq parameter. These results

indicated that Film-2 and Film-3 possess a rougher surface morphology. These

morphological changes can be related to the fact that PLGA µS were successfully

anchored onto the films surface, which formed the multilayered micro-nano

composite.

The porosity of the composite film was examined to estimate the water retention

capacity, as displayed on Figure 2C. It appears that Film-1 without microspheres had

16
a relatively porosity of 35.26%. The Film-2 had highest porosity of 43.47% likely due

to the inclusion of microsphere that increases the roughness of the membrane as per

the SEM images of surface topography (Figure 2A). However, the drug loading

reduced the porosity of Film-3 (29.52%). This may come from the smaller sizes of

PLGA µS (Figure 2A and 2B) and left Cur in Film-3.

The in vitro swelling properties, as an important factor for prospective evaluation

of biomaterials,[45] can be influenced by many factors, such as the hydration ability

of the materials, the temperature of the environment, the ionic strength and pH value

of the media.[10] As shown in Figure 2D, rapid swelling was observed for the films

with PLGA microspheres and Film-1 without PLGA µS showed much slower

swelling rate. Film-1 took 2 d to attain 34% swelling, whereas Film-2 with PLGA µS

and Film-3 with Cur-PLGA µS attained the same degree of swelling by 0.8 d and 1.1

d, respectively. These findings were likely due to the enlargement of the pore size at

the initial swelling period by addition of microspheres, which is likely an important

parameter for cell attachment and growth.[45] Compared with Film-1 groups, there

was a significant difference in the swelling rate of the Film-2 and Film-3 for 2 d.

Moreover, Film-1 reached the equilibrium state of swelling within 2.5 d, whereas

Film-2 and Film-3 did not achieve equilibrium for the tested duration.

17
Figure 3. The degradation performance (A) and release behavior (B) of composite

films. Inset: the abridged general view is the three stages of drug release including

diffusion, swelling and erosion. (C) The tensile strength and Young′s modulus for

composite films was test. (D) Antibacterial performance of Film-1 and Film-3 against

Staphylococcus and Escherichia coli was studied.

The weight loss is important for films as a wound dressing or an artificial skin.

Furthermore, drug release is affected by the degradability of films. Figure 3A showed

the weight loss of the composite films (Film-1, Film-2 and Film-3). The degradation

rate was relatively slow initially for the PLGA µS-embedded composite films. The

weight loss gradually increased from 7 d and 14 d, suggesting that CS and Aloe

degraded gradually by the influence of lysozyme. It is suggested that the PLGA

microspheres constantly exposed to hydrolysis-based degradation upon the

degradation of CS and Aloe. After 28 days of incubation, CS and Aloe matrix of

18
Film-1 collapsed and lost about 47.91 ± 0.40% of its initial weight, whereas the Film-

2 and Film-3 exhibited a significantly lower degradation rate (Figure 3A).

The encapsulation efficiency (EE) and loading efficiency (LE) of Cur within

PLGA microspheres of Film-3 were determined using UV-spectroscopy. The EE and

LE of Cur-loaded Film-3 was 77.56% and 13.85%, respectively. The higher value of

the LE was possibly attributed to the fact that the partially unloaded Cur precipitated

in organic matrix during the film preparation process, and the Cur on the surface was

not completely washed away. Besides the structural control of film, drug controlled

release behavior was a key parameter for an active wound material. The accumulative

release profiles of the Cur from Film-3 were presented in Figure 3B. An initial burst

release of Cur was observed from the Film-3 followed by a sustained release. The

burst release was probably due to fast diffusion of Cur, likely caused by rapid

swelling on the surface.[37] The cumulative release amount of 89.20 ± 0.50% was

observed by 360 hours, suggesting an appropriate sustained release for wound healing

and skin regeneration.

Mechanical strength, sustainability, flexibility and bending properties are

important factors considered for wound dressing and skin repair materials. Hence, the

functional properties of the skin repair materials were evaluated by the tensile

tests.[45] As shown in Figure 3C, the tensile strengths of Film-2 and Film-3 were

significantly lower than that of Film-1. It was possibly due to the disruption of

intermolecular hydrogen bonding and electrostatic interaction between CS and Aloe

caused by addition of PLGA microspheres which reduced the crystallinity. These

19
results were consistent with the XRD patterns. In comparison with Film-2, Film-3

loaded with Cur had significantly higher tensile strength, but lower Young’s modulus,

probably due to the intermolecular interactions between CS and Cur. The wet films

were very soft with variable and controllable shape, which enabled films to contact

with the skin completely. We expect that composite films should be suitable as wound

dressing and have the capability to be used as artificial skin.

Besides the mechanical properties, antibacterial properties would benefit wound

dressing material. Thus we performed a test for antibacterial properties using Gram

negative Escherichia coli and Gram positive Staphylococcus aureus that act as the

predominant pathogenic bacteria for severe skin wound infection.[42] As shown in

Figure 3D, the bacterial colonies around the Film-3 were completely inhibited on

both bacterial strain culture plates after 12 h of contact in contrast to the films without

Cur. Film-1 and Film-2 also showed the bacteria inhibition zones given the reported

antibacterial properties of CS and Aloe.[46, 47] The bacteria growth inhibition zones

with the diameters of 7 ± 1 (E. coli) and 3 ± 1 mm (S. aureus) were observed for the

Film-1. In contrast, a larger antibacterial ring of 6 ± 1 mm diameters was observed

around the Film-3. These results may indicate that the Cur was more sensitive to S.

aureus in regard to its intrinsic anti-inflammatory properties. In addition, the well-

dispersed distribution of Cur-loaded PLGA microspheres made Cur release steadily

from Film-3 over period and ultimately realized long-term antibacterial effects. These

results manifest that the PLGA microspheres had limited effect on the antibacterial

activity of CS when embedded within the organic matrix. Moreover, Film-3

20
embedded Cur-loaded PLGA microspheres possessed an efficient antibacterial effect

that has potential to control dermal bacterial infections during wound healing.

3.2. In vitro assays

Figure 4. The images of AO/EB staining (A) and relative viability (B) for the NIH-

3T3 cells co-cultured with the films after 24, 48 and 72 h. (C) FE-SEM images of

morphology of NIH-3T3 cells attaching to the films for 24 h. The data show

significant statistical differences between the Film-3 groups and the Film-2 group at a

same time point (*P < 0.05). Scale bars = 100 μm.

An AO/EB staining offered a direct and easy observation of cell viability on

composite films in Figure 4A. From 24 to 72 h, seldom dead cells appeared on all the

21
dressing materials. A little dead cell appeared on Film-3 than the other groups at 24 h.

This was likely due to the initial burst release of Cur from the Film-3, consistently

with results in Figure 3B. The increased cellular proliferation was observed from the

gradual increase of the number of cells attached to the surface of films, indicating that

films possessed excellent biocompatibility, and could recruit host cells and promote

cell proliferation. It appeared that the number of the cells attached to the Film-2 and

Film-3 was significantly higher than the Film-1 after 72 h. The results revealed that

PLGA-loaded films appeared to be more efficient in promoting cell proliferation with

viability.

In vitro quantitative cell proliferation was evaluated by cck-8 test.[48] As

presented in Figure 4B, all the films were favorable for cells growth up to 72 hours.

The relative cell viability of Film-3 achieved 112.49%, suggesting Cur

stimulate proliferation. The Cur-loaded film (Film-3) showed the best

biocompatibility among all the groups tested. In parallel, the adhesion properties of

NIH-3T3 cells on films after 24 h were measured by SEM, as displayed on Figure 4C.

The cells growing on all films were in spindle and round shapes. It was known that

the topological structure and matrix components will affect the cell behavior. It can be

seen that more cells grow in Film-2 and Film-3 than in Film-1. The rougher surfaces

on Film-2 and Film-3 (Figure 2A) may benefit the cell attachment and proliferation.

These results indicated that the interactions between cells and biomaterial films via

surface topography and surrounding matrix of materials.

3.3 In vivo assays

22
Figure 5. (A) Establishment of a full-thickness skin injury model and treatment

process in a Wistar rat. (B) Gross observation of excisional gauze, films-treated

wounds after 0, 7 and 14 days after operation. (C) Proportion of the wound area left

after 7 and 14 days of the operation (n = 6). (D) Wound healing rate of the wounds

covered by gauze and different films after surgery for 7 and 14 days. * represents a

statistical difference between the experimental group and control group (P < 0.05),
23
compared to the gauze-treated wound as control. All the scale bars represent 10 mm.

Gauze, a conventional wound dressing, was used as control. The prepared novel

composites films were applied as wound dressing on the full-thickness critical size

skin injury on a rat model to compare the wound healing rate in vivo (Figure 5A).

The full-thickness wound with diameter of 20 mm was made on the dorsal skin, and

covered by the films with the same size. Figure 5B illustrates gross appearance of

skin injury covered with the gauze and composite films at day 0, 7 and 14 after

surgery. The skin wounds treated with composite films exhibited better and faster

healing than the control gauze group after 7 days. Moreover, the less ecchymoses

associated was observed after being treated with Cur-loaded film than other groups. A

noticeable decrease in wound diameter was noticed after 7 days. In addition,

compared with the gauze-treated group, significant wound closure and re-

epithelialization was observed after 14 days in all groups with composite films.

However, Film-1 and Film-2 still displayed the remaining scab compared to no scab

in Film-3 groups. Compared with the ones covered with gauze, the composite films

showed better fluid retention due to the addition of natural moisturizing factor Aloe.

The extent and rate of wound skin repair after treatment were shown in Figure 5C.

Compared the wound size on day 7 and 14 with the original wound size on day 0, the

extent of wound healing was calculated. The smallest wound size was observed on the

rats treated with Film-3 after days 7 (24.01%) and 14 (3.63%). The wound treated

with Film-1 and Film-2 had similar sizes (31.50%, 30.24% on day 7, and 8.15%, 6.70%

on day 14). The wounds treated with Film-1 were not significantly different from the

24
wounds covered with Film-2 on day 7 and 14, resulting in the formation of a rough

and thin skin layer. The wound size of rats treated by the films was significantly

smaller than that treated with gauze on day 7 and 14. Figure 5D showed the rate of

wound healing by the calculation of the ratio of the repaired wound area to original

wound area as a function of time. Wounds treated with Film-3 showed the highest

wound healing rate, indicating that sustained Cur release from the composite film can

effectively restore the epithelization of multipotent epidermal progenitors,

accelerating skin healing and inhibit scar formation, and reducing complications. Cur

and Aloe possibly facilitated host stem/progenitor cells recruitment into the implanted

films and accelerated the wound repair process.[7, 19]

Figure 6. Light microscopy images for HE stained tissue sections of wound on the

Wistar rats after 7 and 14 days. The specific cell types in the histological sections are

indicated by letters (F: fibroblasts; L: inflammatory cell; B: blood vessels; S:

25
squamous epithelial; H: hair follicle). Magnification of images at 7 days (Upper: 10x,

Lower: 20x) and 14 days (Upper: 10x, Lower: 20x).

Wound healing is a complex process requiring the collaborative efforts of many

different tissues and cell lineages.[42] The formation of granulating tissues, re-

epithelialization, tissue hyperplasia and cicatrization and the cooperation played

critical roles in the wound healing process. New capillaries and fibroblasts form

granulating tissue, accompanied by inflammatory cell infiltration. As shown in Figure

6, significant infiltration of inflammatory cells was observed in gauze group at day 7,

while fibroblasts and many inflammatory cells appeared when the wound tissue

covered with the developed composite films. Moreover, more granulation tissue was

observed in Film-3 group， at 14 days after initial treatment, squamous epithelium

appeared in all groups with films. It is noteworthy that skin injuries treated with the

Film-3 were almost completely covered with a new epithelium and many hair follicles

had grown. In contrast, the injuries were still at the late phase of inflammation in the

gauze treatment groups. Results demonstrated that Cur-loaded film (Film-3) could

facilitate the wound repair with a higher level of integrity, and while reduce

inflammation as compared to other groups. This was good consistent with the MTT

results that Cur-loaded composite film can promote primary human fibroblast cell

proliferation (Figure 4B). These results showed that Film-3 could be helpful to the

regeneration of squamous epithelium.

4. Conclusions
26
 A shape-controllable, CS/Aloe film uniformly embedded with Cur-loaded PLGA

microspheres was developed to improve wound healing and skin tissue regeneration.

An optimized method of ultrasonic emulsification achieved a uniform dispersion of

PLGA microspheres in CS/Aloe films. The CS/Aloe-PLGA@Cur films dressing

showed suitable physicochemical properties and flexibility which were suitable for

clinical applications and achieved promising antibacterial activity against both Gram-

positive bacteria and Gram-negative. In addition, the CS/Aloe-PLGA@Cur films

showed higher fibroblasts proliferation and anti-inflammatory properties,

consequently promoting to skin tissue regeneration. Therefore, our CS/Aloe-

PLGA@Cur films dressing may serve as an effective and translational tool for skin

tissue regeneration.

Acknowledgement

This research was supported via National Key Research and Development Progra

m of China (2017YFC1104102), National Natural Science Foundation of China (5150

3040 and 31370958), Key Program of Natural Science Foundation of Fujian Province

(2018Y0056), the Training Program of Fujian Excellent Talents in University (No.10)

, the Outstanding Youth Research Talent Cultivation Plan for Universities of Fujian

Province (601936) and Fujian New Century Excellent Talents in University (No. 31).

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Highlights:
 The composite films were prepared by an easy one step phacoemulsification method.

 PLGA@Cur microspheres were uniformly embedded in CS/Aloe film.

30
 The composite films possessed multiple anti-bacterial effects.
 The composite films have controlled shape to meet the different needs of wounds.
 Extended release property effectively promotes wound healing and skin
regeneration.

Uniform self-assembly PLGA@Cur microspheres in CS/Aloe film was fabricated via

an easy one step phacoemulsification technique. The curcumin-releasing chitosan/aloe
film showed excellent biocompatibility and biological properties, which exhibited a
promising regenerative potential.

31