Accepted Manuscript

Preparation and characterization of gellan gum/glucosamine/

clioquinol film as oral cancer treatment patch

Wanchi Tsai, Huifang Tsai, Yinuan Wong, Juiyen Hong, Shwujen

Chang, Mingwei Lee

PII: S0928-4931(16)31415-1
DOI: doi: 10.1016/j.msec.2017.05.040
Reference: MSC 8022
To appear in: Materials Science & Engineering C
Received date: 21 September 2016
Revised date: 24 April 2017
Accepted date: 4 May 2017

Please cite this article as: Wanchi Tsai, Huifang Tsai, Yinuan Wong, Juiyen Hong, Shwujen
Chang, Mingwei Lee , Preparation and characterization of gellan gum/glucosamine/
clioquinol film as oral cancer treatment patch, Materials Science & Engineering C (2017),
doi: 10.1016/j.msec.2017.05.040

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 ACCEPTED MANUSCRIPT

Preparation and characterization of Gellan gum/Glucosamine/Clioquinol film as oral

cancer treatment patch

Wanchi Tsaia, Huifang Tsaib, Yinuan Wongb, Juiyen Hongb, Shwujen Changc,
Mingwei Leeb,d,*
a
Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical
Universuty, Taiwan
b

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Department of Medical Laboratory and Biotechnology, Chung Shan Medical
University, Taiwan
c
Department of Biomedical Engineering, I-Shou University, Taiwan

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d
Department of Clinical Laboratory, Chung Shan Medical University Hospital,
Taiwan

Abstract
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To administer cancer drugs with improved convenience to patients and to enhance
the bioavailability of cancer drugs for oral cancer therapy, this study prepared Gellan
gum/Glucosamine/Clioquinol (GG/GS/CQ) film as the oral cancer treatment patch.
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GG/GS/CQ film fabricated through the EDC-mediated coupling reactions

(GG/GS/CQ/EDC film). The film of the physicochemical properties and drug release
kinetics were studied. The effectiveness of GG/GS/CQ/EDC film as oral cancer
treatment patch were evaluated with the animal model. The results confirmed that CQ
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can be incorporated via EDC-mediated covalent conjugation to gellan

gum/glucosamine. Mechanical testing revealed that the maximum tensile strength and
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elongation percentage at break were 1.91 kgf/mm2 and 5.01% for GG/GS/CQ/EDC
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film. After a drug release experiment lasting 45 days, 86.8% of CQ was released from
GG/GS/CQ/EDC film. The Huguchi model fit the GG/GS/CQ/EDC drug release data
with high correlation coefficients (R2= 0.9994, respectively). The effect of the CQ
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dose on oral cancer cells (OC-2) was tested, and the IC50 of CQ alone and CQ with 10
μM CuCl2 were 9.59 and 2.22 μM, respectively. The animal testing indicated that
GG/GS/CQ/EDC film was decreased epidermal growth factor receptor (EGFR)
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expression and suppress tumor progression. These findings provide insights into a
possible use for GG/GS/CQ/EDC film for oral ca in clinical practice.The
GG/GS/CQ/EDC film suitable as the dressing for use in the treatment of early-stage
cancer or as wound care after surgery in late-stage of oral cancer treatment.

Keywords
Gellan gum, Glucosamine, Clioquinol, Oral cancer treatment patch, EGFR
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1. Introduction
Every year, nearly half of a million patients worldwide are diagnosed with oral
cancer, and approximately 150,000 oral cancer patients die each year [1]. The normal
oral mucosa is generally pink in color. Leukoplakia and erythroplakia are the two
most common potentially malignant disorders of the oral cavity and are also
considered precancerous conditions. Leukoplakia is a condition in which thick white
and gray patches form inside the mouth or on the tongue as a result of chronic

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irritation. In erythroplakia, abnormal red areas or red spots form on the mucous
membrane lining the mouth, an area that often bleeds easily [2,3]. The treatment for
oral cancer is dependent on the stage of development of the cancer. Staging for oral

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cancer follows a classification into stages 0-4 [4]. In stage 0, tumor cells are localized
inside the oral mucosa epithelium; in stages 1-2, the diameter of the tumor is between

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2 cm and 4 cm, and there are no metastases to the neck lymph node. However, in
stages 3-4, the diameter of the tumor is more than 4 cm, or metastases are found in the
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neck lymph node and other tissues. Chemotherapy and radiotherapy are usually used
in early-stage oral cancer treatment, whereas surgery is the primary treatment in
late-stage oral cancer. An improvement of patient convenience is necessary for
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treatment with cancer drugs. The study aimed to design a gellan gum/glucosamine
(GG/GS) oral cancer treatment patch, which includes the chemotherapy drug
clioquinol (CQ), and that can be used not only in early-stage oral cancer therapy but
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also as a wound care dressing after surgery in late-stage oral cancer treatment.
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More than 100 chemotherapy or chemo drugs are used to treat cancer such as
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doxorubicin, cisplatin, paclitaxel et al [5]. Clioquinol (CQ) has been used for many
years as an antimicrobial agent and more recently as a potential for cancer therapies.
Clioquinol, a lipophilic compound capable of forming stable complexes with copper
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ions, is a potent proteasome inhibitor and inducer of apoptosis [6]. The following
research suggests that copper can be used as a novel selective target for cancer
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therapies. Daniel et al [7] have demonstrated that CQ induces cell death in malignant
cells by inhibiting the proteasome through a dual copper-dependent and -independent
mechanism. In addition, Mao and Schimmer [8] also demonstrated that CQ delays the
growth of tumors in mouse models of malignancy. Moreover, CQ has been used in the
treatment of many types of human cancer, including prostate, breast, colon, lung, and
brain cancer [9, 10]. Thus, we believe that CQ may be a novel anti-oral cancer agent
that could be repurposed for this new indication. CQ given directly to the oral cancer
area is quickly diluted by oral fluid and cleared. To enhance the bioavailability of CQ
for oral cancer treatment, we proposed GG/GS film as the CQ delivery carriers. In the
future, the film can be used for oral cancer therapy application.
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Nature polymers have attracted attention as matrix materials for controlled

release [11, 12] and gene delivery applications [13]. Polymers derived from microbial
source [14-16] are excellent candidate biomaterials due to their exceptional
biodegradability and biocompatibility. There are two major microbial macromolecules,
polyesters and polysaccharides, are used in drug delivery [17] and other medical
applications [18]. Gellan gum (GG) is an anionic heteropolysaccharide produced by
Sphingomonas elodea. Based on its excellent biocompatibility and nontoxicity and its
special physicochemical properties, gellan gum has been widely used as wound

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dressings and as drug delivery materials in previous studies. We previously utilized
gellan gum as a delivery material for anti-inflammatory agents to prevent tissue
adhesion in the postoperative abdomen. In addition, we prepared gellan gum films as

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wound dressings, demonstrated its good biocompatibility, and showed that it was
capable of accelerating wound repair [19]. Due to the brittleness of gellan gum film

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fabricated through EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide)
cross-linking [20], we used a mixture of gellan gum with glucosamine as the CQ
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delivery agent to improve the mechanical properties of gellan gum film. In this study,
CQ can be incorporated via EDC-mediated covalent conjugation to gellan
gum/glucosamine film (GG/GS/CQ/EDC film), the film of the physicochemical
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properties and drug release kinetics were studied [21, 22]. Finally, we developed
animal models for oral cancer and used this model to investigate the effectiveness of
GG/GS/CQ/EDC film as oral cancer treatment patch.
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2. Experimental methods
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2.1. Materials
Gellan gum, Glucosamine hydrochloride, MTT reagent were obtained from Sigma.
1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) was purchased from Acros.
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Clioquinol was purchased from Alfa Aesar. Cell culture medium (DMED), 10X
trypsin-EDTA, fetal bovine serum were purchased from Gibco. Zoletil 50 was
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obtained from Virbac. OCT-Polyethylene glycol was was obtained from Leica. All
chemicals used in this study were of reagent grade. The animal experiments in this
study were approved by the Chung-Shan Medical University Experimental Animal
Center. A rabbit anti-EGFR polyclonal antibody (St John’s Laboratory; subclass IgG)
was used at a 1:200 dilution. Immunohistochemistry (IHC) detection kits were
purchased from Enzo Life Sciences (product: HighDef™ red IHC chromogen (AP)).

2.2. Fabrication of GG/GS/CQ/EDC film

The GG/GS/CQ film was fabricated by mixing 28 mL of an aqueous solution of
gellan gum (1%), 7 mL of an aqueous solution of glucosamine (1%) and 50 mg of CQ
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(dissolved in 2 mL of DMSO) in a glass dish (diameter = 10 cm). This solution was

evaporated at 37°C and 1 atm for 3 days to obtain a dry GG/GS/CQ film. The
GG/GS/CQ film was then cross-linked by immersing them into DDW (distilled
deionized water) containing 15 mM EDC for 24 h at room temperature. The
cross-linked films were washed with DDW three times to remove residual EDC and
then dried at room temperature [23]. After crosslinking, the film was referred to as
GG/GS/CQ/EDC, respectively.

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2.3. Characterization of the GG/GS/CQ/EDC oral cancer treatment patch
We used an FTIR-L396A (Perkin-Elmer) to analyze the properties of the chemical
functional groups of the GG, GG/CQ, GG/CQ/EDC, GG/GS/CQ and

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GG/GS/CQ/EDC films. The analysis of the gel content and water content of the
GG/GS/CQ/EDC film was performed as follows. The GG/GS/CQ/EDC films were

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dried (2 cm x 2 cm pieces). The dry weight (Wd) was measured, and then the dried
films were swelled in phosphate buffered saline (PBS) at 37°C for 24 h. The wet
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weight (Ww) of the film was determined after wiping off excess water using filter
paper. The film was dried again in an oven for 24 h at 50°C, and its subsequent weight
was recorded as Wrd. The gel content and water content ratio were calculated as
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follows [19]:
Gel content % = (Wrd/Wd) x 100
Water content % =【(Wrd-Wd)/ Wrd】x 100
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2.4. Mechanical property measurements

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GG/GS/non-crosslink, GG/GS/EDC, GG/GS/CQ/EDC films were cut into 1 cm × 5

cm pieces. We then used an H1-KS testing machine (Tinius Olsen) with a crosshead
speed of 5 mm/min to measure the mechanical properties of these films and to
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automatically record the mechanical parameters.

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2.5. In vitro release studies

In vitro drug release studies were performed in 15 ml tubes. The GG/GS/CQ/EDC
films (1x1 cm2) were placed into the tubes and immersed in 1 ml of phosphate buffer
(0.02 M, pH 7.2). Samples (n = 5) were incubated at 37°C with shaking for 45 days.
At defined time points, 1 ml of the release buffer was withdrawn and replaced with
fresh buffer. The CQ content was determined spectrophotometrically at 255 nm. The
kinetics of CQ release from GG/GS/CQ/EDC film was determined by finding the best
fit of the dissolution data to one of five distinct models as previously described:
Zero-order、First-order、Second order 、Hixson-Crowell and Higuchi as follows [24,
25]:
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Table 1
Mathematical models used to describe drug dissolution curves
Zero-order Qt = Q0 + K0t
where Qt is the amount of drug dissolved in time t, Q0 is the
initial amount of drug in the solution (most times, Q0 = 0)
and K0 is the zero order release constant expressed in units of
concentration/time.

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First-order log Qt = log Q0 - kt/ 2.303
where k is the first order rate constant, and t is the time
Second order Qt/Q∞(Q∞- Qt)k2t

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where k2 is the second order rate constant, Q∞ is the amount

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of drug dissolved at infinite time
Hixson-Crowell Q01/3 –Qt 1/3 = kst
where ks is a constant incorporating the surface-volume
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relation.
Higuchi Qt = kHt1/2 where kH is the Higuchi dissolution constant
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2.6. Effect of CQ dose on cytotoxicity in oral cancer cells (OC-2)

Human oral cancer cells (OC-2) were plated in 96-well plates with 5,000 cells per
well. Following overnight incubation, cells were treated with 2, 5, or 10 μM of CQ
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with or without 10 μM of CuCl2 for 24 h. DMSO-treated cells served as a control. To

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quantify the cell viability, the medium was replaced with 150 μL of medium
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containing 10% MTT (Sigma-Aldrich). After 1 h of incubation at 37°C, the MTT

solution in wells was removed, and the formazan crystals within cells were
solubilized with 100 μL of DMSO [26]. The absorbance of each sample at 595 nm
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was measured by an enzyme-linked immunosorbent assay reader (Power Wave 340;

BioTek Instruments, Inc.) or a microplate reader (Bio-Rad Laboratories, Richmond,
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CA, USA). Cell viability was determined by normalizing the absorbance value of
each sample to that of DMSO-treated control cells and represented as the mean±S.D.
(standard deviation) of six independent experiments performed in triplicate.

2.7. In vivo evaluation of GG/GS/CQ/EDC film for oral cancer treatment

In vivo experiments were carried out with male Syrian golden hamsters aged 4
weeks obtained from National Laboratory Animal Center (Taipei, Taiwan). The 7,
12-dimethyl-1,2-benz[a]anthracene (DMBA)-induced hamster cheek pouch model of
carcinogenesis was modified from Salley [27]. The animals were treated by painting
the entire mucosal surface of the left cheek pouches three times a week for 14
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continuous weeks with a 0.5% solution of DMBA dissolved in mineral oil. After 14
weeks, we observed 100% oral tumor formation with severe histopathological
abnormalities in all the hamsters treated with DMBA (Fig.1.A). The tumor and
surrounding tissue was removed. The wound was then covered with GG/GS/CQ/EDC
film (Fig.1.B). Hamsters in the control group were not given any oral cancer treatment
patch. The experimental hamsters were sacrificed on the 7th day after surgery, and the
injured site with the film was removed and fixed in a 10% formalin solution. The
tissues were processed according to standard procedures for histological and

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immunohistochemical (IHC) analyses. In this study, we selected epidermal growth
factor receptor (EGFR) expression as the marker for evaluating cancer growth.

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(A) (B)
Fig.1. (A) DMBA-induced oral carcinogenesis in the hamster cheek pouch. (B)
Manipulation of the GG/GS/CQ/EDC film covering the wound after the tumor and
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surrounding tissue were removed.

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2.8 Statistical analysis

Each of the experiments was repeated at least five times, and the values are
expressed as the mean ± standard deviation. For comparison between two groups of
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data, Student’s t-test was performed. The differences were considered to be

statistically significant at P < 0.05.
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3. Results and discussion

3.1. FTIR characterization of gellan gum-based oral cancer treatment patches
Fig.2.A shows the FTIR spectrograms of GG, GG/CQ and GG/CQ/EDC films. In
the FTIR spectrum of GG, the peaks at 3332, 2894, 1600, 1403 and 1017 cm-1 are
attributed to the stretching vibrations of –OH, aliphatic –CH, asymmetric COO-,
symmetric COO- and hydroxylic C-O bonds, respectively. In the FTIR spectrum of
GG/CQ, the CQ introduced one particularly important peak at 1480 cm-1 that was
assigned to the stretching vibrations of C=C and C=N of heterocyclic aromatic ring
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[28,29]. The peak at 1201 cm-1 is due to the aliphatic C-N stretch in CQ [30, 31]. The
N-H groups of CQ are shown as stretching frequencies at 3400-3250 cm-1 and overlap
with the –OH of gellan gum. In the FTIR spectrum of GG/CQ after EDC
cross-linking, two new absorption peaks are visible at 1708 and 1556 cm-1. The
absorption peak at 1708 cm-1 indicates that carboxyl groups on the β-D-glucuronic
acid (Glcp A) of gellan gum can generate an ester bond with –OH groups and
confirms that the gellan gum cross-linking reaction was successful. The absorption
peak at 1556 cm-1 indicates that carboxyl groups on β-D-glucuronic acid (Glcp A) of
gellan gum can generate an N=O bond with the –NH groups of CQ and confirms that

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CQ can be covalently conjugated to gellan gum via an EDC-mediated reaction [32].
The FTIR spectral study was also performed to obtain the conformational

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stretching and bending vibrations of GG/GS/CQ and GG/GS/CQ/EDC films. The
FTIR spectrum of GG/CQ and GG/GS/CQ have similar functional group regions

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because they have similar functional groups. However, in the FTIR spectrum of
GG/GS/CQ, the stretching vibrations of C=C and C=N of the heterocyclic aromatic
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ring in CQ shift from 1480 to 1487 cm-1, and the aliphatic C-N stretch shifts from
1201 to 1197 cm-1. The FTIR spectrum of GG/GS/CQ cross-linked via EDC (Fig.2B)
also shows two new absorption peaks at 1705 and 1571 cm-1. These data confirm that
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CQ can be covalently conjugated to gellan gum but not to glucosamine via EDC [33,
34]. The reason is that CQ and glucosamine have the same functional group (amine),
making it difficult form covalent bonds between CQ and glucosamine. In this study,
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EDC is used to cross-link gellan via hydroxyls and carboxylic acids, and it cross-links
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gellan gum/glucosamine via amines and carboxylic acids. The drug CQ was also
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covalently coupled via EDC to form stable bonds with gellan gum/glucosamine.
Covalent bonding of CQ to gellan-based carriers can allow release for extended times
and reduce the necessary drug loading to help prevent adverse side effects.
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Fig.2.A FTIR spectra of the GG, GG/CQ and GG/CQ/EDC films.
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Fig.2.B FTIR spectra of the GG/GS/CQ/EDC film.

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3.2. Physical properties of GG/GS/CQ/EDC oral cancer treatment patches

Using the EDC-mediated cross-linking process, we fabricated GG/GS/CQ/EDC
oral cancer treatment patch, and we then studied their physical properties. The
average thickness of these films was 22 ± 4 μm. The prepared films were soft, flexible,
transparent, and capable of being fixed in position. The gel content of a film is related
to its crosslinking density [35]. The gel content of the GG/GS/EDC and
GG/GS/CQ/EDC films were 80.5±1.4 and 83.5±0.6 (P value < 0.05), respectively.
The results showed that CQ can increase the cross-linking degree of the gellan gum

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polymer. This is because CQ can form covalent bonds with gellan gum, which
increases the formation of an interpenetrating polymer network structure. Water
content is a basic property of polymer films. In general, the water content of a

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polymer film can be considered to be the sum of strong-affinity water and
weak-affinity water in the film. A film with high water adsorption may absorb excess

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water and expand, causing deformation and rendering it unsuitable as an oral cancer
treatment patch. Table 2. shows the water content of GG/GS/EDC and
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GG/GS/CQ/EDC films. In this system, the presence of CQ or glucosamine increased
the degree of gellan gum cross-linking, but the water absorption capacity of these
films showed a slight decrease. For the statistical test, we found no significant
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difference between the two groups (P value > 0.05). The results showed that the
addition of GS or CQ into gellan gum, which did not affect the water content of gellan
gum.
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For clinical use, the most important mechanical properties of oral cancer treatment
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patches are tensile strength and elongation percentage. Mechanical testing (Table 2)
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revealed that the maximum tensile strength and elongation percentage at break of the
non-cross-linked GG/GS film were 1.73 kgf/mm2 (16.95 MPa) and 3.52%,
respectively. The maximum tensile strength and elongation at break of the
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EDC-cross-linked GG/GS film were 2.11 kgf/mm2 (20.67 MPa) and 5.71%. It was
observed that tensile strength and elongation of the cross-linked film both increased.
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Vijayabaskar et al. [36] indicated that the tensile strength of a polymer is closely
correlated to the density of crosslinking and that cross-linking causes an increase in
the tensile strength and elongation percentage [37]. As shown in Table 2, in the
presence of CQ, the tensile strength and elongation percentage of the GG/GS/CQ
cross-linked film progressively decrease (1.91 kgf/mm2 and 5.01%). CQ is
a hydrophobic drug. The addition of CQ into the GG/GS hydrogel might decrease the
hydrophilic nature of GG and decrease its tensile strength and elongation, which
results in mechanical resistance [38].
A treatment patch for oral cancer therapy is not currently available as a product.
Thus, there is no standard reference for the physical and chemical properties of oral
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cancer treatment patches. In this study, we have completed an efficacy test and a
pharmacokinetic study of the GG/GS/CQ/EDC oral cancer treatment patch in an
animal model. The physical properties of the film can be used as the reference for
clinical applications in the future.
In addition, We have examined the degradation rate of GG/GS/CQ/EDC film and
GG/CQ/EDC film in pH 7.4 PBS solution at 37℃ for 28 days, the degradation rate of
both oral cancer treatment patches were less than 10%. To avoid the bacterial
contamination, the oral cancer treatment patch is recommended to replace every 3-7

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days. Hence, the slow degradation property of GG/GS/CQ/EDC film has
imperceptible effect on being an oral cancer treatment patch.

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Table 2.
Physical properties of the gellan gum-based films.

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sample Gel Water Mechanical Test
content content Maximun Maximun Elongation
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(%) (%) force (gf) strength of break
(kgf/mm2) (%)
GG/GS/non-cross - - 1040±34 1.73±0.05 3.52±0.42
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link
GG/GS/EDC 80.5±1.4 93.5±0.5 1266±78 2.11±0.13 5.71±0.90
GG/GS/CQ/EDC 83.5±0.6 92.4±0.6 1146±11 1.91±0.02 5.01±0.86
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3.3. In vitro release studies

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The in vitro drug release studies for GG/GS/CQ/EDC film was performed in
phosphate buffer (0.02 M, pH 7.2) as a representative medium. Using a
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spectrophotometer, the amount of CQ released was measured by absorbance at 255

nm. Fig.3. shows the percentage of CQ released from the GG/GS/CQ/EDC film over
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time. The experimental results show that the amount of CQ released from
GG/GS/CQ/EDC film increases with time. The plot of CQ release kinetics from
GG/GS/CQ/EDC has a steep slope, which is attributed to the faster degradation rate of
the GG/GS/CQ/EDC drug carrier matrix. After the release experiment had been
carried out for 45 days, the amount of CQ released from the GG/GS/CQ/EDC film
was 86.8%. CQ given directly to the oral cancer area is quickly diluted by oral fluid
and cleared. In this study, with CQ covalently conjugated to gellan gum/glucosamine,
approximately 6-8% (8.0-10.8 μM) of CQ was released from the film every other day,
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and sustained release was observed for up to 45 days. From the MTT cell viability
assay, we also confirmed that the IC50 value of CQ for human oral cancer cell line
OC-2 cells was 9.22 μM. These results indicated that the observed release rate of CQ
from GG/GS/CQ/EDC film meets the requirements for clinical applications.

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Fig.3. Release profile of CQ from the GG/GS/CQ/EDC film in PBS at 37°C.

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Drug release modeling and determination of the critical parameters of carrier

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systems are important for understanding and elucidating mechanical and drug
transport properties. This system uses gellan gum/glucosamine as a carrier of CQ;
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gellan gum is a biodegradable polysaccharide polymer with water-absorbing and

swelling properties. Diffusion, swelling and erosion mechanisms are the most
important rate-controlling mechanisms for controlled-release products. To better
characterize drug release from the polymeric systems studied, five common
pharmacokinetics models were used to analyze the release kinetics of CQ from the
GG/GS/CQ/EDC film. The model that best fits the CQ release data was selected
based on the correlation coefficient (R2) value obtained with various models. The
results are shown in Table 3. The results indicate that the Huguchi model fits the
GG/GS/CQ/EDC release data (Fig.4.) with high correlation coefficients (R2= 0.9994).
In 1961, the first example of a mathematical model aimed to describe drug release
from planar matrix system was proposed by Huguchi. This model is based on
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the hypotheses that (i) the initial drug concentration in the matrix is much higher than
drug solubility; (ii) drug diffusion takes place only in one dimension; (iii) drug
particles are much smaller than the thickness of the system; (iv) matrix swelling and
dissolution are negligible; (v) drug diffusivity is constant; and (vi) perfect sink
conditions are always attained in the release environment [39, 40]. Therefore, this
study confirmed that CQ is released from GG/GS/CQ/EDC film at a constant rate of
diffusion, without a burst effect, and these system are of a suitable design for
achievement of prolonged therapeutic action in vivo.

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Table 3.
Results of the kinetic models applied to the release of CQ from the GG/GS/CQ/EDC

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film.
GG/GS/CQ/EDC film

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Zero order fit linear equation
y=0.08924+0.00865x
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R2=0.9803
First order y=0.03965x-2.27493
R2=0.8767
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Second order y=10.73041-0.25427x

R2=0.63793
Hixson-Crowell y=0.4672+0.00773x
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R2=0.9262
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Huguchi y=0.07362x-0.04693
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R2=0.9994
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Fig.4. The Huguchi model fits the release of CQ from the GG/GS/CQ/EDC film.
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3.4. Effect of the CQ dose on OC-2 cell death

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To evaluate the cytotoxic effect of CQ, cultures of the human oral cancer cell line
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OC-2 were treated with DMSO or various concentrations of CQ for 24 h. To clarify

whether Cu2+ could influence the cytotoxicity of CQ, the CQ-treated cells were
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incubated with or without CuCl2, and then the cell viability was analyzed by an MTT
assay. As shown in Fig.5, exposure to CQ resulted in a dose-dependent decrease of
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cell viability in OC-2 cells, and such inhibition was further enhanced by the presence
of 10 μM of CuCl2. The IC50 of CQ alone and CQ with 10 μM of CuCl2 was 9.59 and
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2.22 μM, respectively. These data demonstrate the anti-cancer potential of CQ and the
synergistic cytotoxicity with copper. From these data, we determined that the amount
of CQ released from GG or GG/GS films reaches doses high enough to kill oral
cancer cells with or without CuCl2.
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Fig.5. Copper can enhance the cytotoxicity of CQ in OC-2 cells. CQ was administered

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at the indicated concentrations with or without 10 μM CuCl2 for 24 h, after which the
cell viability was measured by an MTT assay. Each bar represents the mean±SD (n=6).
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***P <0.001.

3.5. Histological and immunohistochemical analyses

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The epidermal growth factor receptor (EGFR) is the cell-surface receptor for
members of the epidermal growth factor family (EGF-family) of extracellular protein
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ligands [41, 42]. The EGFR signaling pathway is one of the most important pathways
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that regulates growth, survival, proliferation, and differentiation in mammalian cells.

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Overexpression of EGFR has been linked to oncogenic transformation, invasive

growth, metastasis and angiogenesis in multiple cancers. The results of histological
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and immunohistochemical analyses of the control group (without treatment patch),

GG/GS/CQ/EDC film-treated group are shown in Fig.6. The experimental groups all
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displayed oral squamous cell carcinoma (OSCC) and continual unregulated

proliferation and infiltration into the subcutaneous tissue [43-45]. In experimental
group, the cancer tissue areas were clearly smaller, the tumor cells were arranged
more loosely than that of the control group. The inhibition of tumor progression is an
important factor in cancer therapy. Use of the treatment patch at the wound site for
only seven days after the tumor and surrounding tissue were removed did not cause
complete suppression of cancer cell proliferation and migration. To determine the
molecular mechanism of tumor growth inhibition by the GG/GS/CQ/EDC film,
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EGFR expression in a Syrian golden hamster model of cancer tissue was analyzed. In
this study, cancer cells, marked by EGFR-positive immunoreactivity, will be labeled
as red spots. In the control group (Fig.6A.), the immunohistochemical reactivity to
EGFR was significantly increased in the cell membrane and cytoplasm of oral
squamous cells, but EGFR expression was observed at a lower level in cancer cells of
experimental group (Fig.6B). Here, we must explain the anti-tumor activity of CQ
was not because of the potentiation of inhibition EGFR or downstream signaling of

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EGFR [46]. CQ markedly activated apoptosis in the tumor cells. In this study, EGFR
was indicated to be a promising target for cancer therapy [47].The results of the

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animal testing indicate that GG/GS/CQ/EDC film can suppress OSCC progression.

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These results also confirmed that the dose of CQ released from the GG/GS/CQ/EDC
film meets the requirement for in vivo applications.
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(A) (B)
Fig.6. Immunoreactivity to EGFR in (A) control animals (without treatment patch), (B)
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the GG/GS/CQ/EDC film-treated group (200X).

4. Conclusion
The significance of this study is to provide a wound care and suppress cancer cells
proliferation after the tumor resection in the oral cavity. Current wound dressings are
designed for traumatic wound. The problems of wound from tumor resection not only
focus on the healing, but also the carcinogenesis of surrounding tissues. The strength
of our research team is to develop various wound dressings for different purpose, like
wound dressing for diabetes wounds, oral cancer wound patch, skin cancer wound
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patch, etc.
Worldwide, the incidence of oral cancer is approximately 2% - 4% of all cancer
cases with a high death rate. In this study, GG/GS/CQ/EDC film was prepared and
tested for use in the treatment of early-stage cancer or as wound care dressings after
surgery in late-stage of oral cancer treatment. The development of GG/GS/CQ/EDC
patch to replace chemotherapy and radiation therapy for oral cancer is not the main
purpose of this work. We want to improve the convenience of treatment with cancer
drugs for patients and to enhance the bioavailability of CQ for oral cancer therapy.

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The results showed that the film with Huguchi model release kinetics effectively
inhibit oral cancer progression in an animal model. Based on these dates, we consider
that the GG/GS/CQ/EDC film has great potential for future use in clinical

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applications.

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Acknowledgement
This research was supported in part by a grant from National Science Council of
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Taiwan.

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Highlights

 The drug carriers for oral cancer therapy application were prepared
by gellan gum and glucosamine.

 Anti-oral cancer potential of clioquinol (CQ) were confirmed by in

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vitro and in vivo test.

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 The work would give scientists and doctors new insights into the use
of oral cancer treatment patch in clinical practice.

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