Drug Development and Industrial Pharmacy

ISSN: 0363-9045 (Print) 1520-5762 (Online) Journal homepage: http://www.tandfonline.com/loi/iddi20

Development of a topical mupirocin spray for

antibacterial and wound healing applications

Rutthapol Sritharadol, Titpawan Nakpheng, Paul Wan Sia Heng & Teerapol
Srichana

To cite this article: Rutthapol Sritharadol, Titpawan Nakpheng, Paul Wan Sia Heng & Teerapol
Srichana (2017): Development of a topical mupirocin spray for antibacterial and wound healing
applications, Drug Development and Industrial Pharmacy, DOI: 10.1080/03639045.2017.1339077

To link to this article: http://dx.doi.org/10.1080/03639045.2017.1339077

Accepted author version posted online: 05

Jun 2017.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=iddi20

Download by: [Cornell University Library] Date: 07 June 2017, At: 20:40
Development of a topical mupirocin spray for antibacterial and wound
healing applications

Rutthapol Sritharadol a, Titpawan Nakpheng b,c, Paul Wan Sia Heng d,

Teerapol Srichana a, b, c *

a
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences,
Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

b
Nanotech-PSU Excellence Center on Drug Delivery System, Faculty of
Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla 90112,
Thailand

D
c
Drug Delivery System Excellence Center, Faculty of Pharmaceutical Sciences, Prince
of Songkla University, Hat Yai, Songkhla 90112, Thailand

d TE
Department of Pharmacy, Faculty of Science, National University of Singapore, 18
EP
Science Drive 4, Singapore S117543, Singapore
C

*Corresponding author: Teerapol Srichana,

AC

E-mail address: teerapol.s@psu.ac.th

Tel: +66 74288979. Fax: +6674288979.

ST

Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince

of Songkla University, Hat Yai, Songkhla, 90112, Thailand.
JU
Development of a topical mupirocin spray for antibacterial and wound healing
applications

Objective: The aim of this study was to develop mupirocin topical spray using

Eudragit E100 as a film-forming agent for treatment of bacterial skin infections as

well as to promote wound healing.

Materials and methods: Twenty-seven of mupirocin formulations were

formulated containing Eudragit E100 and other excipients. Mupirocin spray was

prepared by aerosol crimping and filling machine using HFA-134a as a

propellant. The formulations were evaluated for their stability and

physicochemical properties. The factorial study was applied to evaluate the

D
effects of glycerol and PEG400 on mupirocin-loaded Eudragit E100 films. The
optimized formulation was assessed of drug release, antibacterial activities, and in

TE
vitro cell line studies in comparison to the ointment formulation.

Results and discussion: Mupirocin sprays were formulated and optimized to

EP
obtain the formulation with excellent physicochemical and mechanical properties
of the dressing film. The formulation had an excellent stability up to a year with
more than 80% of mupirocin content. Mupirocin was released from the film up to
C

90% within 2 h. The formulation had a potent antibacterial effect against S.

AC

aureus and S. epidermidis. The formulation was safe to use as a topical

formulation that had no toxicity to keratinocytes, fibroblasts, and monocytes. The

formulation also had an antiendotoxin effect without stimulating the production
ST

of NO and inflammatory cytokines (IL-1β and TNF-α).

Conclusion: Mupirocin topical spray was successful developed as a topical

JU

formulation and can be used instead of the ointment formulation. Animal

experiments are warranted to further emphasize the safe use in the human skin.

Keyword: Mupirocin, Topical spray, Eudragit E100, Anti-infectives,

Biomaterials, Wound healing

1. Introduction

An open wound is an injury involving an external or internal break of body

tissue, usually involving the skin. The main complication is the risk of infection caused

by entry of microorganism especially bacteria since the outer layer of the skin will be

breached, resulting in loss of the protection barrier against environmental contaminants

(Chiller et al., 2001; Church et al., 2006; Rhody, 2000). It is, therefore, advisable to treat

the wound with a topical antimicrobial agent so as to reduce the likelihood of

opportunistic microorganisms settling down in the wound and to decrease any risk of

systemic infections (Sarabahi, 2012; Spann et al., 2003).

D
The most commonly used topical antibiotics include neomycin, bacitracin, silver

TE
sulfadiazine, and mupirocin (Sarabahi, 2012; Spann et al., 2003). However, there are
EP
contraindications governing their use. Silver nitrate, for example, is limited in burn

wounds because the application of silver over a large body surface area may lead to
C

electrolyte imbalance. Neomycin has been reported to cause allergic contact dermatitis
AC

when applied to intact skin. Bacitracin is not indicated for the treatment of chronic

ulcers because of the increased risk of sensitization while silver sulfadiazine should be
ST

avoided in patients with known hypersensitivity to sulfur-containing drugs (Church et

al., 2006).
JU

Mupirocin is highly active against aerobic Gram-positive cocci such as S. aureus,

S. epidermidis as well as some Gram-negative cocci including methicillin-resistant S.

aureus (MRSA) (Spann et al., 2003). Mupirocin (or pseudomonic acid A) is a fermentation

product of Pseudomonas fluorescens, a compound that exerts antibacterial activity by

inhibiting protein synthesis through preventing the incorporation of isoleucine into

proteins by binding to isoleucyl transfer-RNA synthetase. Mupirocin is commercially

available as a 2% cream or ointment in a hydrophilic base. The indications include

prophylaxis use in ulcers, operative wounds, burns, and treatment of skin infections

(GlaxoSmithKline, 2001).

A topical formulation contains a vehicle or carrier for the drug compound and is

optimized for its application to a particular part of the body or type of skin condition.

The formulation may design to be moisturizing or maximizing the penetration of an

active ingredient into or through the skin. The rate and amount of absorption depend on

the local skin thickness and barrier properties, the physicochemical properties of the

D
drug molecule such as molecular size and lipophilicity, and the concentrations of active

TE
and excipients in the formulation. Topical formulations have been prepared in various
EP
forms including solution, lotion, semi-solid (cream, ointment, gel, paste), aerosol or

spray, powder, solid film, and transdermal patch. However, certain preparations have
C

limitations. Cream, for example, requires a preservative to extend the shelf life, and this
AC

may increase the risk of skin allergy. Stinging sensations are often experienced

following application of lotions or creams, and sometimes, with ointments (Bolognia et

ST

al., 2010).

Spraying a topical formulation onto a wound to form a thin film offers certain
JU

advantages. The film applied directly to the wound with a known drug dose provides

physical protection, and prevents entry of microorganisms from the external

environment. Film flexibility permits adaptation to body contours, and the film

mechanical properties may be increased by repeated spraying to build up several

overlapping layers of the film. Importantly, control over the permeation of air and
moisture is also feasible so as to maintain a favorable environment for wound healing,

and to avoid maceration due to excess moisture accumulation (Cutting and White, 2002;

Jolly and Jolly, 2011). According to the principle of moist wound healing that led to the

development of wound dressings to promote the healing process for all types of wound.

We hypothesized that mupirocin developed as a topical spray using Eudragit

E100 can provide a thin film for wound healing and have superior properties to the

ointment. In this study, the physicochemical and mechanical properties of the films

along with drug stability have been evaluated. The in vitro drug release, antibacterial

activity, cytotoxicity and wound healing potential in cell line studies of the optimized

D
spray formulation were investigated and compared with a conventional ointment

formulation.
TE
EP
2. Materials and Methods
C

2.1 Materials
AC

Mupirocin was purchased from Kirsh Pharma Asia Pacific Pte. Ltd. (International

Business Park, Singapore). Mupirocin ointment (Bactroban, Lot: 4BA12, SmithKline

ST

Beecham, Rizal, Philippines) was acquired from the drugstore. Eudragit E100 was

supplied by Evonik Industries (Essen, Germany). Glycerol was from P. C. Drug Center
JU

Company (Bangkok, Thailand). PEG400 was from Srichand United Dispensary

(Bangkok, Thailand). Absolute ethanol and acetone were purchased from RCI Labscan

(Bangkok, Thailand). HFA-134a propellant was from Mexichem (Cheshire, UK). Glass

aerosol canisters (15 mL volume) were obtained from Schott (Mainz, Germany) and
metering valves from Bespak Europe Ltd (Norfolk, UK). All solvents, chemicals and

biochemicals used were analytical grade.

2.2 Bacterial strains and cell line

Standard bacterial strains including Staphylococcus aureus (S. aureus, ATCC-25923),

Staphylococcus epidermidis (S. epidermidis, ATCC-35983) and Pseudomonas aeruginosa

(P. aeruginosa, ATCC-27853) were purchased from American Type Culture Collection

(ATCC, Manassas, VA, USA). Streptococcus suis (S. suis) was purchased from the

Thailand Institute of Scientific and Technological Research (Bangkok, Thailand).

D
TE
Lipopolysaccharide (LPS) of Escherichia coli (E. coli) was purchased from Sigma-

Aldrich (St. Louis, MO, USA). Human keratinocyte (HaCaT) cell line was purchased
EP
from Cell Lines Service GmbH (Eppelheim, Germany). Human fibroblast (BJ) cell line

and mouse’s macrophage (RAW264.7) cell line were purchased from ATCC.
C
AC

2.3 Preparation of mupirocin spray

A full-factorial design was used to investigate the effect of Eudragit E100 (film-forming
ST

agent), glycerol (humectant and plasticizer) and PEG400 (plasticizer) on the properties

of mupirocin spray formulations using the IBM SPSS Statistics for Windows, version
JU

20.0 software (IBM Corp., USA) for generating a total of 27 formulations. Product

concentrates containing 4% w/v of mupirocin were prepared with three different

concentrations of 2, 4, and 10% w/v of Eudragit E100, respectively, together with

glycerol and PEG400. The components of each product concentrate were dissolved in a

4:1 v/v cosolvent mixture of absolute ethanol and acetone by stirring at 1,500 rpm using
a magnetic stirrer for 30 min in a closed container. Aliquots of each product concentrate

(6 mL) were added to individual glass aerosol canister (15 mL volume, 20 mm neck

diameter), and crimped using a 50 μL metering valve as shown in Fig. 1. HFA-134a (6

mL) was introduced into the canister using the aerosol crimping and filling machine

(model 2016, Pamasol Willi Mäder, Zurich, Switzerland). The final spray formulations

comprised 2% w/v mupirocin solution with 1, 2, or 5% of Eudragit E100 as shown in

Table 1.

2.4 Evaluation of physicochemical properties

D
TE
Physicochemical properties were carried out concurrently with evaluation of films

properties and stability to screen the appropriate formulation for the skin. The best
EP
formulation was tested in antibacterial activities and in vitro cell line studies.
C

2.4.1 pH, density, and viscosity

AC

The pH of mupirocin liquid spray formulations was measured in triplicate using a pH

meter (FE20/FG, Mettler Toledo, Greifensee, Switzerland) with a non-aqueous probe.

ST

The pH meter was calibrated using standard solutions of pH 4, 7, and 10. The fluid

density was determined using a pycnometer and calculated as g/mL. The viscosity of
JU

mupirocin liquid spray formulations was measured at 35 °C using a Brookfield

viscometer (model DV-III, Brookfield Engineering Laboratories, MA, USA) at spindle

rotational speeds of 100, 150, and 200 rpm.

2.4.2 Surface tension and contact angle

The surface tension of mupirocin liquid formulations was measured by drop shape

analysis using a contact angle meter (OCA 15 EC, Dataphysics Instruments GmbH,

Filderstadt, Germany) to investigate the spreadability. A drop of mupirocin solution was

ejected through a 1 mL glass needle where the droplet was photographed, and from its

shape, the surface tension was calculated by the software. The contact angle of the

solution on a cellulose tubular membrane (Cellu Sep, Membrane Filtration Products Inc.,

Seguin, TX, USA) was measured using the contact angle meter. The contact angle was

D
also determined by imaging a water droplet delivered by an automatic syringe on the

film, and the contact angle calculated by the software.

TE
EP
2.5 Preparation of mupirocin-loaded Eudragit E100 films and evaluation

As the sprayed film from the canister was extremely thin, harvesting it or the
C

determination of its mechanical property was not feasible. Therefore, the thicker films
AC

were casted to investigate the effect of spray composition on the properties using

factorial design. Nine formulations of mupirocin-loaded Eudragit E100 film were

ST

prepared for optimization containing 2% w/v mupirocin and 20% w/v Eudragit E100.

The ratios of plasticizer:Eudragit E100 (P:E) were 0.5, 0.75 and 1 whereas
JU

PEG400:glycerol (PEG400:G) was in a ratio of 1:6, 1:1 and 6:1, respectively (Table 2).

These ratios were similar to that of the spray formulations in Table 1 except the amount

of each ingredient in the film increased until the film formed a thick sheet. Therefore a

much thicker film is able to be measured its mechanical properties. This is based on the

assumption that the films properties can be scalable. The films were produced by
solvent evaporation whereby 10 mL of solution was transferred onto an 80 mm x 80

mm polytetrafluoroethylene (PTFE) plate and dried in the fume cupboard at room

temperature for 24 h. The dried uniform films were harvested, rolled with an aluminum

foil and stored at room temperature until needed for testing.

2.5.1 Film’s thickness, surface morphology, and transparency

The film thickness was measured using a micrometer (293-721-30 CE, Mitutoyo,

Kawasaki, Japan). At least three spots from different regions of each film were

measured, and results were averaged. The surface morphology of plasticized and non-

D
plasticized film was examined using a scanning electron microscope (JSM-6010LV,

TE
JEOL, Tokyo, Japan). Film transparency was measured by spectrophotometry (UV-1201,
EP
Shimadzu, Kyoto, Japan). The percentage light transmittance at 600 nm through length-

width of 40 mm x 25 mm sample was recorded.

C
AC

2.5.2 Mechanical properties

The tensile properties of 50 mm x 20 mm film samples were determined using a tensile

ST

testing instrument (EZ Test, Shimadzu, Kyoto, Japan) with a 100 N capacity-loaded cell.

The initial sample gauge length was 40 mm, and the extension speed was 30 mm/min.
JU

The film tensile strength (τ, N/mm2) and percentage elongation at break (ε) were

computed from the stress-strain profile using the following equations, where Lmax is the

maximum force (N), Ai is the initial cross-sectional area of the sample (mm2), ΔIb is the

increase in length at break (mm), and Ii is the initial gauge length (mm).
Tensile strength (τ)

𝐿𝑚𝑎𝑥
𝜏= 𝐴𝑖

Elongation at break (𝜀)

∆𝐼𝑏
𝜀=
𝐼𝑖

2.5.3 Film’s wettability and water content

The film’s wettability was examined by measuring the contact angle of a water droplet

D
on the films using contact angle meter (FTA200, First Ten Angstroms Inc., Portsmouth,

TE
VA, USA). A droplet of water was placed on the film surface with an automatic syringe.
EP
The droplet was then photographed and calculated the contact angle by FTA200

software.
C

The moisture content of the films was determined by Karl Fischer analysis (701
AC

KF Titrino, Metrohm, Herisau, Switzerland). Samples (200 mg) were accurately weighed

and dissolved in methanol before titration. The moisture content was expressed as a
ST

percentage of the film weight.

JU

2.5.4 Optimization of the film’s properties parameters

The MODDE software (Version 10.1.1, Umetric Inc., San Jose, CA, USA) was used to

evaluate the effect of spray composition on film properties of the resultant solid films

and optimize the formulation. Full factorial design was used to produce the total of 9

formulations of mupirocin-loaded Eudragit E100 film and polynomial regression

analysis was applied to study the effect of liquid spray composition (plasticizer:Eudragit

E100 ratio, X1 and PEG400:glycerol ratio, X2) on film tensile strength, elongation at

break, light transmittance, contact angle, and water content. The most promising

mupirocin spray formulation was chosen for further investigation in drug release,

solvent residues, antibacterial properties, and cell line studies.

2.6 Stability evaluations of mupirocin spray

2.6.1 Physical stability and sprays parameters evaluation

D
The physical stability and sprays performance of mupirocin formulations was observed

visually for color change and formulation precipitation. Sprays parameters of the

TE
optimized mupirocin formulation were evaluated for drying time, stickiness, and film on
EP
the forearm skin after spraying from a distance of 10 cm. The water-resistance property

of the optimized sprayed film against washing by rinsing with water for 15 s was also
C

evaluated. Unstable formulations were excluded from further investigation. The

AC

remaining formulations were stored at room temperature, and the physical stability was

assessed at periodic intervals of 3 months, for up to one year.

ST

2.6.2 Content of mupirocin delivered by actuation of the valve

JU

The amount of mupirocin delivered from the spray canister per valve actuation was

determined by actuating spray 10 times into a beaker of 30 mL ethanol and the amount

of drug deposited analyzed using high performance liquid chromatography (HPLC)

(Waters 1500 series, Waters Associates Inc., Milford, MA, USA) according to United

States Pharmacopeia 30 (USP30) monograph for mupirocin. The content uniformity of

the liquid formulation was determined at the initial (3rd-12th doses), middle (101st-110th

doses) and last doses (191st-200th doses) of the canister after a year’s storage under room

temperature. HPLC analysis used a 4.6 mm x 25 cm column containing reverse phase

octadecyl silane chemically bonded to porous silica. The mobile phase comprised a

75:25 phosphate buffer (pH 6.3)-acetonitrile mixture, filtered through a 0.45 μm filter and

degassed. The flow rate was 2 mL/min, and detection by UV at a wavelength of 229 nm

(2998 PDA detector, Waters Associates Inc., Milford, MA, USA).

2.7 In vitro mupirocin release from the optimized mupirocin spray

D
TE
Drug release from mupirocin spray in vitro was measured using Franz diffusion cells

(Variomag Telesystem, Thermo Fisher Scientific Inc., Waltham, MA, USA). The
EP
optimized mupirocin formulation was sprayed once onto a polyamide membrane

(Sartorius Stedim Biotech GmbH, Gottingen, Germany). Fifty milligrams of mupirocin

C

ointment (2% w/w, Bactroban, Lot: 4BA12, SmithKline Beecham, Rizal, Philippines)
AC

contained 1 mg of mupirocin equivalent to that of the spray formulation, was used as a

control formulation. After rinsing the compartments with isotonic phosphate buffer
ST

solution, a 5 mm x 2 mm stirrer bar was loaded into the receptor chambers prior to

filling with isotonic phosphate buffer solution. The polyamide membrane coated with
JU

the optimized formulation or ointment was mounted to cover the receptor compartment,

and the donor compartments were tightly clamped in vertical position. The Franz cells

were retained in a water bath at 37 ̊C (PolyScience, Niles, IL, USA). Samples (1 mL) of

receiver fluid were collected at 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h before analysis by

HPLC. An equal volume of preheated isotonic phosphate buffer solution was added to
the receptor compartment after sampling.

2.8 Analysis of solvent residues following spraying of the optimized mupirocin

spray

The solvent residue following spray discharge was analyzed by gas chromatography-

flame ionization detection (GC-FID) (HP 6850 GC, Agilent, Santa Clara, CA, USA)

using helium as the carrier gas. The optimized mupirocin formulation was sprayed 10

times into a glass container from a distance of 10 cm, before rinsing with acetonitrile

(500 µL) and injecting into the GC-FID instrument with a HP-INNOWax polyethylene

D
glycol capillary column of dimensions of 30 m x 0.25 mm x 0.25 µm. The inlet

TE
temperature was 220 °C, and the column gas flow rate was set at 1 mL/min. The initial
EP
temperature was maintained at 40 °C for 4 min, then ramped to 65 °C at 20 °C/min, held

for 3 min, ramped to 220 °C at 50 °C/min and maintained at 220 °C for 5 min. The
C

detector temperature was held at 220 °C. The sample injection volume was 1 µL with a
AC

50:1 split ratio. Total run time for an injection was approximately 15 min, including

temperature re-equilibration and pre-injection washes. Data were recorded and analyzed
ST

using instrument software to quantify the solvent residues.

JU

2.9 Antibacterial activities

2.9.1 Agar diffusion antibiotic sensitivity testing

The agar diffusion method was employed to determine the zone of inhibition against

bacterial strains of S. aureus, S. epidermidis, S. suis and P. aeruginosa. All strains were

incubated in brain heart infusion (BHI) broth (Becton, Dickinson and Company, Buena,
NJ, USA) at 37 °C for 18 h, and centrifuged at 3,500 rpm for 5 min. The bacterial pellet

was dispersed in 10 mL normal saline solution, and the turbidity was adjusted to 0.5

McFarland with equivalent to 108 CFU/mL. Disc diffusion was performed by placing

four discs on the agar surface, and adding 10 µL of the optimized mupirocin spray or 10

mg of mupirocin ointment onto each disc which equivalent to 200 µg of active

ingredient. The plates were incubated at 37 °C for 18 h before measuring the diameter of

the inhibition zone.

2.9.2 Antiendotoxin effect of mupirocin spray using RAW 264.7 cells

D
Antiendotoxin activity of the optimized mupirocin spray was investigated by assay of

TE
NO and inflammatory cytokine (IL-1β and TNF-α) production by RAW264.7 cells after

stimulation by LPS of E. coli. RAW 264.7 cells were seeded at a density of 1x106
EP
cells/well into a 96-well plate and incubated at 37 °C with 5% CO2. After 24 h, the culture
C

medium RPMI-1640 (Gibco, Grand Island, NY, USA) was replaced with fresh medium.
AC

Cells were stimulated with 5 µg/mL of LPS in the presence (1 mg/mL) or absence of the

optimized mupirocin spray and mupirocin ointment. After incubation for 18 h,

ST

supernatant (50 µL) was collected and assayed for nitric oxide by the Griess reaction.

Griess reagent (100 µL) was mixed with 50 μL of the cell culture supernatant and
JU

incubated at room temperature for 10 min. The absorbance was quantified using a

microplate reader (Biohit 830, Helsinki, Finland) at 540 nm. IL-1β and TNF-α production

was measured using mouse IL-1β and TNF-α ELISA test kits (R&D Systems Inc.,

Minneapolis, MN, USA). The level of cytokine production was also determined by
measuring the absorbance at a wavelength of 450 nm.

2.10 In vitro cell line studies

2.10.1 Cell viability assay

Human keratinocytes (HaCaT) and fibroblasts (BJ cells) were used to examine the effect

of mupirocin liquid spray formulations on cell viability. HaCaT and BJ cells were

cultured in Dulbecco’s Modified Eagle Medium (DMEM) and Eagle’s Modified Eagle

Medium (EMEM), respectively supplemented with 10% fetal bovine serum (FBS) and

D
antibiotics (100 U penicillin and 100 U/mL streptomycin) at 37 °C with 5% CO2. The

TE
cells were trypsinized, centrifuged at 130xg, 4 °C for 5 min, and seeded into a 96-well

plate (Corning Costar, NY, USA) at a density of 1×106 cells/well. The toxicity of the
EP
optimized mupirocin spray and mupirocin ointment was investigated using the methyl

thiazol tetrazolium (MTT) assay. Culture medium was used as a control. Briefly, the
C
AC

optimized mupirocin formulation was sprayed once into 2 mL of culture medium

(containing 1 mg of mupirocin), and mupirocin ointment (50 mg) was dissolved in 2 mL

of medium, respectively to produce a drug concentration of 500 µg/mL. Aliquots (100

ST

µL) were loaded into 96-well plates containing cells that had been seeded for 18 h, and
JU

incubated overnight. The medium was replaced with 100 µL of the fresh medium along

with 50 µL of MTT solution and incubated at 37 ºC under 5% CO2 for 4 h. The media

containing MTT were removed, and 100 µL of DMSO was added. The absorbance was

determined by a microplate reader at a wavelength of 570 nm. The percentage of cell

viability was calculated and compared to control.

2.10.2 In vitro scratch assay

HaCaT and BJ cells were used to evaluate the wound healing effect through spreading

and migration capabilities using the scratch assay. The cells at a density of 1x106

cells/mL were seeded in the 6-well plate. When the monolayer formed, a linear scratch

was generated with a 10 μL sterile pipette tip, then washing with phosphate buffered

saline (PBS). The medium was replaced with 1 mg of mupirocin from the optimized

mupirocin spray and mupirocin ointment which were dissolved in 3 mL of medium.

Cells without any formulations were served as a control. Photographs were taken using a

D
microscope at 0, 12, 24 and 48 h. The images acquired for each sample were further

TE
analyzed quantitatively by using computer software (Image J 1.42q/Java 1.6.0.10). The

distance of each scratch closure (n=10) was determined and the percent migration rate
EP
was calculated using the following equation:
C

𝐴𝑣𝐿day 0 -𝐴𝑣𝐿𝑑𝑎𝑦 2
Percent migration rate = ( ) × 100
𝐴𝑣𝐿day 0
AC

Where 𝐴𝑣𝐿 = average length between scratch

ST

2.10.3 Determination of collagen production

JU

The effect of mupirocin liquid spray formulations on collagen production was

investigated using BJ cells. The cells were cultured as described in section cell viability

and seeded into a 24-well plate at a density of 1×106 cells/well. The amount of soluble

collagen type I was assayed using the soluble collagen assay kit. Mupirocin spray and

ointment were used as experimental groups. Briefly, the optimized mupirocin

formulation was sprayed once into 2 mL culture medium (containing 1 mg of

mupirocin), and mupirocin ointment (50 mg) was dissolved in 2 mL of medium,

respectively, to produce a drug concentration of 500 µg/mL. Aliquots (200 µL) were

added to the seeded cells in 24-well plates and incubated overnight. Culture medium was

used as a control. Next, 100 μL supernatants were collected and mixed with 300 μL of

dye reagent (SircolTM Soluble Collagen Assay, Biocolor Ltd., County Antrim, UK)

under room temperature for 10 min. The samples were centrifuged at 15,000xg for 10

min. The supernatant was removed and the pellet was redissolved in 500 μL of alkali

D
reagent (SircolTM Soluble Collagen Assay, Biocolor Ltd., County Antrim, UK). The

TE
solutions were transferred to a 96-well plate and assayed using a microplate reader at a

wavelength of 540 nm. The amount of collagen was calculated based on a standard
EP
curve of soluble collagen concentration (Biocolor Ltd., County Antrim, UK).
C

2.11 Statistical analysis

AC

All data were presented as mean ± standard deviation (SD) at least three replicates

(unless indicated). The statistical significance was determined by paired or unpaired

ST

Student's t-tests. Significant differences between the means of multiple groups were
JU

identified using one-way analysis of variance (one-way ANOVA) with p-value < 0.05

considered statistically significant.

3. Results and discussion

3.1 Formulations and physicochemical properties of mupirocin spray

A total number of 27 mupirocin-containing liquid spray formulations were prepared

according to the excipient compositions as shown in Table 1. Polymethacrylates were

primarily used as film-coating agents in pharmaceutical dosage forms. Films of

different solubilities can be produced based on the polymer functional groups. Eudragit

E100 composed of polybutyl methacrylate, 2-dimethylaminoethyl methacrylate, methyl

methacrylate in a ratio of 1:2:1. It is widely used in topical formulations and

transdermal drug delivery system as a film-forming agent because of nontoxic,

D
nonirritant, and safe in humans (Hu et al., 2011). In addition, the concentrations of 5 to

TE
20% of the dry polymer can be used to control the release of active substance (Rowe et

al., 2009). A cosolvent of ethanol and acetone was used to dissolve hydrophobic drug
EP
and polymer. Ethanol is widely used as a solvent in pharmaceutical formulations and
C

cosmetics. Topical ethanol can be used in the transdermal drug delivery system as a

penetration enhancer and co-surfactant (Krishnaiah et al., 2008; Kweon et al., 2004).
AC

Acetone is useful to serve as a cosolvent in topical preparations since its high

evaporation rate and acetone showed a preferred combination of solvents where all
ST

components have a good solubility. The 4:1 v/v cosolvent mixture of absolute ethanol

and acetone is suitable for the spray system. This ratio facilitates evaporation time of the
JU

solvents in a few second after discharging from the spray canister to skin, and it was

found that the increase in the ratio of acetone higher than 4:1 (>20% v/v acetone)

resulting in a precipitation of spray formulation.The Glycerol in a concentration less

than 30% serves as a humectant and emollient in topical formulations and also has a

plasticizing effect in film-coating formulations (Rowe et al., 2009). Polyethylene

glycols are widely used in various pharmaceutical formulations. PEG400 is useful as a

plasticizer and essentially nonirritant to the skin (Rowe et al., 2009). Moreover,

copolymer network of polyethylene glycols with poly(methacrylic acid) have been used

as bioadhesive controlled drug delivery formulations (Peppas, 2004). The formulations

were then evaluated for their physicochemical properties such as pH, density, viscosity,

surface tension and contact angle as shown in Fig. 2.

The pH values of the liquid formulations were in the range of 6.1 to 7.4 which is

suitable for human skins (Gohel and Nagori, 2009), thus mupirocin formulations are

suitable for the topical application. For example, the growth of S. aureus is strongly

inhibited under acidic condition (Lambers et al., 2006). Therefore, it is expected that

D
this pH of the formulations will enhance the antibacterial activity.

TE
The densities of the liquid spray formulations were confined to a fairly narrow
EP
range of 0.81 to 0.90 g/mL and were largely unaffected by the spray composition. The

measured viscosity values were between 2.4 to 13.8 cP and increasing excipient
C

concentration, particularly Eudragit E100 (Formulations #19-27), increased viscosity.

AC

The viscosity of the formulations could affect the spray performance, causing difficulty

in spraying and resulting in a low content of available drug. However, the viscosity of

the liquid formulations prepared was still in the range that they could easily be sprayed.
ST

The surface tensions of the formulations were generally low, in the range of 21.0

to 26.0 mN/m2, suggesting good spreadability when applied to the skin (Zheng, 2010).
JU

The cellulose membrane was used to mimic human skin in contact angles measurement

(Tas et al., 2003). The contact angles of liquid formulations were between 10.9 to 26.1

degrees on a cellulose membrane. This range indicates potentially good wettability.

Contact angles less than 50 degrees suggest that the liquid will spread with ease over a
surface. The liquid forms a thin film, when the contact angle approaches a very low

value (Chibowski and Jurak, 2013).

3.2 Preparation of mupirocin-loaded Eudragit E100 films and their properties

According to Table 2, nine liquid formulations with different ratios of P:E and

PEG400:G were obtained as uniform solid films and evaluated

physicochemical properties (Table 3). Film thickness varied from 0.40 to 0.55 mm,

which depended on the amount of solids added to the formulations. Stress-strain curves

of tensile tests serve to quantify film’s mechanical properties. Increasing the PEG400

content of the films resulted in a reduction in tensile strength and an increase in film

D
elongation, demonstrating the plasticizing effect of PEG400 (Chan et al., 2005).

TE
Possibly, the presence of the plasticizer may decrease the number of binding sites

between polymer-polymer contacts, reducing the rigidity of the polymer structure (Chan
EP
et al., 2005). In this study, the results show PEG400 with a greater plasticizing action

than glycerol, and when the ratio of the P:E or PEG400:G increased (Formulations #1-3,
C

4-6 and 7-9).

AC

The contact angle of mupirocin-loaded films decreased as the PEG400 content

increased, indicating improved wettability because of the well-known hydrophilic

ST

property of PEG400. The water contents in films were in the range of 1.4 to 2.2% w/w

and tended to increase with increasing glycerol content, reflecting its good water
JU

absorbing properties (Nashed et al., 2003). A large amount of water also affected the

mechanical properties of the films because of the hydrophobic property of Eudragit

E100. Therefore, films with a large amount of water lead to hard and brittle defined by a

high tensile strength and low elongation at break. On the other hand, desired films with

a little amount of water lead to soft and weak which defined by a low tensile strength

and high elongation at break.

 Uniform and transparent mupirocin-loaded films were obtained when the

PEG400 and glycerol contents were limited to 1% w/v. The film clarity is an advantage

for clinical examination of the underlying wound. However, haziness was observed with

increasing PEG400 content, which adversely affected film transparency as shown in

Table 3.

The cross-sectional surface morphology of mupirocin-loaded films is shown in

Fig. 3. The surface morphology of plasticized films with PEG400 and glycerol appeared

porous, whereas the non-plasticized films were uniform and smooth, indicating hard and

brittle films. The porous structure of plasticized films might be due to the phase

separation that occurred between polymer and plasticizer, and caused haziness of the

D
films (Chan et al., 2005).

3.3 Optimization of mupirocin-loaded Eudragit E100 films

TE
EP
Polynomial linear regressions show the relationship between the main factors P:E ratio
C

(X1) and PEG400:G ratio (X2) fitting to the five variable responses which were tensile
AC

strength, elongation at break, light transmittance, contact angle and water content have

been presented as the following equation:

ST

Tensile strength:

0.06-0.10X1+0.06X12 -0.34X2+0.76X22 +0.11X1X2, r2 = 0.968

JU

Elongation at break:

169.26-52.58X1-8.37X12 +45.32X2+16.99X22 -34.68 X1X2, r2 = 0.988

Light transmittance:
 0.15-0.11X1+0.06X12 +0.02X2+0.19X22 -0.04X1X2, r2 = 0.920

Contact angle:

46.84+0.73X1-0.47X12 -6.88X2+8.32X22 -3.23X1X2, r2 = 0.995

Water content:

1.69+0.28X1-0.15X2, r2 = 0.907

Fig. 4. reveals that the most important parameter is PEG400:G ratio that

D
indicates PEG400 and glycerol gave a different plasticizer effect on the film’s

properties while the other coefficients had less effect. The reasons as described above

TE
showed that coefficient of the P:E ratio (X1) mainly affected to light transmittance while
EP
the coefficient of the PEG400:G ratio (X2) significantly affected to film’s properties.

Hence, the properties of mupirocin-loaded Eudragit E100 films were dependent on both
C

type and ratio of plasticizer.

AC

Response contour plots were analyzed according to set criteria for an optimal

mupirocin-loaded film which represented the mupirocin formulation in Table 1 for

topical drug delivery. The film should be soft and flexible (low tensile strength, high
ST

elongation) with high light transmittance, and the contact angle of water on the films

should be minimized as well as minimal water content to achieve water-resistance. Fig.

JU

5 indicates contour plot analysis identified an optimized P:E and PEG400:G ratio was

around 0.5 and 5:1, respectively (films formulation No. 3, Table 2) which was close to

the sprays formulation No. 19 from Table 1 established as the optimized mupirocin

spray.
3.4 Physical stability, evaluated sprays parameters, and content of mupirocin

delivered by actuation of the valve

The results showed that all formulations have an excellent physical stability and spray

performance without any change in color, formulation precipitation, and immiscibility.

The optimized mupirocin spray formed the film immediately after sprays droplets

reached the skin with a drying time less than 3 min. The stickiness of the film was low.

The sprayed film covered around 300 mm2. The film was flexible and uniform without

any crack (Fig. 6A). The optimized mupirocin spray was intact on the skin and resistant

against washing with water as shown in Fig. 6B. This is an advantage of hydrophobic

D
property of Eudragit E100 (Paradkar et al., 2015; Patra et al., 2017).

TE
The amount of mupirocin delivered by actuation after a year storage is shown in

Fig. 7. The results show the percent pooled content of mupirocin released from 27
EP
formulations at the initial (3rd-12th dose), middle (101st-110th dose) and end (191st-200th

dose) were consistent within a year. The content of each formulation was in the
C

acceptance limit according to USP30 (U.S. Pharmacopeial Convention., 2007), except

AC

formulations #9 and #25 that showed less than 80% and were excluded from further

study. The variation of delivered dose depended on the solvents and HFA-134a

propellant system. High concentration of Eudragit E100 (Formulations #19-27) can lead
ST

to a low drug availability. However, those formulations were homogeneous and stable

resulting in a proper dose.

JU

3.5 In vitro drug release

The cumulative release profiles of mupirocin from the optimized mupirocin spray and

mupirocin ointment are shown in Fig 8. Both delivery systems exhibited as a first-order

kinetic with a rapid ‘burst release’ phase over the first two hours, and the release profile
reached plateau, resulting in no further drug release until the end of test over 24 h. The

drug release from the optimized mupirocin spray was around 90% in 2 h compared with

30% for the ointment formulations, indicating that the topically applied films would

have provided rapid and highly efficient delivery of the sprayed dose into the wound

bed to control bacterial infection. The amount of drug delivered is expected to be

controllable by application of multiple sprayed coats to the wound area, while the

sustained release is feasible by periodic application of further coats. In this system, the

drug was incorporated into the slowly dissolving materials like cellulose, polyethylene

glycols, polymethyl methacrylates, waxes (Wise, 2000). The dissolution rate of coat

depends on the solubility and thickness of the coating. For this reason, spray

D
formulations can generate a thin film which spread over the area resulting in a higher

TE
surface area resulting in a greater drug release than the ointment.
EP
3.6 Antibacterial activities of mupirocin spray
C

The antimicrobial activity of the optimized mupirocin spray and mupirocin ointment
AC

against skin bacterial strains is shown in Table 4. It is estimated that bacterial species in

the environment numbered many hundreds of thousands. A notable species from the

viewpoint of human medicine is S. aureus. It is a frequent colonizer of skin and mucosa

ST

surfaces. A number of extracellular enzymes and exotoxins are responsible for the

clinical symptoms of infections caused by this pathogen. S. epidermidis and other

JU

species are also frequent agents in foreign body infections due to their ability to form

biofilms on the surfaces of inert objects (Sutherland et al., 1985). The inhibition zone

produced by the 20 µg/mL of the optimized mupirocin spray against S. aureus (diameter

41.6 ± 0.2 mm) was larger than that produced by the ointment (diameter 36.9 ± 0.4

mm), S. epidermidis and S. suis were 45.0 ± 0.2 mm and 18.8 ± 1.5 mm, respectively,
while the inhibition zones of the ointment against S. epidermidis and S. suis were 45.0 ±

0.2 mm and 0.0 mm, respectively. The Gram-positive cocci, S. aureus and S.

epidermidis, were susceptible to mupirocin released from both spray and ointment

formulation. The optimized mupirocin spray displayed superior antibacterial activity

against S. suis, while the bacteria were resistant to the ointment. Hence, S. suis was

resistant to mupirocin. The Gram-negative P. aeruginosa was inhibited only at a

concentration of 6,400 µg/mL indicating resistance to both spray and ointment

formulations (Sutherland et al., 1985). The difference in the zone of inhibition between

spray and ointment could be described by the contact angle indicating the spreadability

of the spray formulations, which were close to zero degrees on the skin. Therefore, the

D
formulations tended to spread out to form a thin film on the skin surface, and this was

TE
concluded that the mupirocin spray had been successfully developed for use as
EP
antibacterial skin preparations.

3.7 Evaluation of wound healing and cytotoxicity effect of mupirocin spray

C

Resulted in HaCaT and BJ cells retained 100% viability following exposure for 18 h to
AC

the optimized mupirocin spray and mupirocin ointment at a concentration of 500 µg/mL

compared with the control group indicating that the excipients are non-toxic (Fig. 9).
ST

The GC-FID was employed to quantify the residual amount of acetone and ethanol co-

solvents deposited, following spraying of the mupirocin liquid formulation. The

JU

concentrations of acetone and ethanol analyzed by GC-FID were 0.09 ± 0.00 and 1.56 ±

0.03% v/v, respectively (retention times, 3.95 and 5.36 min, respectively) compared

with the concentrations of acetone and ethanol in the spray canister were of 10 and 40%

v/v, respectively (Table 5). These results indicated that more than 99% of acetone and

96% ethanol evaporated during the spray delivery. Fast evaporating time of the

propellant and solvents after spraying resulted in a thin film can be formed within a
short time causing a low toxicity towards human skin cells in vitro in the present study

and are not expected to cause skin toxicity or irritation when sprayed onto the skin

surface. However, the safety of the formulation needs to be further investigated in

animal models and man.

RAW264.7 are phagocytic cells that play an important role in defense

mechanism. They release NO and inflammatory cytokines (IL-1β or TNF-α) following

the activation of phagocytosis (Shah et al., 2002). Cell viability of RAW264.7 cells

shows that mupirocin spray has a cytotoxic effect less than ointment, but both produces

a percentage of cell viability less than 80% while LPS did not show toxicity to the cells

(Fig. 10). However, the induced NO, IL-1β , and TNF-α of RAW264.7 cells by LPS

D
were evaluated for the antiendotoxin effect of the optimized mupirocin spray as shown

TE
in Fig. 11. NO synthesis occurs during inflammatory wound healing process. Large
EP
amounts of NO produced during cell inflammation have harmful effects on various

cellular functions. However, NO appears to be an important regulator of wound

C

collagen accumulation and may affect collagen synthesis or breakdown in the wound

(Schäffer et al., 1996). The results showed the optimized mupirocin spray did not
AC

stimulate NO production. Moreover, both spray and ointment significantly inhibit the

production of NO by 85.7 and 92.9% compared to LPS inductive group. Unfortunately,

ST

production of collagen by BJ cells exposed to the formulations could not be detected in

culture supernatants. IL-1β expressed in keratinocytes and epidermis is an important

JU

inflammatory mediator in the skin and believed to be the initiator of the inflammatory

response. TNF-α accumulated in epidermal mast cells and expressed by keratinocytes in

response to irritation. The expression of TNF-α induced after challenging with irritants

depends on the release of IL-1β. Both are initial response cytokines, which introduce a

cascade of inflammation reaction. The results show that mupirocin spray and ointment
have an effective reduction in IL-1β and TNF-α, in which spray can reduce IL-1β

production greater than ointment. The optimized mupirocin spray did not stimulate the

production of inflammatory cytokines except the ointment that increases IL-1β level

compared to a control group. IL-1β and TNF-α at a concentration less than 100 ng/mL

were not toxic to keratinocytes (Aramwit et al., 2009). The results confirm that the

safety of mupirocin spray was not toxic to skin cell lines.

The optimized mupirocin spray and mpirocin ointment were evaluated for the

rate of migration of HaCaT and BJ cells. The scratch assay was studied at 0, 12, 24, and

48 h (Table 5 and Fig. 12, 13.) to mimic migration of cells in vitro (Liang et al., 2007).

The scratch assay covered the second phase of wound healing characterized by

D
proliferation and migration of either keratinocytes or fibroblasts (Gurtner et al., 2008;

TE
Schäfer and Werner, 2007). The results show that the presence of mupirocin spray and
EP
ointment did not differ in the proliferation and migration of the cells compared to the

control. It was observed that the formulations did not promote the wound healing by
C

itself since there were no growth factors in the formulations or wound healing
AC

associated cytokines production from the cells such as transforming growth factor-β

(TGF-β), which played important roles in the regulation of cell proliferation, adhesion,

migration, differentiation, extracellular matrix deposition, and involved in regulating

ST

wound healing and fibrotic scar formation (Leask and Abraham, 2004). As shown in

Fig. 12 and 13, an incubation time of 24 h of the HaCaT cells resulted in the complete
JU

closure of the gap occurred in the control group. While mupirocin spray and ointment

showed the length between the scratch mark edges are loosely closed to about 56.6 ±

4.3 µm, respectively. For the BJ cells, all formulations including the control group

showed loosely closure of the gap at 24 h. However, the confluence of the both cells
was seen within 48 h after treatment with all formulations except in the ointment

formulation which was loosely closure of the gap.

Furthermore, wound healing effect is still dependent on the excipient’s

properties containing in the formulations. Glycerol served as a humectant by preventing

the formulation from drying out, resulting in the moist wound environment. The

glycerol proved to be a useful adjunct in facilitating wound healing process with less

scarring by preventing tissue dehydration and cell death. A moist environment can also

accelerate angiogenesis, and increase the breakdown of dead tissue and fibrin (Field and

Kerstein, 1994; Mallefet and Dweck, 2008). Furthermore, the droplets of polymer

solution impinge onto the skin surface then evaporates forming the thin film which

D
facilitates the healing process without the need of wound dressing (Davis et al., 2014).

4. Conclusion
TE
EP
Mupirocin liquid spray formulations were successfully prepared using Eudragit E100 as

the film-forming agent, glycerol as a humectant and plasticizer, and PEG400 as a

C

plasticizer. The optimized mupirocin spray formulation generates the fast formation of
AC

thin film with the rapid release of the drug within 2 h. The formulation displayed

antibacterial activity against S. aureus and S. epidermidis in culture and was non-toxic
ST

towards human skin cells in cell culture. Moreover, the formulation did not stimulate

the production of NO and inflammatory cytokines (IL-1β and TNF-α) by mouse

JU

macrophages. These findings recommend further investigations of mupirocin sprayed

films as a topical application in wound healing.

Acknowledgment

We would like to thank the Drug Delivery System Excellence Center and Nanotech-PSU
Excellence Center on Drug Delivery System for the use of their facilities. This work was

financially supported by the Thailand Research Fund through the Royal Golden Jubilee

Ph.D. Program (PHD/0062/2557 to R. Sritharadol). We would also like to thank the

reviewers of this article and Dr. Brian Hodgson for comments and assistance with the

English.

Conflict of interest

The authors report no declarations of interest.

References

D
Aramwit, P., Kanokpanont, S., De-Eknamkul, W., Kamei, K. and Srichana, T., 2009.

TE
Monitoring of inflammatory mediators induced by silk sericin. J Biosci Bioeng.

107, 556-561.
EP
Bolognia, J. L., Jorizzo, J. L. and Schaffer, J. V., 2010. Dermatology. 1st edition. Chapter

129: Other Topical Medications. Annemarie Uliasz, Mark Lebwohl.

Chan, L. W., Ong, K. T. and Heng, P. W., 2005. Novel film modifiers to alter the physical
C

properties of composite ethylcellulose films. Pharm Res. 3, 476-489.

AC

Chibowski, E. and Jurak, M., 2013. Comparison of contact angle hysteresis of different

probe liquids on the same solid surface. Colloid Polym Sci. 291, 391-399.

Chiller, K., Selkin, B. A. and Murakawa, G. J., 2001. Skin microflora and bacterial
ST

infections of the skin. J Investig Dermatol Symp Proc. 6, 170-174.

Church, D., Elsayed, S., Reid, O., Winston, B. and Lindsay, R., 2006. Burn wound
JU

infections. Clin Microbiol Rev. 19, 403-434.

Cutting, K. F. and White, R. J., 2002. Maceration of the skin and wound bed. 1: Its nature

and causes. J Wound Care. 11, 275-278.

Davis, S. C., Gil, J., Treu, R., Valdes, J., Solis, M., Eberlein, T. and Eaglstein, W. H.,

2014. The healing effect of over-the-counter (OTC) wound healing agents applied

under semiocclusive film dressing. Br. J. Dermatol. 172, 544-546.

Dürrigl, M., Kwokal, A., Hafner, A., Šegvić Klarić, M., Dumičić, A., Cetina-Čižmek, B.

and Filipović-Grčić, J., 2011. Spray dried microparticles for controlled delivery

of mupirocin calcium: Process-tailored modulation of drug release. J

Microencapsul. 28, 108-121.

Field, F. K. and Kerstein, M. D., 1994. Overview of wound healing in a moist

environment. Am. J. Surg. 167, 2S-6S.

GlaxoSmithKline. 2001. Bactroban. Product Monograph. 1-18.

Gohel, M. and Nagori, S., 2009. Fabrication and design of transdermal fluconazole
spray. Pharm Dev Technol. 14, 208–215.
Gurtner, C. G., Werner, S., Barrandon, Y. and Longanker, M. T., 2008. Wound repair and

regeneration. Nature. 453, 314-321.

D
Hu, Y., Wu, Y. Y., Xia, X. J., Wu, Z., Liang, W. Q. and Gao, J. Q., 2011. Development

TE
of drug-in-adhesive transdermal patch for α-asarone and in vivo
pharmacokinetics and efficacy evaluation. Drug Deliv. 18, 84-89.
Jolly, S. and Jolly, S., 2011. Dry wound healing concept using spray-on dressings for
EP
chronic wounds. Advanced Wound Repair Therapies. Woodhead Publishing

Limited. Sawston, Cambridge, UK. 209-226.

C

Krishnaiah Y. S., Kumar, M. S., Raju, V., Lakshmi, M. and Rama, B., 2008.
AC

Penetration-enhancing effect of ethanolic solution of menthol on transdermal

permeation of ondansetron hydrochloride across rat epidermis. Drug Deliv. 15,
227–234.
Kweon, J. H., Chi, S. C. and Park, E. S., 2004. Transdermal delivery of diclofenac using
ST

microemulsions. Arch Pharmacol Res. 27, 351–356.

Lambers, H., Piessens, S., Bloem, A., Pronk, H. and Finkel, P., 2006. Natural skin surface
JU

pH is on average below 5, which is beneficial for its resident flora. Int J Cosmet

Sci. 5, 359-370.

Leask, A. and Abraham, D. J., 2004. TGF-b signaling and the fibrotic response. FASEB.

218, 816–827.
Liang, C. C., Park, A. Y. and Guan, J. L., 2007. In vitro scratch assay: a convenient and

inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2, 329-

333.

Mallefet, P. and Dweck, A. C., 2008. Mechanisms involved in wound healing. J. Biomed.

Sci. 609-615.

Nashed, G., Rutgers, R. P. G. and Sopade, P. A., 2003. The plasticisation effect of glycerol

and water on the gelatinisation of wheat starch. Starch. 55, 131-137.

Paradkar, M., Thakkar, V., Soni, T., Gandhi, T. and Gohel, M., 2015. Formulation and
evaluation of clotrimazole transdermal spray. Drug Dev Ind Pharm. 41, 1718-
1725.
Patra, C. N., Priya, R., Swain, S., Jena, G. K., Panigrahi, K. C. and Ghose, D, 2017.

D
Pharmaceutical Significance of Eudragit: A Review. FJPS.
Peppas, N. A., 2004. Devices based on intelligent biopolymers for oral protein delivery.

Int J Pharm. 277, 11-17.

TE
Rhody, C., 2000. Bacterial infections of the skin. Primary Care. 27, 459-473.
EP
Rowe, R. C., Sheskey, P. J. and Quinn, M. E., 2009. Handbook of Pharmaceutical

Excipients, 6th ed. The Pharmaceutical Press and The American Pharmacists
C

Association, London.
AC

Sarabahi, S., 2012. Recent advances in topical wound care. Indian. J. Plast. Surg. 45, 379-

387.

Schäfer, M. and Werner, S., 2007. Transcriptional control of wound repair. Ann Rev Cell
ST

Dev Biol. 23, 69-92.

Schäffer, M. R., Tantry, U., Gross, S. S., Wasserkrug, H. L. and Barbul, A., 1996. Nitric
JU

oxide regulates wound healing. J Surg Res. 63, 237-240.

Shah, V., Bayeta, E. and S., Lau B. H., 2002. Pycnogenol® augments macrophage

phagocytosis and cytokine secretion. PJN. 1, 196-201.

Spann, C. T., Taylor, S. C. and Weinberg, J. M., 2003. Topical antimicrobial agents in

dermatology. Clin. Dermatol. 50, 407-421.

Tas, C. , Ozkan, Y. , Savaser, A. and Baykara, T., 2003. In vitro release studies of

chlorpheniramine maleate from gels prepared by different cellulose derivatives.

Farmaco. 58, 605-611.

U.S.Pharmacopeial Convention., 2007. USP30-NF25, USP, Rockville, MD.

Wise, D. L., 2000. Handbook of pharmaceutical controlled release technology. 1st ed.

Marcel Dekker, Inc., New York, USA. 183-224.

Zheng, W., 2010. Surface wetting characteristics of rubbed polyimide thin films. Abbass

A Hashim (Ed.). ISBN: 978-953-307-059-9. InTech, Available from:

http://www.intechopen.com/books/polymer-thin-films/surface-wetting-

characteristics-of-rubbed-polyimide-thinfilms.

D
TE
EP
C
AC
ST
JU
JU
ST
AC
C
EP
TE
D
JU
ST
AC
C
EP
TE
D
JU
ST
AC
C
EP
TE
D
JU
ST
AC
C
EP
TE
D
JU
ST
AC
C
EP
TE
D
JU
ST
AC
C
EP
TE
D
JU
ST
AC
C
EP
TE
D
JU
ST
AC
C
EP
TE
D
Table 1. The composition of a final concentration of mupirocin sprays.
Active
Excipients Cosolvent
Formulation ingredient

No. Mupirocin Eudragit

PEG400 Glycerol Ethanol:Acetone
E100 (4:1 v/v)
(g) (g) (g)
(g)
1 1 1.26 1.13

2 1 1.26 2.26

3 1 1.26 5.65

4 1 2.52 1.13

5 1 2.52 2.26

D
6 1 2.52 5.65

8
1

1
6.30

6.30
TE 1.13

2.26
EP
9 1 6.30 5.65

10 2 1.26 1.13
C

2 q.s 100 mL
11 2 1.26 2.26
AC

12 2 1.26 5.65

13 2 2.52 1.13
ST

14 2 2.52 2.26

15 2 2.52 5.65
JU

16 2 6.30 1.13

17 2 6.30 2.26

18 2 6.30 5.65

19 5 1.26 1.13

20 5 1.26 2.26
21 5 1.26 5.65

22 5 2.52 1.13

23 5 2.52 2.26

24 5 2.52 5.65

25 5 6.30 1.13

26 5 6.30 2.26

27 5 6.30 5.65

D
TE
EP
C
AC
ST
JU
Table 2. The composition of mupirocin-loaded Eudragit E100 films for the

optimization process.

Active
ingredien Excipients Cosolvent
Formulati t
on No. Mupiroci Eudrag PEG40 Glycer P:E Ethanol:Aceto
PEG400:
n it 0 ol rati ne
G ratio
(g) (g) (g) (g) o (4:1 v/v)
0.5
1 1.42 8.58 1:6
0
0.5
2 5.00 5.00 1:1
0
0.5
3 8.58 1.42 6:1
0
0.7

D
4 2.14 12.86 1:6
5
5

6
2 20 7.50

12.86
7.50

2.14
0.7
5
0.7
5
TE 1:1

6:1
q.s. 100 mL
EP
1.0
7 2.86 17.14 1:6
0
1.0
8 10.00 10.00 1:1
C

0
1.0
AC

9 17.14 2.86 6:1

0
P:E; Plasticizers (PEG400 and glycerol):Eudragit E100 ratio, and PEG400:G; PEG400:

glycerol ratio.
ST
JU
 Table 3. Film’s thickness, light transmittance, contact angle, water content and
mechanical properties of mupirocin-loaded Eudragit E100 films (Mean ± SD, n=3).
Light Water Tensile
Thickness *Contact Elongation at
Formulations transmittance content strength
(mm) angle (°) 2 break (%)
(%) (%) (N/mm )
1 0.40 ± 0.02 0.5 ± 0.0 58.1 ± 0.3 1.4 ± 0.0 1.50 ± 0.00 160.1 ± 0.0
2 0.52 ± 0.02 0.3 ± 0.0 51.7 ± 0.2 1.6 ± 0.0 0.83 ± 0.00 158.4 ± 0.0
3 0.50 ± 0.03 0.6 ± 0.0 50.5 ± 0.1 1.4 ± 0.0 0.57 ± 0.00 307.3 ± 0.0
4 0.49 ± 0.04 0.3 ± 0.0 62.1 ± 0.3 1.9 ± 0.0 1.13± 0.00 135.1 ± 0.0
5 0.55 ± 0.03 0.3 ± 0.0 56.2 ± 0.3 1.7 ± 0.0 0.77 ± 0.00 142.8 ± 0.0
6 0.52 ± 0.01 0.3 ± 0.0 48.0 ± 0.6 1.4 ± 0.0 0.43 ± 0.00 240.1 ± 0.0
7 0.58 ± 0.02 0.3 ± 0.0 64.9 ± 0.4 2.1 ± 0.0 0.97 ± 0.00 110.8 ± 0.0
8 0.53 ± 0.02 0.2 ± 0.0 59.1 ± 0.7 2.2 ± 0.0 0.60 ± 0.00 118.7 ± 0.0
9 0.45 ± 0.00 0.3 ± 0.0 45.4 ± 0.6 1.8 ± 0.0 0.57 ± 0.00 130.5 ± 0.0

*The contact angle values are an angle in the degree of a water droplet on the films.

D
TE
Table 4. Antimicrobial activity of mupirocin spray and ointment against some standard
skin bacterial strains.
EP
Zone of inhibition diameter (mm) (mean ± SD, n=4)
Samples Staphylococcus Staphylococcus Streptococcus Pseudomonas
aureus* epidermidis* suis** aeruginosa*
C

Mupirocin spray 41.6 ± 0.2 >45.0 18.8±1.5 -

Mupirocin
36.9 ± 0.4 >45.0 - -
AC

ointment

* S. aureus, S. epidermidis and P. aeruginosa were performed using disc diffusion

method with 10 µL of samples.
** S. suis was performed using agar cup diffusion method loaded with 200 µL of
ST

sample.
JU
 Table 5. In vitro scratch assay of mupirocin formulations on HaCaT cells and BJ cells.

Length between the scratch (µm) Migration rate of HaCaT cells (%)
Treatment 48
0h 12 h 24 h 12 h 24 h 48 h
h
Negative control 337.6 ± 12.2 67.1 ± 3.4 CC CC 80.1 ± 0.8 100 100
Mupirocin spray 336.9 ± 3.4 79.3 ± 5.4 LC CC 76.5 ± 1.5 100 100
HaCaT

Mupirocin 56.6 ± 82.7 ±

327.6 ± 5.4 75.0 ± 2.3 LC 77.1 ± 0.6 100
ointment 4.3 1.3
Negative control 322.0 ± 14.7 59.3 ± 7.9 LC CC 81.6 ± 2.5 100 100
Mupirocin spray 332.4 ± 23.1 75.7 ± 15.6 LC CC 77.3 ± 3.6 100 100
BJ

Mupirocin
343.9 ± 14.6 66.5 ± 6.4 LC CC 80.7 ± 1.7 100 100
ointment

D
All data represented in mean ± SD (n = 10); LC: loosely closure; CC: complete closure
of the gap.
TE
EP
C
AC
ST
JU