Electrospun PEGPLA Nanofibrous Membrane for

Sustained Release of Hydrophilic Antibiotics

Xiuling Xu,1 Wen Zhong,1,2 Shufei Zhou,1 Adriana Trajtman,2 Michelle Alfa2
1
Department of Textile Sciences, Faculty of Human Ecology, University of Manitoba, Winnipeg, Manitoba R3T 2N2,
Canada
2
Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3T 2N2,
Canada

Received 7 May 2009; accepted 9 March 2010

DOI 10.1002/app.32415
Published online 21 May 2010 in Wiley InterScience (www.interscience.wiley.com).
ABSTRACT: Reported in this study is the successful incorporation of a hydrophilic antibiotic drug, tetracycline hydrochloride (TCH), into electrospun PEGPLA nanofibrous
membrane without loss of its bioactivity. Degradation behavior of the copolymer was studied in vitro. Release behavior of
TCH from the electrospun membrane and antimicrobial
effects of the TCH-loaded membrane against Staphylococcus
aureus culture were investigated. The medicated nanofibrous
membrane demonstrated sustained release of TCH over
6 days and was found to be effective in inhibiting growth of
S. aureus. In addition, increasing the antibiotic drug content
in the electrospun membranes was found to enhance the

anti-bacterial effectiveness of the medicated fiber mats. And

the combination of mechanical barriers provided by the electrospun biodegradable nanofibrous membranes and their
capability of local sustained delivery of antibiotics made
these membranes more useful in biomedical applications,
particularly as new wound dressings for ulcers caused by diabetes or other diseases, and to provide a better means of
C 2010
treatment for these malignant wounds and ulcers. V

INTRODUCTION

often provide insufficient dosage to the wounded tissues,6 and can barely penetrate ischemic wounds
with little granulation tissue. An effective treatment
to overcome these side effects is topical administration,6,8,9 in which the antibiotic is used in minimal
amounts at the site of infection, and efforts are made
to ensure that the drugs function efficiently at the site.
The rate of drug uptake by the human body may
increase with the decrease in the size of the drug
and its carrier, because of the increasing surface area
of the drug or carrier. Hence, drug delivery systems
have been developed using polymeric materials in
the form of nano or micro particles,10 hydrogels,11 or
micelles.12 Recently, drug-loaded electrospun nanofibers have attracted a great deal of attention,
because they offer higher drug encapsulation efficiency and better structural stability than other drug
carriers.1315 Electrospinning is a simple method of
fabricating nanofiber mats. A polymer solution is
placed into a syringe with a capillary outlet and is
subjected to a high voltage electric field. When the
electric force exceeds the surface tension of the polymer solution, a fiber jet is ejected from the outlet. As
the jet travels through the air, the solvent evaporates, leaving behind ultra-fine fibers that deposit on
a grounded collector. Electrospun nanofibrous mats
have a high porosity and a large specific surface
area and could be used in the development of drugloaded dressings that would allow sustained, site-

Major medical, social, and economic implications

result from lower extremity amputation, a complication of diabetic foot ulcers.14 It has been reported
that approximately 15% of diabetics develop foot
ulcers; non-healing ulcers precede 84% of all lower
extremity amputations in diabetic patients.1
Diabetic foot ulcers are open wounds easily
infected by bacterial contamination. Guidelines for
treating these ulcers suggest that suitable dressings
combining debridement and antimicrobial activity
with moisture control are essential topical treatment
methods.3 Some of the dressings currently used in the
treatment of skin ulcers may cause tissue damage
through shear, friction, and/or pressure to the
wound5; however, wound coverage is important to
help prevent infection, and to treat infected ulcers.6
Although antibiotics are useful in controlling infection, systemic administration can cause undesirable
side effects including renal and liver toxicity,6,7 can

Correspondence to: W. Zhong ([email protected]).

Contract grant sponsors: NSERC (Natural Sciences and
Engineering Research Council of Canada), MHRC
(Manitoba Health Research Council).
Journal of Applied Polymer Science, Vol. 118, 588595 (2010)

C 2010 Wiley Periodicals, Inc.

V

Wiley Periodicals, Inc. J Appl Polym Sci 118: 588595, 2010

Key words: electrospun; nanofibers; sustained release;

antibiotics

ELECTROSPUN PEGPLA NANOFIBROUS MEMBRANE

specific delivery of drugs to wounds, while providing comfort by minimizing shear/friction to the
wounded area.
Usually the physical and chemical properties of
different drugs affect the success of incorporating
them into nanofibers. Specifically, hydrophobic
drugs can be easily encapsulated into hydrophobic
polymers by electrospinning the mixed solution of a
drug and a polymer. However, water-soluble drugs,
including the commonly used broad-spectrum antibiotics such as clindamycin, cephalexin, ciprofloxacin, and cefoxitin sodium, cannot be directly incorporated into hydrophobic polymers. Although
hydrophilic polymers are good carriers for watersoluble drugs, they cannot be used as the drug
delivery system, because they can quickly dissolve
in blood or tissue fluid. Under such conditions, the
drug release rate cannot be controlled. Usually, the
problem is solved by cross-linking the drug-loaded
fibers.16 However, many drugs may lose their bioactivity or even their molecular identity during crosslinking. Moreover, toxic chemicals may become
incorporated into the dressings during the chemical
cross-linking process.
Because of the difficulties as described above, only
a few existing reports on electrospun fibrous mats
for wound dressings1721 are devoted to developing
dressings loaded with anti-infection drugs.22 The
aim of this work is, therefore, to develop electrospun
drug-loaded fibrous dressings that will provide a
locally-controlled release of drugs to the wounds.
Specifically, the current study (1) involves a novel
emulsion-electrospinning process to efficiently
incorporate a hydrophilic antibiotic drug into the
electrospun fibers without losing drug bioactivity
and (2) is designed to demonstrate sustained release
of the drug from the electrospun fibrous dressing, as
well as its ability to inhibit bacterial growth. The
work is expected to contribute to the development
of new wound dressings for ulcers caused by diabetes or other diseases and to provide a better means
of treatment for these malignant wounds and ulcers.
In the present study, TCH, a well-known broad
spectrum antibiotic, is chosen as a model drug,
because it is active against most common pathogens
and will cause only a few allergic reactions.23 The
electrospun fibers of amphiphilic poly(ethylene glycol)-poly(L-lactic acid) (PEGPLA) diblock copolymer are employed as vehicles for the release of this
antibiotic. The viability and bioactivity of the
released drug is examined using in vitro Staphylococcus aureus inhibition tests.
Wound dressings based on biodegradable PLA are
promising for clinical uses, because the acidic environment induced by the degradation of the polymer
helps to reduce bacteria growth24,25 and promote
epithelization.25,26 Furthermore, it is reported that

589

local lactate concentrations can stimulate local collagen synthesis.25,27

EXPERIMENTAL
Materials
Tetracycline hydrochloride (TCH,
95% purity), Fluorescein isothiocyanate (FITC), Polylactide (PLA)
were purchased from Sigma-Aldrich Canada (Oakville, Ontario). Chloroform (analytical grade) was
supplied by Fisher Scientific Canada (Ottawa, Ontario). Tris(hydroxymethyl) aminomethane (Trisbase) was supplied by Sigma-Aldrich, Canada and
was used without further purification to prepare
Tris-HCl buffer solution of pH 8.6. Triethyl benzyl
ammonium chloride (TEBAC), proteinase K and sodium azide were also purchased from SigmaAldrich, Canada. All reagents were used without
further purification.
Diblock copolymer PEGPLA (prepared from
MPEG750 and L-lactide with a feed ratio of 17 : 620)
was synthesized in our own lab. In a typical preparation process, L-lactide (supplied by Purac biomaterials, Lincolnshire, IL) was polymerized at 125 C
with 0.025% w/w tin(II) octoate (Sn(Oct)2) as catalyst and in the presence of PEG (supplied by SigmaAldrich Canada) as macromolecular initiator under
argon atmosphere and anhydrous condition for 50 h.
Then the polymerization product was dissolved in
chloroform, precipitated into anhydrous alcohol, and
dried in a vacuum at 60 C for 48 h. Dried PEGPLA
diblock copolymer is then obtained. Its molecular
weight (Mn) and polydispersity (PD) determined by
gel permeation chromatography (WATERS 410 GPC,
Tetrahydrofuran was used as solvent with 1 mL/
min flow rate, polystyrene as column standard)
were 93,000 and 1.65, respectively.

Preparation and evaluation of water-in-oil emulsions containing TCH

The method to prepare water-in-oil (W/O) emulsions was described in detail by Xu, et al.28 In the
present study, two batches of 1 mL water solution
containing, respectively, 1.0 and 3% w/w of TCH
(with respect to PEGPLA) were emulsified in 20
mL of 7.5% w/w chloroform solution of PEGPLA
in a Silverson L4RTA homogenizer. The homogenizer rotated at  7000 r/min for about 20 min. To
obtain stable and homogeneous W/O emulsions, 5%
w/w of TEBAC (with respect to PEGPLA) was
added as a surfactant to the oily phase prior to
emulsification, to lower surface tension and to
render the aqueous emulsion droplets evenly
distributed.
Journal of Applied Polymer Science DOI 10.1002/app

590

The zero shear viscosities and electrical conductivities of the as-prepared TCH containing emulsions
and PEGPLA control solution were measured at
25 C by a digital Viscometer (Brookfield DV-II
PRO Digital Viscometer) and a conductivity meter
(Orion 4-Star pH/Conductivity Benchtop Meter),
respectively.

Preparation of TCH-loaded electrospun

fibrous mats
The stable and homogenous W/O emulsions were
electrospun using a conventional electrospinning
setup. In the present study, electrospinning parameters were: electric field strength: 1.52.0 kV/cm; air
gap distance: 20 cm; inner diameter of spinneret: 0.4
mm; flow rate of solution: 68 mL/h. In our experience, the optimal concentration of the PEGPLA/
chloroform solution was 7.5% w/w. Accordingly,
this concentration was chosen throughout the electrospinning process in the study. In addition, electrospinning parameters were kept constant, and the
experiment was conducted consistently at room temperature in air. To remove the residual chloroform,
fiber mats collected were freeze-dried for about 48 h
at 50 C under a vacuum of 10 Pa.
As a control, the unloaded PEGPLA fibers were
prepared by electrospinning 7.5% w/w chloroform
solution of PEGPLA. 5% w/w of TEBAC (with
respect to PEGPLA) was added into the PEGPLA/
CHCl3 solutions as a surfactant to improve the electrospinning process. The spinning parameters were
all kept identical with those mentioned above.
A scanning electron microscope (SEM, Cambridge
Stereoscan 120) was used to observe the surface
morphology and size distribution of the electrospun
fibers. Its accelerating voltage was 20 kV. Samples
were mounted on metal stubs using a double-sided
adhesive tape and vacuum-coated with a platinum
layer prior to examination. To determine the average
fiber diameter for each sample, 10 fibers from each
sample were randomly selected in the SEM images,
and fiber diameters were measured at six different
points on each fiber.

Preparation of FITC-loaded electrospun fiber mats

To demonstrate the structure of the drug-loaded
nanofibers fabricated by the emulsion electrospinning, FITC was used as an indicator (in place of
TCH) in the emulsion electrospining as described in
the previous sections. The distribution of FITC in the
nanofibers was investigated under a confocal microscope (Olympus IX-70) with Fluoview Software
using an Argon laser for excitation at 488 nm (green
fluorescence).
Journal of Applied Polymer Science DOI 10.1002/app

XU ET AL.

In vitro degradation studies

For in vitro degradation studies, as-spun PEGPLA
and PLA fiber mats with a thickness of about 0.20.3
mm were cut into 20  20 mm2 pieces. They were
weighed (m0), put into vials (n 3) containing 25
mL of phosphate buffer solution (PBS, pH 7.4), and
then incubated in a rotary shaker at 37 6 0.1 C for
various periods of time. At each specific incubation
time, the samples were withdrawn and washed thoroughly with distilled water. The samples were
wiped and dried in vacuum at room temperature
until a constant weight (md) is obtained. The mass
loss of the samples was calculated with the following equations:
Mass loss percentage m0  md =m0  100
Degradation profiles of PEGPLA and PLA fiber
mats in the presence of an enzyme were obtained by
putting samples in 0.05 mol/L Tris-HCl buffer solution (pH 8.6) containing 6lg/mL of proteinase K at
37 6 0.1 C, following the same procedure as mentioned above.
It should be noted that PLA fiber mats were
employed as a reference for the discussion of PEG
PLA degradation behavior. Its molecular weight
(Mn) and polydispersity (PD) determined by gel permeation chromatography were 102,000 and 1.23,
respectively.
In vitro drug release studies
The released TCH in the buffer solution was monitored by a UV-visible spectrophotometer at the
wavelength of 366 nm. The drug-loaded fiber sample (2030 mg) was incubated at 37 C in 20 mL of
phosphate buffered saline (PBS, pH 7.4). At the
required incubation time, the sample was transferred
to 20 mL of fresh buffer solution, and the released
TCH in the original buffer solution was determined.
The detected UV absorbance of TCH was converted
to its concentration according to the calibration
curve of TCH in the same buffer. Then the accumulative weight and relative percentage of the released
TCH were calculated as a function of incubation
time.
The total content of TCH in the fibers was determined as follows. The three original TCH-loaded
fiber mats were placed into separate vials filled with
20 mL of 0.05 mol/L Tris-HCl buffer solution (pH
8.6) containing 50lg/mL of proteinase K at 37 C. After about 5 h, the fiber mats were found to have all
degraded into small chippings with the dimensions
of the order of micros suspending in the buffer solution. We assumed that TCH had been completely
released into the buffer solution. The cloudy

ELECTROSPUN PEGPLA NANOFIBROUS MEMBRANE

591

Figure 1 SEM images of 1% w/w (a) and 3% w/w (b) TCH-loaded PEGPLA nanofibers and PEGPLA nanofibers without drugs (c).

suspensions were filtered using 0.45 lm filter. Then

the resultant solutions were monitored at the wavelength of 366 nm. Concentrations of released TCH in
the solutions were determined according to the calibration curve of TCH in the same buffer. The total
content of TCH in the fibers was easily calculated
from the average of the three fiber mats. All results
are expressed as the mean 6 SD (standard deviation
of the mean).

Antibiotic activities of TCH-loaded nanofibrous

membranes
A modified Kirby-Bauer antibiotic test was used to
assess the susceptibility of bacteria to drug-loaded
nanofibrous membranes. A commercially available
TCH-sensitive bacteria, S. aureus ATCC 25,923
(American Type Culture Collection, Seattle), was
used for this test. In this study, the tested samples
were divided into four groups: (1) a 1% w/w TCHloaded fibrous disk (30 ug/disk), (2) a commercially
available TCH-containing paper disk (30 ug/disk)
(Antimicrobial Susceptibility Discs, Oxoid, Canada),
(3) a TCH-loaded filter paper disk (30 ug/disk), prepared by means of dropping a TCH water solution
onto a filter paper disk, and (4) negative controls: a
blank PEGPLA fibrous disks and a blank filter paper disk (without TCH). All samples were round
disks of 0.6 cm in diameter. They are sterilized using
UV irradiation before the susceptibility test.
The sample disks were placed on agar plates
streaked with S. aureus suspension and incubated in
a CO2 incubator at 37 C. After 24 h, the inhibitory
effect of each sample disk was evaluated by the diameter of an area of clearing around a disk where
bacteria are not capable of growing (known as the
inhibition zone). Then each sample disk was transferred to a fresh bacterial-streaked agar plate for the
examination of the inhibitory effect provided by the
remaining drug in the disk. Each test of antibacterial
activity of the sample disks against S. aureus was
performed for five consecutive days. The antibacterial experiments were performed in triplicate. The

size of inhibition zone of each tested sample was

measured and averaged from three different trials.
All results are expressed as the mean 6 SD (standard deviation of the mean). Student t-test is used to
determine the significant differences among the
groups. A P value less than 0.05 is considered to be
significant.
RESULTS AND DISCUSSIONS
The morphology of the electrospun TCH-loaded
PEGPLA nanofibers with 1.0, and 3.0 w/w of TCH
loadings is shown in Figure 1(a,b). They appeared
uniform. Their surfaces were smooth and no drug
crystals were detected. Moreover, it seemed that
there were no significant differences in either the
morphology or the average diameter of the composite fibers containing different amounts of tetracycline. Compared with unloaded PEGPLA fibers [in
Fig. 1(c)], they looked much thinner. Their average
diameters were all about 600 nm, while the average
diameter of unloaded PEGPLA fibers was about
730 nm. This was ascribed to the decreasing viscosity of TCH containing emulsions due to high shear
stress of homogenizer during emulsification process.
The viscosities of the W/O emulsions containing 1.0
and 3.0 w/w of TCH and PEGPLA blank solution
were 0.53, 0.51, and 0.62 Pa s, respectively. Another
possible reason for the reduction of the diameters of
TCH-loaded PEGPLA fibers was that the electrical
conductivity of the spinning emulsion was enhanced
because of the presence of TCH in the aqueous
phase. The electrical conductivities of the W/O
emulsions containing 1.0 and 3.0 w/w of TCH and
PEGPLA blank solution were 2.85, 2.94, and 3.12 S
m1, respectively. Therefore, in the present study,
both effects favor the formation of TCH-loaded
fibers with smaller diameters.
A confocal microscope image for FITC-loaded
PEGPLA nanofiber that has been fabricated using
the same method was shown in Figure 2. It can be
seen from the image that the green fluorescent FITC
has been trapped in the core of the nanofiber,
Journal of Applied Polymer Science DOI 10.1002/app

592

Figure 2 Confocal microscope image for FITC-loaded

PEGPLA nanofiber. [Color figure can be viewed in the
online issue, which is available at www.interscience.wiley.
com.]

demonstrating the formation of a sheath/core structure via the emulsion electrospinning, with small
molecular drug in the core and polymer on the
sheath of the nanofiber.
Degradation
The hydrolytic degradation of both PLA and PEG
PLA fiber mats was carried out in PBS at 37 6
0.1 C. Their profiles of degradation were shown in
curves a and b in Figure 3. It can be seen that the
two polymers display similar degradation kinetics.
In PBS, their rates of weight loss remained relatively
low in the first 23 days. By the end of Day 23, the
remaining masses of PLA and PEGPLA fiber mats
were 92.8 6 1.5% and 87.4 6 1.6%, respectively.
Their rates of weight loss increased from Day 23
and raised dramatically from Day 56. By the end of
Day 85, the remaining masses of PLA and PEGPLA
fiber mats reduced to 33.1 6 4.0% and 14.7 6 6.9%,
respectively. It is known that the hydrolysis of PLA
is a process in which ester bonds are randomly cut.
The hydrolysis process can be further autocatalyzed
by the carboxylic acid groups generated during degradation, especially by those carboxylic acid groups
in the center of the fiber mass.2932 As a result, the
degradation of PLA in PBS is heterogeneous with a
fast degrading center and a slowly degrading outer
layer.29 It can be seen that the hydrolysis degradation rate of PEGPLA fiber mats was faster than that
of the PLA ones. The reason is that hydrophilic PEG
blocks allow more water molecules to diffuse into
the interior of the fibers.
The degradation of both PEGPLA and PLA fiber
mats was significantly accelerated in the presence of
proteinase K (curves c and d in Fig. 3). In the presJournal of Applied Polymer Science DOI 10.1002/app

XU ET AL.

ence of proteinase K, the mass losses of PEGPLA

and PLA fiber mats were 72.5 6 3.9% and 89.8 6
2.8%, respectively in only 1.8 days, if compared to
their mass loss of 85.2 6 4.0% and 66.9 6 6.9%,
respectively in 85 days in PBS. This was in agreement with the reports that proteinase K can catalyze
the hydrolysis of PLA molecules.33,34 Furthermore,
in the presence of protainase K, the weight loss of
both PEGPLA and PLA fiber mats increased steadily with the degradation time (Fig. 3, inserted curves
c and d), because the enzymatic degradation is
mainly a surface erosion process. Similar degradation behavior was also shown in other reports,35
which indicated that the enzymatic degradation
itself of PLA electrospun fibers was of zero-order
kinetics.
It was interesting to see that the enzymatic degradation rate of PEGPLA fiber mat was slower than
that of PLA fiber mat, although the former was
more hydrophilic than the latter. For example, by
the end of 1.3 h, 53.3 6 2.2% and 68.2 6 5.1% of
mass loss were monitored for PEGPLA and PLA,
respectively. The interpretation is as follows: the enzymatic degradation takes place in two steps: (1) the
enzyme approaches the fiber surface; (2) it initiates
hydrolysis of the polymer. The second step is determined by the nature and concentration of the
enzyme used, while the first step is determined by
the surface properties of the fibers. It is possible that
PEG blocks enriched on the surface of the PEGPLA
fibers, when PEGPLA fibers contact with water.
However, proper PEG chains can sterically prevent

Figure 3 Weight remaining of PEG750PLA (red curves,

b and c) and PLA (black curves, a and d) polymers as a
function of degradation time in PBS (-l- for PLA and -*for PEGPLA) and in 0.05 mol/L Tris-HCl buffer solution
(pH 8.6) containing 6lg/mL of proteinase K (-!- for PLA
and -D- for PEGPLA) at 37 C. The insert is the magnification of curves c and d. [Color figure can be viewed in the
online issue, which is available at www.interscience.wiley.
com.]

ELECTROSPUN PEGPLA NANOFIBROUS MEMBRANE

593

enzymatic attack to PLA molecules to some

extent.36,37
The degradation behavior of PEGPLA contacting
with open wound in vivo will be studied in our
future work.
Release of TCH from TCH-loaded nanofibers
Figure 4 shows the release profiles of TCH from 1
and 3% w/w TCH-loaded fiber mats, respectively.
Both membranes showed a sustained drug release
behavior, although a certain amount of rapid drug
release was found within the first 12 h (32.5% for 1%
w/w samples vs. 21.8% for 3% w/w samples) in
comparison to the entire 6 days release experiment.
For the delivery of antibiotic drugs, a certain initial
burst release is actually required to achieve enough
initial dosage, because it is important to eliminate
the intruding bacteria before they begin to proliferate. As for the bacteria cells that have survived the
initial stage, sustained drug release is necessary to
prevent their further population.38 It was interesting
to notice that rate of TCH release would decrease
with increased TCH content in the fibers during the
entire process of release. For example, the release
percentages were about 47.8 and 34.4% at Day 1,
72.2 and 54.9% at Day 2, 83.0 and 70.3% at Day 3,
respectively, for the two samples examined. Similar
release behaviors were reported by Xu et al.39 In an
emulsion electrospinning, TCH was well incorporated into PEGPLA fibers, forming core-sheath
structured fibers (Fig. 2) during the electrospinning
of the W/O emulsion containing TCH. This kind of
core-sheath structured fibers is a reservoir-type
drug-loaded system: a greater amount of TCH in the
emulsion for electrospinning resulted in a thinner
core and a thicker sheath of fibers due to the movement of TCH towards the fiber axis and the higher
degree of de-emulsification of the aqueous droplet
with larger TCH concentration. It was found in this
study, however, rate of release from the 1% w/w
TCH-loaded fibers was larger than that from the 3%
w/w fibers. The difference of percentages of daily
release from 3% w/w fibers was not as obvious as
the difference of percentages of daily release from 1%
w/w fibers [Fig. 4(B)], indicating that the 3% w/w
fibers displayed more sustained release due to a
smaller rate of release.
For both samples, daily release of TCH decreased
gradually as the 6-day experiment approached its
end [Fig. 4(B)]: about 47.8, 24.4, and 10.8% of TCH
were released from 1% w/w TCH-loaded fibers
within Days 1, 2, and 3, respectively, but only 3.5,
1.4, and 0.7% of TCH were released in Days 4, 5,
and 6; likewise, release from 3% w/w TCH-loaded
fibers were about 34.4, 20.5, and 15.4% in Days 1, 2,
and 3, but only 6.7, 4.5, and 2.7% in Days 4, 5, and 6,

Figure 4 Profiles of the release of TCH from 1% w/w

and 3% w/w TCH-loaded PEGPLA nanofibers in PBS at
37 C, the data representing the mean 6 SD (n 3): cumulative curve (a) and differential curve (b). [Color figure can
be viewed in the online issue, which is available at
www.interscience.wiley.com.]

respectively. Interpretation can be as follows. First,

TCH in the fibers runs out as the release goes on and
on. Second, TCH molecules located in the center of
the fibers have a longer distance to travel in the
course of diffusing. Another reason is increased crystallinity of PEGPLA as a result of its incubation in
PBS at 37 C. It is known that a small molecule diffuses more slowly through a crystalline polymer than
through an amorphous polymer of the same kind,40
so that release of TCH slows down as the release
goes on and on.

Antimicrobial activities of TCH-loaded nanofiber

membranes
To evaluate the efficacy of the medicated membranes, in this study, use was made of a commercially available TCH-sensitive bacterium, a Grampositive spherical bacterium that is typically found
Journal of Applied Polymer Science DOI 10.1002/app

594

Figure 5 Diameters of inhibition zones in the Staphylococcus aureus susceptibility test (30 ug/disk for all samples).
[Color figure can be viewed in the online issue, which is
available at www.interscience.wiley.com.]

in the skin, nasal passages, and mucous membranes,

and can cause a wide range of suppurative infections.38 The antimicrobial effect of electrospun membranes with and without TCH can be compared by
the sizes of their inhibitory zones. Besides, two positive controls, a commercial TCH-loaded disk and
TCH-impregnated paper, were used to compare the
sustained release of drugs from drug-loaded substrates. There was no inhibitory zone surrounding
the negative control (drug-free electrospun fiber
disk) on the bacterial-inoculated agar plate and bacteria grew robustly. The results of the microorganism susceptibility tests for all samples except the
negative control were shown in Figure 5. It can be
noted that obvious inhibitory zones were observed
around the 1% w/w TCH-loaded fiber mats: average
17.9 mm for Day 1, 15.9 mm for Day 2, and 9.9 mm
for Day 3, respectively. Although the zones of inhibition were much smaller (6.5 mm in average) at Day
4, but the inhibitory effects were still seen on that
day in the antibiotic-containing disk. On Day 5, the
released TCH was not enough to inhibit the bacteria,
so the remaining viable bacteria could proliferate,
and, as a result, no inhibition zone was observed at
Day 5.
One the contrary, one of the positive control (TCH
on the paper) gave the largest inhibition zone (24.1
mm) at Day 1, as shown in Figure 5, indicating that
most of the TCH was released from the paper in the
first day because of the burst release. At Day 2, its
inhibitory effect decreased dramatically to 7.2 mm.
From the third day, inhibition zone was not
observed any longer, because almost all drugs had
been depleted from the paper in the first two days.
As also demonstrated in Figure 5, the other positive control (commercial paper disk) had its inhibitory effect in the first 3 days, and diameters of the
inhibitory zone decreased dramatically (22.5 mm at
Day 1, 16.5 mm at Day 2, and 6.6 mm at Day 3). At
Days 4 and 5, the commercial paper disk lost its
Journal of Applied Polymer Science DOI 10.1002/app

XU ET AL.

antibacterial activity because the TCH had almost

got depleted in the disk after 3 days release.
Statistical analysis reveals that on Days 4 and 5,
the effect of bacteria inhibition of TCH-loaded nanofibers is significantly higher than both the negative
and positive controls (P < 0.01), as indicated in Figure 5. These results were enough to confirm that
TCH released from the medicated membrane will
retain its biological function, or that the activity of
antibiotics will be preserved after emulsification and
electrospinning. And prolonged inhibitory effects of
TCH-loaded fiber mats on S. aureus indicated sustained release of TCH from the medicated membrane, which is consistent with data in Figure 4.
For a comparison with the 1% w/w TCH-loaded
fiber mats, 3% w/w TCH-loaded fiber mats were
tested to clarify how the anti-bacterial effectiveness
of medicated fiber mats was related with increased
TCH content in the electrospun membranes. Obviously, the latter had stronger and more sustained inhibitory effects on S. aureus (Fig. 6). Specifically, even
at Day 5, release of bioactive drug from 3% w/w
TCH-loaded fiber mat was still able to substantially
inhibit bacterial growth, displaying a 12 mm inhibition zone on the agar plate; however, no inhibition
zone was observed in the case of 1% w/w TCHloaded fiber mat at that day (Fig. 6). In addition, the
inhibition zone of 3% w/w TCH-loaded fiber mats
vanished less quickly than that of the 1% w/w TCHloaded fiber mats. These results are in agreement
with what is shown in Figure 4: rate of release of
TCH decreases with increased TCH content in the
fibers, so that 3% w/w TCH-loaded fiber mats are capable of a more sustained TCH release than the 1%
w/w TCH-loaded fiber mats. Statistical analysis also
suggests that all the differences in bacteria inhibitions
are significant (P < 0.01) except for Day 2.

Figure 6 Diameters of inhibition zones in the Staphylococcus aureus susceptibility test of 1% w/w and 3% w/w
TCH-loaded PEGPLA fibrous mat. [Color figure can be
viewed in the online issue, which is available at www.
interscience.wiley.com.]

ELECTROSPUN PEGPLA NANOFIBROUS MEMBRANE

CONCLUSIONS
Medicated biodegradable PEGPLA nanofibrous
membranes containing TCH were fabricated by
means of emulsion-electrospinning. In vitro degradation studies revealed that the PEGPLA nanofiber
mats showed an 85% mass loss by the end of 85
days in PBS. However, the presence of proteinase K
significantly increased the degradation process, indicating the possibility of a different degradation profile in vivo, which will be studied in our future
work. Successfully incorporated into the nanofibers
and evenly released without losing its bioactivity,
the drug proved to be effective in inhibiting S. aureus bacteria growth in 5 consecutive days. Increased
TCH content in the electrospun membranes
enhanced the anti-bacterial effectiveness of these
medicated fiber mats and helped sustain the release.
Our work is expected to lead to the development of
new wound dressings that are capable of controlled
release of therapeutic agents to prevent bacterial
growth, and, as a result, to significantly reduce the
costs of treatment and care of diabetic foot ulcers.
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Journal of Applied Polymer Science DOI 10.1002/app