Antimicrobial activity of PLA/PEG nanofibers containing terpinen-4-ol

against Aggregatibacter actinomycetemcomitans

Neymara C. Nepomuceno,1 Maria ^ ngela A. Barbosa,2,3 Roberta F. Bonan,3 Juliano E. Oliveira,4
2,5
 bio C. Sampaio,
Fa Eliton S. Medeiros 1,2
1
Materials and Biosystems Laboratory (LAMAB), Department of Materials Science and Engineering (DEMAT), Federal University of
Paraıba (UFPB), Joa
~o Pessoa, PB 58.051-900, Brazil
2
Post-Graduation Program in Dentistry (PPGO/CCS/UFPB), Federal University of Paraıba (UFPB), Joa
~o Pessoa, PB C58.051-900,
Brazil
3
Health Sciences Center (CCS), Federal University of Paraıba (UFPB), Joa
~ o Pessoa, PB C58.051-900, Brazil
4
Department of Materials Engineering (DEMat), Federal University of Lavras (UFLA), Lavras-MG, CEP 37200-000, Brazil
5
Social and Clinical Dentistry Department (DCOS), Paraiba Federal University (UFPB), Bucal Biology Laboratory–LABIAL, Joa
~o
Pessoa, PB 58.051-900, Brazil
Correspondence to: E. S. Medeiros (E - mail: eliton@ct.ufpb.br)

ABSTRACT: In this article, nanofibrous mats of poly(lactic acid) (PLA) and poly(ethylene glycol) (PEG) with different PLA/PEG ratios
were prepared by solution blow spinning. Terpinen-4-ol, a major phytoconstituent from tea tree oil (Melaleuca alternifolia) was added
to the fibers and their antimicrobial activity against Aggregatibacter actinomycetemcomitans (ATCC 00078) was tested. Spun mats were
characterized by scanning electron microscopy, thermogravimetric analyses, differential scanning calorimetry, Fourier-transform infra-
red spectroscopy (FTIR), and cell viability tests by biofilm grown on the surface of inhibitory fibers. Fibers had average diameters
dependent on polymer ratio. PEG acted as a plasticizer resulting in a reduction in PLA crystallinity. Addition of PEG lead to a faster
drug release. Fibrous mats with terpinen-4-ol, whose incorporation was verified by FTIR, showed an effective antimicrobial activity
against A. actinomycetemcomitans, similar to those containing 0.12% chlorhexidine gluconate (P < 0.05), used as both the positive
control and the dose level recommended for patient treatment. These results confirm the potential of solution blow spun fibrous
mats containing terpinen-4-ol in the treatment of aggressive periodontitis. V C 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 135,

45782.

KEYWORDS: biomaterials; biopolymers and renewable polymers; fibers; nanostructured polymers; synthesis and processing
techniques

Received 25 April 2017; accepted 11 September 2017

DOI: 10.1002/app.45782

INTRODUCTION destruction observed, and directly proportional to the pres-

Periodontal diseases are complex and multifactorial manifes-
tations characterized by inflammatory lesions mediated by
interactions between the host and microorganisms occurring
in the periodontium. This results in the destruction of the
coating and supportive periodontal structures.1 Several
periodontal diseases, especially in cases of aggressive peri-
odontitis, are associated with Aggregatibacter actinomycetem-
comitans.2 Aggressive periodontitis is a disease with rapid
progression which affects adolescents and young adults, rang-
ing in age from 12 to 30 years. It causes fast vertical loss of
alveolar bone support, usually resulting in appearance of
deep alveolar bone pockets.3 Clinically, the amount of dental
plaque is inconsistent with the degree of periodontal
ence of A. actinomycetemcomitans.4
A. actinomycetemcomitans is a gram-negative coccobacillus of
the Pasteurellaceae family5 capable of producing many virulence
factors to facilitate its colonization, invasion, and destruction of
periodontal tissues. A. actinomycetemcomitans biofilms can be
easily formed on both biotic and abiotic surfaces such as glass,
plastics, and hydroxyapatite, which is an important factor in the
colonization of the oral cavity.6 When structured in the form of
a biofilm, this microorganism has a greater resilience, compared
to their planktonic form, because they respond to local environ-
mental conditions by changing gene expression patterns or con-
ducting physiological activities to adapt to a certain condition,
such as the presence of antimicrobial agents.7 The treatment of

C 2017 Wiley Periodicals, Inc.

V

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ARTICLE WILEYONLINELIBRARY.COM/APP
aggressive periodontitis consists in controlling the periodontal
infection by scaling and root planing, however, there is no suffi-
cient evidence to prove that only periodontal instrumentation is
effective for complete A. actinomycetemcomitans elimination.3
Therefore, it is necessary to look for new adjuvants such as phy-
totherapeutic agents.
There has been growing interest in the use of essential oils and
plant extracts, considering the antimicrobial effectiveness against
a wide variety of microorganisms.8 In this context, melaleuca
(Melaleuca alternifolia) oil, popularly known as tea tree oil
(TTO), is of great medical importance in the treatment of peri-
odontal diseases because it contains phytoconstituents such as
a-terpineol and terpinen-4-ol whose antibacterial, anti-
inflammatory, and antifungal action against several human
pathogens are well known.8–10 Both TTO and terpinen-4-ol
have minimal impact on the development of antimicrobial resis-
tance, which justifies the importance of more studies with this
essential oil and with its isolated compounds.11
In the biomedical field, nanostructures can be used as vehicles
for controlled release of drugs, bioactive molecules such as cyto-
kines, growth factors, anticancer drugs, enzymes, and some vita-
mins as well as hemostatic, scaffolds, and dermal regeneration
devices.12 Among these, nanofibers are one of the most produced
and studied nanostructures given their vast number of applica-
tions and benefits,13 including large surface area, high porosity
with small pore size, and the possibility of controlling their struc-
ture, a requirement in pharmaceutical formulations.13,14
Solution blow spinning (SBS) has recently emerged as a micro
and nanofiber production technique with many potential appli-
cations in the controlled release of drugs. This method uses a
syringe pump to deliver a polymer solution to an apparatus
consisting of concentric nozzles whereby the polymer solution is
pumped through the inner nozzle while a constant, high veloc-
ity gas flow is sustained through the outer nozzle. Pressure dif-
ference and shearing at the gas/solution interface help form
multiple strands of polymer solution towards a collector. Dur-
ing flight, the solvent component of the strands rapidly evapo-
rates forming a web of micro and nanofibers. The effect of
injection rate, gas flow pressure, polymer concentration, work-
ing distance, and nozzle geometry are parameters used to con-
trol fiber morphology and production rate.15 SBS has been
successfully used to produce polymer micro and nanofibers at
high rates and proven to be a promising technique to encapsu-
late essential oils and phototherapeutic agents for the controlled
delivery.16
In this article, poly(lactic acid) (PLA)/poly(ethylene glycol)
(PEG) nanofibers containing terpinen-4-ol, a major component
of TTO and chlorhexidine gluconate (CHX) were produced by
SBS and their inhibitory activity against A. actinomycetemcomi-
tans was studied.
EXPERIMENTAL
Materials
PLA (Mw 5 1.25 3 105 g mol21) was purchased from Biomater
(S~ao Paulo, Brazil) and PEG (Mw 5 8 3 103 g mol21) from
Sigma-Aldrich (Saint Louis, MI). Chloroform (99% purity) and
45782 (2 of 9)
acetone (99.5% purity) were purchased from F. Maia (Cotia-SP,
Brazil). Terpinen-4-ol and CHXwere purchased from Sigma-
Aldrich, and used as antimicrobial agents. For in vitro
tests, cultures of planktonic cells and monospecimen biofilms
of A. actinomycetemcomitans, ATCC 00078, were used.
A. actinomycetemcomitans was kindly supplied by the Oswaldo
Cruz Foundation (Rio de Janeiro, Brazil). Broth brain heart infu-
sion (BHI) (KASVI, Ref.: K25–620008), hemin (Sigma Aldrich,
assay 90%), yeast extract, menadione (Sigma Aldrich, assay
98%), resazurin (Sigma Aldrich, dye content 80%), and defi-
brinated sheep blood (Sigma Aldrich) were used as purchased.
Characterization
Determination of Minimum Inhibitory and Minimum
Bactericidal Concentrations. The minimum inhibitory concen-
tration (MIC) was determined by microdilution technique using
96-well microplates.17 The inoculum was standardized to a 0.5
McFarland scale. The culture medium used was Broth BHI, sup-
plemented with hemin 5 mg mL21 solution, yeast extract 0.005
mg mL21, and menadione 1 mg mL21 solution. Antimicrobial
agents were prepared at concentrations varying from 15 to 400
mL mL21.17 An aliquot containing 100 mL of culture medium,
100 mL of the test substance, and 10 mL of the inoculum were
dispensed into each well. Tests were performed in triplicate and
plates were incubated at 37 8C for 48 h.
Resazurin staining assay followed by visual inspection was used
to confirm the presence of viable microorganisms. 35 mL of
resazurin was added to each well, which was incubated for 1 h.
A color change of resazurin from blue to pink-violet was inter-
preted as presence of viable microorganisms.18 MIC was consid-
ered as the lowest concentration of test substance able to inhibit
visible growth of the microorganisms, that is, without causing
any color changes in resazurin.
The minimum bactericidal concentration (MBC) was considered
as the lowest concentration able to prevent visible growth of
subculture. This test was performed in a Whitley model DG250
anaerobic workstation under an atmosphere of 85% N2, 5%
CO2, and 10% H2. 100 mL aliquots from wells corresponding at
least to MIC were subcultured in BHI agar plates supplemented
with 5% defibrinated sheep blood and incubated for 48 h at
37 8C.19
Production of Fibrous Mats by Solution Blow Spinning. Table
I shows the compositions of spinning solutions used in SBS. All
solutions were prepared by solubilizing PLA (10% w/v) in a
chloroform:acetone (3:1 v/v) mixture. Then, 10, 20, and 30%
w/v PEG was added with respect to PLA. Group 1 (PLA/PEG)
refers to the negative control, in which no antimicrobial agent
was added. For test group (PLA/PEG/terpinen-4-ol), 40% v/w
terpinen-4-ol was added and for positive control group (PLA/
PEG- CHX), 0.12% v/w CHX was added relatively to the total
weight of the PLA/PEG mixture.16 0.12% CHX is used in the
literature as the dose level recommended for patient
treatment.20
The fibrous mats were prepared using a SBS apparatus at a
pressure of 344.74 kPa and injection rate of 100 lL min21. Two
concentric nozzles with protrusion of 0.5 mm (inner nozzle)
J. APPL. POLYM. SCI. 2017, DOI: 10.1002/APP.45782
ARTICLE WILEYONLINELIBRARY.COM/APP

Table I. Composition of Polymer Solutions Used to Produce PLA/PEG Solution Blow Spun Fibers Containing Terpinen-4-ol and Chlorhexidine
Gluconate

Sample Group PLA (%) PEG (%) Antimicrobial agent

PLA/PEG0 I 10 0 —
PLA/PEG10 10 10
PLA/PEG20 10 20
PLA/PEG30 10 30
PLA/PEG0/terpinen-4-ol II 10 0 Terpinen-4-ol (40%)
PLA/PEG10/terpinen-4-ol 10 10
PLA/PEG20/terpinen-4-ol 10 20
PLA/PEG30/terpinen-4-ol 10 30
PLA/PEG0/chlorhexidine III 10 0 Chlorhexidine gluconate (0.12%)
PLA/PEG10/chlorhexidine 10 10
PLA/PEG20/chlorhexidine 10 20
PLA/PEG30/chlorhexidine 10 30

were positioned at a working distance of 20 cm from the rotat-
ing collector (600 rpm) onto which spun mats were deposited
(Figure 1).21,22
Fourier-Transform Infrared Spectroscopic Characterization.
The incorporation of terpinen-4-ol and CHX in the fibrous
mats was verified by Fourier-transform infrared spectroscopy
(FTIR) using compressed KBr disks containing 2% of samples.
Spectra were recorded on a spectrophotometer using the hori-
zontal attenuated total reflectance accessory from Shimadzu,
IRAffinity-1 model/FTIR-8000 series, in the region of 600–
4000 cm21, at a resolution 4 cm21 and 20 standard scans.
Morphological Studies of Mats by Scanning Electron
Microscopy. The morphology of the spun mats was observed
using a LEO 1430 Zeiss scanning electron microscope (SEM).
Samples previously collected onto aluminum foil were metalized
with gold using an Emitech K 550X sputter coater. Fiber diame-
ters were measured using an imaging software (Image J,
National Institutes of Health (NIH), Bethesda, MD, USA). For
each specimen, the mean diameter and its distribution was
determined by analyzing 90 randomly chosen fibers.
Differential Scanning Calorimetry. Calorimetric analyses were
performed on a Differential Scanning Calorimeter, DSC 60 (Shi-
madzu, Japan). Approximately 6 mg samples were heated from
25 to 180 8C under nitrogen atmosphere at a flow of 50
mL min21 and heating rate of 10 8C min21. Glass transition
(Tg), crystallization (Tc), and melting (Tm) temperatures, melt-
ing enthalpy (DHm), and degree of crystallinity (Xc) were calcu-
lated using the following equation:
DHf
Xc ð%Þ5 3100 (1)
DHf0 WPLA
where DHf 0 is the melting enthalpy (J/g) of the component in
its fully crystalline state, DHf is the melting enthalpy determined
for each sample, and WPLA is the weight fraction of PLA in the
blend. A reference value of 93.7 J/g was used for DHf 0 in all
calculations.23

Figure 1. Scheme of the solution blow spinning setup used for fiber production.

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Figure 2. (a) Scheme of the specimen construction and (b) specimen produced by SBS.
Thermogravimetric Analyses. The thermostability of the mats
were performed on a DTG 60H thermogravimetric analyzer
(Shimadzu, Japan) using about 6 mg samples that were heated
from room temperature to 800 8C under nitrogen atmosphere at
a flow of 50 mL min21 and heating rate of 10 8C min21.
Antimicrobial Properties of Fibrous Mats against A.
actinomycetemcomitans. For A. actinomycetemcomitans biofilm
growth, disks of 2.3 cm in diameter were cut from fibrous mats
with a punch [Figure 2(a)]. Samples were exposed to the cul-
ture medium by hanging a weight of acrylic resin with a dental
floss to assure full specimen submersion in the culture medium
[Figure 2(b)]. All tests were performed in triplicate.
The antimicrobial activity of polymer blends was tested using a
static model of bacterial biofilms based on the Guggenheim
model,24 which was chosen due to similarity to natural condi-
tions of periodontal pockets.
After growth and standardization of inoculum at 0.5 McFarland
scale, the specimens previously sterilized by ultraviolet light
were inserted into falcon tubes in upright position25 and
remained incubated under anaerobic workstation in an atmo-
sphere of 85% N2, 5% CO2, and 10% H2 at 37 8C for 24 h.
Only the bacterial biofilms grown on the inhibitory surface of
the nanofibers was considered. Fibrous mats were sonicated
(Ultrasonic Cleaner USC 750; Single Group, S~ao Paulo-SP,
Brazil) for bacteria dispersion and then homogenized in a vor-
tex (Vortex Mixer; Vision Scientific Co. Ltd., Seoul, South
Korea). After specimens were removed, the result of the bacte-
rial content was used for fluorescence analysis.
For cell quantification by fluorescence imaging, a calibration
curve was used as a reference for the readings of the examined
wells. For this purpose, a combination of live (maintained in
saline) and dead A. actinomycetemcomitans bacteria (maintained
in 70% isopropyl alcohol) was prepared at the following con-
centrations: 0, 20, 50, 80, and 100% viable bacteria and the
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complement reverse (100, 80, 50, 20, and 0%) of non-viable
bacteria. Mixtures of 30 mL of live and dead bacteria were trans-
ferred to a 96-well black plate suitable for fluorescence.
Cell viability was quantified by fluorescence imaging using a
mixture of dyes (Syto 9 and propidium iodide) from the cell
viability kit Live/Dead(r) BackLight(tm) (Molecular Probes;
Invitrogen, Carlsbad, CA). After exposure of each well to the
reagent mixture, analysis was carried out on the fluorescence
microplate reader (FluoStar Optima; BMG LabTech, Germany).
Statistical Analyses. Data analyses were performed by descrip-
tive statistics. Tukey Test with a significance level set to P < 0.05
was used to analyze differences between groups, using SPSS 20.0
(2012 IBM Corp. SPSS 20.0 for Windows. Armonk, NY). Data
were presented as mean 6 standard deviation.
RESULTS AND DISCUSSION
Antimicrobial In Vitro Activity of Terpinen-4-Ol
Terpinen-4-ol shows antimicrobial activity against planktonic
strain of A. actinomycetemcomitans with MIC and MBC, respec-
tively, of 25 6 10 and 25 6 5 mg/mL. Values lower than 100 mg/
mL for antimicrobial activity tests through broth microdilution
technique are considered indicative of significant antibacterial
activity.26
Morphology of the Fibers Incorporated with Terpinen-4-Ol and
CHX Gluconate
The microscopic aspects of nanofibers are shown in Figure 3
and the average diameters in Figure 4. Fiber diameter ranged
from 612 (PLA/PEG10) to 1411 nm (PLA/PEG30/CHX). It was
observed that fiber diameter increased as the amount of PEG
increased. All fibrous mats presented a homogeneous aspect,
except the 30% PEG group [Figure 3(c)], because the high con-
tent of PEG in the blends caused some phase separation due to
its immiscible nature. However, this feature could not be
observed for other mats with the same PEG content due to the
plasticizing effect of terpinen-4-ol.
J. APPL. POLYM. SCI. 2017, DOI: 10.1002/APP.45782
ARTICLE WILEYONLINELIBRARY.COM/APP
Figure 3. SEM photomicrographs of PLA/PEG0 (a), PLA/PEG30 (b), PLA/PEG20/terpinen-4-ol (c), and PLA/PEG10–chlorhexidine (d). [Color figure
can be viewed at wileyonlinelibrary.com]
Infrared Spectroscopic Characterization
Figure 5 shows the infrared spectra of PLA/PEG samples with
and without CHX and terpinen-4-ol confirming their incorpo-
ration in the fibers. PLA fibers (PLA/PEG0) shows [Figure 5(a)]
peaks at 1757 cm21, related to stretching of the carbonyl
(C@O) groups; two others at 2995 and 2944 cm21 attributed to
CH stretching; another band at 1453 cm21, referring to CACH3
and three peaks at 1132, 1087, and 1045 cm21, for the ACOCA
groups. The peaks at 756 and 870 cm21 are assigned, respec-
tively, to the crystalline and amorphous phases of PLA,27,28
which are in the same range of PEG, that is, 753 and
Figure 4. Average fiber diameter and standard deviation for PLA/PEG
fibers.
45782 (5 of 9)
664 cm21.29 In Figure 5(b), the band at 2995 decrease in inten-
sity and the 2944 cm21, shifts to 2887 cm21 because of the
presence of terpinen-4-ol molecules sandwiched between poly-
mer chains. As for the positive control, that is, CHX [Figure
5(c)], it was not possible to identify the typical peaks of bigua-
nide groups, around 1550–1450 cm21 due to its small amount
in the formulations.30,31
Differential Scanning Calorimetry
DSC curves for nanofibers with and without terpinen-4-ol are
shown in Figure 6. Neat PLA (PLA/PEG0) [Figure 6(a)] shows
a glass (Tg) transition and melting (Tm) temperature of about
60 and 150 8C, respectively, confirming the values reported in
the literature.16,23 Small shifts in these events are correlated to
processing conditions, such as solvent used and the spinning
technique. Moreover, the addition of PEG, terpinen-4-ol [Figure
6(b)] and CHX [Figure 6(c)] causes some changes at the molec-
ular level affecting Tg and Tm. The influence of the PEG amount
was evident for all three groups. Because of its low molecular
weight, PEG molecules act as a plasticizer to PLA, shifting Tg
and the cold crystallization temperature (Tc), between 70 and
110 8C, to lower values as the concentration increases.
Glass transition, crystallization and melting temperature, melt-
ing enthalpy, and degree of crystallinity Xc (%) for PLA/PEG
blends containing CHX and terpinen-4-ol are shown in Table II.
The degree of crystallinity decreases as the amount of PEG
increases in the blends because PEG chains, sandwiched
between PLA chains, acted as a plasticizer and hindered their
packing into crystalline structures.16,23 On the other hand,
J. APPL. POLYM. SCI. 2017, DOI: 10.1002/APP.45782
ARTICLE WILEYONLINELIBRARY.COM/APP
Figure 5. FTIR spectra of PLA/PEG pristine fibers (a) and fibers containing terpinen-4-ol (b) and chlorhexidine (c).
however, the melting temperature of PLA was not affected by the
addition of PEG, this behavior was somewhat expected due to
the different chemical natures, that is, hydrophobic and hydro-
philic between PLA and PEG. In other words, despite the lower
crystallinity, PLA and PEG may have undergone phase separation
to form single PLA crystalline domains. It is known that the
degree of crystallinity plays an important role in drug release
profile in physiological medium, the results suggest the formation
of a more amorphous nanofiber as PEG is added. This property
is evidenced by the decrease in the reference values for Xc.
The glass transition temperatures of samples containing
terpinen-4-ol were lower than for PLA/PEG samples (Table II),
shift in polymer Tg from 62 to 55 8C. This plasticizing effect is
similar to those reported in the literature for PLA.30,32
The cold crystallization event was not well defined for fibrous
mats without the addition of PEG (PLA/PEG0/terpinen-4-ol).
The benzene bulky groups attached to the methyl groups of
terpinen-4-ol may have inhibited the folding of PLA chains and
hence the formation of crystals.30 Further addition of PEG
(PLA/PEG10-terpinen-4-ol) enhanced even more the plasticizing
effect and reduced the degree of crystallinity.
CHX did not significantly modify the degree of crystallization
or melting temperature (Tm) of the polymer blends (Table II) as
a result of the lower amount and the bulky groups associated
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with CHX that neither acted as a plasticizer nor as a nucleating
agent.28,31 The cold crystallization and glass transition tempera-
tures shift towards lower temperatures due to the plasticizing
effect of PEG.
Thermogravimetric Analyses
TGA curves in Figure 7 shows mass loss around 80–1008 C for
all groups due to water/moisture evaporation. PLA has two
characteristic stages of mass loss. The first, with onset tempera-
ture around 360 8C, mainly related to the splitting of the main
chains and to inter/intramolecular transesterification reactions.16
The second, starting around 400 8C, is related to active pyrolysis
the thermal degradation of PLA chains. Since the onset of PEG
degradation is higher than for PLA, this is shown as a shoulder
that increases in intensity as its proportion increases in the
blend [Figure 7(a)]. Addition of terpinen-4-ol [Figure 7(b)]
reduces the onset of degradation because of the volatilization of
molecules that are trapped between PLA/PEG molecules. On
the other hand, CHX [Figure 7(c)] does not impart any signifi-
cant difference because of its low content and relatively poor
interaction with the blends.31
Cell Viability and Biofilm Formation
The percentage of viable cells after a period of 24 h of cell expo-
sure to solution blow spun PLA/PEG fibrous mats, with and
without terpinen-4-ol and CHX, is showed in Figure 8. All
J. APPL. POLYM. SCI. 2017, DOI: 10.1002/APP.45782
ARTICLE WILEYONLINELIBRARY.COM/APP

Figure 6. DSC curves of PLA/PEG pristine fibers (a) and fibers containing terpinen-4-ol (b) and chlorhexidine (c).

bacteria were dead for all fibers containing CHX. PEG addition PLA/PEG blends also caused a significant change in cell viability
slightly reduced the number of viable cells because of its antimi- with addition of terpinen-4-ol for all concentrations. Among
crobial activity.33 samples containing PEG, fibers with 10 and 20% PEG

Table II. Glass Transition, Crystallization and Melting Temperature, Melting Enthalpy, and Degree of Crystallinity for PLA/PEG Blends Containing
Chlorhexidine and Terpinen-4-ol

Polymer blend Tg (8C) Tm (8C) DHm (J/g) Tcc (8C) Xc (%)

PLA/PEG0 62 149 22 93 22.6
PLA/PEG10 53 150 21 76 19.9
PLA/PEG20 55 150 19 69 15.6
PLA/PEG30 52 150 18 — 12.7
PLA/PEG0/terpinen-4-ol 55 147 19 78 19.2
PLA/PEG10/terpinen-4-ol 53 148 17 82 16.1
PLA/PEG20/terpinen-4-ol 53 147 19 — 15.6
PLA/PEG30/terpinen-4-ol 52 148 15 — 10.6
PLA/PEG0/chlorhexidine 61 150 20 97 21.0
PLA/PEG10/chlorhexidine 53 149 20 75 18.3
PLA/PEG20/chlorhexidine 52 149 18 77 15.2
PLA/PEG30/chlorhexidine 52 149 18 — 12.8

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ARTICLE WILEYONLINELIBRARY.COM/APP
Figure 7. TG curves of PLA/PEG pristine fibers (a) and fibers containing terpinen-4-ol (b) and chlorhexidine (c).
(PLA–PEG10 and 20) were statistically different (P 5 0.05).
Fibers with 20% PEG had higher antimicrobial activity when
compared to other fibers of the same group.
Comparing PLA/PEG/terpinen-4-ol with the positive control
(PLA/PEG/CHX), for all the fibers, there was no statistically
Figure 8. Percentage of viable cells of A. actinomycetemcomitans biofilm
on the surfaces of nanofibers with different concentrations of PEG. *Sta-
tistically significant difference between PEG concentration, while main-
taining set drug group (P < 0.05) (ANOVA Tukey).
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significant difference between the two groups (P 5 0.05).
Accordingly, fibers containing terpinen-4-ol for all PEG concen-
trations, showed similar antimicrobial activity to fibers contain-
ing CHX. In other words, terpinen-4-ol, a phytotherapeutic
agent, shows the same potential in the treatment against A. acti-
nomycetemcomitans as CHX. This is an interesting finding not
only because terpinen-4-ol side effects are potentially fewer but
also because it can be used as an alternative treatment for
patients with intolerance or allergic reactions to CHX.34,35
In a previous study, we evaluated the antimicrobial activity of
PLA blends with poly(vinyl pyrrolidone) (PVP), with PVP con-
centrations ranging from 0, 5, 10, and 20%, incorporated with
copaiba oil against planktonic strains of Staphylococcus aureus
and Escherichia coli using the agar diffusion method.16 The anti-
microbial properties of the blend were directly related to the
amount of PVP, which was attributed to the hydrophilic nature
of this polymer, allowing greater interaction with the culture
medium and thus favoring drug release. However, the same
behavior did not hold true for PLA/PEG blends due to the
lower molecular weight of PEG (8,000 g/mol) as compared to
their PVP used (55,000 g/mol), since after a period of 24 h PEG
completely was readily dissolved carrying all terpinen-4-nol to
the culture medium and therefore reducing cell viability by
exposing them to the phytotherapeutic agent.
J. APPL. POLYM. SCI. 2017, DOI: 10.1002/APP.45782
ARTICLE WILEYONLINELIBRARY.COM/APP
CONCLUSIONS
PLA/PEG nanofibers containing terpinen-4-ol and CHX were
successfully produced by SBS. The addition and PEG increased
the diameter and reduced the degree of crystallinity of the
fibers. Fibers containing terpinen-4-ol exhibited an excellent
antimicrobial activity against A. actinomycetemcomitans, a
behavior comparable to CHX, a substance commonly used in
the treatment of periodontitis caused by these bacteria.
Although there were slight differences in the microbial activity
of PLA/PEG fibers with respect to increasing PEG concentra-
tion, this behavior was attributed to the highly hydrophilic
nature and the low molecular weight of PEG that proportion-
ated a fast release of the phytotherapeutic agent within the 24 h
release time. The produced fibrous mats were highly effective
against A. actinomycetemcomitans, demonstrating their potential
as adjuncts to the treatment of aggressive periodontitis. Further
studies should be performed varying PEG molecular weight and
concentrations of terpinen-4-ol, as well as cytotoxicity tests.
ACKNOWLEDGMENTS
The authors would like to thank the Brazilian national research
council CNPq (Grant Nos. 150325/2015-0, 302044/2015-9, and
403357/2016-0) and Minas Gerais State research council FAPE-
MIG (Grant No. APQ-01505-15) for the financial support pro-
vided, and I. A. P. Farias and M. A. C. Oliveira for their help with
microbiological tests.
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