Poly(anhydride-esters) comprised exclusively of naturally occurring antimicrobials and EDTA: Antioxidant and antibacterial activities

Article abstract of DOI:10.1021/bm500303a

Carvacrol, thymol, and eugenol are naturally occurring phenolic compounds known to possess antimicrobial activity against a range of bacteria, as well as antioxidant activity. Biodegradable poly(anhydride-esters) composed of an ethylenediaminetetraacetic acid (EDTA) backbone and antimicrobial pendant groups (i.e., carvacrol, thymol, or eugenol) were synthesized via solution polymerization. The resulting polymers were characterized to confirm their chemical composition and understand their thermal properties and molecular weight. In vitro release studies demonstrated that polymer hydrolytic degradation was complete after 16 days, resulting in the release of free antimicrobials and EDTA. Antioxidant and antibacterial assays determined that polymer release media exhibited bioactivity similar to that of free compound, demonstrating that polymer incorporation and subsequent release had no effect on activity. These polymers completely degrade into components that are biologically relevant and have the capability to promote preservation of consumer products in the food and personal care industries via antimicrobial and antioxidant pathways.

Full text of DOI:10.1021/bm500303a

Article
pubs.acs.org/Biomac
Open Access on 04/07/2015
Poly(anhydride-esters) Comprised Exclusively of Naturally Occurring
Antimicrobials and EDTA: Antioxidant and Antibacterial Activities
Ashley L. Carbone-Howell, Nicholas D. Stebbins, and Kathryn E. Uhrich*
S
* Supporting Information
ABSTRACT: Carvacrol, thymol, and eugenol are naturally occurring phenolic compounds known to possess antimicrobial
activity against a range of bacteria, as well as antioxidant activity. Biodegradable poly(anhydride-esters) composed of an
ethylenediaminetetraacetic acid (EDTA) backbone and antimicrobial pendant groups (i.e., carvacrol, thymol, or eugenol) were
synthesized via solution polymerization. The resulting polymers were characterized to confirm their chemical composition and
understand their thermal properties and molecular weight. In vitro release studies demonstrated that polymer hydrolytic
degradation was complete after 16 days, resulting in the release of free antimicrobials and EDTA. Antioxidant and antibacterial
assays determined that polymer release media exhibited bioactivity similar to that of free compound, demonstrating that polymer
incorporation and subsequent release had no effect on activity. These polymers completely degrade into components that are
biologically relevant and have the capability to promote preservation of consumer products in the food and personal care
industries via antimicrobial and antioxidant pathways.
thymol, and eugenol are three such phenolic compounds that
are major constituents of oregano, thyme, and clove essential
oils, respectively.4,5 In addition to their antibacterial activity,6
these compounds are potent antioxidants;7 thus, they can
potentially be used to prevent the bacterial spoilage8,9 and
oxidation10,11 of perishable food items (e.g., meat, dairy, fruit)
as well as personal care products.12
Currently, antimicrobials are physically mixed into formula-
tions to prevent bacterial growth. The sustained release of these
bioactive compounds can naturally preserve consumer products
thereby increasing shelf life. To extend bioactive release,
INTRODUCTION
■
Prevention of microbial contamination in the personal care and
food industries is of paramount importance for consumer
protection. However, most commonly used synthetic preserva-
tives, such as parabens, suffer from problems such as
sensitization and toxicity.1 Another commonly used antimicro-
bial, triclosan, is thought to breed bacterial resistance. Triclosan
can potentially disrupt the endocrine system and its level in the
blood correlating to a consumer’s use has been discovered.2
Moreover, parabens, triclosan, and other preservatives have
environmental bioaccumulation issues, with concentrations of
these chemicals detected in aquatic life,3 leading to a call for
safer alternatives. Alternatively, many naturally occurring
phenolic compounds possess known activity against a range
of Gram-positive and Gram-negative bacteria. Carvacrol,
Received: February 26, 2014
Revised: March 25, 2014
Published: April 7, 2014
© 2014 American Chemical Society
1889
dx.doi.org/10.1021/bm500303a | Biomacromolecules 2014, 15, 1889−1895

Biomacromolecules
Article
Scheme 1. Synthesis of Antimicrobial-Containing PAEs (4) and Precursors (3)
researchers have physically incorporated the aforementioned
phenols into polymer matrices. Chitosan nanoparticles loaded
with eugenol or carvacrol were successfully formulated but
suffered from low antimicrobial content (ca. 3% w/w)13 or a
lack of release studies.14 Likewise, carvarcol-containing poly-
ethylene-co-vinylacetate films15 and thymol-containing poly-
caprolactone films16 suffered from low loading at 7 and 10 wt
%, respectively. Eugenol has been physically incorporated into
methacrylate polymers; however, release studies were con-
ducted under nonphysiological conditions (i.e., ethanol).17
Additionally, poly(lactic-co-glycolic acid) (PLGA) films con-
taining carvacrol within the polymer matrix exhibited
antibiofilm and antibacterial activities,18 although the release
is not quantified. Eugenol and carvacrol-loaded PLGA nano-
particles displaying antibacterial activity have been formulated
by multiple groups but the nanoparticles exhibited a burst
release; nearly 50% antimicrobial released in the first 8 h for
eugenol19 and 60% released in 3 h for carvacrol.20
Chemical incorporation of bioactives into a biodegradable
polymer backbone has multiple advantages (e.g., higher drug
loading, sustained release, tunable release rates and geometry
depending on desired application) over the aforementioned
physical incorporation methods.21,22 Chemical incorporation of
eugenol into a polymer backbone has also been achieved, along
with antibacterial activity, but the polymethacrylate was not
degradable, limiting its use in other applications.23 In this work,
we present the synthesis of biodegradable poly(anhydride-
esters) (PAEs) containing one of the three phenolic
compounds (carvacrol, thymol, or eugenol) chemically linked
and subsequently polymerized through EDTA, a widely used
chelator known to prevent oxidation caused by metals,24 to
achieve high drug loading (>50%). Previously, our group has
synthesized polymers chemically, incorporating various bifunc-
tional25,26 and monofunctional27,28 bioactives; however, in this
research the polymers hydrolyze into the phenols and EDTA,
both of which are known preservatives and safe to use in
consumer products.29,30 Furthermore, all ultimate degradation
products are found on the FDA’s generally regarded as safe
(GRAS) list. Chemical structures, thermal properties, and
molecular weights of polymer and precursors were determined
and in vitro release studies confirmed that the polymer
degraded into the free antimicrobials and EDTA. The release
media antioxidant efficacy and antibacterial activity were
determined using a free radical quenching assay and a disk
diffusion assay with a Gram-positive bacterium (Staphylococcus
aureus) and a Gram-negative bacterium (Escherichia coli),
respectively, and compared to activities of the free phenolic
compounds.
EXPERIMENTAL SECTION
■
Materials. Concentrated hydrochloric acid (HCl), 1 N HCl, 1 N
sodium hydroxide (NaOH), poly(vinylidine fluoride), and poly-
(tetrafluoroethylene) syringe filters, and Wheaton glass scintillation
vials were purchased from Fisher Scientific (Fair Lawn, NJ). All other
reagents, solvents, and fine chemicals were obtained from Aldrich
(Milwaukee, WI) and used as received.
1
Structural and Chemical Composition Characterization. H
NMR spectra of all products were recorded on a Varian 400 MHz
spectrophotometer. Samples (5−10 mg) were dissolved in DMSO-d6,
which was also used as the internal reference. Fourier-transform
infrared (FT-IR) absorbance spectra were measured on a Thermo
Nicolet/Avatar 360 FT-IR spectrometer. Samples (1 wt %) were
ground with KBr and pressed into a disc. Each spectrum was an
average of 32 scans. Elemental analyses were performed by QTI
(Whitehouse, NJ).
Molecular Weight. Polymer precursors were analyzed by mass
spectrometry (MS) to determine molecular weights. A Finnigan LCQ-
DUO running Xcalibur software and an adjustable atmospheric
pressure ionization electrospray source (API-ESI Ion Source) was
used. Samples were dissolved in methanol (10 μg/mL) and injected
with a glass syringe. During the experiment, the pressure was 0.8 ×
10−5 Torr and the API temperature was 150 °C. Polymer weight-
averaged molecular weights (Mw) and polydispersity indices (PDI)
were determined by gel permeation chromatography (GPC) on a
PerkinElmer liquid chromatography system consisting of a Series 200
refractive index detector, a Series 200 LC pump, and an ISS 200
autosampler. Automation of the samples and collection and processing
of the data was done using a Dell OptiPlex GX110 computer running
PerkinElmer TurboChrom 4 software using a PerkinElmer Nelson 900
Series Interface and 600 Series Link. Polymer samples were prepared
for autoinjection by dissolving polymer in DCM (10 mg/mL) and
filtering through 0.45 μm poly(tetrafluoroethylene) (PTFE) syringe
filters. Samples were resolved on a Jordi divinylbenzene mixed-bed
GPC column (7.8 × 300 mm, Alltech Associates, Deerfield, IL) at 25
°C, with DCM as eluent at a flow rate of 0.5 mL/min. Molecular
1890
dx.doi.org/10.1021/bm500303a | Biomacromolecules 2014, 15, 1889−1895

Biomacromolecules
Article
weights were calibrated relative to polystyrene standards (Polymer
Source Inc., Dorval, Canada).
(PerkinElmer Lambda XLS spectrophotometer, Waltham, MA) at λ
= 270 nm. Additionally, the bioactive (1) release from diacid (3) was
elucidated under the same conditions listed above, using powdered
diacid samples (15 mg) incubated in 10 mL of PBS (pH 7.4) in 15 mL
centrifuge tubes (BD Falcon, Franklin Lakes, NJ). After centrifugation
at 6000 rpm for 4 min, the degradation media (5 mL) was collected at
predetermined time points and fresh PBS (5 mL) replaced that which
was removed. High performance liquid chromatography (HPLC)
quantified the amount of free phenol in spent media released by
comparison to calibration curves of standard solutions. Media was
analyzed via HPLC using an XTerra RP18 3.5 μm 4.6 × 150 mm
column (Waters, Milford, MA) on a Waters 2695 Separations Module
equipped with a Waters 2487 Dual Absorbance Detector. All samples
were filtered using 0.22 μm poly(vinylidine fluoride) syringe filters and
subsequently injected (20 μL) using an autosampler. The mobile
phase composed of methanol (55%) and 50 mM KH2PO4 with 1%
formic acid in DI water at pH 2.5 (45%) run at 0.8 mL/min flow rate
at ambient temperature. Absorbance was monitored at λ = 270. The
degradation experiments were performed in triplicate.
Antioxidant Activity via Radical Scavenging. To determine
the degradation media antioxidant activity, a 2,2-diphenyl-1-picrylhy-
drazyl (DPPH) radical scavenging assay was used.31 This was
performed by adding a sample (0.1 mL) to a 24 μg/mL DPPH
solution in methanol (3.9 mL). Hour-24 polymer degradation media
samples (0.1 mL) were incubated with the DPPH solution (3.9 mL) at
room temperature with gentle shaking. After 1 h, solutions were
analyzed by UV/vis spectrophotometry at λ = 517 nm. For
comparison, a solution of freshly prepared bioactive (1) at the same
concentration of the 24-h degradation media (as determined at
HPLC) was prepared and analyzed via the same method. DPPH %
radical reduction was calculated by [(Abst0 − Abst)/Abst0] × 100,
where Abst0 is the initial absorbance, and Abst is the absorbance after 1
h. All radical quenching assays were performed in triplicate. Student’s t
tests were used to determine the significant difference of the
antioxidant activity between degradation media and free antimicrobial
(significantly different if p < 0.05)
Antibacterial Testing. Carvacrol, thymol, and eugenol are well-
known antimicrobial agents, exhibiting activity against a range of
bacteria;6 thus, the activity of degradation media against Gram-positive
and Gram-negative bacteria was tested using the disc diffusion
method.32 First, polymer was completely hydrolyzed with 1 N NaOH,
then acidified to pH 2 using concentrated HCl, after which bioactives
were extracted with ethyl acetate. The organic layer was dried over
MgSO4 and concentrated in vacuo. Upon solubilization in 1:1 PBS/
DMSO, the concentration of all bioactives was 10 mg/mL. The three
free bioactives were tested against equal concentrations of bioactives
extracted from the degradation media (10 mg/mL) and tested as
follows: Muller-Hinton agar (Becton Dickinson, Sparks, MD) was
poured into sterile Petri dishes (Fisher, Fair Lawn, NJ) to an even
thickness of 4 mm. Bacteria inocula (S. aureus or E. coli) were
suspended in nutrient broth (EMD Chemicals, Gibbstown, NJ) and
turbidity to give a bacterial count of approximately 108 colony forming
units per mL. The agar plate was inoculated with bacteria broth culture
using a sterile cotton swab (Fisher, Fair Lawn, NJ). Sterile paper discs
(6 mm diameter, Becton Dickinson, Franklin Lakes, NJ) were
impregnated with 25 μL of test solutions (one for each free phenol,
one for each phenol degradation media, one for EDTA, and one for
PBS/DMSO). Discs were then placed onto an agar plate and gently
pressed down. Plates were incubated at 37 °C for 24 h, after which
zones of inhibition were measured with a ruler and rounded to the
nearest millimeter.
Thermal Properties. Differential scanning calorimetry (DSC) was
performed using a Thermal Analysis (TA) DSC Q200 to evaluate
thermal transitions (i.e., melting points of polymer precursors and
glass transition temperatures of polymers). TA Universal Analysis
2000 software was used for automation and data collection on an IBM
ThinkCentre computer. Samples (5−10 mg) were heated under dry
nitrogen gas from −10 to 200 °C at a heating rate of 10 °C/min and
cooled to −10 °C at a rate of 10 °C/min with a two-cycle minimum.
Melting temperatures were calculated at the peak of melting.
Thermogravimetric analyses (TGA) were performed on a
PerkinElmer Pyris 1 system with TAC 7/DX instrument controller.
PerkinElmer Pyris software running on a Dell Optiplex GX110
computer was used for automation and data collection and processing.
Samples (5−10 mg) were heated under dry nitrogen gas from 25 to
400 °C at a heating rate of 10 °C/min. Decomposition temperatures
were measured at the onset of thermal decomposition.
Polymer Precursor (3) Synthesis. Antimicrobial-containing
diacids were synthesized using a modified previously developed
procedure.27 Diacids (3) were prepared by reaction of ethyl-
enediaminetetraacetic acid (EDTA) dianhydride (2) with the
appropriate antimicrobial (1) in the presence of a base (triethylamine)
(Scheme 1). The full characterization of the thymol-based system is
presented as an example. The data for carvacrol- and eugenol-based
systems can be found in the Supporting Information. Thymol (1a; 18
mmol) was dissolved in anhydrous THF (75 mL) and anhydrous
triethylamine (Et3N, 64 mmol). EDTA dianhydride (5; 9 mmol) was
added to the reaction mixture while stirring at room temperature to
yield a suspension. The reaction stirred at room temperature under
nitrogen overnight. Then, the reaction mixture was poured over DI
water (∼500 mL) and acidified to pH 2 using concentrated HCl. The
solid diacid (3a) that formed was isolated via vacuum filtration, washed
with water (3 × 200 mL), and dried overnight under vacuum at room
temperature.
Thymol-Based Diacid (3a). Yield: 76% (off-white powder). 1H
NMR (DMSO-d6, 400 MHz): δ 7.21 (d, 2H, Ar−H); 7.02 (d, 2H,
Ar−H); 6.87 (s, 2H, Ar−H); 4.15 (s, 4H, CH2); 3.77 (s, 4H, CH2);
3.13 (s, 4H, CH2); 2.92 (m, 2H, CH); 2.23 (s, 6H, CH3); 1.08 (d,
12H, CH3). IR (KBr, cm−1): 3400 (OH, COOH), 1760 (CO,
ester), 1712 (CO, COOH). MS m/z = 557.2 [M + 1],
(C30H40N2O8)n (556.7) Calcd: C, 64.14; H, 7.4; N, 4.70. Found: C,
64.73; H, 7.24; N, 5.03. Tm: 113 °C.
Polyanhydride-ester (4) Synthesis. Diacid (3; 5.4 mmol) was
dissolved in 20% (w/v) anhydrous DCM, and triethylamine (24
mmol) was added. Then, triphosgene (6 mmol) dissolved in
anhydrous DCM (15 mL) was added dropwise at 0 °C to the stirring
reaction mixture over 1 h using a syringe pump to yield a suspension.
After stirring for 2 h at 0 °C under nitrogen, the reaction mixture was
poured over diethyl ether (∼400 mL). The solid polymer (4) that
formed was isolated by vacuum filtration, washed with acidic water
(100 mL) and dried overnight under vacuum at room temperature.
Thymol-Based Polymer (4a). Yield: 39% (brown powder). 1H
NMR (DMSO-d6, 400 MHz): δ 7.22 (d, 2H, Ar−H); 7.03 (d, 2H,
Ar−H); 6.82 (s, 2H, Ar−H); 4.41 (s, 4H, CH2); 3.68 (s, 4H, CH2);
2.92 (m, 5H, CH2, CH); 2.24 (s, 6H, CH3); 1.06 (d, 12H, CH3). IR
(KBr, cm−1): 1815 and 1745 (CO, anhydride), 1730 (CO, ester).
Mw = 23200 Da; PDI 1.3. Tg = 77 °C. Td = 223 °C.
In Vitro Bioactive Release. First, the release of diacid (3) from
polymer (4) was evaluated to determine the amount of time required
to hydrolyze anhydride bonds through in vitro degradation in
phosphate buffered saline (PBS). Polymers were ground into powder
using mortar and pestle to obtain particles of ∼300−500 μm, as
determined by standard testing sieves (Aldrich, Milwaukee, WI).
Powdered polymer samples (15 mg) were incubated in 10 mL of PBS
(pH 7.4) in 20 mL Wheaton glass scintillation vials (Fisher, Fair Lawn,
NJ) using a controlled environment incubator-shaker (New Brunswick
Scientific Co., Edison, NJ) at 60 rpm at 37 °C. At predetermined time
intervals, the media was replaced with fresh PBS and the spent media
was analyzed by ultraviolet−visible (UV−vis) spectrophotometry
RESULTS AND DISCUSSION
■
Synthesis and Characterization. The phenolic antimicro-
bials (1) are reacted with EDTA dianhydride (2) in the
presence of triethylamine to yield diacid (3) via a ring-opening
transesterification (Scheme 1). The diacids, 3, were successfully
prepared in high yields (76−85%) with only minor purification
1
necessary. Diacid structures were confirmed by H NMR and
1891
dx.doi.org/10.1021/bm500303a | Biomacromolecules 2014, 15, 1889−1895

Biomacromolecules
Article
1
Figure 1. H NMR spectra of thymol (a), corresponding diacid (b), and polymer (c).
FT-IR spectra, while DSC, MS, and elemental analysis were
used for melting point, molecular weight, and chemical
composition determination, respectively. In the 1H NMR
spectrum of 3a, which is provided as an example (Figure 1), the
disappearance of the phenol signal of 1a at 9.02 ppm and the
appearance of EDTA linker peaks at 4.15, 3,77, and 3.13 ppm
demonstrate successful ring-opening esterification to generate
3a. The diacids 3 were polymerized via solution polymerization
techniques,33,34 using triphosgene as the coupling reagent in the
presence of triethylamine at 0 °C. Solution polymerization was
chosen instead of melt-condensation to prevent potential ring
closure and regeneration of the EDTA dianhydride, 5.34
Polymers were characterized by 1H NMR to confirm structure.
Additionally, FT-IR (Figure 2) confirmed the synthesis of a
poly(anhydride-ester) through the disappearance of the
carboxylic acid stretch at 1712 cm−1 in diacid 4 and the
appearance of the CO anhydride stretches at 1815 and 1745
cm−1 in polymer 5.
The polymers (4) displayed moderate molecular weights
typical for solution polymerization techniques, ranging from
11000 to 23000 Da.33,34 PDI values were narrow (1.3−1.5)
following isolation from the reaction mixture, indicating high
homogeneity. The polyanhydride with the bulkiest antimicro-
bial, eugenol (4c), was the most difficult to polymerize and
displayed the lowest molecular weight. All polymers (4) are
amorphous with no indication of melting temperatures (up to
200 °C), exhibiting only glass transition temperatures in the
range of 65 to 86 °C. With thermal decomposition occurring at
temperatures >220 °C, it is anticipated that processing via melt-
extrusion will be a viable method for film production and
processing at elevated temperatures.
In Vitro Bioactive Release. Bioactive release was carried
out on powdered samples in PBS at physiological conditions
(37 °C, pH 7.4). The degradation rate of polymer into
bioactive via anhydride and ester bond hydrolysis is an
important factor in obtaining controlled antimicrobial release.
Based upon previous knowledge of PAEs, the anhydride bonds
Figure 2. FTIR spectra of thymol-containing diacid 3a (A, top) and
polymer 4a (B, bottom).
1892
dx.doi.org/10.1021/bm500303a | Biomacromolecules 2014, 15, 1889−1895

Biomacromolecules
Article
Scheme 2. Proposed Hydrolytic Degradation Scheme of Antimicrobial-Containing PAEs (4) into Diacid (3) and Free
Antimicrobial (1) and EDTA
are expected to hydrolyze first, followed by the ester bonds
(Scheme 2).35 To obtain polymer degradation lag time (i.e., the
time during which little degradation takes place) under
physiological conditions, degradation of polymer into diacid
was first determined by UV−vis spectrophotometry. All
polymers (4) exhibited a brief lag time (<6 h) before any
degradation was detected, as polyanhydrides commonly exhibit
a degradation lag time due to their surface eroding proper-
ties.35,36 To study complete degradation, bioactive release was
monitored under identical conditions with HPLC. The studies
were concluded upon complete degradation into respective
bioactives and EDTA. Small amounts of diacid were also
observed in degradation media, with the amounts of thymol
increasing over time (e.g., thymol retention time of 14.45 min,
thymol diacid retention time of 4.54 min), thus, proving that
the anhydride bonds hydrolyze first, followed by ester bonds.
All chemically incorporated bioactive was completely released
after 16 days. At the end of the study, a mass balance was
performed (>97% mass accounted for) and the release data was
normalized (Figure 3)
radical quenching assay was used.31 DPPH is often used to
assess antioxidant activity by determining the change in
absorbance at 517 nm via UV−vis spectrophotometry; the
solution color turns from deep purple to light yellow upon free
radical quenching, thus reducing absorbance at 517 nm. Given
that the assay is dependent on antioxidant concentration,37
degradation media at 24 h were analyzed (Figure 4) and
Antioxidant Activity. To ensure bioactive released from
polymers exhibited the same efficacy as free bioactive, a DPPH
Figure 4. DPPH reduction results comparing bioactive released from
polymer at a 24 h time point to free bioactive.
compared to freshly prepared solutions of free bioactives of the
same concentrations (52 μg/mL for carvacrol, 74 μg/mL for
eugenol, and 100 μg/mL for thymol). Student’s t tests were
performed to ascertain significant differences (p < 0.05)
between the released and freshly prepared samples. The
observed activities displayed no significant differences between
released samples and free bioactive for all three phenols; thus
the intermediate degradation products (i.e., diacid) and EDTA
present in the degradation media had a negligible effect on free
radical quenching ability. The DPPH quenching efficacy of the
concentrations tested are consistent when compared to
literature values of 500 μg/mL solutions of eugenol, carvacrol,
and thymol (93, 79, and 82%, respectively).7
Antibacterial Testing. To ensure that polymer degradation
products diffusing from the discs would cause clear zones of
Figure 3. Normalized release of antimicrobials (1) from polymers (4)
is a result of in vitro hydrolytic degradation.
1893
dx.doi.org/10.1021/bm500303a | Biomacromolecules 2014, 15, 1889−1895

Biomacromolecules
Article
Figure 5. Disk diffusion assay results for E. coli (A) and S. aureus (B) showing zones of growth inhibition for 1:1 PBS/DMSO (a), extracted eugenol
(b), extracted thymol (c), extracted carvacrol (d), free eugenol (e), free thymol (f), free carvacrol (g), and EDTA (h).
growth inhibition, the polymer was completely hydrolyzed and
a specific bioactive concentration was prepared from extracted
products and compared to that of equivalent concentrations of
free bioactives; the concentration for all bioactives was kept at
10 mg/mL (greater than the minimum inhibitory concen-
trations (MICs) to ensure a clear zone is observed) in 1:1 PBS/
DMSO. S. aureus, a Gram-positive bacterium, and E. coli, a
Gram-negative bacterium, were both evaluated, since both
strains are commonly encountered and often responsible for
contamination of products leading to spoilage.38 As shown in
Figure 5, the free phenols diffused from the discs and prevented
bacterial growth on the agar. Both the free bioactives (e−g) and
the extracted bioactives (b−d) exhibited nearly the same zones
of inhibition for both strains (Table 1). Neither EDTA (h) nor
and EDTA, are both commonly used as preservatives and are
found on the FDA GRAS list. These polymers are unique in
that the polymer completely breaks down into useful products:
EDTA and an antimicrobial. A sustained release of compounds
with antimicrobial, antioxidant, and chelation abilities can be
beneficial for consumer product protection; the spoilage of
products caused by bacterial contamination and oxidation can
be prevented by polymer degradation products. Furthermore,
bioactives released from polymer display similar antioxidant and
antibacterial activities. PAE properties allow for future
formulations, such as films and microspheres, in addition to
tunable release rates and thermal properties by changing the
dianhydride used.
ASSOCIATED CONTENT
* Supporting Information
Characterization data for carvacrol- and eugenol-based
polymers and precursors are included. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
Table 1. Zones of Growth Inhibition for Extracted and Free
Phenols
S
E. coli
S. aureus
compound
zone of inhibition (mm) zone of inhibition (mm)
PBS/DMSO
extracted eugenol
free eugenol
AUTHOR INFORMATION
Corresponding Author
*E-mail: keuhrich@rutgers.edu. Tel.: (848) 445-0361. Fax:
(848) 445-7036.
■
4
4
8
8
7
7
4
4
7
6
6
6
extracted thymol
free thymol
extracted carvacrol
free carvacrol
EDTA
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
PBS/DMSO (a) controls exhibited any activity against either
strain. Carvacrol and thymol displayed greater activity
compared to eugenol, which is expected owing to eugenol’s
higher MICs against both strains.6 Overall, this assay shows that
the methods used to process the bioactives used did not alter
their antibacterial activity upon release from polymer.
The authors thank the National Institutes of Health and Center
for Advanced Food Technology (Rutgers University) for
financial support, Michelle Moy (Chemistry, Rutgers Univer-
sity) for assistance with the experiments, and Dr. Susan Skelly
(Microbiology, Rutgers University) for assistance with the
antimicrobial studies.
CONCLUSION
■
REFERENCES
■
PAEs containing naturally occurring antimicrobials found in
plant extracts were synthesized using solution polymerization
methods, resulting in products with high drug loading (>50%).
Polymers (4) hydrolytically degraded after 16 days, releasing
free phenolic antimicrobials and EDTA in a controlled manner.
Final polymer degradation products, phenolic antimicrobials
(1) Soni, M. G.; Carabin, I. G.; Burdock, G. A. Food Chem. Toxicol.
2005, 43, 985−1015.
(2) Dann, A. B.; Hontela, A. J. Appl. Toxicol. 2011, 31, 285−311.
(3) Brausch, J. M.; Rand, G. M. Chemosphere 2011, 82, 1518−1532.
(4) Chaieb, K.; Hajlaoui, H.; Zmantar, T.; Ben, K.-N. A.; Rouabhia,
M.; Mahdouani, K.; Bakhrouf, A. Phytother. Res. 2007, 21, 501−506.
1894
dx.doi.org/10.1021/bm500303a | Biomacromolecules 2014, 15, 1889−1895

Biomacromolecules
Article
(5) Lee, S.-J.; Umano, K.; Shibamoto, T.; Lee, K.-G. Food Chem.
2004, 91, 131−137.
(6) Burt, S. Int. J. Food Microbiol. 2004, 94, 223−253.
(7) Lin, C.-W.; Yu, C.-W.; Wu, S.-C.; Yih, K.-H. Yaowu Shipin Fenxi
2009, 17, 386−395.
(8) Cleveland, J.; Montville, T. J.; Nes, I. F.; Chikindas, M. L. Int. J.
Food Microbiol. 2001, 71, 1−20.
(9) Holley, R. A.; Patel, D. Food Microbiol. 2005, 22, 273−292.
(10) Baron, C. P.; Andersen, H. J. J. Agric. Food Chem. 2002, 50,
3887−3897.
(11) Soliva-Fortuny, R. C.; Biosca-Biosca, M.; Grigelmo-Miguel, N.;
Martin-Belloso, O. J. Sci. Food Agric. 2002, 82, 1490−1496.
(12) Varvaresou, A.; Papageorgiou, S.; Tsirivas, E.; Protopapa, E.;
Kintziou, H.; Kefala, V.; Demetzos, C. Int. J. Cosmet. Sci. 2009, 31,
163−175.
(13) Woranuch, S.; Yoksan, R. Carbohydr. Polym. 2013, 96, 586−592.
(14) Chen, F.; Shi, Z.; Neoh, K. G.; Kang, E. T. Biotechnol. Bioeng.
2009, 104, 30−39.
(15) Nostro, A.; Scaffaro, R.; D’Arrigo, M.; Botta, L.; Filocamo, A.;
Marino, A.; Bisignano, G. Appl. Microbiol. Biotechnol. 2012, 96, 1029−
1038.
(16) Sanchez-Garcia, M. D.; Ocio, M. J.; Gimenez, E.; Lagaron, J. M.
J. Plast. Film Sheeting 2008, 24, 239−251.
(17) Peppas, N. A.; am, E. D. J. J. Appl. Polym. Sci. 1997, 66, 509−
513.
(18) Zodrow, K. R.; Schiffman, J. D.; Elimelech, M. Langmuir 2012,
28, 13993−13999.
(19) Gomes, C.; Moreira, R. G.; Castell-Perez, E. J. Food Sci. 2011,
76, N16−N24.
(20) Iannitelli, A.; Grande, R.; Di, S. A.; Di, G. M.; Sozio, P.; Bessa, L.
J.; Laserra, S.; Paolini, C.; Protasi, F.; Cellini, L. Int. J. Mol. Sci. 2011,
12, 5039−5051.
(21) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K.
M. Chem. Rev. (Washington, D. C.) 1999, 99, 3181−3198.
(22) Prudencio, A.; Schmeltzer, R. C.; Uhrich, K. E. Macromolecules
2005, 38, 6895−6901.
(23) Rojo, L.; Vazquez, B.; Parra, J.; Lopez, B. A.; Deb, S.; San, R. J.
Biomacromolecules 2006, 7, 2751−2761.
(24) Cannarsi, M.; Baiano, A.; Sinigaglia, M.; Ferrara, L.; Baculo, R.;
Del, N. M. A. Int. J. Food Sci. Technol. 2008, 43, 573−578.
(25) Erdmann, L.; Uhrich, K. E. Biomaterials 2000, 21, 1941−1946.
́
(26) Rosario-Melendez, R.; Harris, C. L.; Delgado-Rivera, R.; Yu, L.;
Uhrich, K. E. J. Controlled Release 2012, 162, 538−544.
(27) Prudencio, A.; Carbone, A. L.; Griffin, J.; Uhrich, K. E.
Macromol. Rapid Commun. 2009, 30, 1101−1108.
(28) Rosario-Melendez, R.; Yu, W.; Uhrich, K. E. Biomacromolecules
2013, 14, 3542−3548.
(29) Brul, S.; Coote, P. Int. J. Food Microbiol. 1999, 50, 1−17.
(30) Whittaker, P.; Vanderveen, J. E.; Dinovi, M. J.; Kuznesof, P. M.;
Dunkel, V. C. Regul. Toxicol. Pharmacol. 1993, 18, 419−427.
(31) Scherer, R.; Godoy, H. T. Food Chem. 2008, 112, 654−658.
(32) Manual of Clinical Microbiology, 7th ed.; Murray, P. R., Baron, E.
J., Pfaller, M. A., Tenover, F. C., Yolken, R. H.ASM Press: Washington,
DC, 1999.
(33) Domb, A. J.; Ron, E.; Langer, R. Macromolecules 1988, 21,
1925−1929.
(34) Schmeltzer, R. C.; Johnson, M.; Griffin, J.; Uhrich, K. J.
Biomater. Sci., Polym. Ed. 2008, 19, 1295−1306.
(35) Whitaker-Brothers, K.; Uhrich, K. J. Biomed. Mater. Res., Part A
2006, 76A, 470−479.
(36) Gopferich, A.; Tessmar, J. Adv. Drug Delivery Rev. 2002, 54,
911−931.
(37) Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Food Sci.
Technol. (London) 1995, 28, 25−30.
(38) Cosentino, S.; Tuberoso, C. I. G.; Pisano, B.; Satta, M.; Mascia,
V.; Arzedi, E.; Palmas, F. Lett. Appl. Microbiol. 1999, 29, 130−135.
1895
dx.doi.org/10.1021/bm500303a | Biomacromolecules 2014, 15, 1889−1895