Pinosylvin-Based Polymers: Biodegradable Poly(Anhydride-Esters) for Extended Release of Antibacterial Pinosylvin

Article abstract of DOI:10.1002/mabi.201500454

Pinosylvin is a natural stilbenoid known to exhibit antibacterial bioactivity against foodborne bacteria. In this work, pinosylvin is chemically incorporated into a poly(anhydride-ester) (PAE) backbone via melt-condensation polymerization, and characteriz

Full text of DOI:10.1002/mabi.201500454

Macromolecular
Bioscience
Communication
Pinosylvin-Based Polymers: Biodegradable
Poly(Anhydride-Esters) for Extended Release of
Antibacterial Pinosylvin
Stephan Bien-Aime, Weiling Yu, Kathryn E. Uhrich*
Pinosylvin is a natural stilbenoid known to exhibit antibacterial bioactivity against foodborne
bacteria. In this work, pinosylvin is chemically incorporated into a poly(anhydride-ester) (PAE)
backbone via melt-condensation polymerization, and characterized with respect to its physic-
ochemical and thermal properties. In vitro release studies demonstrate that pinosylvin-based
PAEs hydrolytically degrade over 40 d to release pinosylvin.
Pseudo-first order kinetic experiments on model compounds,
butyric anhydride and 3-butylstilbene ester, indicate that the
anhydride linkages hydrolyze first, followed by the ester bonds
to ultimately release pinosylvin. An antibacterial assay shows
that the released pinosylvin exhibit bioactivity, while in vitro
cytocompatibility studies demonstrate that the polymer is
noncytotoxic toward fibroblasts. These preliminary findings
suggest that the pinosylvin-based PAEs can serve as food pre-
servatives in food packaging materials by safely providing
antibacterial bioactivity over extended time periods.
1. Introduction
physically admixed within food matrices of either vege-
table or animal origin.[3–5] The inherent properties of pino-
sylvin and related stilbenoids have generated interest for
extended release applications, from food protection to bio-
active delivery carriers.
Pinosylvin is an analog of resveratrol and a natural stilbe-
noid present in Pinus species.[1,2] Owing to its intrinsic anti-
microbial bioactivity, pinosylvin research has gained great
interest. For instance, several studies have reported on pino-
sylvin antibacterial bioactivity against common foodborne
pathogens such as Gram-positive (Staphylococcus aureus,
Listeria monocytogenes) and Gram-negative (Escherichia
coli, Salmonella) bacteria, even when the compound was
To achieve extended release of stilbenoids, researchers
have physically encapsulated similar stilbenoids into
polymer matrices. In one approach, chitosan micro-
spheres were loaded with resveratrol, yielding however
microspheres with low bioactive loadings (<10%) and a
burst release of the bioactive (over 60% within 30 min).[6]
In another approach, chemical modification of resvera-
trol into a triacetyl ester was attempted,[7] but the ester-
protected resveratrol hydrolyzed completely within 1 h.[7]
As resveratrol and pinosylvin are structurally similar, the
physical and chemical modifications of pinosylvin, as
described above, should result in similar outcomes. Con-
sequently, novel polymer systems capable of achieving
higher bioactive loading and providing extended release
of pinosylvin as a food preservative into common food
packaging materials are of interest; such targeted goals
S. Bien-Aime, Prof. K. E. Uhrich
Department of Chemistry and Chemical Biology
Rutgers University
610 Taylor Road
Piscataway, New Jersey 08854-8087, USA
E-mail: keuhrich@rutgers.edu
Dr. W. Yu
Department of Biomedical Engineering
Rutgers University
599 Taylor Road
Piscataway, New Jersey 08854-8087, USA
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can be achieved by chemically incorporating pinosylvin
into a poly(anhydride-ester) (PAE) backbone.
synthesis was adapted from a methodology employed else-
where.[14] Briefly, 3,5-dimethoxybenzyl bromide (1) was
heated with triethyl phosphite in the presence of (n-Bu)4NI,
then the intermediate reacted with benzaldehyde to yield
3,5-dimethoxystilbene (2) (Figure 1, 2A), followed by dem-
ethylation to generate pinosylvin (3) (Figure 1, 3B). The
chemical composition of pinosylvin was also confirmed
by infrared spectroscopy (IR) and mass spectrometry (MS).
Ring-opening of glutaric anhydride linker molecules to
generate pinosylvin diacid (4) was confirmed by the pres-
ence of the ester (C O) and carboxylic acid (C O) by IR
spectroscopy (Figure S5, Supporting Information) and the
relevant chemical shifts in the hydrogen nuclear magnetic
resonance (1H-NMR) spectra (Figure 1, 4C).
Pinosylvin monomer (5) was formed by acetylation
of diacid 4 in excess acetic anhydride, as confirmed by
nuclear magnetic resonance (NMR) and IR spectroscopy.
As pinosylvin monomer (5) had a moderate melting tem-
perature (Tm = 155 °C) and a high decomposition tempera-
ture (Td = 246 °C), melt-condensation polymerization was
possible. Melt-condensation polymerization of activated
monomer 5 at 170 °C produced polymer 6, as confirmed via
H-NMR (Figure 1, 6D) and IR spectroscopy, which shows
the presence of anhydride carbonyls and ester bonds, the
preservation of the double bonds, and the disappearance
of terminal carboxylic acids C O (Figure S5, Supporting
Information). Polymer 6 had a weight-average molecular
weight of 61 kDa, a number-average molecular weight
of 47 kDa, and a polydispersity index of 1.3. The polymer
had a glass transition and decomposition temperatures of
PAEs are predominantly surface-eroding polymers
with minimal burst release, allowing for controlled
and sustained release of bioactives in a near-zero order
manner.[8,9] Additionally, PAEs can be prepared to achieve
higher bioactive loadings (50%–100%),[10,11] and for-
mulated into different geometries, hence offering the
potential to satisfy diverse applications.[12,13]
In this work, the synthesis of pinosylvin-based polymers
via melt-condensation polymerization, followed by charac-
terization of its physicochemical and thermal properties, is
presented. In vitro degradation studies in phosphate buff-
ered saline (PBS, pH 7.4) were performed over 40 d, and
bioactive release was verified following polymer degrada-
tion. Pseudo-first order kinetic experiments were carried
out on model compounds (butyric anhydride and 3-butyl-
stilbene ester) to understand the hydrolytic degradation
of pinosylvin-based PAEs. The polymer’s cytocompatibility
was assessed in vitro against fibroblast cells, and the
release media confirmed antibacterial bioactivity via disc
diffusion assay against S. aureus and E. coli bacteria.
2. Results and Discussion
2.1. Synthesis and Characterization
Pinosylvin was chemically incorporated in a PAE backbone,
yielding 50 wt% loading as outlined in Scheme 1; pinosylvin
Scheme 1. Synthesis of pinosylvin-based PAEs (6) from pinosylvin (3), an antimicrobial stilbenoid.
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Figure 1. H-NMR spectra highlighting key chemical shifts of A) 3,5-dimethoxybenzyl bromide (2); B) pinosylvin (3); C) pinosylvin diacid (4 ) ,
and D) pinosylvin-based PAEs (6 ) .
50 and 190 °C respectively, exhibited no Tm, and exhibited
a contact angle (CA) value of 84°.
(1)
k = kobserved / water
[
]
where k is the reaction rate constant, kobserved the pseudo-
first order rate constant, and [water] the molar concentra-
tion of water.
2.2. Relative Rate of Hydrolysis: Kinetic Experiments
Pseudo-first order kinetic experiments were carried on two
small molecules representing the PAE structure, namely
butyric anhydride (12) and 3-butylstilbene ester (11), in
order to better comprehend hydrolytic degradation of
pinosylvin PAEs. Hydrolysis rate constants of the model
compounds are shown in Table 1 and determined using
the following formula[15]
In the case of 3-butylstilbene ester (11), hydrolysis
rate constants for 2.5%, 5%, and 10% 1,4-dioxane/PBS
media were determined using Equation (1). Then, hydrol-
ysis rate constants of the ester (11) for 0% 1,4-dioxane/
PBS were extrapolated from the values obtained for
2.5%, 5%, and 10% 1,4-dioxane/PBS media (Figure S7, Sup-
porting Information). As the percentage of 1,4-dioxane
increased, the rate constants decreased: this effect is
likely due to 1,4-dioxane dispersing water molecules
and preventing water from interacting via hydrogen
bonding with charged species in the transition state.
Therefore, the activation energy of the transition state
increased relative to that of water alone, and rate of
ester hydrolysis consequently slowed. This observation
Table 1. Hydrolysis rate constants of model compounds, butyric
anhydride (12), and 3-butylstilbene ester (11), representing degra-
dable linkages of pinosylvin PAEs (6, Scheme 1).
k [L mol−1 s−1]
(30.6 2.24) × 10−6
(56.7 5.01) × 10−7
Compound
Butyric anhydride
3-Butylstilbene ester
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2.3. Hydrolytic Degradation of Pinosylvin-Based PAEs
corresponds to a water-catalyzed hydrolysis, because
for acid- or base-catalyzed hydrolysis reactions, addition
of 1,4-dioxane would increase the hydrolysis rate
constants.[16]
Bioactive release from polymer discs was monitored in
vitro by high-performance liquid chromatography on
polymer discs at physiological conditions (37 °C, pH 7.4).
Polymer degradation through hydrolysis of anhydride and
ester bonds is a primary factor for controlled release of the
stilbenoid. To evaluate the hydrolysis of pinosylvin PAEs
(6), pseudo-first order kinetic experiments were performed
on butyric anhydride (12) and 3-butylstilbene ester (11)
model compounds. Based on the kinetic analysis on these
small molecules, butyric anhydride (12) underwent faster
hydrolysis [k = (30.6 2.24) × 10−6 L mol−1 s−1] than 3-butyl-
stilbene ester (11) [k = (56.7 5.01) × 10−7 L mol−1 s−1]. These
findings suggest the anhydride bonds of pinosylvin PAEs
(6), being more hydrolytically labile,[20,21] hydrolyze first
to yield pinosylvin diacid (4), followed by hydrolysis of
the ester bonds to generate pinosylvin (3).
At 37 °C, butyric anhydride (12) manifested a faster
hydrolysis rate constant [k = (30.6 2.24) × 10−6 L mol−1 s−1]
than 3-butylstilbene ester (11) [k = (56.7
5.01) ×
10−7 L mol−1 s−1]. Numerous studies in the literature
have investigated the hydrolysis of similar anhydrides
and esters, but they were performed under either acid-
or base-catalyzed conditions, at room or higher temper-
atures (>>37 °C).[17–19] Therefore, because the hydrolysis
conditions and parameters are extremely different,
no direct comparisons can be made between the
hydrolysis rate constants obtained in the literature
and the ones from this study. Nonetheless, the experi-
mental data obtained from this work correlate with
the fact that the anhydride bonds, being more labile,
are more susceptible to hydrolysis compared to ester
bonds.[20,21]
As shown in Figure S8 (Supporting Information), a
minor amount (≈5%) of pinosylvin (3) is quickly released
Figure 2. Disc diffusion assay results for A,C) E. coli and B,D) S. aureus showing zones of inhibition (ZH) for free pinosylvin (ZH = 17.5 mm),
extracted pinosylvin (3 , Z= 17.0 mm), monoacid (7 , Z= 10.0 mm), and diacid (4 , Z= 11.0 mm) at 10 mg mL–1 in DMSO.
H
H
H
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into the degradation media of polymer 6. After the first
2 d, polymer 6 exhibited a sustained release of pinosylvin
(3). The polymer is relatively slow degrading, releasing
≈10% of pinosylvin (3) in 40 d. This slow-degrading
behavior of 6 is likely due to the relatively high hydro-
phobicity (CA 84°), and 6 is estimated to release 100% of
pinosylvin (3) in 28 months, on extrapolation (Figure S8,
Supporting Information). At the end of study, a mass
balance was performed (96% mass accounted for) fol-
lowing analysis of bioactive in residual polymer 6.
polymer 6 needed for 250 μg mL–1 therapeutic effect of
pinosylvin (3)). Therefore, pinosylvin polymers (6) are rec-
ommended for external applications, such as preservatives
in food packaging materials.
3. Conclusions
Sustained release of pinosylvin (3) may provide prolonged
antibacterial effects against common foodborne bacteria
when released from polymers in a sustained fashion.
Attempts in the literature for achieving extended release
of pinosylvin (3) and similar stilbenoids by the use of pol-
ymers as delivery vehicles have been unsuccessful.[6,7] As
an alternative, the chemical incorporation of pinosylvin
(3) into a PAE backbone for the extended release of the
bioactive is investigated in this work. Physicochemical
and thermal testing revealed successful synthesis while
in vitro hydrolytic degradation of polymer (6) confirmed
sustained bioactive release of pinosylvin (3) over 40 d. To
understand the chemical degradation of pinosylvin-based
PAEs (6), pseudo-first order kinetic experiments performed
on butyric anhydride (12) and 3-butylstilbene ester (11)
model compounds revealed faster hydrolysis for the anhy-
dride [k = (30.6 2.24) × 10−6 L mol−1 s−1] compared to the
ester [k = (56.7 5.01) × 10−7 L mol−1 s−1]. Pinosylvin (3)
released from polymer 6 retained its antibacterial biolog-
ical activity as observed via a disc diffusion assay. In vitro
cytocompatibility studies demonstrated that polymer
6 is cytocompatible up to 0.5 μg mL–1, which concentra-
tion falls well below pinosylvin’s MIC of 250 μg mL–1.
As a result, pinosylvin PAEs (6) constitute a promising
technology to be employed, as part of future work, for
external applications, such as food additives into common
food packaging materials for food preservation and food
safety.
2.4. Antibacterial Disc Diffusion Assay
To confirm the benign effects of polymerization pro-
cesses on pinosylvin’s (3) antibacterial bioactivity, a
disc diffusion assay was carried out against Gram-
positive S. aureus and Gram-negative E. coli foodborne
bacteria.[22] The concentration of each substance (free
pinosylvin, extracted pinosylvin (3), monoacid (7), and
diacid (4)) in dimethyl sulfoxide (DMSO) was greater
than the minimum inhibitory concentration (MIC) for
pinosylvin (3) (250 μg mL–1) to evaluate inhibition zones
on the inoculated agar plates.[3] From the data collected
(Figure 2), both free and extracted pinosylvin (3) diffused
from the discs and exhibited similar zones of inhibi-
tion against both bacterial strains. Pinosylvin monoacid
(7) and diacid (4) also prevented bacterial growth, with
diacid (4) showing slightly larger zone of inhibition. This
difference may be due to the greater aqueous solubility
of 4 (2.24 mg mL–1) compared to 7 (0.80 mg mL–1), there-
fore allowing diacid (4) to diffuse more readily through
the hydrophilic agar plate. Furthermore, release studies
have shown that diacid (4) breaks down readily into
monoacid (7) and subsequently pinosylvin (3) within
the timeframe the disc diffusion assay was performed
(100% pinosylvin release in 18 h). This observation, cou-
pled with diacid’s aqueous solubility and larger diffusion
area, would explain the greater zone of inhibition for
4. Overall, this assay demonstrates that polymerization
processes did not affect pinosylvin’s (3) antibacterial
bioactivity against two of the most common foodborne
pathogens.
Supporting Information
Supporting Information is available from the Wiley Online
Library or from the author.
Acknowledgements: Damla Kaya (Rutgers University, Biomedical
Engineering) is thanked for experimental assistance. Dr. Ralf
Warmuth (Rutgers University, Chemistry) and Dr. Susan Skelly
(Rutgers University, Microbiology) are also thanked for their
assistance with kinetic experiments and antibacterial assay,
respectively.
2.5. In Vitro Cytocompatibility Studies
All polymers were cytocompatible at 0.5, 0.1, and
0.05 μg mL–1 over 72 h (Figure S3, Supporting Informa-
tion). No significant difference in cell viability was found
between the polymer groups and the media control. How-
ever, polymers at 3, 1.5, and 1 μg mL–1 were cytotoxic
toward fibroblasts. Since pinosylvin’s (3) antibacterial
MIC against common Gram-positive and Gram-negative
bacteria is 250 μg mL–1,[3] pinosylvin polymers (6) will be
toxic at such high concentrations in vivo (500 μg mL–1 of
Received: December 12, 2015; Revised: February 17, 2016;
Published online: ; DOI: 10.1002/mabi.201500454
Keywords: antibacterial; biodegradable; extended release;
pinosylvin; poly(anhydride-esters)
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