Biodegradable ferulic acid-containing poly(anhydride-ester): Degradation products with controlled release and sustained antioxidant activity

Article abstract of DOI:10.1021/bm3018998

Ferulic acid (FA) is an antioxidant and photoprotective agent used in biomedical and cosmetic formulations to prevent skin cancer and senescence. Although FA exhibits numerous health benefits, physicochemical instability leading to decomposition hinders i

Full text of DOI:10.1021/bm3018998

Article
pubs.acs.org/Biomac
Biodegradable Ferulic Acid-Containing Poly(anhydride-ester):
Degradation Products with Controlled Release and Sustained
Antioxidant Activity
Michelle A. Ouimet,† Jeremy Griffin,‡ Ashley L. Carbone-Howell,† Wen-Hsuan Wu,§
Nicholas D. Stebbins,† Rong Di,∥ and Kathryn E. Uhrich*,†,‡
†Department of Chemistry and Chemical Biology, Rutgers University, 610 Taylor Road, Piscataway, New Jersey 08854, United States
‡Department of Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, New Jersey 08854, United States
§Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey 08901, United States
∥Department of Plant Biology and Pathology, Rutgers University, 59 Dudley Road, New Brunswick, New Jersey 08901, United States
S
* Supporting Information
ABSTRACT: Ferulic acid (FA) is an antioxidant and
photoprotective agent used in biomedical and cosmetic
formulations to prevent skin cancer and senescence. Although
FA exhibits numerous health benefits, physicochemical
instability leading to decomposition hinders its efficacy. To
minimize inherent decomposition, a FA-containing biodegrad-
able polymer was prepared via solution polymerization to
chemically incorporate FA into a poly(anhydride-ester). The
polymer was characterized using nuclear magnetic resonance
and infrared spectroscopies. The molecular weight and thermal
properties were also determined. In vitro studies demonstrated
that the polymer was hydrolytically degradable, thus providing
controlled release of the chemically incorporated bioactive with no detectable decomposition. The polymer degradation products
were found to exhibit antioxidant and antibacterial activity comparable to that of free FA, and in vitro cell viability studies
demonstrated that the polymer is noncytotoxic toward fibroblasts. This renders the polymer a potential candidate for use as a
controlled release system for skin care formulations.
inorganic nanohybrid material for controlled release via
diffusion, but the low FA concentrations exhibited minimal
antioxidant activity.12,13 To overcome this issue with physical
incorporation, polymers containing cinnamoyl moieties chemi-
cally conjugated as pendant groups have been used to improve
photostability, but exhibited low drug loading (ca. 50%).14,15
Polymers containing cinnamoyl groups in the main chain and
polymerized with poly(ethylene glycol) derivatives also exhibit
lower drug loading and require enzymatic degradation.16 Other
researchers have been successful with incorporating antiox-
idants into a biodegradable polymer backbone to increase drug
loading (in some cases, the polymer is comprised entirely of the
antioxidant),17−20 and a few examples using FA, specifically, are
known.21,22 Our work is distinguished by the success at both
protecting the unstable bioactive from decomposition and
improving drug loading by chemically incorporating FA into a
hydrolytically degradable polyanhydride backbone, thus
enabling controlled FA release. This type of controlled release
INTRODUCTION
■
Exposure of skin to ultraviolet radiation (UV) causes oxidative
stress, which can result in photosensitivity, senescence, and skin
cancer.1 The prevention of oxidative stress can be achieved
using antioxidants as photoprotective agents that absorb UV
radiation and scavenge free radicals responsible for causing
damage.2 Many phenolic compounds have gained considerable
attention in recent years for their use in preventing oxidative
stress.2 Ferulic acid (FA), in particular, is a phenolic compound
that exhibits anti-inflammatory, antimicrobial, and anticancer
properties.3−6 As a photoprotective agent and antioxidant in
biomedical and cosmetic formulations, FA also prevents
harmful radiation effects both as a UV absorber and a free
radical scavenger.2,7 Although FA exhibits beneficial properties,
it undergoes thermal-, air-, and light-induced decomposition
through a proposed decarboxylation mechanism,8 which
reduces its efficacy.9,10
To improve the physiochemical stability of FA in
formulations, researchers have attempted to protect it from
decomposition using controlled release technologies via
physical incorporation into various types of matrices.11 For
example, FA has been physically incorporated into an organic−
Received: December 11, 2012
Revised: January 15, 2013
Published: January 17, 2013
© 2013 American Chemical Society
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Scheme 1. Synthesis of FA-Containing Poly(anhydride-esters) (5) and FA-Containing Polymer Precursors Including Diacids
(4)
Scheme 2. Proposed Hydrolytic Degradation Scheme of FA-Containing Poly(anhydride-esters) (5) to Yield Diacid (4) and FA
(1)
as the solvent and internal reference. FTIR spectra were obtained
using a Thermo Nicolet/Avatar 360 spectrometer, and samples (1 wt
%) were ground and pressed with KBr into a disc. Each spectrum was
an average of 32 scans.
system can offer advantages compared to conventional
formulations by reducing the frequency of applications and
improving patient compliance.23
Here we report the synthesis of a biodegradable poly-
(anhydride-ester) containing FA in the backbone via solution
polymerization methods and its characterization using proton
and carbon nuclear magnetic resonance (1H and 13C NMR)
and Fourier transform infrared (FTIR) spectroscopies. Thermal
properties were evaluated using differential scanning calorim-
etry (DSC) and thermogravimetric analysis (TGA). Gel
permeation chromatography (GPC) and mass spectrometry
(MS) were used to determine weight-averaged molecular
weight (Mw) of the polymer and molecular weights of all
polymer precursors, respectively. In addition, in vitro FA release
studies were performed to study the polymer hydrolytic
degradation. The antioxidant and antibacterial activities of the
degradation products were evaluated and compared to free FA.
The polymer cytotoxicity toward fibroblasts was assessed in
vitro.
Molecular Weight. Polymer precursors were analyzed via MS to
determine molecular weights. A Finnigan LCQ-DUO equipped with
Xcalibur software and an adjustable atmospheric pressure ionization
electrospray ion source (API-ESI Ion Source) was used with a pressure
of 0.8 × 10−5 and 150 °C API temperature. Samples dissolved in
methanol (<10 μg/mL) were injected with a glass syringe. GPC was
used to determine polymer weight-averaged molecular weight and
polydispersity using a Perkin-Elmer 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
processing of the data were performed using a Dell OptiPlex GX110
computer running Perkin-Elmer TurboChrom 4 software with a
Perkin-Elmer Nelson 900 Series Interface and 600 Series Link.
Polymer samples were prepared for autoinjection by dissolving in
dichloromethane (DCM, 10 mg/mL) and filtering through 0.45 μm
poly(tetrafluoroethylene) 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 the mobile phase at a
flow rate of 1.0 mL/min. Molecular weights were calibrated relative to
broad polystyrene standards (Polymer Source Inc., Dorval, Canada).
Thermal Properties. DSC measurements were carried out on a
TA Instrument Q200 to determine melting (Tm) and glass transition
(Tg) temperatures. Measurements on samples (4−6 mg) heated under
nitrogen atmosphere 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 were performed. TA Instruments Universal Analysis 2000
software, version 4.5A, was used to analyze the data. TGA was utilized
for determining decomposition temperatures (Td) using a Perkin-
Elmer Pyris 1 system with TAC 7/DX instrument controller and
Perkin-Elmer Pyris software for data collection. Samples (5−10 mg)
were heated under nitrogen atmosphere from 25 to 400 °C at a
EXPERIMENTAL SECTION
■
Materials. Silica gel and fetal bovine serum were purchased from
VWR (Radnor, PA) and Atlanta Biologicals (Lawrenceville, GA),
respectively. Hydrochloric acid (HCl, 1 N), concentrated HCl,
poly(vinylidine fluoride) and poly(tetrafluoroethylene) syringe filters,
Wheaton glass scintillation vials, and 96-well plates were purchased
from Fisher Scientific (Fair Lawn, NJ). Mouse areolar fibroblasts were
purchased from ATCC (Manassas, VA). All other reagents, solvents,
and fine chemicals were purchased from Aldrich (Milwaukee, WI) and
used as received.
1H and 13C NMR and FTIR Spectroscopies. H and 13C NMR
1
spectra were recorded on a Varian 400 or 500 MHz spectrometer
using deuterated chloroform (CDCl3) with tetramethylsilane (TMS)
as the internal reference or deuterated dimethyl sulfoxide (DMSO-d6)
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heating rate of 10 °C/min. Decomposition temperatures were
measured at the onset of thermal decomposition.
products were analyzed and quantified via HPLC using an XTerra
RP18 5 μm 4.6 × 150 mm column (Waters, Milford, MA) on a Waters
2695 Separations Module equipped with a Waters 2487 Dual λ
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 was comprised of 50
mM KH2PO4 with 1% formic acid in DI water at pH 2.5 (70%) and
acetonitrile (30%) run at 1 mL/min flow rate at ambient temperature.
Absorbance was monitored at λ = 335. Amounts were calculated from
a calibration curve of known FA standard solutions.
t-Butyl FA (2) Synthesis. To prepare the t-butyl FA (Schemes 1
and 2), a procedure adapted from Hu. et al.24 was used where
Meldrum’s acid (2.5 equiv) was dissolved in toluene (50 mL), tertiary
butanol (2.5 equiv) was added, and the reaction was heated to 100 °C
with stirring for 5 h. Without separation, the reaction was cooled to
room temperature where vanillin was added followed by pyridine (2.5
mL) and piperidine (0.25 mL), and then heated to 75 °C with stirring
for 24 h. The reaction mixture was dried in vacuo, and the residue
obtained was diluted in diethyl ether, washed with saturated aqueous
sodium bicarbonate (2 × 200 mL), 1 N HCl (2 × 200 mL), and
distilled water (1 × 200 mL). The organic layer was then dried
overnight over MgSO4, filtered, and the solvent was removed in vacuo
to yield crude product. This was purified on silica gel via flash
chromatography using 4:1 hexane:ethyl acetate as eluent (see the
Supporting Information for characterization data).
t-Butyl FA-Containing Diacid Intermediate (3) Synthesis. t-
Butyl FA (2) (2 equiv) was dissolved in anhydrous dimethylforma-
mide (DMF), to which sodium hydride (NaH, 2.2 equiv) was added
slowly. After 30 min, adipoyl chloride (1 equiv) dissolved in 10 mL of
DMF was added dropwise at 20 mL/h. Reaction progress was
monitored by thin layer chromatography (4:1 hexane:ethyl acetate as
eluent). Once completed, the reaction mixture was diluted with ethyl
acetate (250 mL) and washed with deionized water (2 × 100 mL).
The organic layer was collected, dried over MgSO4, and the solvents
were removed in vacuo. This was purified on silica gel via flash
chromatography using 4:1 hexane:ethyl acetate as eluent (see the
Supporting Information for characterization data).
FA-Containing Diacid (4) Synthesis. Compound 3 (1 equiv)
was dissolved in anhydrous DCM to which trifluoroacetic acid (TFA)
(40 equiv) was added and left to stir overnight. Solvent was removed
in vacuo, and the residue was triturated with DI water (300 mL),
isolated via vacuum filtration, and dried in vacuo for 24 h (see the
Supporting Information for characterization data).
FA-Containing Poly(anhydride-ester) (5) Synthesis. The
polymer (5) was prepared using a modified version of a previously
described procedure25 (Scheme 1). Diacid 4 (1 equiv) was dissolved in
20 mL anhydrous DCM under argon. After adding triethylamine
(NEt3, 4.4 equiv), the reaction mixture was cooled to 0 °C.
Triphosgene (0.33 equiv) dissolved in 10 mL anhydrous DCM was
added dropwise (20 mL/h). The reaction was allowed to stir at 0 °C
until CO2 evolution ceased (ca. 6 h). The reaction mixture was poured
over chilled diethyl ether (400 mL), and the precipitate was isolated
via vacuum filtration. The residue was dissolved in anhydrous DCM,
washed with acidic water (1 × 250 mL), dried over MgSO4,
concentrated, and precipitated with an excess of chilled diethyl ether
(500 mL). The ether was decanted or filtered off via vacuum filtration,
and the polymer was dried in vacuo at room temperature (see the
Supporting Information for characterization data).
In Vitro FA Release from Polymer. The release of 1 from
polymer (5) was evaluated by in vitro degradation in phosphate-
buffered saline (PBS). Polymer discs (n = 3) were prepared by
pressing ground polymer (45 5 mg) into 8 mm diameter × 1 mm
thick discs in an IR pellet die (International Crystal Laboratories,
Garfield, NJ) with a benchtop hydraulic press (Carver model M,
Wabash, IN). Pressure of 10 000 psi was applied for 5 min at room
temperature. This methodology was preferred, as it minimized
interferences from external effects (e.g., formulation additives) on
polymer degradation. The PBS pH was adjusted to 7.40 using 1 N
sodium hydroxide. All pH measurements were performed using an
Accumet AR15 pH meter (Fisher Scientific, Fair Lawn, NJ).
To measure hydrolytic degradation, the polymer (5) discs were
placed in 20 mL Wheaton glass scintillation vials with 10 mL of PBS
and incubated at 37 °C with agitation at 60 rpm using a controlled
environment incubator-shaker (New Brunswick Scientific Co., Edison,
NJ). The degradation media was collected every 2 days until day 12
and then collected every 4 days until day 30. Media was replaced by
fresh PBS (10 mL), and the spent media was analyzed over 30 days by
high-performance liquid chromatography (HPLC). The degradation
Radical Scavenging (Antioxidant) Activity.26 To assess the FA
antioxidant activity in degradation media, a 2,2-diphenyl-1-picrylhy-
drazyl (DPPH) radical scavenging assay was employed. This was
evaluated by adding sample (0.1 mL) to a 0.024 mg/mL DPPH
solution in methanol (3.9 mL). Day-10 and day-20 polymer
degradation media samples (0.1 mL) from the polymer (see In
Vitro FA Release from Polymer section) were incubated with the
0.024 mg/mL DPPH solution (3.9 mL) at room temperature. After 1
h, solutions were analyzed via UV/vis with a Perkin-Elmer Lambda
XLS spectrophotometer (Waltham, MA) (λ = 517 nm). A free FA
solution was freshly prepared at specific concentrations corresponding
to HPLC data gathered on days 10 and 20. These samples were each
analyzed identically to the aforementioned degradation media samples.
DPPH % radical reduction was calculated by [(Abst0 − Abst)/Abst0] ×
100, where Abst0 is the initial absorbance, and Abst is the absorbance
after a period of time, namely 1 h. Absorbance values from adding PBS
(0.1 mL) to the DPPH solution (3.9 mL) was used as Abst0. All radical
scavenging assays were performed in triplicate. Student’s t tests were
used to determine the significant difference of the antioxidant activity
between free FA and FA degradation media (significantly different if p
< 0.05).
In Vitro Cytotoxicity Assay. Evaluation of the polymer cell
compatibility was performed by culturing NCTC clone 929 (strain L)
mouse areolar fibroblasts in media containing the dissolved polymer.
These L929 fibroblasts are a standard cell type for cytocompatibility
testing as recommended by ASTM.27 The polymer was dissolved in
dimethyl sulfoxide (DMSO; 10 mg/mL) as a stock solution and
serially diluted with cell culture media to two concentrations (0.01
mg/mL and 0.10 mg/mL), based on standard cytotoxicity
protocols.28−31 Cell culture media consisted of Dulbecco’s Modified
Eagle’s Medium, 10% v/v fetal bovine serum, 1% L-glutamate, and 1%
penicillin/streptomycin. The polymer-containing media was distrib-
uted into a 96-well plate and seeded at an initial concentration of 2000
cells per well (n = 3). The media with dissolved polymer was
compared to two controls: DMSO-containing media and media
without the polymer or DMSO.
Cellular morphology was observed and documented at 100×
original magnification (Olympus, IX81, Center Valley, PA) at 48, 72,
and 96 h post seeding. Cell viability was determined by using a
CellTiter 96AQueous One Solution Cell Proliferation Assay
(Promega, Madison, WI). The MTS tetrazolium compound [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4,7-sulfophen-
yl)-2H-tetrazolium, inner salt; MTS)] is bioreduced by cells into a
colored formazan product that is soluble in the tissue culture medium.
Following the appropriate incubation time, 20 μL of the MTS reagent
was added to 100 μL of culture medium and further incubated for 4 h.
The absorbance was then recorded with a microplate reader (Model
680; Bio-Rad, Hercules, CA) at λ = 490 nm. Cell numbers were
calculated based upon a standard curve created 24 h after original cell
seeding.
Cell studies were performed in triplicate, and statistical analysis was
performed with SPSS software (version 15.0 for Windows; Chicago,
IL). ANOVA followed by pairwise comparison with Scheffe’s post hoc
test allowed for comparison of the polymer to the DMSO media
control.
In Vitro Antibacterial Assays. A study supplemental to the
antioxidant assays was performed to ensure that full activity was
maintained after polymer synthesis. To further assess FA bioactivity,
the antibacterial activity was evaluated by hydrolyzing the polymer
with NaOH, and degradation products were extracted using ethyl
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Figure 1. 1H NMR spectra of FA-containing polymer and each intermediate step. t-Butyl FA, 2 (A), t-butyl FA-containing diacid intermediate, 3 (B),
FA-containing diacid, 4 (C), and FA-containing polymer, 5 (D) spectra are illustrated above.
acetate and subsequently dried in vacuo. This extracted powder was
screened against Escherichia coli (E. coli) O157:H7 ATCC 43895, a
model bacterium, and compared to that of free FA. More detailed
methods can be found in the Supporting Information.
(4) was formed after coupling compound 2 to adipoyl chloride
in DMF containing NaH and removing the t-butyl ester with
TFA. The diacid (4) was isolated, and the structure was
1
confirmed by H and 13C NMR and FTIR spectra. DSC and
MS were used for melting point and molecular weight
determination, respectively.
RESULTS AND DISCUSSION
■
1H NMR spectra confirmed all polymer intermediates and
polymer structures shown in Figure 1. Coupling constants
between 15.8 and 16 Hz demonstrated that the double bonds
are in the trans (E) configuration, with signals appearing at 7.84
and 6.83 ppm for 5 (Figure 1D). As depicted in Figure 1B, the
appearance of signals at 2.67 and 1.92 ppm and the
disappearance of the phenol signal (6.06 ppm for 2) indicated
successful acyl chloride coupling to FA in a 1:2 ratio (acyl
chloride:FA). The disappearance of signals at 1.54 ppm (Figure
1C) illustrates the complete removal of the protecting t-butyl
groups. The FTIR spectrum of 4 (Figure 2B) indicates
coupling of the acyl chloride via ester bond formation (1766
cm−1) and the presence of carboxylic acid bands (at 2586 and
1677 cm−1 illustrating the alcohol and carbonyl groups of the
acid).
Polymer Synthesis and Characterization. To overcome
FA limitations regarding its poor physicochemical stability, FA
was chemically incorporated into a polymer backbone as
outlined in Scheme 1. A one-pot Knoevenagel condensation
reaction was utilized by reacting t-butanol with Meldrum’s acid
to form a malonic acid monoester, which was immediately
reacted with vanillin in the presence of pyridine and piperidine
as catalysts to yield the t-butyl FA compound (2).
To synthesize 4, various reaction conditions were explored
by changing temperature, base, and solvent to directly couple
the acyl chloride to the FA phenol, but these conditions
resulted in impure products that could not be further purified.
For sole reaction at the phenol, various groups were used to
protect the carboxylate group, where the t-butyl groups yielded
pure material without compromising the integrity of the FA
structure during protection and deprotection. Ultimately, diacid
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In Vitro FA Release from Polymer. The polymer’s in vitro
degradation was measured by the appearance of FA in
degradation media via HPLC to elucidate release rates from
5 to yield 1 and biocompatible adipic acid (not detected by
HPLC), as depicted in Scheme 2. This degradation is integral
to improving FA stability and delivery, as FA release is
controlled via anhydride and ester bond hydrolysis. To
ascertain whether the polymer would ultimately hydrolyze
and release the covalently attached bioactive, in vitro
degradation was monitored over 30 days using polymer discs.
Hydrolytic degradation of 5 was studied using pressed polymer
discs in PBS and analyzed via HPLC. Previous studies have
indicated more rapid anhydride bond hydrolysis than that of
ester bonds.32 Within this study, however, the relative
hydrolysis rate was not apparent in the studies performed, as
diacid (4) absorbance (retention time of 8.07 min (Figure 3B))
was minimal at the specified wavelength for HPLC studies.
Moreover, the diacid exhibits limited solubility in PBS relative
to ferulic acid. Although there was likely diacid present due to
anhydride bond hydrolysis, minimal amounts were detected
under the conditions studied. FA detection with a 3.71 min
retention time (Figure 3B) indicated further hydrolysis of ester
bonds. The polymer released 6.2% FA over 30 days, as depicted
in Figure 3A. It is important to note that no decomposition
peaks were observed, suggesting the FA compound remains
active. After 30 days, a thinner substrate was observed from the
initial 8 mm diameter × 1 mm thick discs, suggesting that
complete degradation for this polymer is >30 days.
Figure 2. FTIR spectra of FA-containing polymer 5 (A, top) and
diacid 4 (B, bottom).
As previous poly(anhydride-ester) studies suggest polymer
degradation to be pH-dependent,28 the release medium pH for
each sample was monitored to ensure acidic degradation
products did not alter the pH. The media pH throughout
degradation studies became more acidic, yet only deviated from
Due to thermal instability of FA, diacid 4 was used directly
for low-temperature solution polymerization using triphosgene
as the coupling agent in the presence of triethylamine to form
the poly(anhydride-ester) (5). The FA structure within the
polymer was preserved after polymerization as indicated by the
1H NMR spectrum (Figure 1D) and FTIR spectrum (Figure
2A), which illustrates the disappearance of the diacid carboxylic
acid peaks, the presence of the anhydride carbonyls and ester
preservation between 1800 and 1720 cm−1, and preservation of
the double bonds (1600 and 1630 cm−1). The polymer
exhibited a Mw of 21 700 Da with a polydispersity index of 1.7
after isolation. Thermal properties of the polymer are favorable
as it decomposes at 332 °C and exhibits Tg at 82 °C. The
successful FA chemical incorporation into a poly(anhydride-
ester) backbone further allows for high drug loading, where
81% of the polymer is the bioactive, significantly improving
upon other researchers’ methods.12−16
the initial 7.40 reading by −0.19
0.13. Due to these low
values, it is expected that the slightly acidic media did not affect
release rates.
Radical Scavenging (Antioxidant) Activity. To establish
whether polymer processing had an effect on antioxidant
activity of released FA, a DPPH radical scavenging assay was
employed. DPPH is widely used to assess the ability of
antioxidants to scavenge or quench free radicals or donate a
hydrogen atom, causing a color change from violet to pale
yellow and a reduction in absorbance.26,33,34 The degradation
media antioxidant activity from days 10 (containing all products
released from days 8−10) and 20 (containing all products
released from days 16−20) were analyzed. These were
compared to the corresponding free FA (1) using freshly
Figure 3. In vitro FA release from FA-containing poly(anhydride-esters) (5) (FA standard error) (A). HPLC chromatographs demonstrating
appropriate peaks for FA (1) and diacid (4) for day-10 samples (B).
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prepared solutions with concentrations equal to HPLC data on
day 10 (18 1 μg/mL) and day 20 (52 1 μg/mL) (Figure
4). Comparable concentrations were chosen because the
quenching percentage is dependent on the antioxidant
concentration.33
both polymer concentrations (Figure 5), pairwise comparison
with Scheffe’s post hoc test indicated no significant statistical
difference between the polymer and the DMSO media control
at the 48, 72, and 96 h time points (significantly different if p <
0.05).
For all conditions, the cell morphology images demonstrate
the typical proliferation expected of healthy fibroblasts. After 96
h of culture, proliferating viable cells were visible with stellate
morphology and extending filopodia. No visible differences
were observed in fibroblast morphology for the polymer at the
two concentrations and three time points; therefore, under
these specific concentrations and conditions, 5 can be
considered noncytotoxic.
In Vitro Antibacterial Assays. To further assess whether
the polymer processing affected the chemically incorporated FA
and remained bioactive, antibacterial tests were performed
using E. coli, a Gram-negative bacterium that free FA is active
against. Degradation media from the in vitro release studies
yielded FA concentrations too low for antibacterial activity to
be observed. Therefore, the polymer was completely hydro-
lyzed, and a fixed concentration was prepared from the
extracted degradation products and compared to free FA
activity. Samples dissolved in DMSO without added bacteria
were first examined to ensure contamination did not occur with
the solutions; negligible increase of OD630 nm indicated no
bacteria growth and thus no contamination.
A fixed FA concentration (3 mg/mL) was selected to
compare the bacteria susceptibility to free and extracted FA.
The extracted powders from 5 included both FA (82%) and
adipic acid (18%); free adipic acid was therefore analyzed for
antibacterial activity at the concentrations present in extracted
powders (0.66 mg/mL). DMSO concentrations were kept
lower than 0.5% to minimize its inhibitory effect.
As depicted in Figure 6, free FA and the extracted powder
from 5 demonstrated activity against E. coli. Although minimal,
adipic acid also demonstrated inhibitory activity. The observed
activity showed no statistical differences between free FA and
the extracted powder. This assay suggests that the FA retains its
antibacterial activity, further demonstrating that the polymer
degradation products did not decompose and have not been
affected by the synthetic methods utilized. These results also
suggest that the polymer may also be useful as preservative
agents in formulations due to its antibacterial activity, but
additional studies must be performed.
Figure 4. DPPH reduction results for FA in degradation media and
free FA for day 10 and day 20 of the release study. Statistical difference
indicated by * (p < 0.05).
Student’s t tests comparing degradation media to the free FA
solution were performed (significantly different if p < 0.05).
The observed antioxidant activity showed no statistical
differences between degradation media and free FA solution
for day 10, whereas day 20 degradation media demonstrated
significantly higher radical reduction than FA alone, indicating
increased quenching (denoted by asterisk in Figure 4). In both
cases, increased antioxidant activity of degradation media
relative to free FA was observed. This increase may be due
to potential antioxidant activity of the diacid, as it has the ability
to scavenge the free radial similarly to free FA. Adipic acid
(which exhibits minimal antioxidant activity)35 was present in
the degradation media and may have contributed to this
increase as well. Further studies must be performed to elucidate
the diacid antioxidant activity. From these results, the polymer
degradation products sustain antioxidant activity over time and
have not been affected by the synthetic approach utilized.
In Vitro Cytotoxicity Assay. Assessing the polymer
toxicity potential is important if this polymer is to be used
for in vivo applications. Cytotoxicity of 5 was evaluated by
culturing fibroblast cells in polymer-containing media at 0.01
and 0.10 mg/mL, as these concentrations are well above those
seen in vitro and can be used to determine dose dependent
toxicity. Studies were performed over a 96 h time period, in
which cell proliferation and morphology were observed. For
CONCLUSIONS
■
By incorporating FA into a polymer backbone, the
physicochemical stability is improved as FA is protected from
premature degradation. The polymer was found to be
Figure 5. Cell viability/proliferation after 48, 72, and 96 h in culture media with the polymer at concentration of (A) 0.01 mg polymer/mL media
and (B) 0.10 mg polymer/mL media.
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Figure 6. Growth curve of treated cells at OD 630 nm. E. coli O157:H7 with DMSO at 0.5% demonstrating OD readings for FA, extracted powder
from 5, adipic acid, DMSO, and bacterium. Data is presented as an average of triplicate OD measurements from two independent tests where OD
directly correlates with cell count.
hydrolytically degradable, where the degradation products
exhibited comparable antioxidant and antibacterial activity to
that of free FA. The demonstrated controlled release of the
chemically incorporated bioactive with no detectable decom-
position suggests that the frequency of applications of the
antioxidant/photoprotective agent can be reduced. The
polymer was deemed noncytotoxic in mouse fibroblast cultures
at 0.01 and 0.10 mg/mL concentrations, demonstrating the
potential for use in vivo. The polymer design allows for
opportunities to develop tunable FA release profiles; future
studies will focus on increasing the FA release rate by changing
the acyl chloride in polymer synthesis or formulating the
polymer into microspheres to further improve their use for skin
care formulations and to perform skin permeation experiments.
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Corresponding Author
*Mailing address: Department of Chemistry and Chemical
Biology, Rutgers, The State University of New Jersey, 610
Taylor Road, Piscataway, NJ 08854-8087, USA. E-mail address:
keuhrich@rutgers.edu. Tel.: (732) 445-0361. Fax: (732) 445-
7036.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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This work was supported by the National Institutes of Health
(NIH 5 R01DE0132070-09 and NIH 1 R01DE019926-01) and
Center and the U.S. Department of Education Graduate
Assistance in Areas of National Need (GAANN) Fellowship
(MAO, ALC). Dr. Bryan Langowski (Rutgers, Department of
Chemistry & Chemical Biology) is thanked for intellectual
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