Ferulic Acid-Based Polymers with Glycol Functionality as a Versatile Platform for Topical Applications

Article abstract of DOI:10.1021/acs.biomac.5b00824

Ferulic acid-based polymers with aliphatic linkages have been previously synthesized via solution polymerization methods, yet they feature relatively slow ferulic acid release rates (～11 months to 100% completion). To achieve a more rapid release rate as required in skin care formulations, ferulic acid-based polymers with ethylene glycol linkers were prepared to increase hydrophilicity and, in turn, increase ferulic acid release rates. The polymers were characterized using nuclear magnetic resonance and Fourier transform infrared spectroscopies to confirm chemical composition. The molecular weights, thermal properties (e.g., glass transition temperature), and contact angles were also obtained and the polymers compared. Polymer glass transition temperature was observed to decrease with increasing linker molecule length, whereas increasing oxygen content decreased polymer contact angle. The polymers' chemical structures and physical properties were shown to influence ferulic acid release rates and antioxidant activity. In all polymers, ferulic acid release was achieved with no bioactive decomposition. These polymers demonstrate the ability to strategically release ferulic acid at rates and concentrations relevant for topical applications such as skin care products.

Full text of DOI:10.1021/acs.biomac.5b00824

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Article
Ferulic acid-based polymers with glycol functionality
as a versatile platform for topical applications
Michelle A. Ouimet, Jonathan J Faig, Weiling Yu, and Kathryn E. Uhrich
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00824 • Publication Date (Web): 10 Aug 2015
Downloaded from http://pubs.acs.org on August 12, 2015
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Ferulic acid-based polymers with glycol functionality as a versatile platform for
topical applications
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Michelle A. Ouimet †, Jonathan J. Faig†, Weiling Yu‡, and Kathryn E. Uhrich†*
†Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ
08854
‡Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854
*Corresponding author:
Dr. Kathryn E. Uhrich
Department of Chemistry and Chemical Biology
Rutgers, The State University of New Jersey
610 Taylor Road
Piscataway, NJ 08854ꢀ8087, USA
Eꢀmail address: keuhrich@rutgers.edu
Tel.: (848) 445ꢀ0361
Fax: (732) 445ꢀ7036
KEYWORDS: biodegradable, polymer, poly(anhydrideꢀester), ferulic acid, antioxidant,
controlled release
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ABSTRACT
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Ferulic acidꢀbased polymers with aliphatic linkages have been previously
synthesized via solution polymerization methods, yet feature relatively slow ferulic acid
release rates (~ 11 months to 100% completion). To achieve a more rapid release rate
as required in skin care formulations, ferulic acidꢀbased polymers with ethylene glycol
linkers were prepared to increase hydrophilicity and, in turn, increase ferulic acid
release rates. The polymers were characterized using nuclear magnetic resonance and
Fourier transform infrared spectroscopies to confirm chemical composition. The
molecular weights, thermal properties (e.g., glass transition temperature), and contact
angles were also obtained and the polymers compared. Polymer glass transition
temperature was observed to decrease with increasing linker molecule length whereas
increasing oxygen content decreased polymer contact angle. The polymers’ chemical
structures and physical properties were shown to influence ferulic acid release rates
and antioxidant activity. In all polymers, ferulic acid release was achieved with no
bioactive decomposition. These polymers demonstrate the ability to strategically
release ferulic acid at rates and concentrations relevant for topical applications, such as
skin care products.
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INTRODUCTION
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Free radicals have been identified as major sources of oxidative stress in cells
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leading to DNA damage.1 Increased oxidative stress has been implicated in various
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deleterious conditions including cardiovascular diseases,2,
neurodegenerative
diseases,4 and cancer,5 while also contributing to the physiology of ageing.6 The
human body combats oxidative stress by employing antioxidants made in the body or
acquired from diet and/or supplements.7, 8 These antioxidants, however, are usually not
available in sufficient levels to overcome the damage from oxidative stress
accumulation. Therefore, researchers have investigated the development of topical
antioxidants with photoprotective and therapeutic efficacy.9
Figure 1. Chemical structure of the bioactive ferulic acid (1) and ferulic acidꢀbased
polymer (5)
One such antioxidant that has been investigated for topical applications is ferulic
acid (FA; Figure 1, 1), a hydroxycinnamic acid found in plants and a potent antioxidant
due to its phenolic and extended side chain conjugation, which forms a resonanceꢀ
stabilized phenolic radical.10 FA (1) has been studied as an ultraꢀviolet (UV) absorber
for enhanced skin protection against photodamage and approved as a sunscreen in
Japan.11 While FA exhibits beneficial antioxidant properties, its short elimination halfꢀ
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life (less than 2 hours)12 and poor stability (decomposition over time) lower its efficacy in
current formulations.13 To extend elimination and prolong stability, FA formulations can
be improved by incorporating this bioactive into a biodegradable polymer backbone.
Such systems have been thoroughly investigated utilizing the reduced FA derivative
dihydroferulic acid14ꢀ18 and, to a lesser extent FA19ꢀ22 itself. FAꢀbased poly(anhydrideꢀ
esters) have been found to enable controlled bioactive (i.e., FA) release and preventing
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the bioactive functional groups from degradation.22
incorporating FA into a polymer backbone, the FA retained its antioxidant activity when
released via polymer degradation. Further, the FAꢀbased polymer prevented
As described herein, by
discoloration, indicating a promising FAꢀbased topical formulation.22
Although previous studies demonstrated that FA could be stabilized via covalent
incorporation into a polymer backbone, minimal amounts of FA were released over the
time frame studied (i.e., ~7 % over 30 days). For topical applications that require higher
FA concentrations over a shorter time frame (i.e., hours to days), an alternate approach
is needed. One approach to increase polymer degradation rates, thereby increasing FA
release, is to increase hydrophilicity of the formulation by incorporating waterꢀ
solubilizing ethylene glycol functionalities via copolymerization with poly(ethylene glycol)
(PEG)23ꢀ25 or to incorporate ethylene glycol within the polymer.26, 27 In this work,
ethylene glycol groups were employed as the linker molecule between two FA
molecules (see Fig 2), enabling increased polymer hydrophilicity to promote polymer
degradation, and promote faster FA release.
Herein, we describe FAꢀbased
biodegradable polymers by introducing ethylene glycol functionality as a linker
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molecule. The synthesis, characterization, and drug release profiles of the glycolꢀ
modified polymers, in addition to polymer cytotoxicity, are discussed.
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MATERIALS AND METHODS
Materials. 1 N hydrochloric acid (HCl), polytetrafluoroethylene (PTFE), and
poly(vinylidine fluoride) (PVDF) syringe filters, and Wheaton glass scintillation vials were
purchased from Fisher Scientific (Fair Lawn, NJ). All other reagents, solvents, and fine
chemicals were purchased from Aldrich (Milwaukee, WI) and used as received.
1H and 13C NMR and FT-IR spectroscopies. Proton (1H) and carbon (13C)
nuclear magnetic resonance (NMR) spectra were recorded on a Varian 400 or 500 MHz
spectrometer using deuterated chloroform (CDCl3) with tetramethylsilane as internal
reference or deuterated dimethyl sulfoxide (DMSOꢀd6) as solvent and internal reference.
Fourier transform infrared (FTꢀIR) spectra were obtained using a Thermo Nicolet/Avatar
360 spectrometer, with samples (1 wt %) ground and pressed with potassium bromide
(KBr) into a disc or solvent casted via dichloromethane (DCM) to acquire a thin film on
sodium chloride (NaCl) plates. Each spectrum was an average of 32 scans.
Molecular Weight. Polymer precursors were analyzed via mass spectrometry
(MS) to determine molecular weights. A Finnigan LCQꢀDUO equipped with Xcalibur
software and an adjustable atmospheric pressure ionization electrospray ion source
(APIꢀESI Ion Source) was used with a pressure of 0.8 x 10ꢀ5 and 150 °C API
temperature. Samples dissolved in methanol (< 10 ꢁg/mL) were injected via a glass
syringe. Gel permeation chromatography (GPC) was used to determine polymer weightꢀ
averaged molecular weight (Mw) and polydispersity indices (PDI) using a Waters liquid
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chromatography system consisting of a Series 2414 refractive index detector, a 1515
isocratic high performance liquid chromatography (HPLC) pump, and a 717plus
autosampler. Automation of the samples and processing of the data was performed
using a Dell OptiPlex GX110 computer running Waters Breeze Version 3.20 software.
Polymer samples were prepared for autoinjection by dissolving in DCM (10 mg/mL) and
filtering through 0.45 ꢁm PTFE syringe filters. Samples were resolved on a Jordi
divinylbenzene mixedꢀbed GPC column (7.8 x 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).
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Thermal Properties. Differential scanning calorimetry (DSC) measurements
were carried out on TA Instrument Q200 to determine melting (Tm) and glass transition
(Tg) temperatures. Samples (4ꢀ6 mg) were heated under nitrogen atmosphere from –10
°C 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 twoꢀcycle minimum. TA Instruments Universal Analysis 2000 software, version
4.5A, was used to analyze the data. Thermogravimetric analysis (TGA) was utilized for
determining decomposition temperatures (Td) using a PerkinꢀElmer Pyris 1 system with
TAC 7/DX instrument controller and PerkinꢀElmer Pyris software for data collection.
Samples (5ꢀ10 mg) were heated under nitrogen atmosphere from 25 °C to 400 °C at a
heating rate of 10 °C/min. Decomposition temperatures were measured at the onset of
thermal decomposition.
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Scheme 1. Synthesis of FA poly(anhydrideꢀesters) (5) and FAꢀcontaining polymer
precursors including tꢀbutyl FA diesters (3) and FA diacids (4) with varying linkers (a22,
b, c)
t-Butyl FA (2) Synthesis. tꢀButyl FA was prepared following previously
published methods (Scheme 1, 2).22, 28
t-Butyl FA Diester (3) Synthesis. tꢀButyl FA (2) (2 eq) was dissolved in
anhydrous dimethylformamide (DMF, 25 mL) to which sodium hydride (NaH, 2.2 eq)
was added slowly. After 30 minutes, acyl chloride (1 eq) dissolved in DMF (10 mL) was
added dropꢀwise to the reaction mixture at 20 mL/hr. Thin layer chromatography (TLC,
4:1 hexane:ethyl acetate) was used to monitor reaction progress. Following tꢀbutyl FA
consumption, the reaction mixture was diluted with ethyl acetate (250 mL) and washed
with deionized (DI) water (2 x 100 mL). The organic layer was collected, dried over
MgSO4, and concentrated in vacuo. Crude product was purified on silica gel via flash
chromatography using 4:1 hexane:ethyl acetate as eluent.
t-Butyl FA-adipic Diester (3a). Synthesized and characterized following
previously published methods.22
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t-Butyl FA-diglycolic Diester (3b). Diglycolyl chloride (1 eq) was used as the
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acyl chloride. Yield: 72 % (white powder). HꢀNMR (400 MHz, CDCl3): δ 7.56 (d, 2H,
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J=16 Hz, RꢀCH=CHꢀR), 7.08ꢀ7.10 (m, 6H, ArꢀH), 6.34 (d, 2H, J=16 Hz, RꢀCH=CHꢀR),
4.62 (s, 4H, CH2), 3.86 (s, 6H, OCH3), 1.54 (s, 18H, 3CH3). 13CꢀNMR (CDCl3): δ 167.9
(2C, C3), 166.3 (2C, C13), 151.3 (2C, C10), 142.8 (2C, C5), 140.7 (2C, C9), 134.3 (2C,
C6), 123.2 (2C, C8), 121.4 (2C, C7), 120.9 (2C, C4), 111.3 (2C, C11), 80.9 (2C, C2), 68.1
(2C, C14), 56.2 (2C, C12), 28.4 (6C, C1). IR (NaCl, cmꢀ1) 1782 and 1705 (C=O, ester),
1638 and 1600 (C=C). Tm: 222ꢀ223 ºC.
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t-Butyl FA-tetraglycolic Diester (3c). To synthesize the diacyl chloride, 3,6,9ꢀ
trioxaundecanedioic acid (tetraglycolic acid, 1 eq) was reacted neat in thionyl chloride (8
eq) under reflux for 24 hours, after which it is concentrated in vacuo to yield a yellow
liquid, tetraglycolyl dichloride. Tetraglycolyl dichloride (1 eq) was used without further
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purification as the diacyl chloride. HꢀNMR (400 MHz, CDCl3): δ 7.55 (d, 2H, J=16 Hz,
RꢀCH=CHꢀR), 7.06ꢀ7.10 (m, 6H, ArꢀH), 6.32 (d, 2H, J=16 Hz, RꢀCH=CHꢀR), 4.45 (s, 4H,
CH2), 3.84 (s, 6H, OCH3), 3.78 (t, 4H, J=7 Hz, CH2), 3.76 (t, 4H, J=7 Hz, CH2), 1.54 (s,
18H, 3CH3). 13CꢀNMR (CDCl3): δ 168.6 (2C, C13), 166.3 (2C, C3), 151.4 (2C, C10),
142.9 (2C, C5), 141.2 (2C, C9), 134.1 (2C, C6), 123.2 (2C, C8), 121.3 (2C, C7), 120.8
(2C, C4), 111.3 (2C, C11), 80.9 (2C, C2), 71.2 (2C, C16), 70.9 (2C, C15), 68.6 (2C, C14),
56.1 (2C, C12), 28.4 (6C, C1). IR (NaCl, cmꢀ1) 1781 and 1708 (C=O, ester),1637 and
1599 (C=C). Tm: 239ꢀ242 ºC.
FA Diacid (4) Synthesis. Compound 3 (1 eq) was dissolved in anhydrous DCM
(25 mL), cooled to 0 ˚C, and anhydrous trifluoroacetic acid (TFA, 40 eq) added. The
reaction mixture stirred overꢀnight, gradually warming to room temperature. Solvent
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was removed in vacuo and the resulting residue was triturated with DI water (300 mL),
isolated via vacuum filtration, and dried in vacuo for 24 hours.
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FA-adipic Diacid (4a). Synthesized and characterized following previously
published methods.22
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FA-diglycolic Diacid (4b). Yield: 84 % (white powder). 1HꢀNMR (500 MHz,
DMSOꢀd6): δ 12.43 (s, 2H, COOH), 7.58 (d, 2H, J=16 Hz, RꢀCH=CHꢀR), 7.51 (s, 2 H,
ArꢀH), 7.29 (d, 2H, J=8 Hz, ArꢀH), 7.20 (d, 2H, J=8 Hz, ArꢀH), 6.60 (d, 2H, J=16 Hz, Rꢀ
CH=CHꢀR), 4.60 (s, 4H, CH2 ), 3.84 (s, 6H, OCH3). 13CꢀNMR (DMSOꢀd6): δ 168.4 (2C,
C1), 168.3 (2C, C11), 151.6 (2C, C8), 144.0 (2C, C3), 140.8 (2C, C7), 134.3 (2C, C4),
123.8 (2C, C6), 122.1 (2C, C5), 120.5 (2C, C2), 112.7 (2C, C9), 67.9 (2C, C12), 56.8 (2C,
C10). IR (KBr, cmꢀ1): 3000ꢀ2500 (OH, COOH), 1752 (C=O, ester), 1696 (C=O, COOH),
1633 and 1600 (C=C). Tm: 241ꢀ244 ºC. ESIꢀMS m/z: 485 zꢀ1.
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FA-tetraglycolic Diacid (4c). Yield: 98 % (white crystals). HꢀNMR (500 MHz,
DMSOꢀd6): δ 12.40 (s, 2H, COOH), 7.51 (d, 2H, J=16 Hz, RꢀCH=CHꢀR), 7.41 (s, 2 H,
ArꢀH), 7.18 (d, 2H, J=8 Hz, ArꢀH), 7.08 (d, 2H, J=8 Hz, ArꢀH), 6.52 (d, 2H, J=16 Hz, Rꢀ
CH=CHꢀR), 4.35 (s, 4H, CH2), 3.74 (s, 6H, OCH3), 3.62 (t, 4H, J=7 Hz, CH2), 3.54 (t,
4H, J=7 Hz, CH2). 13CꢀNMR (DMSOꢀd6): δ 169.0 (2C, C1), 168.3 (2C, C11), 151.7 (2C,
C8), 144.0 (2C, C3), 140.9 (2C, C7), 134.2 (2C, C4), 123.8 (2C, C6), 122.1 (2C, C5),
120.4 (2C, C2), 112.7 (2C, C9), 70.8 (2C, C14), 70.3 (2C, C13), 68.1 (2C, C12), 56.8 (2C,
C10). IR (KBr, cmꢀ1): 2920 (OH, COOH), 1780 (C=O, ester), 1690 (C=O, COOH), 1630
and 1600 (C=C). Tm: 86ꢀ89 ºC. ESIꢀMS m/z: 597 [z + Na]
FA Polymer (5) Synthesis. Polymer (5) was prepared using a modified
literature procedure (Scheme 1).29 In brief, 4 (1 eq) was suspended in anhydrous DCM
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(20 mL) under argon. After adding triethylamine (TEA, 4.4 eq), the reaction mixture was
cooled to 0 °C. Triphosgene (0.33 eq) dissolved in anhydrous DCM (10 mL) was added
dropꢀwise (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 isolated via vacuum filtration. The residue was dissolved in
anhydrous DCM, washed with acidic water (1 x 250 mL), dried over MgSO4,
concentrated to ~10 mL, and precipitated with an excess of chilled diethyl ether (500
mL). 5 was isolated via vacuum filtration and dried in vacuo at room temperature.
FA-adipic Polymer (5a). Synthesized and characterized following previously
published methods.22
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FA-diglycolic Polymer (5b). Yield: 84 % (white powder). 1HꢀNMR (500 MHz,
DMSOꢀd6): 7.93 (d, 2H, J=16 Hz, RꢀCH=CHꢀR), 7.66 (s, 2 H, ArꢀH), 7.45 (d, 2H, J=8 Hz,
ArꢀH), 7.28 (d, 2H, J=8 Hz, ArꢀH), 6.94 (d, 2H, J=16 Hz, RꢀCH=CHꢀR), 4.63 (s, 4H, CH2
), 3.86 (s, 6H, OCH3). 13CꢀNMR (DMSOꢀd6): δ 168.3 (2C, C1), 163.4 (2C, C11), 151.8
(2C, C8), 148.7 (2C, C3), 141.8 (2C, C7), 133.6 (2C, C4), 124.0 (2C, C6), 123.2 (2C, C5),
118.0 (2C, C2), 113.4 (2C, C9), 67.9 (2C, C12), 56.9 (2C, C10). IR (KBr, cmꢀ1): 1780ꢀ
1710 (C=O, anhydride and ester region), 1630 and 1600 (C=C). Mw = 18,300 Da, PDI =
1.3. Tg = 108 °C. Td = 338 °C.
FA-tetraglycolic Polymer (5c).
Yield: 90 % (light beige powder). 1HꢀNMR
(500 MHz, DMSOꢀd6): 7.87 (b, 2H, J=16 Hz, RꢀCH=CHꢀR), 7.58 (b, 2 H, ArꢀH), 7.38 (b,
2H, ArꢀH), 7.20 (b, 2H, ArꢀH), 6.86 (b, 2H, J=16.0 Hz, RꢀCH=CHꢀR), 4.42 (b, 4H, CH2),
3.81 (b, 6H, OCH3), 3.66 (b, 4H, CH2), 3.48 (b, 4H, CH2). 13CꢀNMR (DMSOꢀd6): δ 168.9
(2C, C1), 163.3 (2C, C11), 151.8 (2C, C8), 148.6 (2C, C3), 141.8 (2C, C7), 133.4 (2C, C4),
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124.0 (2C, C6), 123.1 (2C, C5), 117.9 (2C, C2), 113.2 (2C, C9), 70.8 (2C, C14), 70.3 (2C,
C13), 68.1 (2C, C12), 59.8 (2C, C10). IR (KBr, cmꢀ1): 1779 and 1708 (C=O, anhydride),
1759 (C=O, ester), 1630 and 1600 (C=C). Mw = 15,400 Da, PDI = 1.4. Tg = 49 °C. Td
= 251 °C.
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Diacid (4) log P Determination.30, 31 Owing to the poor solubility of FA Diacids
(4) in the release media, log P, partitioning of respective compounds between water and
oil phase, studies were conducted to understand the diacid relative hydrophobicity.
HPLC equipped with a reverseꢀphase C18 (RP18) column was used to measure diacid
hydrophobicity. Studies were performed on an XTerra® RP18 5 ꢂm 4.6x150 mm
column (Waters, Milford, MA) on a Waters 2695 Separations Module equipped with a
Waters 2487 Dual λ Absorbance Detector. A mobile phase consisting of 50 mM
KH2PO4 with 1 % formic acid in DI water at pH 2.5 (65 %) and acetonitrile (35 %) run at
1 mL/min flow rate at ambient temperature using λ = 255 and 275 nm for reference
samples and λ = 320 nm for diacid samples was used for the study. All samples were
prepared in phosphate buffered saline (PBS) and filtered using 0.22 ꢂm PVDF syringe
filters prior to autoinjection (20 ꢁL). The phase preference is expressed by capacity
factor k’, which k’ can be calculated using Equation 7.1, where tR and t0 are the retention
times of the sample and PBS, respectively.
k’ = (tR – t0) / t0
Equation 1
log Po,w = slope * log k’ + yꢀintercept
Equation 2
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Toluene, thymol, anisole, and benzyl alcohol were used as reference samples
and their retention times obtained. A calibration curve was generated using published
log Po,w values31 for the reference samples plotted on the yꢀaxis and calculated log k’
values for the xꢀaxis. This curve was used to find the extrapolated log P diacid values
of the regression line using Equation 2.
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Contact Angle Measurements. To ascertain the relative hydrophobicity of the
final polymers, static contact angles were measured by dropping DI water onto pressed
polymer discs using a RaméꢀHart Standard Goniometer Model Number 250ꢀ00 156
(Mountain Lakes, NJ) outfitted with a Dell Dimension 3000 computer with DROPimage
Advanced software. Measurements were taken after equilibrium was attained (typically,
20 seconds).
Polymer Swelling Measurements. Polymer discs were prepared by pressing
ground polymer (50 ± 5 mg) into 8 mm diameter x 1 mm thick discs in an IR pellet die
(International Crystal Laboratories, Garfield, NJ) with a benchꢀtop hydraulic press
(Carver model M, Wabash, IN). Pressure of 10,000 psi was applied for 10 min at room
temperature. All weights were measured gravimetrically. Using Wheaton glass
scintillation vials (20 mL), discs were submerged in PBS (10 mL, pH 7.4) and incubated
at 37 ˚C with mild agitation (60 rpm) using a controlled environment incubatorꢀshaker
(New Brunswick Scientific Co., Edison, NJ). Media was removed at regular time
intervals after which polymer discs were removed from vials, blotted with Kimwipes®
and weighed to obtain the wet weight. Samples were then dried at room temperature
under vacuum until a constant dry weight was recorded. Swelling was calculated
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according to Equation 3 where ww refers to wet weight and wd refers to dry weight. All
studies were performed in triplicate.
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[(ww – wd ) / wd] * 100
Equation 3
In Vitro FA Release. Polymer degradation was measured as a function of FA
release. Polymer discs were prepared by pressing ground polymer (50 ± 5 mg) into 8
mm diameter x 1 mm thick discs in an IR pellet die (International Crystal Laboratories,
Garfield, NJ) with a benchꢀtop hydraulic press (Carver model M, Wabash, IN). Pressure
of 10,000 psi was applied for 10 min at room temperature. This methodology was
preferred as it minimized interference from external effects (e.g., formulation additives)
and provided similar polymer surface areas that would be exposed to the aqueous
buffer environment.
FA release was monitored by placing polymer discs into 20 mL Wheaton glass
scintillation vials with 10 mL of PBS (pH 7.4) and subsequently incubating samples at
37 ºC with mild agitation (60 rpm) using a controlled environment incubatorꢀshaker (New
Brunswick Scientific Co., Edison, NJ). Media was collected every 24 hours for 20 days
and replaced with fresh PBS (10 mL). Spent media was immediately analyzed via
HPLC. The degradation products were analyzed and quantified via HPLC using an
XTerra® RP18 5 ꢂm 4.6x150 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 ꢂm PVDF syringe filters and subsequently injected (20
ꢁL) using an autosampler. The mobile phase was comprised of 50 mM KH2PO4 with 1
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% formic acid in DI water at pH 2.5 (65 %) and acetonitrile (35 %) run at 1 mL/min flow
rate at ambient temperature. Absorbance was monitored at λ = 320 nm. Amounts were
calculated from known concentrations of standard FA solutions.
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Antioxidant Activity.22, 32 The degradation products’ antioxidant activity was
assessed and compared to that of free ferulic acid using a 2,2ꢀdiphenylꢀ1ꢀpicrylhydrazyl
(DPPH) radical scavenging assay. Antioxidant activity was evaluated by adding sample
(0.1 mL) to a 0.024 mg/mL DPPH solution in methanol (3.9 mL). Dayꢀ10 polymer
degradation media samples (0.1 mL) were mixed with the 0.024 mg/mL DPPH solution
(3.9 mL) at room temperature. After 1 hour, solutions were analyzed via UV/Vis with a
PerkinꢀElmer Lambda XLS spectrophotometer (Waltham, MA) (λ = 517 nm). Fresh FA
solutions prepared at concentrations corresponding to dayꢀ10 HPLC data were
analyzed identically to the aforementioned degradation media samples. DPPH %
radical reduction was calculated following Equation 4 where Abst0 is the initial
absorbance and Abst is the absorbance after 1 hour. Abst0 values were determined by
adding PBS (0.1 mL) to the DPPH solution (3.9 mL) and analyzing the resulting
absorbance (λ = 517 nm). All radical scavenging assays were performed in triplicate.
Student’s tꢀtests were performed to determine significant differences between free FA
and FA degradation media antioxidant activity (p < 0.05).
[(Abst0 – Abst)/Abst0] * 100 Equation 4
Cytotoxicity. In vitro cytocompatibility studies were performed by culturing 3T3
mouse fibroblasts in cell media (Dulbecco’s Modified Eagle Medium supplemented with
10% Fetal Bovine Serum, 1% Penicillin Streptomycin) containing the FAꢀdiglycolic and
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FAꢀtetraglycolic polymers. Polymers were first sterilized under UV at λ = 254 nm for 900
s (Spectronics Corporation, Westbury, NY), dissolved in DMSO to yield 10 mg/mL
solutions, and then diluted with cell media to reach concentrations of 0.1 mg/mL, 0.01
mg/mL and 0.001 mg/mL. Aliquots of cell media containing polymers were then added
to allocated wells in a 96ꢀwell plate with 2000 cells/well and incubated at 37 °C. DMSO
(1 %) in cell media was used as a negative control.
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Cell viability was determined using CellTiter 96® Aqueous One Solution Cell
Proliferation Assay. After 24 h, 48 h, and 72 h incubation with polymers, 20 ꢂL of (3ꢀ
(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀcarboxymethoxyphenyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolium)
(MTS) reagent was added to each well and further incubated for 4 h at 37 °C. The
absorbance was then recorded with a microplate reader (Coulter, Boulevard Brea, CA)
at 492 nm.
RESULTS AND DISCUSSION
Synthesis and Characterization. FA was successfully incorporated into
polymer backbones via ester and anhydride bonds. The commercial availability of
diglycolyl chloride simplified the FA (diglycolyl) polymer synthesis as shown in Scheme
1. As tetraglycolyl chloride was not commercially available, tetraglycolic acid was
chlorinated using thionyl chloride to obtain the diacyl chloride molecule. The acyl
chlorides (both tetraglycolic and diglycolic) were separately reacted with tꢀbutyl FA to
yield 3 (Figure 2A), and subsequently deprotected using TFA. The synthesized diacid
(4, Figure 2B) underwent solution polymerization using triphosgene as a coupling agent
to yield polymer 5 (Figure 2C). Structures of compounds 3 were confirmed via the
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appearance of linker methylene protons in H NMR (Figure 2A and 2B) and the ester
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functionality in both C NMR and FTIR spectra. Subsequent deprotection to acquire 4
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was validated by the absence of the tꢀbutyl peak at 1.54 ppm (Figure 2A and 2B) in H
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13
NMR and carboxylic acid carbonyl group in FTIR and C NMR spectra. MS confirmed
the mass of both polymer precursors. The composition of polymers’ (5) was confirmed
by 13C NMR spectra, and FTIR analysis depicted the absence of the carboxylic acid and
formation of anhydride bond.
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Figure 2. Representative 1H NMR spectra of FAꢀdiglycolic polymer (5) and
intermediates. In this example, spectra for tꢀbutyl FAꢀdiglycolic diester 3b (A), FAꢀ
diglycolic diacid 4b (B), and FAꢀdiglycolic polymer, 5b (C) are presented.
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1
Figure 3. Representative H NMR spectra of FAꢀtetraglycolic polymer (5) and
intermediates. As a specific example, spectra of tꢀbutyl FAꢀtetraglycolic diester 3c (A),
FAꢀtetraglycolic diacid 4c (B), and FAꢀtetraglycolic polymer, 5c (C) are illustrated above.
Polymers were obtained with MW values ranging from 15,400 to 21,700 Da with
1.3 – 1.7 PDI values (Table 1). As the linker mass (or length) increased, the mass
percent of FA chemically incorporated in the polymer backbone decreased giving a 5a >
5b > 5c trend for FA loading. Glass transition temperature decreased with increasing
linker chain length (5b > 5a > 5c), likely due to the enhanced polymer flexibility.33, 34
Thus, the polymer with longest chain length should display the highest flexibility, for
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example 5c exhibited the lowest Tg value (Tg = 44 ºC). As Tg exceeds physiological
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temperature (37 ˚C) in all cases, all polymers are suitable for topical applications.
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Table 1. Polymer characterization including drug loading percentage, MW, PDI, Tg, Td,
and contact angle measurements.
Drug loading
(%)
Contact angle
(degrees)
Mw (Da)
PDI
Tg (°C)
Td (°C)
5a22
5b
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67
21,700
18,300
15,400
1.7
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1.4
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5c
Diacid (4) Log P Determination. Measuring the diacid (4) water solubility
proved difficult as the diacid (4b and 4c) quickly degraded into FA. Therefore, a log P
extrapolation method via HPLC provided quantitative insight to diacid hydrophobicity.31
Capacity factor, k’, values were calculated by Equation 1 after the diacid retention times
were obtained using HPLC (Table 2). Although not a direct correlation with solubility
limits, the log P value indicates the waterꢀsolubility of a compound relative to oil (or
organic solvent).30 The calculated log P values in Table 2 illustrate the trend where 4a
< 4b < 4c, indicating increased hydrophilicity as diacid oxygen content increases.
Table 2. Calculated log P values for diacid molecules to elucidate their relative
hydrophobicity
Sample
4a
Retention time (min)
Calculated log P values
19.19
2.2
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4b
4c
15.35
12.92
2.0
1.8
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Contact Angle Measurements. To evaluate relative polymer hydrophobicity,
static contact angles were measured by dropping DI water onto pressed polymer disc
surfaces. The polymers were found to be relatively hydrophilic with contact angles
ranging from 52 – 17 degrees (Table 1). As oxygen content increased, relative
hydrophilicity increased as indicated by the lowered contact angle, which is in
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agreement with other published glycolꢀcontaining polyanhydrides.23
During
measurements, the water droplet gradually expanded on the pressed polymer 5c disc,
whereas the water droplets for 5a and 5b remained stagnant, further indicating
increased hydrophilicity with more glycol functionality. These results correlated to diacid
log P values (Table 2), as the diacid log P value decreased, the polymer contact angle
measurement became more hydrophilic. The enhanced hydrophilicity should influence
the polymer degradation rate and subsequent bioactive release as water penetration
into the polymeric matrix is an important factor in polyanhydride degradation.35, 36
Polymer 5c exhibited the lowest contact angle value and also displayed the fastest
release rates relative to polymers 5b and 5a, as will be discussed later.
Polymer Swelling Measurements. Polymer swelling was obtained according to
Equation 3. As shown in Figure 4, polymers 5a and 5b displayed similar swelling
capabilities, reaching ~15% swelling. The extent of swelling for these polymers
continued to increase until 18 h after which swelling plateaued. Swelling in 5c was
highest throughout the study, presumably due to its higher hydrophilicity.27 We
hypothesized that extensive bulk erosion was occurring in 5c, as confirmed by the loss
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of structural integrity at 18 h during the swelling study. Swelling acts as a mechanism
by which polymer degradation within inner layers of the polymer matrix is activated,
enabling bulk erosion.26
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Figure 4. Swelling studies of FAꢀbased polymers
In Vitro FA Release. Polymer degradation was measured by quantifying FA in
degradation media as FA’s appearance is indicative of both anhydride and ester bond
hydrolysis. Discs were used to enable uniform polymer degradation without additional
formulation steps that may alter polymer degradation and FA release rates. Relative
hydrolysis rates of anhydride vs. ester bonds were not apparent as diacid absorbance
(Rt = 15.35 and 12.92 min for 4b and 4c, respectively) was minimal at the observed
wavelengths. Additionally, diacid solubility in PBS varied with glycol content. Detection
of 1 (Rt = 2.98 min) indicated complete ester and anhydride bond hydrolysis with no
decomposition peaks observed. Collectively, FAꢀtetraglycolic polymer degraded the
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fastest, fully hydrolyzing to FA and tetraglycolic acid in 12 days followed by FAꢀdiglycolic
and FAꢀadipic polymers. This trend of increasing ethylene oxide units culminating in
faster polymer degradation is consistent with polymer hydrophilicity, as indicated by
contact angle measurements. While the FAꢀdiglycolic and FAꢀadipic polymer exhibited
nearꢀlinear release curves, FAꢀtetraglycolic possessed a sigmoidal curve, which we
hypothesis is due to polymer disc swelling.
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Figure 5. FAꢀbased polymer release profiles, cumulative FA release into degradation
media from polymer discs in physiological conditions
An initial state of swelling was observed for the FAꢀtetraglycolic samples.
Swelling refers to water uptake into the matrix that results in increased volume.37 Water
can permeate the FAꢀtetraglycolic polymer more rapidly due to ethylene oxide groups’
ability to hydrogen bond. The initial, slower FA release observed for polymer 5c is due
to this initial water uptake and subsequent polymer degradation, resulting in increased
polymer porosity.38 The increased porosity facilitates hydrolytic degradation within the
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polymer interior, leading to an accumulation of degradation products. Once the
degradation products were released from the disc, a larger internal surface area was
available for erosion, leading to the observed increased release rate. Differences in FA
release were attributed to increased polymer hydrophilicity via the ethylene glycol
moiety within the polymer backbone. Complete FA release was obtained by 5c and 5b
(in 12 and 20 days, respectively), which is a drastic improvement over 5a (calculated to
fully degrade in 11 months). By adjusting the glycol content, it is possible to fineꢀtune
the extent of FA release.
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Antioxidant Assay. A radical quenching assay was used to determine polymer
degradation media antioxidant activity to ensure that the synthetic methods and release
conditions did not influence FA activity. The DPPH reduction percentage was expected
to increase with increasing antioxidant activity, which is directly correlated to antioxidant
concentration. As illustrated by Figure 6, FA bioactivity was not adversely influenced by
incorporation into the polymer backbone.
Furthermore, radical quenching was
confirmed to be concentrationꢀdependent as the faster degrading polymers (5b and 5c)
reduced DPPH significantly more than 5a, which is consistent with previous literature.39
Therefore, by modifying the extent of glycol content in FA polymers, the extent of radical
scavenging can be controlled.
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Figure 6. Antioxidant activity of day 10 FAꢀcontaining release media compared to free
FA. Statistical difference indicated by * (P < 0.05)
Cytotoxicity. In vitro cytotoxicity studies were conducted to ensure that glycol
modified FA polymers (5b and 5c) would be suitable for topical use. Cytotoxicity was
conducted using 3T3 mouse fibroblasts in the presence of polymerꢀcontaining media at
0.1, 0.01, and 0.001 mg/mL, as these are therapeutically relevant values, whereas
DMSOꢀcontaining media was used as a control. Studies were performed over a 72 h
duration, monitoring cell viability every 24 h.
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Figure 7. Glycolꢀmodified FA polymers cytotoxicity after 24, 48, and 72 h in cell culture
media with polymer concentrations of 0.1, 0.01, and 0.001 mg/mL
Collectively, all polymers were cytocompatible at 0.01 mg/mL and 0.001 mg/mL
over 72 hours (i.e., no significant difference in cell viability was found between the
polymer groups and the control (Figure 7)). At 0.1 mg/mL, both polymers were found to
decrease cell viability after 48 h although this concentration is much higher than
expected for in vivo applications. Thus, at concentrations of 0.01 mg/mL and below, we
consider the glycolꢀmodified FA polymers as cytocompatible and potentially suitable for
topical use.
CONCLUSION
Antioxidants, including FA, exhibit beneficial therapeutic properties to combat
oxidative stress, but need better storage stability and delivery methods. Such
antioxidant delivery systems are finding significant and diverse applications ranging
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from biomaterials to consumer products. In previous work, FAꢀadipic polymers
displayed good antioxidant activity; however, the slow release profile limited its
applicability in topical applications. In this work, glycol groups were incorporated into
the polymer backbone to enhance the FA release rate. Increasing ethylene glycol
content promoted faster polymer degradation, higher antioxidant activity and influenced
polymer thermal properties. These biodegradable polymers offer a tunable means of
FA stabilization and delivery for a variety of skin care formulations.
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SUPPORTING INFORMATION AVAILABLE
Polymer and polymer precursors 13C NMR spectra as well as polymer thermal DSC and
TGA traces are included. This information is available free of charge via the Internet at
http://pubs.acs.org/.
ACKNOWLEDGEMENTS
This work was supported by the U.S. Department of Education Graduate Assistance in
Areas of National Need (GAANN) Fellowship (MAO) and the National Institutes of
Health (NIH). Dr. Bryan Langowski (Rutgers, Department of Chemistry & Chemical
Biology) is thanked for intellectual discussions.
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