Ferulic Acid-Based Polymers with Glycol Functionality as a Versatile

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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

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 08854-8087, USA E-mail address: [email protected] Tel.: (848) 445-0361 Fax: (732) 445-7036

KEYWORDS: biodegradable, polymer, poly(anhydride-ester), ferulic acid, antioxidant, controlled release

ACS Paragon Plus Environment

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ABSTRACT Ferulic acid-based 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 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. bioactive decomposition.

In all polymers, ferulic acid release was achieved with no 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 Free radicals have been identified as major sources of oxidative stress in cells leading to DNA damage.1 Increased oxidative stress has been implicated in various deleterious conditions including cardiovascular diseases,2,

3

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 acid-based 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 resonancestabilized phenolic radical.10 FA (1) has been studied as an ultra-violet (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 half-


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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 acid14-18 and, to a lesser extent FA19-22 itself. FA-based poly(anhydrideesters) have been found to enable controlled bioactive (i.e., FA) release and preventing the bioactive functional groups from degradation.22

As described herein, by

incorporating FA into a polymer backbone, the FA retained its antioxidant activity when released via polymer degradation.

Further, the FA-based polymer prevented

discoloration, indicating a promising FA-based 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 watersolubilizing ethylene glycol functionalities via copolymerization with poly(ethylene glycol) (PEG)23-25 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 FA-based

biodegradable polymers by introducing ethylene glycol functionality as a linker


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molecule. The synthesis, characterization, and drug release profiles of the glycolmodified polymers, in addition to polymer cytotoxicity, are discussed.

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. 1

H and

C NMR and FT-IR spectroscopies. Proton (1H) and carbon (13C)

13

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 (DMSO-d6) as solvent and internal reference. Fourier transform infrared (FT-IR) 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 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 x 10-5 and 150 °C API temperature. Samples dissolved in methanol (< 10 µg/mL) were injected via a glass syringe. Gel permeation chromatography (GPC) was used to determine polymer weightaveraged 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 µm PTFE syringe filters.

Samples were resolved on a Jordi

divinylbenzene mixed-bed 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). Thermal Properties.

Differential scanning calorimetry (DSC) measurements

were carried out on TA Instrument Q200 to determine melting (Tm) and glass transition (Tg) temperatures. Samples (4-6 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 two-cycle 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 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 °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(anhydride-esters) (5) and FA-containing polymer precursors including t-butyl FA diesters (3) and FA diacids (4) with varying linkers (a22, b, c)

t-Butyl FA (2) Synthesis.

t-Butyl FA was prepared following previously

published methods (Scheme 1, 2).22, 28 t-Butyl FA Diester (3) Synthesis.

t-Butyl 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 drop-wise to the reaction mixture at 20 mL/hr. Thin layer chromatography (TLC, 4:1 hexane:ethyl acetate) was used to monitor reaction progress. Following t-butyl 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 acyl chloride. Yield: 72 % (white powder).

1

H-NMR (400 MHz, CDCl3): δ 7.56 (d, 2H,

J=16 Hz, R-CH=CH-R), 7.08-7.10 (m, 6H, Ar-H), 6.34 (d, 2H, J=16 Hz, R-CH=CH-R), 4.62 (s, 4H, CH2), 3.86 (s, 6H, OCH3), 1.54 (s, 18H, 3CH3).

13

C-NMR (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, cm-1) 1782 and 1705 (C=O, ester), 1638 and 1600 (C=C). Tm: 222-223 ºC. t-Butyl FA-tetraglycolic Diester (3c). To synthesize the diacyl chloride, 3,6,9trioxaundecanedioic 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 purification as the diacyl chloride.

1

H-NMR (400 MHz, CDCl3): δ 7.55 (d, 2H, J=16 Hz,

R-CH=CH-R), 7.06-7.10 (m, 6H, Ar-H), 6.32 (d, 2H, J=16 Hz, R-CH=CH-R), 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).

13

C-NMR (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, cm-1) 1781 and 1708 (C=O, ester),1637 and 1599 (C=C). Tm: 239-242 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 over-night, 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. FA-adipic Diacid (4a).

Synthesized and characterized following previously

published methods.22 FA-diglycolic Diacid (4b). Yield: 84 % (white powder).

1

H-NMR (500 MHz,

DMSO-d6): δ 12.43 (s, 2H, COOH), 7.58 (d, 2H, J=16 Hz, R-CH=CH-R), 7.51 (s, 2 H, Ar-H), 7.29 (d, 2H, J=8 Hz, Ar-H), 7.20 (d, 2H, J=8 Hz, Ar-H), 6.60 (d, 2H, J=16 Hz, RCH=CH-R), 4.60 (s, 4H, CH2 ), 3.84 (s, 6H, OCH3).

13

C-NMR (DMSO-d6): 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, cm-1): 3000-2500 (OH, COOH), 1752 (C=O, ester), 1696 (C=O, COOH), 1633 and 1600 (C=C). Tm: 241-244 ºC. ESI-MS m/z: 485 z-1. FA-tetraglycolic Diacid (4c).

Yield: 98 % (white crystals). 1H-NMR (500 MHz,

DMSO-d6): δ 12.40 (s, 2H, COOH), 7.51 (d, 2H, J=16 Hz, R-CH=CH-R), 7.41 (s, 2 H, Ar-H), 7.18 (d, 2H, J=8 Hz, Ar-H), 7.08 (d, 2H, J=8 Hz, Ar-H), 6.52 (d, 2H, J=16 Hz, RCH=CH-R), 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).

13

C-NMR (DMSO-d6): 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, cm-1): 2920 (OH, COOH), 1780 (C=O, ester), 1690 (C=O, COOH), 1630 and 1600 (C=C). Tm: 86-89 ºC. ESI-MS 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 drop-wise (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 FA-diglycolic Polymer (5b). Yield: 84 % (white powder).

1

H-NMR (500 MHz,

DMSO-d6): 7.93 (d, 2H, J=16 Hz, R-CH=CH-R), 7.66 (s, 2 H, Ar-H), 7.45 (d, 2H, J=8 Hz, Ar-H), 7.28 (d, 2H, J=8 Hz, Ar-H), 6.94 (d, 2H, J=16 Hz, R-CH=CH-R), 4.63 (s, 4H, CH2 ), 3.86 (s, 6H, OCH3).

13

C-NMR (DMSO-d6): 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, cm-1): 17801710 (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).

1

H-NMR

(500 MHz, DMSO-d6): 7.87 (b, 2H, J=16 Hz, R-CH=CH-R), 7.58 (b, 2 H, Ar-H), 7.38 (b, 2H, Ar-H), 7.20 (b, 2H, Ar-H), 6.86 (b, 2H, J=16.0 Hz, R-CH=CH-R), 4.42 (b, 4H, CH2), 3.81 (b, 6H, OCH3), 3.66 (b, 4H, CH2), 3.48 (b, 4H, CH2).

13

C-NMR (DMSO-d6): 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, cm-1): 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. 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 reverse-phase C18 (RP18) column was used to measure diacid hydrophobicity.

Studies were performed on an XTerra® RP18 5 µm 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 µm PVDF syringe filters prior to autoinjection (20 µL). 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’ + y-intercept


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 y-axis and calculated log k’ values for the x-axis. This curve was used to find the extrapolated log P diacid values of the regression line using Equation 2. 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 Ramé-Hart Standard Goniometer Model Number 250-00 156 (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 bench-top 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 incubator-shaker (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.

[(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 bench-top 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 incubator-shaker (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 µm 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 µm PVDF syringe filters and subsequently injected (20 µL) 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. Antioxidant Activity.22,

32

The degradation products’ antioxidant activity was

assessed and compared to that of free ferulic acid using a 2,2-diphenyl-1-picrylhydrazyl (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).

Day-10 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 Perkin-Elmer Lambda XLS spectrophotometer (Waltham, MA) (λ = 517 nm). Fresh FA solutions prepared at concentrations corresponding to day-10 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 t-tests 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 FA-diglycolic and


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FA-tetraglycolic 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 96-well plate with 2000 cells/well and incubated at 37 °C. DMSO (1 %) in cell media was used as a negative control. 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 µL of (3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (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 t-butyl 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 1H NMR (Figure 2A and 2B) and the ester functionality in both

13

C NMR and FTIR spectra. Subsequent deprotection to acquire 4

was validated by the absence of the t-butyl peak at 1.54 ppm (Figure 2A and 2B) in 1H NMR and carboxylic acid carbonyl group in FTIR and

13

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 intermediates.

1

H NMR spectra of FA-diglycolic polymer (5) and

In this example, spectra for t-butyl FA-diglycolic diester 3b (A), FA-

diglycolic diacid 4b (B), and FA-diglycolic polymer, 5b (C) are presented.


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Figure 3. Representative

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1

H NMR spectra of FA-tetraglycolic polymer (5) and

intermediates. As a specific example, spectra of t-butyl FA-tetraglycolic diester 3c (A), FA-tetraglycolic diacid 4c (B), and FA-tetraglycolic 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 temperature (37 ˚C) in all cases, all polymers are suitable for topical applications.

Table 1. Polymer characterization including drug loading percentage, MW, PDI, Tg, Td, and contact angle measurements. Drug loading (%)

Mw (Da)

PDI

Tg (°C)

Td (°C)

Contact angle (degrees)

5a22

81

21,700

1.7

82

332

52

5b

79

18,300

1.3

108

235

34

5c

67

15,400

1.4

49

262

17

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 water-solubility 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

Retention time (min)

Calculated log P values

4a

19.19

2.2


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15.35 12.92

4b 4c

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2.0 1.8

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 agreement

with

other

published

glycol-containing

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

Figure 4. Swelling studies of FA-based 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, FA-tetraglycolic polymer degraded the


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fastest, fully hydrolyzing to FA and tetraglycolic acid in 12 days followed by FA-diglycolic and FA-adipic 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 FA-diglycolic and FA-adipic polymer exhibited near-linear release curves, FA-tetraglycolic possessed a sigmoidal curve, which we hypothesis is due to polymer disc swelling.

Figure 5. FA-based polymer release profiles, cumulative FA release into degradation media from polymer discs in physiological conditions

An initial state of swelling was observed for the FA-tetraglycolic samples. Swelling refers to water uptake into the matrix that results in increased volume.37 Water can permeate the FA-tetraglycolic 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 fine-tune the extent of FA release. 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 concentration-dependent 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 FA-containing 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 polymer-containing media at 0.1, 0.01, and 0.001 mg/mL, as these are therapeutically relevant values, whereas DMSO-containing 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. Glycol-modified 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 glycol-modified 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.

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In previous work, FA-adipic 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.

SUPPORTING INFORMATION AVAILABLE Polymer and polymer precursors

13

C 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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57x20mm (600 x 600 DPI)


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Ferulic Acid-Based Polymers with Glycol Functionality as a Versatile

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