Enzymatic Polymerization of an Ibuprofen-Containing Monomer and Subsequent Drug Release

Article abstract of DOI:10.1002/mabi.201500030

Novel ibuprofen-containing monomers comprising naturally occurring and biocompatible compounds were synthesized and subsequently polymerized via enzymatic methods. Through the use of a malic acid sugar backbone, ibuprofen was attached as a pendant group,

Full text of DOI:10.1002/mabi.201500030

Full Paper
Enzymatic Polymerization of an Ibuprofen-
Containing Monomer and Subsequent Drug
a
Release
Nicholas D. Stebbins, Weiling Yu, Kathryn E. Uhrich*
Novel ibuprofen-containing monomers comprising naturally occurring and biocompatible
compounds were synthesized and subsequently polymerized via enzymatic methods. Through
the use of a malic acid sugar backbone, ibuprofen was attached as a pendant group, and then
subsequently polymerized with a linear aliphatic diol (1,3-propanediol, 1,5-pentanediol, or 1,8-
octanediol) as comonomer using lipase B from
Candida antarctica, a greener alternative to tradi-
tional metal catalysts. Polymer structures were
elucidated by nuclear magnetic resonance and infra-
red spectroscopies, and thermal properties and
molecular weights were determined. All polymers
exhibited sustained ibuprofen release, with the longer
chain, more hydrophobic diols exhibiting the slowest
release over the 30 d study. Polymers were deemed
cytocompatible using mouse fibroblasts, when eval-
uated at relevant therapeutic concentrations. Addi-
tionally, ibuprofen retained its chemical integrity
throughout the polymerization and in vitro hydrolytic
degradation processes. This methodology of enzy-
matic polymerization of a drug presents a more
environmentally friendly synthesis and a novel
approach to bioactive polymer conjugates.
1
. Introduction
Lipase-catalyzed polycondensations have garnered atten-
tion due to their chemoselectivity, non-toxicity, recycla-
N. D. Stebbins, Prof. K. E. Uhrich
Department of Chemistry and Chemical Biology, Rutgers
University, 610 Taylor Road, Piscataway, New Jersey 08854-8087,
USA
[1]
bility, andderivationfromrenewableresources. Onesuch
lipase, Novozym 435 (N435, lipase B from Candida
antarctica immobilized on acrylic resin), has been used
to synthesize a wide range of polymers with different
properties. Aliphatic polyesters of diol/diacid mono-
E-mail: keuhrich@rutgers.edu
W. Yu
Department of Biomedical Engineering, Rutgers University, 599
Taylor Road, Piscataway, New Jersey 08854-8087, USA
[2,3]
[4,5]
[6]
mers,
terpolymers of diacids and diols
in addition to polycarbonates.
diol/diester monomers,
hydroxyacids, and
have been synthesized,
[
7–9]
a
Supporting Information is available from the Wiley Online Library or
[10]
from the author.
Silicone-containing
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DOI: 10.1002/mabi.201500030 1115

N. D. Stebbins et al.
www.mbs-journal.de
[
11,12]
[13]
[34]
polymers
and polyamides
have also been synthe-
resources
Thus eliminating the phrase, – the work
sized using N435, indicating it’s broad applicability. Addi-
tionally, someenzymaticallysynthesizedpolymerscontain
pendant groups for future modification (i.e., coupling a
described herein incorporates many of these principles. A
high priority of this research is inclusion of renewable
resources, as such, L-malic acid is a naturally occurring
dicarboxylic acid found in many fruits such as apples,
[6,7,9]
bioactive or targeting moiety);
however, limited
[35,36]
examples of bioactive-containing monomers polymerized
grapes, and berries and serves as the polymer back-
bone. Thediolcomonomerschosenareusedincosmeticand
[14]
via enzymatic methods are known.
Using polymers for delivery of bioactives, such as drugs,
[37,38]
pharmaceutical formulations.
panediol is derived from biological sources.
Additionally, 1,3-pro-
[15]
[39]
has been widely studied;
however, the majority of
In addition
research has focused on physically incorporating drugs into
polymers or chemically conjugating drugs to a pre-existing
polymer backbone where the polymer may – or may not –
be degradable. Drug-containing monomers can allow for
higher drug loading and tunability of release rates and
other polymer properties not possible with using pre-
to the novelty of a polymer comprised of renewable
monomers, the enzymatic polymerization of such a drug-
containing monomer with hydrolytically degradable bio-
[
14]
active pendantester groups has few literature examples.
In summary, this paper presents one of the few examples of
synthesizing drug-containing monomers to make biocom-
patible polymers via green methods including enzyme-
based polymerization; the only other literature examples
exhibit low drug loading (<25 wt%) and utilize toxic
reagents/solvents. Further,thesecompletelybiodegradable
polymers are anticipated to be significant because of the
environmentally sustainable synthetic methodology used
to make environmentally sensitive polymers.
[16]
existing polymers.
Among such researched drugs,
ibuprofenisawidelyusednon-steroidalanti-inflammatory
drug (NSAID) to treat pain, inflammation, and fever, yet it
suffers from severe gastrointestinal side-effects at higher
[
17,18]
doses.
Physical incorporation of ibuprofen into a
polymer has been achieved and exhibits limited drug
[19–26]
loadings(<30%)andaburstreleaseprofile.
Insystems
wherethedrugischemicallyincorporatedintothepolymer,
non-biodegradable polymers are often used, leading to
potential adverse effects when used in vivo as the polymer
Polymer and precursor structures were determined by
1
13
proton and carbon nuclear magnetic resonance ( H and
C
NMR) and Fourier transform infrared (FT-IR) spectroscopies.
Thermalpropertieswereevaluatedbydifferentialscanning
calorimetry (DSC) and thermogravimetric analysis (TGA).
Mass spectrometry (MS) and gel permeation chromatog-
raphy (GPC) were used to determine molecular weights
[
27–30]
may remain.
In previous work, an ibuprofen-con-
taining polyester was prepared using N435 but had low
drug loading (3–13%) and a burst release; 35% of ibuprofen
[31]
was released after 18 d, but not thereafter.
Polymers of
sebacic acid, glycerol, poly(ethylene glycol), and ketoprofen
have similarly been developed using N435; however, low
drug loading (<25%) and the use of toxic, expensive vinyl
moieties remain issues. Between 10 and 25% of ketoprofen
was slowly released over 14 d; however, the release rate
(M ) of precursors and polymers, respectively. To evaluate
w
drug release from polymer, in vitro studies were performed
under physiological conditions. Polymer cytocompatibility
was determined using fibroblasts, and chemical structures
of released drugs were confirmed.
[14]
after that time was not studied.
Additionally, ibuprofen
[32]
pro-drug micelles were developed;
drug loading was
between 7 and 41 wt%, but toxic reagents were used for
synthesis. To overcome low drug loadings, Rosario-Melen-
dez et al. reported on the synthesis of ibuprofen and tartaric
acid-based polymers using 1,8-octanediol as comonomer
and stannous octoate as catalyst toyield polymerswith 65–
2. Experimental Section
2
.1. Materials
Dichloromethane (DCM) (ꢀ99.8%), methanol (MeOH) (HPLC grade,
99.9%), L-malic acid (ꢀ99%, Fluka), dimethylformamide (DMF)
(anhydrous, ꢀ99.8%), sodium carbonate (Na CO ) (ꢀ99.5%), benzyl
ꢀ
[33]
6
7 wt% ibuprofen.
In this work, the utility of enzyme-
2
3
mediated polymer synthesis is expanded; specifically,
the development of a drug-containing monomer and the
utilization of enzymes (N435) to generate three different
ibuprofen and malic acid-based polyesters using diols as
comonomers is described.
bromide (BnBr) (98%), magnesium sulfate (MgSO
ꢀ99.5%), sodium bicarbonate (NaHCO ) (ꢀ99.7%), deuterated
chloroform (CDCl
(99.8%, 0.03% TMS), ibuprofen (ꢀ98%),
-dimethylaminopyridine (DMAP) (ꢀ99%), pivalic anhydride
4
) (anhydrous,
3
3
)
4
(
99%), cyclopentylmetyl ether (CPME) (anhydrous, ꢀ99.9%), palla-
diumoncarbon(Pd/C)(10 wt%loading),Celite(512medium),lipase
The synthetic methodology herein utilizes ‘‘greener,’’
more sustainable processes when compared with more
traditional polycondensation methods. Of the twelve
principles of green chemistry, this work focuses on
acrylic resin from Candida antarctica (Novozym 435, ꢀ5,000 U/g),
1,3-propanediol (98%), 1,5-pentanediol (96%), 1,8-octanediol (98%),
diphenyl ether (Ph
phosphate buffered saline (PBS) (tablets), monobasic potassium
phosphate (KH PO
2
O) (ꢀ99%), chloroform (CHCl
3
)
(ꢀ99.8%)
(i) replacement of environmentally hazardous chemicals
2
4
) (ꢀ99%), acetonitrile (HPLC grade, ꢀ99.9%),
with safer materials, (ii) solvent elimination, (iii) product
biodegradation, (iv)useofcatalystsand(v)useofrenewable
concentrated hydrochloric acid (HCl) (37%), sodium hydroxide
(NaOH) (ꢀ98%), dimethyl sulfoxide (DMSO) (ꢀ99.7%) were
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Enzymatic Polymerization of an Ibuprofen-Containing Monomer
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3
5
̊
obtained from Sigma–Aldrich (Milwaukee, WI) and used as
Samples were eluted through two PL gel columns 10 and 10 A
received. Hydrogen (H
2
) was obtained from Airgas (Piscataway,
(Polymer Laboratories) in series at 25 8C, with DMF with 0.1%
ꢃ1
NJ). Dulbecco’sModified EagleMedium(DMEM)wasobtainedfrom
Corning (Corning, NY). (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) reagent
was obtained from Promega (Madison, WI). (Poly(vinylidine
fluoride) (PVDF) and polytetrafluoroethylene (PTFE) syringe filters,
and Wheaton glass scintillation vials were purchased from Fisher
Scientific (Fair Lawn, NJ).
trifluoroacetic acid as eluent at a flow rate of 0.8 mL ꢂ min
.
Molecular weights were calibrated relative to narrow polystyrene
standards.
2
.4. Thermal Analysis
DSC was performed using a TA DSC Q200 to evaluate melting (T
m
)
and glass transition (T
g
) temperatures. TA Universal Analysis 2000
software was used for automation and data collection on an IBM
2
.2. Structural Characterization
ThinkCentre computer. Samples (5–10 mg) were heated under dry
ꢃ1
NMR spectra of all compounds were recorded on a Varian 400 or
nitrogen gas to 200 8C at a heating rate of 10 8C ꢂ min and cooled
m
ꢃ1
1
ꢃ1
5
00 MHz spectrophotometer. Samples (ꢁ5 mg ꢂ mL
for H;
40 mg ꢂ mL for C) were dissolved in deuterated chloroform
), with tetramethylsilane as the internal reference. Each
to –50 8C at a rate of 10 8C ꢂ min with a two-cycle minimum. T
ꢃ1
13
ꢁ
was calculated at the peak of melting and T
midpoint of the curve.
g
was defined as the
(
CDCl
3
1
spectrum was an average of 16 scans for H NMR and 256 scans for
TGA was performed on a Perkin-Elmer Pyris 1 system with TAC
7/DX instrument controller. Perkin-Elmer Pyris software running
on a Dell Optiplex GX110 computer was used for automation and
data collection and processing. Samples (5–10 mg) were heated
1
3
C NMR. FT-IR absorbance spectra were measured on a Thermo
Nicolet/Avatar 360 FT-IR spectrometer. Samples (1–3 wt%) were
dissolved in dichloromethane (DCM) and solvent-cast onto NaCl
plates. Each spectrum was an average of 32 scans.
under dry nitrogen gas from 25 to 600 8C at a heating rate of 10 8C
ꢃ1
ꢂ min . Decomposition temperatures (T
d
) were measured at the
onset of thermal decomposition.
2
.3. Molecular Weight
Polymer precursors were analyzed by MS to determine molecular
weights. A Finnigan LCQ-DUO running Xcalibur software and an
adjustable atmospheric pressure ionization electrospray source
2
.5. Dibenzyl-l-Malate (2) Synthesis
[40]
Using the same methodology as Guo et al.,
L-malic acid (1, 1 eq)
(
(
API-ESIIonSource)wereused.Samplesweredissolvedinmethanol
was dissolved in anhydrous DMF and stirred in a round-bottomed
flask under nitrogen as shown in Scheme 1. Sodium carbonate (2.4
eq) was added to the reaction to form a white suspension that was
stirred for 30 min. Benzyl bromide (3 eq) was then added and
reaction was heated to 40 8C with stirring overnight. The reaction
mixturewasthenconcentratedin vacuo, dilutedwithethylacetate
(EtOAc), and washed with saturated sodium bicarbonate (3ꢄ),
deionized (DI) water, and brine. The organic layer was dried over
ꢃ1
10 mg ꢂ mL ) and injected with a glass syringe. During the
ꢃ5
experiment, the pressure was 0.8 ꢄ 10 Torr and the API temper-
ature was 150 8C. Polymer weight-averaged molecular weights
(M
w
) and polydispersity indices (PDI) were determined by GPC on a
Waters (Watford, MA) liquid chromatography system consisting of
a 515 HPLC pump and 2414 refractive index (RI) detector with
Empower software. Polymer samples were prepared for auto-
injection by dissolving polymer in dimethylformamide (DMF,
MgSO
4
, filtered, and the solvent removed in vacuo to yield
1
0 mg ꢂ mLꢃ1) and filtering through 0.45 mm PTFE syringe filters.
compound 2.
Scheme 1. Synthesis of polymer precursors and poly(ibuprofen-L-malate) polyesters using aliphatic diols of different lengths (a, b, c).
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N. D. Stebbins et al.
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1
Yield: 95% (off-white oil). H NMR (400 MHz, CDCl
3
): d 7.28–7.38
remove lipase. Filtrate was concentrated in vacuo to yield
(
m, 10H,Ar–H);5.10–5.20(split, 4H,CH );4.54(s, 1H,CH);3.23(s, 1H,
2
compound 7.
1
3
1
OH); 2.82–2.94 (split, 2H, CH
2
). C NMR (125 MHz, CDCl
3
): d 173.4,
Poly(octylene ibuprofen-L-malate) (7a) Yield: 82% (tan paste). H
ꢃ1
1
70.5,135.4,135.4,128.9,128.6,68.0,67.6,67.0,39.0.IR(NaCl,cm ):
NMR (500 MHz, CDCl
(b, 1H, CH); 3.62–4.22 (b, 5H, CH, CH
CH ); 1.84 (b, 1H, CH); 1.17–1.71 (b, 15H, CH
3
): d 7.21 (b, 2H, Ar–H); 7.09 (b, 2H, Ar–H); 5.47
); 2.84 (b, 2H, CH ); 2.44 (b, 2H,
, CH , CH , CH ) 0.89 (b,
3 3
H, CH ). CNMR(125 MHz, CDCl ):d178.2, 174.5(2C), 139.9, 137.1,
n ¼ 3 480 (w; OH), 1 740 (s; C¼O, ester). MS (ESI): m/z ¼ 315.1
2
2
þ
[
M þ 1] . T
d
¼ 248 8C.
2
3
2
2
2
1
3
6
1
29.9, 128.1, 68.9, 45.3, 44.7, 35.5, 31.1, 29.6 (2C), 22.3, 18.9, 17.2 (2C).
ꢃ1
IR (NaCl, cm ): n ¼ 1 741 (s; C¼O, ester). M
w
¼ 8.1 kDa, PDI ¼ 1.4.
2
.6. Ibuprofen Dibenzyl-l-Malate (4) Synthesis
T
g
¼–35 8C, T
d
¼ 221 8C.
[
41]
Asolvent-freeesterificationreportedbySakakuraetal. wasused
to couple the drug to compound 2. Ibuprofen (3, 1.1 eq),
Poly(pentylene ibuprofen-L-malate) (7b) Yield: 78% (brown
1
paste). H NMR (500 MHz, CDCl
3
): d 7.19 (b, 2H, Ar–H); 7.08 (b,
4
-(dimethylamino) pyridine (DMAP; 2 mol%), and dibenzyl-L-
2H, Ar–H); 5.47 (b, 1H, CH); 3.78 (b, 1H, CH); 2.75 (b, 2H, CH ); 1.83 (b,
2
1
3
malate (2, 1 eq) were combined in a round-bottomed flask under
nitrogen. Then, pivalic anhydride (1.1 eq) was added. Reaction was
heated to 55 8C and stirred overnight. Reaction was then diluted
with EtOAc and washed with saturated sodium bicarbonate (3ꢄ),
3 3
1H, CH); 1.52 (b, 3H, CH ); 0.88 (b, 6H, CH ). C NMR (125 MHz,
CDCl
3
): d 179.1, 172.7 (2C), 139.7, 136.0, 128.4, 126.3, 67.2, 44.0, 43.8,
ꢃ1
35.1, 29.1 (2C), 28.7, 27.0, 21.3 (2C), 17.5, 17.1. IR (NaCl, cm ): n ¼ 1
743 (s; C¼O, ester). Mw ¼ 6.4 kDa, PDI ¼ 1.4. T ¼–8 8C, T ¼ 215 8C.
g
d
DI water, and brine. The organic layer was dried over MgSO
4
,
Poly(propylene ibuprofen-L-malate) (7c) Yield: 81% (brown
1
filtered, and concentrated in vacuo to yield compound 4.
3
paste). H NMR (500 MHz, CDCl ): d 7.20 (b, 2H, Ar–H); 7.08 (b,
1
Yield: 85% (off-white oil). H NMR (400 MHz, CDCl
3
): d 7.28–7.38
2H, Ar–H); 5.47 (b, 5H, CH , CH); 3.70–4.20 (b, 1H, CH); 2.91 (b, 2H,
2
(
m, 10H, Ar–H); 7.15 (m, 2H, Ar–H); 7.04 (m, 2H, Ar–H); 5.52 (m, 1H,
CH); 4.90–5.20 (split m, 4H, CH ); 3.72 (m, 1H, CH); 2.88 (split m, 2H,
CH );2.40(dd, 2H, CH );1.81(m, 1H, CH); 1.46(dd, 3H, CH ); 0.87(dd,
). C NMR (125 MHz, CDCl ): d 173.9, 170.5, 169.0, 140.8,
CH ); 2.44 (b, 2H, CH ); 1.79–2.12 (b, 3H, CH , CH); 1.51 (b, 3H, CH );
2
2
3
2
3
1
2
0
.89 (b, 6H, CH
3
). C NMR (125 MHz, CDCl
3
): d 174.9, 172.6 (2C),
2
2
3
139.7, 136.0, 128.4, 126.3, 68.2, 45.3, 44.8, 35.8, 30.4 (2C), 22.6 (2C),
13
18.6, 18.4. IR (NaCl, cmꢃ1): n ¼ 1 743 (s; C¼O, ester). Mw ¼ 4.8 kDa,
3
1
6
H, CH
3
3
37.2, 135.3, 129.5 (2C), 128.8 (6C), 128.6 (6C), 127.5 (2C), 68.7, 67.6,
PDI ¼ 1.3. T
g
¼–5 8C, T ¼ 212 8C.
d
7.0, 45.1, 38.9, 36.3, 30.4, 22.6, 18.6. IR (NaCl, cmꢃ1): n ¼ 1 743 (s;
þ
C¼O, ester). MS: m/z ¼ 502.3 [M þ 1] . T
d
¼ 268 8C.
2
.9. In Vitro Drug Release from Polymer
Drug release from polymers (7a–c) was evaluated by in vitro
degradationin phosphatebufferedsaline(PBS)underphysiological
conditions. Polymersamples(30mgseach, n ¼ 3)wereincubatedin
10 mLPBS(pH7.4)in20 mLWheatonglassscintillationvials(Fisher,
Fair Lawn, NJ) using a controlled environment incubator-shaker
(New Brunswick Scientific Co., Edison, NJ) at 60 rpm at 37 8C. At
predetermined time intervals throughout the 30 d of the study,
media (5 mL) was collected and replaced with fresh PBS (5 mL) and
the spent media was analyzed by high-performance liquid
chromatography (HPLC). Analysis was performed using an XTerra
2
.7. Ibuprofen L-Malic Acid (5) Synthesis
Ibuprofen dibenzyl-L-malate (4, 1 eq) was dissolved in anhydrous
ꢃ1
cyclopentylmethylether(CPME, 10 mL ꢂ g )and10%palladiumon
carbon (Pd/C, catalytic amount) was added. The reaction flask was
evacuated under vacuum and purged with hydrogen gas (3ꢄ), then
allowedtostiratroomtemperatureunderhydrogenovernight. The
mixturewasfilteredthroughCelitetoremovePd/C. Thefiltratewas
concentrated in vacuo to yield pure compound 5.
1
Yield: 91% (light tan paste). H NMR (400 MHz, CDCl
3
): d 7.19 (t,
2
2
H, Ar–H); 7.08 (dd, 2H, Ar–H); 5.47 (split, 1H, CH); 3.78 (m, 1H, CH);
.75 (split, 2H, CH ); 1.83 (m, 1H, CH); 1.52 (d, 3H, CH ); 0.88 (d, 6H,
). C NMR (CDCl , 125 MHz): d 174.9, 173.9 (2C), 141.0, 136.9,
RP18 3.5 mm 4.6 ꢄ 150 mm column (Waters, Milford, MA) on a
2
2
3
Waters 2695 Separations Module equipped with a Waters 2487
Dual Absorbance Detector. All samples were filtered using 0.22 mm
PVDF syringe filters and subsequently injected (20 mL) using an
13
CH
3
3
ꢃ1
1
1
29.5, 127.5, 67.8, 45.0, 44.8, 35.9, 22.6, 18.6. IR (NaCl, cm ): n ¼ 3
00—3 300 (w; OH, acid), 1 743 (s; C¼O, ester), 1 719 (s; C¼O, acid).
autosampler. The mobile phase, which was developed as a
[42]
–
MS: m/z ¼ 321.1 [M – 1] . T
d
¼ 211 8C.
modification of a published procedure,
was comprised of
acetonitrile (70%) and 10 mM KH
2
PO
4
in DI water at pH 2.5
ꢃ1
(
30%) run at 0.5 mL ꢂ min flow rate and ambient temperature.
Absorbance was monitored at l ¼ 223, one of the absorption
wavelengths for ibuprofen. Amounts were calculated from a
calibration curve of known standard solutions.
2
.8. Ibuprofen L-Malic Acid Polymer (7) Synthesis
Ibuprofen-L-malic acid (5, 1 eq) and diol (6, 1 eq) were added to a
round-bottomed flask with N435 (10 wt% of total monomers, dried
at room temperature and 2 Torr for 24 h). Diphenyl ether (200 wt%
of total monomers) was added. Stirring (200 rpm) was initiated and
reaction was performed in three sequential steps: (i) reaction
stirred 80 8C for 4 h at atmospheric pressure under nitrogen, (ii)
reaction stirred at 80 8C under vacuum (2 Torr) for 24 h, and (iii)
reactionwasstirredat95 8Cundervacuum(2 Torr)foranadditional
2
.10. Structural Characterization of Released
Ibuprofen
Polymers (40 mgs) were placed in 20 mL Wheaton scintillation
vials, then PBS (10 mL) and 1 N NaOH (2 mL) added to the vials,
which were incubated in a controlled environment incubator-
shaker at 37 8C with 60 rpm agitation. After complete polymer
2
4 h. Upon completion, the reaction was brought to room temper-
ature, dissolved in chloroform (CHCl
3
), and gravity-filtered to
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Enzymatic Polymerization of an Ibuprofen-Containing Monomer
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degradation, solutions were acidified to pH 2 using concentrated
HCl and ibuprofen was extracted from the samples with DCM
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium) (MTS) reagent was added to each well
and further incubated for 4 h at 37 8C. The absorbance was then
recorded with a microplate reader (Coulter, Boulevard Brea, CA) at
(
4
10 mL, 3ꢄ). The organic layer was dried over MgSO , filtered, and
concentrated in vacuo.
492 nm.
2
.11. Cytocompatibility Studies
3
. Results and Discussion
In vitro cytocompatibility studies were performed by culturing 3T3
mouse fibroblasts in cell media (DMEM supplemented with 10%
FBS, 1% pen/strep) containing the three different polymers.
Polymers were first sterilized under UV at l ¼ 254 nm for 900 s
3
.1. Synthesis and Characterization
To prevent unwanted side reactions from occurring, the
carboxylic acid groups of L-malic acid were protected before
ibuprofen was coupled to the malic acid alcohol group.
Thus, the selective benzyl protection of malic acid
carboxylic acid groups was adapted from a previously
published procedure using benzyl bromide and sodium
(
Spectronics Corporation, Westbury, NY), dissolved in DMSO to
ꢃ1
yield 10 mg ꢂ mL solutions, and then diluted with cell media to
ꢃ1
reach concentrations of 0.01 and 0.001 mg ꢂ mL . Cell media
containing polymers were then added to allocated wells in a 96-
well plate with 2 000 cells/well and incubated at 37 8C. DMSO (1%)
in cell media was used as control. Cell viability was determined
using CellTiter 96 Aqueous One Solution Cell Proliferation
Assay. After 24, 48, and 72 h incubation with polymers, 20 mL of
[40]
carbonate.
The appearance of benzylic and aromatic
1
signals in the H NMR spectrum (Figure 1) and the
Figure 1. 1H NMR spectra of compounds 2, 4, 5, and 7b showing benzyl protection, drug coupling, deprotection, and subsequent
polymerization.
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N. D. Stebbins et al.
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ꢃ1
preservation of the FT-IR band (Figure 2) at 3 480 cm (O-H)
of 2 confirmed that successful reaction occurred at the acid
groups without affecting the secondary alcohol. Thereafter,
ibuprofen was coupled to the alcohol via a solvent-free
esterification using catalytic DMAP and pivalic anhydride
to afford 4 in high yield. The significant chemical shift of
malic acid backbone protons in the NMR spectra (Figure 1)
MeOH, as it is less toxic, less volatile, and less likely to
[43]
form peroxides.
Low volatility is preferable when
choosing solvents to minimize exposure to air and
[
34,43]
laboratory workers, especially in plant-scale labs.
Diacid 5 was obtained in high yield after filtration through
Celite and removal of solvent in vacuo. NMR spectrum
(Figure 1) indicated the disappearance of benzylic protons
while IR spectrum (Figure 2) displayed the preserved
of 4 along with the formation of an IR band (Figure 2) at 1
ꢃ1
ꢃ1
7
43 cm (ester, C¼O) and disappearance of the alcohol
pendant ester linkage at 1 743 cm and appearance of an
ꢃ1
ꢃ1
band at 3 480 cm indicated successful coupling of drug to
additional carbonyl band at 1 719 cm (–COOH). At each
2
. Subsequent deprotection via palladium-mediated hydro-
step, mass spectrometry was used to confirm product
13
genolysis selectively cleaved the benzyl esters while
leaving the pendant ester group intact. During this step,
CPME was used as a greener alternative to traditional
solvents such as tetrahydrofuran, dioxane, DCM, and
molecular weight, and C NMR spectroscopy was used to
confirm chemical structure. All polymer precursors were
viscous oils or foams that did not display melting
temperatures.
Figure 2. IR spectra of precursors 2 (red, upper), 4 (blue, mid-upper), and 5 (green, mid-lower), and polymer 7b (violet, lower) presented as
examples; key IR bands are labeled on each spectrum.
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Enzymatic Polymerization of an Ibuprofen-Containing Monomer
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Polyester synthesis was performed using a modified
Table 1. Summary of thermal properties, molecular weights, and
polydispersity indices of polymers 7a–c.
[2]
polymerization procedure of aliphatic diacids and diols.
The polyesterification of equimolar amounts of ibuprofen-
L-malic acid anddiol was catalyzed by N435. Thisparticular
enzyme is readily dispersed in the reaction mixture and
known to be activated by heat. In a prior study using a
different dicarboxylic acid and diol, the N435 enzyme
produced polymers of the same molecular weight and was
a)
b)
c)
c)
Polymer
T
g
[8C]
T
d
[8C]
M
w
[kDa]
PDI
7
7
7
a
b
c
–5
–9
212
215
221
4.8
6.4
8.1
1.4
1.4
1.3
–35
[
44]
stable at 70–90 8C.
Diphenyl ether, a hydrophobic, high-
a)
b)
Determined on second heating cycle of DSC; determined by TGA
boiling solvent that has been shown to be effective for such
reactions, was chosen as solvent. Upon exposure to the
initial temperature of 80 8C, the reaction formed a mono-
phasic mixture that remained for the reaction duration.
After 48 h of polymerization time, the reaction mixture
became solid and product was isolated after dissolution in
chloroform, filtration of lipase beads, and solvent removal
in vacuo. No further purification or precipitation steps was
used;however,duringenzymeremovalviafiltration, notall
product was dissolved after 10 min, thus 100% conversion
was not obtained. The lack of monomer present in the
reaction mixture was confirmed by NMR and IR spectros-
copies. Chloroform was chosen because it is the preferred
c)
as the onset of thermal decomposition; determined by GPC.
ꢃ1
carbonyl peak at 1 743 cm , indicative of an ester in
1
Figure2. HNMRspectraofpolymers7aand7ccanbefound
in Supporting Information (Figure S1). Polymer T
were low (<0 8C), and decreased with increasing aliphatic
diolchainlength;noT transitionswereexhibited(Table1).
valueswere>200 8C, confirmingthatthepolymershould
g
values
m
T
d
be relatively stable to temperatures at which these
polymers would be reacted or processed.
[45]
solvent to terminate the lipase reactions; despite the use
of this solvent in this one step, our overall synthetic
methods are greener than traditional methods; other
methods to achieve similar reactions utilize excess
3
.2. In Vitro Bioactive Release
In vitro degradation of the polymers was determined by
appearance of ibuprofen in degradation media samples in
PBS at physiological conditions (37 8C, pH 7.4). At prede-
termined times, an aliquot of release media was collected
and analyzed via HPLC. The degradation rate of polymer
into bioactive via ester bond hydrolysis (Scheme 2) is an
important factor in obtaining controlled ibuprofen release;
during degradation, the ultimate hydrolytic degradation
productswerethediol(6), L-malicacid(8), andibuprofen(3).
As an example, ibuprofen (3), ibuprofen-malic diacid (5),
and malic acid (8) all exhibited unique retention times at
5.70, 4.29, and 2.79 min, respectively (Figure 3B). Polymers
7a–c exhibited controlled, sustained ibuprofen release
throughout the 30 d of the study. Increased aliphatic chain
of the diol used decreased the drug release rate; after 30 d,
42, 58, and 82% of total ibuprofen was released from the
[
46]
[31]
carbodiimides,
chlorinated solvents,
and metal cata-
[
47–49]
lysts often used
in contrast to solvent-free, catalytic
reactions, safer solvents, and enzyme catalyst. GPC analysis
indicated that Mw values were moderate and PDI values
narrow (Table 1) substantiating that an additional purifi-
cation step was unnecessary. The longer chain length diol,
1
,8-octanediol, resulted in polymers with slightly higher
[2,3]
M
w
, as expected based on previously published results.
of these enzymatically synthesized
Notably, the M
w
polyesters are similar to other malic acid-containing
[
9]
[50]
polymers; due to malic acid’s high acidity,
polymers
1
rarely reach higher than 10 kDa. H NMR spectroscopy of
polymers 7a–c displayed all expected peaks; 7b is provided
as an example in Figure 1. IR spectra display the only
Scheme 2. Complete hydrolysis of polymer 7 into diol (6), L-malic acid (8), and ibuprofen (3).
Macromol. Biosci. 2015, 15, 1115–1124
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N. D. Stebbins et al.
www.mbs-journal.de
Figure 3. A. Cumulative release of ibuprofen as determined by HPLC data; B. HPLC chromatogram depicting the unique retention times of
L-malic acid, 8 (R 2.79 min), diacid, 5 (R 4.29 min), and ibuprofen, 3 (R 5.70 min).
t
t
t
1
octylene, pentylene, and propylene polymers, respectively
Figure 3A). The release rate correlates to the water
under vacuum and analyzed. The H NMR spectrum
(Figure S2) of extracted drug showed all peaks at the same
chemical shifts as free ibuprofen.
(
solubility of the diols; the less water-soluble, more hydro-
phobic 1,8-octanediol (log P ¼ 1.44) displays the slowest
release followed by intermediate 1,5-pentanediol (log
P ¼ 0.19), and 1,3-propanediol (log P ¼–0.68). After 30 d,
the polymers were completely hydrolyzed according to the
methods described above. Extracted ibuprofen was quan-
tified in all samples to ensure a mass balance of the drug in
the remaining polymer residue. Complete polymer degra-
dation and 100% ibuprofen release is expected in 8–15
months for all three polymers. Additionally, to ensure that
polymer processing did not affect the drug molecule
structure, the ibuprofen extracted from media was dried
3
.3. Cytocompatibility Studies
Based on the therapeutic plasma concentration of ibupro-
fen, two different concentrations of polymer were
[51,52]
tested.
All polymers were cytocompatible at 0.01
ꢃ1
and 0.001 mg ꢂ mL over 72 h, i.e., no significant difference
in cell viability was found between the polymer groups and
ꢃ1
the media alone control. The 0.01 mg ꢂ mL concentration
[
51]
is comparable to the commonly used ibuprofen dosing.
This data demonstrated that these ibuprofen-based
Figure 4. Cytocompatibility of polymers after 24, 48, and 72 h incubation. All groups contained 1% DMSO in cell media and control group
has no polymer. Absorbance at 490 nm after MTS treatment is proportional to cell viability. Data presented as mean ꢅ standard deviation.
n ¼ 6 in each group.
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Enzymatic Polymerization of an Ibuprofen-Containing Monomer
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polymers are cytocompatible at clinically relevant concen-
trations and, thus, promising candidates for biomedical
applications (Figure 4).
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metal-mediated polymerization, produced three polyesters
with varying aliphatic chain length with varying release
characteristics. All polymers released ibuprofen over 30 d
underphysiologicalconditions;thereleaseratecanbetuned
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