Biodegradable electroactive polymers for electrochemically-triggered drug delivery

Article abstract of DOI:10.1039/c4tb00355a

We report biodegradable electroactive polymer (EAP)-based materials and their application as drug delivery devices. Copolymers composed of oligoaniline-based electroactive blocks linked to either polyethylene glycol or polycaprolactone blocks via ester bo

Full text of DOI:10.1039/c4tb00355a

Journal of
Materials Chemistry B
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PAPER
Biodegradable electroactive polymers for
electrochemically-triggered drug delivery†
Cite this: J. Mater. Chem. B, 2014, 2,
6809
ab
*
John G. Hardy,
David J. Mouser,a Netzahualcoyotl Arroyo-Curras,c
´ ´
Sydney Geissler,ab Jacqueline K. Chow,a Lindsey Nguy,a Jong M. Kima
and Christine E. Schmidt
ab
*
We report biodegradable electroactive polymer (EAP)-based materials and their application as drug delivery
devices. Copolymers composed of oligoaniline-based electroactive blocks linked to either polyethylene
glycol or polycaprolactone blocks via ester bonds were synthesized in three steps from commercially
available starting materials and isolated without the need for column chromatography. The
physicochemical and electrochemical properties of the polymers were characterized with a variety of
techniques. The ability of the polymers to deliver the anti-inflammatory drug dexamethasone phosphate
on the application of electrochemical stimuli was studied spectroscopically. Films of the polymers were
shown to be degradable and cell adhesive in vitro. Such EAP-based materials have prospects for
integration in implantable fully biodegradable/bioerodible EAP-based drug delivery devices that are
capable of controlling the chronopharmacology of drugs for future clinical application.
Received 2nd March 2014
Accepted 19th August 2014
DOI: 10.1039/c4tb00355a
www.rsc.org/MaterialsB
Stimuli-responsive drug delivery systems are particularly
attractive for the treatment of such conditions because of their
Introduction
potential for the controlled delivery of precise quantities of
drugs at specic locations and times. Materials responding to
stimuli such as enzymes, light, pH, temperature, ultrasound
and electric/magnetic elds have been developed for use as
drug delivery devices.2,3,6,15–30 An exciting new research area is
the development of electroactive materials capable of control-
ling either cell behavior or the delivery of drugs.31–35 Here we
report the rst application of biodegradable EAP-based mate-
rials for electrochemically-triggered drug delivery.
Systems capable of precisely controlling levels of drugs in
specic tissues or the blood stream potentially allow mainte-
nance of the drug above its minimum effective level and below
the level at which its use results in unwanted side effects.
Furthermore, as our understanding of chronobiology has
deepened, we have become aware of its importance in a variety
of conditions including Alzheimer's disease, cancer, cardio-
vascular diseases, diabetes, epilepsy, pain, Parkinson's disease
and infectious diseases.1–3 Such conditions can be treated most
effectively by drugs with chronopharmacologies synchronized
with the chronobiology of the specic condition.2–6 Chronobi-
ological processes that are governed on a genetic level by clock
genes display rhythmic endogenous oscillations driven by the
circadian clock (i.e. circadian rhythms). Perturbations of circa-
dian rhythms are involved with a variety of conditions
(including cancers, depression, diabetes, and sleep disorders),
as discussed in detail in a selection of excellent reviews,1,7–11 and
such conditions are attractive targets for chrono-specic
treatments.12–14
EAPs are of scientic interest because of their potential for
application in the electronics36–38 and biomedical31–33,35,39–59
industries. Although there are
a
number of different
EAPs,36–39,56,60,61 those most commonly investigated for biomedical
applications are derivatives of polyaniline, polypyrrole, poly-
thiophene and poly(3,4-ethylenedioxythiophene).31–33,50,56,57,62 The
highly conjugated backbone of EAPs synthesized via conven-
tional means (e.g., electrochemical polymerization of the
constituent monomers at the surface of an electrode, or the
solution/solid state polymerization of the monomers in the
presence of a catalyst) is responsible for their high conductivity,
and the lack of enzymatically cleavable moieties (e.g., amides or
esters) renders them non-biodegradable. Clearly, non-biode-
gradable EAPs are best suited for devices that will be implanted
for long periods such as electrodes for the recording or stimu-
lation of neuronal activity,51,52 whereas, biodegradable EAPs are
ideal for devices implanted for comparatively short durations
such as drug delivery devices or tissue scaffolds.33,35,63–67 The
Miller group reported the rst conceptually biodegradable EAPs
aDepartment of Biomedical Engineering, The University of Texas at Austin, Austin, TX
78712, USA. E-mail: johnhardyuk@gmail.com
bJ. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida,
Biomedical Sciences Building JG-53, P.O. Box 116131, Gainesville, FL 32611-6131,
USA. E-mail: schmidt@bme.u.edu
cCenter for Electrochemistry, Chemistry Department, The University of Texas at Austin,
Austin, TX 78712, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4tb00355a
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in 1995.68 The multiblock copolymers were composed of elec-
troactive oligothiophenes and non-electroactive aliphatic spacers
connected via ester bonds; however, iodine (a toxic oxidizing
agent) was necessary to render them electroactive.68 Analogous
structures described in the literature incorporated electroactive
oligomers that were insoluble or aggregated in water.69 This
approach is not ideal for clinical applications as the insoluble/
aggregated oligomers are likely to remain in the body aer
degradation of the bulk material. Rivers and co-workers were the
rst to report the preparation of fully biodegradable electroactive
polyesters incorporating a water soluble electroactive oligomer
(pyrrole–thiophene–pyrrole) displaying carboxylic acids at their
termini,70 however, similar to earlier work, these polymers were
only electroactive aer oxidation with iodine. Guimard and co-
workers subsequently described analogous systems in which the
length of the electroactive oligomer was increased from 3 to 4
heterocycles that could be doped with biocompatible anions
(either chloride, Clꢀ, or perchlorate, ClO4ꢀ) and the resulting
polymers were suitable for the attachment and proliferation of
Schwann cells in vitro.71 The groups of Albertsson and Wei have
reported a variety of biodegradable electrically EAPs through
various combinations of short water soluble oligomers of aniline
and biodegradable polymers of natural or synthetic origins.35,72–84
Electrical stimulation of cells attached to the surfaces of cam-
phorsulfonic acid-doped (CSA, Fig. 1) materials was demon-
strated to enhance neurite extension from rat neuronal
pheochromocytoma (PC12) cells,84 and increase levels of early
markers of osteogenic differentiation of preosteoblastic MC3T3-
E1 cells.85 Researchers have begun to address the cytotoxic
degradation products of these polymers seen in vitro,86–88 via the
synthesis of oligoaniline analogues with reduced toxicities.86,87
The interested reader is directed towards the following recent
reviews for a more thorough discussion of the potential of such
materials for biomedical applications.32,33,35,50,51,55,56
Scheme 1 Synthesis of electroactive polyesters 1 and 2 via the Steglich
esterification. (a) DCC, DMAP, NMP, 72 h, r.t.
reviews.55,58 It is noteworthy that all of the previously described
systems use non-biodegradable EAPs as the stimulus-respon-
sive matrix from which to deliver the drug. Here we report the
rst biodegradable EAP-based drug delivery devices, employing
solution-processable EAPs that facilitate the preparation of
materials with high drug loadings (16 to 31 wt%).
Results and discussion
Our prototypical biodegradable electroactive polyesters are
multiblock copolymers composed of electrochemically respon-
sive blocks of oligoaniline terminated with carboxylic acid
moieties to ensure their water solubility, linked to alcohol-
terminated blocks of either FDA-approved polyethylene glycol
(PEG) or polycaprolactone (PCL) via ester bonds. The molecular
weight of each of the individual blocks is below 3 kDa, well
below the renal ltration threshold of 70 kDa.99–101
The Miller group was also the rst to report the application
of EAPs for drug delivery, demonstrating the ability to trigger
the release of glutamate anions from polypyrrole lms upon
reduction of the polypyrrole.89 Since this exciting report, a
variety of other biologically relevant anionic, cationic and
neutral drugs with either low/high molecular weights have been
shown to be deliverable using EAP-based materials (e.g., aden-
osine triphosphate,90 dexamethasone phosphate (DMP,
Fig. 1),91–93 DNA,94 dopamine,95,96 nerve growth factor,97 N-
methylphenothiazine98), as described in more detail in excellent
The carboxylic acid-terminated aniline pentamers (AP) were
obtained in analytically pure form using minor modications to
Fig. 1 Camphorsulfonic acid (CSA) and dexamethasone phosphate Scheme 2 Synthesis of electroactive polyesters 3 and 4 via the
(DMP).
Steglich esterification. (a) DCC, DMAP, NMP, 72 h, r.t.
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Table 1 Surface and physicochemical properties of the polyester films
Tg (ꢁC) Tc (ꢁC) Tm (ꢁC) Conductivity, s (S cmꢀ1
)
a
c
c
c
Ra (mm)
Rq (mm)
RZDIN (mm)
Xc
Glass substrate
Polyester 1 undoped
0.730 ꢂ 0.153
1.474 ꢂ 1.449
0.905 ꢂ 0.182 3.8303 ꢂ 2.04
4.312 ꢂ 3.684 12.099 ꢂ 11.493 0.0
0.0
N/Ab N/Ab
N/Ab
N/Ab
N/Ab
72.4
51.5
N/Ab,c
N/Ab,c
22.7
N/Ab,c
N/Ab,c
53.1
1.81 ꢃ 10ꢀ9 ꢂ 6%
3.38 ꢃ 10ꢀ8 ꢂ 1%
4.01 ꢃ 10ꢀ10 ꢂ 14%
8.34 ꢃ 10ꢀ8 ꢂ 2%
1.80 ꢃ 10ꢀ9 ꢂ 6%
6.14 ꢃ 10ꢀ9 ꢂ 3%
6.04 ꢃ 10ꢀ10 ꢂ 14%
2.20 ꢃ 10ꢀ8 ꢂ 2%
Polyester 1 doped with CSA 0.461 ꢂ 0.421
0.530 ꢂ 0.485
1.109 ꢂ 0.710
Polyester 2 undoped
Polyester 2 doped with CSA 0.449 ꢂ 0.061
Polyester 3 undoped
0.303 ꢂ 0.051
Polyester 3 doped with CSA 0.668 ꢂ 0.082
Polyester 4 undoped
11.49 ꢂ 1.26
12.926 ꢂ 1.089 44.196 ꢂ 3.155
49.8 N/Dd
0.572 ꢂ 0.099
0.479 ꢂ 0.182
1.111 ꢂ 0.178
0.315 ꢂ 0.218
0.727 ꢂ 0.147
2.717 ꢂ 0.523
1.669 ꢂ 0.535
2.649 ꢂ 0.874
0.747 ꢂ 0.542
1.674 ꢂ 0.173
13.4 N/Dd
28.4
53.4
0.0
0.0
0.0
0.0
50.5
37.6
41.8
39.7
N/Ab,c
N/Ab,c
N/Ab,c
N/Ab,c
N/Ab,c
N/Ab,c
N/Ab,c
N/Ab,c
0.273 ꢂ 0.191
Polyester 4 doped with CSA 0.598 ꢂ 0.097
a
d
As determined by XRD. b Not applicable. c Amorphous. Not determined (below 0 ꢁC).
the methodology described in the literature (Scheme S1†).102 differential scanning calorimetry (DSC) (Fig. S3† and Table 1).
Polyesters 1–4 were synthesized by adaptation of the literature.81 IR spectra of the CSA-doped lms (Fig. S4†) show peaks char-
In short, the carboxylic acids displayed at the termini of the acteristic of electroactive AP blocks, either the PEG or PCL
electroactive AP blocks were activated with dicyclohexyl- blocks respectively, and the esters interspersed between them.
carbodiimide (DCC) in the presence of a catalytic amount of 4- UV-visible spectra of thin lms of the polymers cast on quartz
dimethylaminopyridine (DMAP) and a stoichiometric quantity slides (Fig. 2) show peaks at ca. 330 nm corresponding to the
of alcohol-terminated PEGs (Scheme 1) or PCLs (Scheme 2), benzenoid p–p* transition in the AP blocks and are evident for
incorporating PEG-400, PEG-2000, PCL-530 or PCL-2000 to all samples; the peaks at ca. 442 nm evident in the CSA-doped
prepare polyesters 1, 2, 3 or 4 respectively. The syntheses and lms correspond to the polaron band; the broad absorption
purications of the polyesters are simple and scalable, aer ca. 866 nm conrms the presence of emeraldine salts of
requiring only three steps from commercially available starting the AP blocks, and the broad absorption aer ca. 1000 nm
materials and no column chromatography. The success of the corresponds to the “free carrier tail” reported for analogous
condensation polymerization was conrmed by the presence of electroactive block copolymers.88,105
the characteristic peaks for esters in the IR spectra (at ca. 1732
cmꢀ1) and 13C NMR spectra (at ca. 174 ppm), and thermogra-
vimetric analysis (TGA) conrmed that the diacid and diol
blocks were present in a 1 : 1 ratio (Fig. S1 and Table S1†).
However, poor solubility (Table S2†) meant that determination
of the molecular weight distributions of polyesters 1–4 via
standard techniques such as GPC/SEC, light scattering or
MALDI-TOF mass spectrometry was not possible.
We prepared CSA-doped lms of the polyesters to compare
their physicochemical properties with analogous oligoaniline-
based materials reported in the literature. Black lms of ca. 20
mm thickness (as determined by prolometry) were prepared by
casting solutions of the polymers and CSA in hexa-
uoroisopropanol (HFIP). Vacuum dried lms of polyesters 1, 3
and 4 were glossy, whereas those of polyester 2 were matte with
spherulites owing to the crystallization of the PEG blocks. The
wide angle XRD patterns for the lms composed of polyesters 1,
3 and 4 are very broad, characteristic of polymers that are
amorphous in the solid state (Fig. S2†). The wide angle XRD
patterns for the lms composed of polyester 2 contain peaks at
ꢁ
ꢁ
˚
2q ¼ 19.1 and 23.3 , corresponding to d-spacings of 4.6 A and
˚
3.8 A respectively, which are characteristic of crystalline PEG
chains (Fig. S2† and Table 1).103 In agreement with the litera-
ture, doping with bulky CSA anions reduced the crystallinity (Xc)
of lms of polyester 2 (Table 1).104 CSA doping also diminished
the thermal stability of the lms, as revealed by TGA (Fig. S1†)
and reductions in glass transition temperatures (Tg) of poly-
Fig. 2 UV-visible spectra of thin polyester films on quartz slides. (A)
Polyester 1 undoped, black line; polyester 1 doped with CSA, dashed
black line; polyester 2 undoped, grey line; polyester 2 doped with CSA,
esters 1, 3 and 4, and the melting and crystallization tempera- dashed grey line; (B) polyester 3 undoped, black line; polyester 3
doped with CSA, dashed black line; polyester 4 undoped, grey line;
polyester 4 doped with CSA, dashed grey line.
tures (Tm and Tc respectively) of polyester 2 as measured by
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Cyclic voltammograms of HCl-doped solutions of polyesters at v ¼ 50 mV sꢀ1 (Fig. S8–S11† for polyesters 1–4), concomitant
1–4 (Fig. S5†), presented redox processes at ca. 0.26 V (leucoe- with increasing absorbance of the PBS at the characteristic
meraldine to emeraldine 1 transition), ca. 0.42 V (emeraldine 1 absorbance maximum of DMP (242 nm) as observed via UV
to emeraldine 2 transition), and ca. 0.6 V (emeraldine 2 to spectroscopy. Pulsatile release from lms deposited on glassy
pernigraniline transition), in agreement with the literature76,78 carbon substrates was studied by chronoamperometry using 1
and depicted in Scheme S2.† Potentials are reported vs. a Ag/ minute of electrical stimulation followed by 14 minutes of rest
AgCl reference electrode calibrated with ferrocenemethanol (Fig. 3A) aer which the quantity of DMP in solution was
prior to every experiment (Fig. S6†).
quantied by UV spectroscopy. The medium was unchanged
The conductance of lms of polyesters 1–4 were measured in between cycles. The data are reported as cumulative release as a
accordance with protocol IPC-TM-650, number 2.5.17.2 of the percentage of the total mass of drug in the lm and compared to
Institute for Interconnecting and Packaging Electronic Circuits. DMP release from unstimulated lms. Voltammetry scans and
Undoped polyesters had low conductivities on the order of chronoamperometry pulses were always initiated at the open
10ꢀ10 or 10ꢀ9 S cmꢀ1. Doping with CSA moderately higher circuit potential of the system. Potential cycling was carried out
conductivities, typically by an order of magnitude to 10ꢀ9 or between 0.7 V and ꢀ0.5 V, rst sweeping in the positive direc-
10ꢀ8 S cmꢀ1 (Table 1), that are somewhat lower than analogous tion of the potential scale at 50 mV sꢀ1. Passive release of DMP
oligoaniline-based structures.35,72,75,77,78,88 Polyesters incorpo- from unstimulated lms was low over the course of the exper-
rating the low molecular weight diols (polyesters 1 and 3) had iment (1.5 hours), ca. 8% for polyester 1, ca. 3% for polyester 2
lower conductivities than the polyesters incorporating higher and ca. 2% for polyesters 3 and 4 (Fig. 3B). Over the period of 24
molecular weight diols (polyesters 2 and 4), probably because days this increased to ca. 83% for polyesters 1 and 2, ca. 48% for
the higher molecular weight diols in polyesters 2 and 4 phase polyester 3 and ca. 35% for polyester 4 (Fig. S12†). Passive
separate into PEG- or PCL-rich phases and AP-rich phases. release from the PEG-based polyester lms (i.e. polyesters 1 and
Other researchers have concentrated on developing biode- 2) was higher than from the PCL-based polyester lms (i.e.
gradable/bioerodible tissue scaffolds with conductivities polyesters 3 and 4) due to the more hydrophilic nature of the
mimicking bodily tissues (typically in the range of 10ꢀ4 S cmꢀ1 PEG backbones. In contrast, electrochemically-triggered release
or higher)106–108 to deliver biomimetic electric currents (in the mA of the drugs by potential cycling (Fig. 3B) resulted in the release
regime) to electrically stimulate cells.109–114 However, for our
envisioned application as implantable devices for electro-
chemically-triggered drug delivery, conductivities lower than
mammalian tissues are preferable, and allow triggered drug
release at specic points in time upon the application of an
electrical potential directly to the EAP, thereby facilitating
synchronized control of the chronopharmacology of the drug in
line with the chronobiology of the specic condition.
We studied the capability of the oligoaniline-based polymers
to deliver an anti-inammatory drug dexamethasone phosphate
(DMP, MW 490 Da) into phosphate buffered saline (PBS).
Dexamethasone was chosen because it is used in the clinic
(oen in multiple doses) and it is simple to monitor its release
via UV spectroscopy. Films composed of polyesters 1–4 and
DMP (of ca. 3 to 4 mg) were prepared on bioinert conductive and
non-conductive substrates (glassy carbon electrodes and glass,
respectively). DMP loadings were at a mole ratio of 1 : 1
DMP : AP (ca. 31 wt% for polyesters 1 and 3, and ca. 16 wt% for
polyesters 2 and 4). The experimental setups are depicted in
Fig. S7.† The setup depicted in Fig. S7A† (using lms deposited
on glassy carbon substrates) is akin to that used for electrodes
implanted for stimulation of the central nervous system in
which the tissue surrounding the implant is used as a counter
electrode.51,52,59,115,116 The setup depicted in Fig. S7B† (using
lms deposited on glass substrates) is a simple closed circuit
similar to those proposed to power some electroactive tissue
scaffolds.32–34
Fig. 3 Electrochemically-triggered drug delivery via potential cycling.
(A) Electrical stimulation paradigm: 6 cycles of 1 minute on, 14 minutes
off. (B) Release of DMP from films of 1–4 in PBS as determined by UV
spectroscopy. Electrochemically-triggered DMP release (solid bars) or
passive DMP release without electrical stimulation (checked bars).
Polyester 1, red. Polyester 2, blue. Polyester 3, green. Polyester 4,
Electrochemically-triggered release (i.e., de-doping) of DMP
from the lms was studied by voltammetry for lms deposited
on glassy carbon substrates. Evidence of the electrochemical de-
doping process was clear from the sequentially diminished
current densities during repetitive potential cycling of the lms purple.
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very slowly over a period of 10 days, and the presence of the
esterase moderately increased the rate at which this occurs
(Fig. S13†). We conclude that the lms would degrade slowly if
administered in vivo (over the period of several months) in line
with other PEG- and PCL-based materials.119,120
As noted above, non-toxic FDA-approved PEG and PCL
derivatives were chosen as the non-electroactive units for
inclusion in the biodegradable polyesters, and degradation of
the polyesters would lead to the release of low molecular weight
polyethylene glycols or caprolactone derivatives that are easily
cleared via the renal system. The toxicity of the electroactive
oligoaniline incorporated in the polymers was rst reported by
Wei and co-workers using the rat glial tumor C6 cell line, in a
study that showed the aniline pentamer displays little/no cyto-
toxicity below 20 mg mLꢀ1, 90% cell viability at 40 mg mLꢀ1
,
and approximately 50% cell viability at 100 mg mLꢀ1
.
121
Subsequent studies by Wei and co-workers showed the aniline
pentamer to be more toxic towards adenocarcinomic human
alveolar basal epithelial A549 cells, with little/no cytotoxicity
below 20 mg mLꢀ1, 90% cell viability at 40 mg mLꢀ1, and
approximately 50% cell viability at 100 mg mLꢀ1; furthermore,
shorter oligoanilines were less cytotoxic.86,87
Pertinently, a study of PCL-based polyesters incorporating
the aniline pentamer from Guo and Albertsson investigating the
toxicity of the water soluble extracts collected aer incubating
the polyesters in medium typically showed human keratinocyte
HaCaT cells to be ca. 90% viable using a variety of polymer
compositions and architectures.72 We adapted this method-
ology to investigate the toxicity of the water soluble extracts
collected aer incubating polyesters 1–4 in medium for 5 days,
using pristine medium as a positive control experiment and
Fig. 4 Electrochemically-triggered drug delivery via a potential step.
(A) Electrical stimulation paradigm: 3 cycles of 0.5 minutes on, 29.5
minutes off. (B) Release of DMP from films of 1–4 in PBS as determined
by UV spectroscopy. DMP release with electrical stimulation (solid
bars) or without electrical stimulation (checked bars). Polyester 1, red.
Polyester 2, blue. Polyester 3, green. Polyester 4, purple.
of ca. 20–65% of the drug during the initial cycle. Subsequent pristine medium containing 15 vol% of ethanol as a negative
cycles were observed to release 5–10% per cycle for the rst six control experiment. Aer exposure of human dermal broblast
cycles, aer which the majority of the drug had been released (HDF) cells to 15 vol% of ethanol we found them to be ca. 11%
from all of the lms.
viable (i.e. cell viability was adversely affected), as depicted in
We also studied the electrochemically-triggered release of Fig. 5. By comparison, HDFs incubated in either the pristine
DMP from lms deposited on glass substrates. A potential step media or the media containing any water soluble extracts from
of +0.6 V was applied to each lm for 30 seconds, followed by polyesters 1–4 began to proliferate (i.e. cell viability was not
29.5 minutes of rest (Fig. 4A) aer which the quantity of DMP in adversely affected).
solution was quantied by UV spectroscopy. The rst stimula-
tion released 5–25% of the drug from the lms (Fig. 4B), the rst
and second stimulation released 25–50%, and the third stim-
ulation released between 50 to 90% of the drugs from the lms,
all of which are clearly distinguishable from the passive release
proles (Fig. 4B and S12†).
Although it is impossible to accurately reproduce the
conditions that materials encounter when implanted in vivo in
the laboratory (particularly the complex tissue-specic distri-
bution of enzymes, or patient-specic immune response),
laboratory-based in vitro degradation studies are useful to
conrm the potential for materials to degrade upon exposure to
enzymes found in vivo. To demonstrate the susceptibility of the
polymers to enzymatic/hydrolytic degradation in vitro we incu-
bated lms of polyesters 1–4 in PBS in the absence or presence
of a high concentration of cholesterol esterase (4 units per mL)
which is an enzyme known to hydrolyze ester bonds in poly-
human dermal fibroblasts as determined by AlamarBlue® assay. Day
esters.71,117,118 We observed the mass of each lm to decrease (0) Black bars. Day (2) Grey bars. Day (4) White bars.
Fig. 5 Assessment of the cytocompatibility of the water soluble
extracts from polyesters 1–4 incubated in media for 5 days towards
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Fig. 7 Assessment of the cell viability of human dermal fibroblasts on
various surfaces after 8 days in culture as determined using a LIVE/
DEAD® Viability/Cytotoxicity Kit. (Black bar) Tissue-culture treated
Corning® Costar® tissue culture plates. (Grey bar) CSA-doped poly-
ester 2. (White bar) CSA-doped polyester 4.
Analogous CSA-doped materials have been shown to support
the adhesion of a variety of cells including C6 cells,122 kerati-
nocytes,72 MC3T3-E1 cells,85 osteoblasts,122 PC12 cells121 and
Schwann cells.122–124 We found that lms of polyesters 1 and 3
were prone to fracture as a consequence of exposure to dynamic
shear forces encountered during multiple medium changes at
37 ꢁC. Films of polyesters 2 and 4 were more robust and we
observed that both HDFs and human mesenchymal stem cells
(HMSCs) adhered to the surface of the lms with morphologies
analogous to those on commercially available conductive
indium tin oxide (ITO) slides (Fig. S14†). Longer term HDF
proliferation studies were complicated by poor adhesion of the
lms of polyesters 2 and 4 to either ITO slides or tissue-culture
treated Corning® Costar® tissue culture plates, and their
mechanical stability, yet HDFs were observed to adhere and
remain viable over 4 days (Fig. S15†). Aer 8 days in culture
HDFs had proliferated on their surfaces (Fig. 6) and a LIVE/
DEAD® Viability/Cytotoxicity Kit for mammalian cells demon-
strated that they were highly viable (Fig. 7).
Together, these results suggest that such copolymers repre-
sent valuable lead structures for the development of clinically
relevant electroactive biomaterials, with potential for in vivo
implantation for a variety of applications (e.g., drug eluting
coatings on completely biodegradable implants).
Conclusions
Electrically triggered drug release from polyesters 1–4 provides
a novel platform for drug delivery. The synthesis and purica-
tion of the polymers is simple and scalable, requiring only 3
steps from commercially available starting materials. The
polymers are solution processable, thus facilitating the prepa-
ration of materials with a high drug loading of 16 or 31 wt%.
Although we used the anti-inammatory DMP as a clinically
applied model drug (primarily because it is straightforward to
Fig. 6 Adhesion of human dermal fibroblasts on various surfaces after
8 days in culture. (A) Tissue-culture treated Corning® Costar® tissue
culture plates. (B) CSA-doped polyester 2. (C) CSA-doped polyester 4.
Live cells were stained green by calcein and dead cells were stained
red by ethidium using a LIVE/DEAD® Viability/Cytotoxicity Kit. Scale quantify its release using UV spectroscopy), it is noteworthy that
bars represent 100 mm.
a wide variety of other biologically-active molecules (such as
those for the treatment of pain) could be delivered using the
6814 | J. Mater. Chem. B, 2014, 2, 6809–6822
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Journal of Materials Chemistry B
stimulation paradigms described here. Furthermore, it would
be possible to modulate the release proles of these molecules
via simple modications of the electrical input, allowing the
release prole to be tailored to treat the condition in the most
therapeutically effective way. Both the experimental paradigms
described have potential to be used for the manufacture of
completely biodegradable drug delivery systems.125 The poten-
tial cycling-induced release paradigm could be powered wire-
lessly.117 The potential step-induced release paradigm could be
powered using a sacricial metal such as magnesium although
this may result in the build-up of hydrogen in vivo (which can be
diminished using zinc alloys).54,126–128 A safer option to power
the potential step-induced release paradigm would be the use of
a biodegradable battery, which is capable of providing poten-
tials of up to 0.6 V as used in this study.129 The other requisite
circuit components such as sensors, switches and wires could
be manufactured from biodegradable/bioerodible EAP-based
materials,130–133 all of which would be embedded within a non-
conductive biodegradable polymer capsule. Moreover, the
biodegradable and cytocompatible nature of the polyesters
suggests that analogous materials may be of use as tissue
scaffolds. Consequently, we foresee great potential for the
development of biodegradable EAP-based drug delivery devices
integrated into tissue scaffolds,33,35 and, moreover, the
exciting prospect of systems capable of controlling the chro-
Synthesis of carboxylic acid-terminated aniline pentamers
(APs). The synthesis of the carboxylic acid-terminated aniline
pentamers was adapted from the method of Wei and co-
workers.102 N-phenyl-1,4-phenylenediamine (9.2 g, 50 mmol)
and succinic anhydride (5.0 g, 50 mmol) were dissolved in
dichloromethane (300 mL) and stirred overnight at room
temperature. The product was isolated by ltration, washed
with diethyl ether until the diethyl ether was clear and colorless,
and dried under vacuum for 24 hours, yielding 12.0 g (42 mmol,
84% yield) of blue-grey solid (a succinic acid-capped aniline
dimer). This blue-grey solid (2.9 g, 10 mmol) and p-phenylene-
diamine (0.54 g, 5 mmol) were dissolved in dimethylformamide
ꢁ
(DMF, 15 mL) and the solution cooled to 0 C on ice. A cooled
solution of DMF (30 mL), water (25 mL) and concentrated
hydrochloric acid (5 mL) was added. A solution of ammonium
persulfate (2.28 g, 10 mmol) in aqueous hydrochloric acid
solution (50 mL, 1 M) was added slowly and the reaction
ꢁ
mixture stirred quickly for 1 hour at 0 C. Aer this time the
reaction mixture was added to water (300 mL) resulting in the
precipitation of a solid, which was isolated by ltration. The
product was reduced by stirring a suspension of the product in
aqueous ammonia (300 mL, 1 M) overnight, aer which the pH
was lowered to 2–3 by addition of aqueous hydrochloric acid (1
M), and the product isolated by ltration. The product was dried
under vacuum at 45 ꢁC for 48 hours. A solution of the crude
product (3.0 g) in DMF (15 mL) was slowly added to ethanol (150
mL) resulting in the precipitation of a solid material that was
isolated by ltration and dried under vacuum. The product was
puried by Soxhlet extraction with 1,2-dichloroethane followed
by THF. The succinic acid-capped aniline pentamer (AP)
(Scheme S1†) was dried under vacuum for 48 hours, aer which
1.8 g (2.7 mmol, 54% yield) was isolated in an analytically pure
form in accordance with the literature.102 1H NMR (400 MHz,
DMSO-d6) dH 9.80 (s, 2H, CONH), 7.46–7.44 (m, 4H, Ar-H), 7.36
(s, 2H, Ar-H), 7.23 (s, 2H, Ar-H), 7.18 (m, 4H, Ar-H), 7.11 (s, 2H,
Ar-H), 7.04–6.95 (m, 6H, Ar-H), 2.62–2.55 (m, 8H, CH2); 13C NMR
(100 MHz, DMSO-d6) dC 177.24 (COOH), 173.94 (CONH), 140.81
(Ar-C), 138.30 (Ar-C), 137.12 (Ar-C), 135.65 (Ar-C), 132.32 (Ar-C),
120.44 (Ar-C), 119.47 (Ar-C), 118.36 (Ar-C), 117.50 (Ar-C), 115.38
(Ar-C), 31.00 (CH2), 29.21 (CH2). ESI-MS (m/z) calculated for
nopharmacology of drugs in
fashion.10,30,134–136
a
clinically relevant
Experimental
Materials
Unless otherwise stated, all chemicals for synthesis and physi-
cochemical analysis were of ACS grade, purchased from Sigma-
Aldrich and used as received without further purication.
Reagents for cell culture were purchased from Invitrogen
(Carlsbad, CA) unless otherwise noted. Human dermal bro-
blasts (HDFs) and human mesenchymal stem cells (HMSCs)
were purchased from Lonza (Gaithersburg, MD). Tissue-culture
treated Corning® Costar® tissue culture plates were purchased
from Sigma Aldrich and pristine indium tin oxide (ITO) slides
were purchased from Ted Pella, Inc. (Reading, CA).
C
38H35N6O6 [M + H]+ requires 672.25; found, 672.25. IR (ATR)
nmax cmꢀ1 3293 (NH, amide), 1712 (C]O, acid).
General methodology for the synthesis of the electroactive
polyesters. The synthesis of the electroactive polyesters adapted
from the method of Wei and co-workers.81 In short, alcohol-
terminated poly(ethylene glycol)s or poly(caprolactone)s (3
mmol) were dissolved in N-methyl-2-pyrrolidone (NMP, 20 mL),
to which was added AP (2.0 g, 3.0 mmol), dicyclohex-
ylcarbodiimide (DCC, 1.9 g, 9.0 mmol) and 4-dimethylamino-
pyridine (DMAP, 0.2 g, 2.0 mmol). The reaction mixture was
stirred at room temperature under an inert atmosphere of
argon. Aer 72 hours the reaction mixture was ltered and
added drop-wise to diethyl ether (1.5 L) that was stirred to
assure the precipitation of a ne powder of the respective
polymer. The stir bar was removed and the product was allowed
to settle to the bottom of the container (typically 15–30
Synthetic/analytical methods
1H and 13C NMR spectra were recorded on a Varian Mercury 400
MHz NMR spectrometer, using residual solvent 1H peaks as
internal references for the 1H NMR spectra and the solvent 13
C
peaks as references for the 13C NMR spectra. The following
notation is used for the 1H NMR spectral splitting patterns:
singlet (s), doublet (d), multiplet (m), broad (br). Mass spectra
were recorded on an Agilent 6530 QTOF mass spectrometer in
electrospray ionization mode. Infrared spectroscopy was carried
out on a Thermo Scientic Nicolet 380 FT-IR Spectrometer
(Thermo Fisher Scientic Inc., USA). Spectra were recorded in
ATR mode at 21 ꢁC, with a 1 cmꢀ1 resolution and 16 scans
(corrected for background and atmosphere using OMNIC so-
ware provided with the spectrometer).
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minutes). The diethyl ether was removed via pipette suction, molecular weight of 2000 Da, and 1.0 g of 4 (Scheme 2) was
and the polymer-rich layer at the bottom of the container was isolated via this procedure, in a yield of 25% by mass. 1H NMR
concentrated with a rotary evaporator to yield a thick oil. The oil (400 MHz, DMSO-d6): dH ¼ 10.19 (s, CONH), 9.89 (s, CONH),
was dissolved/dispersed in chloroform (10 mL), aer which it 7.68 (br, NH), 7.53–7.45 (m, Ar-H), 7.35 (s, Ar-H), 7.22 (d, Ar-H),
was re-precipitated in diethyl ether, and this process of resus- 7.19–6.90 (m, Ar-H), 4.11 (m, CH2O, PCL), 3.75–3.55 (m, CH2O,
pension in chloroform followed by re-precipitation in diethyl diethyleneglycol spacer), 3.52–3.48 (m, CH2O, diethyleneglycol
ether was repeated two more times. The resulting polymers were spacer), 2.69–2.55 (m, CH2), 1.66 (m, CH2), 1.55 (m, CH2), 1.29
dried under high vacuum for 24 hours. The polymers could be (m, CH2); 13C NMR (100 MHz, DMSO-d6): solubility too low. IR
reduced to the leucoemeraldine state via brief exposure to (ATR) nmax cmꢀ1 3293 (NH, amide), 1732 (C]O, ester).
aqueous hydrazine for ca. 15 minutes (aer which no further
gas was observed to evolve) followed by dialysis against ultra-
pure water in a cellulose dialysis tube with a molecular weight
Film preparation and characterization
cutoff of ca. 3500 Da, and dried under high vacuum for 48
hours.
Film preparation. Films were prepared by casting solutions
of the polymers in hexauoroisopropanol (HFIP, typically 0.1 g
Synthesis of electroactive polyester 1. Polyester 1 was mLꢀ1) onto HFIP insoluble substrates, (e.g., microscope slides
synthesized using poly(ethylene glycol) with an average molec- with dimensions of 2.5 cm ꢃ 5 cm or glassy carbon electrodes
ular weight of 400 Da, and 1.6 g of 1 (Scheme 1) was isolated via with surface areas of 0.0314 cm2). The solvent was allowed to
1
this procedure, in a yield of 50% by mass. H NMR (400 MHz, evaporate in a fume hood, and the lms were subsequently
DMSO-d6): dH ¼ 10.20 (br s, CONH), 9.90 (br s, CONH), 7.68 (br, dried under vacuum for 48 hours at room temperature. The
NH), 7.53–7.45 (m, Ar-H), 7.36 (s, Ar-H), 7.24 (d, Ar-H), 7.19–6.90 polymers were doped by the addition of camphorsulfonic acid
(m, Ar-H), 3.75–3.55 (m, CH2O), 3.52–3.48 (m, CH2O), 2.62–2.55 (CSA) to the HFIP solution prior to casting on microscope slides.
(m, CH2); 13C NMR (100 MHz, DMSO-d6): dC ¼ 174.3 (CO2R), Unless otherwise stated CSA doping was at a mole ratio of 3 : 1
173.9 (CONH), 173.7 (CONH), 140.7 (Ar-C), 138.3 (Ar-C), 137.6 CSA : AP.72
(Ar-C), 135.5 (Ar-C), 134.3 (Ar-C), 126.4 (Ar-C), 125.1 (Ar-C),
Prolometry. Prolometry was carried out using a Veeco
120.44 (Ar-C), 119.2 (Ar-C), 118.8 (Ar-C), 117.5 (Ar-C), 115.4 (Ar- Dektak 6M Stylus Prolometer (Veeco Instruments Inc., NY)
C), 72.7 (CH2O), 70.2 (CH2O), 25.0 (CH2), 24.7 (CH2). IR (ATR) tted with a 12.5 mm stylus tip. The prolometer was isolated on
nmax cmꢀ1 3293 (NH, amide), 1732 (C]O, ester).
an air table to reduce ambient vibrations. The prolometer was
Synthesis of electroactive polyester 2. Polyester 2 was operated at 10 mg of stylus force, and used to record proles of
synthesized using alcohol-terminated poly(ethylene glycol)s distances of ca. 1 cm, recording data points every 555 nm. Data
with an average molecular weight of 2000 Da, and 4.25 g of 2 analysis was carried out with the soware provided by the
(Scheme 1) was isolated via this procedure, in a yield of 53% by manufacturer, which allowed the determination of the thick-
1
mass. H NMR (400 MHz, DMSO-d6): dH ¼ 10.20 (br s, CONH), ness and roughness of the lms. The surface roughness
9.90 (br s, CONH), 7.68 (br, NH), 7.53–7.45 (m, Ar-H), 7.36 (s, Ar- parameters are analyzed and reported in accordance with the
H), 7.24 (d, Ar-H), 7.19–6.90 (m, Ar-H), 3.75–3.55 (m, CH2O), ISO 25178 series. The average roughness (Ra) is the arithmetic
3.52–3.48 (m, CH2O), 2.62–2.55 (m, CH2); 13C NMR (100 MHz, average of the deviation from the mean line, and is the most
DMSO-d6): dC solubility too low. IR (ATR) nmax cmꢀ1 3293 (NH, used international parameter of roughness, and the root-mean-
amide), 1732 (C]O, ester).
square roughness (Rq) is based upon this. The average height
Synthesis of electroactive polyester 3. Polyester 3 was difference between the ve highest peaks and the ve lowest
synthesized using poly(caprolactone) diol with an average valleys (RzDIN) was determined in accordance with DIN 4768/1
¨
molecular weight of 530 Da, and 2.2 g of 3 (Scheme 2) was as specied by the Deutsches Institut fur Normung. Doping the
isolated via this procedure, in a yield of 62% by mass. 1H NMR lms with CSA altered the surface roughness in all cases
(400 MHz, DMSO-d6) dH 10.19 (s, CONH), 9.89 (s, CONH), 7.68 because protonation of the oligoaniline blocks by the CSA
(br, NH), 7.53–7.45 (m, Ar-H), 7.35 (s, Ar-H), 7.22 (d, Ar-H), 7.19– caused rearrangement of the polymers on the nanoscale.104
6.90 (m, Ar-H), 4.11 (m, CH2O, PCL), 3.75–3.55 (m, CH2O,
Conductivity determination. The conductance of lms of 1–4
diethyleneglycol spacer), 3.52–3.48 (m, CH2O, diethyleneglycol were measured in accordance with protocol IPC-TM-650,
spacer), 2.69–2.55 (m, CH2), 1.66 (m, CH2), 1.55 (m, CH2), 1.29 number 2.5.17.2 described by the Institute for Interconnecting
(m, CH2); 13C NMR (100 MHz, DMSO-d6) dC 174.4 (CO2R), 174.2 and Packaging Electronic Circuits. Films supported on glass
(CO2R), 173.2 (CONH), 140.7 (Ar-C), 138.3 (Ar-C), 137.6 (Ar-C), slides were examined by chronoamperometry using a CHI900C
135.5 (Ar-C), 134.3 (Ar-C), 126.4 (Ar-C), 125.1 (Ar-C), 120.44 (Ar- electrochemical workstation (CHI instruments, Austin, TX).
C), 119.2 (Ar-C), 118.8 (Ar-C), 117.5 (Ar-C), 115.4 (Ar-C), 70.2 Chronoamperometric measurements were made with a two-
(CH2O, diethyleneglycol spacer), 68.7 (CH2O, diethyleneglycol point probe system (copper alligator clips), by connecting
spacer), 63.9 (CH2O, PCL), 33.8 (CH2), 32.6 (CH2), 32.3 (CH2), counter and reference electrodes together. Briey, two thin
31.4 (CH2), 30.6 (CH2), 29.4 (CH2), 28.2 (CH2), 25.3 (CH2), 25.0 strips of adhesive-backed copper tape (Ted Pella, Inc., Redding,
(CH2), 24.7 (CH2). IR (ATR) nmax cmꢀ1 3293 (NH, amide), 1732 CA) were attached to the lms, parallel to one another, sepa-
(C]O, ester).
rated by a distance of 0.5 cm. The working and counter elec-
Synthesis of electroactive polyester 4. Polyester 4 was trodes were clipped on the strips of copper tape, and the current
synthesized using poly(caprolactone) diol with an average measured for 50 seconds during a potential step experiment at
6816 | J. Mater. Chem. B, 2014, 2, 6809–6822
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Journal of Materials Chemistry B
10 V. The electrodes were moved to different positions aer phosphate buffered saline (PBS, 50 mL, pH 7.0) that was
each measurement, and the current passed was recorded in at prepared by bubbling nitrogen through it for 10 minutes. The
least ve different positions. The resistance (R, U) of the lms mixture was stirred for 5 minutes under nitrogen prior to
was determined in accordance with eqn (1):
ltration to remove the stir bar and any insoluble polymer
before the electrochemical measurements were made. A three-
electrode system consisting of two Pt meshes as working and
counter electrodes, and an Ag/AgCl reference electrode. Unless
otherwise stated, the scan rate was 20 mV sꢀ1. Solutions of the
polyesters were metastable, and there was evidence of aggre-
gation of the polymers within the duration of the experiment.
Those polymers incorporating polyethylene glycol chains
between the oligoanilines (1 and 2) were somewhat less prone to
aggregation than those incorporating polycaprolactone chains
between the oligoanilines (3 and 4).
R ¼ V/I
(1)
The resistivity (U cmꢀ1) of the lms was determined in
accordance with eqn (2):
r ¼ Rwt/L
(2)
In which: w corresponds to the width of the lm in cm (2.5
cm); t corresponds to the thickness of the lm in cm (as
determined via prolometry); and L corresponds to the length
of the lm in cm (0.5 cm). The conductivity (S cmꢀ1) of the lms
was determined in accordance with eqn (3):
X-ray diffraction (XRD). XRD data was collected on a Rigaku
R-Axis Spider diffractometer with an image plate detector using
˚
a graphite monochromator with CuKa radiation (l ¼ 1.5418 A)
at room temperature. The instrument was controlled using
Rapid/XRD diffractometer control soware (Rapid/XRD Version
2.3.8., Rigaku Americas Corporation, The Woodlands, TX). The
s ¼ 1/r
(3)
Differential scanning calorimetry (DSC). DSC experiments integration of the two dimensional data into a one dimensional
were carried out with a DSC Q100 (TA Instruments, USA), using pattern was accomplished using 2DP (2DP Version 1.0., Rigaku
airtight aluminum pans. Films were precisely weighed into Americas Corporation, The Woodlands, TX). Percentage crys-
aluminum pans (TA Instruments, USA), and analyses were tallinities were determined by taking the ratio of the crystalline
carried out under a nitrogen atmosphere (ow rate of 50 mL area to the amorphous area in the XRD spectrum. Areas were
minꢀ1). The samples were treated as follows: heated from room determined by prole tting of the crystalline peaks and
temperature to 200 ꢁC (10 ꢁC minꢀ1), cooled to 0 ꢁC (10 ꢁC amorphous humps. A constant linear background was used in
minꢀ1), heated from 0 ꢁC to 200 ꢁC (5 ꢁC minꢀ1), cooled to 0 ꢁC the prole tting to eliminate potential variations in area
ꢀ1
(5 C min ), heated from 0 C to 200 C (10 ꢁC minꢀ1), and determination caused by subjective selection of shape and level.
ꢁ
ꢁ
ꢁ
nally cooled to 0 C (10 C minꢀ1).
All XRD pattern analysis was performed using JADE soware
ꢁ
ꢁ
Thermogravimetric analysis (TGA). TGA was conducted on a (MDI, v 8.1).
TA Instruments TGA Q500 thermogravimetric analyzer (TA
Instruments, USA), using a ramp rate of (10 ꢁC minꢀ1) under
In vitro drug delivery, degradation and cell culture studies
nitrogen gas. Weight loss from the polymers occurring below
ꢁ
200 C was ascribed to the evaporation of solvents, and above
Preparation of drug doped lms. Films of ca. 3–4 mg were
ꢁ
200 C it was ascribed to the decomposition of the polymers. prepared by casting solutions of 1–4 and DMP (at a mole ratio of
The polyesters synthesized via the condensation of AP with low 1 : 1 DMP : AP) in hexauoroisopropanol (typically 0.1 g mLꢀ1
)
molecular weight diols, polymers 1 (PEG of ca. 400 Da) and 3 onto a glassy carbon electrode (0.0314 cm2, CH Instruments,
(PCL of ca. 530 Da) respectively, were observed to decompose in Inc.) or a glass slide. The solvent was allowed to evaporate in a
two steps, rst the diol blocks decomposed between 200–300 ꢁC, fume hood, and the lms were subsequently dried under
ꢁ
followed by the decomposition of AP above this temperature. By vacuum for 48 hours at 60 C.
comparison, those polyesters synthesized via the condensation
Drug delivery studies. Voltammetry experiments were
of AP with higher molecular weight diols, polymers 2 (PEG of ca. carried out using a CHI6273C electrochemical analyzer (CH
2000 Da) and 4 (PCL of ca. 2000 Da) respectively, displayed Instruments, Inc.). Phosphate buffered saline (PBS, pH 7.0) was
somewhat greater thermal stabilities, and the diol blocks deoxygenated for 10 minutes with argon before the electro-
ꢁ
decomposed between 200–410 C, followed by the decomposi- chemical measurements were made.
tion of the aniline pentamer above this temperature.78
Electrochemically-triggered release (i.e., de-doping) of DMP
UV-visible absorption spectra of lms. UV-visible absorption from the lms deposited on glassy carbon substrates by
spectra of very thin lms supported on quartz microscope slides potential cycling, was achieved using a three-electrode system
(with dimensions of 2.5 cm ꢃ 1 cm, supplied by Ted Pella, Inc.) consisting of one polymer lm-coated glassy carbon working
were recorded at room temperature on a Beckman Coulter electrode, a Pt mesh counter electrode, and an Ag/AgCl refer-
DU720 general purpose UV-visible spectrophotometer (Beck- ence electrode in 4 mL PBS. Prior to each experiment there was a
man Coulter Inc., CA).
10 s “quiet time”, the initial potential was 0 V, the high potential
Voltammetry. Voltammetry experiments were carried out was 0.7 V, the low potential was ꢀ0.5 V, the initial scan was
using a CHI6273C electrochemical analyzer (CH Instruments, positive, the current was measured at intervals of 0.001 V, the
Inc.). Polymers (5 mg) were dissolved in DMSO (0.5 mL), doped scan rate used in all experiments was 50 mV sꢀ1, and this
with hydrochloric acid (HCl, 50 mL, 1 M), added to deoxygenated stimulation lasted 62 seconds. The lms were allowed to rest for
This journal is © The Royal Society of Chemistry 2014
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14 minutes aer which the quantity of DMP in solution was at 37 ꢁC in 2 mL of HDF/HMSC growth medium was composed
quantied by UV spectroscopy. The medium was unchanged of: high glucose Dulbecco's Modied Eagle Medium (DMEM,
between cycles, and the data are reported as cumulative release 440 mL); fetal bovine serum (50 mL); antibiotic–antimycotic
as a percentage of the total mass of drug in the lm over the (5 mL); non-essential amino acids (5 mL), and 2 ng mLꢀ1 basic
period of the experiment. These data are compared to passive broblast growth factor. The media was ltered using a sterile
DMP release from unstimulated lms measured every 15 Millipore 220 nm syringe lter and used for subsequent cell
minutes. For experiments using multiple scans, a scan from 0.7 culture experiments as follows.
V to ꢀ0.5 V and back to 0.7 V was regarded as one additional
Commercially available tissue-culture treated Corning®
Costar® 96 well tissue culture plates were used as substrates
cycle and each cycle lasted 48 seconds (see Fig. S7–S10†).
Electrochemically-triggered release (i.e., de-doping) of DMP upon which HDF cells were seeded at 10 000 cells per cm2 under
from lms deposited on glass substrates using a potential step 100 mL of medium (one of the following: medium that had
was achieved by connecting two thin strips of adhesive-backed previously been incubated with the polymers; pristine medium
copper tape (Ted Pella, Inc.) were attached to the lms, parallel as a positive control experiment; or pristine medium containing
to one another, separated by a distance of 1.1 cm. The counter 15 vol% ethanol as a negative control experiment), and incu-
ꢁ
and reference electrodes were connected together and clipped bated at 37 C, 95% humidity, and a CO2 content of 5%.
to copper tape on one side of the lm, and the working electrode
Aer 2 days the cells were washed gently with PBS, followed
was clipped to copper tape on the other side of the lm. A by the addition of pristine medium containing 10% v/v of the
polycarbonate well with a square hole (sides of 0.9 cm) was AlamarBlue® reagent. Aer 2.5 hours of culture, the medium
attached to the lm and copper tape using vacuum grease and was aspirated and replaced with medium (either medium that
binder clips to assure a rm seal, with PBS (0.5 mL) added to the had previously been incubated with the polymers, or pristine
well. A potential step of +0.6 V was applied to each lm for 30 medium for control experiments). The aspirated medium con-
seconds, followed by 29.5 minutes of rest aer which the taining the AlamarBlue® reagent was placed in another 96 well
concentration of DMP in solution was quantied by UV plate, and the uorescence was measured with a uorimeter
spectroscopy.
(Synergy HT Multi-Mode Microplate Reader, Biotek US,
DMP release was quantied by UV spectroscopy using a Winooski, VT). Two controls were considered during the
BioTek Epoch® plate reader (BioTek US, Winooski, VT) equip- measurement of the uorescence: the rst was wells containing
ped with a Take3 Micro-volume Plate and Gen5 v2.04 Soware medium alone (i.e. no cells or AlamarBlue® reagent), which was
supplied with the plate reader. Two samples of 2 mL were not uorescent; and the second was wells that contained the
removed from the release medium at specic time points and AlamarBlue® reagent but no cells (used for baseline correction).
absorbance readings were carried out at 242 nm (the charac- Numbers of cell adhered to the various surfaces studied herein
teristic absorbance band of DMP). Absorptions were corrected are reported relative to their initial seeding density of 10 000
by subtracting the reading of PBS alone from each sample. A cells per cm2, which was assigned an arbitrary value of 100%.
standard calibration curve for DMP was plotted to dene the Aer another 2 days (i.e. at 4 days aer initial seeding) the
quantitative relationship between the observed absorbance and AlamarBlue® assay was repeated. Results presented are the
the concentration of DMP. Prior to the experiment, the mass of average of eight samples.
the drug-doped polymer lm on the substrate was determined
Cell adhesion studies. Commercially available conductive
by subtracting the mass of the substrate from the mass of the ITO slides were used for control experiments. Clean ITO slides
polymer-coated substrate. Data are plotted as % DMP release and ITO slides coated with polyester lms doped with CSA (at a
relative to the quantity of DMP theoretically in the lm at the mole ratio of 1 : 1 CSA : AP) were inserted in tissue culture
beginning of the experiment, and all data are the average of at plates and sterilized by incubation in 70% ethanol solution,
least three samples.
followed by exposure to UV for 30 minutes. Aer sterilization,
Degradation studies. Films (of ca. 4 mg) were incubated in the slides (coated and uncoated) were incubated for 30 minutes
ꢁ
PBS (1 mL) at 25 C, in the absence or presence of cholesterol under 3 mm of medium. HDF/HMSC growth medium was
esterase (4 units per mL, Sigma Aldrich, USA). At specic time composed of: high glucose Dulbecco's Modied Eagle Medium
points the buffer was removed, the lms were carefully washed (DMEM, 440 mL); fetal bovine serum (50 mL); antibiotic–anti-
with deionized water, and the lms were then dried under high mycotic (5 mL); non-essential amino acids (5 mL), and 2 ng
vacuum for 22 hours, aer which the mass of the lm was mLꢀ1 basic broblast growth factor. Medium was aspirated and
determined on a high precision balance. The buffer (with or replaced prior to HDF or HMSC seeding. Cell viability before
without enzymes) was replaced, and the mass of the lm was starting the experiment was determined by the Trypan Blue
recorded over a period of several days. Films were undoped to (Sigma, USA) exclusion method, and the measured viability
help differentiate degradation from dopant leaching. The mass exceeded 95% in all cases. Cells were seeded at 10 000 cells per
loss proles are the average of at least three samples.
cm2 under 3 mm of medium, and incubated at 37 ꢁC, 95%
Studies of the cytotoxicity of the degradation products of the humidity, and a CO2 content of 5%. Aer 2 days the medium
polymers. The polymers (200 mg) were sterilized by incubation was aspirated and the lms were washed gently with PBS. Cells
in 70% ethanol solution, followed by exposure to UV for 30 were xed with 4% paraformaldehyde in PBS for 15 min, per-
minutes and allowed to dry in a sterile tissue culture hood meabilized with 0.1% Triton X-100 (Fluka) and 2% bovine
overnight. The polymers were subsequently incubated for 5 days serum albumin (BSA) in PBS buffer for 5 min, followed by
6818 | J. Mater. Chem. B, 2014, 2, 6809–6822
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Journal of Materials Chemistry B
blocking with 2% BSA in PBS buffer for 30 min at room and becomes integrated into the DNA strands. Fluorescence
temperature. Actin laments and cell nuclei within cells were images of cells were captured using a color CCD camera
stained with Alexa Fluor 488® Phalloidin (Life Technologies, (Optronics® MagnaFire, Goleta, CA, USA) attached to a uo-
USA) for 30 min and 40,6-diamidino-2-phenylindole (DAPI, rescence microscope (IX-70; Olympus America Inc.). Cells were
Invitrogen, USA) for 5 min. The cells were then washed three counted with the cell counter tool (plugin) in the open source
times with PBS buffer and stored at 4 ꢁC until images were program ImageJ, all cells on all images were counted. Results of
acquired. Fluorescence images of cells were captured using a AlamarBlue® assays presented are the average of four samples
colour CCD camera (Optronics® MagnaFire, Goleta, CA, USA) and ethidium/calcein stained images are representative of 3
attached to a uorescence microscope (IX-70; Olympus America samples.
Inc.). Images are representative of 3 samples.
Cell proliferation studies. The cell proliferation studies were
carried out in accordance with previously reported method-
Acknowledgements
ology137,138 employing the AlamarBlue® assay. Commercially We thank Andrea P. Carranza, YoungIn Jun, Min Joo Sohn and
available tissue-culture treated Corning® Costar® tissue culture Pei-Yun Tseng for early work related to the synthesis of well-
plates were used for control experiments. Polyester lms doped dened oligomers of aniline, and David Aguilar Jr for assistance
with CSA (at a mole ratio of 1 : 1 CSA : AP) were cast in tissue with early work related to the drug delivery studies. We thank
culture plates, dried under high vacuum for 48 hours at room the University of Texas at Austin for nancial support of Jong
temperature and sterilized by incubation in 70% ethanol solu- Min Kim, David James Mouser and Min Joo Sohn in the form of
tion, followed by exposure to UV for 30 minutes. Aer sterili- Undergraduate Research Fellowships. At the Department of
zation, lms were incubated for 30 minutes under 3 mm of Chemistry at the University of Texas at Austin we thank Prof.
medium. HDF/HMSC growth medium was composed of: high Christopher W. Bielawski for the loan of quartz cuvettes for
glucose Dulbecco's Modied Eagle Medium (DMEM, 440 mL); dynamic light scattering experiments, Dr Karin M. Keller and Dr
fetal bovine serum (50 mL); antibiotic–antimycotic (5 mL); non- Ian Riddington for electrospray ionization mass spectra, Prof.
essential amino acids (5 mL), and 2 ng mLꢀ1 basic broblast Michael J. Krische for access to an IR spectrometer, Prof. C.
growth factor. Medium was aspirated and replaced prior to HDF Grant Willson for access to DSC and TGA apparatus, and Dr
seeding. Cell viability before starting the experiment was Vincent M. Lynch for assistance with X-ray diffraction data
determined by the Trypan Blue (Sigma, USA) exclusion method, collection. At the Department of Chemical Engineering at the
and the measured viability exceeded 95% in all cases. Cells were University of Texas at Austin we thank Chase Cornellison for
seeded at 10 000 cells per cm2 under 3 mm of medium, and assistance with image processing. We thank the National
ꢁ
incubated at 37 C, 95% humidity, and a CO2 content of 5%.
Science Foundation for Grant no. 0741973 that was used to
Aer 2 days the cells were washed gently with PBS, followed purchase the Rigaku R-Axis Spider diffractometer. We thank Dr
by the addition of fresh medium containing 10% v/v of the Yohannes H. Rezenom of the Laboratory for Biological Mass
AlamarBlue® reagent. Aer 2.5 hours of culture, the medium Spectrometry in the Department of Chemistry at Texas A&M
was aspirated and replaced with fresh medium, and 100 mL of University in College Station, TX, for MALDI-TOF mass spectra.
the aspirated medium containing the AlamarBlue® reagent was We thank the University of Florida for nancial support in the
placed in a 96 well plate, and the uorescence was measured form of startup resources.
with a uorimeter (Synergy HT Multi-Mode Microplate Reader,
Biotek US, Winooski, VT). Two controls were considered during
the measurement of the uorescence: the rst was wells con-
Notes and references
taining medium alone (i.e. no cells or AlamarBlue® reagent),
which was not uorescent; and the second was wells that con-
tained the AlamarBlue® reagent but no cells (used for baseline
correction). Numbers of cell adhered to the various surfaces
studied herein are reported relative to their initial seeding
density of 10 000 cells per cm2, which was assigned an arbitrary
value of 100%. Aer another 2 days (i.e. at 4 days aer initial
seeding) this process was repeated. The medium was aspirated
and replaced once more at 6 days aer initial seeding, and
nally aer a total of 8 days in culture the viability of the cells
was evaluated using a LIVE/DEAD® Viability/Cytotoxicity Kit for
mammalian cells (Molecular Probes, Eugene, OR). The medium
was removed and cells on the surfaces were incubated with 4
mM ethidium and 2 mM calcein AM in PBS for 15 min at 37 ꢁC in
the dark. Live cells were stained green because of the cyto-
plasmic esterase activity, which results in reduction of calcein
AM into uorescent calcein, and dead cells were stained red by
ethidium, which enters the cells via damaged cell membranes
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