Journal of Biomaterials and Nanobiotechnology, 2011, 2, 18-27
doi:10.4236/jbnb.2011.21003 Published Online January 2011 (http://www.SciRP.org/journal/jbnb)
Simultaneous Release of a Hydroxy-Methylglutaryl
Coenzyme A Reductase Inhibitor and a
Glycoprotein IIb/IIIa Antagonist from a
Thermoresponsive NiPAAm/NtBAAm Copolymer
System
J. A. Hickey¹, I. Lynch², K. A. Dawson², D. Cox³, A. K. Keenan¹
¹UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland; ²Irish Centre for Col-
loid Science and Biomaterials, UCD School of Chemistry and Chemical Biology, University College Dublin, Ireland; ³Molecular and
Cellular Therapeutics, R.C.S.I., Dublin, Ireland
Received September 13th, 2010; revised September 25th, 2010; accepted September 30th, 2010
ABSTRACT
While deployment of intracoronary stents has been shown to reduce restenosis, stenting can also damage the endothe-
lial monolayer lining the vessel wall, leading to possible in-stent thrombosis. Local drug delivery from stent surfaces
represents a means of delivering therapeutic doses of drug directly to the target site. The aim of this study was to elute
fluvastatin, which can inh ibit vascular smooth muscle cell proliferation, and xemilofib an, which p revents p latelet adhe-
sion and aggregation, together in bioactive concentrations from the same copolymer system. Combined elution from
thermoresponsive N-isopropylacrylamide (NiPAAm)/N-tert-butylacrylami de (NtBAAm)-derived copolymer systems was
achieved using microgels (NiPAAm/NtBAAm 65/35 wt/wt) randomly dispersed in 85/15 matrices. Fluvastatin elution
from 5  m films over a 14-day period showed initial burst release, which leveled off. Of the total incorporated (8.33 ±
0.21 nmol, n = 4), 68.5% was eluted during this period. Xemilofiban relea se was measured in terms of its ability to in-
hibit platelet adhesion, using a microfluidic system. To investigate the influence of location and hydrophobicity on elu-
tion of bioactivity, three separate systems were employed. While elution of anti-adhesive activity from the system con-
taining xemilofiban-loaded matrices was more dramatic in the short term, a more sustained level of inhibition was
achieved when xemilofiban had been incorporated into microgels. All samples investigated for anti-adhesive activity
also decreased human coronary artery smooth muscle cell proliferation. Therefore xemilofiban has potential as an
agent for preventing in-stent thrombos is. Our study has demonstrated the feasibility o f using this novel matrix/microg el
system to regulate simultaneous release of both agents in bioactive concentrations.
Keywords: Fluvastatin, Xemilofiban, Nipaam/Ntbaam, Microgels, Local Drug Delivery
1. Introduction
The use of intravascular coronary stents during angio-
plasty procedures was first reported by Ulrich Sigwart in
1986 [1]. With the utilization of a stent there is the possi-
bility of increased neointimal hyperplasia, as the metallic
stent can cause physical injury to the endothelium, which
can result in in-stent restenosis (ISR) and in-stent
thrombosis (IST). Those higher risk patients with com-
plex lesions (small vessel, bifurcation lesions) have an
in-stent restenosis (ISR) occurrence rate of 30-60%,
while in those with less complex lesions the rate is
reduced to 15-20% of patients [2]. IST has an occurrence
rate of ≈ 1% [3]. Drug-eluting stents (DES) have had a
major influence on the reduction of in-stent restenosis.
To prevent restenosis, a clear understanding of events
responsible for growth factor- or cytokine-mediated vas-
cular smooth muscle cell (VSMC) proliferation and mi-
gration (intimal hyperplasia) has led to a precise selec-
tion of potential therapeutic candidates [4]. However, the
current leading agents, sirolimus and paclitaxel, can in-
hibit endothelial cell as well as VSMC proliferation,

Simultaneous Release of a Hydroxy-Methylglutaryl Coenzyme A Reductase Inhibitor and a Glycoprotein IIb/IIIa
Antagonist from a Thermoresponsive NiPAAm/NtBAAm Copolymer System
19
thereby affecting the re-endothelialisation process which
is central to vessel recovery. HMG CoA reductase in-
hibitors (statins) could be an option for the inhibition of
neointimal hyperplasia, since these agents inhibit VSMC
proliferation by a mechanism independent of cholesterol
synthesis [5]. In an attempt to prevent the problem of IST,
patients are placed on a strict regime of anti-platelet
agents. However despite these adjunct therapies, stent
thrombosis still occurs in 1% of patients [6]. The residual
occurrence of IST may be caused by lack of penetration
of such agents in adequate concentrations to the desired
site of action following oral or systemic administration.
Local delivery of agents designed to inhibit VSMC pro-
liferation and platelet aggregation via a copolymer-
coated stent platform is an attractive strategy for offset-
ting the incidence of ISR and IST.
Poly (n-isopropylacrylamide) (NiPAAm) is a polymer
that can become inversely soluble upon heating. This
change in state occurs at what is known as the lower
critical solution temperature (LCST) [7]. Below the
LCST the polymer chains are extended, separated and
surrounded by water, but above the LCST the polymer
becomes insoluble and precipitates out of solution [8].
The temperature range at which the soluble-insoluble
shift occurs in relation to NiPAAm is between 31-34˚C.
The addition of N-tert-butyl-acrylamide (NtBAAm) al-
ters the relative hydrophobicity/hydrophilicity of NiPA-
Am thereby giving a means for regulating the elution of
drugs for local delivery on the basis of their polarity. In
this study, a range of copolymers exhibiting increasing
hydrophobicities were synthesised in varying (w/w) ra-
tios of NiPAAm/NtBAAm (85/15, 65/35 and 50/50).
The potential of NiPAAm/NtBAAm-derived copoly-
mers to act as drug delivery vehicles for small molecules
like colchicine [9-10] and therapeutic proteins like vas-
cular endothelial growth factor (VEGF) [11] has been
previously investigated. Another approach is to cross-
link NiPAAm/NtBAAm copolymer microgel particles
and disperse these through a bulk copolymer matrix. At
their LCST the network volume changes from an ex-
panded water-containing network, to a collapsed network
where the water has been expelled. As the temperature
increases the particles shrink and become hard and
sphere shaped. [12] These microgels are then dispersed
through the non cross-linked NiPAAm/NtBAAm matri-
ces and can be used for drug loading and elution. Our
group has previously shown that fluvastatin could be
eluted from these matrix/microgel copolymer systems for
up to 60 days with retention of bioactivity.
In this study, dual drug release from 3 different sys-
tems was investigated. System A was comprised 65/35
microgels containing fluvastatin embedded in a 50/50
matrix containing xemilofiban, while System B com-
prised 65/35 microgels containing fluvastatin embedded
in an 85/15 matrix containing xemilofiban. Finally, Sys-
tem C contained 65/35 microgels incorporating xemilofiban,
embedded in an 85/15 copolymer containing fluvastatin.
Bioactivity of fluvastatin was assessed in terms of effects
on human coronary artery smooth muscle cell (HCA-
SMC) and human coronary artery endothelial cell (HCA-
EC) proliferation while the bioactivity of xemilofiban
was evaluated in terms of effects on platelet adhesion
under conditions of flow.
2. Materials and Methods
2.1. Materials
N-isopropylacrylamide (NiPAAm; Acros Organics, USA)
and N-tert-butylacrylamide (NtBAAm; Fluka, Switzer-
land) were recrystallized twice from n-hexane and dried
at room temperature under vacuum. N, N-Azobisisobu-
tyronitrile (AIBN; Phase Separation, UK) was recrystal-
lized from methanol. Solvents were reagent grade and
were purified by conventional methods. 14C-fluvastatin
(specific activity 56.9 Ci/mmol) was generously donated
by Novartis, Hanover. Xemilofiban (SC-54701) was
kindly donated by Dr. Dermot Cox, RCSI. HCASMC
with the corresponding growth medium (Medium 231)
were purchased from Cascade Biologics (Belgium). HC-
AEC with the corresponding medium (Endothelial Cell
Growth Medium) were purchased from Promocell. 5-
Bromo-2’-deoxyuridine (BrdU) was obtained from Roche
Diagnostics (Germany). All other chemicals and reagents
were of the highest grade commercially available.
2.2. Preparation of NiPAAm/NtBAAm
Copolymers
The copolymers were synthesized through free radical
polymerisation of the corresponding monomers with
AIBN used as an initiator. The methodology was as out-
lined by McGillicuddy et al. [13] Briefly, mixtures of
NiPAAm and NtBAAm monomers in different ratios
were dissolved in benzene with AIBN then added. The
removal of oxygen was achieved by bubbling N2 gas
through the solution for 30 min. Polymerisation then
proceeded at 60˚C for 24 h. Benzene was evaporated off
and the copolymer was dissolved in acetone and precipi-
tated in n-hexane. This whole procedure was repeated
three times. The purified copolymer was dried at room
temperature under vacuum.
2.3. Preparation of Microgels
Microgels were synthesized using the method of disper-
sion polymerisation. [14] In their desired w/w ratios

Simultaneous Release of a Hydroxy-Methylglutaryl Coenzyme A Reductase Inhibitor and a Glycoprotein IIb/IIIa
Antagonist from a Thermoresponsive NiPAAm/NtBAAm Copolymer System
20
(85/15, 65/35, 50/50, total 0.2 g) NiPAAm/NtBAAm and
bisacrylamide (0.02 g) were dissolved in 36 ml of water.
One ml of 0.1 wt% Triton solution was added and heated
to 70˚C. The solution was degassed by bubbling with N2.
Ammonium persulphate (0.02 g) was dissolved in 4 ml
of water, degassed, and added slowly to the stirring
monomer solution, under an atmosphere of N2. The reac-
tion was left for 12 h at 70˚C and the resulting microgel
dispersion (1 wt% in water) cleaned by dialysis, and
freeze-dried before use.
2.4. Preparation of, and Drug Incorporation into
Matrix/Microgel Copolymer Systems
Fluvastatin was incorporated into 65/35 microgel parti-
cles (5 mg) by incubation with 1 mL of an ethanolic so-
lution of 5mM drug containing tracer levels of
[14C]-fluvastatin for 24 h at 4˚C. The microgel/drug sus-
pension was then centrifuged at 5000 rpm for 10 min, the
supernatant containing unincorporated drug was removed,
and the microgels resuspended in 0.5 ml ethanol. Drug-
loaded microgel suspensions were subsequently added to
0.5 mL of a 10% 50/50 or 85/15 matrix copolymer solu-
tion containing 2.5 mM xemilofiban. Alternatively xe-
milofiban was incorporated into 65/35 microgel particles
which were subsequently incorporated into a 50/50 ma-
trix containing fluvastatin. Aliquots (27 µl) of the result-
ing suspensions were applied evenly to the wells of 24-
well tissue culture plates and were allowed to dry over-
night in an ethanolic atmosphere. All manipulations in
aqueous medium were done at 37˚C which is above the
LCST and therefore all copolymer systems were in the
collapsed/insoluble state.
2.5. Drug Release from Copolymer Films
Dried films were washed with prewarmed PBS (37˚C) to
retain the collapsed state of the copolymer, prevent its
dissolution, and remove any surface-bound drug. Drug
elution from the resulting films at 37˚C was monitored
(as [14C]-fluvastatin) every 24 h by removing, storing and
replacing the PBS solution. The amount of [14C]-
fluvastatin eluted into the overlying solution was deter-
mined by scintillation counting against a suitable stan-
dard curve, from which total eluted fluvastatin was de-
termined.
2.6. Cell Culture
HCASMC that had been isolated from a 21-year old
Caucasian male by Cascade Biologics were received as
cryopreserved cultures (passage 2) from Cytotech Ltd.
(Denmark). The cells were maintained in their appropri-
ate medium, Medium 231, supplemented with foetal bo-
vine serum (4.9%), basic fibroblast growth factor (2ng/
ml), epidermal growth factor (0.5g/ml), heparin (5ng/ml),
insulin (5µg/ml), BSA (0.2µg/ml), gentamicin (10µg/ml)
and amphotericin B (0.25µg/ml). The cells were grown
to confluency in 75cm2 filter top tissue culture flasks and
were maintained at 37˚C in a humidified atmosphere
containing 95% O2 and 5% CO2. Subcultures were cre-
ated by passaging using a trypsin/EDTA (T/E) (0.025
%/0.01%) mixture in phosphate buffer saline (PBS),
harvested by centrifugation (3 min at 433 × g) and
seeded at stated densities. Cells of passages 4-10 were
used for experiments.
HCAEC were purchased from Promocell (Germany).
The cryopreserved cells (passage 2) received had been
isolated from a 63-year old Caucasian male. The appro-
priate endothelial cell growth medium was used to main-
tain the HCAEC. The medium was supplemented with 20
% heat-inactivated foetal calf serum (FCS), endothelial
cell growth supplement/heparin (2 ml), human recombi-
nant epidermal growth factor (5 µg/500 µl), hydrocorti-
sone (500 µg/500 µl), gentamicin (10 µg/ml) and am-
photericin B (0.25 µg/ml). The cells were grown to con-
fluency in 75 cm2 filter top tissue culture flasks and were
maintained at 37˚C in a humidified atmosphere contain-
ing 95% O2 and 5% CO2. Subcultures were created by
passaging using a T/E (0.025%/0.01%) mixture in PBS,
harvested by centrifugation (3 min at 433 × g) and
seeded at stated densities. Cells of passages 4-10 were
used for experiments. Cells were also routinely tested for
the presence of mycoplasm.
2.7. Measurement of Cellular Proliferation
(BrdU ELISA)
HCASMC or HCAEC (3 × 104 cells/ml/well) were seed-
ed into 96-well plates and left to adhere overnight. Cells
were then incubated with samples of fluvastatin eluted
from the various copolymer systems and left for 48h.
Proliferation was subsequently assessed by measurement
of BrdU incorporation into the DNA of proliferating cells
using a colorimetric ELISA.
2.8. Measurement of Platelet Adhesion under
Conditions of Flow
A novel microflow system consisting of a novel syringe
pump with microfluidic biochip and flow sensor con-
trolled by a PC using dedicated software was used to
assess the effects of eluted samples of xemilofiban.
Blood was collected from healthy volunteers who were
not taking any medication and were free from aspirin and
other anti-platelet agents within the previous 2 weeks.
The blood was drawn by venupuncture into tubes con-
taining a 1:10 volume of 3.8% (wt/vol) trisodium citrate
and gently mixed. Each microgel channel of the novel

Simultaneous Release of a Hydroxy-Methylglutaryl Coenzyme A Reductase Inhibitor and a Glycoprotein IIb/IIIa
Antagonist from a Thermoresponsive NiPAAm/NtBAAm Copolymer System
21
microflow system was coated overnight in humid condi-
tions at 4˚C with fibrinogen (2 µg/ml). All channels were
then coated with BSA (10 µg/ml) to saturate non-specific
binding sites and left for approximately 30 min. Prior to
the shear experiments all channels were washed through
with JNL. Aliquots of whole blood were incubated for 5
or 10 min with the day 1, 3, 9 and 14 samples released
from the following copolymer films:
A) 65/35 microgels containing fluvastatin embedded
in a 50/50 matrix containing xemilofiban
B) 65/35 microgels containing fluvastatin embedded in
an 85/15 matrix containing xemilofiban
C) 65/35 microgels containing xemilofiban embedded
in an 85/15 copolymer containing fluvastatin.
Whole blood and sample were together infused into
the fibrinogen-coated channels under a shear stress of 32
dyne cm-2 for 3 min. Phase contrast images at the indi-
cated shear stress levels were captured using MetaMorph
imaging software for analysis at a later time. The images
were then used to quantify platelet adhesion by counting
the number of platelets within each illustrated quadrant.
2.9. Data Analysis
Data are presented as means ± SEM of the indicated
number (n) of determinations. Statistical analysis of dif-
ferences between groups was performed by ANOVA,
followed by Bonferroni’s multiple comparisons post-test.
The statistical package PRISM was used for all analyses.
Differences between means were considered significant
when p < 0.05.
3. Results
3.1. Drug Elution from Copolymer Films
For normalization purposes, all release data were graphed
as a percentage of total drug incorporated into copolymer
films. The elution of fluvastatin from fluvastatin-loaded
65/35 microgels embedded in a xemilofiban-containing
85/15 matrix saw a release of 12.69 ± 0.74% on day 1
with a total release of 68.55 ± 1.54% by the final day
(Figure 1(a)). After the 14-day elution a distribution
graph was calculated to show the amounts eluted, re-
maining and total amount incorporated in the system
(Figure 1(b)). A total of 8.33 ± 0.21 nmol was incorpo-
rated, 5.71 ± 0.13 nmol were eluted and 2.58 ± 0.12 nmol
remained upon completion.
3.2. Effect of Native and Eluted Xemilofiban on
Platelet Adhesion as Assessed Using a
Microfluidic System
Xemilofiban had a statistically significant effect on
(a)
(b)
Figure 1. (a) Cumulative release of fluvastatin from fluvas-
tatin-loaded 65/35 microgels embedded in an 85/15 copoly-
mer matrix containing xemilofiban over a 14-day period.
The microgels were pre-incubated with 5 mM fluvastatin
while the matrix was pre-incubated with 2.5 mM xemilofi-
ban and left overnight. Copolymer films were then cast in
24-well plates and after 24 h PBS was added to the wells.
On the final day the copolymer films were dissolved and
total drug incorporated was calculated. Cumulative release
was subsequently expressed as a % of total drug incorpo-
rated into the films. Data are representative of mean ±
S.E.M. of 4 individual copolymer films. (b) Distribution
graph of drug- eluting films releasing fluvastatin. The total
amount of drug initially loaded into the copolymer films
was determined after dissolving the copolymer films. Data
are representative of mean ± S.E.M. of 4 individual co-
polymer films.
platelet adhesion at concentrations of 0.01 and 0.1 µM,
with adhesion being reduced to 56.06 ± 7.8% and 3.44 ±
1.43% of controls, respectively (Figure 2). A group of
controls were used in the experiment with a “high” xe-
milofiban concentration (1 µM), a “low” xemilofiban
concentration (0.01 µM) and a “low” concentration of
fluvastatin (0.1 µM). All samples were mixed with whole
blood and subjected to shear. Of the controls the “high”
xemilofiban concentration significantly inhibited platelet
adhesion, reducing it to 5.81 ± 1.59%. “Low” xemilofi-
ban reduced platelet adhesion to 65.25 ± 9.91% and
“low” fluvastatin to 57.83 ± 19.13%. The phase contrast
images from which these results were extrapolated, by
quantifying the number of adhered platelets, also show
clearly the decrease in platelet adhesion using the high
xemilofiban concentration (Figure 3). System A (the

Simultaneous Release of a Hydroxy-Methylglutaryl Coenzyme A Reductase Inhibitor and a Glycoprotein IIb/IIIa
Antagonist from a Thermoresponsive NiPAAm/NtBAAm Copolymer System
22
Figure 2. Effect of xemilofiban on platelet adhesion as as-
sessed using a microfluidic system. Whole blood which had
been treated with increasing concentrations of xemilofiban
was passed through microcapillary channels on a biochip
under shear stress. Numbers of platelets adhered were
counted using MetaMorph. Results were calculated as a %
of control (no drug) and values are presented as mean ±
S.E.M., n = 3, ***p < 0.001 w.r.t. control.
Figure 3. The effects of xemilofiban and fluvastatin on
whole blood assessed using the microfluidic system. Whole
blood was mixed with either drug and then passed through
the fibrinogen coated biochannels and subjected to shear
rate, phase contrast images were captured using Meta-
Morph. Images are as follows: (a) whole blood alone; (b)
high xemilofiban, 1 µM; (c) low xemilofiban, 0.01 µM; (d)
fluvastatin 0.1 µM.
most hydrophilic system of the 3) showed significant
decreases for day 1 and 3 with adhesion reduced to 7.18
± 3.02% and 15.46 ± 5.62%, respectively; thereafter
platelet adhesion increased to 62.43 ± 20.4% and 82.08 ±
24.97% respectively. System B (the more hydrophobic
system) showed a significant decrease for day 1 with a
reduction to 8.74 ± 4.57%. On days 3, 9 and 14, there
was no significant difference in reductions; however there
was an apparent reduction in platelet adhesion to half that of
the control on day 14. Finally, with system C in which xe-
milofiban was incorporated into the microgels, there was
a significant decrease seen for days 1, 3, 9 and 14, with
reductions to 8.96 ± 5.14%, 47.97 ± 11.89%, 50.54 ± 15.37%
and 51.11 ± 10.26% respectively (Figure 4).
Figure 4. Effect of samples eluted from copolymer films on
platelet adhesion assessed using the microfluidic system.
System A composition was as follows 65/35 microgels con-
taining fluvastatin embedded in a 85/15 matrix containing
xemilofiban, System B was 65/35 microgels containing flu-
vastatin embedded in an 85/15 matrix containing xemilofi-
ban and System C was comprised of 65/35 microgels con-
taining xemilofiban embedded in an 85/15 copolymer con-
taining fluvastatin Copolymer films were cast into 24-well
plates and overlaid with PBS and eluted samples were col-
lected daily. Whole blood was treated with these eluted
samples and passed through the microfluidic system. The
amount of platelets adhered was counted using MetaMorph.
Results were calculated as a % of control (no drug) and
values are presented as mean ± S.E.M., n = 5, ***p < 0.001
w.r.t. cont r ol .
3.3. Effect of Eluted Fluvastatin on HCASMC
Proliferation
All samples eluted from each system significantly inhib-
ited HCASMC proliferation (Figure 5). It could be seen
that while proliferation was inhibited in the presence of
each sample, a steady loss in anti-proliferative activity
was seen over time. Samples eluted from System A de-
creased proliferation to 12.86 ± 4.55% of control, using
the Day 1 sample, with proliferation recovering to 62.29

Simultaneous Release of a Hydroxy-Methylglutaryl Coenzyme A Reductase Inhibitor and a Glycoprotein IIb/IIIa
Antagonist from a Thermoresponsive NiPAAm/NtBAAm Copolymer System
23
Figure 5. Effects of samples eluted from system A-C on
HCASMC proliferation as assessed using the BrdU assay.
Each sample was incubated with smooth muscle cells for 48
h. Results were calculated as % of control (no drug) and
values are presented as mean ± S.E.M., n = 3, ***p < 0.001
w.r.t. cont r ol.
± 8.17% by day 14. With regard to Systems B and C,
proliferation was reduced to 8.17 ± 0.6% and 8.32 ± 0.6
% of control respectively, on day 1. By day 14 HCA-
SMC proliferation was 77.74 ± 9.44% with System B,
and 65.22 ± 7.69% with System C. Upon comparison of
anti-proliferative activity with that obtained with native
fluvastatin, it can be approximated that fluvastatin was
present in concentrations between 0.01 and 1.4 µM in
samples tested from these systems.
3.4. Effect of Eluted Fluvastatin on HCAEC
Proliferation
When endothelial cells were treated for 48 h with the
eluted samples, there was no statistically significant ef-
fect on proliferation seen in the presence of any sample
from any of the 3 systems (Figure 6).
Figure 6. Effects of samples eluted from system A-C on
HCAEC proliferation as assessed using the BrdU assay.
Each sample was incubated with endothelial cells for 48 h.
Results were calculated as % of control (no drug) and val-
ues are presented as mean ± S.E.M., n = 3, N.S. w.r.t. con-
trol.
4. Discussion
The overall aim of this study was to control release and
deliver bioactive concentrations of a HMG-CoA reduc-
tase inhibitor (fluvastatin) and glycoprotein IIb/IIIa an-
tagonist (xemilofiban) from a NiPAAm/NtBAAm ma-
trix/microgel drug delivery system. Using the thermore-
sponsive properties of the matrices and microgels, the
potential of such systems for the elution of these drugs
has been demonstrated.
The anti-proliferative nature of statins has been exten-
sively studied. Statins have been reported to inhibit smooth
muscle cell proliferation both in vitro and in vivo [15].
Lovastatin, simvastatin and cerivastatin were found to
significantly inhibit VSMC proliferation when treated
with concentrations of 0.1-50 µM. [16,17].
Takeda et al. [18] found that simvastatin significantly
inhibited cell proliferation in bronchial VSMC while
having no effect on cell viability. An in vitro study by
Corpataux et al. [19] found that the reduction in smooth
muscle cell proliferation produced by fluvastatin was

Simultaneous Release of a Hydroxy-Methylglutaryl Coenzyme A Reductase Inhibitor and a Glycoprotein IIb/IIIa
Antagonist from a Thermoresponsive NiPAAm/NtBAAm Copolymer System
24
significantly greater than that with other statins such as
simvastatin, lovastatin, atorvastatin, cerivastatin and pra-
vastatin. Jaschke et al. [17] showed that after treatment
with cerivastatin (0-100 nM) only the highest concentra-
tion had a significant effect on endothelial cell prolifera-
tion. Thus, although fluvastatin was used to treat both
HCAEC and HCASMC in this study, the latter were
much more sensitive to these anti-proliferative effects.
Therefore fluvastatin was selected as the statin of choice
in the present project for local drug delivery due to its
active and lipophilic nature, both essential requirements
for a potential candidate for local drug delivery.
We have previously reported that NiPAAm/NtBAAm
copolymers can elute fluvastatin for up to 60 days while
retaining its bioactivity, as assessed under both static and
perfusion conditions [20]. The present study has further
shown that it is possible to co-elute bioactive fluvastatin
with a second drug type incorporated in the system. In
System A fluvastatin was eluted from 65/35 microgels
and then released through a hydrophilic 85/15 matrix.
Upon completion of this release experiment there was <
20% of the drug remaining in the system. It is important
to note here that the pH value of blood may have an ef-
fect on drug elution in vivo; however such an investiga-
tion in vitro was beyond the scope of the present work.
Also, while every step in the preparation of the mi-
crogel/copolymer systems was carefully carried out,
there is the possibility that small quantities of drug were
lost. However, such losses were deemed minimal, since
subsequent experiments showed comparable amounts of
activity eluted in replicate experiments. While the inhibi-
tion of vSMC proliferation steadily decreased with each
of the daily samples used, proliferation at 48 h was still
inhibited. Samples eluted from the other two systems
showed an inhibition of proliferation resembling that of
System A.
In Systems A and B, fluvastatin was incorporated into
the microgels and then eluted through hydrophilic (Sys-
tem A) or hydrophobic (System B) matrices. This may
explain the slight differences between these systems i.e.
System A samples showed slightly greater inhibiton of
proliferation and the drug was eluted faster. With regard
to System C fluvastatin was dispersed in the matrix and
thus only had one barrier to pass through; the results here
were similar to those for System A. This could be due to
the fact that hydrophobic drug was eluted from microgels
into a hydrophilic matrix in one instance and straight
from a hydrophobic matrix in the other. It is possible that
the time course of elution from a hydrophobic matrix is
comparable to that of elution from microgels and a hy-
drophilic matrix together. However it is also important to
note that major differences may only be seen with exten-
sion of the 14-day period (Figure 6). The comparable
levels of overall inhibition seen between systems could
also be due to the fact that each of the systems is eluting
a similar concentration of fluvastatin on the relevant
days.
None of the samples eluted from system A-C had a
significant effect on HCAEC proliferation after 48 h,
which suggests that re-endothelialisation in vivo would
not be impeded up to this point.
The rationale for using the GpIIb/IIIa antagonist xe-
milofiban in this study was based on the fact that GpIIb/
IIIa antagonists have been used successfully as intrave-
nous anti-platelet agents post-surgery (PTCA) [21] fol-
lowing their development of oral agents, mixed results
coronary syndromes and long-term management of pa-
tients [22]. However with 5 large-scale trials completed
by 2001, which included over 42,000 patients (EXCITE,
[23] OPUS, [24] SYMPHONY 1 and 2, [25,26] BRAVO
[27]), it was consistently found that the GpIIb/IIIa agents
xemilofiban, orbofiban, sibrafiban and lotrafiban were no
more effective than aspirin when given post-surgery.
However, another study showed that their use during
PCA procedures was effective and in fact improved
in-hospital survival rates [28]. Heer et al. [29] have also
offered evidence of their effectiveness during primary
angioplasty. Therefore their effectiveness as an intrave-
nous treatment in conjunction with PCA gives an indica-
tion that these agents could be used for local delivery
from a stent platform.
The rationale for elution of an anti-platelet agent has
been strengthened by reports demonstrating elution of
GpIIb/IIIa receptor monoclonal antibody from polymer-
coated stents. Yin et al. [30] showed that the GpIIb/IIIa
receptor antibody eluted from I-PLA polymer-coated
stents inhibited platelet aggregation. Aggarwal et al. [31]
showed that GpIIb/IIIa antibody could be eluted for up to
14 days, with antibody still remaining after that time un-
der conditions of flow.
Our study firstly showed that xemilofiban concentra-
tions of 0.01 and 0.1 µM had a statistically significant
effect on platelet adhesion (Figure 3). This gives a good
indication that xemilofiban is capable of exerting its ef-
fects under conditions of flow and that platelets in circu-
lating blood are more susceptible to the anti-adhesive
action of xemilofiban in this model.
Under conditions of flow System A showed that on
day 1 and 3, samples decreased platelet adhesion signifi-
cantly, and the results were in fact on a comparable level
with the control “high xemilofiban” concentration; how-
ever day 9 and 14 samples had only a minor effect on
adhesion equivalent to that of the “low xemilofiban”
concentration (Figures 4 and 5). As this was a hydro-

Simultaneous Release of a Hydroxy-Methylglutaryl Coenzyme A Reductase Inhibitor and a Glycoprotein IIb/IIIa
Antagonist from a Thermoresponsive NiPAAm/NtBAAm Copolymer System
25
philic matrix it could be inferred that the hydrophobic
xemilofiban was expelled rapidly and therefore only had
a significant effect at day 1 and 3. It may be possible to
prolong the elution of the drug from this matrix type by
incubating the matrix or microgels with a higher concen-
tration of xemilofiban. Alternatively using a more hy-
drophobic matrix could also potentially prolong the re-
lease of xemilofiban. It should be noted here that meas-
urement of the exact concentration of xemilofiban re-
leased was beyond the scope of the present study.
System B had 65/35 microgels containing fluvastatin
embedded in a 50/50 matrix containing xemilofiban; this
is the more hydrophobic system and therefore it could be
anticipated that elution would be prolonged. With this
system a statistically significant effect was seen on day 1
with a reduction to a level similar to “high xemilofiban”.
This correlated well with the burst release that is usually
seen on Day 1 with all systems. While the remaining
samples did not elicit a statistically significant effect it
could be seen that there was a trend towards continued
inhibition of platelet adhesion (Figure 5). Thus this sys-
tem gave an indication that the inhibitory effect would
have continued beyond 14 days.
System C saw the incorporation of xemilofiban into
65/35 microgels, which were then embedded in a 50/50
matrix. This final system showed release of bioactive
drug over an extended time interval. In this case drug
would have firstly been released from the microgels and
then passed through the hydrophobic matrix. Each of the
daily samples had a significant effect on platelet adhe-
sion, with the day 1 sample having the greatest effect
(Figure 5).
The contrasts between the profiles of each of the de-
livery systems can be explained in terms of their compo-
sition. With regard to Systems A and B, xemilofiban is
contained in a hydrophilic and hydrophobic matrix re-
spectively. The drug appears to be eluted faster from
System A as a greater effect can be seen on Days 1 and 3,
after which the anti-adhesive effect is reduced. The hy-
drophobic matrix of System B retards the release of xe-
milofiban slighty, thereby resulting in a greater anti-ad-
hesive effect being seen at Day 14 compared with Sys-
tem A. Another point to note with these two systems is
that as xemilofiban is contained in the matrix, it only has
one diffusion barrier prior to release, thereby resulting in
faster elution. With System C however, xemilofiban is
contained in the microgel component, and therefore has
two diffusion barriers prior to release. Comparing Sys-
tem C with Systems A and B, it can be noted that a
steady elution rate appears to be maintained, which may
reflect more tightly controlled release. With all three sys-
tems the Day 1 eluate had the most significant anti-ad-
hesive activity, which is characteristic of the burst release
seen with these systems [20].
5. Conclusions
Overall, this study showed that it is possible to elute two
bioactive drug types from one copolymer system, result-
ing in three points of information. Firstly alteration of the
composition of the copolymer systems i.e. the hydropho-
bic or hydrophilic nature of the matrix or microgels can
affect elution. Also placement in either matrix or mi-
crogels can alter the period of elution i.e. the incorpora-
tion of drug into the microgels can prolong the effect of
the drug. The study finally shows that xemilofiban re-
mains bioactive and can affect platelet adhesion under
flow conditions. The use of thermoresponsive copoly-
mers is relevant as they allow for the incorporation of
drug into a soluble mixture followed by precipitation
above the LCST onto, potentially, a stent. Therefore the
thermoresponsive properties coupled with the relative
hydrophobicities and hydrophilicities of the NiPAAm/
NtBAAm copolymers adds to their potential for the de-
livery of numerous drugs for extended periods. Therefore
this study has shown that two drugs eliciting different
actions can be eluted for up to 14 days in bioactive con-
centrations with the potential for extended release. Also
fluvastatin did not alter platelet adhesion at concentra-
tions eluted nor did xemilofiban affect cell proliferation
at estimated concentrations eluted.
6. Acknowledgements
We would like to acknowledgement the financial support
provided by the Irish Heart Foundation without whom
this work would not have been possible.
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