Reduction-sensitive functionalized copolyurethanes for biomedical applications

Abstract

In the present paper we combine functionalization and biodegradation in the rational design of polymers that can be used as carrier systems for drug delivery in the colon. Functionalization of new polyurethanes (PUs) was achieved by thiol–ene coupling reactions, a simple and straightforward procedure included among the so-called click reactions, which are currently accepted as one of the most powerful tools in organic chemistry. Enhancement of the degradability of the new materials by the introduction of disulfide linkages into the polymer backbone has led to a new group of stimulus-responsive sugar-based polyurethanes able to be degraded by tripeptide glutathione under physiological conditions. Atomic Force Microscopy (AFM) on solid-supported multilayered dry polymer films—prepared by spin-coating from dimethylsulfoxide solutions—was used to study the morphology of the polymers and the degradation process in reductive environments. Matrix systems containing polymers selected according to their rheological properties were also investigated as modulated methotrexate-release systems.

Full text

Reduction-sensitive functionalized
copolyurethanes for biomedical applications
Cristina Ferris,
a
M. Violante de Paz,
a
´
Angela Aguilar-de-Leyva,
b
Isidoro Caraballo
b
and Juan A. Galbis*
a
In the present paper we combine functionalization and biodegradation in the rational design of polymers
that can be used as carrier systems for drug delivery in the colon. Functionalization of new
polyurethanes (PUs) was achieved by thiol–ene coupling reactions, a simple and straightforward
procedure included among the so-called click reactions, which are currently accepted as one of the
most powerful tools in organic chemistry. Enhancement of the degradability of the new materials by the
introduction of disulfide linkages into the polymer backbone has led to a new group of stimulus-
responsive sugar-based polyurethanes able to be degraded by tripeptide glutathione under physiological
conditions. Atomic Force Microscopy (AFM) on solid-supported multilayered dry polymer films—
prepared by spin-coating from dimethylsulfoxide solutions—was used to study the morphology of the
polymers and the degradation process in reductive environments. Matrix systems containing polymers
selected according to their rheological properties were also investigated as modulated methotrexate-
release systems.
Introduction
The emergence of novel biomedical technologies such as gene
therapy, regenerative medicine, tissue engineering, controlled
drug delivery, and bionanotechnology has meant that the
development of biodegradable polymeric materials for those
applications has become a hot topic. Recent efforts in polymer
chemistry have been made to promote the synthesis and design
of a wide range of natural or synthetic polymers with particular
biomedical applications, capable of undergoing degradation by
hydrolytic or enzymatic mechanisms.
1
Among the synthetic materials used in biomedicine, poly-
urethanes (PUs) are being widely investigated due to their low
toxicity, potential biodegradability, biocompatibility, and
versatile structures.
2
Because of their excellent biocompatibility
and low thrombogenicity, biodegradable polyurethanes can be
used as non-permanent devices. Numerous articles and reviews
have reported exhaustive studies on degradability of poly-
urethanes, mainly following processes involving hydrolytic,
enzymatic, and oxidative pathways.
3–7
The introduction of other hydrolyzable linkages besides the
urethane bonds can enhance the degradation rates of the
materials.
8–10
To synthesize more biodegradable PUs, we
demonstrated in a previous work that the introduction of
disulde bonds into polyurethane skeletons as readily available
hydrolyzable linkages could accomplish this goal.
11
Disulde
bonds are prone to rapid cleavage in a reductive environment by
the action of the natural tripeptide glutathione (g-gluta-
mylcysteinylglycine, GSH).
11–14
This quick-response chemical
degradation contrasts sharply with common hydrolytically
degradable mechanisms for polymers such as aliphatic poly-
esters and polycarbonates, which usually display gradual
degradation kinetics inside the body.
15–17
At the same time, the rational design of polymers tailored to
exert distinct biological functions plays an important role in the
development of controlled drug delivery systems. Thus, the
controlled release of therapeutic molecules can be achieved by
anchoring the active agent to polymer structures by means of
physical interactions or by covalent linkages. Hence, the func-
tionalization of polymers, and more specically, PU mate-
rials,
18–21
becomes a topic of great interest. We proposed in
previous studies a simple and straightforward procedure to
prepare highly functionalized new PUs
22,23
via click chemistry
(CC) reactions, which are currently accepted as a very powerful
tool in organic chemistry.
24–26
Focusing on the development of materials as vehicles, colon-
targeting drug delivery is one of the most grown elds in
pharmaceutical technology. These systems can be employed for
both local-acting drugs and active principles intended for
systemic absorption. In this latter case, the colon offers the
advantage of its non-aggressive physicochemical environment
and the absence of digestive enzymes. Therefore, it is possible
to deliver active molecules such as polypeptides, proteins, and
a
Dpto. Qu´
ımica Org´
anica y Farmac´
eutica, Facultad de Farmacia, Universidad de
Sevilla, 41012-Sevilla, Spain. E-mail: jgalbis@us.es; Fax: +34 954556737; Tel: +34
954556736
b
Dpto. Farmacia y Tecnolog´
ıa Farmac´
eutica, Facultad de Farmacia, Universidad de
Sevilla, 41012-Sevilla, Spain. E-mail: caraballo@us.es; Fax: +34 954556085; Tel:
+34 954556136
Cite this: Polym. Chem.,2014,5,2370
Received 8th November 2013
Accepted 16th December 2013
DOI: 10.1039/c3py01572f
www.rsc.org/polymers
2370 |Polym. Chem.,2014,5, 2370–2381 This journal is © The Royal Society of Chemistry 2014
Polymer
Chemistry
PAPER

selected drugs to the colon as an alternative to other costly and
patient-uncomfortable routes.
In the present paper, we aim to unite two features for the
rational design of polymers tailored for use as drug delivery
vehicles in the colon: functionality via CC, and enhanced
degradability via the introduction of disulde linkages in
reductive environments. Thus, polyurethanes that integrate
both attributes could be highly promising functional biomate-
rials with enormous potential in formulating sophisticated drug
and gene delivery systems.
In order to investigate the possibility of employing PUs as
release-controlling polymers for colon targeting, we prepared a
sustained-release formulation of methotrexate, a chemotherapy
drug. This was subjected to release assays using the official USP
dissolution apparatus and a new method combining the pH-
gradient and a reductive environment to simulate the progress
of the dosage form through the gastro-intestinal tract.
Experimental
Materials and methods
Commercial reagents and solvents were purchased from Aldrich
Chemical Co. and used as received. The following materials
were used in the preparation of the tablet: Fast Flo lactose
(Foremost, USA), silicon dioxide (Aerosil®) (Degussa, Germany),
magnesium stearate (Fagron, Spain), Eudragit FS 30 D (Evonik,
Essen, Germany), and methotrexate (Sigma-Aldrich, Spain).
When necessary, solvents were dried and puried by appro-
priate standard procedures.
Elemental analyses were determined in the Microanalysis
Laboratories of the IIQ Service, cicCartuja, Seville. IR spectra
were recorded on a JASCO FT/IR-4200 spectrometer. Nuclear
Magnetic Resonance (NMR) spectra were recorded at 300 K on
either a Bruker Advance AV-500 or a Bruker AMX-500. Chemical
shis(d) are reported as parts per million downeld from
Me
4
Si. Gel permeation chromatography (GPC) analyses were
performed using a Waters apparatus equipped with a Waters
2414 refractive-index detector and two Styragel® HR columns
(7.8 300 mm) linked in series, thermostatted at 60 C, using
N-methylpyrrolidone (NMP) as the mobile phase, at a ow rate
of 0.5 mL min
1
. Molecular weights were estimated against
polystyrene standards. Intrinsic viscosity was determined in
dichloroacetic acid (DCA) with an AMVn Automated Micro-
viscometer from Anton Paar. The thermal behavior of the
polyurethanes was examined by Differential Scanning Calo-
rimetry (DSC), using a TA DSC Q-200 Instrument calibrated with
indium. DSC data were obtained from samples of 4–6mgat
heating/cooling rates of 10 C min
1
under a nitrogen ow. The
glass transition temperatures were determined at a heating rate
of 20 C min
1
from rapidly melt-quenched polymer samples.
Thermogravimetric analyses (TGAs) were performed under a
nitrogen atmosphere (ow rate 100 mL min
1
) with a Universal
V4.3A TA Instrument at a heating rate of 10 C min
1
. AFM
images were obtained with a Molecular Imaging PicoPlus 2500
(Agilent Technologies). A standard silicon cantilever from
NanoWorld with a resonance frequency around 320 kHz was
used for performing the tapping mode. The scanning speed was
0.4 Hz.
The polymerization reactions were performed in the absence
of humidity, under an inert atmosphere. All glassware was
heated overnight at 80 C before use, and aer assembly was
further heated under vacuum to eliminate the surface moisture.
The diol monomers 2,3,4-tri-O-allyl-L-arabinitol (ArAll
3
,1),
22
3,4-
di-O-allyl-2-methyl-L-arabinitol (ArAll
2
,2)
22
and 2,3,4-tri-O-{3-[2-
(tert-butoxycarbonylamino)ethyl]thiopropyl}-L-arabinitol (Ar–
SNHBoc, 3)
23
were dried under high vacuum for at least 3 days.
1,6-Hexamethylene diisocyanate (HMDI) and 4,40-methylenebi-
s(phenyl isocyanate) (MDI) were stored at 4 C, and handled
under an inert atmosphere. Anhydrous N,N-dimethylforma-
mide (DMF) was used as the polymerization solvent. The other
reactants for the polymerizations were stored in a desiccator
under an inert atmosphere until required.
Synthesis of allyl-based copolyurethanes
Copolyurethane PU{[(ArAll
3
)
50
-DiT
50
]HMDI} (4). The diol
monomer ArAll
3
(1,
22
0.15 g, 0.55 mmol) was loaded into a
round-bottom ask with an argon–vacuum inlet. The system
was treated with three cycles of vacuum–argon before the
addition, via cannula, of dried DMF (3 mL) and 2,20-dithiodie-
thanol (DiT, 67 mL, 0.55 mmol). The mixture was stirred to
homogenization, and the diisocyanate (HMDI, 177 mL, 1.10
mmol) was added under an argon atmosphere, followed by the
catalyst (dibutyltin dilaurate, one drop). The polymerization
solution was stirred at 25 C for 5 h under an argon atmosphere.
Methanol (0.2 mL) was added, the mixture was stirred for 30
min, and the solution was added dropwise into cold diethyl
ether (250 mL), where the polymer PU{[(ArAll
3
)
50
-DiT
50
]HMDI}
(4) precipitated. The polymer was dried under vacuum for 2 days
and stored in a desiccator (0.39 g, 93%).
M
w
10 500; M
n
7000; M
w
/M
n
1.5. Intrinsic viscosity: 0.11 dL
g
1
. IR: n(cm
1
) 3320 (N–H), 3065 (C–H vinyl), 1680 (C]O
urethane), 1531 (N–H, N–C]O, urethane), 1256 (C–N), 1067 (C–
O–C).
1
H NMR (DMSO-d
6
, 500 MHz): d(ppm) 1.42 (bs, 8H, CH
2
-
c), 1.83 (bs, 8H, CH
2
-b), 2.89–3.10 (m, 12H, CH
2
-a/2CH
2
CH
2
S),
3.18–3.76 (m, 3H, H-2/H-3/H-4), 3.86–4.82 (m, 14H, H-1/H-5/
2CH
2
CH
2
S/3CH
2
CH]), 5.23–5.47 (m, 6H, 3CH]CH
2
), 5.84–
6.03 (m, 3H, 3CH]CH
2
), 6.76 (bs, 4H, NH).
13
C NMR (DMSO-d
6
,
125 MHz): d(ppm) 25.7 (C–c), 29.7 (C-b), 38.2 (2CH
2
CH
2
S), 40.6
(C-a), 61.4 (2CH
2
CH
2
S), 63.2, 63.5 (C-1/C-5), 70.2, 72.9
(3CH
2
CH]), 76.5, 78.2, 79.1 (C-2/C-3/C-4), 114.5, 116.8 (3CH]
CH
2
), 135.2, 135.4 (3CH]CH
2
), 156.5 (C]O).
Anal. calcd for (C
22
H
36
N
2
O
7
)
50
(C
12
H
22
N
2
O
4
S
2
)
50
: C, 53.52; H,
7.66; N, 7.34; S, 8.41. Found: C, 53.46; H, 7.95; N, 7.46; S, 8.48.
Copolyurethane PU{[(ArAll
3
)
20
-DiT
80
]HMDI} (5). This was
obtained from ArAll
3
(1, 0.13 g, 0.48 mmol), DiT (234 mL, 1.91
mmol) and HMDI (284 mL, 2.39 mmol) in DMF (3 mL), following
the procedure described previously for 4,at25C. The reaction
was worked up as described above to give the title compound as
a solid (0.79 g, 96%).
M
w
15 600; M
n
9500; M
w
/M
n
1.6. Intrinsic viscosity: 0.13 dL
g
1
. IR: n(cm
1
) 3321 (N–H), 3072 (C–H vinyl), 1682 (C]O
urethane), 1530 (N–H, N–C]O, urethane), 1254 (C–N), 1066
This journal is © The Royal Society of Chemistry 2014 Polym. Chem.,2014,5,2370–2381 | 2371
Paper Polymer Chemistry

(C–O–C).
1
H NMR (DMSO-d
6
, 500 MHz): d(ppm) 1.23 (bs, 8H,
CH
2
-c), 1.37 (bs, 8H, CH
2
-b), 2.84–3.13 (m, 12H, CH
2
-a/
2CH
2
CH
2
S), 3.38–3.79 (m, 3H, H-2/H-3/H-4), 3.84–4.36 (m, 14H,
H-1/H-5/2CH
2
CH
2
S/3CH
2
CH]), 5.02–5.33 (m, 6H, 3CH]CH
2
),
5.75–6.02 (m, 3H, 3CH]CH
2
), 6.80, 7.12 (bs, 4H, NH).
13
C NMR
(DMSO-d
6
, 125 MHz): d(ppm) 25.8 (C-c), 29.2 (C-b), 37.1
(2CH
2
CH
2
S), 40.1 (C-a), 61.5 (2CH
2
CH
2
S), 62.5, 63.0 (C-1/C-5),
71.4, 72.6 (3CH
2
CH]), 76.8, 77.6, 77.9 (C-2/C-3/C-4), 115.9,
116.3 (3CH]CH
2
), 135.1, 135.5 (3CH]CH
2
), 155.8 (C]O).
Anal. calcd for (C
22
H
36
N
2
O
7
)
20
(C
12
H
22
N
2
O
4
S
2
)
80
: C, 48.59; H,
7.22; N, 8.09; S, 14.83. Found: C, 48.65; H, 7.41; N, 8.11; S, 14.62.
Copolyurethane PU{[(ArAll
2
)
20
-DiT
80
]HMDI} (6). This was
obtained from ArAll
2
(2,
22
0.18 g, 0.74 mmol), DiT (345 mL, 2.96
mmol) and HMDI (598 mL, 3.72 mmol) in DMF (3 mL), following
the procedure described previously for 4,at25C. The reaction
was worked up as described above to give the title compound as
a solid (1.12 g, 89%).
M
w
35 000; M
n
18 200; M
w
/M
n
1.9. Intrinsic viscosity: 0.26 dL
g
1
. IR: n(cm
1
) 3320 (N–H), 3055 (C–H vinyl), 1681 (C]O
urethane), 1530 (N–H, N–C]O, urethane), 1254 (C–N), 1067 (C–
O–C).
1
H NMR (DMSO-d
6
, 500 MHz): d(ppm) 1.25 (bs, 8H, CH
2
-
c), 1.39 (bs, 8H, CH
2
-b), 2.87–3.10 (m, 12H, CH
2
-a/2CH
2
CH
2
S),
3.26–3.76 (m, 6H, H-2/H-3/H-4/CH
3
), 3.88–4.39 (m, 12H, H-1/H-
5/2CH
2
CH]/2CH
2
CH
2
S), 5.06–5.31 (m, 4H, 2CH]CH
2
), 5.80–
5.97 (m, 2H, 2CH]CH
2
), 6.82, 7.14 (bs, 4H, NH).
13
C NMR
(DMSO-d
6
, 125 MHz): d(ppm) 26.4 (C-c), 29.8 (C-b), 37.7
(2CH
2
CH
2
S), 40.7 (C-a), 57.6 (CH
3
), 62.0 (2CH
2
CH
2
S), 62.5, 63.0
(C-1/C-5), 72.1, 73.4 (2CH
2
CH]), 77.3, 78.1, 79.0 (C-2/C-3/C-4),
116.7, 116.8 (2CH]CH
2
), 135.7, 135.9 (2CH]CH
2
), 156.4, 156.6
(C]O).
Anal. calcd for (C
20
H
34
N
2
O
7
)
20
(C
12
H
22
N
2
O
4
S
2
)
80
: C, 47.92; H,
7.22; N, 8.22; S, 15.05. Found: C, 48.05; H, 7.20; N, 8.16; S, 14.76.
Copolyurethane PU{[(ArAll
3
)
50
-DiT
50
]MDI} (7). This was
obtained from ArAll
3
(1, 0.15 g, 0.55 mmol), DiT (67 mL, 0.55
mmol) and MDI (0.28 g, 1.10 mmol) in DMF (3 mL), following
the procedure described previously for 4,at25C. The reaction
was worked up as described above to give the title compound as
a solid (0.46 g, 91%).
M
w
25 200; M
n
13 700; M
w
/M
n
1.8. Intrinsic viscosity: 0.39 dL
g
1
. IR: n(cm
1
) 3315 (N–H), 3042 (C–H vinyl), 1703 (C]O
urethane), 1522 (N–H, N–C]O, urethane), 1216 (C–N), 1063 (C–
O–C).
1
H NMR (DMSO-d
6
, 500 MHz): d(ppm) 3.05 (t, 4H,
2CH
2
CH
2
S, J¼6.4 Hz), 3.64–3.87 (m, 7H, PhCH
2
Ph/H-2/H-3/H-
4), 3.90–4.54 (m, 14H, 3CH
2
CH]/H-1/H-5/2CH
2
CH
2
S), 5.06–
5.32 (m, 6H, 3CH]CH
2
), 5.83–5.98 (m, 3H, 3CH]CH
2
), 7.03–
7.18 (m, 4H, Arom.), 7.29–7.47 (m, 8H, Arom./NH).
13
C NMR
(DMSO-d
6
, 125 MHz): d(ppm) 37.4 (2CH
2
CH
2
S), 40.3
(PhCH
2
Ph), 62.4 (2CH
2
CH
2
S), 63.2, 64.1 (C-1/C-5), 70.7, 72.0,
73.4 (3CH
2
CH]), 76.9, 77.2, 78.3 (C-2/C-3/C-4), 116.8, 116.9,
117.0 (3CH]CH
2
), 118.8, 119.0, 129.3, 136.0, 137.4 (Arom.),
135.5, 135.6, 135.8 (3CH]CH
2
), 153.7, 153.9 (C]O).
Anal. calcd for (C
29
H
34
N
2
O
7
)
50
(C
19
H
20
N
2
O
4
S
2
)
50
: C, 62.19; H,
5.87; N, 6.04; S, 6.92. Found: C, 62.08; H, 5.64; N, 6.37; S, 6.63.
Copolyurethane PU{[(ArAll
3
)
20
-DiT
80
]MDI} (8). This was
obtained from ArAll
3
(1, 0.10 g, 0.37 mmol), DiT (180 mL, 1.47
mmol) and MDI (0.46 g, 1.84 mmol) in DMF (3 mL), following
the procedure described previously for 4,at25C. The reaction
was worked up as described above to give the title compound as
a solid (0.73 g, 93%).
M
w
12 200; M
n
7400; M
w
/M
n
1.6. Intrinsic viscosity: 0.11 dL
g
1
. IR: n(cm
1
) 3321 (N–H), 3037 (C–H vinyl), 1700 (C]O
urethane), 1525 (N–H, N–C]O, urethane), 1219 (C–N), 1068 (C–
O–C).
1
H NMR (DMSO-d
6
, 500 MHz): d(ppm) 3.05 (bs, 4H,
2CH
2
CH
2
S), 3.49–3.88 (m, 7H, PhCH
2
Ph/H-2/H-3/H-4), 3.89–
4.51 (m, 14H, 3CH
2
CH]/H-1/H-5/2CH
2
CH
2
S), 5.08–5.30 (m,
6H, 3CH]CH
2
), 5.83–5.98 (m, 3H, 3CH]CH
2
), 7.01–7.16 (m,
4H, Arom.), 7.26–7.49 (m, 8H, Arom./NH).
13
C NMR (DMSO-d
6
,
125 MHz): d(ppm) 37.3 (2CH
2
CH
2
S), 41.6 (PhCH
2
Ph), 62.4
(2CH
2
CH
2
S), 63.8, 64.1 (C-1/C-5), 70.7, 72.0, 73.4 (3CH
2
CH]),
76.9, 77.1, 78.3 (C-2/C-3/C-4), 116.8, 116.9 (3CH]CH
2
), 118.8,
119.0, 129.3, 137.3, 137.4 (Arom.), 135.3, 135.6, 135.8 (3CH]
CH
2
), 153.7 (C]O).
Anal. calcd for (C
29
H
34
N
2
O
7
)
20
(C
19
H
20
N
2
O
4
S
2
)
80
: C, 58.91; H,
5.37; N, 6.54; S, 11.98. Found: C, 58.88; H, 5.09; N, 6.53; S, 12.12.
Copolyurethane PU{[(ArAll
2
)
20
-DiT
80
]MDI} (9). This was
obtained from ArAll
2
(2, 0.09 g, 0.36 mmol), DiT (177 mL, 1.44
mmol) and MDI (0.45 g, 1.80 mmol) in DMF (3 mL), following
the procedure described previously for 4,at25C. The reaction
was worked up as described above to give the title compound as
a solid (0.69 g, 91%).
M
w
73 900; M
n
36 100; M
w
/M
n
2.0. Intrinsic viscosity: 0.37 dL
g
1
. IR: n(cm
1
) 3316 (N–H), 3033 (C–H vinyl), 1699 (C]O
urethane), 1522 (N–H, N–C]O, urethane), 1216 (C–N), 1069 (C–
O–C).
1
H NMR (DMSO-d
6
, 500 MHz): d(ppm) 2.94–3.10 (bs, 4H,
2CH
2
CH
2
S), 3.24–3.85 (m, 10H, H-2/H-3/H-4/PhCH
2
Ph/CH
3
),
3.95–4.51 (m, 12H, H-1/H-5/2CH
2
CH]/2CH
2
CH
2
S), 5.01–5.33
(m, 4H, 2CH]CH
2
), 5.77–5.98 (m, 2H, 2CH]CH
2
), 6.94–7.17
(m, 8H, Arom./NH), 7.22–7.45 (m, 4H, Arom.).
13
C NMR (DMSO-
d
6
, 125 MHz): d(ppm) 36.7 (2CH
2
CH
2
S), 40.0 (PhCH
2
Ph), 57.0
(CH
3
), 61.9 (2CH
2
CH
2
S), 63.3, 64.9 (C-1/C-5), 71.5, 71.6
(2CH
2
CH]), 76.5, 77.4, 78.3 (C-2/C-3/C-4), 116.0, 116.5 (2CH]
CH
2
), 118.3, 118.4, 128.8, 135.6, 136.8 (Arom.), 134.7, 135.3,
(2CH]CH
2
), 153.2 (C]O).
Anal. calcd for (C
27
H
32
N
2
O
7
)
20
(C
19
H
20
N
2
O
4
S
2
)
80
: C, 58.50; H,
5.34; N, 6.62; S, 12.13. Found: C, 58.70; H, 5.12; N, 6.78; S, 12.09.
Synthesis of protected amine-based copolyurethanes
Copolyurethane PU{[Ar(S-NHBoc)
50
-DiT
50
]HMDI} (10). The
diol monomer 2,3,4-tri-O-{3-[2-(tert-butoxycarbonylamino)
ethyl]thiopropyl}-L-arabinitol (3,
23
0.20 g, 0.25 mmol) was loaded
into a round-bottom ask with an argon–vacuum inlet. The
system was treated with three cycles of vacuum–argon before
the addition, via cannula, of dried DMF (1 mL) and 2,20-
dithiodiethanol (DiT, 30 mL, 0.25 mmol). The mixture was stir-
red to homogenization, and the diisocyanate (HMDI, 80 mL, 0.50
mmol) was added under an argon atmosphere, followed by the
catalyst (dibutyltin dilaurate, one drop). The polymerization
solution was stirred at 25 C for 5 h under an argon atmosphere.
Methanol (0.1 mL) was added, the mixture was stirred for 30
min, and the solution was added dropwise into cold diethyl
ether (250 mL), where the polymer PU{[Ar(S-NHBoc)
50
-DiT
50
]
HMDI} (10) precipitated. The polymer was dried under vacuum
for 2 days and stored in a desiccator (0.26 g, 82%).
2372 |Polym. Chem.,2014,5,2370–2381 This journal is © The Royal Society of Chemistry 2014
Polymer Chemistry Paper

M
w
25 400; M
n
12 700; M
w
/M
n
2.0. Intrinsic viscosity: 0.33 dL
g
1
. IR: n(cm
1
) 3323 (N–H), 2928 (C–H), 1694 (C]O urethane),
1529 (N–H, N–C]O, urethane), 1251 (C–N), 1050 (C–O–C).
1
H
NMR (DMSO-d
6
, 500 MHz): d(ppm) 1.24 (bs, 8H, CH
2
-c), 1.38
[bs, 35H, CH
2
-b/3C(CH
3
)
3
], 1.74 (bs, 6H, 3CH
2
-e), 2.48–2.61 (m,
12H, 3CH
2
-f/3CH
2
-g), 2.89–3.14 (m, 18H, CH
2
-a/2CH
2
CH
2
S/
3CH
2
-h), 3.37–3.86 (m, 9H, H-2/H-3/H-4/3CH
2
-d), 4.05–4.30 (m,
8H, H-1/H-5/2CH
2
CH
2
S), 5.70, 6.87, 7.15 (bs, 7H, NH).
13
C NMR
(DMSO-d
6
, 125 MHz): d(ppm) 26.4 (C-c), 28.2, 31.3 (3C-g/3C-f),
28.7 [3C(CH
3
)
3
], 29.7 (C-b), 30.4 (C-e), 37.7 (2CH
2
CH
2
S), 39.9,
40.1, 40.2 (3C-h), 40.6 (C-a), 62.0 (2CH
2
CH
2
S), 62.8, 63.5 (C-1/C-
5), 68.1, 69.5, 71.0 (3C-d), 77.6, 78.4, 79.7 (C-2/C-3/C-4), 78.1
[3C(CH
3
)
3
], 155.9, 156.4 (C]O).
Anal. calcd for (C
43
H
81
N
5
O
13
S
3
)
50
(C
12
H
22
N
2
O
4
S
2
)
50
: C, 51.02;
H, 8.02; N, 7.57; S, 12.38. Found: C, 50.92; H, 8.03; N, 7.80; S,
12.34.
Copolyurethane PU{[Ar(S-NHBoc)
20
-DiT
80
]HMDI} (11). This
was obtained from 2,3,4-tri-O-{3-[2-(tert-butoxycarbonylamino)
ethyl]thiopropyl}-L-arabinitol (3, 0.18 g, 0.22 mmol), DiT (110
mL, 0.89 mmol) and HMDI (180 mL, 1.11 mmol) in DMF (1 mL),
following the procedure described previously for 10,at25C.
The reaction was worked up as described above to give the title
compound as a solid (0.38 g, 76%).
M
w
20 700; M
n
12 000; M
w
/M
n
1.7. Intrinsic viscosity: 0.31 dL
g
1
. IR: n(cm
1
) 3322 (N–H), 2 924, 2856 (C–H), 1682 (C]O
urethane), 1530 (N–H, N–C]O, urethane), 1251 (C–N), 1068 (C–
O–C).
1
H NMR (DMSO-d
6
, 500 MHz): d(ppm) 1.25 (bs, 8H, CH
2
-
c), 1.38 [bs, 35H, CH
2
-b/3C(CH
3
)
3
], 1.73 (bs, 6H, 3CH
2
-e), 2.45–
2.62 (m, 12H, 3CH
2
-f/3CH
2
-g), 2.89–3.14 (m, 18H, CH
2
-a/
2CH
2
CH
2
S/3CH
2
-h), 3.17–3.79 (m, 9H, H-2/H-3/H-4/3CH
2
-d),
4.05–4.33 (m, 8H, H-1/H-5/2CH
2
CH
2
S), 5.71, 6.85, 7.13 (bs, 7H,
NH).
13
C NMR (DMSO-d
6
, 125 MHz): d(ppm) 26.4 (C–c), 28.3,
31.3 (3C-g/3C-f), 28.7 [3C(CH
3
)
3
], 29.8 (C-b), 30.4 (C-e), 37.7
(2CH
2
CH
2
S), 39.7, 40.5 (3C-h), 40.7 (C-a), 62.0 (2CH
2
CH
2
S), 62.8,
63.4 (C-1/C-5), 68.1, 69.4, 70.9 (3C-d), 79.8, 80.1, 80.4 (C-2/C-3/C-
4), 78.2 [3C(CH
3
)
3
], 156.0, 156.4 (C]O).
Anal. calcd for (C
43
H
81
N
5
O
13
S
3
)
20
(C
12
H
22
N
2
O
4
S
2
)
80
: C, 48.32;
H, 7.53; N, 7.53; S, 15.59. Found: C, 48.59; H, 7.49; N, 8.12; S,
15.28.
Copolyurethane PU{[Ar(S-NHBoc)
50
-DiT
50
]MDI} (12). This
was obtained from 2,3,4-tri-O-{3-[2-(tert-butoxycarbonylamino)
ethyl]thiopropyl}-L-arabinitol (3, 0.10 g, 0.12 mmol), DiT (15 mL,
0.12 mmol) and MDI (0.06 g, 0.24 mmol) in DMF (1 mL),
following the procedure described previously for 10,at25C.
The reaction was worked up as described above to give the title
compound as a solid (0.14 g, 80%).
M
w
8800; M
n
6600; M
w
/M
n
1.3. Intrinsic viscosity: 0.10 dL g
1
.
IR: n(cm
1
) 3311 (N–H), 2921 (C–H), 1692 (C]O urethane),
1530 (N–H, N–C]O, urethane), 1220 (C–N), 1066 (C–O–C).
1
H
NMR (DMSO-d
6
, 500 MHz): d(ppm) 1.39 [bs, 27H, 3C(CH
3
)
3
],
1.66–1.83 (m, 6H, 3CH
2
-e), 2.43–2.64 (m, 12H, 3CH
2
-f/3CH
2
-g),
2.97–3.16 (m, 10H, 2CH
2
CH
2
S/3CH
2
-h), 3.22–3.88 (m, 13H, H-2/
H-3/H-4/3CH
2
-d/PhCH
2
Ph), 4.00–4.68 (m, 8H, H-1/H-5/
2CH
2
CH
2
S), 5.33, 6.85, 7.13 (bs, 7H, NH), 7.00–7.18; 7.27–7.49
(m, 16H, Arom.).
13
C NMR (DMSO-d
6
, 125 MHz): d(ppm) 28.3,
31.3 (3C-g/3C-f), 28.6 [3C(CH
3
)
3
], 30.4 (C-e), 37.5 (2CH
2
CH
2
S),
40.1 (PhCH
2
Ph), 40.5, 41.7 (3C-h), 62.5 (2CH
2
CH
2
S), 63.1, 63.7
(C-1/C-5), 68.1, 69.4, 70.8 (3C-d), 77.4, 77.6, 78.2 (C-2/C-3/C-4),
78.2 [3C(CH
3
)
3
], 119.0, 129.3, 135.7, 137.0 (Arom.), 153.7, 156.0
(C]O).
Anal. calcd for (C
50
H
79
N
5
O
13
S
3
)
50
(C
19
H
20
N
2
O
4
S
2
)
50
: C, 56.81;
H, 6.84; N, 6.72; S, 10.99. Found: C, 56.79; H, 6.77; N, 6.75; S,
10.98.
Synthesis of amine-based copolyurethanes
Copolyurethane PU{[(Ar(S–NH
2
)
3
)
50
-DiT
50
]HMDI} (13). A
solution of PU{[Ar(S-NHBoc)
50
-DiT
50
]HMDI} (10, 0.15 g, 0.69
meq. NHBoc) in a 1 : 2 mixture of 37% HCl–THF (2 mL) was
stirred at 25 C for 4 h.
23
The mixture, neutralized with a diluted
solution of sodium carbonate, was added dropwise into cold
diethyl ether (200 mL) to give the title copolyurethane as a solid
(0.11 g, 96%).
M
w
22 900; M
n
11 100; M
w
/M
n
1.7. Intrinsic viscosity: 0.20 dL
g
1
. IR: n(cm
1
) 3327 (N–H), 2932 (C–H), 1692 (C]O urethane),
1518 (N–H, N–C]O, urethane), 1250 (C–N), 1072 (C–O–C).
1
H
NMR (DMSO-d
6
, 500 MHz): d(ppm) 1.26 (bs, 8H, CH
2
-c), 1.39
(bs, 8H, CH
2
-b), 1.76 (bs, 6H, 3CH
2
-e), 2.51–2.76 (m, 12H, 3CH
2
-
f/3CH
2
-g), 2.74–3.07 (m, 12H, CH
2
-a/2CH
2
CH
2
S/3CH
2
-h), 3.27–
3.81 (m, 9H, H-2/H-3/H-4/3CH
2
-d), 3.87–4.40 (m, 14H, H-1/H-5/
2CH
2
CH
2
S), 7.12, 8.11 (bs, 10H, NH/3NH
2
).
13
C NMR (DMSO-d
6
,
125 MHz): d(ppm) 26.6 (C–c), 28.7, 30.4 (3C-g/3C-f), 29.9 (C-b),
30.0 (C-e), 37.9 (2CH
2
CH
2
S), 39.8 (3C-h), 40.5 (C-a), 58.9, 62.6 (C-
1/C-5), 62.1 (2CH
2
CH
2
S), 69.3, 70.0, 71.1 (3C-d), 77.7, 78.4, 79.9
(C-2/C-3/C-4), 156.5 (C]O).
Anal. calcd for (C
28
H
57
N
5
O
7
S
3
)
50
(C
12
H
22
N
2
O
4
S
2
)
50
: C, 48.31;
H, 8.01; N, 9.86; S, 16.12. Found: C, 48.62; H, 8.25; N, 9.87; S,
16.41.
Copolyurethane PU{[(Ar(S–NH
2
)
3
)
20
-DiT
80
]-HMDI} (14). This
was obtained from PU{[Ar(S-NHBoc)
20
-DiT
80
]HMDI} (11, 0.15 g,
1.00 meq. NHBoc) in a 1 : 2 mixture of 37% HCl–THF (3 mL),
following the procedure described previously for 13. The reac-
tion was worked up as described above to give the title
compound as a solid (0.12 g, 90%).
M
w
17 200; M
n
11 800; M
w
/M
n
1.5. Intrinsic viscosity: 0.15 dL
g
1
. IR: n(cm
1
) 3320 (N–H), 2935 (C–H), 1677 (C]O urethane),
1531 (N–H, N–C]O, urethane), 1260 (C–N), 1068 (C–O–C).
1
H
NMR (DMSO-d
6
, 500 MHz): d(ppm) 1.24 (bs, 8H, CH
2
-c), 1.38
(bs, 8H, CH
2
-b), 1.75 (bs, 6H, 3CH
2
-e), 2.49–2.66 (m, 12H, 3CH
2
-
f/3CH
2
-g), 2.71–3.05 (m, 12H, CH
2
-a/2CH
2
CH
2
S/3CH
2
-h), 3.25–
3.78 (m, 9H, H-2/H-3/H-4/3CH
2
-d), 3.87–4.37 (m, 14H, H-1/H-5/
2CH
2
CH
2
S), 7.15, 8.15 (bs, 10H, NH/3NH
2
).
13
C NMR (DMSO-d
6
,
125 MHz): d(ppm) 26.4 (C–c), 28.4, 30.5 (3C-g/3C-f), 29.7 (C-b),
30.1 (C-e), 37.7 (2CH
2
CH
2
S), 39.7 (3C-h), 40.7 (C-a), 59.9, 62.4 (C-
1/C-5), 62.0 (2CH
2
CH
2
S), 68.3, 69.6, 71.0 (3C-d), 77.6, 78.2, 79.7
(C-2/C-3/C-4), 156.4 (C]O).
Anal. calcd for (C
28
H
57
N
5
O
7
S
3
)
20
(C
12
H
22
N
2
O
4
S
2
)
80
: C, 46.53; H,
7.45; N, 9.28; S, 17.98. Found: C, 47.01; H, 7.30; N, 9.45; S, 17.34.
Copolyurethane PU{[(Ar(S–NH
2
)
3
)
50
-DiT
50
]MDI} (15). This
was obtained from PU{[Ar(S-NHBoc)
50
-DiT
50
] MDI} (12, 0.18 g,
0.72 m eq. NHBoc) in a 1 : 2 mixture of 37% HCl–THF (1.0 mL),
following the procedure described previously for 13. The reac-
tion was worked up as described above to give the title
compound as a solid (0.12 g, 83%).
This journal is © The Royal Society of Chemistry 2014 Polym. Chem.,2014,5, 2370–2381 | 2373
Paper Polymer Chemistry

M
w
8800; M
n
6800; M
w
/M
n
1.3. Intrinsic viscosity: 0.10 dL g
1
.
IR: n(cm
1
) 3312 (N–H), 2922 (C–H), 1702 (C]O urethane),
1509 (N–H, N–C]O, urethane), 1226 (C–N), 1071 (C–O–C).
1
H
NMR (DMSO-d
6
, 500 MHz): d(ppm) 1.77 (bs, 6H, 3CH
2
-e), 2.48–
2.68 (m, 12H, 3CH
2
-f/3CH
2
-g), 2.99–3.15 (m, 10H, 2CH
2
CH
2
S/
3CH
2
-h), 3.25–3.91 (m, 13H, H-2/H-3/H-4/3CH
2
-d/PhCH
2
Ph),
4.01–4.68 (m, 8H, H-1/H-5/2CH
2
CH
2
S), 5.33, 7.13, 8.20 (bs, 7H,
NH/3NH
2
), 7.01–7.19, 7.30–7.50 (m, 16H, Arom.).
13
C NMR
(DMSO-d
6
, 125 MHz): d(ppm) 28.0, 28.3 (3C-g/3C-f), 30.2 (C-e),
37.4 (2CH
2
CH
2
S), 40.2 (PhCH
2
Ph), 41.7 (3C-h), 62.4
(2CH
2
CH
2
S), 63.0, 63.9 (C-1/C-5), 68.0, 69.6, 70.8 (3C-d), 77.4,
77.7, 78.4 (C-2/C-3/C-4), 119.0, 129.3, 135.7, 137.0 (Arom.), 153.7
(C]O).
Anal. calcd for (C
35
H
55
N
5
O
7
S
3
)
50
(C
19
H
20
N
2
O
4
S
2
)
50
: C, 55.98;
H, 6.53; N, 8.46; S, 13.84. Found: C, 55.78; H, 6.95; N, 8.60; S,
13.48.
Degradation of allyl and amine-based copolyurethanes with
glutathione
Preparation of polymer disks. Five polymer disks were made
from each polymer studied. The disks were prepared by the
application of 10 ton cm
1
pressure over the powdered polymer
(20 mg) for 5 minutes. The disks were then dried at 45 C for
3 days.
Degradation conditions. Every polymer disk was submerged
in a reduced glutathione solution (5 mL buffer solution, 5 mM),
at pH 7.40. An argon ow was passed through the solution for 5
minutes and the vial was then sealed. The solutions were heated
at 37 0.5 C and stirred with an orbital stirrer (200 rpm) to
ensure access of the tripeptide to the whole surface of the
polymer samples. The solutions were changed every two days
and replaced with freshly prepared solutions, to avoid loss of
activity with time. The process was monitored for an exact
period of time (1, 2, 4, 7, or 10 days).
Quenched degradation experiments. The vial content was
poured into a ask provided with a stirring bar. THF (10 mL),
sodium hydrogen carbonate (252 mg, 3 mmol), and methyl
iodide (0.44 mL, 7 mmol) were added sequentially. The mixture
was stirred at 25 C for 5 h. The liquid phase was eliminated and
the disks were washed with distilled water (3 10 mL) and then
dried. The molecular weights (M
n
and M
w
) and polydispersity
indices (M
w
/M
n
) of the degraded polymers were studied by gel
permeation chromatography (GPC).
Drug delivery studies
Preparation and coating of tablets. In a typical procedure,
500 mg of a powder blend containing 98% of Fast Flo lactose, 1%
of aerosil, and 1% of magnesium stearate were blended for 5
minutes in a Turbula mixer (Willy A. Bachofen, Basel, Switzer-
land). This blend was then suspended in THF (0.5 mL). Mean-
while, a solution of methotrexate (MTX) (12.5 mg, 2.75 10
2
mmol) and polymer 14 (18 mg, 2.75 10
2
meq.–NH
2
)inTHF
(0.5 mL) was prepared. The suspension and the solution were
then mixed in a glass vial and gently stirred for 4 h at 25 C. The
THF was removed by a continuous ow of argon at 25 C over-
night and then under vacuum for a further 24 h. The nal
mixture was then converted into a disk by the application of 10
ton cm
1
pressure for 1 min, at 25 C. The tablet obtained was
later coated with Eudragit FS 30 D up to a nal weight gain of 1%.
Drug release study. An in vitro dissolution study was carried
out using the paddle method in a Sotax AT7 Smart USP disso-
lution apparatus (Allschwil, Switzerland). During the test, the
formulation was exposed to four phases simulating, in subse-
quent order, the stomach, jejunum, distal ileum, and proximal
colon. The pH gradient is based on the method reported by
Schellekens et al.
27
The specications of these phases are shown
in Table 1.
At the end of each phase, a switch solution was added to
obtain the required composition of the next phase. Table 2
shows the composition of these switch solutions. The rotation
speed of the paddle was 50 rpm and the temperature of the
dissolution medium was kept at 37 0.5 C. In order to have a
better simulation of the physicochemical conditions in the
colon, we modied the method reported by Schellekens et al.,
27
creating a reductive environment during phase IV, corre-
sponding to the colon. For this purpose the experiments were
carried out under an argon atmosphere, and glutathione was
added to the media up to a nal concentration of 5 mM.
Several samples (5 mL each) were withdrawn at 30, 60, 90,
120, 150, 180, 210, 240, 250, 260, 270, 285, 300, 315, 330, 345,
360, 1440, and 3060 minutes and measured in an Agilent 8453
UV/VIS spectrophotometer (California, USA). The methotrexate
content was calculated using a previously determined calibra-
tion curve at 303 nm.
28
Results and discussion
Synthesis of functionalized copolymers
Reduction-sensitive biodegradable polymers and conjugates
have emerged as a fascinating class of biomedical materials that
can be elegantly applied to drug delivery systems, as shown in
the interesting review of Zhong et al.
12
Such polymers incorpo-
rate disulde linkage(s) that can be reduced by GSH in the body.
In order to use those polymers as drug or gene carriers, an
essential feature is a reversible (physical or chemical) linkage
between the polymer and the drug to be delivered in the human
body. For this purpose, there are two main options: a covalent
hydrolyzable union (e.g., an ester or urethane linkage), or ionic
interactions between oppositely charged moieties (e.g., carbox-
ylates and ammonium ions). Both ester and urethane bonds can
be easily cleaved by enzymes.
29–31
In aqueous solutions, and if
Table 1 Specifications of the four phases of in vitro dissolution assays
Phase GI-segment Vol. (mL)
Residence
time (h) pH
I Stomach 500 2.0 1.2
II Jejunum 575 2.0 6.8
III Ileum (distal) 842 0.5 7.5
IV Colon
(proximal)
922 1.5 6.0
2374 |Polym. Chem.,2014,5,2370–2381 This journal is © The Royal Society of Chemistry 2014
Polymer Chemistry Paper

the drug has been anchored to the polymer by ionic-type
interactions, the high dielectric constant of water will be able to
lower the attractive forces between the charged particles, and
thereby enable modulation of the drug release in the body.
To obtain reduction-sensitive biodegradable polymers that
can be used as carriers of negatively charged drugs or gene
material, we aimed to synthesize novel multiamine-based
copolyurethanes that contain disulde linkages in the polymer
backbone. Furthermore, the introduction of polar moieties into
the new carriers is of interest, since they can boost the hygro-
scopic nature of the polymers and, hence, their degradation rate
under physiological conditions in the human body.
11
The copolymerization procedure, as well as the degradability,
was initially tested by preparing various multiallyl disulde-
containing copolyurethanes. The two functional allyl diol
monomers 2,3,4-tri-O-allyl-L-arabinitol (ArAll
3
,1) and 3,4-di-O-
allyl-2-O-methyl-L-arabinitol (ArAll
2
,2), and the commercial 2,20-
dithiodiethanol (DiT) were the starting diol materials for the
preparation of the novel linear multiallyl-based copolyurethanes
(4–9, Scheme 1). Thus, mixtures of the L-arabinitol-based
monomers (1or 2) and 2,20-dithiodiethanol (DiT) with lower or
equal proportion of the sugar-based monomer related to DiT
were used. The incorporation of aromatic or aliphatic segments
due to the chosen diisocyanate (HMDI or MDI in all cases) may
impart diverse properties, and hence, specic applications—
from industrial to biomedical ones. The copolymerization reac-
tions took place under an argon atmosphere at 25 C in DMF for
5 h to ensure the total consumption of diol monomers. The
recovered multiallyl copolyurethanes (4–9) were isolated by
precipitation in diethyl ether with excellent yields (>85%).
To synthesize the copolymers PU{[(Ar(S–NH
2
)
3
)
50
-DiT
50
]-
HMDI}] (13), PU{[(Ar(S–NH
2
)
3
)
20
-DiT
80
]-HMDI} (14), and PU
{[(Ar(S–NH
2
)
3
)
50
-DiT
50
]-MDI} (15) (Scheme 2), the starting
material used was the sugar-based diol monomer 2,3,4-tri-O-{3-
[2-(tert-butoxycarbonylamino)ethyl]thiopropyl}-L-arabinitol (3),
which was freshly prepared according to the thiol–ene strategy
described previously by us.
23
A priori, the thiol–ene reaction between a precursor of the
diol 3, the fully protected 2,3,4-tri-O-allyl-1,5,-di-O-trityl-L-ara-
binitol, and the unprotected cysteamine could work; the
subsequent detritylation would render the expected diol 2,3,4-
tri-O-[3-(2-aminoethyl)thiopropyl]-L-arabinitol which, however,
could not be used as the diol monomer in the polymer synthesis
because signicant cross-linking side reactions could take place
due to the amine groups. The copolymerization procedure used
(Scheme 2) was analogous to the method utilized for the mul-
tiallyl-based copolyurethanes; the choice of compound 3(with
the amine groups conveniently protected) prevented those side
reactions.
The amount of amino pendant groups in the copolymer
backbone was controlled by the molar ratio of the L-arabinitol-
based monomers (1or 2) in the feed.
The synthesized polymers were fully characterized by GPC,
FTIR spectra, NMR experiments, and elemental analysis. The
experimental copolymer compositions were studied by
elemental microanalysis. These data reveal that the copolymer
content in the L-arabinitol monomer and DiT units is in good
agreement with that of the corresponding feed.
Properties of new copolyurethanes
Thermal properties. The thermal properties of these mate-
rials were investigated by Differential Scanning Calorimetry
(DSC) and Thermogravimetric Analysis (TGA). Molecular
weights, polydispersities, and thermal properties of PUs are
shown in Table 3.
The DSC analysis of the polymers studied in the present
study revealed that the MDI-based PUs are essentially amor-
phous and the HMDI-based PUs have semicrystalline struc-
tures. MDI-based polymers are also stiffer than the HMDI
counterparts, as can be inferred from their T
g
values. The T
g
of
HMDI-based PUs are below the physiological temperature,
ranging from 14 Cto34C, whereas in the case of MDI-based
PUs the values range from 51 Cto91C.
Fig. 1 displays the heat ow vs. temperature curve for the
polymer PU{[Ar(S-NHBoc)
20
-DiT
80
]HMDI} (11). It shows two endo-
thermic peaks at 20 C and 117 C (corresponding to T
g
and T
m
,
respectively), and an exothermic peak at 80 C, due to the partial
crystallization of the melted amorphous phase into crystallites and
the subsequent growth of the crystalline fraction in the sample.
Table 2 Composition of the switch solutions
From To Contents
Switch solution I Phase I Phase II 2–37 g potassium dihydrogen
phosphate, 17.44 mL sodium
hydroxide 2.0 M, demineralized
water up to 75 mL
Switch solution II Phase II Phase III 1.75 g potassium dihydrogen
phosphate, 10.30 mL sodium
hydroxide 2.0 M, demineralized
water up to 267 mL
Switch solution III Phase III Phase IV 12 mL hydrochloric acid 3.0 M,
demineralized water up to 80 mL,
28.3 g of glutathione, inert
atmosphere
This journal is © The Royal Society of Chemistry 2014 Polym. Chem.,2014,5, 2370–2381 | 2375
Paper Polymer Chemistry

Scheme 2 Synthesis of multiamine disulfide-containing copolyurethanes. (i) HMDI, DMF, dibutyltin dilaurate, 25 C; (ii) MDI, DMF, dibutyltin
dilaurate, 25 C; (iii) 37% HCl–THF 1 : 2 mixture, 25 C, neutralization.
Scheme 1 Polymerization reactions leading to multiallyl disulfide-containing copolyurethanes. (i) HMDI, DMF, dibutyltin dilaurate, 25 C; (ii) MDI,
DMF, dibutyltin dilaurate, 25 C.
2376 |Polym. Chem.,2014,5, 2370–2381 This journal is © The Royal Society of Chemistry 2014
Polymer Chemistry Paper

Fig. 2 exemplies how the functionalization of the sugar
moiety in the polymer greatly affects its T
g
; thus, DSC traces of
selected, chemically related L-arabinitol-based copolyurethanes
(with various functional pendant groups, allyl, -NHBoc, and
amino groups) are displayed.
The replacement of allyl groups in PU{[(ArAll
3
)
50
-DiT
50
]
HMDI} (4) by 3-[2-(tert-butoxycarbonylamino)ethylthio]propyl
groups in the polymer PU{[Ar(S-NHBoc)
50
-DiT
50
]HMDI} (10) led
to a signicant reduction in T
g
(from 34 Cto13C), probably
due to the high-bulk pendant groups in the polymer chains.
However, once the amino groups were deprotected, a stiffer
material was obtained (PU{[(Ar(S–NH
2
)
3
)
50
-DiT
50
]HMDI} (13), T
g
32 C), probably because of the increased number of
hydrogen bonds. The same trend was observed in most cases for
batches of three chemically related polymers (with allyl, NHBoc,
and amino pendant groups, Table 3).
Comparing the T
m
values of polymers PU{[(ArAll
3
)
50
-DiT
50
]
HMDI} (4) and PU{[(ArAll
3
)
20
-DiT
80
]HMDI} (5) with their corre-
sponding functionalized counterparts PU{[(Ar(S–NH
2
)
3
)
50
-DiT
50
]
HMDI} (13) and PU{[(Ar(S–NH
2
)
3
)
20
-DiT
80
]-HMDI} (14), respec-
tively, a shiof less than 4 C was observed, whereas there was a
signicant decrease in DH
m
when the allyl-containing pendant
chains in copolymers 4and 5were replaced by longer amino-
derivative side chains. As expected, for the semicrystalline
copolymers, the smaller the length of the side chains, the higher
the crystalline fraction in the polymeric structure, as conrmed
by the DH
m
values. In addition, the NHBoc-based polyurethanes
10 and 11 showed a fall in T
m
of over 10 C in relation to 4and 5,
respectively, as well as a considerable reduction in their fusion
enthalpies. In summary, it seems that the presence of long
aliphatic side chains in 10,11,13, and 14 decreases the ratio of
the crystalline phase in the materials.
Another parameter that exhibited a marked impact on
melting temperatures was the polymer DiT content. So, when
the DiT diol was the main monomer in the polymerization feed
(80% molar ratio in polymers 5,11, and 14) compared with the
Table 3 Molecular weights and thermal properties of synthesized polyurethanes
Polymer
GPC DSC TGA
M
wa
M
w
/M
na
T
m
/DH
b
T
gc
Td
0d
Td
e
DW
f
(%)
PU{[(ArAll
3
)
50
-DiT
50
]HMDI} (4) 10 500 1.5 88/90 34 261 293/307/464 44/34/12
PU{[(ArAll
3
)
20
-DiT
80
]HMDI} (5) 15 600 1.6 128/53 14 263 265/346/458 53/27/10
PU{[(ArAll
2
)
20
-DiT
80
]HMDI} (6) 35 000 1.9 129/50 14 275 278/352/454 61/24/8
PU{[(ArAll
3
)
50
-DiT
50
]MDI} (7) 25 200 1.8 —81 271 278/328/402 42/40/18
PU{[(ArAll
3
)
20
-DiT
80
]MDI} (8) 12 200 1.6 —85 222 245/300/551 45/40/5
PU{[(ArAll
2
)
20
-DiT
80
]MDI} (9) 73 900 2.0 —91 207 233/300/467 43/39/9
PU{[Ar(S-NHBoc)
50
-DiT
50
]HMDI} (10) 25 400 2.0 77/42 13 238 254/360/449 24/67/4
PU{[Ar(S-NHBoc)
20
-DiT
80
]HMDI} (11) 20 700 1.7 117/33 20 230 249/354/425 48/45/6
PU{[Ar(S-NHBoc)
50
-DiT
50
]MDI} (12) 8800 1.3 —51 218 225/309/348 38/44/17
PU{[(Ar(S–NH
2
)
3
)
50
-DiT
50
]HMDI} (13) 22 900 1.7 89/20 32 235 241/324 22/61
PU{[(Ar(S–NH
2
)
3
)
20
-DiT
80
]-HMDI} (14) 17 200 1.5 124/24 24 259 271/340 43/48
PU{[(Ar(S–NH
2
)
3
)
50
-DiT
50
]MDI} (15) 8800 1.3 —77 209 246/319/356 25/51/19
a
Determined by GPC analysis against polystyrene standards using NMP as the mobile phase.
b
The melting temperatures (C, T
m
) and their
respective enthalpies (DH) were measured by DSC at heating rates of 10 C min
1
.
c
The glass-transition temperatures (C, T
g
) were taken as the
inection point of the heating DSC traces of melt-quenched samples recorded at 20 C min
1
.
d
Onset temperature (C): temperature at which a
10% weight loss was observed in the TGA traces recorded at 10 C min
1
.
e
Decomposition temperatures (C) measured at the peaks of the
derivative curves; major peaks in bold.
f
Weight loss at the end of the decomposition step.
Fig. 1 Heat flow vs. temperature curve of polymer 11.
Fig. 2 DSC traces of selected, chemically related L-arabinitol-based
copolyurethanes.
This journal is © The Royal Society of Chemistry 2014 Polym. Chem.,2014,5, 2370–2381 | 2377
Paper Polymer Chemistry

polymers that present a 50% molar ratio (PUs 4,10, and 13,
respectively), T
m
values increased around 40 C. In contrast,
choosing the diallyl- or triallyl-diol monomer for the prepara-
tion of new polymers had no clear effect on the DSC traces of the
resulting materials. So, when the DSC traces of PU{[(ArAll
3
)
20
-
DiT
80
]HMDI} (5) and PU{[(ArAll
2
)
20
-DiT
80
]HMDI} (6) were
superimposed for comparison, they showed no major
differences.
Thermal stability of the copolyurethanes was evaluated by
thermogravimetry (TGA) under an inert atmosphere. Detailed
temperatures and weight loss data for all the studied materials
are given in Table 3. The copolymers were stable to thermal
degradation up to 200 C.
The TG curves of the copolymers show a two- or three-step
degradation process. In the rst stage, which is the main tran-
sition in most cases, the copolymer degradation is related to the
disulde segments. This step occurred at 225–293 C, with an
associated weight loss ranging from 22% to 61%. The thermal
cleavage of a disulde bond in proteins is the result of b-elim-
ination from the cystine residue, and could lead to thiocysteine
residues (and a possible loss of elemental sulfur), cysteine
residues, and dehydroalanine.
32
This trend backs up the notion
that b-elimination is a general mechanism of disulde
compounds, as observed in the case of cystine. The degradation
may involve cleavage of the disulde bond by b-elimination and
the formation of elemental sulfur and terminal alkenes. As
expected, the weight loss associated with the degradation of
disulde bonds increased with the DiT ratio (from 50% to 80%)
in the polymers. Thus, for the HMDI-based copolyurethanes, it
increased from 44% to 53% for the allyl-containing copolymers,
from 24% to 48% for the NHBoc-containing copolymers, and
from 22% to 43% for the amine-functionalized ones.
As previously reported,
11
the decomposition of disulde
bonds occurred at different temperatures for each polymer.
Generally speaking, the copolymers with the highest DiT content
in the feed exhibited a decrease in the decomposition temper-
atures in every pair of related copolymers—feed sugar : DiT
ratios 20 : 80 and 50 : 50—with the exception of the amine
functionalized copolymers, where the trend was reversed. The
derivative thermogravimetric analysis (DTG) curves provide a
better visualization of the phenomena mentioned above. Thus,
in Fig. 3 the curves of derivative of weight vs. temperature for
polymers PU{[(ArAll
3
)
20
-DiT
80
]HMDI} (5) and PU{[(ArAll
3
)
50
-
DiT
50
]HMDI} (4) are displayed, with a shiin their rst
maximum decomposition rate from 265 C to 293 C.
Fig. 4 displays representative TGA traces of the MDI-based
copolymers 7,12, and 15. In general, the stability of the copol-
ymers functionalized with (protected or free) amino groups
decreased when compared with that of those bearing allyloxy
pendant groups in their structures, leading to materials of lower
thermal resistance. The presence of those (protected or free)
amino groups in the side chains had a marked effect on the
degradation proles, as can be seen by the fall in the onset
temperatures.
In addition, the degradation proles of allyl-functionalized
polyurethanes display another major difference with those of
copolymers functionalized with (protected or free) amino
groups: the degradation step at 455 C—probably associated
with the allyloxy groups in the former batch of polymers—was
not observed in amino-derivative PUs. Lastly, the presence of a
higher density of allyl groups in PU{[(ArAll
3
)
20
-DiT
80
]HMDI} (5)
compared with PU{[(ArAll
2
)
20
-DiT
80
]HMDI} (6) did not show
major hein TGA studies.
Glutathione-mediated degradation of new polyurethanes. As
mentioned above, disulde bonds are prone to rapid cleavage in
a reductive environment through the fast and readily reversible
thiol–disulde exchange reactions.
13,14
In a previous work, we
investigated the glutathione-mediated degradation pathway of
2,20-dithiodiethanol-based copolyurethanes where the second
diol monomers used were 2,3,4-tri-O-benzyl-L-arabinitol and
2,3,4-tri-O-methyl-L-arabinitol.
11
It was observed that the lysis
rate depends not only on the content of disulde bonds in the
polymer backbone but also on the crystallinity of the nal
macromolecule. Hence, although in principle the higher the
DiT mol percent in the feed was, the faster the lysis rate would
be, this effect can be partially overshadowed by the presence of
well-packed semicrystalline regions (with the DiT units
involved) which are of difficult access to the reducing agent (the
hydrophilic reduced glutathione).
Fig. 3 DTG profiles of two multiallyl HMDI-based copolyurethanes.
Fig. 4 Degradation profiles of three chemically related copolymers
performed by TG.
2378 |Polym. Chem.,2014,5,2370–2381 This journal is © The Royal Society of Chemistry 2014
Polymer Chemistry Paper

The degradation behavior in reductive environments (37 C,
pH 7.40, [GSH] ¼5 mM) of the new multiallyl-, NHBoc-, and
amino-based PUs was investigated. The synthesized polymers—
even those with enhanced hydrophilic character—were not
soluble in the aqueous incubation media. In these assays, no
signicant weight loss of the samples was observed. Degraded
copolyurethanes were monitored by GPC to determine the
changes in M
w
that the samples underwent during the incuba-
tion period. We can assume that the small size of the tripeptide
glutathione (the most abundant low-molecular-weight biolog-
ical thiol) facilitates its dispersion into the polymer disk
throughout the trials, with degradation effects not only on the
surface of the polymer disks but also within them.
Fig. 5 shows the evolution of M
w
of selected copolyurethanes
(4,5,7, and 14) during the reductive lyses. The data refer to DM
w
(the M
w
remaining aer degradation, as a percentage). All of
them showed the same degradation trend during the incuba-
tion period; thus, the degradation proles are characterized by a
rapid decrease in molecular weight during the rst two days,
followed by a second trend in which degradation proceeds at
lower rates.
The degradation curves of PU{[(ArAll
3
)
50
-DiT
50
]HMDI} (4)
and PU{[(ArAll
3
)
20
-DiT
80
]HMDI} (5) are almost identical, with
the degradation rate of polymer 4showing a slight improve-
ment aer 7 days. These results conrm that an increase of the
disulde content from 50 to 80 mol percent does not have a
marked effect on the cleavage of HMDI-based copolymers in
reductive environments.
As expected, the nature of the diisocyanate used played an
important role in the degradation rate. HMDI-based copolyur-
ethanes showed higher degradability than MDI-based polymers.
For instance, at the end of ten days, PU{[(ArAll
3
)
50
-DiT
50
]MDI}
(7) showed a weight loss of 30% compared with the 50% loss for
PU{[(ArAll
3
)
50
-DiT
50
]HMDI} (4). Circumstances able to obstruct
the access of the reducing agent (the hydrophilic reduced
glutathione) to the DiT moieties will decelerate the degradation
process. Thus, not only well-packed semicrystalline regions
(with the DiT unit involved), but also the presence of hydro-
phobic, highly hindered moieties (such as DiT-MDI repeating
units) in the polymer, can brake the degradation process.
Therefore, even though the MDI-based copolymers of the
present study are essentially amorphous, they are harder to
cleave than HMDI-based PUs, despite their ample DiT mol
percent (50% and 80%).
The amine-based copolymers exhibited enhanced degrada-
tion trends. The best degradation rates were obtained for the
amino-based copolymer PU{[(Ar(S–NH
2
)
3
)
20
-DiT
80
]-HMDI} (14)
with an associated weight loss close to 90% aer ten days. This
is probably due to its greater hydrophilicity compared with 4
and 5.
AFM studies. Degradation-induced changes in the surface
morphologies of lms of PU{[(ArAll
3
)
20
-DiT
80
]HMDI} (5) and PU
{[(Ar(S–NH
2
)
3
)
20
-DiT
80
]-HMDI} (14) (before and aer degrada-
tion) were observed using Atomic Force Microscopy (AFM). On
the microscopic scale, AFM is an ideal tool for visualizing the
topography of the lms. For the preparation of polymer lms by
spin-coating, polymers 5and 14 were rstly dissolved in dime-
thylsulfoxide at a weight/volume ratio of 0.05 : 1 (g mL
1
)ina
glass vial. The polymer solutions were dropped onto a mica
surface (10 mL) and the disks were subjected to four cycles of
1000 rpm (1 min each) under high vacuum, which resulted in
homogeneous polymer lms.
For degradation studies, the polymer lms were submerged
in a reduced glutathione solution (10 mL, 5 mM) at pH 7.4. An
argon ow was passed through the solutions for 5 min, and the
vials were then sealed, gently stirred, and incubated at 37 C for
an exact period of time. Once the degradation had taken place,
the samples were isolated, washed and dried, and then studied
by AFM. Images recorded in Fig. 6a–d and Fig. 6e–f are of 4 mm
4mm scans and 2.35 mm2.35 mm scans, respectively, and
represent typical areas of the resulting polymer lms.
Fig. 6a and b display the morphology of lms of polymer 5
before and aer incubation, respectively. When the lm was
prepared by dissolving polymer 5in DMSO and then subjected
to spin-coating, the topography image displayed the
morphology of a polymer with a singular neuron-like appear-
ance (Fig. 6a), which evolved to a slightly spotted surface aer
10 days of incubation in the reductive media (Fig. 6b).
In contrast, the preparation of the lm for polymer 14 by
spin-coating under identical conditions resulted in a complete
substrate coverage, and the deposited lm showed signicant
height variations. As expected, and in concordance with the
DM
w
values obtained, the polymer semblance of 14 (Fig. 6c and
e) showed signicant changes when degraded (Fig. 6d and f)—
when the lm made with polymer 14 was exposed to a reductive
environment, a marked decrease in z-axis values was achieved,
conrming the enhanced degradability feature of PU 14
compared with PU 5.
Controlled delivery of methotrexate from PU 14-based
tablets. Methotrexate (MTX) (Fig. 7) is an antimetabolite and
antifolate drug that works by inhibiting the metabolism of folic
acid. MTX is effective for the treatment of a number of cancers,
including breast, head and neck, leukemia, lymphoma, lung,
osteosarcoma, bladder, and trophoblastic neoplasms.
33
This
drug contains two carboxylic acid groups in its structure, which
are ionized at physiological pH, and can be linked to poly-
cationic materials by ionic interactions.
Hence, PU{[(Ar(S–NH
2
)
3
)
20
-DiT
80
]-HMDI} (14) is a poly-
cationic PU under physiological conditions; it shows noticeable
Fig. 5 Degradation rates of selected PUs (4,5,7, and 14).
This journal is © The Royal Society of Chemistry 2014 Polym. Chem.,2014,5,2370–2381 | 2379
Paper Polymer Chemistry

degradability in a reductive environment, being resistant to
oxidative degradation and insoluble in water. These properties
make this polymer a potential candidate for the preparation of
colon-targeted drug delivery systems (for example, in anticancer
therapies).
In vitro dissolution studies of tablets containing the anti-
cancer drug MTX were carried out and, during the test, the
formulations were exposed to four phases simulating, in
subsequent order, the stomach, jejunum, distal ileum, and
proximal colon. Fig. 8 shows the results of the drug release
assays. As can be observed, 20% of methotrexate was released
by the end of phase I (pH 1.2). This fact indicates that the
coating layer of Eudragit FS 30 D, which is insoluble in water at
pH below 7, failed to keep its integrity. Therefore, in formula-
tions intended for colon targeting, tablets should be coated with
a thicker layer of Eudragit FS 30 D to prevent premature drug
release during this phase.
In the second phase, the amount of drug released increased
from below 20% to above 50% (see Fig. 8). The shape of the
release prole in this second phase shows an increasing release
rate. This kind of kinetics is typical of degradation-driven release
processes. Even though the rate of drug release in the formula-
tion tested was not the most appropriate for use in the colon, this
can be adjusted by the inclusion of channeling agents in the
formulation, i.e., water-soluble substances not wetted with the
polymer solution. These channeling agents would act by
enhancing water penetration once the Eudragit coating layer was
dissolved, increasing the drug release rate in the colon.
Fig. 8 also shows the impressive ability of PU{[(Ar(S–
NH
2
)
3
)
20
-DiT
80
]-HMDI} (14) to control the drug release, despite
its low concentration in the tablet. Even below 5% w/w, this
polymer is able to control the release of methotrexate from a
tablet containing 92% w/w lactose. This is an outstanding result
and indicates that this polyurethane is a good candidate to act
as a drug-release-controlling polymer in several types of drug
delivery device—not only for colon targeting, but also for other
administration routes. For example, in the standard oral route,
this polymer can provide a sustained release of the drug in the
upper gastro-intestinal tract, with an increased release rate in
the colon. This kind of release prole is usually desired in order
to compensate for the absorption capacity of the colon being
lower than that of the small intestine. Due to the degradation
properties of PU{[(Ar(S–NH
2
)
3
)
20
-DiT
80
]-HMDI} (14), another
interesting eld of application could be in parenteral dosage
forms—for example, sustained release injectable suspensions.
Conclusions
The preparation of novel reduction-sensitive biodegradable
multiallyl- and multiamine-based copolyurethanes, useful as
Fig. 6 AFM topography images of polymer 5before and after
degradation (6a and 6b), polymer 14 before degradation (6c and 6e)
and polymer 14 after degradation (6d and 6f). (6a–d) 4 mm4mm
scans and height section analysis (left side, in nm) of polymer-coated
mica substrates. The scale bar in the topography image is 0.8 mm. (6e–
f) 2.35 mm2.35 mm scans and height section analysis (left side, in nm)
of polymer 14-coated mica substrate. The scale bar in the topography
image is 0.47 mm.
Fig. 7 Chemical structure of methotrexate (MTX).
Fig. 8 Cumulative amount of methotrexate (MTX) released vs. time.
2380 |Polym. Chem.,2014,5, 2370–2381 This journal is © The Royal Society of Chemistry 2014
Polymer Chemistry Paper

carriers of anionic drugs (at physiological pH) or gene materials,
was successfully achieved. The TGA of the new materials
revealed that the copolymers functionalized with amino groups
(protected or free) exhibited lower thermal stability than those
bearing allyloxy pendant groups. The DSC analysis of these
polymers revealed that the MDI-based PUs were essentially
amorphous while the HMDI-based PUs were semicrystalline
materials. MDI-based polymers were also stiffer than their
HMDI counterparts, as could be inferred from their respective
T
g
values. Functionalization of the sugar moieties greatly
affected the T
g
of the polymer precursors. When the DiT diol
was the main diol monomer in the polymerization feed—80%
molar ratio in polymers 5,11, and 14—T
m
values increased
about 40 C compared with the polymers containing just a 50%
molar ratio (PUs 4,10, and 13). The best degradation rates in
reductive environments (37 C, pH 7.40, [GSH] ¼5 mM) were
obtained for the amino-based copolymer PU{[(Ar(S–NH
2
)
3
)
20
-
DiT
80
]-HMDI} (14) with an associated weight loss close to 90%
aer ten days. In AFM studies, when the lm of polymer 14 was
exposed to a reductive environment, there was a marked
decrease in z-axis values, conrming the enhanced degrad-
ability feature of PU 14 compared with PU {[(ArAll
3
)
20
-DiT
80
]-
HMDI} 5. Polymer 14 was used in the preparation of colon-
targeted drug delivery systems. An in vitro study of tablets
containing the anticancer drug MTX demonstrated an impres-
sive ability of PU 14 to control the drug's release, despite its low
concentration in the tablet (below 5% w/w).
Acknowledgements
We thank the Ministerio de Econom´
ıa y Competitividad (Grant
MAT2012-38044-C03-01) of Spain for nancial support. We also
thank the Ministerio de Educaci´
on Cultura y Deporte for the
predoctoral grant FPU (Formaci´
on de Profesorado Uni-
versitario) for Cristina Ferris. We would also like to thank Dr
Consuelo Cerrillos, of the Microscopy Service (CITIUS, Seville),
for help with the AFM experiments.
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