Applied Radiation and Isotopes 63 (2005) 423–431
Thermoresponsive polymers as promising new materials
for local radiotherapy
M. Hruby´
a,
, V. Sˇubr
a
, J. Kucˇka
b
, J. Kozempel
b
, O. Lebeda
c
, A. Sikora
a
a
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovske´ho Sq. 2,
162 06 Prague 6, Czech Republic
b
Department of Organic & Nuclear Chemistry, Faculty of Science of the Charles University, Hlavova 2030,
128 43 Prague, Czech Republic
c
Nuclear Physics Institute, Academy of Sciences of the Czech Republic, 250 68 Rˇezˇ near Prague, Czech Republic
Received 31 March 2005; received in revised form 15 May 2005; accepted 18 May 2005
Abstract
We describe a novel thermoresponsive polymeric drug delivery system based on poly(N-isopropylacrylamide) with
isotopically labellable end groups [
L
-tyrosinamide or diethylenetriaminepentaacetic acid (DTPA)] designed for local
radiotherapy. The polymers are readily soluble in isotonic aqueous sodium chloride at room temperature and the phase
separation is complete at body temperature as proved by DSC measurements. Sufficent binding capacity for
radionuclides and chemical stability are demonstrated on
125
I and
90
Y-labelled polymers.
r
2005 Elsevier Ltd. All rights reserved.
Keywords: Thermoresponsive; Polymer; Poly(N-isopropyl acrylamide); DTPA; Tyrosinamide; Local radiotherapy; Yttrium-90;
Iodine-125
1. Introduction
Water-soluble polymers are ofsteadily increasing
interest for biomedical applications at present. They
are used in drug formulations (
), as blood plasma substituents
(
), as carriers in drug delivery
systems (
) and numerous other
applications.
Thermoresponsive, sometimes also called thermosen-
sitive polymers based on poly(N-isopropylacrylamide)
(
), poly(N-isopropylmethacrylamide)
(
), elastin-like peptides (
), Pluronics (
) and similar systems, that are soluble in
aqueous media at low temperature and reversibly
precipitate in a very narrow temperature range, are
promising candidates for medical applications. Most
experiments performed in this field were to target
chemotherapeutics by local hyperthermia (
), some experiments were carried out to create a
depot ofa chemical drug at a target site (
), e.g. urine bladder, or in
covalently cross-linked gels with temperature-controlled
swelling and drug release (
) and in
thermoresponsive micelles (
).
There are some advantages in using radiopharmaceu-
ticals in polymeric drug delivery systems in comparison
with chemical drugs (
). Above all, the
ARTICLE IN PRESS
www.elsevier.com/locate/apradiso
0969-8043/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apradiso.2005.05.043
Corresponding author. Tel.: +420 296 809 212;
fax: +420 296 809 410.
E-mail address: mhruby@centrum.cz (M. Hruby´).
effective chemical amount of the radionuclide is much
lower than that ofthe chemical drug so that, a very
small quantity ofa polymer carrier is sufficient. It is also
not necessary to release the radionuclide from the carrier
to accomplish the task.
Application ofa radionuclide-carrying thermorespon-
sive polymer to a joint damaged by inflammation or
arthrosis may decrease pain [beneficial effects of local
radiotherapy in such cases are well known (
)] since it suppresses
macrophage activity and this may subsequently decrease
the consumption ofanalgetics and other drugs (of
ten
immunosuppressants such as cyklophosphamide or
azathioprine). Excess ofthe precipitated polymer may
serve as a lubricant, lowering mechanical damage ofthe
cartilage and neighbouring tissues. Another use ofthis
system could be intratumoral application or adjuvant
radiotherapy after surgical removal of malignant tissues
(
;
) in order to
prevent a relapse caused by residual cancer cells. To our
knowledge, no such thermoresponsive system has been
described for local radiotherapy in literature so far.
In this article we describe the synthesis ofa new type
ofazo initiator enabling the introduction ofthiazolidine-
2-thione reactive groups to the polymer chain during
polymerization and a new thermoresponsive system
(see
for preparation) based on poly(N-
isopropylacrylamide) carrying a labellable phenol (re-
acting with iodine radioisotopes) or chelating grouping
diethylenetriaminepentaacetic acid (DTPA) (reacting
with polyvalent metal radioisotope cations) at the
ends ofpolymer chains. These groups enable binding
to most medical radionuclides. These polymers are
readily soluble in water at room temperature, which
allows facile isotope labelling and administration via
injection, and they precipitate slightly below the
body temperature so they should persist at the site of
injection.
ARTICLE IN PRESS
CN
HOOC
N
CN
COOH
(Z)
I
DCC
S
NH
S
DMAP
THF
CN
N
CN
O
O
N
S
S
S
S
(Z)
II
THF
60
°
C
O
H
N
H
O
N
S
S
CN
III
R
R
O
n
R =
or
N
N
N
N
H
Fig. 1. Synthesis ofreactive polymer III.
M. Hruby´ et al. / Applied Radiation and Isotopes 63 (2005) 423–431
424
2. Materials and methods
2.1. Materials
N-isopropylacrylamide,
L
-tyrosinamide, ethylenedia-
mine, 4, 4
0
-azobis (4-cyanopentanoic acid), thiazolidine-
2-thione and diethylenetriaminepentaacetic dianhydride
(DTPAA) were purchased from Sigma-Aldrich Ltd.
(Czech Republic) and other chemicals were purchased
from Lachema (Czech Republic). Tetrahydrofuran
(THF) was kept over sodium and distilled before use,
N-isopropylacrylamide was recrystallized from hexane
and other chemicals were used without additional
purification.
Carrier-free Na
125
I and
90
YCl
3
aqueous solutions
were purchased from Lacomed Ltd. (Czech Republic).
Dialysis tubing Spectra/Por 3 (molecular weight cut-
off 3500 Da) was purchased from Serva Electropho-
resis GmbH (Germany), PD-10 columns and Sephadex
G 25 were obtained from Amersham Biosciences
(Sweden).
2.2. Characterization of polymers
1
H NMR spectra were measured in THF-d
8
on a
Bruker Avance MSL 200 MHz NMR spectrometer
(Bruker Daltonik, Germany). IR spectra were recorded
in KBr pellets on a Perkin–Elmer Paragon 1000 PC FT-
IT spectrometer (Perkin–Elmer Inc., USA). Gel permea-
tion chromatography (GPC) was performed in a mixture
of acetate buffer (pH 6.5, 0.3 mol L
1
) and methanol
(20:80 v/v) as a mobile phase on a TSK 3000 column
(Polymer Laboratories Ltd., UK) using a HPLC System
A¨KTA Explorer (Amersham Biosciences; Sweden)
equipped with RI, UV and multiangle light-scattering
DAWN DSP-F (Wyatt, USA) detectors. The refractive
index increment (dn/dc) ofpoly(N-isopropylacrylamide)
in this solvent is 0.153
70.002 mL g
1
(as measured on a
Brice–Phoenix visual laboratory type differential re-
fractometer BP-2000-V, Phoenix Precision Instrument
Co., USA); the effect of end groups on the refractive
index increment was neglected.
2.3. Preparation of 3, 3
0
-azobis (4-cyano-4-methyl-1-
oxobutane-1.4-diyl)bis(thiazolidine-2-thione)
(TT–ABIC; II)
4, 4
0
-azobis (4-cyanopentanoic acid) (2.00 g, 7.14 mmol),
thiazolidine-2 thione (1.87 g, 15.7 mmol) and 4-dimethyla-
minopyridine (96 mg, 0.79 mmol) were dissolved in THF
(20 mL) and dicyclohexylcarbodiimide (DCC; 3.88 g,
18.8 mmol) was dissolved in THF (5 mL). Both solutions
were cooled to 10 1C. The cold solutions were mixed
together and kept at 10 1C f or 1 h and then at 5 1C f or
24 h. Acetic acid (0.1 mL) was added and the mixture was
stirred for an additional 1 h at room temperature. The
precipitated dicyclohexylurea was filtered-off and THF
was evaporated in vacuo. The oily residue was dissolved in
dichloromethane and crystallized from a dichlorometha-
ne–diethyl ether mixture.
ARTICLE IN PRESS
III
...
O
N
S
S
...
O
N
S
S
H
2
N
NH
2
III
IV
...
O
N
H
CONH
2
OH
V
...
O
NH
NH
2
...
O
NH
NH
O
N
HOOC
N
N
HOOC
COOH
HOOC
VI
L-tyrosinamide
DTPA dianhydride
Fig. 2. Modifications ofthe end groups ofpolymer III to isotopically labellable moieties.
M. Hruby´ et al. / Applied Radiation and Isotopes 63 (2005) 423–431
425
Yield 3.0 g (87%), m.p. 126–130 1 C. Elemental analysis:
calculated/found: C 44.79/45.16, H 4.59/4.74, N 17.41/
16.95,
S
26.57/26.47.
Molar
absorption
coefficient
305 nm
¼
20; 500 L mol
1
cm
1
(methanol).
1
H NMR: s ¼
1:56 (s, 6 H), 2.75 (m, 4 H), 2.95 (m, 4 H), 3.19 (m, 4 H),
4.04 (m, 4 H).
2.4. Preparation of poly(N-isopropylacrylamide) with
L
-
tyrosinamide end groups (IV)
N-isopropylacrylamide
(500 mg,
4.42 mmol)
and
TT–ABIC (II, see
for the initiator weight to
obtain the corresponding polymer IV) were dissolved in
THF (4 mL) and polymerization was carried out under
nitrogen at 60 1C for 12 h.
L
-tyrosinamide (180 mg,
1.00 mmol) was then added and the mixture was heated
with stirring for another 3 h at 60 1C. The mixture was
filtered and the polymer was then precipitated from the
filtrate with diethyl ether (Et
2
O; 40 mL). The precipi-
tated polymer IV was collected, redissolved in THF
(5 mL) and precipitated again by addition ofEt
2
O
(50 mL). The combined precipitates ofpolymer IV were
dried in vacuo.
Crude polymer IV was dissolved in methanol (10 mL),
water (20 mL) was added and the mixture was dialyzed
against distilled water at room temperature for 72 h. The
aqueous solution ofthe polymer was freeze dried.
The lyophilized powder was redissolved in water
(20 mL) and the appropriate polymer fraction was
separated on a Sephadex G-25 column (120 mL) using
distilled water as a mobile phase. The aqueous polymer
solution was freeze dried to obtain pure IV (see
for yields). The
L
-tyrosinamide content was assayed
spectrophotometrically (l ¼ 275 nm) in water. See
for
L
-tyrosinamide contents in polymers IV-1, IV-3
and IV-5.
2.5. Preparation of poly(N-isopropylacrylamide) with
primary amino end groups (V)
N-isopropylacrylamide
(1000 mg,
8.84 mmol)
was
polymerized to the solution of III as described above.
Ethylenediamine (590 mL, 8.84 mmol) was added and the
mixture was heated with stirring at 60 1C for 2 h. The
polymer V was precipitated from the filtrate with diethyl
ether (Et
2
O; 80 mL). The precipitated polymer V was
collected, redissolved in THF (10 mL) and precipitated
again by addition ofEt
2
O (100 mL). The combined
precipitates ofpolymer V were dried in vacuo.
The crude polymer V was dissolved in water (20 mL)
and the polymer fraction was separated on a Sephadex
G-25 column (120 mL) using distilled water as a mobile
phase. The polymer solution was freeze dried to obtain
pure V. See
for yields.
2.6. Preparation of poly(N-isopropylacrylamide) with
DTPA end groups (VI)
Polymer V (300 mg) was dissolved in dimethylaceta-
mide
(6 mL)
and
ethyldiisopropylamine
(500 mL,
2.92 mmol)
and
4-dimethylaminopyridine
(50 mg,
0.41 mmol) were added. The resulting solution was
heated up to 50 1C, DTPAA (214 mg, 0.6 mmol) was
added and the mixture was stirred at 50 1C for 3 h. The
resulting solution was stirred for 12 h at room tempera-
ture and then quenched to water (30 mL). Ammonium
sulfate (15 g) was dissolved in this mixture and the
precipitate ofpolymer VI was collected. The salting-off
procedure was repeated once again. The crude polymer
VI was dissolved in water (20 mL) and purified by gel
filtration on a Sephadex G-25 column (120 mL) using
aqueous ammonium acetate (0.8 mol L
1
) as a mobile
phase. The polymer solution was desalted by dialysis
against distilled water for 72 h. The polymer solution
was freeze dried to obtain pure VI (see
for
yields).
The content ofDTPA groups (see
) was
determined by chelatometric titration in ammonium
hydroxide–ammonium chloride buffer (0.2 mol L
1
, pH
9.25) with zinc acetate (0.25 mmol L
1
) and Eriochrome
Black T as an indicator.
2.7. Differential scanning calorimetry (DSC)
DSC measurements were performed on a Perkin–
Elmer DSC Pyris 1 (Perkin–Elmer, USA). The temperature
ARTICLE IN PRESS
Table 1
Polymers with
L
-tyrosinamide end groups (IV-1, IV-3 and IV-5)
Polymer
IV-1
IV-3
IV-5
Initiator (molar %)
1.0
3.0
5.0
Initiator (mg)
21
64
107
Initiator (mmol)
0.044
0.133
0.221
Yield (%)
52
37
46
Functional group
content (mmol g
1
)
9.0
26.6
41.5
M
n
(kDa)
9.3
7.5
6.7
M
w
(kDa)
20.9
18.3
17.2
Table 2
Polymerization conditions and characteristics ofpolymers V-1,
V-3 and V-5
Polymer
V-1
V-3
V-5
Initiator (molar %)
1.0
3.0
5.0
Yield (%)
74
76
85
M
n
(kDa)
7.9
7.0
5.5
M
w
(kDa)
18.6
18.5
14.3
M. Hruby´ et al. / Applied Radiation and Isotopes 63 (2005) 423–431
426
and power scales ofthe calorimeter were calibrated
using indium and cyklohexene as standards. The
measurements were carried out in a helium atmosphere
with liquid nitrogen as the coolant. The heating and
cooling rate was 5 1C min
1
, the measurement tempera-
ture interval was 278–323 K. The sample (ca. 10 mg) was
placed in a sealed Al pan.
2.8. Labelling of polymer IV-5 with
125
I
Water (200 mL), phosphate-buffered saline (PBS, pH
7.4;
50 mL),
aqueous
solution
ofpolymer
IV-5
(20 mg mL
1
; 20 mL), aqueous chloramine T solution
(10 mg mL
1
;
5 mL)
and
aqueous
Na
125
I
solution
(9 MBq mL
1
; 5 mL) were placed in this order into a
1.5 mL Eppendorftest tube and incubated f
or an
appropriate time (1, 1.5, 15, 30 and 60 min) at 20 1C.
Iodination was then quenched by addition ofPBS (pH
7.4; 200 mL) and aqueous
L
-ascorbic acid (10 mg mL
1
;
50 mL). The high-molecular-weight fraction was then
separated from iodine on a PD-10 desalting column
using water as a mobile phase. 1.5 mL fractions were
taken and their radioactivity was measured with
a NaI(Tl) scintillation counter, 74038 type (RFT,
Germany). The relative errors ofthe radioactivity
counts were within 5%.
The effect of pH on the labelling yield of polymer IV-5
with
125
I was estimated using PBS buffers of pH 4.8 and
7.4 and an incubation time of30 min.
The effect of temperature on the labelling yield of
polymer IV-5 with
125
I was estimated using an incuba-
tion time 30 min and incubation temperatures of20 1C,
37 1C and 50 1C.
The effect of polymer concentration on the labelling
yield ofpolymer IV-5 with
125
I was estimated using an
incubation time 30 min and polymer concentrations in
solutions were 0.50, 2.0 and 10 mg mL
1
.
2.9. Labelling of polymer VI-5 with
90
YCL
3
Aqueous
90
YCl
3
solution (12 MBq mL
1
; 10 mL) was
incubated with a solution ofpolymer VI-5 (10 mg mL
1
;
200 mL) in aqueous ammonium acetate (0.5 mol L
1
) at
20 1C for 10 min. The polymer fraction was separated by
gel filtration on a PD-10 column using aqueous
ammonium acetate (0.5 mol L
1
) as a mobile phase.
1.5 mL fractions were collected and their activity was
measured on an ionizing chamber Bqmetr 4 (BQM;
Czech Republic), or radiometrically via a calibrated
Contamat FHT 111 M (ESM Eberline Instruments
Strahlen- und Umweltstechnik GmbH; Germany) in a
defined geometry.
Aqueous
90
YCl
3
(12 MBq mL
1
) and
90
Y-DTPA
complex, prepared by mixing a saturated solution of
DTPA in water (20 mL) and aqueous
90
YCl
3
(10 mL;
12 MBq mL
1
), were used as standards for calibration
ofretention volumes ofnonpolymeric
90
Y species (see
for a typical separation).
The saturation binding capacity ofpolymer VI-5 for
90
Y
3+
was estimated by mixing decreasing volumes ofa
solution ofpolymer VI-5 (10 mg mL
1
) in aqueous
ammonium acetate (0.5 mol L
1
) with 10 mL of an
aqueous
90
YCl
3
solution (3.7 GBq mL
1
) until approxi-
mately 50% labelling yield was obtained (5 mL offered
57% labelling yield, which corresponds to the capacity
420 MBq mg
1
; the polymer fraction was separated as
described above).
2.10. Estimation of in vitro stability of the
90
Y-labelled
polymer VI-5
The main polymer fraction containing
90
Y-labelled
polymer VI-5 after separation on a PD-10 column
(800 mL) was incubated with the solution ofcompeting
ions (200 mL ofaqueous solution containing 750
mmol L
1
NaCl, 50 mmol L
1
MgSO
4
, 5.0 mmol L
1
CaCl
2
and 5.0 mmol L
1
KH
2
PO
4
) at 37 1C for 24 h.
Samples (500 mL) were taken after 1 and 24 h as follows:
the incubation mixture was cooled to 20 1C and, after
ARTICLE IN PRESS
Table 3
Characteristics ofpolymers VI-1, VI-3 and VI-5
Polymer
VI-1
VI-3
VI-5
Yield (%)
84
84
79
Functional group
content (mmol g
1
)
6.9
9.3
11.5
M
n
(kDa)
11.7
8.9
7.8
M
w
(kDa)
19.9
18.4
17.9
120
100
80
60
40
20
0
1
7
8
9
2
3
4
5
6
10 11 12
volume fraction #
radioactivity (%)
90YCI3
90Y - DTPA
VI-5 - DTPA
Fig.
3. Separation
of
90
Y-labelled
polymer
IV-5
from
90
Y-DTPA and
90
YCl
3
on a PD-10 column.
M. Hruby´ et al. / Applied Radiation and Isotopes 63 (2005) 423–431
427
complete dissolution ofthe polymer, a sample was
taken. The polymer fraction was separated from the
sample and its radioactivity was measured as described
above. The remaining solution was then returned to the
thermostatted bath (37 1C).
3. Results and discussion
3.1. Preparation of the polymers
The polymers with end groups that could be
isotopically labelled were prepared by free-radical
polymerization of N-isopropylacrylamide, using a reac-
tive initiator carrying activated carboxyl groups and
subsequent transformation of these reactive end groups
with amines. The initiator, TT–ABIC (II), was prepared
by acylation ofthiazolidine-2-thione with a commer-
cially available radical initiator containing carboxylic
acid groups 4, 4
0
-azobis(4-cyanopentanoic acid) using
DCC as the condensation agent (see
) and
4-dimethylaminopyridine as the acylation catalyst. The
thiazolidine-2-thione-activated carboxyl reactive groups
serve as an acylation agent, highly selective for acylation
ofamines in the presence ofwater and alcohols, much
more selective than e.g., succinimidyl esters widely used
for similar purposes (
;
).
Because polymerization (see
) carried out in
DMSO led to isolation problems, THF was used as
solvent, since poly(N-isopropylacrylamide) could be
easily separated from THF solution by precipitation
with diethyl ether. THF does not react with the
thiazolidine-2-thione amide, but behaves in radical
polymerization as a transfer agent, which somehow
decreases the molecular weight ofthe polymer and
decreases the content ofend groups from the initiator in
the product to approximately 0.1–0.3 groups per chain.
Since this effect was not strong enough to significantly
hinder the possibility ofusing the product f
or the
intended purpose, we have performed all polymeriza-
tions in THF. To avoid undesired hydrolysis ofthe
thiazolidine-2-thione amides during separation and
purification, the reactive polymeric intermediate III
was not isolated but immediately aminolysed with a
proper amine to a corresponding stable amide.
The poly(N-isopropylacrylamide) with
L
-tyrosinamide
end groups (IV) was prepared by in situ aminolysis of III
with
L
-tyrosinamide (see
). The polymer was
isolated from the solution by precipitation into diethyl
ether and purified from low-molecular-weight impurities
and oligomers by dialysis against water. Purification of
polymer VI was finished by GPC on Sephadex G25
using water as a mobile phase and lyophilization.
The determined molecular weights ofpolymers IV are
shown in
. The M
n
ofall polymers IV is ca 8 kDa
and M
w
is ca 18 kDa, with a polydispersity index
I ¼ M
w
=M
n
ofabout 2.4. The molecular weights of
polymers IV slightly decrease with increasing initiator
concentration in the polymerization mixture (see
for comparison of polymers IV-1, IV-3 and IV-5, which
were prepared using 1, 3 and 5 molar % initiator), which
together with the rather low concentration offunctional
end groups shows a relatively significant transfer
properties ofTHF. The content of
L
-tyrosinamide in
polymer IV-5, however, is fully sufficient for the
intended use (the theoretical labelling capacity for
131
I
is 50 GBq mg
1
, and 6.7 GBq mg
1
for
125
I).
The polymer VI with DTPA end chelating groups was
prepared in a two step reaction involving aminolysis of
III with ethylenediamine and subsequent acylation
reaction with DTPAA (see
). As both ethylene-
diamine and DTPAA are bifunctional agents, their
excess is necessary to suppress an increase in molar
weight during the reaction. The aminolysis ofreactive
polymer III to amine–amide V was performed analo-
gous to the
L
-tyrosinamide polymer IV. The highest
yield ofacylation ofpolymer V to VI with DTPA
dianhydride without degradation ofthe polymer was
obtained when the reaction was carried-out in dimethy-
lacetamide with the use oftertiary base ethyldiisopro-
pylamine and 4-dimethylaminopyridine as an acylation
catalyst (see
, the maximum DTPA content
was 11.5 mmol g
1
, which corresponds to a theoretical
capacity for
90
Y 20.8 GBq mg
1
). The excess off
ree
DTPA was removed by twice salting off the polymer
from solution and GPC on Sephadex G-25. The mobile
phase must contain a salt (ammonium acetate was used
in this case) to avoid ionic interactions between the
charged polymer and the column.
Since molecular weights ofpolymers V (see
and VI (see
) are not higher than those of
polymer IV with
L
-tyrosinamide end groups, which
cannot crosslink during aminolysis ofpolymer III, the
excess amounts ofethylenediamine and DTPAA used
are sufficient to avoid crosslinking.
3.2. Differential scanning calorimetry (DSC)
The phase separation behaviour ofthermoresponsive
(co)polymers is studied mostly by UV–VIS spectro-
photometry (measurement ofturbidity;
) and DSC (
).
The method based on the measurement ofturbidity
that increases by several orders ofmagnitude above the
cloud point is facile and allows one to use low
concentrations ofthe polymer and slower heating or
cooling rate, but it is less accurate in quantitative
estimation ofthe precipitated concentrated phase.
This is because turbidity is not a linear function of
concentration ifparticle size is not constant during the
course ofthe experiment due to aggregation. Also the
ARTICLE IN PRESS
M. Hruby´ et al. / Applied Radiation and Isotopes 63 (2005) 423–431
428
exact temperature in the UV–VIS cuvette is difficult to
control precisely. On the other hand, DSC, which
measures the heat absorbed by endothermic phase
separation, is much more precise in the estimation of
fraction of the polymer that separates from the solution
at a particular temperature, which is the most important
parameter, considering the potential use ofthese
polymers as local radiopharmaceuticals. This is why
we decided to use DSC to study the phase separation
behaviour ofthermoresponsive polymers described in
this paper.
Most measurements were performed in isotonic
(0.15 mmol L
1
) aqueous NaCl as a model ofphysiolo-
gical environment. Important DSC data for phase
separation ofpolymers IV-1, IV-3, IV-5, VI-1, VI-3
and VI-5 from their aqueous solutions by heating are
summarized in
; see
for a typical DSC
curve. These data include the peak onset (beginning of
phase separation, the most important parameter),
maximum (maximum rate ofphase separation) and
end (phase separation completed). The heating rate
5 1C min
1
was used for all the described experiments,
because measurements using lower heating rates (0.5, 1
and 2 1C min
1
) offered the same results while the
sensitivity and signal response significantly dropped.
As the content of
L
-tyrosinamide end groups, which are
less polar than the polymer poly(N-isopropylacryla-
mide), in polymer IV increases, the phase separation
temperature onset slightly decreases (see
).
On the other hand, with increasing content ofDTPA
end groups, which are more polar than the polymer
poly(N-isopropylacrylamide),
in
polymer
VI
the
phase separation temperature onset slightly increases
(see
). This is consistent with the
hydrophobic nature ofthe phase separation phenomen-
on and also in accord with data obtained with other
thermoresponsive systems for the drug delivery of
chemotherapeutics (
).
A very important fact from the application point of
view is that both polymers with the highest content of
labellable groups (IV-5 and VI-5) are completely
precipitated below human body temperature (37 1C).
Concerning the effect of ionic strength on the phase
separation behaviour (see
), the increase in ionic
strength from 0 (distilled water) to 0.15 mol L
1
generally decreases the phase separation temperatures
ofall thermoresponsive polymers, independently ofthe
end groups, by the salting-off effect resulting from the
partial hydrophobic nature ofthe polymer chain.
Phase separation exhibits a certain degree ofhyster-
esis, which means that although the phase separation is
reversible, the precipitate starts to dissolve on cooling
with a certain temperature delay. As shown on polymer
IV-5 (see
), the precipitate starts to dissolve on
cooling approximately at the temperature when it starts
to precipitate on heating. This is important, e.g., for
local application onto peripheral tissues such as knee,
where a temporary local hypothermia may occur.
An increase in the polymer concentration also decreases
the phase separation onset temperature (see
3.3. Labelling of polymer IV-5 with
125
I
The thermoresponsive poly(N-isopropylacrylamide) IV
with
L
-tyrosinamide end groups was labelled by electro-
philic iodination with
125
I as a model iodine radioisotope
using the chloramine T method (
).
Only the polymer with the highest
L
-tyrosinamide group
content (IV-5) was used for radiolabelling experiments.
The polymer fraction was separated after labelling from
the low-molecular-weight iodine species by gel filtration
using PD-10 desalting columns. The columns have a
certified bed volume of8.3 mL, making the separation
procedure fully reproducible.
We studied the effects of time, pH, temperature and
polymer concentration on the labelling yield (in % of
ARTICLE IN PRESS
Table 4
Phase separation behaviour ofpolymers in aqueous NaCl
(0.15 mol L
1
)
Polymer
Onset (1C)
Maximum (1C)
End (1C)
IV-1
29.6
31.5
37.5
IV-3
29.3
31.2
35.7
IV-5
28.9
31.1
33.2
VI-1
28.2
31.5
35.2
VI-3
29.3
29.7
34.9
VI-5
29.7
31.2
33.0
The polymer concentration was 5 mg mL
1
as measured by
DSC.
0
10
20
30
40
50
60
70
80
90
100
28
29
30
31
32
33
34
35
36
37
38
temperature (
°
C)
response (%)
differential
integral
Fig. 4. The DSC curves (differential and integrated) of polymer
IV-5 (5 mg mL
1
) in aqueous NaCl (0.15 mmol L
1
) normalized
to maximal value with corrected background.
M. Hruby´ et al. / Applied Radiation and Isotopes 63 (2005) 423–431
429
total radioactivity added to the labelling mixture as
Na
125
I). Labelling by electrophilic aromatic iodination is
very fast at room temperature, especially at the
beginning ofthe reaction, then slows down and reaches
steady state after 30 min (see
), giving a yield
of77%. To obtain sufficiently high labelling yields, it
is crucial to work in the absence ofeven traces offree
L
-
tyrosinamide. The content of
L
-tyrosinamide in polymer
IV-5 is relatively low, so the presence ofan impurity of
free
L
-tyrosinamide in the concentration tenths percent
is able to decrease the labelling yield ofthe polymer
below 25% as a result ofpreferential iodination offree
L
-tyrosinamide.
We found that a decrease in pH from 7.4 to pH 4.8,
which improves iodination yields ofmany other com-
pounds, decreased the labelling yield from 77% to 51%.
The pH 7.4 buffer, suitable for parenteral application of
such a system, was thus used for other labelling
experiments. Especially interesting is the effect of
temperature on the labelling yield. We found that the
labelling yield decreases with increasing labelling tem-
perature (20 1C–77%; 37 1C–72%; 50 1C–64%), which is
a reversed trend compared with most chemical reactions
including
iodination
oflow-molecular-weight
com-
pounds. This is most likely due to the decreased reactivity
ofphenolic groups in the precipitated polymer compared
with that in the solution, due to the diffusion barrier.
As it could be expected, when the polymer concentra-
tion in solution increases, the labelling yield slightly
increases as well (0.50 mg mL
1
–71%; 2.0 mg mL
1
–79%;
10 mg mL
1
–86%).
3.4. Labelling of polymer VI-5 with
90
YCL
3
The chelating DTPA-containing polymer was labelled
quantitatively (499%) with
90
Y
3+
ions in ammonium
acetate buffer. The gel filtration on a PD-10 column was
effective in separation of the polymer fraction from free
90
YCl
3
and
90
Y-DTPA complex (see
). While
90
YCl
3
is partly adsorbed in the column (
90
YCl
3
peak
tailing),
90
Y—DTPA and
90
Y—VI-5 were not signifi-
cantly adsorbed in aqueous ammonium acetate (sharp
peak resolution and no significant radioactivity re-
mained in the column after separation). The labelling
capacity was determined from labelling of the decreasing
amounts ofpolymer VI-5 with a concentrated
90
Y
solution. The obtained labelling capacity 420 MBq mg
1
is sufficient for local radiotherapy. The quantitative
yield ofradiolabelling also permits application ofthe
polymer system without separation ofthe polymer
fraction after labelling on a PD-10 column.
ARTICLE IN PRESS
Table 5
Phase separation behaviour ofpolymers used for radioisotope labeling studies in aqueous environment
Polymer
End group
Solvent
Onset (1C)
Maximum (1C)
End (1C)
IV-5
L
-tyrosinamide
water
31.4
33.3
38.0
IV-5
L
-tyrosinamide
aq. NaCl (0.15 mol L
1
)
28.9
31.1
33.2
VI-5
DTPA
water
32.0
34.3
37.1
VI-5
DTPA
aq. NaCl (0.15 mol L
1
)
29.7
31.2
33.0
The polymer concentration was 5 mg mL
1
, as measured by DSC.
Table 6
Temperature hysteresis ofphase separation ofpolymer IV-5 in
aqueous NaCl (0.15 mol L
1
)
Polymer
concentration
(mg mL
1
)
Onset
(1C)
Maximum
(1C)
End (1C)
Heating
5
30.3
32.2
36.2
10
29.3
32.7
36.8
20
27.8
31.2
36.1
Cooling
5
30.4
29.4
27.3
10
31.0
28.6
26.3
20
30.7
29.2
26.2
Polymer concentrations were measured by DSC.
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
t (min)
labelling yield (%)
Fig. 5. The dependence ofthe labelling yield ofpolymer IV
with
125
I on time.
M. Hruby´ et al. / Applied Radiation and Isotopes 63 (2005) 423–431
430
In humans, however, numerous ions are present that
may compete with the complexation of
90
Y
3+
by DTPA.
They include, above all, Ca
2+
and Mg
2+
(complexed by
DTPA) and phosphates (complexing Y
3+
). This is why
we tested the stability ofthe
90
Y- labelled polymer in the
environment containing these ions, in concentrations
similar to those in blood plasma (Ca
2+
1 mmol L
1
;
Mg
2+
10 mmol L
1
; phosfate 1 mmol L
1
). There was
no significant leakage of
90
Y from the labelled polymer
(stability 497%) after 24 h incubation at 37 1C, which is
consistent with very high in vivo stabilities ofDTPA-
radiolabelled complexes bound, e.g., to antibody, which
were reported by
4. Conclusions
We described novel thermoresponsive polymers based
on poly(N-isopropylacrylamide) with isotopically label-
lable end groups designed for local radiotherapy. The
polymers are readily soluble in isotonic (9 mg mL
1
)
aqueous sodium chloride at room temperature and their
phase separation is complete at body temperature, as
proved by DSC measurements. Sufficient binding
capacity for radioisotopes was demonstrated using
125
I
and
90
Y.
Acknowledgements
The authors gratefully thank the Grant Agency of the
Academy ofSciences ofthe Czech Republic for financial
support (Grant no. B4050408).
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