A cancer-targetable copolymer containing tyrosine segments for labeling radioactive halogens

Article abstract of DOI:10.1016/j.reactfunctpolym.2010.12.009

A series of cancer-targetable copolymers containing rhodamine and tyrosine segments were synthesized and further labeled with isotope ¹²⁵I. Copolymers with different molecular weights (PRTH-1, PRTH-2 and PRTH-3) formed aggregates in water with average diameters of 66 nm, 191 nm and 137 nm, respectively. The cancer cell targeting properties of the copolymers were investigated by comparing BEL-7402 liver cancer cells and L-02 human normal liver cells. The results indicate that their targeting properties are related to the average diameters of the PRTH copolymers that self-assembled in water. PRTH-2 copolymers were selected as having the best targeting properties for cancer cells. The in vivo study of HepA mouse models based on single-photon emission computed tomography (SPECT) also showed good tumor-targeting properties of PRTH-2 labeled with ¹²⁵I.

Full text of DOI:10.1016/j.reactfunctpolym.2010.12.009

Reactive & Functional Polymers 71 (2011) 390–394
Contents lists available at ScienceDirect
Reactive & Functional Polymers
journal homepage: www.elsevier.com/locate/react
A cancer-targetable copolymer containing tyrosine segments for labeling
radioactive halogens
⇑
⇑
Yu Qi, Najun Li , Qingfeng Xu, Xuewei Xia, Jianfeng Ge, Jianmei Lu
Key Laboratory of Organic Synthesis of Jiangsu Province, College of Chemistry, Chemical Engineering and Materials Science, Suzhou University, Suzhou, Jiangsu 215123, China
a r t i c l e i n f o
a b s t r a c t t a b s t r a c t
Article history:
Received 13 July 2010
Received in revised form 12 December 2010
Accepted 13 December 2010
Available online 21 December 2010
A series of cancer-targetable copolymers containing rhodamine and tyrosine segments were synthesized
and further labeled with isotope 125I. Copolymers with different molecular weights (PRTH-1, PRTH-2 and
PRTH-3) formed aggregates in water with average diameters of 66 nm, 191 nm and 137 nm, respectively.
The cancer cell targeting properties of the copolymers were investigated by comparing BEL-7402 liver
cancer cells and L-02 human normal liver cells. The results indicate that their targeting properties are
related to the average diameters of the PRTH copolymers that self-assembled in water. PRTH-2 copoly-
mers were selected as having the best targeting properties for cancer cells. The in vivo study of HepA
mouse models based on single-photon emission computed tomography (SPECT) also showed good
tumor-targeting properties of PRTH-2 labeled with 125I.
Keywords:
Copolymer
Synthesis
Tyrosine
Isotope
Ó 2010 Elsevier Ltd. All rights reserved.
Targeting
1. Introduction
tron emission tomography (PET), single-photon emission com-
puted tomography (SPECT) and high-resolution autoradiography
Biocompatible polymers, such as poly(ethylene glycol)s (PEGs),
polyacrylic acids (PAAs), poly(ethylene imine)s (PEIs) and poly(a-
mido-amine)s (PAMAMs), have attracted great attention in the
field of cancer diagnosis and therapy [1–6]. The synthetic polymers
can easily achieve important properties, such as enhanced perme-
ability and retention (EPR) effects and membrane destabilizing ef-
fects. The EPR effects enable polymeric molecules to be collected in
tumor tissues [7–11]. The membrane destabilizing effects can pre-
vent polymers and loaded drug molecules from being decomposed
in lysosomes. Generally, such membrane destabilizing effects
could be achieved by the pH-induced conformational change of an-
ionic carboxylate polymers [12–16] or through the proton sponge
effect [17–21], although the latter has been called into question
[22,23]. In addition, the synthetic polymers should be traceable
during the diagnostic procedures. The most frequent method for
detecting the polymers utilizes conjugates of a fluorescent dye to
emit intense fluorescent light in cells or tumors. However, the
method used to detect the fluorescent signals suffers from many
problems in vivo, especially when investigating deep and absorbing
tissues such as the lung, spleen or liver [24].
[26–30]. It is chemically stable for the radioactive halogen reagents
to be covalently labeled onto a tyrosine moiety. In addition, the
amino acid tyrosine has a better biocompatibility than various
molecules used as ligands for radioactive metal ions. Therefore, it
is attractive to introduce tyrosine moieties into the polymer side
chains to combine the advantages of functional polymers and iso-
topic diagnosis.
In this paper, copolymers containing both rhodamine chro-
mophores and tyrosine segments (PRTHs, see Scheme 1) were syn-
thesized for the purpose of targeting cancer cells. At the same time,
another copolymer without tyrosine segments (PRH) was synthe-
sized for comparison. The rhodamine segments enabled the
copolymer to be detected by fluorescence analysis, and the tyro-
sine segments were introduced for labeling radioactive 125I for
SPECT. Both PRTHs and PRH have properties that allow them to tar-
get cancer cells. The in vitro and in vivo studies verified the cancer
cell targeting properties and tumor tissue targeting properties of
PRTH, respectively. The copolymer PRTH would be potentially
applicable for in vivo cancer diagnosis and therapy.
Radionuclide imaging has been widely applied to diagnosis and
therapy [25]. In radiochemistry, tyrosine moieties can be labeled
with radioactive halogens, such as 18F, 123I, 125I, and 131I for posi-
2. Experimental
2.1. Materials
⇑
Corresponding authors. Tel./fax:+86 512 65880367 (N. Li), Tel./fax: +86 512
65882875 (J. Lu).
2-Hydroxyethyl acrylate (HEA) was purchased from Aldrich and
purified twice by passing it through a column filled with basic
E-mail addresses: linajun@suda.edu.cn (N. Li), lujm@suda.edu.cn (J. Lu).
1381-5148/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.reactfunctpolym.2010.12.009

Y. Qi et al. / Reactive & Functional Polymers 71 (2011) 390–394
391
Scheme 1. Synthetic routes of PRTH and PRH.
alumina to remove the inhibitor. The initiator 2,20-azobis(isobuty-
ronitrile) (AIBN, A.R.) was purified by recrystallization with meth-
anol. Rhodamine B (A.R.), thionyl chloride (A.R.), methacrylic
obtained by using a Panasonic DMC-FX30 camera on an Olympus
fluorescence microscope with an exposure time of 2.5 s. The
images of the HepA mouse models were obtained using Philips
Skylight single-photon emission computed tomography (SPECT).
anhydride, L-tyrosine ethyl ester hydrochloride, chloramine-T and
Na2S2O5 were purchased from Sinopharm Chemical Reagent Co.,
Ltd. NaI-125 was provided by the Jiangsu Institute of Nuclear Med-
icine. The copolymer PRH was synthesized in our lab. All other re-
agents and solvents were purchased from commercial sources and
used as received. Bovine Calf Serum (BCS) and FCS-RPMI-1640
solution were purchased from Gibco and used as received. The
BEL-7402 liver cancer cells and L-02 human normal liver cells were
purchased from the Chinese Academy of Sciences Cell Bank. The li-
ver cancer HepA mouse models and pelltobarbitalum natricum
were provided by the Jiangsu Institute of Nuclear Medicine.
2.3. Cellular uptake and imaging
The cells were incubated in a FCS-RPMI-1640 solution contain-
ing 10% BCS at 37 0.5 °C for 25 h to obtain a 70% cell climbing ra-
tio. The media was changed to 200 lL of 1640 solution, and PRTH
or PRH was added into the cell culture to obtain a definite final
concentration. The cells were incubated for 30 min at 37 °C unless
otherwise stated. The fluorescence images of BEL-7402 liver cancer
cells and L-02 human normal liver cells were obtained by using a
Panasonic DMC-FX30 camera on an Olympus fluorescence micro-
scope with an exposure time of 2.5 s.
2.2. Characterizations
1H NMR spectra were measured by an INOVA 400 MHz NMR
spectrometer using CDCl3 or DMSO-d6 as solvents at ambient tem-
perature. Elemental analysis was performed by an Italian 1106 FT
analyzer. The molecular weights and polydispersity of the copoly-
mers were measured using Waters 1515 Gel Permeation Chroma-
tography (GPC) with DMF as the mobile phase at a flow rate of
1 mL minꢀ1 and a column temperature of 30 °C. The size distribu-
tion values of the polymer aggregates were obtained on an
Hpps501-high performance particle size analyzer. The morphology
examination of the PRTH aggregates was performed on a Philips
CM120 transmission electron microscope (TEM) with an accelerat-
ing voltage of 100 kV. The sample was prepared by placing a drop
of the 0.2 mg mLꢀ1 PRTH solution onto a mesh copper grid and dry-
ing the sample in air before measurement. The cell images were
2.4. In vivo experiments
The HepA liver cancer cells were inoculated into the abdominal
cavity of a normal mouse. The ascitic fluid was extracted and di-
luted by physiological saline to obtain the cancer cell concentra-
tion of 1 ꢁ 108 mLꢀ1. Then 0.2 mL of cancer cell solution was
inoculated into the left axillary of ICR mice. Tumor tissues with a
diameter of about 0.5 cm were obtained after 6 days. The HepA
mouse models obtained were used in this study to investigate
the accumulation behavior of PRTH in tumor tissues. PRTH-2 la-
beled with 125I was dissolved in saline water and diluted to a con-
centration of 100 lCi. The PRTH-2 solution was injected through
the tail vein at a dosage of 0.2 mL of solution per mouse. After
the experiment, the mice were anesthetized via injection of pellto-

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Y. Qi et al. / Reactive & Functional Polymers 71 (2011) 390–394
barbitalum natricum. The images were obtained on a Skylight
SPECT.
5 h. The reaction mixture was added dropwise into dichlorometh-
ane with stirring to precipitate the copolymer. The copolymer was
purified with a Soxhlet extractor for 14 days and dried in vacuum
at room temperature to a constant weight. In comparison, PRH
was copolymerized using the same procedure above without the
MTyr monomer.
2.5. Synthesis
2.5.1. Rhodamine B (2-hydroxyethyl acrylate) ester (MR)
Rhodamine B (2.1 mmol) was dissolved in 1,2-dichloroethane
(15 mL), then thionyl chloride (12.7 mmol) was added slowly at
room temperature. The reaction mixture was refluxed for 8 h. After
evaporation of the solvent in a vacuum to obtain rhodamine B acid
chloride, 2-hydroxyethyl acrylate (9.6 mmol) was added dropwise
to the rhodamine B acid chloride solution in dichloromethane and
stirred for 24 h at room temperature. The crude product was puri-
fied by column chromatography (CH2Cl2/MeOH = 30/1, v/v) to ob-
tain 0.9 mmol of product MR (yield; 42.9%). 1H NMR (CDCl3):
d = 8.31 (d, 1H, J = 7.8 Hz), 7.84 (t, 1H, J = 7.5 Hz), 7.76 (t, 1H,
J = 7.7 Hz), 7.32 (d, 1H, J = 7.4 Hz), 7.08 (d, 2H, J = 9.5 Hz), 6.93 (d,
2H, J = 9.5 Hz), 6.81 (s, 2H), 6.38 (d, 1H, J = 17.3 Hz), 6.05 (m, 1H),
5.87 (d, 1H, J = 10.4 Hz), 4.29 (m, 2H), 4.17 (m, 2H), 3.66 (q, 8H,
J = 7.2 Hz), 1.34 (t, 12H, J = 7.1 Hz). Anal. calcd. for C33H37ClN2O5:
C 68.68, H 6.46, N 4.85. Found: C 68.47, H 6.31, N 4.81.
2.6. Radiochemistry
PRTH-2 (25
0.2 mol Lꢀ1, pH = 8.0) and then 3
of chloramine-T (3 mg mLꢀ1) were added to the mixture, which
was shaken for 1 min. Na2S2O5 (20
L, 5 mg mlꢀ1) was added,
and mixture was shaken for an additional 3 min. The solution
lg) was dissolved in 25
lL of phosphate buffer (PB,
L of NaI-125 (1 mCi) and 10 lL
l
l
was diluted with 200 lL of PB solution and purified by passing
through a PD10 (Sephadex G25) column to obtain 125I-labeled
PRTH-2.
3. Results and discussion
3.1. Characterization of PRTH
A typical 1H NMR spectrum of PRTH-1 is shown in Fig. 1. The
resonant signal at d = 4.01 ppm is assigned to the methylene pro-
tons in the hydroxyethyl segments, and the chemical shift at
d = 9.17 ppm is the characteristic signal of phenolic hydroxy pro-
tons in the tyrosine segments. The content of the tyrosine seg-
ments in PRTH can be calculated from the integral area ratio of
the peaks at d = 9.17 and 4.01 ppm [31]. Because the integral area
of the rhodamine segments is too small to accurately calculate its
content in the copolymer chain, it should be determined by using
the UV–Vis absorption of the MR monomer as a Ref. [32]. First,
the standard ethanol solutions of MR were tested to obtain the
standard linear curve of absorption intensity (Iabs, A.U.) versus con-
centration (CMR, ꢁ10ꢀ6 mmol mLꢀ1): Iabs = 0.0134 ꢁ CMR ꢀ 0.0552.
Then, the concentration of rhodamine segments in the 0.2 mg mLꢀ1
ethanol solution of PRTH or PRH was tested, and the content of
rhodamine segments in PRTH or PRH was calculated (see Table
1). The number-average molecular weight and polydispersity in-
dex (PDI) of PRTH and PRH were determined by GPC and are sum-
marized in Table 1.
2.5.2. Ethyl 2-amino-3-(4-hydroxyphenyl)propanoate (Tyr-OEt)
L-Tyrosine ethyl ester hydrochloride (11.4 mmol) and NaHCO3
(5.0 g) were added in water (75 mL). Dichloromethane (50 mL)
was slowly added into the suspension and stirred slowly for 4 h.
The dichloromethane solution was separated and evaporated to
obtain a yellow solid. The crude product was washed by ethyl ether
to obtain 9.1 mmol of Tyr-OEt (yield; 79.8%). 1H NMR (CDCl3):
d = 9.15 (s, 1H), 6.95 (d, 1H, J = 8.2 Hz), 6.65 (d, 1H, J = 8.4 Hz),
4.01 (q, 1H, J = 7.1 Hz), 3.46 (m, 1H), 2.70 (m, 1H), 1.70 (m, 1H),
1.12 (t, 1H, J = 7.1 Hz). Anal. calcd. for C11H15NO3: C 63.14, H
7.23, N 6.69. Found: C 63.06, H 7.25, N 6.63.
2.5.3. Ethyl 3-(4-hydroxyphenyl)-2-(methacrylamido) propanoate
(MTyr
)
Tyr-OEt (7.2 mmol) and triethylamine (7.9 mmol) were dis-
solved in dichloromethane (40 mL). The methacrylic anhydride
(7.8 mmol) in dichloromethane (40 mL) was added, and the solu-
tion was stirred at 0 °C for 24 h. After reaction, the solution was
washed with NaHCO3 (0.06 g mLꢀ1
) solution and NaH2PO4
(0.06 g mLꢀ1) solution. The dichloromethane solution was sepa-
rated and evaporated to obtain a yellow oil. The crude product
was dissolved in tetrahydrofuran (THF, 10 mL). The solution was
then added dropwise into water to obtain 6.1 mmol MTyr yellow
oil (yield; 84.7%). 1H NMR (CDCl3): d = 9.24 (m, 1H), 8.19 (d, 1H,
J = 7.7 Hz), 7.02 (m, 1H), 6.64 (m, 1H), 5.63 (m, 1H), 5.35 (m, 1H),
4.36 (dd, 1H, J = 8.3 Hz, J = 14.5 Hz), 4.05 (m, 1H), 2.90 (m, 1H),
1.79 (m, 1H), 1.12 (t, 1H, J = 7.1 Hz). Anal. calcd. for C15H19NO3: C
64.97, H 6.91, N 5.05. Found: C 64.74, H 6.89, N 4.98.
Fig. 2 shows the diameter distribution of PRTH aggregates in
water solution. The average diameters of PRTH-1, PRTH-2 and
PRTH-3 aggregates are 66 nm, 191 nm and 137 nm, respectively.
It can be seen that the aggregates acquire a spherical morphology.
Because the tyrosine segments are hydrophobic, PRTH containing
hydrophobic and hydrophilic segments could form micelles or
2.5.4. Copoly-(MR–MTyr–HEA) (PRTH) and copoly-(MR–HEA) (PRH)
To prepare PRTH, MTyr, MR, HEA and AIBN were dissolved in
cyclohexanone with the mole ratio summarized in Table 1. The
solution was degassed using dry nitrogen and stirred at 70 °C for
Table 1
The characterization data of PRTHs.
Samples Reactant mol
ratio HEA/MR/
Composition [HEA]/ Mn
[MR]/[MTyr
Polydispersity
index (PDI)
]
MTyr/AIBN
PRTH-1
PRTH-2
PRTH-3
PRH
100:1:5:1
100:1:5:2
100:1:5:5
100:1:0:1
97.94%:0.15%:1.91% 25,100 2.6
98.14%:0.15%:1.71% 38,800 3.6
98.23%:0.14%:1.63% 31,300 2.5
99.67%:0.33%:0%
23,700 2.7
Fig. 1. 1H NMR (DMSO-d6) spectra of PRTH-1.

Y. Qi et al. / Reactive & Functional Polymers 71 (2011) 390–394
393
Fig. 4. Average fluorescent intensity in the BEL-7402 and L-02 cells images. I7402
and IL-02 are the average fluorescent intensity in 7402 cells and L-02 cells,
respectively.
of PRTH aggregates might be correlated to their cancer cell target-
ing degree.
In regards to the cellular internalization mechanism of the poly-
mers, endocytosis is considered to be the general entry mecha-
nism. Endocytosis belongs to the energy-dependent uptake,
which would be hindered at a low temperature [33]. As shown in
Fig. 5, a significant reduction in cellular uptake at 4 °C for PRTH-
2 was observed. This result indicates endocytosis to be the inter-
nalization mechanism for the uptake of PRTH. It has been previ-
ously reported that some synthetic polymers without special
recognition units on main or side chains can be taken up by cells,
mainly by fluid-phase or adsorptive endocytosis [34], and can fur-
ther target the cancer cells [35]. However, no systematic studies
have been found so far to clarify the details of the mechanism for
the uptake of conjugates without receptor-specific recognition
units. This mechanism should be studied further.
Fig. 2. Diameter distributions and TEM images (the inserted scale span is 200 nm)
of PRTHs in water solution (0.2 mg mLꢀ1).
micelle-like aggregates in water solution. The reason for the differ-
ent sizes of PRTH aggregates should be attributed to the different
average molecular weights of each polymer because the composi-
tion of PRTHs is similar from polymer to polymer.
3.2. Cell culture studies
Fig. 3 shows the images of PRH and PRTH internalized by BEL-
7402 liver cancer cells and L-02 human normal liver cells. Fig. 4 re-
veals the degree of cancer cell targeting properties, which were
valued by comparing the fluorescent intensity of BEL-7402 with
L-02 cells (I7402/IL-02). A high I7402/IL-02 value means a high cancer
cell targeting degree. Both PRH and PRTH can target BEL-7402 can-
cer cells. The values of I7402/IL-02 in Fig. 4 reveal that the cancer cell
targeting degrees are in the sequence of PRTH-2 > PRTH-3 > PRTH-
1, which coincides with the sequence of the average diameters of
PRTH aggregates in water (Fig. 2). The results indicate that the size
3.3. In vivo studies
Although PRH can target BEL-7402 cancer cells, the optical
imaging diagnosis method based on detecting the fluorescent sig-
nals of PRH passing through the tissues suffers some problems
in vivo. Similar to other optical imaging methods, fluorescence
imaging is hampered by surface reflectance, absorption, scattering
and autofluorescence [24]. The fluorescent signals are affected by
the thickness and optical properties of the tissues. These disadvan-
tages limit the applications of PRH. On the other hand, radionuclide
imaging, which is commonly devised into two general modalities,
such as SPECT and positron emission tomography (PET), can over-
come these problems and has already been widely used. Tyrosine
Fig. 3. Images of BEL-7402 and L-02 cells. The BEL-7402 cells were incubated in
1640 solution containing 100
(D), for 30 min. The L-02 cells were incubated in 1640 solution containing
100
g mLꢀ1 PRTH-1 (E), PRTH-2 (F), PRTH-3 (G) and PRH (H).
l
g mLꢀ1 PRTH-1 (A), PRTH-2 (B), PRTH-3 (C) and PRH
Fig. 5. Optical, fluorescent and merged images of 7402 cells incubated in 1640
l
solution containing 200 l
g mLꢀ1 PRTH-2 at 37 °C (A) and 4 °C (B).

394
Y. Qi et al. / Reactive & Functional Polymers 71 (2011) 390–394
Acknowledgements
This work is supported by the National Natural Science Founda-
tion of China(20876101, 20902065), the Project of Ministry of Edu-
cation (20070285003), the project supported by the Major
Fundamental Research Program of the Natural Science Foundation
of Jiangsu Higher Education Institutions (08KJA430004) and the
Innovative Research Team of Advanced Chemical and Biological
Materials, Suzhou University.
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4. Conclusion
In summary, we have developed a novel cancer-targetable PRTH
copolymer containing rhodamine and tyrosine segments. The rho-
damine segments enable PRTH to be traced by fluorescent signals.
However, many obstacles limit the application of the methods
based on the detection of fluorescent signals. Tyrosine segments
were introduced to label radioactive halogens, such as 125I, for iso-
tope detection. The BEL-7402 cancer cell targeting degree of PRTH
is related to the size of the micelle-like aggregates formed in aque-
ous solution. The in vivo studies prove the ability of PRTH to target
some tumor tissues and also reveal the availability of 125I-labeled
PRTH in SPECT. Therefore, PRTH can be detected by both fluores-
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