Synthesis and characterization of folic acid modied water-soluble chitosan

derivatives for folate-receptor-mediated targeting

Huihui Jing, Zhide Guo, Wenyan Guo, Wenjiang Yang, Peng Xu, Xianzhong Zhang

Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, 19 Xinjiekou Outer St., Beijing 100875, China
a r t i c l e i n f o
Article history:
Received 18 December 2011
Revised 7 March 2012
Accepted 29 March 2012
Available online 4 April 2012
Keywords:
Water-soluble chitosan
Folic acid
Technetium-99m
DOTA
Biodistribution
a b s t r a c t
Three chemical modication methods of carboxymethylation, quaternization and hydroxypropylation
were used to synthesize water-soluble chitosan derivatives. In order to study the feasibility of these
chitosan derivatives as backbones of nuclear imaging agents, folic acid (FA) and Technetium-99m were
introduced onto the water-soluble chitosan chains. The bifunctional chelating agent 1,4,7,10-tetraazacy-
clododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the folate grafted chitosan derivatives
for chelating with radionuclides such as
64
Cu and
68
Ga. The structures of these new ligands were charac-
terized with multiple methods. The solubility and stability of the
99m
Tc-complexes were both favorable.
Further study of their radiochemical and biological properties will be performed to evaluate the useful-
ness of these water-soluble chitosan derivatives for nuclear imaging agent design.
2012 Elsevier Ltd. All rights reserved.
Chitosan (poly-b-1,4-2-amino-2-deoxy-b-D-glucan) is a linear
polysaccharide composed of repeating units of glucosamine and
N-acetylglucosamine, which is produced from chitin by deacetyla-
tion.
13
Chitosan, as well as its derivatives, is widely used in water
treatment, food industry, agriculture and cosmetics.
4,5
Because of
the low toxicity, biodegradability and biocompatibility, chitosan
and its derivatives are well accepted in biomedical eld, especially
in drug and gene delivery.
611
Recently, some researchers
1218
also
tried to apply chitosan in radiopharmaceuticals. Only a fewradiola-
beled chitosan derivatives for nuclear imaging and radiotherapy
have been reported by far (
153
Sm/
166
Ho labeled chitosan complex
(
153
Sm/
166
Ho-CHICO),
12,13 153
Sm/
166
Ho labeled N-(g-propanoylva-
lin)-chitosan and
153
Sm/
166
Ho labeled N-(g-propanoyl-aspartic
acid)-chitosan (
153
Sm/
166
Ho-CHICO-asp/CHICO-val),
14 99m
Tc la-
beledGalactosyl-methylatedchitosan(
99m
Tc-GMC),
15 99m
Tc labeled
hydrazinonicotinamide-galactosylated chitosan (
99m
Tc-HGC),
16 18
F
labeled galactosylated chitosan (
18
F-FB-GC),
17 99m
Tc labeled folate
conjugated chitosan (
99m
Tc-CSFA),
18 99m
Tc labeded chitosan-folate
dithiocarbamate (
99m
Tc-CSFADTC),
18
etc.). Although chitosan has
many good biological properties, some unfavorable physical charac-
ters of chitosanlimit its applicationin biomedical andradiopharma-
ceutical elds. One major problem is that chitosan is insoluble in
neutral and alkaline pH because of its stable crystalline structure
formed by strong hydrogen bond, and the larger the molecular
weight is, the poorer the solubility becomes.
19
Chitosan contains
abundant reactive amino and hydroxyl groups, both of which allow
chemical modication to improve the solubility of chitosan under
mild reaction conditions. Thus, various functional groups have been
introduced onto chitosan chain to form water-soluble derivatives,
such as poly(ethylene oxide) (PEO) grafted chitosan,
20
thiolated
chitosan,
21
glycol chitosan,
22
carboxymethyl chitosan,
8,23
quatern-
ized chitosan,
9
isobutyl chitosan,
24
etc. Besides, we also considered
to introducing different functional molecules into the chainof chito-
san at the same time, which might lead to the discovery of a new
type of multi-modal imaging agents. Most of the reported multi-
modal imaging agents were nanoparticles coated with some kind
of molecule contained reactive functional groups.
2527
However, it
is still not quite clear that whether it is safe to apply nano materials
in human. Considering the favorable biological properties of chito-
san, we believe the water-soluble chitosan derivatives could be-
come valuable linear backbones for both radiopharmaceuticals
and multi-modal imaging agents.
In this study, chitosan was chemical modied in the three ways
of carboxymethylation, quaternization and hydroxypropylation. As
a result, we obtained three water-soluble chitosan derivatives with
improved solubility, as well as good bioactivity, stability and aque-
ous solution property. Furthermore, folic acid (FA) and bifunctional
chelating agent DOTA were conjugated to chitosan chain for target-
ing and radiolabeling purpose. We use chitosan (CS, Sigma
Aldrich, US) with molecular weight of 50 kDa as the initial reagent
in order to provide enough reactive units to conjugate with FA and
DOTA molecules, at the same time to avoid forming oversize parti-
cles with too large molecular weight.
First, three water-soluble chitosan derivatives were synthesized
herein (Scheme 1). We obtained O-carboxymethyl chitosan (CMC)
0960-894X/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.102

Corresponding author. Tel.: +86 10 58802038; fax: +86 10 62205562.

E-mail addresses: zhangxzh@bnu.edu.cn, zhangxzh@gmail.com (X. Zhang).
Bioorganic & Medicinal Chemistry Letters 22 (2012) 34183424
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
j our nal homepage: www. el sevi er. com/ l ocat e/ bmcl
through the reaction between CS and chloroacetic acid (P99.5%,
Tianjin Fuchen Chemical Reagent Factory, China) at room temper-
ature after alkalization of CS for hours,
28,32
N-trimethyl chitosan
(TMC) by quaternizing CS with CH
3
I (Beijing Chemical Reagent
Company, China) in reuent N-methyl pyrrolidone (P99.0%, Tian-
jin Jinke Institute of Fine Chemicals, China),
29,33
and hydroxypropyl
chitosan (HPC) through the addition of propylene oxide (P99.0%,
Sinopharm Chemical Reagent Co., Ltd, China) on to CS with the
catalysis of tetramethylammonium hydroxide (TMAH, Tianjin Yon-
gda Chemicals Research and Development Center, China).
30,34
All
the three products have improved solubility compared with the
raw material chitosan.
Overexpression on numerous tumor types and limited expres-
sion in normal tissues make folate receptor a valuable tumor target
for folate-based radiopharmaceuticals;
10
hence, in this study, folic
acid (96102%, ACROS ORGANICS, US) was selected as a model tar-
geting molecule for folate-receptor-mediated targeting imaging.
We conjugated FA to the reactive amino groups of the chitosan
derivatives obtained above in the presence of N-hydroxy-
succinimide (NHS, >98%, Alfa Aesar, UK) and N-(3-dimethylamino-
propyl)-N
0
-ethylcarbodiimide hydrochloride (EDC: 99%, Energy
Chemical, China).
18,35
As shown in Scheme 2, three chitosan deriv-
atives grafted with folate were harvested accordingly: folate conju-
gated O-carboxymethyl chitosan (CMCF), folate conjugated N-
trimethyl chitosan (TMCF) and folate conjugated hydroxypropyl
chitosan (HPCF). To compare with the reported complex
99m
Tc-
CSFA, we also labeled these modied chitosan derivatives with
99m
Tc (6 h half-life, 140 keV) by direct labeling method, since there
were remaining reactive amino groups on chitosan chains could
chelate with
99m
Tc.
In order to make it possible to label the chitosan derivatives
with metal radionuclides like
64
Cu and
68
Ga, bifunctional chelating
agent DOTA (Shanghai Majin Chemical Technology Co., Ltd, China)
was introduced into folic acid modied chitosan derivatives. Three
chitosan derivatives conjugated with both DOTA and folate were
synthesized through a two-step procedure (Scheme 3): rst, DOTA
was grafted to the water soluble chitosan derivatives backbone
with the activation of EDC and NHS; second, activated FA solution
in DMSO was added to nish the FA conjugation. After purication
and lyophilization, three nal products were harvested: DOTA and
folate conjugated O-carboxymethyl chitosan (DCMCF), N-trimethyl
chitosan (DTMCF) and hydroxypropyl chitosan (DHPCF).
36
In fur-
ther study, these chitosan derivatives with both folate and DOTA
O

OH
O
H
2
N
O
O
OH
O
N
H
O
O

x
y
CMC
COOH
COOH
O

HO
OH
NH
2
O
O
HO
OH
NH
O
O

x
y
O
O
OH
OH
N
O
O
OH
OH
N
H
O
O

w
y
TMC
O
OH
OH
H
2
N

x
-
w
CS
O

OH
O
H
2
N
O
O
OH
O
N
H
O
O

x
y
OH
OH
HPC
a
b
c
Scheme 1. Synthesis route of water-soluble chitosan derivatives: CMC, TMC and
HPC. Reagents and conditions: (a) Chloroacetic acid, KOH, isopropanol, rt, 5 h; (b)
CH
3
I, N-methyl pyrrolidone, NaOH, 60 C, 6 h; (c) propylene oxide, TMAH, isopro-
panol, NaOH, rt, 1 h, then 60 C, 8 h.
O
O
HO
OR
X
O
O
HO
OR
NH
O
O

w
y
O
HO
OR
NH
2

x-w
CMC: X=-NH
2
, R=-CH
2
COOH
TMC: X=-
+
N(CH
3
)
3
, R=-H
TMCF: X=-
+
N(CH
3
)
3
, R=-H
HPC: X=-NH
2
, R=-CH
2
CH(OH)CH
3
Water-soluble chitosan derivatives
O
O
HO
OR
X
O
O
HO
OR
NH
O
O
w
y
O
HO
OR
NH
2
x-w-m
Folate conjugated water-soluble chitosan derivatives
O
HO
OR
NH
O
m
O
NH
O
N
H
N
N
HN
N H
2
N
O

d
99m
Tc-CMCF/TMCF/HPCF
e
CMCF: X=-NH
2
, R=-CH
2
COOH
HPCF: X=-NH
2
,R=-CH
2
CH(OH)CH
3

O
OH
Scheme 2. Synthesis route of folate-modied chitosan derivatives: CMCF, TMCF and HPCF. Reagents and conditions: (d) FA, EDC, NHS, DMSO, 30 C, 16 h; (e)
99m
TcO
4

, SnCl
2
,
HAcNaAc buffer, 100 C, 30 min.
H. Jing et al. / Bioorg. Med. Chem. Lett. 22 (2012) 34183424 3419
will be labeled with
64
Cu or
68
Ga for PET imaging, or
153
Sm for
radiotherapy, or Gd(III) for MR imaging.
All the products were determined by element analysis (Elemen-
tal analyzer, Vario EI, Elementar Company, Germany). Moreover,
the three water-soluble chitosan derivatives of CMC, TMC and
HPC were characterized by
1
H NMR spectra (in D
2
O, 400 MHz,
Bruker spectrometer, Switzerland), the six folate conjugated chito-
san derivatives of CMCF, TMCF, HPCF, DCMCF, DTMCF and DHPCF
were analyzed with UV spectroscopy (UVVisible Spectrometer,
UT-1901, Beijing Purkinje General Instrument Co., Ltd, China),
and the three DOTA modied chitosan derivatives of DCMC, DTMC
and DHPC were chelated with plenty of Gd(III)(GdCl
3
aqueous
solution, derived from the reaction of Gd
2
O
3
(99.90%, ACROS
ORGANICS, US) and 6 mol/L HCl), then analyzed with Inductive
Coupled Plasma Emission Spectrometer (ICP: ULTIMA, JY Company,
France) to determine the amount of DOTA conjugated to chitosan
skeleton. The degree of the substitution (DS) of carboxymethyl
groups on 6-O- of CMC is calculated from the results of acidbase
titration and element analysis. The calculation of the degree of
quaternization of TMC and the DS of hydroypropyl groups on
HPC was performed by using the
1
H NMR spectra. The DS of FA
on the six folate conjugated chitosan derivatives were calculated
by comparing their absorbance with the standard curve of folic
acid obtained at 363 nm by UV spectrophotometer.
18
According to the element analysis data of the raw material
chitosan (C/N ratio: 5.533, N: 7.407%, C: 40.98%, H: 6.693%), the de-
gree of deacetylation (DD) of CS was calculated as 77.26%, which
falls in the range (7585% deacetylated) provided by the product
supplier. Further, calculated from the element analysis data of
CMC and HPC (Shown in Table 1), the DS of carboxymethyl groups
on CMC and hydroxypropyl groups on HPC were 52.64% and
188.1% (the hydroxypropyl groups must have been grafted onto
both 3-OH and 6-OH of chitosan), respectively. Because of the exis-
tence of N-monomethylated and N-dimethylated units, it is impos-
sible to calculate the degree of quaternization of TMC from the
element analysis data accurately.
O
O
HO
OR
X
O
O
HO
OR
NH
O
O

w
y
O
HO
OR
NH
2

x-w
CMC: X=-NH
2
, R=-CH
2
COOH
TMC:X=-
+
N(CH
3
)
3
, R=-H
HPC: X=-NH
2
, R=-CH
2
CH(OH)CH
3
O
O
HO
OR
X
O
O
HO
OR
NH
O
O
w
y
O
HO
OR
NH
2

x-w-a
O
HO
OR
NH
O
a

Water-soluble chitosan derivatives

O
N
N
N
N
COOH
COOH
HOOC
O
O
HO
OR
X
O
O
HO
OR
NH
O
O
w
y
O
HO
OR
NH
2
x-w-a-b
O
HO
OR
NH
O
a

O
N
N
N
N
COOH
COOH
HOOC
O
HO
OR
NH
O
b
O

HN
O
NH
N
N
NH
N
NH
2
O
f
g
DCMC: X=-NH
2
, R=-CH
2
COOH
DTMC: X=-
+
N(CH
3
)
3
, R=-H
DHPC: X=-NH
2
, R=-CH
2
CH(OH)CH
3
DCMCF: X=-NH
2
, R=-CH
2
COOH
DTMCF: X=-
+
N(CH
3
)
3
, R=-H
DHPCF: X=-NH
2
, R=-CH
2
CH(OH)CH
3
O
OH
Scheme 3. Synthesis route of DOTA and folate modied chitosan derivatives: DCMCF, DTMCF and DHPCF. Reagents and conditions: (f) DOTA, EDC, NHS, MES, PBS, rt,
overnight; (g) FA, EDC, NHS, DMSO, 30 C, 16 h.
Table 1
Structure analysis data of chitosan derivatives
Content Element analysis data DD or DS (%) Number of FA Number of DOTA
C/N ratio N (%) C (%) H (%)
a b a c a d
CS 5.533 7.407 40.98 6.693 77.26 / / / / /
CMC 6.435 6.565 42.25 6.733 52.64 51.9 / / / /
TMC 7.738 5.434 42.05 7.545 / 29.6 / / / /
HPC 10.37 4.561 47.31 7.950 188.1 / / / / /
CMCF 5.436 7.802 42.41 6.874 / / 19.5 18.9 / /
TMCF 5.422 8.367 45.37 7.909 / / 27.2 25.6 / /
HPCF 7.981 6.070 48.44 7.766 / / 17.7 16.4 / /
DCMC 5.781 7.244 41.88 6.570 / / / / 20.2 21.1
DTMC 6.837 6.096 41.68 7.427 / / / / 19.3 20.0
DHPC 8.388 5.412 45.39 7.186 / / / / 29.3 30.3
DCMCF 5.113 8.443 43.17 6.984 / / 19.6 18.5 20.2 21.1
DTMCF 6.106 6.851 41.84 7.795 / / 10.2 9.1 19.3 20.0
DHPCF 6.928 6.583 45.61 6.814 / / 18.5 17.4 29.3 30.3
a
Calculated from the data of element analysis.
b
Calculated from the data of
1
H NMR or acidbase titration.
c
Calculated from the data of UV absorbance.
d
Calculated from the ICP results.
3420 H. Jing et al. / Bioorg. Med. Chem. Lett. 22 (2012) 34183424
Based on the results of element analysis obtained above, the
amounts of folic acid and DOTA conjugated to the other six chito-
san derivatives were also calculated from their element analysis
data: there are about 19.5 FA molecules in each CMCF molecule,
about 19.6 FA molecules and 20.2 DOTA molecules in each DCMCF
molecule, about 27.2 FA molecules in each TMCF molecule, about
10.2 FA molecules and 19.3 DOTA molecules in each DTMCF mole-
cule, about 17.7 FA molecules in each HPCF molecule, and about
18.5 FA molecules and 29.3 DOTA molecules in each DHPCF
molecule.
Figure 1 is the
1
H NMR spectra of CMC, TMC and HPC in D
2
O.
The resonances in the region between 3.7 and 4.5 ppm in Figure
1a are the protons of 3-O- and 6-O-subsitituted carboxymethyl
(OCH
2
COOD) of CMC, and no sharp signal of the resonance of
the protons from NCH
2
COOD can be found around 3 ppm, which
indicates that carboxymethlation only took place on the hydroxyl
groups but not on the amino groups of CS.
28
The DS of TMC can
be observed from the quaternized peak at 3.2 ppm and the tertiary
peak at 2.9 ppm in Figure 1b.
29
The degree of quaternization of
TMC was calculated as 29.6% according to the following formula:
DS
Z
peak at 3:2 ppm
=
Z
peak at 5:0 ppm

Z
peak at 5:3 ppm

9 1
The peaks at 5.0 and 5.3 ppm are used as internal standards.
The strong resonance signal at 1.0 ppm in Figure 1c suggests
that a large number of hydroxypropyl groups have been introduced
into the CS molecule successfully.
The DS of carboxymethyl groups on CMC was also measured by
an acidbase titration method.
31
A CMC solution (0.500 g CMC in
50 mL 0.1 mol/L HCl standard solution) was titrated with
0.1 mol/L sodium hydroxide standard solution under stirring. Re-
cord the volume of sodium hydroxide consumed at pH 2.1 and
pH 4.3 as V
1
and V
2
, respectively. Calculation of the DS of caboxym-
ethyl groups on CMC was performed according to the following
formula:
DS 0:203A=1 0:058A 2
A V
2
V
1
C=W 3
W is the weight of the CMC sample (g); C is the concentration of the
sodium hydroxide standard solution (mol/L).
In our test, W is 0.500 g, V
1
is 22.55 mL, V
2
is 34.01 mL, and C is
0.09627 mol/L. The corresponding DS of caboxymethyl groups on
CMC was calculated as 51.9%, this result is agreed well with the
conclusion of element analysis.
The UV spectroscopy of CMC, TMC, HPC and their derivatives
are demonstrated in Figure 2a, b and c, respectively. It is obvious
that folate conjugated molecules have much stronger UV absor-
bance than those without folate, which is a proof of the successful
coupling of folic acid molecules to the chitosan derivatives. When
the concentration of folic acid is between 5.15 and 30.9 lg/mL, it
has good linear relationship with its UV absorbance at 363 nm.
Hence, a standard curve of folic acid was obtained by measuring
absorbance of different concentrations of folic acid at 363 nm with
a UV spectrophotometer.
18
Accordingly, the DS of folic acid can be
calculated by comparing the absorbance of each folate conjugated
chitosan derivative (after subtracted the absorbance of the corre-
sponding chitosan derivative backbone) at 363 nm with the stan-
dard curve of folic acid (Fig. 2d). The DS is calculated as 0.125 for
CMCF, 0.109 for DCMCF, 0.184 for TMCF, 0.058 for DTMCF, 0.087
for HPCF and 0.081 for DHPCF. Further calculation shows that there
are about 18.9 FA molecules in each CMCF molecule, about 18.5 FA
molecules in each DCMCF molecule, about 25.6 FA molecules in
each TMCF molecule, about 9.1 FA molecules in each DTMCF mol-
ecule, about 16.4 FA molecules in each HPCF molecule and about
17.4 FA molecules in each DHPCF molecule, which are all close to
the conclusions of element analysis. To determine the amount of
DOTA grafted on water-soluble chitosan derivatives, the excess of
GdCl
3
was added to the solution of DOTA grafted chitosan
Figure 1.
1
H NMR spectra of (a) CMC, (b) TMC and (c) HPC in D
2
O.
H. Jing et al. / Bioorg. Med. Chem. Lett. 22 (2012) 34183424 3421
derivatives. After the reaction, DTPA was used to remove the unre-
acted Gd(III) ion. In this condition, it is generally believed that the
mole ratio of DOTA and Gd(III) is 1:1 because of their strong chela-
tion ability. By measuring the weight ratio of Gd(III) in the
Table 2
The biodistribution results of
99m
Tc-complexes in normal mice (% ID/g, mean SD, n = 5)
Tissues Post-injection time
99m
Tc-complexes
99m
Tc-CMC
99m
Tc-CMCF
99m
Tc-TMC
99m
Tc-TMCF
99m
Tc-HPC
99m
Tc-HPCF
Heart 1 h 4.64 1.31 2.57 1.31 1.02 0.26 0.53 0.14 0.40 0.09 0.31 0.17
4 h 3.10 0.58 1.20 0.24 1.15 0.47 0.53 0.06 0.32 0.0.23 0.31 0.19
Liver 1 h 13.12 1.59 28.47 7.14 59.46 13.16 104.38 4.96 82.48 12.68 81.39 16.01
4 h 17.83 2.60 30.07 6.63 60.86 9.18 108.63 19.81 85.53 13.27 89.78 11.35
Lung 1 h 12.36 2.60 5.41 1.09 32.76 19.30 9.93 1.24 1.79 0.78 1.18 0.38
4 h 7.32 3.60 1.81 0.46 11.19 7.69 12.47 0.61 0.91 0.27 0.63 0.42
Kidney 1 h 14.88 1.07 12.84 3.46 14.84 3.74 2.86 0.83 1.80 0.84 0.54 0.26
4 h 15.52 2.42 15.34 2.83 14.40 2.97 3.08 0.15 1.19 0.04 0.75 0.35
Spleen 1 h 7.03 1.84 3.42 0.71 207.41 78.65 73.00 23.74 118.27 52.80 96.87 55.19
4 h 7.30 1.94 3.37 0.99 196.18 73.38 116.10 18.46 68.29 39.46 74.17 23.50
Stomach 1 h 1.78 0.36 1.29 0.67 6.01 2.94 0.89 0.52 1.37 0.94 0.66 0.19
4 h 1.95 0.87 1.19 0.27 4.83 2.52 1.77 1.22 0.72 0.22 1.21 0.58
Bone 1 h 4.39 0.27 2.37 0.25 8.01 2.78 1.79 0.22 2.04 0.71 0.83 0.40
4 h 6.33 0.99 2.73 0.63 7.88 2.42 2.97 0.33 0.80 0.19 1.80 1.00
Muscle 1 h 0.97 0.11 0.40 0.04 0.54 0.24 1.01 0.13 0.50 0.0.70 0.17 0.10
4 h 0.84 0.22 0.42 0.15 0.94 0.50 0.26 0.05 0.19 0.14 0.21 0.18
Intestines 1 h 3.45 0.77 1.77 0.16 2.04 0.89 0.65 0.47 3.60 1.19 1.59 0.42
4 h 3.25 0.72 1.16 0.25 1.88 0.62 1.28 0.35 4.13 2.64 1.76 0.95
Blood 1 h 27.09 3.19 9.32 1.85 3.11 1.01 0.52 0.14 0.62 0.60 0.29 0.12
4 h 14.92 1.69 4.09 0.99 1.38 0.31 0.33 0.05 0.07 0.02 0.07 0.02
Figure 2. UV analysis of (a) CMC and its derivatives, (b) TMC and its derivatives and (c) HPC and its derivatives; (d) standard curve of folic acid measured at 363 nm by UV
spectrophotometer.
3422 H. Jing et al. / Bioorg. Med. Chem. Lett. 22 (2012) 34183424
complexes with ICP, the number of DOTA coupled to each chitosan
derivative can be calculated accordingly.
11
In this study, the weight
ratio of Gd(III) (subtracted the chelation of Gd(III) with the water-
soluble chitosan derivative backbone) was 4.6%, 4.3% and 4.8%
in [Gd(III)]DCMC, [Gd(III)]DTMC and [Gd(III)]DHPC, respectively.
Calculation from these results suggests that there are about 21.1
DOTA molecules in each DCMCF molecule, about 20.0 DOTA mole-
cules in each DTMCF molecule and about 30.3 DOTA molecules in
each DHPCF molecule, which coincide well with the conclusions of
element analysis.
The radiolabeling conditions of CMC, CMCF, TMC, TMCF, HPC and
HPCF with
99m
Tc were also studied, and the optimized procedure
was performed as shown in Scheme 2e.
37
These
99m
Tc-complexes
(
99m
Tc-CMC,
99m
Tc-CMCF,
99m
Tc-TMC,
99m
Tc-TMCF,
99m
Tc-HPC
and
99m
Tc-HPCF) were analyzed with instant thin-layer chromatog-
raphy (ITLC)-SG chromatographic strips (PALL Life Sciences, US)
with ACD buffer (pH 5.0, a mixture of 0.068 mol/L citrate and
0.074 mol/L glucose) as the mobile phase. In this system, the R
f
of
all these
99m
Tc-complexes were 00.1, while the R
f
of
99m
TcO
4

and
99m
TcO
2
H
2
O were 0.91.0. The labeling yields of
99m
Tc-CMC,
99m
Tc-TMC,
99m
Tc-HPC and
99m
Tc-HPCF were more than 95%, while
the labeling yields of
99m
Tc-CMCF and
99m
Tc-TMCF were a little
lower, but still over 90%. After puried using a HiTrap desalting col-
umn (Sephadex G100, GE Healthcare, US), the radiochemical purity
of all the synthesized
99m
Tc-complexes can be improved to over
95%.
The stabilities of the six
99m
Tc-complexes were tested in water
solution and mouse plasma by determining the radiochemical pur-
ity (TLC method, under ITLC-SG/ACD system) at different time
points after preparation or incubating in the solution of 0.5 mL
murine plasma at 37 C. Plasma proteins were precipitated by add-
ing 100 lL acetonitrile and removed by centrifugation at 6000 rpm
for 5 min at room temperature. The radiochemical purity of the six
99m
Tc-complexes all remained above 90% after 6 h in water solu-
tion at room temperature and in murine plasma at 37 C, indicating
that these six
99m
Tc-complexes have good stability.
The partition coefcient (lg P) of each
99m
Tc-complex was mea-
sured by the method reported by Guo et al.
18
The partition coef-
cient values of
99m
Tc-CMC,
99m
Tc-CMCF,
99m
Tc-TMC,
99m
Tc-TMCF,
99m
Tc-HPC and
99m
Tc-HPCF were 1.48 0.006, 1.66 0.003,
1.92 0.17, 1.23 0.01, 1.58 0.01 and 2.00 0.04, respec-
tively, suggesting that all these
99m
Tc-complexes are hydrophilic.
Compared with
99m
Tc-CSFA (lg P = 1.68 0.14), it seems that
the solubility of these water-soluble chitosan derivatives have no
signicant improvement. However, one fact that should not be ig-
nored is that the chitosan used to synthesize the reported
99m
Tc-
CSFA has the molecular weight of 8 kDa, while the molecular
weight of the raw material chitosan in this study is much higher,
about 50 kDa, which leads to a signicantly poorer solubility. From
this point of view, the result that the solubility of the nal products
herein close to that of
99m
Tc-CSFA has proved the modication
methods in this study has achieve the goal of solubility
improvement.
The biodistribution studies of the six
99m
Tc-complexes were
performed in normal Kunming female mice (1820 g), and the re-
sults are shown in Table 2. All the biodistribution studies were car-
ried out in compliance with the national laws related to the
conduct of animal experiments and the animals were not observed
any adverse reactions in our biological experiments. As described
in Table 2,
99m
Tc-TMC,
99m
Tc-TMCF,
99m
Tc-HPC and
99m
Tc-HPCF
were mainly accumulated in liver and spleen, while the uptake
of
99m
Tc-CMC and
99m
Tc-CMCF in liver and spleen were much low-
er. However, all of them hardly showed any elimination from kid-
ney, liver and spleen. Although there were some initial
accumulation of
99m
Tc-CMC,
99m
Tc-CMCF and
99m
Tc-TMC in lung,
their clearance from lung were fast. The high liver and spleen up-
take may caused by the large particles formed by
99m
Tc-complexes
in physiological environment, and these particles did not tend to
be absorbed by kidney. To conrmwhether these folate conjugated
99m
Tc complexes can target at folate receptor or not, further stud-
ies with animal models bearing tumors rich in folate receptors will
be conducted.
In summary, three chemical modied water soluble chitosan
derivatives have been synthesized, on the basis of which three fo-
late grafted chitosan derivatives were obtained successfully, and
their corresponding
99m
Tc-complexes were prepared in high yields.
The solubility of these chitosan derivatives have been improved
greatly, and their
99m
Tc-complexes possess good stability. Three
chitosan derivatives containing both folate and DOTA were also
synthesized. With different chelating metal, the complexes can
be used for different purpose, such as
64
Cu and
68
Ga for PET imag-
ing,
67
Ga and
111
In for SPECT imaging,
153
Sm and
177
Lu for radio-
therapy, Gd(III) for MR imaging, etc. If chelating with two kinds
of such metals, it is possible to develop these chitosan derivatives
into dual modal imaging agents. The favorable results and charac-
teristics demonstrated in this study as well as the potential pros-
pect prove the feasibility of these water-soluble chitosan
derivatives as nuclear imaging agent backbones, which makes a
push for the further studies of their radiochemical and biological
properties.
Acknowledgments
This study was nancially supported by the Fundamental Re-
search Funds for the Central Universities of China, and partially
by the National Natural Science Foundation of China (20871020),
Beijing Natural Science Foundation (2092018) and the Scientic
Research Foundation for the Returned Overseas Chinese Scholars,
State Education Ministry.
References and notes
1. Agrawal, P.; Strijkers, G. J.; Nicolay, K. Adv. Drug Delivery Rev. 2010, 62, 42.
2. Dash, M.; Chiellini, F.; Ottenbrite, R. M.; Chiellini, E. Prog. Polym. Sci. 2011, 36,
981.
3. He, P.; Davis, S. S.; Illum, L. Int. J. Pharm. 1998, 166, 75.
4. Felse, P. A.; Panda, T. Bioprocess Biosyst. Eng. 1999, 20, 505.
5. Hu, Y.; Jiang, H.; Xu, C.; Wang, Y.; Zhu, K. Carbohydr. Polym. 2005, 61, 472.
6. Mansouri, S.; Cuie, Y.; Winnik, F.; Shi, Q.; Lavigne, P.; Benderdour, M.;
Beaumont, E.; Fernandes, J. C. Biomaterials 2006, 27, 2060.
7. Martin, L.; Wilson, C. G.; Koosha, F.; Tetley, L.; Gray, A. I.; Senel, S.; Uchegbu, I. F.
J. Controlled Release 2002, 80, 87.
8. Wang, F.; Zhang, D.; Duan, C.; Jia, L.; Feng, F.; Liu, Y.; Wang, Y.; Hao, L.; Zhang, Q.
Carbohydr. Polym. 2011, 84, 1192.
9. Zheng, Y.; Cai, Z.; Song, X.; Yu, B.; Bi, Y.; Chen, Q.; Zhao, D.; Xu, J.; Hou, S. Int. J.
Pharm. 2009, 382, 262.
10. Yang, S. J.; Lin, F. H.; Tsai, K. C.; Wei, M. F.; Tsai, H. M.; Wong, J. M.; Shieh, M. J.
Bioconjugate Chem. 2010, 21, 679.
11. Nam, T.; Park, S.; Lee, S. Y.; Park, K.; Choi, K.; Song, I. C.; Han, M. H.; Leary, J. J.;
Yuk, S. A.; Kwon, I. C.; Kim, K.; Jeong, S. Y. Bioconjugate Chem. 2010, 21, 578.
12. Shin, B. C.; Park, K. B.; Jang, B. S.; Lim, S. M.; Shim, C. K. Nucl. Med. Biol. 2001, 28,
719.
13. Cho, B. C.; Kim, E. H.; Choi, H. J.; Kim, J. H.; Roh, J. K.; Chung, H. C.; Ahn, J. B.; Lee,
J. D.; Lee, J. T.; Yoo, N. C.; Sohn, J. H. Yonsei Med. J. 2005, 46, 799.
14. Marques, F.; Gano, L.; Batista, M. K. S.; Gomes, C. A. R.; Gomes, P.; Santos, I. J.
Labelled Compd. Radiopharm. 2009, 52, 79.
15. Kim, E.-M.; Jeong, H.-J.; Park, I.-K.; Cho, C.-S.; Kim, C.-G.; Bom, H.-S. J. Nucl. Med.
2005, 46, 141.
16. Kim, E. M.; Jeong, H. J.; Kim, S. L.; Sohn, M. H.; Nah, J. W.; Bom, H. S.; Park, I. K.;
Cho, C. S. Nucl. Med. Biol. 2006, 33, 529.
17. Yang, W.; Mou, T.; Guo, W.; Jing, H.; Peng, C.; Zhang, X.; Ma, Y.; Liu, B. Bioorg.
Med. Chem. Lett. 2010, 20, 4840.
18. Guo, W.; Jing, H.; Yang, W.; Guo, Z.; Feng, S.; Zhang, X. Bioorg. Med. Chem. Lett.
2011, 21, 6446.
19. Zhang, J.; Xia, W.; Liu, P.; Cheng, Q.; Tahi, T.; Gu, W.; Li, B. Mar. Drugs 1962,
2010, 8.
20. Choi, J.; Lee, S.; Kang, H. J.; Lee, J.; Kim, J.; Yoo, H. O.; Stratton, T.; Applegate, B.;
Youngblood, J.; Kim, H.; Ryu, K. Macromol. Res. 2010, 18, 504.
21. Bernkop-Schnrch, A.; Steininger, S. Int. J. Pharm. 2000, 194, 239.
22. Knight, D. K.; Shapka, S. N.; Amsden, B. G. J. Biomed. Mater. Res. A 2007, 83A,
787.
H. Jing et al. / Bioorg. Med. Chem. Lett. 22 (2012) 34183424 3423
23. Mathew, M. E.; Mohan, J. C.; Manzoor, K.; Nair, S. V.; Tamura, H.; Jayakumar, R.
Carbohydr. Polym. 2010, 80, 442.
24. Li, D.-H.; Liu, L.-M.; Tian, K.-L.; Liu, J.-C.; Fan, X.-Q. Carbohydr. Polym. 2007, 67,
40.
25. Louie, A. Chem. Rev. 2010, 110, 3146.
26. Ali, Z.; Abbasi, A. Z.; Zhang, F.; Arosio, P.; Lascialfari, A.; Casula, M. F.; Wenk, A.;
Kreyling, W.; Plapper, R.; Seidel, M.; Niessner, R.; Knoll, J.; Seubert, A.; Parak,
W. J. Anal. Chem. 2011, 83(8), 2877.
27. Yang, X. Q.; Hong, H.; Grailer, J. J.; Rowland, I. J.; Javadi, A.; Hurley, S. A.; Xiao, Y.
L.; Yang, Y. N.; Zhang, Y.; Nickles, R. J.; Cai, W. B.; Steeber, D. A.; Gong, S. Q.
Biomaterials 2011, 32, 4151.
28. Chen, X.-G.; Park, H.-J. Carbohydr. Polym. 2003, 53, 355.
29. Thanou, M. M.; Kotz, A. F.; Scharringhausen, T.; Lueen, H. L.; de Boer, A. G.;
Verhoef, J. C.; Junginger, H. E. J. Controlled Release 2000, 64, 15.
30. Shi, Y.; Li, J.; Chen, Y.; He, G. Text. Aux. 2006, 23, 5.
31. Zhao, A.; Yuan, X.; Chang, J. Polym. Bull. 2004, 4, 5.
32. Synthesis of CMC: Briey, 5 g chitosan, 5.75 g potassium hydroxide (in 5 mL
H
2
O) and 40 mL isopropanol were added into a ask to swell and alkalize for
over 5 h. 2.5 g monochloroacetic acid was add slowly in to the reagent mixture
in ice bath. The reaction was continued for 5 h at room temperature with
stirring. The solid was ltered and then solved in water. After adjusting the pH
to 78 with hydrochloric acid, plenty of ethanol was added to precipitate. The
precipitation was ltered and rinsed in 70% ethanol, 95% ethanol and acetone
successively to desalt and dewater, and vacuum dried at room temperature to
obtain CMC.
33. Synthesis of TMC: Briey, 1 g chitosan in 40 mL N-methyl pyrrolidone was
mixed with 7 mL 5% (w/v) sodium hydroxide at 60 C. Six milliliter
methyliodide was added and the reaction was allowed to reux at 60 C for
6 h with stirring. The product was precipitated with ethanol/ether (1:1),
subsequently isolated by ltration and thoroughly washed with the mixture of
ethanol and ether. The product was dried under vacuum.
34. Synthesis of HPC: Briey, 2 g chitosan in 20 mL isopropanol was stirred for
30 min at room temperature to swell. Two milliliter 33% sodium hydroxide
was added and stirred for 1 h at room temperature followed by freezing
overnight to alkalize. Twenty milliliter propylene oxide was added to the
reagent mixture. The reaction was carried out with 1 mL TMAH as catalyst at
room temperature for 1 h with stirring, and continued for another 8 h at 60 C.
Then the pH of the mixture was adjusted to neutral. The solvent was removed
through rotary evaporation. Then the solvent residue was redissolved in some
water and puried by dialyzing in D45 mm dialysis bags (Closure 800012000)
against distilled water at room temperature for 4 days. The nal product was
harvested by lyophilization.
35. Synthesis of CMCF, TMCF and HPCF conjugates: Briey, 88 mg (0.2 mmol) FA,
57 mg EDC (0.3 mmol) and 34 mg (0.3 mmol) NHS were dissolved in 3 mL
dimethylsulfoxide (DMSO) and stirred for 1 h at 30 C in dark. The mixture was
then added to a solution of 100 mg water soluble chitosan (CMC or TMC or
HPC) in 10 mL phosphate buffer (0.05 mol/L, pH 7.4). The reaction was allowed
to be stirred for 16 h at 30 C and shielded from light. Next, the solution was
dialysed in D45 mm dialysis bags (Closure 8000120000) against phosphate
buffer (0.05 mol/L, pH 7.4) for 3 days and followed by distilled water for
another 3 days to remove the unreacted small molecules and desalt. The crude
products of folate conjugated chitosan derivatives were obtained by
lyophilization. The further purication was performed by passing the
solution of crude products in 2% acetic acid through a Sephade G-100
column, then freeze-dried again to get the rened products of folate
conjugated water soluble chitosan derivatives.
36. Synthesis of DCMCF, DTMCF and DHPCF conjugates: The synthesis of these three
conjugates was carried out with a two-step procedure. First, graft DOTA
molecules onto the chitosan derivatives backbones. Briey, 808 mg (2 mmol)
DOTA was activated with 575 mg (3 mmol) EDC and 345 mg (3 mmol) NHS in
10 mL 0.02 mol/L 2-(N-morpholino)ethanesulfonic acid buffer (MES, pH 5.5,
>99%, Amresco, US) for 30 min at room temperature. 500 mg water soluble
chitosan (CMC or TMC or HPC) in 15 mL distilled water was then added in the
activated DOTA solution. Adjust pH to 88.5 with 1 mol/L sodium hydroxide,
and stir the mixture overnight at room temperature. The solution was then
dialysed in D45 mm dialysis bags (Closure 8000120000) against phosphate
buffer (0.05 mol/L, pH 7.4) for 3 days. Second, dialysed solution in the rst step
react with folic acid and then puried by the method similar with that
described in the synthesis of folate conjugated water soluble chitosan
derivatives above. After lyophilization the nal products of chitosan
derivatives modied with both DOTA and folate were obtained.
37.
99m
Tc-labeling of CMC, CMCF, TMC, HPC and HPCF: Briey, a solution of 1 mg
chitosan derivatives in 0.2 mL HAcNaAc buffer (pH23 for CMC and CMCF, pH
4 for TMCF, pH 6.5 for TMC, HPC and HPCF) was mixed with 40 lL stannous
chloride solution (2 mg/mL in 0.1 mol/L HCl) in a vial. Then 0.5 mL Na
99m
TcO
4
solution (about 37555 MBq) eluted from a
99
Mo/
99m
Tc generator (HTA Co.,
Ltd, China) was added to the above reagent mixture. After heated in boiling
water bath for 30 min, the nal
99m
Tc-complexes (
99m
Tc-CMC,
99m
Tc-CMCF,
99m
Tc-TMC,
99m
Tc-TMCF,
99m
Tc-HPC and
99m
Tc-HPCF) were obtained.
3424 H. Jing et al. / Bioorg. Med. Chem. Lett. 22 (2012) 34183424