Development of 153Sm-folate-polyethyleneimine-conjugated chitosan nanoparticles for targeted therapy

Article abstract of DOI:10.1002/jlcr.3305

The aim of this study was to develop biocompatible, water-soluble ¹⁵³Sm-labeled chitosan nanoparticles (NPs) containing folate and polyethyleneimine functionalities i.e. chitosan-graft-PEI-folate (CHI-DTPA-g-PEI-FA), suitable for targeted thera

Full text of DOI:10.1002/jlcr.3305

Research article
Received 26 January 2015,
Revised 13 April 2015,
Accepted 20 April 2015
Published online 2 June 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3305
1
53
Development of Sm-folate-
polyethyleneimine-conjugated chitosan
nanoparticles for targeted therapy
a
b
b
Esmail Mollarazi, Amir R. Jalilian, * Fariba Johari-daha,
a,c
and Fatemeh Atyabi
1
53
The aim of this study was to develop biocompatible, water-soluble
Sm-labeled chitosan nanoparticles (NPs) containing
folate and polyethyleneimine functionalities i.e. chitosan-graft-PEI-folate (CHI-DTPA-g-PEI-FA), suitable for targeted therapy.
The physicochemical properties of the obtained NPs were characterized by dynamic light-scattering analysis for their mean
1
size, size distribution, and zeta potential; scanning electron microscopy for surface morphology; and H-NMR, FT-IR analyses
for molecular dispersity of folate in the NPs. NPs were spherical with mean diameter below 250 nm, polydispersity of below
1
53
0
.15, and positive zeta potential values. The NP complex ( Sm-CHI-DTPA-g-PEI-FA) was stable at 25 °C (6–8 h, >90%
radiochemical purity, instant thin layer chromatography (ITLC)). Binding studies using fluorescent NPs for internalization
also demonstrated significant uptake in MCF-7 cells. MCF-7 cell internalization was significantly greater for 4T1. In blocking
studies, both MCF-7 and 4T1 cell lines demonstrated specific folate receptor (FR) binding (decreasing 45%). In vivo
1
53
biodistribution studies indicated major excretion of NPs metabolites and/or free Sm through the kidneys. The preliminary
imaging studies in 4T1 tumor-bearing mice showed minor uptake up to 96 h. The present folic acid that functionalized
chitosan NP is a candidate material for folate receptor therapy.
153
Keywords: chitosan nanoparticles; Sm; biodistribution; folate receptor; MCF-7; 4T1; breast cancer; SPECT
Introduction
Conjugation of chitosan with polyethyleneimine (PEI) has
been shown to effectively increase cell uptake rely on excess
cationic charge to mediate cellular entry due to its buffering
The advances in nanoparticle (NP) science application in
molecular medicine have led to the development of many
nanoconstructs for therapy and diagnosis in human diseases.
Radiolabeled NPs based on various inorganic nanocores
12
capacity. PEI exists as a branched polymer, as well as in linear
form. The branched form of PEI shows a theoretical ratio of
primary to secondary to tertiary amine groups of 1:2:1. These
1
including paramagnetic metal oxides, and organic/biological
amines have pKa values spanning the physiological pH range,
2
3
polymers such as chitosan, aggregated albumin, and so on
have been reported for targeted therapy/diagnosis based on
the size and molecular surface decorations.
Because of its excellent effects, internal radiation therapy is in
widespread use for the treatment of a variety of diseases. Such
13
resulting in buffering capacity.
On the other hand, many
studies have addressed concerns about the toxicity of
conventional PEI. The cytotoxicity of PEI is dependent on its
molecular weight. A lower molecular weight PEI has a lower
14,15
cytotoxicity.
Because of negatively charged plasma
4
5 153
diseases include cancer and rheumatoid arthritis.
Sm, which
components, cellular entry mechanism based on charge
has a suitable half-life (46.7 h) and β-emitting property along
with γ-ray for imaging, represents an interesting candidate for
developing therapeutic molecules.
For many years, complexes combining radioactive metals with
diethylene triamine pentaacetic acid (DTPA) have been widely
a
6
7–9
Food and Drug Control Laboratories and Food and Drug Laboratory Research
used in molecular imaging and radiotherapy.
many DTPA lanthanides analogs have proved stable enough
Moreover,
Centre, MOHME, Tehran, Iran
10
b
for use in a physiological medium as radiopharmaceuticals.
So immobilization of these auxiliary agents on polymeric carrier
Radiation Application Research School, Nuclear Science and Technology
Research Institute (NSTRI), Tehran, 14155-1339, Iran
1
1
matrix is essential.
c
Medical Nanotechnology Research Centre, Tehran University of Medical
Chitosans, a family of linear binary polysaccharides, comprised Sciences, Tehran, Iran
of beta (1–4)-linked 2-amino-2-deoxy-β-D-glucose (GlcN; D-unit)
*
Correspondence to: Amir R. Jalilian, Radiation Application Research School,
and the N-acetylated analog (GlcNAc; A-unit), have been
proposed as biocompatible alternative cationic polymers that
are suitable for drug delivery.
Nuclear Science and Technology Research Institute (NSTRI), Tehran, 14155-
1
E-mail: ajalili@aeoi.org.ir
339, Iran.
J. Label Compd. Radiopharm 2015, 58 327–335
Copyright © 2015 John Wiley & Sons, Ltd.

E. Mollarazi et al.
153
interaction is likely to be ineffective for in vivo drug delivery of
vectors administered intravenously.
Finally, samarium-153 chitosan-graft-PEI-folate ( Sm-CHI-g-
PEI-FA) NPs were prepared at optimized conditions followed by
The folate receptor is a high-affinity membrane folate-binding stability, human tumor cell binding, and biodistribution studies
protein, overexpressed in a wide variety of human tumors. in normal and tumor-bearing animals using dissection and
Meanwhile, normal tissue distribution of folate receptor is highly Single Photon Emission Computed Tomography (SPECT) studies.
restricted, making it a useful marker for targeted drug delivery to
1
6,17
tumors.
Folate conjugates have been shown to be taken into
Experimental
receptor-bearing tumor cells via folate receptor-mediated
endocytosis. Folate conjugation, therefore, presents a useful Chitosan (medium molecular weight; deacetylation degree, 87.7%),
method for receptor-mediated drug delivery into receptor- sodium periodate, sodium borohydride (granular, 10–40 meshes, 98%),
branched PEI 1800 Da, folic acid dihydrate and diethylene triamine
pentaacetic acid-dianhydride (DTPA-DA), dicyclohexylcarbodiimide
positive tumor cells.
In this study, in the first part, PEI-folate (PEI-FA) was
(
DCC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-
synthesized according to the reported method using a simple
conjugation step via a water elimination reaction (Figure 1).
In the next step, the chitosan-graft-PEI-folate (CHI-g-PEI-FA)
NPs were prepared by an imine reaction between periodate-
oxidized chitosan and the aforementioned low molecular weight
Aldrich Chemical Co. (Germany); (99%). All other reagents were
153
commercially available and used as received. Production of
Sm was
152
performed at the Tehran research reactor using
Sm (n, gamma)
Sm with purity of >98% was obtained from
153
152
Sm nuclear reaction.
ISOTEC Inc., USA. Radiochromatography was performed by counting of
PEI to reduce cytotoxicity and enhance the cell nucleus Whatman no. 2 (GE, England) using a thin layer chromatography scanner,
membrane transfer efficiency (Figure 2).
Bioscan AR2000 (Paris, France). Calculations were based on the 103-keV
153
peak for Sm. All values were expressed as mean ± standard deviation,
and the data were compared using student’s t-test. Statistical
significance was defined as P < 0.05. Animal studies were performed in
accordance with the United Kingdom Biological Council’s Guidelines on
the Use of Living Animals in Scientific Investigations, 2nd edition. Cell
lines were purchased from the Institute Pasteur of Iran.
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Production and quality control of
SmCl solution
3
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The
Sm was produced by neutron irradiation of 100 μg of enriched
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52
18
Sm
of 5 × 10 n cm
7.75 GBq/mg. The irradiated target was dissolved in 200 μL of 1.0 mol/
2 3
O according to reported procedures at a thermal neutron flux
1
3
ꢀ2 ꢀ1
1
5
3
s
for 5 days. Specific activity of the
Sm was
2
1
53
Figure 1. Proposed reaction scheme for synthesis of PEI-FA.
3
L HCl, to prepare SmCl and diluted to the appropriate volume with
Figure 2. Proposed reaction scheme for synthesis CHI-DTPA-g-PEI-FA.
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 327–335

E. Mollarazi et al.
ultra pure water, to produce a stock solution. The mixture was filtered Synthesis of copolymer
through a 0.22 μm of biological filter and sent for use in the radiolabeling
The CHI-DTPA-g-PEI-FA copolymer was synthesized in two steps. In the
first step, periodate-oxidized chitosan was prepared by a modified
step. Radionuclidic purity of the solution was tested for the presence of
other radionuclides using beta spectroscopy and High Purity Germanium
2
0
method of Y. Jia et al. Briefly, 300 mg of CHI-DTPA was dissolved into
0 mL of acetic acid (1%, v/v), and the obtained clear solution was
degassed by purging N for 30 min, and then 1.5 mL of sodium periodate
NaIO ) solution (10 mg/mL) was added to the solution in a light-
(HPGe) spectroscopy to detect various interfering beta and gamma
2
emitting radionuclides. The radiochemical purity was also checked by
Whatman no. 1 chromatography paper, and developed in a mixture of
2
(
4
10 mmol/L DTPA solution and also 10% ammonium acetate/methanol
protected glass vessel. After mixing the solutions, the reaction was
performed for 30 min at 50 °C with occasional shaking. In order to
quench the reaction, 0.1 mL of glycerine of reaction medium was added.
Then the solution was dialyzed (MWCO = 3500 Da) against deionized
water. In the second step, 200 mg of PEI-FA was reacted with the
(
1:1) as mobile phases.
Preparation of low molecular weight chitosan
Chitosan (2 g, medium molecular weight, 400,000 g/mol) was dissolved periodate-oxidized chitosan solution with magnetic stirring for 6 h at
in 100 mL of acetic acid (6%, v/v) and depolymerized at room
70 °C. Subsequently, the solution was treated with sodium borohydride
temperature under stirring with 10 mL of NaNO solutions in water at
2
(0.4 g NaBH /g chitosan), dialyzed against deionized water (MWCO = 5–
4
definite concentration (12 g/L), to obtain the desired final molecular 8 kDa), to remove unreacted PEI-FA. After dialysis, the copolymer was
weight of 9000 g/mol. Afterwards, chitosan was precipitated by the
addition of 4 M of NaOH until pH 9 was reached. The resulting precipitate
was filtered and washed with cold acetone. The white yellowish solid
lyophilized.
was filtrated, washed thoroughly with acetone, and re-dissolved in a Characterization of CHI-DTPA-g-PEI-FA conjugate
minimum volume of acetic acid of 0.1 N (40 mL), and purification was
carried out by subsequent dialysis against demineralized water. (Sigma
dialysis tubes, molecular weight cutoff, 5–8 kDa). The dialyzed product
was lyophilized using a LyoTrap plus Freeze dryer (LTE Scientific,
1
H-NMR spectrum of the prepared CHI-DTPA-g-PEI-FA was recorded on
™
Avance 500 spectrometer (Bruker, Germany) operating at 500 MHz
using D O and DMSO-d as solvents. Chemical shifts (δ) were given in
2 6
parts per million using tetramethylsilane as an internal reference. The
Fourier transform infrared spectrum of products was recorded using
KBr pellet at room temperature.
Oldham, UK), and the yellowish lyophilized product was then stored at
4
°C until use. The average molecular weights of the prepared chitosans
were determined by a gel permeation chromatography. The molecular
weight of the product was checked according to the published
1
9
procedure.
Size distribution of NPs
The mean diameter and size distribution of the NPs were determined by
dynamic light scattering using Zetasizer® (Nano-ZS, Malvern, Instruments,
Malvern, UK). All dynamic light-scattering measurements were carried
out at a wavelength of 633 nm at 25 °C with an angle detection of 90°.
The samples were diluted in acetic acid (16 μmol/L) in deionized water,
three subsequent measurements were determined for each sample,
and the result was expressed as mean size ± standard deviation.
Synthesis of CHI-DTPA conjugate
The conjugated chitosan-diethylene triamine pentaacetic acid (CHI-
DTPA) was synthesized by the coupling reaction of free carboxyl group
of DTPA-DA with amine group of chitosan in the presence of DCC and
NHS. Briefly, to a 60 mg of DTPA-DA dissolved in a 5 mL of hot ethanol
were added 25 mg of DCC and 13 mg of NHS, respectively, and mixed
for 1 h. After elapsed time, they were then mixed together under
mechanical stirring. The coupling reaction was carried out for 5 h at
Determination of zeta potential
70 °C under mechanical stirring with 300 rpm.
After the reaction, the reaction solution was dialyzed against The zeta potential measurements were performed by Laser Doppler
deionized water. About 160 mg of low molecular weight chitosan,
prepared as described earlier, was dissolved in 15 mL of acetic acid
Electrophoresis using Zetasizer (Nano-ZS). To maintain a constant ionic
strength, the samples were diluted (1:50 v/v) in 1 mmol/L (pH 6.5) of NaCl.
21
(
1%, v/v). The mixture was dialyzed against water using a dialysis Each sample was measured three times.
membrane (molecular weight cut-off (MWCO): 5–8 kDa) for 48 h with
successive exchange of fresh deionized water to remove water-soluble
by-products. The reaction solution was then lyophilized. Dialyzed
product was freeze-dried and stored at ꢀ20 °C until use.
Radiolabeling of CHI-g-PEI-FA and the effect of
concentration of the chitosan-conjugate solution and pH on
the labeling yield
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53
The Sm-CHI-g-PEI-FA NP suspension was prepared by adding 16 μL of
Synthesis of folate-PEI
1
53
152
3 3
SmCl (1 mCi, obtained by neutron activation of SmCl ) solution to
Before reaction of PEI, an activated ester of folate, NHS-folate, was
20 μg of lyophilized modified chitosan (CHI-DTPA-PEI-FA) particles, which
synthesized. Folic acid (240 mg) was dissolved in 10 mL of dimethyl were dispersed in 0.5–1.0 mL of phosphate buffer saline (PBS) solution
sulfoxide (DMSO) along with addition of 200 mg excess of DCC and followed by thorough stirring in a lead-shielded sonicator. The resulting
20 mg excess of NHS into the solution. The reaction mixture was stirred suspension was incubated for 1 h at 37 °C. The contents were transferred
1
for 2 h in darkness at room temperature. The insoluble by-product,
dicyclohexylurea, was removed by filtration through glass wool. The
into an evacuated sterile sealed vial for subsequent use. In order to
investigate the effects of various factors on the labeling yield of the
filtrate containing the DMSO solution of the NHS-folate product was complex, the pH of the reaction mixture was varied in the range of
stored at ꢀ20 °C until use in further synthesis. Folate-PEI conjugate was
synthesized by reacting 400 mg of PEI dissolved in 5 mL of DMSO with
the NHS-folate synthesized earlier, as shown in Figure 1. The product
was then diluted in two volumes of deionized water and then poured MeOH/H
4.5–7.5 by adding an appropriate volume of 0.5 N of HCl and 0.5 N of
NaOH to the chitosan solution. The labeling yield was measured by
instant thin layer chromatography using ITLC with a solvent system of
1
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153
2
O/HOAc (50:50:5). The R
f
values of free
Sm and
Sm-CHI-
into over an excess amount of acetone.
The suspension was decanted, and the precipitate was re-dissolved in
g-PEI-FA NP complex were 0.5 and 0.0, respectively. The stability of
Sm-CHI-g-PEI-FA derivative was examined by temporally measuring
1
53
deionized water. The reaction solution was then lyophilized, and product the radiochemical purity of the complex by ITLC at 1, 2, 4, and 24 h after
was freeze-dried and stored at ꢀ20 °C until use.
preparation.
J. Label Compd. Radiopharm 2015, 58 327–335
Copyright © 2015 John Wiley & Sons, Ltd.
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E. Mollarazi et al.
1
53
Radiolabeling of chitosan by
biodistribution studies
Sm for control
Internalization studies of fluorescent NPs using fluorescent
imaging microscopy
2
2
The complex was prepared according to the recently published method.
Briefly,
For qualitative uptake studies, the MCF-7 cells were seeded in the
1
53
Sm-chitosan complex was prepared by dissolving of chitosan chambered glass system (Lab-Tek; Nunc International Co., Naperville, IL,
(35 mg) in 3.5 mL of 1% acetic acid aqueous solution following the addition USA). Cells were washed four times after incubation with FA-loaded
of ascorbic acid (15 mg), and the mixture was stirred at room temperature NPs for 2 h and then fixed by a cold mixture of methanol/acetone
until a transparent solution was formed. To the aforementioned mixture, (50:50 v/v) for 15 min at room temperature. The cells were washed twice
153
2
96–370 MBq (in 0.5 mL) of SmCl
3
was added followed by stirring for
with PBS and mounted in mounting medium consisting of Na2 HPO4 and
5
min and standing for 30 min at room temperature. For measuring acetic acid (pH 5.5)/glycerol (50:50 v/v) to be observed by fluorescence
1
53
radiochemical purity and radiolabeling yield, 1 μL sample of the
Sm-
microscope (λext: 540 nm and λem: 580 nm; BX40; Olympus, Tokyo,
chitosan complex was spotted on a chromatography paper (Whatman no. Japan). The fluorescent images were taken by DP70 digital imaging
1
), and developed in a mixture of methanol/water/acetic acid (4:4:2) as system (Olympus, Tokyo, Japan) and analyzed by Olysia imaging
153 153
f
the mobile phase. The R values of free Sm and Sm-chitosan complex software (Olympus, Tokyo, Japan).
were 0.45 and 0.0, respectively.
Cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT)
1
53
153
Biodistribution of
Sm-CHI-g-PEI-FA,
Sm-chitosan, and
1
53
SmCl in Balb/c mice
3
MCF-7 cells were seeded in clear 96-well plates at a density of 4000 cells
per well for 16 h to reach in suitable confluent monolayer. After time
interval, the cells were subsequently incubated with 200 μL/well of no
or 1, 2, 5, and 10 μg, μCi, and μg/μCi, respectively, of CHI-g-PEI-FA,
1
53
153
Biodistribution of Sm-conjugates and free SmCl
-month-old mice weighing 35–42 g. One hundred microliters of sterile
radiolabeled copolymer (concentration: 2 mg/mL) was administered
through the tail vein of each mouse, (injected dose: 100 μCi). The animals
3
was studied in 2–
3
1
53
153
SmCl
the incubator for 24, 72, and 96 h. After incubation for time intervals at
7 °C, the cells were incubated for 4 h with a 0.5 mg/mL solution of
3
, and
Sm-CHI-g-PEI-FA complex, and the cells were kept in
2
were sacrificed by CO gas at different time intervals, and different
3
organs (bone, blood, liver, spleen, kidney, muscle, lung, heart, bladder
gastric, and intestinal) were removed, washed with normal saline, and
dried in paper folds. The radioactivity in each organ was counted using
NaI(Tl) well-type gamma counter, and the counts were recorded in the
gamma spectrometer (Canberra, Meriden, USA), and expressed as
percent injected dose per gram of the organs. Further, specific counts
per gram of blood were calculated for each time interval.
MTT, which was disposed afterwards and replaced with 200 μL of equal
parts of DMSO. Absorbance at 570 nm was measured. Data shown are
based on five different experiments, and the results were expressed as
mean ± standard deviation.
In vivo imaging of the radiolabeled NPs
The animal experiments were carried out using female Balb/c mice
weighing 18–21 g each (5–7 weeks old) from Pasteur Institute, Tehran,
Iran. The animals were inoculated subcutaneously with suspended 4T1
Cell culture
153
The cell binding and internalization of Sm conjugates into MCF-7 and
T1 breast cancer cell lines as folate receptor positive (FR(+)) and CHO
Chinese hamster ovarian) cell lines as folate receptor negative (FR(ꢀ))
were studied. The cell lines were grown at 37 °C in a humidified
atmosphere containing 5% CO in a special Roswell Park Memorial
6
cells (1 × 10 /0.1 mL) in the left loin region. The radiotracer studies were
4
(
153
carried out 2 weeks after 4T1 cell injection. Sm-CHI-g-PEI-FA NPs were
administered intravenously via a tail vein to the animals. We then
performed scintigraphy and recorded the 24-h, 48-h, and 96-h images
by using gamma camera. The in vivo work was approved by the ethical
committee of the Pharmaceutical Research Centre, Faculty of Pharmacy,
Tehran University of Medical Sciences.
2
Institute (RPMI) 1640 culture media without folic acid with 10% fetal
bovine serum, L-glutamine, and antibiotic in tissue culture flasks. Twenty
hours prior to each experiment, the cells were seeded in 24-well plates
5
(
10 cells per well) to form confluent monolayers overnight.
Results and discussion
In vitro cell binding and internalization
Preparation of conjugate and characterization
For cell binding experiments, the monolayers of cells were rinsed with
ice-cold PBS pH 7.4. Pure special RPMI medium (without fetal bovine
serum/L-glutamine/antibiotic) only (1 mL), or medium (500 μL), and a
folic acid solution (for blocking study, 1 mM, 500 μL) were added into
the corresponding wells.
The well plates were pre-incubated at 37 °C for 40 min. The solutions
of the complexes (8 μCi, 1 mCi/mL), purified by column chromatography
using Sephadex G-25, were added in each well, and the well plates were
The key steps in the synthesis were the functionalization of DTPA
into chitosan and the conjugation of PEI-FA functions to chitosan
by a rarely used method. PEI contains primary amino groups,
which account for 25% of the nitrogen atoms, through which a
desired targeting ligand may be attached, either directly or via
a spacer. CHI-g-PEI is more positively charged; therefore, they
are likely to interact more effectively with the negatively charged
incubated again at 37 °C for 1 h and then rinsed with ice-cold PBS pH 7.4 cell surface via nonspecific charge interaction.
and 300 μL of stripping buffer (aqueous solution of 0.1 M of acetic acid
After synthesis of CHI-DTPA and PEI-FA, grafting of this two
and 0.15 M of NaCl) for 5 min, respectively, to remove bound complex polymers was carried out using periodate ion as an oxidizing
2
3
from the FR on the cell surface. The monolayers were dissolved in 1 N
of NaOH (1000 μL) and internalized (acid-resistant), radioactivity was
measured in a γ-well counter. Then the cells were pelleted by
centrifugation at 800 g and resuspended in full media. The percentage
agent, which splits the carbon–carbon bond of vicinal diols to
give the dialdehyde group. The study by Nicolet and Shinn
26
suggested that this oxidation can be extended to cases in which
23
153
hydroxyl is replaced by primary or secondary amines. Figure 3
of cell uptakes of the
Sm-NPs was calculated as percentage
shows the Fourier transform infrared spectra of chitosan (a), CHI-
DTPA (b), and CHI-DTPA-g-PEI-FA (c). From the chitosan
spectrum, it was found that distinctive absorption bands appear
uptake = [pellet activity (cpm)]/[pellet activity (cpm) + supernatant
activity (cpm)] × 100, and internalizations of each sample were
determined. The percent of internalized radioactivity was calculated by
dividing the internalized radioactivity by the total radioactivity associated at 1668 cm
ꢀ
1
ꢀ1
(amide I), 1575 cm
(–NH
bending), and
2
2
4,25
ꢀ1
ꢀ1
with the cells (surface-bound + internalized) and multiplying by 100.
1405 cm
(amide III). The absorption bands at 1154 cm
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 327–335

E. Mollarazi et al.
δ = 4.69 ppm (H
, H
), and δ = 1.87 ppm (ꢀNCOCH
NMR of CHI-DTPA shown in Figure 4a was as follows:
δ = 4.50 ppm (H ), δ = 3.54–3.70 ppm (H , H , H ), δ = 2.87–
.98 ppm (protons of CH –CH ), δ = 3.21 ppm, and δ = 3.41–
.46 ppm (related to protons of CH near carbonyl group). The
1
), δ = 2.89 ppm (H
2
), δ = 3.63–3.89 ppm (H
3 4
, H ,
1
H
5
6
3
) (data not shown). H-
1
4
5
6
2
3
2
2
2
assignment of chemical shifts of PEI was shown in Figure 4b in
which the proton peaks of PEI (–NHCH CH –) appeared at
.44–2.58 ppm. In Figure 4c, the chemical shifts at δ = 2.40–
.81 ppm (CH CH – of PEI) and δ = 6.75, 7.65 ppm (correspond
2
2
2
2
2
2
to the para-aminobenzoic acid from the folate), and 8.58 ppm
corresponds to the pteridine moiety proton from the folate)
(
1
obviously showed the conjugation of PEI-FA. In the H-NMR
spectra illustrated in Figure 4d, appearance of new proton
signals at δ = 3.06–3.53 ppm and the signals at 6.37, 7.39, and
8.58 ppm, all indicated that PEI-FA was grafted to the chitosan
chain and confirmed the formation of CHI-DTPA-g-PEI-FA
1
polyplex. FT-IR together with H-NMR measurements pointed
to the conclusion that the different groups have successfully
conjugated onto the chitosan via
subsequent reduction reaction.
a condensation and
Figure 3. FT-IR spectra for chitosan (a), CHI-DTPA (b), and CHI-DTPA-g-PEI-FA (c).
ꢀ
1
(
1
asymmetric stretching of the C–O–C bridge), 1070 cm , and
ꢀ
1
033 cm (skeletal vibration involving the C–O stretching) are Size determination
the characteristics of its carbohydrate structure. Compared with
ꢀ
1
The CHI-DTPA-g-PEI-FA NPs prepared in white, porous,
lyophilized powder were used for material experiments. Figure 5
represents the particle size distribution of CHI-DTPA-g-PEI-FA
particles, where the majority of particles (88.4%) have diameters
in the range of 200–220 nm. Particle size distribution was
measured by using Coulter Counter (Coulter Multisizer, Coulter
that of chitosan, the peak at 1646 and 1404 cm (amide groups
characteristic) in spectra (b) appears sharper compared with that
ꢀ
1
of chitosan. Also, the peak at 1735 cm
is related to the
carbonyl group of carboxyls in DTPA. These results indicated
the derivation reaction of DTPA that took place at the N-position,
ꢀ
ꢀ
1
1
and –NH–CO– groups have been formed. The peak at 1115 cm
(
(
(
vibration of C–N in PEI), strong peaks at 2873 and 2926 cm
related to methylene groups in PEI), and peaks at 2099–2182
of R–N¼C– bonds in pteridine moiety from the folate) show
the conjugation of PEI-FA to chitosan derivative.
1
H-NMR spectra of chitosan before and after conjugation with
DTPA and grafting with PEI-FA are shown in Figure 4. The
1
H-NMR assignments of chitosan resonances are as follows:
Figure 5. Zeta potential distribution of CHI-DTPA-g-PEI-FA nanoparticles recorded
up to 200 mV.
1
Figure 4. H-NMR spectra of chitosan-DTPA (a), PEI (b), PEI-FA (c), and CHI-DTPA-
Figure 6. Size distribution of CHI-DTPA-g-PEI-FA nanoparticles using Coulter
g-PEI-FA conjugate in D
2
O.
Counter.
J. Label Compd. Radiopharm 2015, 58 327–335
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www.jlcr.org

E. Mollarazi et al.
Electronics Inc., Hialeah, FL, USA). A degree of self-aggregation subsequent experiments. Based on the aforementioned results,
was also observed (Figure 6).
To investigate the shape of CHI-DTPA-g-PEI-FA NPs, scanning of
it was concluded that the optimal procedure for the preparation
153
Sm-CHI-g-PEI-FA with a high labeling yield (96%) is as
electron microscopy was performed using a Jeol T330A (Jeol USA follows: 16 mg of chitosan (MW = 11 kD) was dissolved in 1 mL
Inc., Peabody, MA, USA). Scanning electron microscopy images reveal of PBS pH 5.7 of the solution. The solution was then mixed with
153
153
that these particles have a spherical shape, as shown in Figure 7. 1 mCi of the
3
SmCl . A high degree of stability of the
Sm-
chitosan-conjugate was retained for at least 24 h after labeling,
as shown in Figure 8.
The labeling yield was determined by instant thin layer
1
53
Effect of various factors on the formation of
complex
Sm-CHI-g-PEI-FA
153
The Sm-CHI-g-PEI-FA complex solution was prepared by mixing chromatography. Each point represents mean ± standard
the SmCl solution (1 mCi) and the chitosan solution, which were deviation (n = 3).
3
153
prepared by dissolving chitosan in normal saline. The pH of the
reaction mixture also varied as described in the Experimental
Biodistribution studies
153
Section. The R values of
Sm-chitosan-conjugate and free
f
153
SmCl3 in the solvent system were 0.1–0.3 and 0.8–1.0, Radioactivities in the major organs of mice at 2, 4, 72, and 96 h
153
153
respectively. Figure 4 shows the effect of various factors on the after the tail vein administration of
Sm-CHI-g-PEI-FA,
Sm-
153
153
labeling yield (%) of Sm-chitosan. Figure 4b shows the effect of chitosan, and SmCl at a dose of (0.1 mCi)/100 μL are shown
3
153
chitosan (MW = 11 kD) concentration versus Sm activity (1, 2, 4, in Figures 9, 10 as percent of the injected dose per gram of
153
and 8 mg: 1 mCi Sm) on the labeling yield. The yield did not tissues (%ID/g).
153
increase significantly with increasing the chitosan concentration
For
Sm cation, the biodistribution was mainly in the liver,
range of 1–8 mg/mL; however, it shows that the labeling yield kidney, and bone. The free cation is mainly soluble in water
reached 96% when the concentration reached 16 mg/mL.
Figure 8A shows that the labeling yield reaches a maximum of
and it can be excreted via urinary tract. Because the metallic
153
Sm is transferred in plasma in protein-bond form, the major
9
0% in the pH of 5–6 and chitosan concentration of 2 mg/mL. As final accumulation was showed to be liver. After 48 h, the
a result, the pH of the reaction mixture was adjusted to 5.7 in the metabolites and/or free cation were excreted from liver into
intestines via hepatobiliary tract resulting significant activity in
this tissue, but it was not significant at 96 h. Trace accumulation
was also observed in spleen.
1
53
Figure 7. Scanning electron microscopy image of CHI-DTPA-g-PEI-FA
nanoparticles.
Figure 9. Percentage of injected dose per gram (%ID/g) of
Balb/c mice tissues at 2–96-h post-injection (n = 3).
Sm-chitosan in
1
53
Figure 8. Effect of pH of the reaction mixture (A), concentration of conjugated chitosan solution (B) on the labeling yield of the Sm-chitosan complex and stability of
labeled compound in elapsed times (C).
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J. Label Compd. Radiopharm 2015, 58 327–335

E. Mollarazi et al.
In vitvo discrimination between MCF-7, 4T1 FR(+) tumors,
and CHO FR(ꢀ) cell lines
Folate receptor binding of the derivatives was performed using
in vitro experiments with MCF-7 human breast cancer cell line
(overexpressing the FRs) and 4T1, as another FR(+) and invasive
breast cancer cell line. CHO cells were also seeded as FR(ꢀ)
studies to confirm the preferential uptake of complex by folate
receptor. Internalization into tumor cells via receptor binding is
important for therapy/scintigraphy of folate receptor-expressing
lesions. After the removal of surface-bound radioactivity with
stripping acidic buffer, internalization studies were performed
153
to determine the degree of internalization of
FA NPs in MCF-7, 4T1, and CHO cells (Figure 11).
Sm-CHI-g-PEI-
1
53
Figure 10. Percentage of injected dose per gram (%ID/g) of Sm-CHI-g-PEI-FA
complex in normal Balb/c mice 2–96-h post-injection (n = 3).
Studies showed that the internalization of radioactive NPs in
MCF-7 cells was significantly greater than 4T1 cells (due to less
FR on the cell surface) at all time intervals (using approximately
Sm-chitosan, among the organs examined, liver and 200,000 cells) even better observed after 90 to 180 min. In
1
53
For
spleen, lung showed much higher levels of radioactivity counts contrast with FR(+) cells, CHO (FR(ꢀ)) cells showed also higher
153
153
than the other organs. The major route of excretion of
CHI-g-PEI-FA is urinary tract as shown in Figure 10, about 80%
Sm- accumulation of Sm-CHI-g-PEI-FA complex.
The higher efficiency of nonspecific uptake was probably
of the whole activity was excreted from the kidneys into the because of high positive charge of polymer along with
urine in 4 h. Because NPs with a diameter of 250 nm would not increasing internalization efficiency of related PEI immobilized
be excreted through the kidneys, it must be breakdown onto chitosan. It seems that the type and nature of cells may also
products. The high water solubility of the complex is a major be responsible for different cellular internalization characteristics
153
cause of this behavior. Other minor accumulated tissues are liver of Sm-NPs.
1
53
and bone; however, it is believed that free
Sm cation is the
Blocking study was carried out where excess FA was added
153
dominant species accumulating in these organs.
into medium 40 min prior to incubation of
Sm-CHI-g-PEI-FA.
The accumulation of radioactivity in reticuloendothelial system The nonspecific binding in the presence of a molar excess of folic
including liver (about 1% ID/g organ), spleen (less than 0.5% ID/g acid after 30 min showed no significant difference in the
153
organ), and lung (averagely 0.5% ID/g organ) was low at all time accumulation of
Sm-CHI-g-PEI-FA in MCF-7 and 4T1 cells
points. A low uptake of radioactivity was observed in the stomach (Figure 12). However, after 180 and 330 min, displacement of
until 96 h (less than 0.2% ID/g), indicating a minimal in vivo the superficial activity in MCF-7 and 4T1 (internalization up to
153
decomposition of the radioligands to form free Sm.
50%), in respect of their related blocked forms MCF-7 + folic acid
153
Figure 11. Percentage of cell internalization and uptakes of radioactivity in MCF-7, 4T1, and CHO cell lines after 30, 90-min, 180-min, and 330-min incubation with Sm-
CHI-g-PEI-FA complex.
J. Label Compd. Radiopharm 2015, 58 327–335
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

E. Mollarazi et al.
1
53
Figure 14. Imaging of intravenous administration of Sm-NPs into 4T1 tumor-
bearing mice.
and internalizations drop averagely to 45%; and (iii) the
remaining uptake of NPs to MCF-7 and 4T1 cells and the lower
uptake of NPs to the CHO cells can be attributed to the some
nonspecific tumor affinity of NPs.
Figure 12. Percentage of cell internalization of radioactivity in unblocked MCF-7,
T1, and related blocked cells after 30-min, 180-min, and 330-min incubation with
Sm-CHI-g-PEI-FA.
4
1
53
153
153
The comparison of
Sm-CHI-g-PEI-FA and
Sm-chitosan
and 4T1 + folic acid, was observed (averagely to 45%), uptake among tissues demonstrated significant difference
153
demonstrating that the internalization of the radiolabeled folate among the accumulating pattern, in case of
Sm-chitosan,
analog into the tumor cells was partially through FR-mediated the major accumulation is taking place in the liver and spleen
endocytosis suggesting the involvement of folate receptor. It due to the insolubility of the unprocessed chitosan at normal
seems that CHI-g-PEI-FA copolymer is likely to interact more serum pH.
99m
effectively with the negatively charged cell surface via
Recent studies on the production and quality control of
Tc-
nonspecific charge interaction. Combination of an increase in chitosan-folate as a possible diagnostic agent for noninvasive
the positive charge of the corresponding copolymer and imaging of folate receptor-positive cells have been reported;
2
4
presence of PEI moiety leads to the elevated cellular uptake however, no in vivo details were presented.
153
and more efficient endosome release of the internalized Sm-
Recently, water-soluble derivatives of chitosan were labeled
99m
CHI-g-PEI-FA, respectively.
with
Tc as possible targeted delivery for nuclear imaging;
Internalization results of the labeled Fluorescein however, they mostly were accumulated in the liver and
isothiocyanate (FITC)-NPs were shown in Figure 13. The targeted excreted via the kidneys because of the water-soluble nature of
NPs can readily enter the cells, and the NPs were localized inside chitosan derivatives. The targeting was based on the particle size
25
endosome. On the other hand, the intensity of FITC was rather than any biological targeting. On the other hand, in the
significantly increased (green clusters inside the cell).
present study, the labeled compound is almost exclusively
Tumor accumulation of Sm-CHI-g-PEI-FA NPs was estimated excreted through the kidneys.
153
using gamma camera imaging by the injection of radiolabeled
On the other hand, many efforts have been carried out on the
NPs (100 μCi/100 μL) via tail vein after 24, 48, and 96 h, which development of folate-based radiotracers for malignancy
demonstrated signal of radioactivity into tumor after 96 h imaging. In one case, radiolabeled super paramagnetic folate
(
Figure 14).
was developed and successfully used in the imaging; however,
Results suggest that (i) the main delivery pathway for Sm- the water solubility remained a disadvantage. Also, various
153
27
CHI-g-PEI-FA complex into the FR+ cells is via folate receptor, folate-based conjugates have been developed using SPECT
which mediated NPs endocytosis; (ii) NPs’s uptake can be radionuclides; however, they lack suitable biodistribution
28
inhibited by folic acid in the case of MCF-7 and 4T1 cells (FR+), properties such as stability and in vivo stability. The present
Figure 13. Fluorescent images of MCF-7 cells incubated with FITC-Chi-PEI-FA nanoparticles for 4 h.
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J. Label Compd. Radiopharm 2015, 58 327–335

E. Mollarazi et al.
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The authors did not report any conflict of interest.
J. Label Compd. Radiopharm 2015, 58 327–335
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org