Vol.2, No.1, 51-61 (2010)
Copyright © 2010 http://www.scirp.org/journal/HEALTH/
Openly accessible at
Bioengineering functional copolymers. XII. Interaction of
boron-containing and PEO branched derivatives of
poly(MA-alt-MVE) with HeLa cells
Mustafa Türk1, Zakir M. O. Rzayev2,*, Gülcihan Kurucu2
1Department of Biology, Faculty of Science and Art, Kırıkkale University, Yahsihan, 71450 Kirikkale, Turkey
2Department of Chemical Engineering, Faculty Engineering, Hacettepe University, Beytepe, 06800 Ankara, Turkey; Correspondence
author: zmo@hacettepe.edu.tr
Received 20 October 2009; revised 20 November 2009; accepted 11 November 2009.
Novel boron-containing bioengineering copoly-
mer and its α-hydoxy-ω-methoxy-poly(ethy-
lene oxide (PEO) macrobranched derivatives
were synthesized by (1) partially amidolysis of
poly(maleic anhydride-alt-methyl vinyl ether)
wıth ethanolamine ester of diphenylboronic acid
and (2) esterification of synthesized B-con-
taining copolymers with PEO. They had a com-
bination of hydrophilic/hydrophobic linkages,
free carboxylic groups, positive charges and an
ionized organoboron linkage as antitumor sites,
along with an ability to interact with HeLa cells.
The structure, composition and properties (cy-
totoxicity and antitumor activity) of synthe-
sized copolymers were investigated. In vitro
cytotoxicity results, obtained by the fluore
scence microscopy measurements indicate that
unlike the virgin copolymer, boron-containing
and PEO macrobranched derivatives exhibit
higher antitumor activity. It was found that
organoboron copolymer exhibits the most apo-
ptotic and necrotic effects against HeLa cells
whereas a minor effect relative to cancer cells
was observed on L929 Fibroblast cells.
Keywords: Synthesis; Organoboron Copolymers;
Structure-Property Relations; Cytotoxicity;
Antitumor Activity; Apoptotic Effect
The bioengineering functional polymers exhibit the
characteristics of 1) alternating and random copolymers
of maleic anhydride (MA) and 2) poly(ethylene oxide)
(PEO), as well as 3) PEO grafted functional macro-
molecules. They are of great interest for many researchers
due to their nontoxic, cell-compatible, biodegradable,
stimuli-responsive properties, and therefore, a wide range
of biomedical and bioengineering applications exist as
drug or enzyme carriers and biomacromolecular conju-
gates both in diagnostics and chemotherapy as effective
antitumor agents [1-8]. It is known that these copolymers
can be regarded as pre-activated polymers due to the
presence of anhydride moieties susceptible to the reaction
with a primary amine of a biomolecule [9]. The alternat-
ing copolymers of maleic anhydride (MA) with methyl
vinyl ether (MVE) or divinyl ether (DVE) were utilized in
various applications in diagnostics [10,11] and in che-
motherapy as effective antitumor agents [8]. Poly
(MA-alt-DVE), known as pyran copolymer is one of the
well known bioengineering polymers having a wide
range of biological activity. It processes antitumor, anti-
viral, antibacterial and antifungal activities, induces in-
terferon formation, and acts as an anticoagulant and
anti-inflammatory agent [8,12-17]. Hirano et al. [18,19]
reported that the poly (MA-alt-DVE) conjugated with
bovine erythrocyte superoxide dismutase (SOD) is re-
sistant against the proteolytic enzymes in serum, and
shows a prolonged half-life in vivo. They established an
increase in half-life after intravenous injection, as well as
its decreased immunogenicity [19]. It was demonstrated
that the copolymer-SOD conjugate shows anti-inflam-
matory effect against rat re-expansion pulmonary edema
at the first step of leukocyte adhesion [15]. Maeda [20]
discussed the development and therapeutic potential of
prototype macromolecular drugs for use in cancer che-
motherapy an artificial bioconjugate of neocarzinostatin
(NCS) and poly(maleic acid-alt-styrene) copolymer. The
biological response-modifiying effects, the mechanism of
a tumor “enhanced permeability and retention” effect and
the tumor-targeting mechanism of NCS-copolymer con-
jugate were also discussed. According to the author, a
principal advantage in the use of this bioconjugate is the
potential for a reduction or elimination of toxicity.
The copolymers of fumaric, citraconic and itaconic
M. Türk et al. / HEALTH 2 (2010) 51-61
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acid and their derivatives as isostructural analogues of
MA, as well as copolymers of some N-substituted
maleimides can also included to class of bioengineering
polymer systems. Cam et al. [21] evaluated the in vitro
cytotoxicities of glycinylmaleimide (GMI) copolymers
using K-562 human leukemia cells and HeLa cells. They
also evaluated the in vitro antitumor activities of co-
polymers against mice bearing sarcome 180. Monomeric
GMI and its copolymers showed higher antitumor activ-
ity than well known 5-fluorouracil (5-FU) at any dosage
One the other hand, growıng interest and much effort
have been also focused on the synthesis of organoboron
low molecular-weight functional compounds, biopolymer
and drugs with boron ligands and evaluation of their
suitability for the bioengineering applications. Aromatic
boronic acid and its functional derivatives, and some
functionalized carboranes have become an very important
class organic compounds, which are utilized in a variety
of biological and medical applications, such as carbohy-
drate recognition [22], neutron capture therapy for cancer
treatment as effective tumour-targeting agents [23,24],
especially for brain tumours [25,26], and protease enzyme
inhibition [27]. Kataoka et al. [28-32] synthesized a novel
water-soluble polymer with lectin-like function by in-
troducing phenylboronates, as sugar -recognizing moie-
ties, into the side-chain of poly (N,N-dimethylacrylamide)
[28,29]. According to the authors, at physiological pH
medium, phenyl-boronates form an appreciably stable
complex with sialic acid (Neu5Ac), a chacteristic anionic
carbohydrate on the surface of the plasma membranes
[30,31]. Authors suggested that boronate-containing pol-
ymer may be an effective immune-adjuvant for the indu
ction of lymphokine-activatd killer (LAK) cell [31]. They
also demonstrated that the copolymers of 3-acry- lami-
dophenylboronic acid and dimethylacrylamide with dif-
ferent compositions coated onto solid substrates support
function as synthetic mitogens for mouse lymphocytes
However, a wide range of functional polymer synthesis
techniques can be utilized for the design of more effective
synthetic routes to prepare new B-containing bioengi-
neering polymers, especially copolymerization of or-
ganoboron monomer and chemical modification of bio-
compatible polymers with organoboron reactive com-
pounds and monomers. Several researchers synthesized
some bioengineering copolymers containing phenylbo-
ronic acid linkages by radical copolymerization and
chemical modification methods, which are exhibit
glugose-, RNA- and DNA-sensitive behavior [33-36].
Recently, we report the synthesis and chracterization of
organoboron copolymers by complex-radical copoly-
merization of p-vinylphenylboronic acid with N-isopro-
pylacrylamide (NIPA), maleic and citraconic anhydrides,
maleimide and chemical modification of poly(NIPA-
rand-MA)s with organoboron amine, as well as synthesis
of supramacromolecular poly(ethylene imine) macro-
complexes and PEO long branched derivatives of or-
ganoboron copolymers having stimuli-responsive and
high HeLa cell transfection behavior [37-40].
The objective of this work is to develop novel boron-
containing functional copolymers with antitumor activity.
In the present article, results of synthesis and characteri-
zation of a new generation of biocompatible boron-con-
taining functional macromolecules having a combination
of hydrophilic and hydrophobic segments, free carbox-
ylic groups, positive charges and an ionized linkage as
antitumor sities, along with an ability to conjugate with
cancer cells were described and discussed. These or-
ganoboron copolymers were synthesized by 1) partially
amidolysis of bioengineering alternating copolymer of
maleic anhydride (MA) and methyl vinyl ether (MVE)
with ethanolamine ester of diphenylboronic acid (EAPB)
and 2) chemical modification (esterification) of synthe-
sized organoboron copolymer with α-hydoxy-ω-meth-
oxy-poly (ethylene oxide (PEO). Special attention was
paid to the role of structural effects, especially to the
influence of organoboron linkage, for the interaction of
organoboron functional copolymers with HeLa cells and
to the evaluation of citotoxisity and antitumour activity
by using a combination of various methods such as sta-
tistical, hematoxylen/eosin staining, apoptotic and ne-
crotic cell indexes, and M30 immunostaining analyses.
2.1. Materials
Ethanolamine ester of diphenyl boronic acid (EAPB)
(Sigma-Aldrich, Germany) was purified by recrystall-
ization from anhydrous ethanol: m.p. 193.5oC (by DSC).
1H NMR spectra (δ, ppm) in CHCl3-d1: CH2-O 1.49,
CH2-NH2 2.96, and 7.38-7.40 (1H), 7.19-7.24 (2H) and
7.13-7.16 (2H) for protons of p-, o-and m-positions in
benzene ring, respectively. Poly(maleic anhydride-alt-
methyl vinyl ether), poly(MA-alt-MVE) (C1) (Sigma-
Aldrich, Germany): Mn 80,000 g.mol-1, Tg 148oC (by
DSC); 1H NMR spectra (δ, ppm) in DMSO-d6: CH2 1.23,
CH-O 2.11, O-CH3 2.08 and CH-CH 3.38. α-Hydox y-ω-
methoxy-poly(ethylene oxide) (Fluka; PEO, Mn 2000
g.mol-1):1H NMR spectra (δ, ppm) in CHCl3-d1: CH2-O
3.75-3.45, OH end group 2.61 and O-CH3 end group 2.16.
Human cervix epithelioid carcinoma cell line (HeLa)
was obtained from the tissue culture collection of the SAP
Institute (Turkey). Cell culture flasks and other plastic
material were purchased from Corning (USA). The
growth medium, which is Dulbecco Modified Medium
(DMEM) without L-glutamine supplemented fetal calf
serum (FCS), and Trypsin-EDTA were purchased from
Biological Industries (Israel). M30 CytoDEATH antibody
M. Türk et al. / HEALTH 2 (2010) 51-61
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2.2. Synthesis
Boron-contaning copolymer (C1-B) was synthesized by
the partially amidolysis of succinic anhydride units of
alternating copolymer C1 with EAPB, containing a pri-
mary amine group, in the 1,4-dioxane solution at 40oC for
3 h under nitrogen atmosphere at molar ratio of C1:EAPB
= 2:1. Approbriate quantities of C1 and EAPB, solvent
were placed in a standard Pyrex-glass tube and flushed
with dried nitrogen gas for at leeast 3 min, then placed in
a carousel type microreactor with a thermostated heater
and magnetic mixer. The resulting copolymer C1-B was
isolated from reaction mixture by precipitating with di-
ethyl ether. Purification of copolymers was done by
dissoving in dioxane and reprecipitating in diethyl ether,
extraction with hexane and draying under vacuum at 50oC
until constant weight.
PEO macrobranched copolymer (C1-B-PEO) was syn-
thesized by the esterification of anhydride unıts of par-
tially amidolysed C1-B copolymer with PEO, containing
an end hydoxyl group, in the same conditions using in our
previous publications [5,37].
2.3. Characterization
FTIR spectra of the organoboron copolymers (KBr pellet)
were recorded with FT-IR Nicolet 510 spectrometer in the
4000-400 cm-1 range, where 30 scans were taken at 4 cm-1
resolution. 1H {13C} NMR spectra were performed on a
JEOL 6X-400 (400 MHz) spectrometer with DMSO-d6 as
a solvent at 25oC.
The differential scanning calorimetry (DSC) analysis
was performed on a Shimadzu calorimeter (Japan) at a
heating rate of 5oC/min, under nitrogen atmosphere. The
X-ray diffraction (XRD) patterns were obtained from a
Rigaku D-Max 2200 powder diffractometer. The XRD
diffractograms were measured at 2θ, in the range 1-50o,
using a Cu-Kα incident beam (λ = 1.5406 Å), mono-
chromated by a Ni-filter. The scanning speed was 1 o/min,
and the voltage and current of the X-ray tubes were 40 kV
and 30 mA, respectively.
The number of living and dead cells were counted with
a haemacytometer (C.A. Hausse & Son Phluila, USA) at
X200 magnification. The number of apoptotic and ne-
crotic cells were determined by Fluorescence Inverted
Microscope (Olympus IX70, Japan). The cell images
were also recorded using the both above mentions mi-
croscopes. Statistical analyses were performed using
Student’s t-test for unpaired data and P values of less than
0.05 were considered significant. Data are presented as
means ± SEM (standard errors of the mean).
2.3. Cytotoxicity
For cytotoxicity experiments, HeLa cells and L929 Fi-
broblast cells respectively. (25 x103 cells per well) were
placed in DMEM by using 24-well plates. Different
amounts of copolymers (C1, C1-B and C1-B-PEO)
(about 50-500 g.mL-1 in aqueous solutions) were put
into wells containing cells, respectively. The plates were
kept in the CO2 incubator (37 C in 5% CO2) for 2-24 h;
the medium was replaced with fresh medium, and incu-
bated at the same conditions for 24 h. Following of this
incubation, HeLa cells and L929 Fibroblast cells were
harvested with trypsin-EDTA, and then were dyed with
trypan blue [41]. The viable cells were counted with a
haemacytometer (C.A. Hausse & Son Phluila, USA),
using light microscope.
2.4. Hematoxylen/Eosin Staining
HeLa cells and L929 Fibroblast cells (25x103 cells per
well) were placed in DMEM by using 24-well plates.
After treating with different amount functional copoly-
mers (C1, C1-B and C1-B-PEO) (about 50-500 g.mL-1
in aqueous solutions) for 2-24 hours period, the medium
was removed, the cells washed with distilled water and
fixed in ethanol, and stained with Hematoxylen/Eosin.
After staining, the cells were observed by light micros-
copy. By this way, cellular and nuclear morphology have
been shown in cultured cells stained with Hematoxy-
2.5. Analysis of Apoptotic and Necrotic
Double staining were performed to quantify the number
of apoptotic cells in culture on basis of scoring of apop-
totic cell nuclei. HeLa cells and L929 Fibroblast cells
(25x103 cells per well) were placed in DMEM by using
24-well plates. After treating with different amount
functional copolymers (C1, C1-B and, C1-B-PEO) (about
50-500 g.mL-1 in aqueous solutions) for 2-24 hours
period, both attached and detached cells were collected,
then washed with PBS and stained with Hoechst dye 3342
(2 g.mL-1), propodium iodide (PI) (1 g.mL-1) and
DNAse free-RNAse (100 g.mL-1) for 15 min at room
temperature. After that 10-50 L of cell supension was
smeared on slide and coverslip for examination by fluo-
rescence microscopy [42,43]. The nuclei of normal cells
were stained light blue but apoptotic cells were stained
dark blue by the hoechst dye. The apoptotic cells were
identified by their nuclear morphology as a nuclear
fragmentation or chromatin condensation. Necrotic cells
were staining red by PI. Necrotic cells lacking plasma
membrane integrity and PI dye cross cell membrane, but PI
dye don’t cross non necrotic cell membrane. The number
of apoptotic and necrotic cells in 10 randomly chosen
microscopic fields were counted and the result expressed
as a ratio of apoptotic and necrotic to normal cells.
2.6. M30 Immunostaining
The percentage of apoptotic cells was determined by M30
CytoDEATH antibody [44]. This is a monoclonal mouse
immunoglobulin (Ig) G2b antibody (clone M30; Roche,
M. Türk et al. / HEALTH 2 (2010) 51-61
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Openly accessible at
Mannheim, Germany) that binds to a caspase-cleaved,
formalin-resistant epitope of cytokeratin 18 cytoskeletal
protein. The immunoreactivity of the M30 antibody is
confined to the cytoplasm of apoptotic cells. HeLa cells
(25x103 cells per well), treated with C1, C1-B and,
C1-B-PEO copolymers (about 50-500 g. mL-1 in aque-
ous solutions) for about 2-24 h, were fixed in 10% neu-
tral-buffered formalin for 15 min, treated with 0.3% hy-
drogen peroxide in methanol for 10 min to block the
endogenous peroxidase activity, washed in the standard
phosphate buffer solution, and then incubated with M30
antibody at room temperature for 1 h. In negative controls,
preimmune mouse serum instead of primary antibody
was used. Immunoreactions were revealed by the
avidin-biotin complex technique using diaminobenzidine
(DAB) as substrate. We counted the number of M30-
positive cytoplasmic staining cells in all fields found at
x400 final magnification. For each image, three randomly
selected microscopic fields were observed, and at least
100 cells/field were evaluated. M30 CytoDEATH anti-
body was not sensetive to L929 Fibroblast. On account of
this reason, M30 CytoDEATH antibody did not applied to
L929 Fibroblast cells.
3.1. Synthesis and Characterization
of Organoboron Functional Copolymers
Boron-containing bioengineering functional copolymer
(C1-B) and its α-hydoxy-ω-methoxy-poly(ethylene oxide)
(PEO) long branched derivatives were synthesized by (1)
amidolysis of succinic anhydride units of biocompatible
poly(MA-alt-MVE) alternating copolymer (C1) with
EAPB containing a primary amine group, and (2) esteri-
fication (grafting) of free anhydride unıts of partially
amidolysed C1-B copolymer with PEO, containing an
end hydoxyl group, respectively. General scheme of
synthesis of the organoboron functıonal copolymer and
its PEO branched derivative can be represented as follows
(Scheme 1).
The synthesized boron-containing copolymers contain
a combination of hydrophilic/ hydrophobic linkages, free
carboxylic groups, positive charges and ionized organo-
boron linkage as antitumor sities, along with an ability to
interact with canser biomacromolecules, especially with
HeLa cells. The chemical and physical structure, com-
position and properties (temperature-responsiveness,
glass-transition, melting and degradation temperatures,
andantitumor activity and cytotoxicity) of synthesized
copolymers were characterized by spectroscopy (FTIR,
1H and 13C NMR), viscometry, DSC, X-ray diffraction
and Fluorescence microscopy analyses.
The results of chemical structural analysis of the syn
thesized organoboron copolymers FTIR (KBr pellet) and
(1H and 13C) NMR spectroscopy (in DMSO-d6 solution)
were summarized in Table 1 (FTIR analysis data for
C1-B) and illustrated in Figure 1 (NMR spectra of C1-B)
and Figure 2 (FTIR spectra of C1-B-PEO). The forma-
tion of amide, carboxyl and organoboron groups in the
structure of C1-B copolymer as results of amidolysis
reaction was confirmed by apearance of the correspoded
characteristic absorption bands for each monomer unit
and diphenylboronic fragment in the spectra. Absorption
Scheme 1. Schematic representation of the synthesis routes of
organoboron functional copolymers (C1-B and C1-B-PEO) by
the amidolysis of poly(MA-alt-MVE) (C1) with organoboron
amine (AEPB) and esterification of poly (MA-alt-MVE)-g
-AEPB) (C1-B) with PEO, respectively.
Table 1. The results of FTIR analysis organoboron functional
copolymer: Poly(MA-alt-MVE)-g-AEPBA) (C1-B).
Absorption bands (cm-1) Band assignments
MA unit
1980-1925 (w) C=O (overtones)
1864 (m-s), 1781 (vs) C=O stretching (anhydride)
1227 (s, broad), 1094 (s) C–O and C–O–C bands
650 (w) CH (in chain backbone)
MVE unit
2942 (m-s) CH3 C–H stretching
2854 (m) CH2 C–H (chain backbone)
1475-1416 (m) CH2 and CH3 deformation
1372 (m) CH3 deformaton ( in O–CH3)
975 (m) CH3 rocking
926 (vs) C–O deformation
735 (m-w), 720 (w) CH2 and CH3 deformation
Maleamide unit
1736 (m-s) C=O strtchıng (in -COOH)
1575-1510 (m-w) COO stretching (H-bonding)
1650 (m),1720 (m-w) NH–C=O amide I band
1315 (w) amide III band
Organoboron linkage
3240 (w), 3100,1600 (m) CH= (in aromatic ring)
1545 (m-w) B–O stretching
1443 (m), 1420 (w) B–Ph aromatic ring
1180 (m) CH in-put-bending
770 (w) CH out-put-bending
702 (m) O-B-Ph aromatic ring
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bands at 1864 and 1781 cm-1, relating to C=O groups of
free anhydride units, indicated the partially amidolysis of
these units as shown in Scheme 1.
From the comparative analysis of 1H and 13C NMR
spectra of virgin alternating copolymer (C1) and its or-
ganoboron derivative (C1–B) (Figure 2a, 2b), the fol-
lowing changes of the characteristic signals were ob-
served: unlike the spectra of C1 copolymer having the
peaks from chemical shifts of the CH and CH2 backbone
and CH3 (in methoxy group) protons new signals from
protons of amide NH, COOH and phenyl groups (in or
ganoboron linkage) were appeared in the spectra of or-
ganoboron copolymer (C1–B). More detailed informa-
tions about micristructure of C1–B copolymer were
pre-pared by analysis of 13C NMR spectra (Figure 2c).
The following chemical shifts (, ppm) of carbon atoms
were observed in the spectra: 174.4 (–C=O of the
maleamide and anhydride units), 128-136 (–B–C6H5
mono-substitued benzene ring), 77.2 (–CH–NH in or-
ganoboron linkage), 58.01 (–CH–O), 49.08 (–CH–
CH-chain backbone), 30.3 (–CH3-O), and 30.15 (–CH2).
Chemical structure of C1–B–PEO long branched co-
polymer was confirmed by the appearance in the FTIR
spectra (Figure 3) the following characteristic absorption
bands (cm-1): 3400 (strong broad peak for OH in H-
bonded carboxyl groups), 2933-2735 for C–H stretching
in CH2 and CH3), 2667 and 2600 (C-H stretching in
CH2–O of PEO branched segments), 2280 and 2135
(Fermi doublet for C–N band), 1986 and 1966 (overtones
of C=O), 1746 (C=O of ester groups), 1710 (C=O of
carboxyl groups), 1630 (NH–C=O amide I band), 1592
(phenyl groups), 1558 (H-bonded COO stretching), 1545
(weak peak for B–O stretching), 1490 (C-H deformation
for CH2–O in PEO branches), 1480 and 1466 (CH2 de-
formation), 1450 (B–Ph aromatic ring), 1405 (amide III
band), 1372 and 1352 (CH3 deformation in O–CH3), 1115
(broad peak for C–O band in CH2–O and CH3–O of PEO
and MVE units, respectively), 948 (strong peak for C–O
deformation in PEO branchs), and etc.
The comparative analysis of the XRD patterns of alter-
nating copolymer and its organoboron derivative show a
significant difference between physical structures of
these copolymers (Figure 3). C1 copolymer has an
amorphous structure, while C1-B copolymer exhibits
pseudo-crystallinity behavior (without re-crystallization
process due to macromolecular physical interactions via
H-bonding, hydrophobic-hydrophilic interactions, etc.)
with degree of pseudo-crystallinity χc = 26.2 % (by XRD
analysis), glass-transition Tg and pseudo-melt phase
transition Tm at 84.2oC and 136.3 oC, respectively (by
DSC analysis). It can be proposed that the producing the
amphiphilic organoboron linkages in side chain of co-
polymer causes a formation of hydrophilic/hydrophobic
balance, more polar amide and carboxyl groups, which
are able to form strong H-bonded segments, and therefore,
self-assembled suramacromolecular structure of C1-B
copolymer as in other organoboron polymer systems [37].
3.2. Cytotoxicity of the Copolymer and its
B-Containing and PEO Branched
In this study, the comparative analysis of HeLa cells
(cancel cells) and L929 Fibroblast cells (normal cells)
has been investigated. The cytotoxicities of C1 co-
polymer and corresponding C1-B, C1-B/PEO deriva-
tives were inquired about the utility for antitumor drugs.
Figures 4 and 5 give the number of viable cancer and
normal cells in each group after incubation of the cells
with copolymer and organoboron copolymers at their
different concentrations for 24 h incubating time in cell
culture media, respectively. Under the same conditions,
the wells containing cells without copolymers were also
studied as a control. The following important results can
be drawn from this graph which is illustrated in these
figures. The C1 copolymer does not exhibit any ob-
servable toxicity in the chosen range of copolymer
concentration. The toxicity of polymers containing bo-
ron (C1-B and C1-B-PEO) was significant, most
probably due to hydrogen bonding supramacromolecu-
lar structure of these copolymers containing a combi-
nation of hydrophilic/hydrophobic linkages, free car-
boxylic groups, which are formed after partial amido-
lysis of anhydride containing copolymer C1 and full
hydrolysis of free anhydride units in the chosen
physiological medium where positive charges and ion-
ized organoboronoxy groups also exist as antitumor sites
along with an ability to interact with cells.
It was observed that an increase of C1-B and C1-
B-PEO concentrations in each well caused higher degree
of dying cells as compared to virgin C1 copolymer tested
under the same conditions. C1-B copolymer exhibits
relatively higher in vitro cytotoxicity than C1-B-PEO
branched copolymer which can be explained by the
higher content of organoboron linkages in C1-B co-
polymer. It is important to note that the boron containing
side chain linkages, rather than the individual copolymers,
increase the cytotoxicity more profoundly; an important
feature which has a significant role in leading us to the
present study. C1 copolymer had less toxicity compared
to cultured cells at various quantities and different incu-
bation times. On the contrary, the toxicity of C1-B and
C1-B-PEO organoboron copolymers towards the HeLa
cells increased by increasing their quantity from 50 to 500
µg.mL-1, whereas, no significant change was observed
with varying time. According to Figure 4, C1 did not
show high toxicity at all although the copolymer amount
was increased from 50 to 500 µg.mL-1 whilst, a signifi-
cant toxicity of C1-B andC1-B-PEO (100 µg.mL-1 and
above) started to be observed when cancer and normal
cells (Figure 5) were incubated for about 4 h. As the
amount of boron con taining polymers and their incubtion
M. Türk et al. / HEALTH 2 (2010) 51-61
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Fıgure 1. 1H NMR spectra of (A) C1 copolymer and (B) C1-B organoboron copolymer; (C) 13C NMR spectra of C1-B
time increased, toxicity to cultured cells was increased.
C1-B-PEO and especially C1-B showed higher toxicity at
500 µg. mL-1. Thus, it can be concluded that virgin C1
alternating copolymer does not exhibit any toxic effect on
cultured HeLa cells, whereas, its organoboron and PEO
branched derivatives are definitely toxic to cells. In par-
ticular, C1-B copolymer containing relatively high
amount of organoboron linkages exhibits high toxicity
toward cancer cells compared to normal cells at 500
µg.mL-1 for 24 h.
3.3. Staining Results
The important observations can be summarized as fol-
lows: we checked for apoptosis or necrosis with double
staining (Hoescht 3342 and PI), M30 immunostaining for
cancer cells. For the morphological observations, cancer
and normal cells were stained by hematoxylen-eosin.
3.4. Hematoxylen-Eosin Staining Results
In this study, C-1 copolymers treated cancer and normal
Openly accessible at
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Figure 2. FTIR spectra of PEO macrobranched organoboron
copolymer (C1-B-PEO).
Figure 3. XRD patterns of (A) alternating copolymer (C1) and
(B) its branched organoboron derivative.
cells have intact nucleus of about 50-200 μg.mL-1 con-
centration during 2-14 h incubation. Cell morphology has
not been changed at the same concentration for 2-14 h
(Figure 6b). While C1-B and C1-B-PEO copolymers
Figure 4. In vitro cytotoxicity of C1, C1-B and C1-B-PEO
copolymers with different amount at 24 h incubation. Number
of viable HeLa cells in wells. Results are presented as
means ± SEM. *Significant difference from control (p < 0.05).
Figure 5. In vitro cytotoxicity of C1, C1-B and C1-B-PEO
copolymers with different amount at 24 h incubation. Number
of viable L929 Fibroblast cells in wells. Results are presented as
means ± SEM. *Significant difference from control (p < 0.05).
treated HeLa cells has no morphological changes at
50-200 μg.mL-1 concentration for about 2-4 h, they have
vacuole formation in their cytoplasms with C1-B co-
polymer between 6-12 hours (Figure 6c). Vacuole for-
mations determined rarely in normal cells (Figure 6e, f).
In addition, cell membranes have lysed with C1-B co-
polymer around 12-24 h but, there was no change in their
nuclei of cancer and normal cells. Moreover, some of the
cells (30% and 15% for HeLa and fibroblast, respectively)
have been detached from the well. Unaffected cells dis-
played similar morphological characteristics as with un-
treated (control) cells.
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Openly accessible at
3.5. Double Staining and M30
Immunostaining Results
In this study, if the HeLa and L929 Fibroblast cells treated
by C1, C1-B, C1-B-PEO copolymers at low conce ntra-
tion for a short time, the number of apoptotic and necrotic
cells was not high (Tables 2 and 3). However, if the
polymer concentration and incubation time were in-
creased, the number of apoptotic and necrotic cells was
increased as well. Especially, the number of apoptotic and
necrotic cells was increased when they were treated by
C1-B copolymer at 500 μg.mL-1 concentration in cancer
cell culture for 24 h (Table 2). The number of apoptotic
and necrotic Fibroblast cells was not increased according
to HeLa cells at the same concentration (Table 3). If cells
were treated by the other copolymer under similar con-
ditions, their apoptotic index was below 30 %. The results
obtained at 500 μg.mL-1 concentration for 24 h are shown
in Table 2. Meanwhile, apoptotic HeLa cells were im-
munostained by M30 antibody (Figure 7a, 7b). The
double staining and M30 immunostaining results were
similar to each other in HeLa cells. Apoptotic indexes of
HeLa cells for M30 immunostaining were 12% for C1, 45
% for C1-B, 23 % for C1-B-PEO at 500 μg.mL-1 and 24 h
incubation. Apoptotic L929 Fibroblast cells were stained
only double staining method (Table 3). In addition to
these polymers, especially boron containing polymers
had toxic effects towards cancer and normal cells. But
toxic effect of boron containing polymers was lower to
normal cells than cancer cells. After an incubation at
50–500 µg.mL-1 for 24 h period, C1 resulted in less
apoptosis, while incubation with C1-B and C1-B-PEO at
the same concentration and incubation time led to high
apoptosis of HeLa cells compared to L929 Fibroblast
cells. Both C1-B and C1-B-PEO may well inhibit cell
growth and viability in HeLa (Figure 7b, c, d) and L929
Fibroblast cells (Figure 7e, f). One the other hand, around
50-500 μg.mL-1 of C1-B and C1-B-PEO copolymer
contents for 24 h gave rise to an increase in necrosis
stained with PI dye (Figure 7c, e and Tables 2 and 3). It is
important to note that incubation for 24 h with 500 μg.
mL-1 C1-B produced apoptosis supporting its high toxicity
and necrotic effect. Furthermore, incubation without
polymers as control cells resulted in a few PI-positive
cells. Whereas, cells exposed to C1-B and C1-B-PEO-
became highly PI-positive, suggesting that they were in
necrosis. HeLa cells incubated with a high dose of boron
containing copolymers resulted in rupture of cell mem-
brane at around 12-24 h incubation period. Cell cyto-
plasm was discharged out of HeLa cells. On the other-
hand, great of number vacuole originated in most of HeLa
cells cytoplasma. It may have given rise to metabolic-
changes of cells, affected by boron containing copoly-
mers. C1-B copolymer was more toxic than virgin
counder the testing conditions determined by us.
Figure 6. Light microscope image of (A) non stained HeLa cell
culture as a control, (B) C1-B-PEO copolymer/HeLa cells
conjugate (stained with hematoxilen-eosin dye); dense spots
were showed nucleus of cells, and distinct violet were indicated
cytoplasma of cells as a control, (C) Light microscope image of
vacuole of HeLa cells cytoplasma; dense spots were showed
nucleus of cells in C1-B copolymers (500 g.mL-1 consantration)
at 24 h incubation. Light microscope image of (D) non stained
L929 Fibroblast cell culture as a control, (B) C1-B-PEO co-
polymer/L929 Fibroblast cells conjugate (stained with hema-
toxilen-eosin dye); dense spots were showed nucleus of cells,
and distinct violet were indicated cytoplasma of cells as a con-
trol, (C) Light microscope image of vacuole of L929 Fibroblast
cells cytoplasma; dense spots were showed nucleus of cells in
C1-B copolymers (500 g.mL-1 consantration) at 24 h incuba-
tion. Images (A) and (D) taken under X200 magnification,
others images taken under x400 magnification.
This work has attempted to develop novel bioengineering
functional organoboron copolymers (C1-B and C1-B-
PEO), namely, amphiphilic macromolecules of which
contained hydrophilic/hydrophobic fragments, ethylene
amidodiphenylborinate linkages, long branched PEO
segments and free carboxylic groups with an ability to
conjugate with cancer HeLa cells. These copolymers
were synthesized by amidolysis and esterification of
anhydride units of poly(MA-alt-MVE) (C1) as a bio-
M. Türk et al. / HEALTH 2 (2010) 51-61
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Table 2. The comparative analysis of apoptotic and necrotic
HeLa cell index for copolymer (C1), organo-boron (C1-B) and
organoboron PEO branched (C1-B-PEO) copolymers at 24 h
Polymer amount
cells (%)
cells (%)
3 ± 2
4 ± 2
50 3 ± 1 5 ± 1.5
100 6 ± 1.5 9 ± 3
250 8.5 ±1 13.8 ± 1.5
500 15 ± 2 27 ± 3
control 5 ± 3 2 ± 1
50 13 ± 2 20 ± 3
100 18 ± 2.5 31 ± 4
250 32 ± 1 41 ± 2
500 43 ± 2 53 ± 3
control 3.5 ± 1.5 5 ± 1.5
50 9 ± 1 11 ± 1.5
100 12 ± 1 16 ± 2
250 15 ± 2 25 ± 3
500 28 ± 2.5 43.5 ± 5
Table 3. The comparative analysis of apoptotic L 929 Fibroblast
cells index for copolymers (C1), organoboron (C1-B) and or-
ganoboron PEO branched (C1-B-PEO) copolymers at 24 h
Polymer amount
cells (%)
cells (%)
3 ±1
50 3±1 3±1.5
100 4±1 6±2
250 7±1 11±1.5
500 9±1.5 17±2
control 2±1 2±1
50 7±2 12±2
100 12±2.5 17±3
250 16±1 25±2
500 27±2 38±2
control 3±2 3±1
50 4±1 8±1.5
100 6±1 11±2
250 9±2 16±3
500 17±2.5 30±1
compatible and non toxic polymer matrix with organo-
boron amine and PEO, respectively. Chemical and
physical structure of organoboron copolymers were con-
firmed by FTIR and 1H (13C) NMR spectroscopy and
X-ray powder diffraction methods. The comparative
analysis of novel organoboron functional copolymers
with antitumor acivity towards cancer and normal cells
was achieved. It was found that unlike the virgin amor-
phous C1 copolymer, organoboron copolymer (C1-B)
exhibited semi-crystalline phase transition behaviour due
to the formation of self-assembled supramacromolecular
structures through strong intra-and intermolecular hy-
Figure 7. Light microscopy images of (A) virgin (nonapoptotic)
HeLa cells as a control group (stained with M30 immu-
nostaining kit), and (B) organoboron copolymer C1-B copoly-
mer/HeLa cells conjugate (stained with M30 immunostaining
kit), where brown cytoplasma of cells image indicates the for-
mation of apoptotic cells; Fluorescence microscopy image of (C)
nucleus of HeLa cells (stained with PI), where formation of red
spots demostrates nucleus of necrotic cells, and (D) nucleus of
HeLa cells (stained with Hoescht 3342), where dense spots
indicates nucleus of apoptotic cells. Fluorescence microscopy
image of (E) nucleus of L929 Fibroblast cells (stained with PI),
where formation of red spots demostrates nucleus of necrotic
cells and green spots demostrates nucleus of living cells, and (F)
nucleus of L929 Fibroblast cells (stained with Hoescht 3342),
where dense spots indicates nucleus of apoptotic cells. İmages
of Cand D were recorded with x400 magnification, others image
were recorded with x200 magnification.
drogen bonding. The interactions of these copolymers
with HeLa cells were investigated by using a combination
of different methods such as cytotoxicity, statistical, he-
matoxylen/eosin staining, apoptotic and necrotic cell
indexes, M30 immunostaining, double staining and M30
immunostaining, light and fluorescence microscopy
analyses. In vitro cytotoxicities and antitumor activities of
organoboron copolymers (C1-B and C1-B-PEO) against
human cervix epithelioid carcinoma cell line (HeLa) was
as well evaluated. It was observed that organoboron co-
polymers exhibited the most apoptotic and necrotic ef-
fects against HeLa cells whereas a minor effect relative to
cancer cells was observed on L929 Fibroblast cells. Thus
the obtained results allow us to propose that synthesized
organoboron copolymers containing a combınation of
non toxic and biocompatibile polymer matrix and long
branched PEO segments with functional groups as anti-
tumor sities, can be utilized as therapeutic potential
functional copolymer drugs, which are able to form an
artificial bioconjugate with HeLa cells, in cancer che-
The supports of the Turkish National Scientific and Technical Council
(TÜBİTAK) through project TBAG-2486 and HU Scientific Research
M. Türk et al. / HEALTH 2 (2010) 51-61
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Openly accessible at
Foundation (BAB) through the BAB-2601006 project are kindly ac-
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