Bioengineering functional copolymers. XII. Interaction of boron-containing and PEO branched derivatives of poly(MA-alt-MVE) with HeLa cells

doi:10.4236/health.2010.21009

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Vol.2, No.1, 51-61 (2010)

doi:10.4236/health.2010.21009

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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.

ABSTRACT

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

1. INTRODUCTION

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

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

tested.

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

[32].

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. MATERIALS AND METHODS

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

(Roche).

M. Türk et al. / HEALTH 2 (2010) 51-61

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-

len/Eosin.

2.5. Analysis of Apoptotic and Necrotic

Cells

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

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. RESULTS AND DISCUSSION

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

M. Türk et al. / HEALTH 2 (2010) 51-61

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

Derivatives

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

Fıgure 1. 1H NMR spectra of (A) C1 copolymer and (B) C1-B organoboron copolymer; (C) 13C NMR spectra of C1-B

copolymer.

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

M. Türk et al. / HEALTH 2 (2010) 51-61

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.

M. Türk et al. / HEALTH 2 (2010) 51-61

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.

4. CONCLUSIONS

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

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

incubation.

Polymer amount

(µg.mL-1)

Apoptotic

cells (%)

Necrotic

cells (%)

control

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

C1-B

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

C1-B-PEO

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

incubation.

Polymer amount

(µg.mL-1)

Apoptotic

cells (%)

Necrotic

cells (%)

control

2±1

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

C1-B

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

C1-B-PEO

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-

motherapy.

5. ACKNOWLEDGEMENTS

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

Openly accessible at

Foundation (BAB) through the BAB-2601006 project are kindly ac-

knowledged.

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