Synthesis of glycopolymers at various pendant spacer lengths of glucose moiety and their effects on adhesion, viability and proliferation of osteoblast cells

Article abstract of DOI:10.1039/c4ra05436a

Multivalent glycopolymers containing three different glucose pendant spacer lengths in the polymer backbone having various weight percentages of covalently bonded glucose moieties were obtained by the deacetylation of acetylated polymers synthesized via reversible addition-fragmentation chain transfer (RAFT) process. This allowed us to control the packing density of the glucose moieties in one unit of the glycopolymer. The biological responses of these macromolecules in terms of cytotoxicity of glucose moieties at various spacer lengths and concentrations were investigated without perturbing the intrinsic chemical nature of functional moiety by employing in vitro osteoblast cells. Osteoblast cell adhesion, viability and proliferation response with synthesized glycopolymers revealed that the higher glycopolymer concentration tolerance limit was associated with an increase in pendant spacer length of glucose moiety in the glycopolymer. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra05436a

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PAPER
Synthesis of glycopolymers at various pendant
spacer lengths of glucose moiety and their effects
on adhesion, viability and proliferation of
osteoblast cells†
Cite this: RSC Adv., 2014, 4, 37400
Mummuluri Trinadh,a Govindaraj Kannan,b Tota Rajasekhar,a Annadanam V. Sesha
a
b
*
*
Sainath and Marshal Dhayal
Multivalent glycopolymers containing three different glucose pendant spacer lengths in the polymer
backbone having various weight percentages of covalently bonded glucose moieties were obtained by
the deacetylation of acetylated polymers synthesized via reversible addition-fragmentation chain transfer
(RAFT) process. This allowed us to control the packing density of the glucose moieties in one unit of the
glycopolymer. The biological responses of these macromolecules in terms of cytotoxicity of glucose
moieties at various spacer lengths and concentrations were investigated without perturbing the intrinsic
chemical nature of functional moiety by employing in vitro osteoblast cells. Osteoblast cell adhesion,
viability and proliferation response with synthesized glycopolymers revealed that the higher
glycopolymer concentration tolerance limit was associated with an increase in pendant spacer length of
glucose moiety in the glycopolymer.
Received 7th June 2014
Accepted 30th July 2014
DOI: 10.1039/c4ra05436a
www.rsc.org/advances
distribution of glyco-moieties in glycans have advantages in
developing new materials for biomedical applications.33,34 The
1. Introduction
initial study by Schnaar et al. demonstrated the adhesion of
chicken hepatocytes to poly(acrylamide) gels, which were
derivatized with N-acetylglucosamine glycopolymeric coat-
ings.35 The broblast cell adhesion and proliferation on
hyperbranched glycoacrylate lms and glycopolymer coatings
on polystyrene carrying N-acetylglucosamine residues were
studied.36,37 The biocompatibility of sugar-modied extracel-
lular matrices of glycopolymers had been extensively studied in
liver cells by varying carbohydrate moieties in polymeric back-
bone architectures.38–42
Synthetic glycopolymers are a useful tool, which allows the
precise control of various biological functions of a cell during
cell/surface interactions. The development of new methods for
the synthesis of tailor-made glycopolymers, as well as glycan
derivatives, has given access to targeted architectures and
modications with carbohydrate moieties.43,44 Previously,
glucose moiety containing glycopolymer (poly-[3-O-(40-vinyl-
benzyl)-D-glucose]) was used for culturing erythrocytes and
observations conrmed that the presence of reducing glucose
moieties was crucial for specic cell attachment.45,46 Despite all
of these biologically important functions, there has been very
little attention paid towards the use of glycopolymers having
different pendant alkyl chain lengths of glucose functional
moiety on polymer backbone in cell and tissue engineering.
To the best of our knowledge, the osteoblast cell adhesion,
viability and proliferation with glycopolymers, consisting of
The unique properties of glycopolymers, which consist of a
synthetic polymeric backbone and pendant glyco-moieties have
been applied in clinical diagnostics, drug and gene delivery
systems,1–6 specic molecular recognition,7–16 surface modi-
ers17–19 and separation of biomolecules.20–23 Hence, the inter-
ests of researchers have been focused on dealing with the
design and development of suitable glycopolymeric materials
by tailoring macromolecular chains and testing their biological
performance. Well-dened glycopolymeric materials provide
structural and functional support to cells/tissues due to their
hydrophilic character, better biocompatibility and ability to
mimic glycoproteins in the biological systems.24–27 The carbo-
hydrate units in glycopolymers act as ligands in a variety of
biological processes and play a signicant role in biomolecule
recognition and determine the interaction with cells for desir-
able tissue growth and repair.28–32 Moreover, glycans are specic
for the regulation of certain cellular functions due to their
multivalent nature and exibility while the varying spatial
aPolymers and Functional Materials Division, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500007, Andhra Pradesh, India. E-mail: avssainath@yahoo.
com; Fax: +91 40 27160591; Tel: +91 40 27192500
bClinical Research Facility, CSIR-Center for Cellular and Molecular Biology,
Hyderabad 500007, Andhra Pradesh, India. E-mail: marshal@ccmb.res.in
† Electronic supplementary information (ESI) available: Detailed characterisation
of glycopolymer and cellular response of free glucose. See DOI:
10.1039/c4ra05436a
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various pendant alkyl chain of glucose moiety has not been necked, round-bottomed ask, followed by 150 mL of acetic
examined yet. However, previously, osteoblast cell adhesion to anhydride and reuxed for 4 h. Aer the solution became clear,
various implant materials, such as metal oxides, bioinert metals, it was poured into a conical ask which contained crushed ice.
¨
metal alloys, bioceramics, synthetic polymers and composites, has The generated crystals were ltered with a Buchner funnel and
been extensively studied.47–54 Formerly, poly(pentauorostyrene) dried. Yield: 75%. M.p. 113–115 ꢁC. 1H NMR (CDCl3, 300 MHz):
based glycopolymer coated substrates were used to study the d 5.73 (d, j ¼ 7.93 Hz, 1H), 5.26 (m, 1H), 5.14 (t, j ¼ 9.06, 7.55 Hz,
attachment of 3T3 broblasts and MC3T3-E1 preosteoblasts.55 2H), 4.30 (m, 1H), 4.11 (d, j ¼ 11.71 Hz, 1H), 3.86 (d, j ¼ 7.18 Hz,
It was expected that the studies of osteoblast cell response at 1H) and 2.09–2.02 (m, 15H). ESI-MS: m/z: 413 (M + Na)+. IR (KBr,
various concentrations of glycopolymers having different cmꢀ1): n 2969 (–CH–), 1746 (–O–C]O–), 1375 (–O–C–, ester) and
pendant alkyl chain lengths of glucose functional moieties on 1226 (–C–O–C–).
acrylate macromolecular chain could help in designing bone
implant coatings that modulate cell adhesion and proliferation 2.3 Preparation of 2,3,4,6-tetra-O-acetyl-D-glucopyranoside
within the multicellular environment of the human body. Thus, (TAGP)
the study of osteoblast cell interactions with glycopolymers is of
A solution of PAGP (5.0 g, 0.0128 mol) and benzylamine (2.1 mL,
great interest for hard tissue engineering and understanding
0.0192 mol) in 30 mL of THF was taken in a 100 mL two-necked,
bone formation process.56
round-bottomed ask and stirred overnight at room tempera-
Here we used the RAFT process for the syntheses of glyco-
ture.60 The mixture was diluted with cold water and extracted
polymers containing three different glucose pendant alkyl chain
with chloroform (3 ꢂ 200 mL). The extracted layer was succes-
lengths in the polymer backbone. The RAFT process is a
sively washed with ice cold dilute HCl solution, saturated
commonly used and reliable method for the synthesis of glyco-
NaHCO3 solution, saturated NaCl solution and water. The
polymers to achieve controlled macromolecular architecture
mixture was dried over anhydrous Na2SO4 and concentrated in
vacuum. The residue was puried by column chromatography
from a wide range of functional monomers.57–59 In this paper, we
report the usefulness of functional moiety with different pendant
with ethyl acetate and n-hexane (2/3, v/v) mixture to afford 3.5 g
alkyl chain lengths in glycopolymers to control the response of
of TAGP, which is a viscous liquid. Yield: 80%. 1H NMR (CDCl3,
different cellular functions such as adhesion, viability and
300 MHz): d 5.55 (t, j ¼ 9.44 Hz, 1H), 5.43 (s, 1H), 5.07 (t, j ¼ 9.06
proliferation of mouse osteoblast (MC3T3) cells in vitro.
Hz, 2H), 4.87 (d, j ¼ 9.44 Hz, 1H), 4.37 (s, 1H), 4.24 (m, 1H), 4.08
(s, 1H) and 2.17–2.01 (m, 12H). ESI-MS: m/z: 371 (M + Na)+. IR
(KBr, cmꢀ1): n 3328 (–OH), 2956 (–CH–), 1750 (–O–C]O–), 1432,
2. Experimental
2.1 Materials
1370 (–O–C–, ester) and 1231 (–C–O–C–).
D-Glucose anhydrous (Fisher Scientic, Qualigens), acetic
anhydride and sodium acetate (Sisco Research Laboratories Pvt.
Ltd.), benzylamine (Finar Chemicals Limited), methyl ethyl
ketone (MEK), acryloyl chloride, triethylamine (Et3N) and azo-
bisisobutyronitrile (AIBN) (Avra Synthesis Pvt. Ltd.), 1,4-buta-
nediol, 1,6-hexanediol, boron triuoride diethyl etherate
2.4 Synthesis of 4-hydroxybutyl acrylate (HBA)
HBA was prepared as reported in literature.61 1,4-Butanediol
(26.9 g, 0.299 mol) was reacted with acryloylchloride (6.92 g,
0.0765 mol) in the presence of triethylamine (10.9 g, 0.108 mol).
The obtained mixture was puried by column chromatography
using hexane/ethyl acetate (v/v, 1 : 1) as eluent. Note that HBA
(BF3$Et2O)
and
2-cyano-2-propyldodecyltrithiocarbonate
1
was liquid in nature. H NMR (CDCl3, 300 MHz): d 6.40 (d, j ¼
˚
(CPDTC) (Sigma-Aldrich) and molecular sieves 4 A (SDFCL, s d
ne-chem Limited) were used as received. Dichloromethane
(DCM) (Finar Chemicals Ltd.) was dried over calcium hydride by
stirring overnight and distillation. Tetrahydrofuran (THF)
(Finar Chemicals Limited) was distilled over sodium wire with
benzophenone under a nitrogen atmosphere. The osteoblast
cells (MC3T3) were obtained from American type culture
collection (ATCC), USA, and their subclones were maintained in
a complete medium (CM). The CM was made of fresh a-
minimum essential medium (MEM) (Invitrogen, USA) having
10% fetal bovine serum (FBS) (Invitrogen, USA) and 100 U mLꢀ1
penicillin, 50 mg mLꢀ1 streptomycin and 50 mg mLꢀ1 gentamy-
cin (Invitrogen, USA). The cells were sub-cultured using trypsin-
ethylenediaminetetraacetic acid (EDTA) (Invitrogen, USA).
17.18 Hz, 1H), 6.20–6.03 (m, 1H), 5.82 (d, j ¼ 10.39 Hz, 1H), 4.19
(m, 2H), 3.67 (m, 2H) and 1.88–1.54 (m, 4H). ESI-MS: m/z: 145
(M + H)+. IR (KBr, cmꢀ1): n 3425 (–OH), 2951 (–CH–), 1723
(–O–C]O–), 1410, 1370 (–O–C–, ester) and 1194 (–C–O–C–).
2.5 Preparation of 60-hydroxyhexyl-2,3,4,6-tetra-O-acetyl-D-
glucopyranoside (HHTAGP)
A solution of PAGP (10 g, 0.026 mol) in dichloromethane (80
mL) was stirred for 2 h with molecular sieves (4 g, 0.4 mm)
under nitrogen atmosphere.62 Then, BF3$Et2O (6.5 mL, 0.051
mol) was subsequently added to the mixture, immediately fol-
lowed by 1,6-hexanediol (3.32 g (0.028 mol) in 15 mL dichloro-
methane). Aer 10 days, the mixture was poured into saturated
NaHCO3 solution (100 mL). The organic layer was separated and
the aqueous phase was extracted with dichloromethane 3 times,
each time with 40 mL of dichloromethane. The total collected
organic layer was washed twice with water, each time with 40
2.2 Preparation of 1,2,3,4,6-penta-O-acetyl-D-
glucopyranoside (PAGP)
Well-ground mixture of 20 g (0.11 mol) of D-glucose and 25 g mL of water. Dichloromethane was evaporated under vacuum
(0.304 mol) of sodium acetate was taken in a 500 mL two- and the resulting syrup was puried by column
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chromatography (hexane/ethyl acetate, 7 : 3 (v/v)). Note that pressure, the yellow syrup was puried by ash column chro-
HHTAGP was a viscous liquid. Yield: 40%. 1H NMR (CDCl3, 300 matography using hexane and ethyl acetate mixture (7 : 3, v/v).
MHz): d 5.55–5.32 (d, j ¼ 32.86 Hz, 1H), 5.15–4.92 (m, 2H), 4.87– Yield: 45%. 1H-NMR (CDCl3, 500 MHz): d 6.47–6.40 (dd, j ¼
4.72 (d, j ¼ 11.71 Hz, 1H), 4.36–4.16 (d, j ¼ 11.71 Hz, 1H), 4.14– 1.07 Hz, 1H), 6.16–6.06 (m, 1H), 5.93–5.88 (dd, j ¼ 1.07 Hz, 1H),
3.88 (m, 4H), 3.76–3.61 (broad s, 1H), 3.52–3.34 (broad s, 1H), 5.58–5.51 (t, j ¼ 9.77 Hz, 1H), 5.15–5.06 (m, 2H), 4.96–4.91 (dd,
2.24–1.88 (m, 12H) and 1.73–1.15 (m, 8H). ESI-MS: m/z: 471 (M + j ¼ 3.66 Hz, 1H), 4.31–4.17 (m, 2H), 4.16–3.99 (m, 4H), 3.77–3.69
Na)+. IR (KBr, cmꢀ1): n 3328 (–OH), 2956 (–CH–), 1750 (–O–C] (m, 1H), 3.51–3.39 (m 1H), 2.09–1.97 (m, 12H), 1.81–1.53 (m,
O–), 1432, 1370 (–O–C–, ester) and 1231 (–C–O–C–).
2H) and 1.31–1.20 (m, 2H). 13C NMR (CDCl3, 75 MHz): d 20.59,
20.91, 25.27, 25.80, 29.64, 61.37, 61.96, 63.95, 67.24, 67.78,
68.10, 68.46, 69.21, 69.82, 70.07, 70.53, 70.96, 72.68, 95.70,
127.37, 132.41, 165.09, 169.56, 170.17, 170.63 and 171. IR (KBr,
cmꢀ1): n 2949 (–CH–), 1750 (–O–C]O–), 1634 (–C]C–), 1409,
1369 (–O–C–, ester) and 1227 (–C–O–C–).
2.6 Preparation of glycoacrylates, acryl-2,3,4,6-tetra-O-acetyl-
D-glucopyranoside (ATAGP) and 60-(acryloxy)hexyl-2,3,4,6-
tetra-O-acetyl-D-glucopyranoside (AHTAGP)
ATAGP and AHTAGP were synthesized according to a typical
standard procedure as follows. A solution of TAGP (18 g, 0.0517
mol) in 180 mL of MEK and 14.39 mL (0.1035 mol) of Et3N was
2.8 Polymerization
taken in a 500 mL two-necked, round-bottomed ask equipped All the monomers, i.e. ATAGP, AHTAGP and ABTAGP, were
with a magnetic stirrer and placed in an ice bath. Acryloyl polymerized via the RAFT method. A typical ATAGP polymeri-
chloride (5.55 mL (0.0568 mol) in 25 mL of MEK) was added zation procedure is as follows. A mixture of acryl-2,3,4,6-tetra-O-
drop by drop with constant stirring without raising the acetyl-D-glucopyranoside (1.7 g, 0.0042 mol), AIBN (18 mg,
temperature above 0 ꢁC. The reaction mixture was stirred for a 0.00012 mol) and RAFT agent, CPDTC (58 mg, 0.00016 mol) in
period of 5 h aer removing from the ice bath. The quaternary 20 mL of THF was deoxygenated by freeze and thaw cycle for
ammonium salt (precipitate) was ltered. The supernatant was three times. Then, the ask was sealed and heated to 80 ꢁC with
washed with cold distilled water and dried with anhydrous stirring. It was allowed to polymerize for 30 h. Aer that, the
Na2SO4. ATAGP was isolated by evaporating the solvent and polymerization was stopped by the addition of 0.5 mL of
puried by column chromatography technique. Yield: 75%. methanol and exposed to air. The polymer was isolated by
M.p. 59-62 ꢁC. 1H NMR (CDCl3, 300 MHz): d 6.59–6.35 (m, 1H), precipitating in cold methanol twice and once in 30% ethyl
6.25–6.03 (m, 1H), 6.01–5.90 (t, j ¼ 10.20 Hz, 1H), 5.81–5.71 (d, j acetate in hexane. The yield of poly(acryl-2,3,4,6-tetra-O-acetyl-D-
¼ 7.93 Hz, 1H), 5.53–5.41 (t, j ¼ 9.82 Hz, 1H), 5.30–5.06 (m, 1H), glucopyranoside) (P(ATAGP)) was about 55%. P(ATAGP):
4.33–4.19 (m, 1H), 4.17–3.99 (d, j ¼ 3.40 Hz, 1H), 3.92–3.79 (d, j 1H NMR (CDCl3, 300 MHz): d 6.40–6.16, 5.80–5.57, 5.52–5.35,
¼ 7.93 Hz, 1H) and 2.12–1.87 (m, 12H). 13C NMR (CDCl3, 75 5.34–5.20, 5.19–4.68, 4.51–4.18, 4.17–3.98, 3.96–3.72, 2.73–2.24,
MHz): d 20.24, 20.37, 20.41, 20.51, 61.31, 67.68, 67.80, 69.10, 2.21–1.82, 1.79–1.06 and 0.90–0.75. 13C NMR (CDCl3, 75 MHz):
69.76, 70.05, 72.55, 72.61, 89.18, 91.81, 132.92, 133.34, 163.61, d 67.73, 69.77, 70.12, 72.70, 89.17, 89.65, 169.23, 169.32,
169.07, 169.23, 169.53, 169.89, 170.0 and 170.42. ESI-MS: m/z: 169.98, 170.47 and 170.56. IR (KBr, cmꢀ1): n 2962 (–CH–), 1756
425 (M + Na)+. IR (KBr, cmꢀ1): n 2963 (–CH–), 1750 (–O–C]O–), (–O–C]O–), 1634 (–C]S–), 1439, 1374 (–O–C–, ester) and 1226
1634 (–C]C–), 1409, 1370 (–O–C–, ester) and 1221 (–C–O–C–). (–C–O–C–). Poly(40-(acryloxy)butyl-2,3,4,6-tetra-O-acetyl-D-gluco-
AHTAGP: yield: 75%, viscous liquid. 1H NMR (CDCl3, 500 MHz): pyranoside) (P(ABTAGP)): yield: 46%. 1H NMR (CDCl3, 300
d 6.47–6.36 (d, j ¼ 17.0 Hz, 1H), 6.19–6.05 (m, 1H), 5.82–5.78 (d, j MHz): d 6.47–6.16, 5.89–5.56, 5.53–5.37, 5.37–5.25, 5.25–4.90,
¼ 10.58 Hz, 1H), 5.27–5.15 (t, j ¼ 9.44 Hz, 1H), 5.15–5.04 (m, 4.56–4.23, 4.20–4.01, 3.98–3.82, 2.85–2.29, 2.26–1.78, 1.56–0.95
1H), 5.04–4.92 (m, 1H), 4.52–4.46 (d, j ¼ 7.93 Hz, 1H), 4.32–4.22 and 0.92–0.82. 13C NMR (CDCl3, 75 MHz): d 20.68, 61.09, 65.11,
(m, 4H), 4.20–4.08 (d, j ¼ 7.18 Hz, 1H), 3.94–3.81 (m, 1H), 3.76– 67.64, 69.24, 69.62, 72.35, 72.58, 72.67, 89.17, 169.00, 169.17,
3.64 (d, j ¼ 9.44 Hz, 1H), 3.55–3.41 (m, 1H), 2.15–2.19 (m, 12H), 169.37, 169.90 and 169.98. IR (KBr, cmꢀ1): n 2962 (–CH–), 1755
1.73–1.50 (m, 4H) and 1.45–1.29 (s, 4H). 13C NMR (CDCl3, 75 (–O–C]O–), 1635 (–C]S–), 1437, 1371 (–O–C–, ester) and 1226
MHz): d 20.35, 20.47, 61.27, 67.64, 69.06, 69.67, 70.01, 72.47, (–C–O–C–). Poly(60-(acryloxy)hexyl-2,3,4,6-tetra-O-acetyl-D-gluco-
72.54, 91.74, 126.87, 133.29, 163.56, 169.01, 169.18, 169.83 and pyranoside) (P(AHTAGP)): yield: 68%. 1H NMR (CDCl3, 300
170.34. ESI-MS: m/z: 525 (M + Na)+. IR (KBr, cmꢀ1): n 2962 (–CH–), MHz): d 6.40–6.24, 5.84–5.59, 5.53–5.37, 5.37–5.21, 5.21–4.95,
1745 (–O–C]O–), 1633 (–C]C–), 1409, 1376 (–O–C–, ester) and 4.48–4.20, 4.20–4.03, 3.99–3.79, 3.76–3.54, 2.74–2.25, 2.19–1.94,
1230 (–C–O–C–).
1.93–1.92 and 0.93–0.81. 13C NMR (CDCl3, 75 MHz): d 20.45,
68.26–67.27, 70.46–69.60, 72.61, 91.75, 169.11, 169.32,
169.30, 170.51 and 170.61. IR (KBr, cmꢀ1): n 2963 (–CH–), 1756
(–O–C]O–), 1634 (–C]S–), 1437, 1372 (–O–C–, ester) and 1226
(–C–O–C–).
2.7 Preparation of 40-(acryloxy)butyl-2,3,4,6-tetra-O-acetyl-D-
glucopyranoside (ABTAGP)
PAGP (10 g, 0.026 mol) and 4-hydroxybutyl acrylate (3.32 g, 0.023
mol) were dissolved in 70 mL of DCM and BF3$Et2O (10.92 g,
0.077 mol) was subsequently added via a syringe. Note that the
mixture was sonicated for 45 min. The reaction mixture was
2.9 Deacetylation of pendant 2,3,4,6-tetra-O-acetyl-D-
glucopyranoside moieties
washed with saturated brine solution and the organic layer was The deacetylated polymers, poly(acryl-D-glucopyranoside) (GP1),
dried over MgSO4. Aer removing the solvent under reduced poly(40-(acryloxy)butyl-D-glucopyranoside) (GP2) and poly(60-
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(acryloxy)hexyl-D-glucopyranoside) (GP3), were synthesized solutions of the glycopolymers were prepared for each concen-
according to a typical procedure as follows. tration, and it was in such a manner that only 4 mL of solution
The P(ATAGP) (200 mg) was dissolved in anhydrous meth- was added to achieve all the desired nal concentrations. All cell
anol and chloroform (2 : 1) (total volume: 10 mL) and stirred culture experiments were performed in triplicates with cells not
under nitrogen atmosphere. A solution of NaOMe (101 mg) in exceeding ten passages post revival. The statistical analysis for
anhydrous methanol (2 mL) was added through a syringe and in vitro cell assay was performed based on three experiments for
the reaction mixture was le stirring for 90 min. Then, the each condition in which a minimum of three samples were
solvent was removed by vacuum evaporation and neutralized used. Microso Excel soware was used for calculating mean
with 2 M HCl solution by stirring overnight. Aer that, the and standard deviation values for all assays.
solution was passed through basic alumina to neutralize the
Cell adhesion assay. This assay was carried out by plating
solution. Excess water was removed by lyophilisation, and the similar number of cells in the plastic tissue culture plate wells.
polymer was recovered as a white powder by precipitation in Aer 4 h, non-adherent cells were washed off and 10 mL of 2 mg
excess acetone. GP1: IR (KBr, cmꢀ1): n 3425 (–OH–), 2927 mLꢀ1 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(–CH–), 1735 (–O–C]O–), 2200 (–CN) and 1628 (–C]S–). GP2: bromide) was added to the remaining cells of each condition,
IR (KBr, cmꢀ1): n 3416 (–OH–), 2962 (–CH–), 1750 (–O–C]O–), followed by incubation at 37 ꢁC and 5% CO2 for 4 h. Aer
2200 (–CN) and 1620 (–C]S–). GP3: IR (KBr, cmꢀ1): n 3425 removing CM, the reaction was stopped by adding dimethylsulf-
(–OH), 2927 (–CH–), 1755 (–O–C]O–), 1635 (–C]S–), 1430, oxide and read at 595 nm by ELISA reader.
1383 (–O–C–, ester) and 1239 (–C–O–C–).
Cell viability assay. The assay was performed by plating
similar number of cells of all conditions in triplicates and
incubated. Aer 24 h of incubation, MTT assay was performed
as described above to calculate the percentage of cell viability.
Cell proliferation assay. The assay was performed by plating
similar number of cells of all conditions in triplicates and
incubated at dissimilar time periods, namely, 4, 24, 48 and 72 h.
At the specied time periods cell number at each concentration
was calculated by MTT assay as described above.
Immunouoresence staining. Different concentrations of
glycopolymer were added to the cells plated in a 6-well plate and
incubated for 24 h. Aer this, the cells were xed with 4%
formaldehyde (Sigma, USA) for 5–6 min. Cells were washed with
PBS for 3 times and permeabilization was performed with 0.1%
Triton X-100 for 5–6 min aer washing with PBS for 3 times.
Blocking was performed with 3% bovine serum albumin (BSA,
Sigma) for 1 h, and cells were washed with PBS once. Rodamine-
phalloidin (Molecular Probes, USA) diluted in 1% BSA to 1 : 200
ratio was added on to the cells and incubated for 1 h. Finally, cells
were washed once with PBS and mounted using uoroshield 40,6-
diamidino-2-phenylindole (DAPI) (Sigma, USA). Cells were
imaged with uorescent microscope (Axio Imager.Z1, Carl Zeiss,
Jena Germany). Integrin a5 and talin co-staining was carried out
as follows: aer 24 h, cells on the above substrates were xed in
4% paraformaldehyde for 10 min at room temperature, per-
meabilized with 0.1% Triton-X-100 for 10 min and blocked with
2.10 Estimation of weight percentage of covalently bonded
glucose moiety in glycopolymer
Covalently bonded glucose moiety in glycopolymer was calcu-
lated to determine their packing density in one unit of glyco-
polymer. The degree of polymerization (Dp) of glycopolymer was
estimated using eqn (1).
È
ꢀ
É
ꢁ
GpðMwÞ ꢀ 345
MðFwÞ
Dp ¼
(1)
where Gp(Mw): molecular weight of deacetylated glycopolymer
1
by H NMR, M(Fw): formula weight for deacetylated monomer
and 345 is a constant, representing the molecular weight of
terminal ends of macromolecular chain generated by RAFT
agent. Hence, glucose moiety (GM) wt% was calculated using
eqn (2).
ꢀ
ꢁ
GMðFwÞ
GpðMwÞ
GMðwt%Þ ¼ 100 ꢂ Dp
(2)
where GM(Fw) is glucose moiety formula weight. All weight
percentages of covalently bonded glucose moiety on three gly-
copolymers were calculated using the above equations.
2.11 In vitro cell assays
Cell culture and statistical analysis. The adherent osteoblast 3% BSA for 30 min. The cells were treated with rabbit anti-mouse
cells (MC3T3) were maintained in CM at 37 ꢁC in 5% CO2. The integrin a5 antibody at 1 : 200 (Santa Cruz, USA) for 1 h at room
CM was prepared using a-MEM medium, supplemented with temperature, followed by 1 h incubation with goat anti-rabbit
10% FBS and 100 U mLꢀ1 penicillin, 50 mg mLꢀ1 streptomycin FITC (uorescein isothiocyanate) at 1 : 200 (GeneI, Bangalore,
and 50 mg mLꢀ1 gentamycin. The cells were sub-cultured aer India). The same procedure and conditions were repeated for
reaching 80% conuency using trypsin-EDTA. In a 96-well plate, talin staining with mouse, anti-mouse talin antibody, followed
104 cells were plated in each well and different concentrations by a 1 h incubation with goat anti-mouse TRITC in the dark at
of glycopolymer were added to the cells. The stock solution of room temperature. Images were captured using a 40ꢂ and 63ꢂ
different concentrations of all three glycopolymers were objective using AxioVision imaging system (Zeiss, Germany).
prepared in phosphate buffered saline (PBS) and sterilized by
ꢁ
autoclaving at 121.5 C for 20 min. Final concentrations of the
glycopolymer ranging from 1 nM to 10 000 mM were achieved by
2.12 Measurements
adding 4 mL of stock solution of different desired concentra- The FT-IR (Thermo Nicolet Nexus 670 spectrometer) spectra
tions in 96 mL of CM for each set of experiments. Separate stock were recorded at a resolution of 4 cmꢀ1 using KBr optics at
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room temperature and a minimum of 32 scans were signal
averaged. The proton nuclear magnetic resonance (1H NMR)
and carbon nuclear magnetic resonance (13C NMR) spectra were
recorded on AVANCE-300 or INOVA-500 spectrometer in CDCl3
or D2O (Aldrich) depending on the solubility of the products
with tetramethylsilane as an internal standard. Mass spectra
were recorded on FINNGAN LCQ Advance Max in methanol. Gel
permeation chromatography (GPC) was performed by using a
Shimadzu system built-in with one (300 ꢂ 7.5 mm) PLgel 5 mm
3. Results and discussion
3.1 Glycopolymers characterization
The synthesized glycoacrylates, acryl-2,3,4,6-tetra-O-acetyl-D-gluco-
pyranoside (ATAGP), 40-(acryloxy)butyl-2,3,4,6-tetra-O-acetyl-D-glu-
copyranoside (ABTAGP) and 60-(acryloxy)hexyl-2,3,4,6-tetra-O-
acetyl-D-glucopyranoside (AHTAGP), were polymerized by RAFT
process using 2-cyano-2-propyldodecyltrithiocarbonate as RAFT
agent with azobisisobutyronitrile as a radical initiator to produce
homopolymers, poly(acryl-2,3,4,6-tetra-O-acetyl-D-glucopyranoside)
(poly(ATAGP)), poly(40-(acryloxy)butyl-2,3,4,6-tetra-O-acetyl-D-gluco-
pyranoside) (poly(ABTAGP)) and poly(60-(acryloxy)hexyl-2,3,4,6-
tetra-O-acetyl-D-glucopyranoside) (poly(AHTAGP)) with a dodecyl
alkyl terminal end, respectively. The resulted polymer pendant
units, 2,3,4,6-tetra-O-acetyl-D-glucopyranoside, were deacetylated
in the presence of sodium methoxide/chloroform/methanol
mixture to obtain poly(acryl-D-glucopyranoside) (GP1), poly-
(4-(acryloxy)butyl-D-glucopyranoside) (GP2) and poly(6-(acryloxy)-
hexyl-D-glucopyranoside) (GP3) glycopolymers, respectively. The
structures of the polymers before and aer deacetylation are
shown in Scheme 1.
3
˚
10 ꢂ 10 A column (VARIAN) and evaporative light-scattering
(PL-ELS) detector (Polymer Laboratories). The eluent was DMF
with a ow rate of 0.5 mL minꢀ1 at 30 ꢁC. Calibration for
detector response was gained using a single narrow PS standard
(molecular weights: 550, 1480; 3950; 10 680 and 31 420 from
Polymer Labs). 1 mg of polymer was dissolved in 1 mL of DMF.
Values of Mn and Mw/Mn were determined using LC Solution for
Windows soware. CO2 incubator was purchased from Thermo
Scientic, USA. Spectrophotometric measurements of adhesion
and proliferation were taken from Molecular Devices – Spectra
Max – 190, USA.
Aer deacetylation, number average molecular weights (Mn),
10 829, 9856 and 4266 g molꢀ1 for GP1, GP2 and GP3, respectively
Scheme 1 Schematic representation of the syntheses of glycopolymers (0, 4 and 6 indicate spacer length) via the RAFT process and the
deacetylation of the pendant 2,3,4,6-tetra-O-acetyl-D-glucopyranoside.
Table 1 Characteristics of acetylated and deacetylated glycopolymers
Acetylated
Deacetylated
GPCa
GPCa
Polymer
Mn
Mw/Mn
Polymer
Mn
Mw/Mn
Molecular weightc (cal.)
b
Tg /ꢁC
b
Tg /ꢁC
P(ATAGP)
P(ABTAGP)
P(AHTAGP)
15 345
18 450
10 959
1.09
1.13
1.07
86
97
71
GP1
GP2
GP3
10 829
9856
4266
1.15
1.16
1.03
9880
13 582
7917
1
4
7
a
In DMF at room temperature. b From DSC curve on second heating. c Molecular weight ¼ Mw(GPC value of acetylated glycopolymer) ꢀ {[Mw (GPC)
ꢀ 345] O monomer molecular weight} ꢂ 168}, where 345 and 168 are the macromolecular terminal ends molecular weight and total pendant
acetates molecular weights.
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with Mw/Mn: ꢃ1.15 (Table 1) were obtained by gel permeation values of the polymers were in the range of 1–7 ꢁC, whereas
ꢁ
chromatography studies in DMF with polystyrene standards. The acetylated polymers showed 71–97 C as shown in Table 1.
deacetylated glycopolymers, GP1, GP2 and GP3, which were
insoluble in water prior to deacetylation, were now soluble in
water. Signal integrations of the glucose moieties in pendant
3.2 Cell adhesion
acetylated polymers, 2,3,4,6-tetra-O-acetyl, appeared at d 1.8–2.3 Fig. 2 shows cell adhesion on tissue culture plastic (TCP)
ppm in 1H NMR spectrum, as shown in Fig. 1a. Aer deacetylation, surfaces at various concentrations (100 nM, 1 mM, 10 mM and
the acetyl peaks of the polymers disappeared and new signals 100 mM) of GP1, GP2 and GP3 in the cell culture media. To carry
corresponding to the OH groups of the glucose moieties63 emerged out quantitative analysis of adhesion % of osteoblast cells, we
at d 3.8–3.92 ppm (Fig. 1b).
used colorimetric cell-surface adhesion assay in which cells
The FT-IR analyses of poly(ATAGP), poly(ABTAGP) and poly- were incubated with MTT aer 4 h of cell adhesion on surface.
(AHTAGP) exhibited characteristic strong absorptions for C]O Non-adhered cells were washed off by removing the culture
stretching centred at about 1756 cmꢀ1 while the C]O stretch- media. Dimethylsulfoxide was added to the surface adhered
ing peaks in GP1, GP2 and GP3 shied to 1735 cmꢀ1 with lower cells, and then the absorbance was measured at 595 nm as
intensities (ESI, Fig. S7 and S8†). The OH signals in GP1, GP2 described previously.51 This method is well accepted for
and GP3 showed characteristic strong absorption centred at measuring the number of adherent cells.64
3425 cmꢀ1. Aer deacetylation, the glass transition temperature
The use of GP1 at various concentrations did not exhibit any
signicant change on osteoblast cell adhesion. However, the %
Fig. 1 1H-NMR spectra of (a) poly(AHTAGP) in CDCl3 and (b) GP3 in D2O.
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osteoblast cell adhesion up to 300 mM of covalently bonded
glucose moiety and a further increase in concentration showed
a decrease in osteoblast cell adhesion. Whereas, GP3, which has
the longest pendant alkyl chain length of glucose moiety in
glycopolymer, showed relatively better osteoblast cell adhesion
at similar concentrations of covalently bonded glucose moiety
of GP1 and GP2. To further validate the above results, free
glucose (FG) at various concentrations was added to the culture
media, and there was no signicant difference on cell adhesion
observed with increasing FG concentrations (ESI, Fig. S11†).
3.3 Immunouoresence staining
Fig. 2 Osteoblast cell adhesion after 4 h at (a) without the use of
glycopolymers, (b) 100 nM, (c) 1 mM, (d) 10 mM and (e) 100 mM of
glycopolymers.
It is well established that cell spreading and movement occur,
although the process of the binding of cell surface integrin
receptors to extracellular matrix adhesion molecules. The
complex at the focal adhesions consists of several proteins, such
as vinculin, talin, a-actin and paxillin, at the intracellular face of
the plasma membrane. Thus, for a better understanding on cell
adhesion and cytoskeletal organization, integrin a5 and talin
co-stained microscopic images of osteoblast cells on plastic
tissue culture surfaces of all three glycopolymers at various
concentrations were obtained. Talin and integrin a5 immu-
nostaining were performed at 1 mM of GP1, GP2 and GP3 and
cell cytoskeletal organization was compared with control, which
was without glycopolymer on plastic tissue culture surfaces, as
shown in Fig. 4. From the observations, it was clearly indicative
that cell spreading was observed in the presence of different
glycopolymers, and the relative number of the focal contact
points decreased with an increase in pendant spacer length of
glucose moiety in glycopolymers from GP1 to GP3. Merged
immunostained images clearly showed that talin co-localized
with integrin a5. The integrin a5 staining images showed a
functional role of the cytoplasmic domain of the alpha subunit
of the integrin, which played an important role in determining
cell surface interaction.65 Optical images of the cells at 1 mM of
GP1, GP2 and GP3 were obtained and are shown in Fig. 5. A
decrease in the number of cells was observed with increasing
glycopolymers concentrations (ESI, Fig. S12†). To investigate
cell motility and cytoskeletal organization at various concen-
trations of GP1, GP2 and GP3, integrin a5 and talin immunos-
taining were performed and the results are shown in ESI,
Fig. S13–15†, respectively. A signicant difference was observed
at various concentrations of glycopolymer with increasing
pendant spacer length of glucose moiety in glycopolymers. GP2
of cell adhesion was a little less than the cell adhesion achieved
on TCP without the use of GP1. The use of GP3 at 100 mM
showed about 6% increase in cell adhesion compared to GP2.
Fig. 3 shows cell adhesion on TCP surfaces at higher concen-
trations (10 mM to 2000 mM) of GP1, GP2 and GP3 and these
results were compared to the one without glycopolymers. It has
been shown that the use of 10 and 100 mM of GP1 and GP2 did
not cause any signicant change on osteoblast cells adhesion.
However, a further increase in the concentration (1000 and 2000
mM) of GP1 and GP2 showed a rapid decrease in osteoblast cell
adhesion. On the other hand, GP3, which has the longest
pendant alkyl chain of glucose moiety in glycopolymer we used,
showed relatively better osteoblast cell adhesion at concentra-
tions similar to those of GP1 and GP2.
These observations clearly indicate that the decrease in
pendant alkyl chain length of glucose moiety in glycopolymer
can reduce cell adhesion at higher concentrations. To have a
better understanding on the role of pendant alkyl chain length
of glucose moiety on osteoblast cells, an estimation of glucose
moiety weight percentage (wt%) in each glycopolymer was
calculated from 1H NMR (see Experimental, eqn (1) and (2)).
The increase in the pendant spacer length (0, 4 and 6 alkyl chain
length) of glycopolymer decreased the covalently bonded
glucose moiety packing density in glycopolymer (71, 56 and 48
wt%, respectively). Hence, cell adhesion was plotted against
covalently bonded glucose moiety concentration in glycopol-
ymers (ESI, Fig. S11†). The GP1 and GP2 exhibited similar
Fig. 3 Osteoblast cell adhesion after 4 h at (a) without the use of glycopolymer, (b) 10, (c) 100, (d) 1000, and (e) 2000 mM of glycopolymers.
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Fig. 4 Fluorescent microscopic images of osteoblast cells stained with integrin a5 and talin at 1 mM of GP1, GP2, GP3 and control (without the
use of glycopolymer).
(ESI, Fig. S14†) and GP3 (ESI, Fig. S15†) showed better cell
Actin staining on TCP was used for the qualitative analysis of
spreading as compared to GP1 (ESI, Fig. S13†) at similar cell spreading in terms of cytoskeleton protein expression at
concentrations.
various concentrations of GP1, GP2 and GP3, and the results are
shown in Fig. 6. These observations clearly indicate that the
concentration of glycopolymers have effects on cell morphology
and spreading. The use of low concentrations (<100 mM) of all
three glycopolymers did not result in any observable differences
in actin staining, and the results were similar to the staining
obtained on TCP without glycopolymers (ESI, Fig. S16†).
Concentrations at and above 1000 mM of GP1 and GP2 showed
reduced focal contact points, which led to a shrinkage in the cell
size. Although the obtained microscopic actin stained cell
images at higher concentrations (up to 2000 mM) of GP3
maintained a signicantly good number of focal points, no
shrinkage in the cell size was observed. Integrin a5 and talin
staining on TCP of glycopolymers at higher concentrations were
obtained and a signicant reduction was observed in cell
spreading. Similar results were observed for integrin a5 and
talin for the use of glycopolymers at higher concentrations and
a representative result of immunostained osteroblast cell at
1000 mM of GP3 was given in ESI, Fig. S17.†
Fig. 5 Brightfield microscopic images of osteoblast cells (a) control
(without the use of glycopolymer) and at 1 mM of (b) GP1, (c) GP2 and
(d) GP3.
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Fig. 6 Fluorescent microscopy images of actin stained osteoblast cells for assessing the expression of cytoskeleton protein at (a) 10, (b) 100, (c)
1000, and (d) 2000 mM of glycopolymers. Insets are magnified images.
higher cell viability (ꢃ95%) for concentrations up to 300 mM as
3.4 Osteoblast cell viability and proliferation
compared to the cell viability obtained for GP1 and GP3. A
There was no signicant difference in cell viability observed at
the lower concentrations of covalently bonded glucose moiety
of GP1 and GP3 as compared to the cell viability (ꢃ70%)
further increase (>300 mM) in the concentration of covalently
bonded glucose moiety of GP1 and GP2 showed a steep
decrease in cell viability. However, the use of the GP3 at
without the use of the glycopolymers (Fig. 7). GP2 showed
Fig. 7 Osteoblast cell viability after 24 h at various concentrations of covalently bonded glucose moiety in glycopolymers. Cells viability values
plotted in (a) logarithmic scale which highlights relatively low concentrations and (b) normal scan which highlights response at higher
concentrations. In logarithmic scale the values at 0.0001 represent cell viability without addition of the glycopolymers.
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concentrations above 300 mM showed a relatively slow decrease polymeric scaffolds at in vitro and in vivo conditions.66–70 Hence,
in cell viability. The osteoblast cells were able to maintain their we expect to use this macromolecular architecture design for
normal viability at higher concentrations (up to 2000 mM) for developing glycopolymers coatings on metal implants in the
the use of covalently bonded glucose moiety of GP3, the future.
glucose moiety of which has the longest pendant alkyl chain
length compared to the GP1 and GP2.
4. Conclusions
Osteoblast cell proliferation was estimated at 24, 48 and 72 h
aer plating the cells at various concentrations (0–3000 mM) of In conclusion, glycopolymers were synthesized by introducing
covalently bonded glucose moiety of glycopolymers, and the different alkyl chain spacer lengths between glucose moiety and
results are shown in Fig. 8. At lower concentrations (0 to 300 polymer backbone and dodecyl alkyl chain at one terminal end
mM), the cell proliferation did not exhibit any signicant of the macromolecular chain via the RAFT process. The effects
difference with variations in alkyl chain length of glucose of pendant spacer lengths of functional moieties in glycopoly-
moiety in glycopolymers. Cell proliferation was reduced rapidly mers on osteoblast cell adhesion, viability and proliferation
with a further increase in the concentration of covalently were investigated. It was demonstrated that the cytotoxicity
bonded glucose moiety of GP1 and GP2 and most of the cells limits of osteoblast cells depend on the pendant spacer length
lost their viability at the concentration of 1000 mM. Interest- of glucose moieties in glycopolymer. Among the synthesized
ingly, we did not observe any adverse effects on osteoblast cell glycopolymers, the glycopolymer with six pendant spacer
viability at 2000 mM of covalently bonded glucose moiety of GP3. lengths of glucose moiety exhibited better osteoblast cell
As per our knowledge and available literature, this is the rst adhesion and proliferation at relatively higher concentrations.
report in which the effects of covalently bonded spacer length of These glycopolymers may have a potential role in orthopaedic
glucose in glycopolymer on osteoblast cells have been investi- applications as a biologically active coating material on
gated. However, previous studies have shown the improved cell implants.
response on metal surfaces having micro/nano-topography and
Acknowledgements
AVSS and MD thank the Council for Scientic and Industrial
Research, India, for nancial support in network projects CSC
0134 and BSC-0112, respectively. We thank CSIR-CCMB for the
microscopy facility. MT, TR and GK thank CSIR, UGC and CSIR
network project for providing fellowships, respectively. MD is
thankful to Mr N R Chakravarthi, Microscopy Facility of CCMB,
for his help in obtaining brighteld images of cells.
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