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alcohol) copolymers
Alessandro F. Martins
a,
, Pedro V. A. Bueno
a
, Heveline D. M. Follmann
a,b
, Samara R. Nocchi
b
,
Celso V. Nakamura
b
, Adley F. Rubira
a
, Edvani C. Muniz
a
a
Grupo de Materiais Polimricos e Compsitos, GMPC, Departamento de Qumica, DBS Bloco B-08, Universidade Estadual de Maring UEM, Av. Colombo 5790,
CEP 87020-900 Maring, Paran, Brazil
b
Laboratrio de Inovao Tecnolgica no Desenvolvimento de Frmacos e Cosmticos, DBS Bloco B-08, Universidade Estadual de Maring UEM, Av. Colombo 5790,
CEP 87020-900 Maring, Paran, Brazil
a r t i c l e i n f o
Article history:
Received 20 August 2012
Received in revised form 13 November 2012
Accepted 15 November 2012
Available online xxxx
Keywords:
Chitosan
N-trimethyl chitosan
Poly(vinyl alcohol)
Copolymers
Graft
a b s t r a c t
N-trimethyl chitosan-graft-poly(vinyl alcohol) (TMC-g-PVA) copolymers were prepared. The grafting
reactions were conducted in water changing the feed ratios of poly(vinyl alcohol)/6-O-succinate-N-tri-
methyl chitosan (PVA/STMC). The structure of TMC-g-PVA copolymers was characterized through
1
H
NMR spectroscopy, thermogravimetric analysis (TGA/DTG), wide-angle X-ray scattering (WAXS) and
scanning electron microscopy (SEM). The quaternization degree (DQ) and substitution degree (DS) of
N-trimethyl chitosan (TMC) and 6-O-succinate-N-trimethyl chitosan (STMC) were determined by
1
H
NMR, being the spectroscopy 14.0 and 5.5 mol-% found, respectively. The viability of HCT-116 cancerous
cells was investigated at different concentrations. The effect of PVA/STMC ratios on the cytotoxicity of the
TMC-g-PVA copolymers was examined and the CC
50
values determined for every case.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Chitosan (CHT) is a non-toxic and biocompatible cationic poly-
saccharide produced by partial deacetylation of chitin.
1,2
The phar-
maceutical and medical areas are the main strands that employ the
excellent properties of CHT for production of biomaterials.
1,2
This
polymer possesses ability to facilitate paracellular transport of
drugs across mucosal barriers. Consequently, hydrogels of CHT
are widely used as devices for controlled releasing of drugs.
3
How-
ever, the CHT is insoluble at physiological pH but active as an
absorption enhancer as soluble in acidic environments.
46
The
mechanism of action of CHT was attributed to combination of bio-
adhesion and transient opening of tight junctions between epithe-
lial cells. Nevertheless, this absorption enhancing ability was
evident only under acidic conditions, where most of the amino
groups were protonated.
7
N-trimethyl chitosan (TMC) is a permanently quaternized CHT-
derivative, which has been proven to be highly soluble over a wide
pH range.
810
Additionally, studies conrmed that the TMC has
good property of mucoadhesion and its ability to transport pro-
teins and peptides is elevated, as compared to CHT.
7
These proper-
ties render TMC more attractive and interesting for the preparation
of biomaterials. At present, it is preferred to replace the CHT by
TMC in the hydrogel matrix (lms, nanoparticles, microparticles
and beads) because TMC has some properties that surpass those
found in CHT.
1114
On the other hand, according to Jintapattanakit
et al.,
6
the structural modication on the amino groups leads to
considerable decreases in biocompatibility of TMC. To overcome
this problem, TMC/polyethylene glycol copolymers (TMC-g-PEG)
15,16
and TMC/poly(N-isopropylacrylamide) (TMC-g-PNIPAAm)
17
were
synthesized in an attempt to increase the biocompatibility of TMC.
However, the grafting of polyethylene glycol (PEG) and poly(N-
isopropylacrylamide) (PNIPAAm) in TMC chains occurred through
the amino groups remaining in TMC structure.
1517
This fact allow
for decreasing the biocompatibility of these copolymers, since this
property is related to existence of unmodied NH
2
groups in TMC
molecules.
6
Moreover, the TMCemployedbyMaoet al.,
15,17
andGer-
mershaus et al.,
16
has also considerable proportions of O-methyl-
ated groups in its chains. Thus, due to the O-methylation process,
the solubility and biocompatibility of the copolymers obtained by
Mao et al.,
15,17
and Germershaus et al.,
16
were reduced. The O-meth-
ylation process and the high structural heterogeneity of TMC
chains
1517
may affect the physicochemical and biological proper-
ties of TMC. Verheul et al.,
7
described a simple method to synthesize
TMC avoiding O-methylation. Such authors showed that the non-O-
methylated TMC with quaternization degree (DQ < 24 mol-%)
presented low cytotoxicity but modest solubility, as compared to
O-methylated TMC (DQ < 24 mol-%).
7
0008-6215/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.11.014
) and 50 lg ml
1
gentamycin, in incubator set at
37 C, 5% CO
2
and 95% relative humidity. The cells were expanded
when monolayer reached conuence after 3 1 days. After reach-
ing 80% conuence, cells were digested by using 0.25% trypsin
(Gibco
H
2
1
6
100 2
DQ%
CH
3
H
2
1
9
100 3
DS%
9HaDQ=100
4CH
3
100 4
DS%
3HaDA=100
4CH
3
100 5
where [(CH
3
)
2
] is the integral of dimethylated amine group peak at
2.87 ppm; [(CH
3
)
3
] is the integral of trimethylated amine group
peak at 3.35 ppm; [CH
3
] is the integral of methyl groups of the acet-
amide moiety (NHCOCH
3
) peak at 2.07 ppm; [H2] is the integral of
the H2 peak at 3.10 ppm (signal related to the hydrogen atoms
bound to the C2 of TMC molecules) and [Ha] is the integral of Ha
peak at 2.42 ppm (signal related to the methylenic hydrogen atoms
bound to the carbon atoms (Ca) of STMC molecules, as represented
in Scheme 2). DQ and DA terms in the Eqs. 4 and 5 represent,
respectively, the quaternization degree and acetylation degree of
TMC molecules.
810
2.4.2. Electrical conductivity of TMC-g-PVA copolymer solutions
The electrical conductivity of TMC and TMC-g-PVA solutions
was performed using a conductivity meter fabricated by Shanghai
REX Instrument Factory (Model DDS-11A). The conductivity of the
various samples was measured at room temperature (25 C) and
also at 40 C, using solutions of 2.0 mg ml
1
in acetic acid 0.25%
(vol/vol).
2.4.3. Thermal analysis through TGA/DTG
Thermal-gravimetric analysis (TGA) was carried out on a ther-
mogravimetric analyzer (Netzsch, model STA 409 PG/4/G Luxx,
USA) at a rate of 10 C min
1
under nitrogen gas owing at
20 ml min
1
in the range of 25600 C.
2.4.4. Wide angle X-ray scattering (WAXS)
WAXS proles were recorded on a diffractometer Shimadzu
(model XRD-600, Japan) equipped with a Ni-ltered Cu-K
a
radia-
tion. The WAXS proles were collected in a scattering range (2h)
from 5 to 70, with resolution of 0.02, at a scanning speed of
2 min
1
. The analyses were performed by applying an accelerating
voltage of 40 kV and a current intensity of 30 mA.
2.4.5. Water solubility testing
Solubility of TMC and TMC-g-PVA copolymers was measured at
different pHs (range of 2.011.0) at room temperature (25 C).
Briey, the samples were suspended in a 0.25% acetic acid solution
(2.0 mg ml
1
) and the pH of each solution was adjusted to a de-
sired value using NaOH and HCl solutions (1.0 and 0.1 M). Thus,
the transmittance of the solutions at 600 nm was recorded on an
ultravioletvisible (UVVis) spectrophotometer (Femto, Brazil)
Scheme 2. Synthetic routes of TMC-g-PVA copolymers. Reagents and conditions: (step I) NMP, succinic anhydride, 100 C, 24 h; (step II) H
2
O, PVA, 70 C, 8 h.
Table 1
PVA/STMC compositions used for obtaining the copolymers
PVA/STMC ratios (wt%/wt%)
Samples (g) 1/2 2 5 11
PVA 0.2 0.4 0.5 0.55
STMC 0.4 0.2 0.1 0.05
A. F. Martins et al. / Carbohydrate Research xxx (2013) xxxxxx 3
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model 800Xi. Curve of transmittance as a function of pH was con-
structed for each polymer. The TMC-g-PVA copolymers were con-
sidered insoluble when the transmittance was less than 90%,
compared to the transmittance of distilled water.
17
2.4.6. Scanning electron microscopy (SEM)
The morphology of TMC-g-PVA copolymers was investigated
through analysis of SEM images (Shimadzu, model SS 550). The
samples were sputter-coated with a thin layer of gold for allowing
the SEM visualization. Images were taken by applying electron
beam accelerating voltage of 1015 kV.
2.4.7. Cytotoxicity assays
The cytotoxicity activities of compounds against HCT-116 cells
were determined by sulforhodamine B assays, as described by Ske-
han et al.,
23
The cells were seeded in 96-well tissue plates (TPP -
Techno Plastic Products, Trasadingen, Switzerland) at a density of
8.0 10
5
cell ml
1
in 100 ll medium for 24 h in the CO
2
incubator.
The compounds were dissolved in dimethyl sulfoxide (1.0%-wt/vol
nal concentration) and added to the medium at various concen-
trations. After incubation for 48 h, the cell monolayers were
washed with 100 ll phosphate buffered saline (PBS), xed with tri-
chloroacetic acid and stained for 30 min with 0.4% wt/vol of sulfo-
rhodamine B (SRB-Sigma Chemical Co., St. Louis, MO, USA)
dissolved in 1.0% vol/vol acetic acid aqueous solution. The excess
of dye was removed by four washes with 1.0% vol/vol acetic acid
aqueous solution, and the protein-bound dye was extracted with
10 mmol unbuffered Tris base [tris(hydroxymethyl)aminometh-
ane] for determination of optical density in a computer-interfaced,
96-well microtiter plate reader (Power Wave XS, BIO-TEK
, Winoo-
ski, VT, USA).
3. Results and discussion
3.1. Characterization of N-dimethyl chitosan (DMC), N-trimethyl
chitosan (TMC) and 6-O-succinate-N-trimethyl chitosan (STMC)
by
1
H NMR
Figure 1 shows the
1
H NMR spectra of CHT, DMC, TMC and
STMC. All the signals were attributed and related with the polysac-
charide structures drawn in Schemes 1 and 2. The
1
H NMR spec-
trum of CHT shows signals at 2.05 ppm, referring to the methyl
hydrogen atoms of the acetamide groups at 3.15 ppm (H2), 3.51
4.25 ppm (H3H6) and H1 at 4.69 ppm, respectively (Fig. 1a). All
these signals were assigned to different hydrogen atoms present
in the saccharide units of CHT (Scheme 1).
10,24,25
The dimethylation degree (DD) of DMC was determined
through Eq. 2 from the integral of H2 peak (at 3.10 ppm) and inte-
gral of peak at 2.87 ppm (Fig 1b) related to hydrogen atoms of N-
dimethylated sites [N(CH
3
)
2
]. The DD of DMC was ca 86 mol-%.
So, this datum conrms the complete N-dimethylation of NH
2
groups, since the CHT is 15 mol-% acetylated. The N-dimethylated
groups were labeled as ND sites (Scheme 1). The appearance of
new peaks at 2.87 and at 3.35 ppm conrms the obtaining of
TMC (Fig. 1c).
3,7,26,27
The TMC presented DQ of 14 mol-%. The DQ
was calculated through Eq. 3 from the integral of H2 peak (at
3.10 ppm) and the integral of peak at 3.35 ppm (Fig. 1c) related
to hydrogen atoms of N-trimethylated sites [N(CH
3
)
3
]. The N-
trimethylated groups were labeled as
+
NT sites (Schemes 1 and 2).
Figure 1d shows the
1
H NMR spectrum of STMC. Comparing the
1
H NMR spectra of TMC and STMC a new peak was observed at
2.42 ppm. This signal was attributed to the hydrogen atoms Ha
of succinate chains
28
(see Scheme 2). The DS of STMC was calcu-
lated through Eqs. 4 and 5, employing the calculated values of
DA and DQ of TMC as input data. Therefore, the DS was calculated
through relation between the integral of Ha signal, related to
hydrogen atoms of
+
NT sites at 3.35 ppm, and the integral of signal
at 2.07 ppm, due to methyl hydrogen atoms of acetamide groups
(Fig. 1d). The average value of DS found was 5.5 mol-%. The signals
of hydrogen atoms H1, H2, H3, H4, H5, and H6 also occur in the
spectra of Figures 1bd.
3.2. Graft content and electrical conductivity
The PVA/STMC ratios used for obtaining the copolymers are
shown in Table 1. The graft content of poly(vinyl alcohol) (PVA)
in N-trimethyl chitosan (TMC) chains increased with the raise of
PVA/STMC ratios employed in feed for the synthesis of TMC-g-
PVA copolymers. When the PVA/STMC ratios were xed at 1/2, 2,
5, and 11 the PVA grafted in TMC chains was 27%, 158%, 432%
and 958%, respectively (Table 2). The grafting degree (DG%) was
calculated through Eq. 1.
21,22
The strong increase of the PVA
amount grafted in STMC chains was due to the high hydrophilicity
of the polymers (STMC and PVA). The PVA chains strongly interact
with
+
NT, ND and OH groups of STMC chains through ion-dipole
forces and intermolecular interactions (dipoledipole and H-bond-
ing), resulting in polymeric aggregates (see Scheme 3).
Data collected from measurements of electrical conductivity at
25 and 40 C of TMC and TMC-g-PVA copolymer solutions are also
shown in Table 2. Conductivity analyses were processed at pH 3.0.
In this condition the ND sites of TMC and STMC chains are
in protonated form, according to the ionization reaction
ND + H
3
O
+
?
+
ND + H
2
O.
10
This event should increase electrical
conductivity of the samples. The TMC solution presented electrical
conductivity of ca 466 lS cm
1
(Table 2). At pH 3.0 the
+
ND and
+
NT sites are positively charged providing higher mobility to TMC
molecules.
On the other hand, the electrical conductivity of TMC-g-PVA
solutions decreased, as compared to TMC solution (Table 2). For
the sample of DG = 27 wt-% the electrical conductivity was ca
Figure 1.
1
H NMR spectra of CHT (a), DMC (b), TMC (c) and STMC (d).
Table 2
PVA graft content in the STMC chains and electrical conductivity of the TMC and TMC-
g-PVA copolymer solutions (2.0 mg ml
1
) at 25 C and 40 C as a function of the graft
content (n = 2)
Samples (g) (DG wt-%) lS cm
1
(25 C) lS cm
1
(40 C)
TMC 465.8 2.2 466.3 2.4
TMC-g-PVA(1/2)
27
27 150.5 2.5 149.3 1.4
TMC-g-PVA(2)
158
158 10.7 1.5 9.8 1.2
TMC-g-PVA(5)
432
432 4.7 1.1 3.9 1.2
TMC-g-PVA(11)
958
958 3.3 1.3 3.4 1.6
4 A. F. Martins et al. / Carbohydrate Research xxx (2013) xxxxxx
Please cite this article in press as: Martins, A. F.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2012.11.014
150 lS cm
1
. In contrast, the samples with DG of 158, 432 and
958 wt-% presented electrical conductivity practically insignicant
(Table 2). It is worth to mention that the increase in temperature
from 25 to 40 C did not affect seriously the electrical conductivity
of the solutions. These results reinforce the existence of intra and
intermolecular interactions among the chain segments of the poly-
mers (see Scheme 3). Such interactions were maintained invariable
even at 40 C and are responsible by formation of polymeric
aggregates.
Therefore, the high intensity of intra and intermolecular inter-
actions among PVASTMC and PVAPVA chain segments, explains
the elevated DG of the TMC-g-PVA samples. Therefore, due to low
DS, bigger amounts of PVA grafted in STMC chains were same ex-
plained by strong association of the polymers (STMC and PVA),
represented in Scheme 3. In addition, only a small proportion of
PVA molecules should react through esterication reaction
22
with
the precursor STMC (see Scheme 2). Huang and Fang
22
synthesized
copolymers based on 6-O-succinate-N-phthaloyl-chitosan and PVA
through esterication reaction. So, the high intensity of intra and
intermolecular interactions inhibited the mobility of STMC mole-
cules. These factors contributed for the elevated DG values, as
found in this study. As a result, the electrical conductivity of the
samples prepared with excess of PVA was insignicant (see
Table 2).
3.3. Characterization of TMC-g-PVA copolymers through
1
H
NMR
Figure 2 shows
1
H NMR spectra of TMC-g-PVA copolymers. The
occurrence of new and large peaks at 1.521.81 range, 2.10 and
4.03 ppm, conrms the graft of PVA in the STMC chains (Fig. 2).
These signals were attributed to the methylenic hydrogen
atoms
2830
(Ha, Hb, Hc and Hc
0
) present in the PVA structure (see
Scheme 2). The signal at 1.92 ppm refers to the hydrogen atoms
(Hd) of the acetylated residues of PVA molecules.
The increase of signal intensity of hydrogen atoms Ha, Hb, Hc,
Hc
0
and Hd were dependent on the PVA/STMC ratios. The PVA/
STMC ratios employed in the feed solutions for the synthesis of
copolymers (TMC-g-PVA) also affected the chemical shift (d) of
hydrogen atoms attributed to the ND sites (compare the Figs. 1
and 2). The peak of Ha was observed in the spectra of sample pre-
pared with excess of TMC (Fig. 2). On the other hand, when the
PVA/STMC ratios were increased to 2, 5 and 11 the Ha signal disap-
peared, while the signals intensity of Hb, Hc, Hc
0
and Hd increased
substantially. This fact occurs due to the elevated DG and low DS.
The signal of hydrogen atoms, related with the
+
NT sites (Fig. 2)
still appears at 3.35 ppm, as observed in Figure 1.
3.4. Characterization of TMC-g-PVA copolymers by TGA/DTG
analysis
Figure 3 shows the TGA curves of TMC, STMC, PVA and TMC-
g-PVA copolymers. The materials (TMC and TMC-g-PVA) were sub-
jected to the same drying process. The acquisitions of the curves
shown in Figure 3 were performed immediately after lyophiliza-
tion process of such materials, using ca 6.0 mg of each sample, ex-
cept the TGA curve of the PVA that was obtained immediately after
receiving the polymer from the supplier, SigmaAldrich.
The rst event of weight loss occurred in TMC and STMC be-
tween 25 and 125 C and comprises the region where the samples
showed a weight loss ranging from 2.2 to 14.1 wt-% (Table 3). So,
the rst stage of weight loss was attributed to evaporation of water
and volatile compounds (Fig. 3). It should be important to notice
that those samples showed different content of water. TMC pre-
sented the highest water content (14.1 wt-%), STMC an intermedi-
ate amount (11.7 wt-%), while PVA showed the smallest value
(2.2 wt-%). TMC
31
and STMC molecules present hydroxyl, ND and
Scheme 3. Physical structure of TMC-g-PVA copolymers depicting the major
interactions (intramolecular and intermolecular interactions) that occur among the
segments of polymers chains (STMCPVA and PVAPVA segments).
Figure 2.
1
H NMR spectra of TMC-g-PVA copolymers.
Figure 3. TGA curves of the PVA, TMC, STMC and TMC-g-PVA copolymers.
A. F. Martins et al. / Carbohydrate Research xxx (2013) xxxxxx 5
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+
NT groups in their structures, which are highly hydrophilic and
could strongly interact with water molecules through intermolec-
ular interactions and ion-dipole forces. On the other hand, hydro-
xyl groups on PVA provided dipoledipole and H-bond
interactions with the water molecules. Additionally, the strongest
PVAPVA interactions should be responsible for decreasing the
water content in this sample related to TMC and STMC.
The content of water in the copolymer samples ranges from 3.1
up to 6.2 wt-%. Certainly, such values are dependent on the hydro-
philicity of both polymers that constitute the TMC-g-PVA copoly-
mers. In addition, the weight loss corresponding to the
volatilization of water in the samples was also dependent on the
PVA/STMC ratio. The TMC-g-PVA(1/2)
27
sample presented 6.2 wt-
% of water (Table 3). In general, the increase of PVA/STMC ratio
provides a decrease in water content of the TMC-g-PVA copoly-
mers. The TMC-g-PVA(11)
958
sample presented only 3.1 wt-% of
adsorbed water, while TMC-g-PVA(5)
432
and TMC-g-PVA(2)
158
samples showed similar values of water content, 3.9 wt-% (Table 3).
The approximation of STMC segments and PVA chains is favored by
the excess of PVA. The excess of PVA favors strong association of
the polymer chains that occurs due to the existence of
+
NT, ND
and OH groups in STMC structure, thus the PVA molecules prefer
interacting with the precursor STMC through H-bonds, ion-dipole
and dipoledipole interactions (Scheme 3), instead being solvated
by water molecules. Therefore, this fact explains the low amount
of adsorbed water found on copolymer samples prepared using
high ratio PVA to STMC.
The degradation of TMC, STMC, PVA and TMC-g-PVA copoly-
mers occurs in second stage of weight loss and it is clearly shown
in TGA curves of Figure 3. The exact value of temperature in which
the second thermal event occurs, for a given sample, is also pre-
sented in Table 3 and it was obtained through rst derivative
(DTG) of respective TGA curve (Fig. 3). TMC and STMC showed sim-
ilar thermal stability. However, the PVA is thermally more stable as
compared to TMC and STMC. The lower thermal stability of TMC
and STMC was due to the huge amount of methyl groups bonded
to the nitrogen atoms.
9,31
The insertion of methyl groups decreased
the intensity of intra-chain interactions, especially of H-bonds
existing among TMCTMC and STMCSTMC structures.
9,31
The increase on the thermal stability of TMC-g-PVA copolymers
occurred with the increase of PVA/STMC ratio. Therefore, the great-
er PVA/STMC ratio favored a close approach of the polymer chains,
thus materials with higher thermal stability compared to raw TMC
and STMC were obtained. An increase in the thermal stability of
ca 92 C was observed, when TGA curves of the samples
TMC-g-PVA(11)
958
and pure TMC are compared (Fig. 3). The
copolymers formed using excess of PVA presented temperature
of degradation higher than the raw PVA. On the other hand,
TMC-g-PVA(1/2)
27
sample obtained with TMC excess presented
two events of degradation, more precisely at 251 and at 295 C (Ta-
ble 3). In general, increasing the ratio of PVA/STMC provided an in-
crease in thermal stability and also a decrease in the water content.
This result was attributed to strong association of the polymers
that favored the occurrence of intra and intermolecular interac-
tions among PVAPVA and STMCPVA chain segments (see
Scheme 3).
3.5. WAXS analysis
Figure 4 shows the WAXS proles of TMC, STMC, PVA and TMC-
g-PVA copolymers. Signicant differences between the WAXS pro-
les of the samples can be observed.
The WAXS prole of PVA exhibits well dened diffraction peaks
at 2h = 19.6, 22.7, 40.6 and small one at 31.7, suggesting the
occurrence of crystalline regions with different periodic distances
in this material.
32,33
The WAXS proles of TMC, STMC and TMC-
g-PVA copolymers exhibited a broad diffraction peak with low
intensity in the range (2h) from 15 to 30 that characterizes its
low crystallinity (Fig. 4). The TMC-g-PVA(1/2)
27
copolymer pre-
sents diffraction peaks of low intensity at 2h = 21.3 and 23.7 sug-
gesting the formation of crystalline regions. The PVA incorporation
in the STMC chains maintained the amorphous characteristic of the
copolymers relative to the pure TMC. However, the decreased crys-
tallinity of TMC-g-PVA copolymers compared to the pure PVA was
evident. The formation of copolymer promotes reduction in inter-
actions among PVAPVA segments and consequent destabilization
of ordered regions, commonly observed in WAXS prole of PVA
(Fig. 4). As already discussed, the strongest intra and intermolecu-
lar interactions on TMC-g-PVA copolymers should be responsible
for decrease in the water content in these samples as compared
to raw TMC (Fig. 3). Thus, on TMC-g-PVA the polymer chains are
closer enough and the strength of interactions among PVATMC
chains should be higher than those among water/PVA or water/
TMC through ion-dipole or dipoledipole interactions. Therefore,
the existence of
+
NT and ND sites in STMC chains contributed to
the low crystallinity of the copolymers, related to pure PVA
(Fig. 4). In this way, the intra and intermolecular interactions,
occurring among the polymers chains (STMC and PVA), were not
sufcient in strength, to establish crystalline domains on TMC-g-
PVA copolymers (Scheme 3). In general, copolymer samples of
Figure 4. WAXS proles of the PVA, TMC, STMC and TMC-g-PVA copolymers.
Table 3
Temperatures for the rst and second thermal events of TMC, STMC, PVA and TMC-g-
PVA copolymers, from the TGA curve (Fig. 3)
Samples Temperature (C) Weight loss (wt-%)
Range Peak (DTG)
First stage
PVA 25125 2.2
TMC 25125 14.1
STMC 25125 11.7
TMC-g-PVA(1/2)
27
25125 6.2
TMC-g-PVA(2)
158
25125 3.9
TMC-g-PVA(5)
432
25125 3.9
TMC-g-PVA(11)
958
25125 3.1
Second stage
PVA 200269 307 29.3
TMC 200312 231 22.7
STMC 200230 230 18.8
TMC-g-PVA(1/2)
27
200295 251/295 14.1/40.7
TMC-g-PVA(2)
158
200285 315 33.8
TMC-g-PVA(5)
432
200288 317 33.4
TMC-g-PVA(11)
958
200297 323 29.7
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Please cite this article in press as: Martins, A. F.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2012.11.014
TMC-g-PVA presented low crystallinity, this characteristic being
independent of PVA/STMC feed ratios used for the synthesis of
copolymers.
3.6. Water solubility of TMC-g-PVA copolymers
The solubility of TMC in acidic media is compared to CHT. But in
neutral and alkaline media it is more soluble as compared to CHT.
79
However, this property depends on quaternization degree (DQ),
molecular weight and methodology of CHT-derivative synthesis.
26
Verheul et al.,
7
established a limiting DQ to the TMC free of O-
methylated groups to be soluble in water at a given concentration.
The solubility in water (pH 7) of O-methyl free TMC (2.5 mg ml
1
)
with DQ < 24 mol-% was lower as compared to O-methylated TMC
with the same DQ. On the other hand, O-methyl free TMC with
DQ > 33 mol-% had a good solubility in water at pH 7.
7
Therefore, the increase in the DQ favors higher solubility of
O-methyl free TMC.
7,26
When the DQ is further increased, the elec-
trostatic repulsion of the positively charged groups becomes more
intense. This will lead to a decrease in intra and intermolecular
interactions, leading to unfolding of the polymer chains and to
higher solubility at pH 7. However, the drastic increase of DQ re-
duces considerably the biocompatibility of TMC.
6
Therefore, one
of the aims of our present study was to improve the solubility of
the TMC of low quaternization degree (DQ = 14 mol-%). As such,
the solubilities of the copolymers in aqueous media were evalu-
ated under different pH conditions (Fig. 5). TMC-g-PVA copolymer
solutions prepared with high PVA to STMC ratio in the feed re-
mained transparent over the entire pH range (from 2 to 11). The
PVA segments grafted on STMC chains increased the hydrophilicity
of the copolymers, thus leading to improved solubility. Therefore,
the TMC-g-PVA(1/2)
27
copolymer prepared with excess of STMC
remained soluble only up to pH 9 (Fig. 5). Also, the TMC solution
of 2.0 mg ml
1
conc. was only partially soluble at pHs higher than
6. The solubility of the TMC-g-PVA copolymers was higher than
TMC in every case. The high solubility of TMC-g-PVA copolymers
at intestinal pH conditions (pH 7.4) enhances the application of
these new materials, as for example in the eld of drug controlled
release.
3.7. Morphology evaluation through scanning electron
microscopy (SEM)
Figure 6 shows micrographs of TMC-g-PVA copolymers at dif-
ferent magnications. The morphology of the TMC-g-PVA(1/2)
27
sample (Fig. 6ac) showed greater fragility as compared to the
samples formed with high PVA/STMC ratios. In general, the excess
of PVA enhanced the formation of bers, as observed in the micro-
graphs of TMC-g-PVA(5)
432
sample (Fig. 6bd). Thus, the higher
Figure 5. Transmittance curves of TMC and TMC-g-PVA copolymer solutions
(concentration: 2.0 mg ml
1
).
Figure 6. SEM images of TMC-g-PVA(1/2)
27
(ac) and TMC-g-PVA(5)
432
(bd).
A. F. Martins et al. / Carbohydrate Research xxx (2013) xxxxxx 7
Please cite this article in press as: Martins, A. F.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2012.11.014
content of PVA favored the formation of more organized copolymer
aggregates. According to TGA/DTG data the increasing of the ratio
(PVA/STMC) provided more thermally stable materials.
3.8. Cytotoxicity
The cytotoxic effect on the viability of HCT-116 cancerous cells
was investigated by analyses of the curves presented in Figure 7.
The half cytotoxicity concentrations (CC
50
) were calculated in
every case. The cytotoxicity assays clearly display strong decrease
in cell viability at concentrations of 1000 lg ml
1
(Fig. 7). In gen-
eral, TMC-g-PVA(1/2)
27
, TMC-g-PVA(2)
158
and TMC-g-PVA(5)
432
copolymers and raw TMC showed lower cytotoxicity effect on
the HCT-116 cells in conc. below 500 lg ml
1
(Fig. 7). On the other
hand, when compared to other samples, the data from cell viability
indicated that the TMC-g-PVA(11)
958
copolymer presented high
cytotoxic effect on the HCT-116 cells in concentration above
100 lg ml
1
(Fig. 7). Nevertheless, it is worthy to mention that
TMC-g-PVA(1/2)
27
, TMC-g-PVA(2)
158
and TMC-g-PVA(5)
432
copoly-
mers offered low and similar cytotoxic effects on the HCT-116 can-
cerous cells when associated to raw TMC in concentration of
750 lg ml
1
(Fig. 7). Coincidentally, this concentration is close to
the CC
50
values for TMC, TMC-g-PVA(1/2)
27
, TMC-g-PVA(2)
158
and TMC-g-PVA(5)
432
samples. So, the CC
50
values found for differ-
ent materials, TMC, TMC-g-PVA(1/2)
27
, TMC-g-PVA(2)
158
, TMC-g-
PVA(5)
432
and TMC-g-PVA(11)
958
, were 751.3, 745.9, 753.9, 750.9
and 573.6 lg ml
1
, respectively. As expected, the PVA graft in
STMC chains increased the solubility and practically maintained
the same cytotoxic activity of TMC on the HCT-116 cancerous cells.
4. Conclusions
N-trimethyl chitosan (TMC) free of O-methylation was synthe-
sized through the N-methylation of chitosan (CHT) and character-
ized by
1
H NMR spectroscopy. TMC-g-PVA copolymers based on
TMC and poly(vinyl alcohol) (PVA) were prepared at different
PVA/6-O-succinate-N-trimethyl chitosan (PVA/STMC) ratios. The
solubility and physicochemical properties of the TMC-g-PVA
copolymers were dependent on PVA/STMC and the samples were
characterized through
1
H NMR, TGA/DTG, WAXS and SEM. The
PVA/STMC feed ratios in solutions used for preparing the TMC-
g-PVA strongly inuence some properties of the obtained copoly-
mers. From TGA/DTG curves, it was veried that the thermal
stability of TMC-g-PVA is higher as higher is the PVA/STMC ratio
used for preparing the copolymers. WAXS proles of TMC-g-PVA
showed alterations related to WAXS of pure PVA and showed that
the crystallinity of TMC-g-PVA was lower as compared to raw PVA.
Finally, as expected, the grafting of PVA in STMC chains for produc-
ing the TMC-g-PVA increased the solubility compared to TMC
(possessing DQ = 14 mol-%) in pHs higher than 6 and practically
maintained the same cytotoxic activity of TMC on the HCT-116
cancerous cells.
Acknowledgments
A.F.M. thanks CNPq for doctorate fellowship. A.F.R. and E.C.M.
thank CNPq for nancial support (Proc. 481424/2010-5).
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Figure 7. Effect of the TMC and TMC-g-PVA copolymers on the viability of HCT-116
cancerous cells at different concentrations (0.11000 lg ml
1
). Error bars represent
the standard deviation of two measurements.
8 A. F. Martins et al. / Carbohydrate Research xxx (2013) xxxxxx
Please cite this article in press as: Martins, A. F.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2012.11.014