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ISSN 1070-4272, Russian Journal of Applied Chemistry, 2012, Vol. 85, No. 11, pp. 1771−1779. © Pleiades Publishing, Ltd., 2012. Original Russian Text © T.L. Yurkshtovich, N.V. Golub, N.K. Yurkshtovich, V.A. Alinovskaya, R.I. Kosterova, 2012, published in Zhurnal Prikladnoi Khimii, 2012, Vol. 85, No. 11, pp. 1867−1875.

MACROMOLECULAR COMPOUNDS AND POLYMERIC MATERIALS

Synthesis, Structure, and Physicochemical Properties of Gel-Forming Dextran Phosphates T. L. Yurkshtovich, N. V. Golub, N. K. Yurkshtovich, V. A. Alinovskaya, and R. I. Kosterova Research Institute of Physical Chemical Problems of the Belarusian State, Minsk, Belarus e-mail: [email protected] Received June 26, 2012

Abstract—Dextran phosphates with the degree of substitution of 0.29–1.09 with phosphoric acid groups and of 0.14–0.83 with carbamate groups were prepared in the orthophosphoric acid–urea system. The effect of the component ratio in the esterifying mixture, temperature, and pressure in the reaction zone on the structure and physicochemical properties of dextran phosphate hydrogels and on the gel fraction yield was examined. DOI: 10.1134/S1070427212100237

Preparation of hydrogels based on synthetic and natural polymers for use in medicine is a promising and actively developing direction of macromolecular chemistry. Hydrogels are used in formulations for producing soft contact lenses, drug forms with prolonged release of the active substance, transdermal therapeutic systems, sorbents, materials for fabricating endoprostheses, etc. In the past decades, dextran occupied a particular place among natural polymers for preparing hydrogels used in medicine. Dextran is a water-soluble polysaccharide. It consists of molecules containing D-glucose residues linked mainly by the α-1,6-D-glucoside bond and, to a considerably lesser extent, by the α-1,2 and α-1,3 bonds. Growing interest in synthesis and physicochemical properties of hydrogels based on physically and chemically cross-linked dextran and its derivatives (methacrylate, hydroxyethyl methacrylate, dextran oligolactates, etc.) is due to the possibility of using them in pharmacology as carrier polymers for immobilization and controlled release of drugs, including proteins [1−4]. This paper deals with preparation of gel-forming dextran phosphates in the system orthophosphoric acid (Н3РО4)–urea [СO(NH2)2] and with a study of their structure and physicochemical properties. The advantages of using phosphoric acid polysaccharide esters for preparing hydrogels for medicine are that

they are nontoxic, meet the biocompatibility and resolution criteria, and exhibit cation-exchange and complexing properties [5−8]. Numerous papers deal with the synthesis of polysaccharide phosphates [5−15]. One of the most widely used procedures for polysaccharide phosphorylation with pentavalent phosphorus derivatives is esterification with orthophosphoric acid in the presence of urea. Relationships of polysaccharide esterification with orthophosphoric acid have been studied mainly for starch and cellulose [7, 9−12]. Data on esterification of dextran in the orthophosphoric acid–urea system are lacking. There are data [14] on preparation of hydrogels based on dextran phosphates using another reagent, phosphorus oxychloride. However, the use in the reaction of an aggressive agent and of a toxic organic solvent (pyridine or benzene), and also degradation of the polysaccharide restrict the possibilities of practical use of this procedure for preparing hydrogels based on dextran phosphates. EXPERIMENTAL As starting materials we used dextran [molecular weight 60 000 Da, ND RB (Standard Document of Belarus Republic) 0221S-2008], orthophosphoric acid [GOST (State Standard) 6552–80, ρ420 = 1.698 g cm–3, с = 85.4%], and urea (chemically pure grade).

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YURKSHTOVICH et al.

The phosphorylation was performed as follows: 20 g of dextran was mixed with 14.8–29.8 g of urea. Then the required volume of orthophosphoric acid (2.6–8.6 ml) was added in small portions with stirring, and the mixture was kept at 100–135°С under a pressure of 0.005–0.10 MPa for 2–3 h. After cooling the mixture to room temperature, distilled water was added, and the esterification product was converted to the Na form: a NaCl solution was added (30 g per 1 l of 70% ethanol), and the solution was alkalized with NaOH to pH 11.5–12.0 at the bath ratio of 1 : 20 g ml–1 and kept at room temperature for several hours. The resulting Na form of dextran phosphate (DP) was washed with a 50% water–ethanol mixture (bath ratio 1 : 20 g ml–1) to remove the residual amounts of the esterifying mixture and NaCl. The washing completeness was checked by determining urea and Cl– ions in the wash solutions. The DP samples were dried at 50°С and residual pressure of 0.01 MPa for 5 h in an HSPT-200 vacuum oven. To perform potentiometric titration, samples of the DP Na salt were converted to the H form by treatment with 0.2 M HCl in a water–ethanol mixture (mole fraction of alcohol 0.7), followed by washing with 70% aqueous ethanol solution in a Soxhlet apparatus and drying at 50°С and residual pressure of 0.01 MPa for 5 h (HSPT200 vacuum oven). Analysis of DP samples for P and N was performed according to [16, 17], respectively. The degree of substitution (DSP) of DP with phosphoric acid groups was calculated by the formula 162.15сР DSР = —–——————– , 1000 – 43cN – 80cP

where сР is the phosphorus content and cN is the nitrogen content (mmol g–1). The degree of substitution (DSN) of PD with carbamate groups was calculated by the formula 162.15сN DSN = —–——————– . 1000 – 43cN – 80cP

The degree of swelling and gel fraction yield were determined gravimetrically [8]. The 31P NMR spectra of the samples in D2O were recorded in the quantitative mode with an Avance-500 spectrometer (Bruker, Germany) without proton decoupling (operating frequency 162 MHz, external reference

85% H3PO4, accumulation number 64). The dynamic viscosity μ (Pa s) of 2% DP solutions was determined at 25 ± 0.2°С with a Bookfield LVDV-II+Pro viscometer (USA) using an SC4-13R(P) temperature-controlled adapter for small samples, at a shear rate of 3.3 s–1. The weight-average molecular weight Mw of watersoluble samples of the initial and modified dextrans was determined by gel permeation chromatography with an Agilent 1200 chromatograph equipped with a refractometric detector. Separation was performed with a PLAquagel-OH 40 column. As mobile phase we used phosphate buffer solution (pH 7.0) with the addition of 0.94% NaCl and 0.02% NaN3. For the calibration, we used standard samples of dextrans (Sigma) with molecular weights of 1, 5, 12, 25, 50, 80, 150, and 270 kDa. The weight-average molecular weight was calculated with Agilent ChemStation software. The IR spectra of the samples pelletized with KBr were recorded with a Thermo Nicolet FT-IR Nexus spectrophotometer. Potentiometric titration of DP samples was performed with 0.1 N NaOH solution at a solution ionic strength of 0.05 [18]. The morphology of structural elements constituting particles of the initial and modified dextrans was examined with a LEO 1420 electron microscope (Germany). Depending on the conditions, esterification of dextran with orthophosphoric acid in a urea melt yields dextran phosphate esters that either are water-soluble or form microgels in water. The influence of the synthesis conditions on the functional composition and yield of the gel fraction of dextran phosphates is illustrated by Fig. 1 and Table 1. The phosphorus content of the polysaccharide monotonically increases with an increase in the concentration of orthophosphoric acid in the phosphorylating mixture and reaches 9.7% (i.e., 0.75 P atom per glucopyranose unit, GPU) at the GPU : Н3РО4 ratio of 1 : 1.0. At a fixed content of orthophosphoric acid, an increase in the CO(NH2)2 : GPU molar ratio from 2 : 1 to 4 : 1 does not affect the amount of the bound phosphorus but favors an increase in the yield of reaction products in the form of gel fraction. It is known [9] that urea and its complex with phosphoric acid decompose at the heat treatment temperature with the release of ammonia, which acts as a neutralizing agent. Hence, one of the factors affecting the yield of

RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 85 No. 11 2012

SYNTHESIS, STRUCTURE, AND PHYSICOCHEMICAL PROPERTIES

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Table 1. Conditions and results of dextran phosphorylation in the orthophosphoric acid–urea systema

a

Phase state

Т, °С

Content of phosphoric acid groups cP, mmol g–1

DSР

DSN

135

1.5

0.30

0.21

125

2.1

0.49

0.25

1.0 : 0.6 : 4.0

125

2.2

0.48

0.42

10.2

1.0 : 0.8 : 4.0

125

2.0

0.47

0.19

4.5

1.0 : 0.8 : 4.0

125

2.2

0.50

0.21

4.5

1.0 : 0.8 : 4.0

100

2.0

0.46

0.27

4.5

1.0 : 0.8 : 4.0

115

2.1

0.49

0.28

4.5

1.0 : 0.8 : 4.0

135

2.5

0.54

0.30

10.2

1.0 : 1.0 : 4.0

125

3.2

0.72

0.18

4.5

1.0 : 1.0 : 4.0

0.005–0.01

125

3.6

1.09

0.57

4.5

1.0 : 1.0 : 4.0

0.03–0.05

125

3.0

0.67

4.5

1.0 : 1.0 : 4.0

0.10

125

3.1

0.69

Dextran moisture content, %

GPU : H3PO4 : CO(NH2)2 molar ratio

10.2

1.0 : 0.3 : 4.0

10.2

1.0 : 0.6 : 4.0

4.5

Р, MPA

0.005–0.01

0.10

sol gel fraction fraction 100

Degree of swelling Q, g g–1

–

–

29.8

70.2

160.9

2.5

97.5

180.0

100 55.4

– 44.6

170.0

100

–

–

100

–

–

76.5

95.7

–

–

5.4

94.6

41.9

0.25

15.1

84.9

39.7

0.22

33.7

66.3

188.2

23.5 100

Under the synthesis conditions used, the yield of reaction products in the form of gel and sol fractions is relatively high and practically the same: 95–98% of the theoretically possible yield, depending on the esterification conditions. Esterification time 3 h.

the gel fraction of dextran phosphates in the orthophosphoric acid–urea system can be an increase in pH of the esterifying mixture. This assumption is confirmed by the results of studying how pH of esterifying solutions of sodium dihydrogen and disodium hydrogen phosphates affects the structure of starch phosphates [15]. These results show that, in weakly acidic aqueous media, the major products are monoesters, whereas alkaline medium favors formation of starch diesters and of a cross-linked structure. At the same time, in esterification of dextran in the orthophosphoric acid–urea system, the CO(NH2)2 : Н3РО4 molar ratio should be above 3 : 1, because at a lower urea content the polysaccharide intensely degrades with the accumulation of colored reaction products. Table 1 shows how the composition and phase state of DP is influenced by the heat treatment temperature and of the pressure in the reaction zone at various molar ratios of the mixture components. As can be seen, under the examined reaction conditions the strongest effect on the increase in the phosphorus content is exerted by

a decrease in the pressure, an increase in the heat treatment temperature exerts a weaker effect, and the moisture content of the initial polysaccharide does not noticeably affect the phosphorus content. P, %

Fig. 1. Bound phosphorus content as a function of the H3PO4 : GPU molar ratio N. Phosphorylation conditions: 125°С, 0.10 MPa, 3 h. GPU : CO(NH2)2 : (1) 1 : 2, (2) 1 : 3, and (3) 1 : 4.


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YURKSHTOVICH et al. (a)

δ, ppm (b)

δ, ppm Fig. 2. 31Р NMR spectra of the phosphorylating mixture, H3PO4 : CO(NH2)2 molar ratio (a) 0.6 : 4.0 and (b) 1.0 : 4.0, after heat treatment at 125°С and a pressure of 0.10 MPa. (δ) Chemical shift; the same for Fig. 4.

At 135°С, the samples become dark already after 1 h of heat treatment, which suggests intense degradation of the polysaccharide. The optimal temperature of the dextran esterification with the minimal yield of the gel fraction

of the reaction products is within 120–130°С. The yield of the DP gel fraction depends on the moisture content of the initial polysaccharide and on the rate of the water removal from the phosphorylation zone, which can be increased by decreasing pressure and increasing reaction temperature (Table 1). Presumably, the water concentration in the esterifying system affects the structure of the polysaccharide phosphates obtained (ratio of mono- and diesters) and, as a consequence, the number of intermolecular cross-links and the weight-average molecular weight Mw of the macromolecules. Table 2 shows how the esterifying mixture composition and heat treatment temperature affect Mw of watersoluble dextran phosphates. As can be seen, first, under the synthesis conditions used, Мw of water-soluble DPs exceeds Мw of the initial polysaccharides, i.e., Мw of DP samples is determined to a greater extent by cross-linking of macromolecules, rather than by their thermal degradation. Second, the dextran esterification temperature significantly affects Мw of the reaction products and the dynamic viscosity of their 2% solutions. For example, Мw of DPs with the same content of phosphoric acid groups but different synthesis temperatures (125 and 100°С) differs by a factor of approximately 10. The observed effect can be attributed to an increase in the content of diesters in DP. To examine the phosphorylation mechanism and determine the chemical structures of DPs, we recorded the 31Р NMR spectra of mixtures of orthophosphoric acid and urea with different component ratios after heat treatment at different pressures. As seen from Fig. 2, in the 31Р NMR spectra of the esterifying mixture, the phosphorus atoms give

Table 2. Influence of reaction temperature on the weight-average molecular weight Mw and dynamic viscosity of 2.0% dextran phosphate solutions. Esterification conditions: pressure 0.10 MPa, time 3 h GPU : H3PO4 : CO(NH2)2 molar ratio

T, °С

1.0 : 0.4 : 4.0 1.0 : 0.6 : 4.0 1.0 : 1.0 : 4.0

125

1.0 : 0.6 : 2.0 1.0 : 0.8 : 4.0

115

1.0 : 0.8 : 4.0 1.0 : 0.8 : 3.0 1.0 : 0.6 : 3.0

100

DSР

DSN

Mw × 10–5, g mol–1

Viscosity μ × 103, Pa s

0.29

0.15

4.38

9.0

0.45

0.19

8.55

130.8

0.72

0.18

8.21

141.6

0.35

0.14

2.18

5.4

0.49

0.28

1.34

5.6

0.46

0.27

0.89

5.0

0.42

0.21

0.83

4.6

0.38

0.16

0.87

4.9



four groups of signals: singlets at δ = 3.2 and –6.1 ppm and multiplets in the ranges from –5.0 to –5.4 and from –20.1 to –22.9 ppm. Comparison of the spectrum obtained with the major signals of the 31Р NMR spectra [19, 20] of disodium hydrogen phosphate (singlet at δ = 3.1 ppm), sodium pyrophosphate (singlet at δ = –5.6 ppm), and sodium tripolyphosphate (doublet at δ = –4.9 and triplet at δ = –19.1 ppm) suggests the presence of pyrophosphate and triphosphate anions, along with a relatively small amount of the unchanged orthophosphate. Certain shift of the 31Р signals of the esterifying mixture relative to the signals of the reference 31Р NMR spectra of sodium pyrophosphate (Na4P2O7) and triphosphate (Na5P3O10) is probably due to the presence of urea, which forms donor–acceptor bonds with phosphates and thus affects the electron density at the magnetic nuclei of P [21]. Furthermore, in contrast to the 31Р NMR spectra of sodium disphosphate and triphosphate, in the 31Р NMR spectrum of the phosphorylating mixture the phosphorus signals are multiplets consisting not only of well-resolved doublet (from –5.3 to −5.4 ppm) and triplet (from –20.1 to –20.4 ppm), but also of a series of ill-defined components that are difficult to interpret in detail, i.e., the esterifying mixture may contain not only the above salts, but also salts of more condensed polyphosphoric acids (e.g., tetraphosphoric acid) and ammonium salts of polyphosphoric acids of different basicities.

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With an increase in the orthophosphoric acid content in the mixture, and also on heating under reduced pressure (0.005–0.03 MPa), the ratio of the integral intensities of the signals in the ranges from –5.3 to –5.4 and from –20.1 to –22.9 ppm to that of the signal at –6.1 ppm increases (Fig. 2b), suggesting formation of ammonium polyphosphates [22] 2H3PO4 + CO(NH2)2 = 2/n (NH4PO3)n + CO2 + H2O,

whose yield and molecular weight depend on the orthophosphoric acid concentration and on the moisture removal rate. Based on the 31Р NMR data and on the mechanism of esterification of low-molecular-weight alcohols with polyphosphoric acids, suggested in the literature, the following mechanism can be suggested for dextran phosphorylation in the orthophosphoric acid–urea system [23] Scheme 1. In accordance with the suggested scheme, high concentrations of pyrophosphoric and triphosphoric acid salts in the esterifying mixture favor formation of DP in the form of a mixture of phosphoric acid monoesters I and diesters V and of diphosphates II. With tetraphosphoric acid, the formation of monoesters of phosphoric I, pyrophosphoric II, and triphosphoric III acids and of diesters of phosphoric V and pyrophosphoric IV acids is

T, %

ν, cm–1 Fig. 3. IR spectra of (1) initial and (2) phosphorylated dextrans (DSР = 1.09, DSN = 0.57). (T) Transmittance and (ν) wavenumber. RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 85 No. 11 2012

1776

YURKSHTOVICH et al. Scheme 1.

O

O R

OH + XO

P

P

O

OX

O R

O

O

P

OX + HO

OX

O

P

OX

OX

O

O

P

OX

O

OX

O

P

O

OX + XO

OX + R

P

OX

O

P

O O

OX

OX

R

O

ROH O

O

P

OX + XO

OX

P

OX

OX

ROH O

P

R

OX

O

P

O O

R + R

O

OX

OX

O OX + XO

P OX

P

OX ,

OX

ROH is the polysaccharide, and X is H or NH4. Scheme 2.

O R

O

P

O OH

R

O

OH I

O

P

O

OH II O R

O

P

O

P

OH

R

O

OH

OH IV

P

O

OH

O O

P

O

O P OH III

O

P

OH

OH

O O

OH

possible [20] (Scheme 2). The structure of the phosphates obtained was determined by 31Р NMR and IR spectroscopy and by potentiometric titration. New bands, absent in the IR spectrum of the initial dextran, appear in the product of dextran esterification with orthophosphoric acid in molten urea (Fig. 3): a shoulder at 840 cm–1 (Р–О vibrations), a shoulder at 955 cm–1, a band near 1050 cm–1 (Р–ОН stretching vibrations), and a shoulder at 1210 cm–1 (Р=О stretching vibrations), indicative of incorporation of phosphoric acid groups [8–10]. The IR spectra of all the samples also contain a strong band near 1710 cm–1, originating from asymmetric

R

R

O

P

O

R

OH V

stretching vibrations of C=O bonds of carbamate groups. Figure 4 shows the 31Р NMR spectrum of water-soluble dextran phosphate. As can be seen, the 31Р spectrum becomes considerably less resolved, with signal broadening, which is mainly due to hindered segmental mobility of the polymer chains of dextran phosphate as a result of intra- and intermolecular hydrogen bonding [21]. In the 31Р NMR spectrum of dextran phosphate, there is a doublet at 2.2–3.5 ppm, which can be assigned on the basis of published data to monoesters [19]. The presence of two broad signals belonging to the phosphate monoester suggests that two hydroxy groups of dextran GPU can undergo esterification. Also, all the dextran phosphate



1777

δ, ppm Fig. 4. 31Р NMR spectra of DP with DSP = 0.67, DSN = 0.25.

samples contain small amounts of diesters, as indicated by the presence of a weak signal at δ = 0.7 ppm. The results of a 31Р NMR study of the chemical structure of water-soluble dextran phosphates are confirmed by the results of potentiometric titration. Table 3 shows that DPs obtained by esterification in a mixture of orthophosphoric acid and urea are mixtures of monoand dibasic phosphates and diphosphates whose percent content depends on the synthesis conditions. Monoesters (structure I, dibasic phosphoric acid groups) are major components of the water-soluble samples. This is indicated by the 31Р NMR data and by the fact that the total exchange capacity (TEC) of the watersoluble phosphates is two times higher than the exchange capacity of the cation exchange for the first equivalence point (EC1). However, with an increase in the orthophosphoric acid content and in the heat treatment temperature and with a decrease in the moisture content of the initial dextran and in the pressure in the reaction zone, i.e., when the reaction conditions become more favorable for the formation of triphosphoric acid, the content of monobasic phosphates increases (structure III). For example, one

VNaOH, ml Fig. 5. Potentiometric titration curves of dextran phosphate samples with different content of phosphoric acid groups. (VNaOH) Titrant volume. сР, mmol g–1: (1) 0.5, (2) 2.1, and (3) 3.6.

equivalence point in the potentiometric titration curve of DP hydrogels suggests the presence of mainly diesters (Fig. 5). The presence of diphosphates (structure II) in some samples is confirmed by data in Table 3 (sample nos. 5 and 6), according to which the phosphorus content (determined colorimetrically) considerably exceeds the value calculated from the potentiometric titration data assuming exclusive formation of diesters (monobasic phosphoric acid groups). The structure of phosphorylated dextran granules was examined by electron microscopy (Fig. 6). As can be seen, treatment of dextran granules in the orthophosphoric acid–urea system leads to their full disintegration into fragments differing in the length and width. The water-soluble samples and thinly cross-linked hydrogels with high degree of swelling (180.1 g g–1) have loose (micropore size varies within 1–2 μm) porous structure

Table 3. Results of potentiometric titration of dextran phosphate Potentiometric titration data Sample GPU : H3PO4 : no. CO(NH2)2 molar ratio

Р, MPa

сР, mmol g–1

DSР

DSN

EC1

TEC

mg-equiv g–1

рK1

рK2

Q, g g–1

1

1.0 : 0.4 : 4.0

0.10

1.5

0.29

0.15

1.5

3.0

2.7

6.5

Water-soluble

2

1.0 : 0.6 : 4.0

0.10

2.3

0.45

0.19

2.3

4.6

2.8

6.6

The same

3

1.0 : 0.8 : 4.0

0.10

3.1

0.69

0.22

3.0

6.0

2.9

6.8

188.2

4

1.0 : 0.6 : 4.0

0.005–0.010

2.2

0.48

0.42

2.2

4.0

2.8

6.5

180.1

5

1.0 : 0.8 : 4.0

0.005–0.010

3.0

0.78

0.83

2.1

2.1

3.1

–

78.8

6

1.0 : 1.0 : 4.0

0.005–0.010

3.6

1.09

0.57

2.3

2.3

3.0

–

41.9


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YURKSHTOVICH et al.

(1a)

(1b)

(2a)

(2b)

(3a)

(3b)

Fig. 6. Electron micrographs of (1a, 1b) initial dextran and (2a, 2b, 3a, 3b) dextran phosphates with different degrees of substitution with phosphoric acid and carbamate groups. (2a, 2b) DSР = 0.48, DSN = 0.42; (3a, 3b) DSР = 0.69, DSN = 0.22.

(Fig. 6, images 2a, 2b). With a decrease in the degree of swelling of hydrogels based on dextran phosphates, the structure gradually becomes denser, and the micropores disappear (Fig. 6, images 3a, 3b). CONCLUSIONS (1) Dextran phosphates with the degree of substitution of 0.28–1.09 with phosphoric acid groups and 0.14–0.83 with carbamate groups were prepared in the form of viscous solutions and hydrogels with the degree of swelling in the range 39.7–188.2 g g–1 and high yield of the gel fraction (up to 98%). (2) The yield of dextran phosphate in the form of gel fraction depends on the moisture content of the initial polysaccharide and on the reaction conditions: reactant ratio, temperature, and pressure.

(3) A study by 31P NMR spectroscopy and potentiometric titration showed that dextran phosphate is a mixture of dibasic and monobasic phosphates and diphosphates whose percent content depends on the synthesis conditions. (4) Hydrogels of dextran phosphates are cation exchangers with the total exchange capacity in the range 2.1–6.0 mg-equiv g–1. They can be used in medicine as polymeric carriers for drugs. REFERENCES 1. Tomme, S.R. van and Hennink, W.E., Expert Rev. Med. Devices, 2007, vol. 4, pp. 147–164. 2. Dijk-Wolthuis, W.N.F. van, Franssen, O., Talsma, H., et al., Macromolecules, 1995, vol. 28, pp. 6317–6322. 3. Maia, J., Ferreira, L., Carvalho, R., et al., Polymer, 2005,



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