Selective carboxypropionylation of chitosan: synthesis, characterization, blood compatibility, and degradation

Carbohydrate Research 346 (2011) 1217–1223

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Selective carboxypropionylation of chitosan: synthesis, characterization, blood compatibility, and degradation Wen-yue Xiong a, Yu Yi a, Hua-zhang Liu b,⇑, Hong Wang a, Jin-hua Liu a, Guo-qing Ying a,⇑ a

College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, Chaowang Road 18, 310014, PR China Institute of Catalysis, State Key Laboratory Breeding Base of Green Chemistry-Synthesis Technology, Zhejiang University of Technology, Hangzhou, Chaowang Road 18, 310014, PR China b

a r t i c l e

i n f o

Article history: Received 12 October 2010 Received in revised form 23 March 2011 Accepted 29 March 2011 Available online 3 April 2011 Keywords: Chitosan Water-soluble polymers Compatibility Anticoagulant Degradation

a b s t r a c t Two water-soluble chitosan (WSC) derivatives of N-succinyl-chitosan (NSCS) and N,O-succinyl-chitosan (NOSCS) with a degree of substitution (DS) that ranged form 0.28 to 0.61 were selectively synthesized by varying the molar ration of succinic anhydride and chitosan. The chemical structure and physical properties of the chitosan derivatives were characterized by FT-IR, 1H NMR, and XRD. XRD analysis showed that the derivatives were amorphous. The lysozyme enzymatic degradation results revealed that the NSCS was of higher susceptibility to lysozyme. The degradation rate and the solubility of the chitosan derivatives were strongly determined by the degree of substitution and the position of the substitution. The results of antithrombotic properties, hemolytic properties and anticoagulant properties of WSCs indicated that the blood compatibility was dramatically improved, and the carboxyl group introduced on the C-6 or C-2 hydroxyl group appeared to impact anticoagulant activity in different ways. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

1. Introduction Chitosan is the deacetylated form of one of the most abundant natural polymer, chitin, or poly(b-(1-4)GlcNAc), which is extracted from exoskeletons, arthropods, crustaceans, insects, and arachnids.1 It has widely drawn interests as a promising biomaterial because of its biodegradability, nontoxicity, antimicrobial, and certain extent of biocompatibility and so on.2,3 Biocompatibility consists of hemocompatibility and histocompatibility. However, chitosan with positive charges tends to adsorb erythrocytes and thrombocytes carrying negative charges on surfaces to form thrombus or cause hemolysis.4 Another drawback that stands in its way to application is that chitosan is insoluble in either water or most of the organic solvent. It is soluble only in diluted solutions such as formic, acetic, hydrochloric acid, and so on at pH below 6.5.5 The reactive amino and hydroxyl groups on chitosan can be chemically or enzymatically altered to improve its properties. On the purpose of improving the solubility and hemocompatibility of chitosan, a special emphasis has been focused on the chemical modifications. For example, Lee et al.6 reported preparation of N-acylated chitosan derivatives, among which N-hexanoyl chito⇑ Corresponding authors. Tel.: +86 571 88320063; fax: +86 571 88320259 (H.-z.L.); tel./fax: +86 0571 88871029 (G.-q.Y.). E-mail addresses: [email protected] (H.-z. Liu), [email protected], [email protected] (G.-q. Ying).

san derivative had the best hemocompatibility. And the N-hexanoyl chitosan derivative prepared by Shigehiro et al.7 happened to be water-soluble with DS of 0.58. Huang et al.8 have chemically modified chitosan sulfate into N-propanoyl-, N-hexanoyl-, and N,O-quaternary substituted chitosan sulfate, and the results showed that the anticoagulant activity of the polysaccharides had been prolonged. N-Succinyl chitosan (NSCS) was obtained from simply reaction between chitosan and succinic anhydride.9 NSCS exhibits several biological properties as nontoxicity.10 As such, NSCS has been explored as a drug carrier and an immobilizer.11,12 Lima et al.13 prepared NSCS, N,O-sucinyl-chitosan in position 2 and 6, and N,O-succinyl-chitosan in position 2, 3, and 6, with low solubility in water and did not mention the hemocompatibility. Actually, synthesis of O-succinyl-chitosan (OSCS) was achieved only by Zhang et al.14 through protecting N-phthaloylchitosan as an intermediate. However, this method needs several steps for the protection and deprotection of N-phthaloyl groups. Most important, it would be a problem to maintain the O-succinyl-chitosan linkage under the heating treatment with hydrazine or other basic conditions. We have tried and ended with failure. Therefore, in this work, we use the selective O-succination of chitosan with MeSO3H system (Scheme 1) to prepared NOSCS, which is studied by Sashiwa et al.15–17 The NSCS and NOSCS were characterized by 1 H NMR, FTIR, and X-ray diffractometry. The in vitro anticoagulant were evaluated by activated partial thromboplastin time (APTT),

0008-6215/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2011.03.037

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2.3. General methods

O 4 6 HO

OH

OH

O

O 5 2 1 O NH2 3 Chitosan

O

O

O

HO

n

n

NSCS

O

NHCOCH2CH2COOH

2.3.1. NMR spectroscopy 1 H NMR spectra were carried out on a Bruker AV600 MHz (Bruker, Rneinstetten, Germany). NSCS and OSCS were dissolved in D2O as a solvent.

O MeSO3H

O OCOCH2CH2COOH (Major) O O

HO

n

NHCOCH2CH2COOH (Minor) NOSCS Scheme 1. Synthesis of NSCS and NOSCS.

prothrombin time (PT), thrombin time (TT), and anticoagulantic index (BCI) using normal rabbit blood and plasma. And the antihemolytic ability was evaluated by hemolysis rate (HR). Further more, other physicochemical properties such as molecular weight and solubility are described here. 2. Experimental section 2.1. Materials Chitosan: 90% degree of N-deacetylation MW 200,000 (determined through Ubbelohde viscometer) (Zhejiang Aoxing Biotechnology Co., Ltd, Yuhuan, China). All commercially available solvents and reagents were used without further purification. 2.2. Synthesis of chitosan derivative 2.2.1. Synthesis of N-succinyl-chitosan (NSCS) Chitosan (1.0 g) was dissolved in 0.5 M acetic acid (35 mL) and then this solution was diluted with acetone (25 mL). Pyridine was added to the solution and stirred to form a homogeneous system. Certain amount of succinic anhydride (0.5, 1.5, 2.0 equiv/glucosamine unit of chitosan) which had been dissolved in 10–15 mL acetone beforehand was added to the system and stirred at room temperature. After stirring for 3 h, the white suspension was poured into EtOH (300 mL). The precipitate filtered was dispersed in 30 ml of water, followed by adding an adequate amount of NaOH to give a clear solution of pH 8–10, dialyzed (membrane tubing, molecular weight cutoff 12,000–14,000, Spectrum Laboratories, Savannah, GA, USA) and finally freeze-dried. Different DS of NSCS were produced with succinyl substitution degree 0.28, 0.35, and 0.53, respectively. 2.2.2. Synthesis of N,O-succinyl-chitosan (NOSCS) 1 g chitosan (5.6 mmol/L calculated as glucosamine unit) was dissolved in MeSO3H (15 mL) at room temperature for 30 min. The succinic anhydride (0.5, 1.5, 3.0 equiv/glucosamine unit of chitosan) was added into this solution. The mixture was stirred at 0 °C temperature for 4 h. After that the mixture was poured into 50 g of crushed ice in order to stop the reaction. The acidic mixture was dialyzed (membrane tubing, molecular weight cutoff 12,000–14,000, Spectrum Laboratories, Savannah, GA, USA) for 1 day to remove most of the acid, followed by neutralization with 2.0 mol/L NaOH. Finally, the mixture was dialyzed again for more than 3 days and lyophilized. Different DS of NOSCS were produced with succinyl substitution degree 0.32 (0.08 on amino), 0.51 (0.12 on amino) and 0.61 (0.20 on amino), respectively.

2.3.2. IR spectroscopy All IR spectra were obtained from samples in KBr pellets using a Thermo Nicolet Avator 370 FT-IR spectrometer (USA). 2.3.3. X-ray diffraction spectrometry X-ray diffraction spectrometry was obtained by using XD-3A powder diffraction meter with Cu Ka radiation in the range of 5–50° (2h) at 40 kV and 30 mA. 2.3.4. Determination of degree of substitution (DS) A degree of substitution (DS mol %) of NSCS and NOSCS were calculated using Eq. 1:

DS ¼

IH2—H6ðmethylene protons of the succinylÞ  14 IH2—H6ðchitosan bacbonesÞ  16

ð1Þ

2.3.5. Determination of the intrinsic viscosity The intrinsic viscosity of the chitosan derivative in 0.2 mol/L CH3COOH/0.1 mol/L NaCl was determined using an Ubbelohdetype viscometer (Schott-Gerate, Mainz, Germany) with a capacity of 15–20 mL. The viscometer was suspended in a thermostatically controlled water bath (Model E200, Lauda Dr. R. Wobser GmbH & Co., KG, Germany) maintained at 30.0 ± 0.1 °C. Flow times of different concentrations of each sample were recorded electronically using photoreceptors mounted on the viscometer stand that could detect the passage of the solution meniscus and the solvent flow time ratio of the kinematic relative viscosity was thus obtained. Because of the low concentrations used (between 0.2 and 1.0 mg/mL), the density corrections for the different solutions, the experiment was carried out in triplicate. 2.3.6. Apparent molecular weight (MW) determination of chitosan derivatives The apparent MWs of chitosan derivatives were examined by size-exclusion chromatography-evaporative light scattering (SECELS) using a Shimadzu LC-20A series HPLC system (Shimadzu Co., Ltd, Tokyo) apparatus equipped with a TSKgel G4000PWxl column. Alltech 2000ES were used as an evaporative light scattering detector. Chitosan derivatives were dissolved in 0.4 mol/L CH3COOH/ 0.15 mol/L CH3COONa aqueous solution (pH 4.0) at 5 mg/L and 20 lL of these samples was applied on the HPLC-ESLD instrument after filtering through 0.45 mm Millipore filters. Gel permeation chromatography (GPC) using dextran at standard MW markers was used for MW analysis of the NSCSs and NOSCSs. This GPC was performed with a Shimadzu LC-20AD pump at flow rate of 0.8 mL/min at rt, with a column TSKgel G4000PWxl (7.5 mm  300 mm; Tosoh Co.; Tokyo, Japan). This GPC method is abbreviated as GPCG4000PWxl. The elution solvent was 0.4 mol/L CH3COOH/0.15 mol/L CH3COONa aqueous solution (pH 4.0). Dextrans having MWs 4.32  103, 1.26  104, 6.06  104, 1.10  105, 2.89  105, 5.21  105 (RM international trade center, China) established by absolute MW determination were dissolved in 0.4 mol/L CH3COOH/0.15 mol/L CH3COONa aqueous solution at 5 mg/L. Eluent and dextrans solutions were filtered through 0.45 mm Millipore filters. And the weight-average molecular weight was calculated by the following equation:

lg ðMW Þ ¼ 0:6387V e þ 9:7878 ðR2 ¼ 0:9968Þ

ð2Þ

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W.-y. Xiong et al. / Carbohydrate Research 346 (2011) 1217–1223 Table 1 The solubility of NSCS and NOSCS in water of various pHsa Sample

Solubilityb

DS pH:

b c

3

5

7

9

11

13

Chitosan

0.00

0.0

NSCS

0.28

7.0

0.53

50.0

0.32

5.0

0.61

100.0

NOSCS

a

1

Solubilityc(g/L)

Solid sample (100 mg) was dispersed in H2O (20 mL). The pH of the solution was adjusted with 1.0 mol/L aqueous HCl and NaOH. White bar, soluble; black bar, insoluble. Solid sample was dispersed in pure water (5 mL) until the transmittance of the polymer solution was less than 50% of the transmittance for pure water.

where MW is weight-average molecular weight ranging from 1 kD to 700 kD and Ve is the elution volume. 2.4. Solubility test The solubility of WSC in different pHs was evaluated was as follows: solid sample (100 mg) was dispersed in H2O (20 mL).18 The pH of the solution was adjusted with 1.0 mol/L HCl and NaOH. Then according to the Cho’s method,19 the transmittance of each solution was measured at 600 nm using a UV–visible spectrophotometer (UV-1800, Shimadazu, Japan), the WSC is considered insoluble when the transmittance of the polymer solution is less than 50% of the transmittance for aqueous media at different pHs. The biggest solubility of WSC in pure water was measured as follows: different solid sample was dispersed in H2O (5 mL). The transmittance of each solution was measured at 600 nm, the WSC is considered insoluble when the transmittance of the polymer solution is less than 50% of the transmittance for pure water. The results are summarized in Table 1. The experiment was carried out in triplicate.

was recorded. For the thrombin time (TT) assay citrated normal rabbit plasma (90 lL) was mixed with 10 lL of a solution of NSCS and NOSCS with different concentrations and incubated for 3 min at 37 °C. Then, TT assay reagent (100 lL), preincubated for 3 min at 37 °C, was added and clotting time was recorded. The tests were repeated three times for each sample. 2.7. Determination of blood compatibility The antithrombotic properties of chitosan and modified chitosan films were determined by spectrophotometry.21 A film was laid on the bottom of a flask at 37 °C on a constant temperature bath and 0.25 mL of rabbit blood (using EDTA as anticoagulant) was added to the center of the membrane. With careful shaking, 0.02 mL of 0.025 mol/L CaCl2 solution was added and maintained for 10 min. Then 50 mL of normal physiological saline solution was carefully added. Finally the absorbance (AS) of the solution was determined by a UV-1800 spectrophotometer (SHIMADAZHU, Japan) at 540 nm and the blood anticoagulant index (BCI) of the sample was calculated at least three times from Eq. 3:

AS  100% AW

2.5. Determination of isoelectric point (IP)

BCI ¼

The IP of WSC was evaluated was as follows: solid sample (50 mg) was dispersed in H2O (50 mL). The pH of the solution was adjusted with 1.0 mol/L HCl and NaOH with stirring. Then the transmittance of each solution was measured at 600 nm using a UV–visible spectrophotometer (UV-1800, Shimadazu, Japan), the pH was considered as the IP of the sample when the transmittance of the polymer solution was the smallest.

where AW was the absorbance of the solution in which 0.25 mL of sheep blood was mixed with 50 mL of physiological saline solution. The hemolytic properties of chitosan and WSC films were examined by spectrophotometry as well,22 5 mL of rabbit blood was dispersed in 10 mL of physiological saline solution at 37 °C, and the film was soaked in this solution for 4.0 h. After the film was removed, the suspension was centrifuged at 750g for 5 min, and the supernatant liquid containing heme was collected to determine the absorbance (AS) at 540 nm. With the same method, the absorbance (AN) of the supernatant liquid in which 5 mL sheep blood was dispersed in 10 mL of physiological saline solution, as well as the absorbance (AS) in which 5 mL of sheep blood was dispersed in 10 mL of distilled water, were determined, respectively. The hemolytic ratio (HR) was calculated at least three times as the following Eq. 4:

2.6. Anticoagulant activity The in vitro coagulation times, including activated partial thrombin time (APTT) and prothrombin time (PT) were determined using an automated blood coagulation analyzer (CA-50, Sysmex Corp., Kobe, Japan). In brief, coagulation times were measured as follows: citrated normal rabbit plasma (90 lL) was mixed with 10 lL of a solution of NSCS and NOSCS (0, 1.0, 2.0, 3.0, 4.0, 5.0 lg) and incubated for 1 min at 37 °C. Then, APTT assay reagent (100 lL) was added to the mixture and incubated for 3 min at 37 °C. Thereafter, 20 mmol/mL CaCl2 (100 lL) was added and the clotting time was recorded. For the prothrombin time (PT) assay citrated normal rabbit plasma (90 lL) was mixed with 10 lL of a solution of NSCS and NOSCS with different concentrations and incubated for 0.5 min at 37 °C. Then, PT assay reagent (100 lL), preincubated for 0.5 min at 37 °C, was added and clotting time

HR ¼

AS  AN  100% AP  AN

ð3Þ

ð4Þ

2.8. In vitro enzymatic degradation The in vitro enzymatic degradation of the chitosan derivatives was studied by decreasing in weight of the polymer solution in the presence of lysozyme (hen egg white) varying from the method

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of Hsieh and Tanuma.23,24 The NSCS and NOSCS derivatives with different DS were dissolved in 10 mL vials containing 5 mL of 0.05 mol/L, pH 7.4, phosphate-buffered saline (PBS) for prescribed days with or without 1.0 mg/mL lysozyme (20,000 U/mg, Biotech, China).25 Then the samples were placed in an Orbital Shakers (SO-701, GMB Co.) with temperature set at constant 37 °C. Samples were removed from the medium by adjusting the pH to the corresponding IP at predetermined time (2, 5, 10, 15, and 20 days), and centrifuged at 10,000 rpm, finally the precipitate was vacuum dried and weighed. For the sample of NOSCS with DS of 0.61, the sample was removed from the medium by adding isovolumetric ethanol at predetermined time (2, 5, 10, 15, and 20 days), and centrifuged at 10,000 rpm, finally the precipitate was vacuum dried and weighed. The degree of in vitro degradation was calculated at least three times by the Eq. 5:

Weight loss ð%Þ ¼

w0  wt  100 w0

ð5Þ

where w0 and wt were the absolute weight of sample dissolved in the PBS with or without lysozyme for the prescribed days. 3. Results and discussion 3.1. The synthesis and characterization of NSCS, NOSCS The synthesis of NSCS and NOSCS was shown in Scheme 1. Figure 1(I) shows the FTIR spectra of (a) chitosan, (b) NSCS, and (c) NOSCS. From the chitosan spectrum, it was found that the characteristic peaks at around 1650, 1599, and 1384 cm1 were assigned to amide I, amine II, and amide III absorption bands of chitosan, respectively. The absorption band at 1156 cm1 was the asymmetric stretching of the C–O–C bridge. Bands at 1073 and 1033 cm1 were assigned to the skeletal vibration of C–O stretching.26 Compared with that of chitosan, 2925 cm1 appears (stretching of –CH2–), the peaks at 3440 and 1599 cm1 (amino group characteristics) decrease dramatically, and the peak at 1650 cm1 (amide I) and 1384 cm1 (amide III) increase, the above results indicated the succinyl derivation reaction took place at the N-position (Scheme 1) and –NH–CO– groups have been formed. From the NOSCS spectrum, the new peak at 1731 cm1 was attributed to the carbonyl group of ester group (C@O of OCOR), which indicated the succinyl derivation reaction took place at the O-position (Scheme 1) and –O–CO– groups have been formed.

H NMR spectrum is the most sensitive and precise technique and results in the most accurate data for determining the DS.27 The 1H NMR spectrum of the NSCS, NOSCS was given in Figure 1(II). The 1H NMR assignments of NSCS were as follows: 1H NMR (D2O) d = 1.96 (NHCOCH3); d = 2.84 (H2); d = 3.46–3.77 (H1, H3, H4, H5, H6); d = 2.37 (NHCOCH2CH2COOH); d = 2.45 (NHCOCH2CH2COOH); The 1H NMR assignments of NOSCS were as follows: d = 1.96 (NHCOCH3); d = 2.79 (H2); d = 2.79 (H6 of CH2OCH2CH2COOH); d = 3.57–3.80 (H1, H3, H4, H5, H6 of CH2OH); d = 2.42 (NHCOCH2CH2COOH); d = 2.55 (OCOCH2CH2COOH). The 1H NMR assignments of NOSCS were as follows: d = 1.96 (NHCOCH3); d = 2.79 (H2); d = 3.57–3.80 (H1, H3, H4, H5, H6); d = 2.42 (OCOCH2CH2COOH); d = 2.55 (OCOCH2CH2COOH). The singlet peak showing up at 2.30 ppm in Figure 1(II) probably was attributed to the protons of N–O cross-linking (between C(6)–OCH2–CH2COOH and C(2)–NH2 groups), according to Jeong et al.28 Chitosan with the molecular weight higher than 5000 cannot dissolve in water because of the strong intermolecular hydrogen bonding. X-ray diffraction spectra of chitosan, NSCS, and NOSCS (Fig. 1(III)) show that chitosan exhibits two reflection fall at 2h = 5°, 2h = 20°. It is reported that the reflection fall at 2h = 5° was assigned to crystal form I and the strongest reflection appears at 2h = 20° which corresponds to crystal forms II.29 However, the XRD spectrum of NSCS and NOSCS have only one broad peak at around 2h = 20°, which indicates that crystal forms have been destroyed in NSCS and NOSCS macromolecules. This result suggests that the intramolecular hydrogen bonding in NSCS has been greatly decreased and the intermolecular hydrogen bonding in NOSCS has been greatly increased after chemical modification in comparison with that of chitosan. As a result, the solubility of NSCS and NOSCS is better than chitosan. The broad peak at around 2h = 20° in the XRD spectrum suggests that weak intramolecular H-bonds still existed. 3.2. Aqueous solubility

2000

Solubility of chitosan, NSCS, and NOSCS was measured at various pH values. As shown in Table 1, NSCS with low DS can dissolve only in acidic region at pH lower than 5.6, which is better than the raw material chitosan that can only dissolve in the aqueous solution at pH lower than 4.5. Because of the massive introducing of carboxypropionyl group into N-position, the NSCS with higher DS can dissolve in much wider pH region (pH <4.5 and pH >6.5, respectively). On one hand, the methylenes of carboxypropionyl group can disrupt the intramolecular hydrogen bond of chitosan, while the carboxyl group could endow chitosan with intermolecular hydrophilicity on the other hand. According to Ying’s work,30 NSC was water-soluble at DS values above 0.47. The NOSCS with low DS value can dissolve only in dilute acid aqueous solution, while the NOSCS with higher DS can dissolve in aqueous solution with all pH regions. However, when the NOSCS with DS value of 0.61 dissolved in basic aqueous solution, within a few minutes the transparent solution changed into subtransparent, interestingly.

1500

3.3. Intrinsic viscosity

1000

Figure 2 displays the relationship between DS and intrinsic viscosity ([g]) and the relationship between intrinsic viscosity and molecular weight for chitosan derivatives of NSCS and NOSCS in 0.2 mol/L CH3COOH/0.1 mol/L NaCl at 30.0 ± 0.1 °C (pH 3.0) and 0.4 mol/L CH3COOH/0.15 mol/L CH3COONH4 (pH 4.0) at rt, respectively. In the present study, the initial increase in the succinic anhydride to NH2 ratio from 0.5:1 to 3:1 resulted in the increase in the DS value from 0.28 to 0.61. However, at the succinic anhydride to NH2 ratio above 3:1, the value leveled off or decreased.

4000 3500 3000

Intensity/a.u.

1

2500

500 0

(III)

0

10

20

30

40

50

60

2θ º

Figure 1. (I) FTIR spectra of (a) chitosan, (b) NSCS, (c) NOSCS; (II) 1H NMR spectra of (b) NSCS, (c) NOSCS; (III) WAXD patterns of (a) chitosan, (b) NSCS, (c) NOSCS.

1221

12000

120

10000

100

8000

80

6000

60

4000

40

2000

20

30

2500

25

2000

20 1500 15 1000 10 500

0

0.28

0.35

0.53

0.60

0

apparent Mw (kD)

140

[ η] (mL/g)

14000

apparent Mw (kD)

[η] (mL/g)

W.-y. Xiong et al. / Carbohydrate Research 346 (2011) 1217–1223

5

0

0 0.32

0.51

0.58

0.61

DS

DS

Figure 2. Effects of DS on intrinsic viscosity (bars) and apparent MW (circles). The intrinsic viscosity of NSCS (open bars) and NOSCS (filled bars) are determined in 0.2 mol/L CH3COOH/0.1 mol/L NaCl at 30.0 ± 0.1 °C (pH 3.0); The MW of NSCS (4) and NOSCS (h) are determined in 0.4 mol/L CH3COOH/0.15 mol/L CH3COONa (pH 4.0) at rt.

It is postulated that, the inhomogeneous mixture caused by the overdose of succinic anhydride and the acid reaction condition could be the reason for the observed stagnation or decrease in the DS value (data not shown). For NOSCS with different DS, the intrinsic viscosity leveled off around 20 dL/g and the apparent MW leveled off around 20 kD, which probably due to the violent fragment of b-1,4 glycosidic bonds in MeSO3H. For a similar DS, the intrinsic viscosity of NSCS first increased and reached the maximum at the DS of 0.53 and then decreased, whose trend matched the trend of the apparent MW. The attachment of succinic anhydride attributed most in the increasing apparent MW at the DS below 0.53 for NSCS. However the decreasing of apparent MW caused by the increasing acid succinic anhydride prevailed since then. A matter of fact, it was also found that the intrinsic viscosity could probably decrease followed by the formation of the new bond.31

zero DS and chitosan derivatives showed no obvious hemolytic ability, it was clear that the HR values of both NSCS and NOSCS declined with the increasing DS. The reason why raw chitosan causes certain extent of hemolysis is due to the electrostatic attraction between chitosan and erythrocyte, which caused the curvature of the erythrocyte membrane and ultimately lead to the release of hemoglobin.32 As for the NSCS and NOSCS, apparently, the reason why the HR values declined with the increasing DS was simply due to the negative charges carrying on. The NOSCS with the proximity DS showed lower HR value than NSCS, which probably attributed to the less amount of amino groups NSCS got. The reaction between chitosan and succinic anhydrides in the solvent of MeSO3H mainly happened on –OH at C-6 position and –NH2 at C-2 position,15,16 thus with the similar DS, NOSCS carrying more amino groups exhibited lower HR.

3.4. Blood compatibility

3.5. Anticoagulant activity

The blood compatibility of NSCS and NOSCS were estimated by BCI and HR. The relationship between BCI or HR and the DS of the derivatives was demonstrated in Figure 3. The BCI value of both the NSCS and NOSCS increased with the raising DS, which was due to the increasing negative charges on the carboxypropionyl groups. There are negative charges on the surface of the blood cell, thus, the negative charges of the derivatives repel the blood cell from coming close because of the electrostatic repulsion, thereby avoiding the coagulation of the blood cells. And there was no obvious distinction between NSCS and NOSCS. Though both chitosan with

The blood coagulation system consists of the intrinsic pathway, the extrinsic pathway, and the common pathway. APTT and PT are used to examine mainly the intrinsic and extrinsic pathway. TT is used to examine the thrombin activity or fibrin polymerization. The anticoagulant properties of the derivatives of NSCS and NOSCS were summarized in Table 2. The APTT of chitosan in this experiment was 328 s at 3 mg/mL, and increased with the increasing concentration, which is also in accord with the results reported by Huang,33 while the APTT of control assay was ca. 34 s. The PT and TT of chitosan at different concentrations in this experiment

100

NOSCS NSCS

9 8 NSCS NOSCS

90

7

HR(%)

BCI(%)

6 80 70

5 4 3 2

60

1 50 0.00

0.20

0.40

DS

0.60

0.80

0 0.00

0.20

0.40

0.60

0.80

DS

Figure 3. The influence of DS on BCI and HR. The BCI (%) of NSCS (j) and NOSCS (N) are determined at 540 nm as well as the HR (%) of NSCS (N) and NOSCS (j).

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Table 2 APTT, TT, and PT of normal rabbit platelet-poor plasma containing NSCS and NOSCS with different DS Samples

APTT (s) at different concentrations (mg/ mL) 1.0

3.0

TT (s) at different concentrations (mg/ mL)

1.0

3.0

5.0

1.0

3.0

5.0

NSCS

DS = 0.28 DS = 0.53

33.2 49.0

48.2 53.4

50.5 55.9

11.8 12.1

12.0 12.0

12.0 12.2

18.0 18.0

18.2 18.1

18.2 18.3

NOSCS

DS = 0.31 (0.08 on amino) DS = 0.61 (0.20 on amino)

88.0 44.4

125.3 61.8

234.7 63.2

12.0 11.9

12.1 12.1

12.1 12.1

18.3 18.5

18.6 20.2

19.0 22.5

Control assay

5.0

PT (s) at different concentrations (mg/ mL)

33.8

12.0

Percentage of weight loss (%)

were almost the same with control assay. It is reported that the carboxymethylation or carboxypropionylation of N-position and N,O-positions in sulfated chitosan imparted better anticoagulant activity.33,34 The anticoagulant activity of the above was largely dependent on the density of negative groups.35 However the effect of carboxypropionylation of chitosan in N-position or N,O-position APTT was not mentioned elsewhere. Results in Table 2 showed that the APTT was prolonged by NSCS and NOSCS, whereas no clotting inhibition was observed in PT assay at the same concentration. And slight clotting inhibition was observed in TT assay at 3 mg/mL and 5 mg/mL of NOSCS with DS of 0.61, while NSCS with different DS did not show any inhibition. NSCS and NOSCS prolonged APTT suggested that they exhibited the activity of inhibiting the intrinsic and/or common pathway probably because of the existing of amino groups. Slight prolongation of TT was observed only when NOSCS was used which indicated that the carboxypropionylation in C-2 position endowed the derivatives with antithrombin activity or antifibrin polymerization. No effect of the anticoagulant on PT observed indicated that it did not inhibit extrinsic pathway of coagulation. Chitosan sulfate can largely prolong the APTT, PT, and TT, because it has high level of negative charge density produced by the sulfate groups. Different from chitosan sulfate, our results could be concluded, firstly, that –NH2 on C-2 position should be the reason prolonging APTT, thus the carboxypropionylation in C2 position actually made the APTT short and leaned to normal level; secondly, that the carboxypropionyl group introduced on the amino group did not need to be present to a high DS to have the APTT around 50 s; thirdly, that the carboxypropionyl group introduced on the C-6 hydroxyl group did not appear to impart antico-

18.1

agulant activity; and fourthly, that the carboxypropionylation in amino group did not endow NSCS with anticoagulant activity in TT assay, while carboxypropionylation in C-6 position endowed NOSCS with slight anticoagulant activity. 3.6. Enzymatic degradation

45

NOSCS 0.31 NOSCS 0.61

40

NSCS 0.28

In human body, chitosan and its derivatives are considered as a biodegradable polymer because of their susceptibilities to lysozyme. Thus, the in vitro degradation behavior of chitosan has been usually investigated using hen egg white lysozyme to cleavage the b-1,4 glycosidic bond.24,36 In this study, the degradation behavior of the NOSCS and NSCS in the presence and in the absence of lysozyme was investigated in PBS at 37 °C, and the method of adjusting pH into corresponding IP was chosen to precipitate the chitosan derivatives of NSCS and NOSCS at different DS (except for NOSCS with DS of 0.61). The IPs of NSCS with the DS of 0.28 and 0.53 were 5.6 and 5.1, respectively. And the IP of NOSCS with the DS of 0.32 was 5.5. However the NOSCS with the DS of 0.61 could not precipitate at any pH, which was probably due to the relatively low MW around 20 kD. Figure 4 shows the percentage of weight loss of NOSCS and NSCS. As shown in Figure 4, the weight loss of raw material chitosan remained 1.1% even after 20 days in PBS with 1.0 mg/mL lysozyme. However, an increasing in DS on amino group had a rising loss of weight and the percentage of weight loss. As a matter of fact, the percentage of weight loss of NSCS with the DS of 0.53 reached 41.7% at the end of the 20th day, which indicated that the substitution on amino group possessed the key to degradation other than the carboxypropionylation in C-6 position. The work of Zhang et al.14 partly supported the results revealed in this paper. Compared to chitosan with different degree of deacetylation (DD) reported by Ren et al.,37 NSCS obviously was more susceptible to lysozyme.

35

NSCS 0.53 CS

4. Conclusion

30 25 20 15 10 5 0

0

5

10

15

20

25

Time (day) Figure 4. Enzymatic degradation of NSCS and NOSCS in PBS at pH 7.4 with lysozyme. The percentage of weight loss (%) of CS (-), NSCS with DS of 0.28 (N), NSCS with DS of 0.53 (), NOSCS with DS of 0.31 (d), and NOSCS with DS of 0.61 (j) are at predetermined time (2, 5, 10, 15, and 20 days).

In this study, water-soluble N,O-succinyl chitosan and N-succinyl chitosan were prepared successfully. The chemical structures and physical properties of NOSCS and NSCS were characterized by FTIR, 1H NMR and XRD. Both chitosan derivatives show much higher solubility in aqueous solution than chitosan. NSCS showed much higher percentage of weight loss, because the carboxypropionylation in amino group was the reason that chitosan derivatives synthesized here could be susceptible to lysozyme. The study of anticoagulantic index (BCI) and hemolysis rate (HR) indicate that the blood compatibility of NOSCS and NSCS has been improved. The anticoagulant properties were assessed by activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) assays using normal rabbit plasma. Compared to control assay, APTT was prolonged, TT was prolonged slightly, while the PT was not affected. APTT decreased with the increasing substitution on amino group and leaned to normal.

W.-y. Xiong et al. / Carbohydrate Research 346 (2011) 1217–1223

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