Highly Efficient One-Step Purification of Sulfated Polysaccharides via

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Highly Efficient One-step Purification of Sulfate Polysaccharides via Chitosan Microspheres Adsorbents Xueqin Wei, Jiangjiang Duan, Xiaojuan Xu, and Lina Zhang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b02975 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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ACS Sustainable Chemistry & Engineering

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Highly Efficient One-step Purification of Sulfate Polysaccharides via

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Chitosan Microspheres Adsorbents

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Xueqin Wei, Jiangjiang Duan, Xiaojuan Xu*, Lina Zhang*

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College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072,

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China

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* Corresponding authors

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Tel.: +86-27-87219274; Fax: +86-27-68762005.

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E-mail: [email protected]; [email protected]

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ACS Paragon Plus Environment


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Abstract

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Sulfated polysaccharides from marine biomass exhibit various biological activities,

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however, traditional purification approaches are time- and money-consuming,

25

particularly involving the usage of toxic chemical reagents. Herein, novel chitosan

26

microspheres cross-linked by genipin (CSGs) were prepared from chitosan solution in

27

alkali/urea aqueous system as multi-functional adsorbents for deproteinization and

28

decoloration by one step. The compression fracture stress of chitosan hydrogels

29

fabricated from the alkaline solution system was over 10 times that of the hydrogels

30

prepared by traditional acid method, suggesting a significantly improved mechanical

31

strength. The robust CSGs with mean diameter of 100 µm exhibited mesoporous

32

structure. The adsorption/desorption performance of CSGs for sulfate polysaccharides

33

was investigated by using heparin as a model, showing significantly high adsorption

34

capacity, over 3-fold of that from commercial Q Sepharose Fast Flow. Moreover,

35

CSGs selectively adsorbed heparin in the presence of BSA at pH 4.6 by adding 0.5 M

36

NaCl to screen electrostatics between CSGs and proteins. Furthermore, CSGs

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adsorbents were practically applied in purification and fractionation of crude fucoidan

38

by

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deproteinization ratio of 96% and decolorization ratio of 94%. This work provided a

40

simple, green and highly efficient approach for purification and fractionation of

41

sulfated polysaccharides.

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Keywords: Chitosan, Scale-up purification, Fractionation of fucoidan, Green

stepwise

elution

with

higher

salt

concentrations,

2


showing

maximum

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approach, Multi-functional adsorbent

44

Introduction

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With nearly three years expedition, the Tara Oceans consortium, a team of

46

multinational scientists, has found many unidentified microorganisms genes in the

47

world oceans, suggesting the mysterious of marine biomass and more efforts should

48

be focused on them.1 Nowadays, it has been recognized that natural sulfated

49

polysaccharides, such as fucoidan from brown algae, are one of the most promising

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glycan-based drugs candidates,2 which reportedly display a wide range of biological

51

activities, such as antitumor, antiviral, anticoagulant, and immunomodulatory effect.3-4

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However, they are still far from clinical applications due to the ambiguous

53

structure-activity relationships resulted from the impurity, the unknown fine structure,

54

and so forth.5 In our laboratory, we have accumulated experiences on the purification,

55

chain conformation and structure-bioactivity relationship of fungus6-7 and yeast

56

polysaccharides.8-9 The issues of purification of polysaccharides should be firstly

57

resolved. However, routine purification protocols including the Sevag method with a

58

mixture of 1-butanol/chloroform to remove proteins and 30% H2O2 to decolor 10-11 are

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not only toxic, but also may cause the degradation and structure alteration, thereby

60

influencing bioactivities.12 Although chromatography packed with ion-exchange or

61

gel-ﬁltration materials have been widely used, the time- and money-consuming

62

coupled with low capacity are not ignorable.13 Therefore, seeking an efficient, green

63

and cost-efficient purification method by using novel adsorbents is extremely urgent. 3



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Adsorbents for selective adsorption of target biomolecules are sustaining

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innovations.14-15 In our laboratory, novel absorbents based on chitin for removal of

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metal ions, dyes and oil have been reported.16-18 For negative charged sulfated

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polysaccharides, polycation would be a good candidate for their adsorption.19

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Chitosan, with cationic nature, is produced from the de-acetylation of chitin,

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consisting of β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine. Owing to its

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abundant hydroxyl and amino groups, chitosan has been proved as an eco-friendly

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adsorbent material for environmental and biomedical applications.20-21 However, it is

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an undeniable fact that chitosan materials prepared from traditional acid system suffer

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from poor mechanical properties.22-23 In our recent findings, high strength chitosan

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hydrogels with a nanofibrous structure obtained from chitosan solution in alkali/urea

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aqueous system at low temperature via physical cross-linking display show

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unexpectedly high compression fracture stress about 100 times as much as that

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prepared from traditional acidic solvent.24 Therefore, we believe that chitosan

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fabricated in alkali system should be an ideal material for selective adsorption of

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sulfated polysaccharides through electrostatic interactions between protonated −NH2

80

groups on chitosan and negatively charged sulfate groups from sulfated

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polysaccharides.

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In the present work, novel chitosan microspheres adsorbents were prepared from

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chitosan solution in LiOH/KOH/urea aqueous system at low temperature using W/O

84

emulsion and then cross-linked by genipin to improve the stability. The adsorption 4


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equilibrium and kinetics of heparin by chitosan microspheres (CSGs) were firstly

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tested. The selectivity of CSGs was studied and discussed with the separation of

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heparin from a model heparin-BSA mixture. Finally, the application of CSGs in

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purification of crude fucoidan was evaluated and compared to that of chemical and

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chromatographic method. This work would provide a simple, green and highly

90

efficient method to separate and purify sulfated polysaccharides in large-scale.

91 92

Experimental section

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Materials. Commercial grade chitosan (degree of deacetylation=89%) was

94

purchased from Ruji Biotechnology Co., Ltd. (Shanghai, China) and used without

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further purification. Crude commercial fucoidan (coded as FN0) from Laminaria

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japonica was purchased from Rizhao Jiejing Ocean Biotechnology Development Co.,

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Ltd. To improve the purity, FN0 was purified by using the classical methods as

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follows. Firstly, 20 g of FN0 powder was subjected to the Sevag method to remove

99

proteins and treated with 30% H2O2 to eliminate pigments followed by exhaustively

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dialyzing, concentrating and finally lyophilizing to gain the purified fucoidan named

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as FN1. Next, 4.4 g FN1, dissolved in 250 mL ultrapure water, was subjected to

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fractionation by anion-exchange chromatography on the DEAE Sepharose CL-6B

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column (φ5 cm×30 cm) and stepwise elution with 0.5, 1 and 2 M NaCl, respectively,

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to obtain three fractions coded as SF0.5, SF1 and SF2 accordingly (Figure S1).

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Genipin was attained from Linchuan Zhixin Biotechonlogy Co., Ltd. (Jiangxi, 5



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China). Heparin (sodium salt with activity 160 U/mg, Mw = 6000～20000) was bought

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from Toyobo (Japan). The chemical structures of genipin and heparin are shown in

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Figure S2. Q Sepharose Fast Flow, with a mean diameter of 90 µm, BET surface area

109

of 195 m2/g and mesoporous structure (Figure S3), was bought from Pharmacia

110

(Sweden). Other chemical reagents from commercial sources were of analytical grade

111

and used without further purification.

112

Preparation of chitosan microspheres. To prepare the chitosan solutions, chitosan

113

powder

was

firstly

dispersed

into

the

alkaline

114

LiOH/KOH/urea/H2O (8:7:8:77 by weight), and was frozen at −35℃ for 6 h. Then,

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the frozen solid was thoroughly thawed by stirring vigorously at ambient temperature.

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After freezing-thawing twice, 4 mL epichlorohydrin as cross-linker was added into

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200 g of the chitosan solution at −20℃ with stirring for 2 h. The obtained solution

118

was centrifuged at 7000 rpm for 10 min at 5℃ to remove air bubbles, and the

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transparent chitosan solution with the concentration of 4 wt % was thus obtained,

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labeled as TCS solution. Subsequently, a well-mixed suspension containing 450 mL

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isooctane and 4.5 mL Span 85 was dispersed in a round-bottomed flask with

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three-neck at 0℃ and was stirred for 10 min. Then, 150 mL TCS solution was rapidly

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dropped into the suspension which was kept stirring for 30 min in water bath at 0℃

124

to achieve stable water/oil emulsion droplets. After removing the water bath, the

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suspension was continued to stir for 4 h at room temperature. Afterwards, the

126

suspension was promptly poured into the solution of ethanol/water (3:2 by volume) 6


aqueous

solvent

of

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and stirred for 1 h to obtain chitosan microspheres coded as CSs. The microspheres

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were then washed repeatedly with ethanol and deionized water for removing all

129

residual reagent. For further cross-linking CSs, 100 mL of the drained microspheres

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were introduced into 40 mL of 5 mg/mL genipin solution, which was stirred at 40℃

131

for 3 h until microspheres started to turn dark blue. The final chitosan microspheres

132

were cleaned with distilled water, coded as CSGs.

133

Characterization of CSGs. The structure and morphology of CSGs were

134

characterized by field-emission scanning electron microscopy (FE-SEM, Zeiss,

135

SIGMA). Surface area and pore size distribution were evaluated using nitrogen

136

adsorption (Micromeritics, AsAp2020, USA). The specific surface area was

137

calculated from the nitrogen adsorption isotherm using the Branauer-Emmett-Teller

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(BET) equation and the pore size distribution using the Barrett-Joyner-Halenda (BJH)

139

model. The zeta potentials of CSGs were measured by a Zetasizer NanoZS (ZEN

140

3600, Malvern, UK) following the method described by Yan et al.25

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Adsorption performance of CSGs. Herein, heparin, the complex mixture of

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highly sulfated glycosaminoglycan,26 was used as a model of sulfated polysaccharide

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to investigate the adsorption characteristics of CSGs. Generally, 0.66 g of drained

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CSGs were mixed with 20 mL heparin solution. The mixed solutions were then kept

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in shaking at ambient temperature for 12 h. The amount of heparin in the supernatant

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was measured by acid oxidation with 90% H2SO4 contained 25 mM borax,27 then

147

monitored by a UV-Vis recording spectrophotometer (UV-61, Meipuda, Shanghai, 7



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China) at 298nm. For the batch adsorption experiment, the amount of heparin

149

adsorbed onto CSGs (qe, mg/g) at equilibrium was calculated by the following

150

formula: qe =

Vi (c0 - ce) (1) mad

151

where c0 and ce are the initial and equilibrium concentration of heparin in the solution,

152

respectively (mg/L). Vi is the volume of the heparin solution (L) and mad is the mass

153

of adsorbent (g).

154

For the adsorption kinetics experiment, CSGs were mixed with 1 mg/mL aqueous

155

heparin and then shaken at ambient temperature. At desired time intervals, the

156

remaining amount of heparin in the aqueous solution was then determined.

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Separation by column packed with CSGs. Using slurry packing method, CSGs

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suspended in the loading buffer were packed in a glass column (φ10 mm×600mm)

159

to form a 15-cm bed, which was eluted with the loading buffer at flow rate of 1

160

mL/min for 24 h at room temperature for equilibrium and stability. The sample

161

solution was added onto the top of the column, followed by an additional 150 mL of

162

loading buffer. Stripping of sample was next initiated by stepwise elution with buffers

163

containing 1, 2 and 4 M NaCl, respectively. Fractions of 3 mL were collected and

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monitored by UV-Vis spectrophotometer at 280 nm for BSA and 298 nm (acid

165

oxidation with 90% H2SO4) for polysaccharides, respectively.

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Characterization of sulfated polysaccharide. To characterize the weight-average

167

molecular weight (Mw) and hydrodynamic radius (Rh) of sulfated polysaccharides in 8


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0.15 M NaNO3 aqueous solution, the static and dynamic light scattering (SLS and

169

DLS) measurements were carried on the light scattering spectrometer (ALV/CGS-3,

170

ALV GmbH, Germany).28 The specific refractive index increments (dn/dc) was

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determined with a differential refractometer (Optilab T-rEX, Wyatt Technology Co.)

172

after the sulfated polysaccharides were dialyzed against the solvent for 72 h. Protein

173

content was quantified using bicinchoninic acid (BCA) assay with bovine serum

174

albumin (BSA) as the standard.29 The deproteinization ratio was calculated by the

175

following equation:

176

Deproteinization ratio (%) = (m0-me)/m0×100

(2)

177

where m0 and me are the contents of proteins before and after deproteinization,

178

respectively. The decoloration ratio was determined by the following equation:

179

Decoloration ratio (%) = (A0-Ae)/A0×100

(3)

180

where A0 and Ae are the absorbance of the polysaccharides dissolved in 27% acetone

181

at 340 nm determined by UV-Vis spectrophotometer before and after decoloration,

182

respectively. Ester sulfate content was measured after acid hydrolysis (1 M HCl at

183

100 ℃ for 6 h,) using the turbidimetric method.30 The total uronic acid content was

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colorimetrically determined using the m-hydroxydiphenyl assay31 with the glucuronic

185

acid as the standard. Neutral sugars composition analysis of the final samples was

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performed by gas chromatography (6890N, Agilent, USA).8

187 188

Results and discussion 9



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Morphology, structure and properties of CSGs. The chitosan microspheres (CSs)

190

prepared from chitosan solution were further cross-linked by genipin (CSGs),

191

resulting in the color change of microspheres from light yellow to dark blue (Figure

192

S4). The crosslinking of CSGs was confirmed by FTIR (Figure S5). As shown in

193

Figure 1a, CSGs exhibited a spherical shape with a mean diameter of 100 µm and

194

both the surface and cross-section (Figure 1c-e) of the CSGs displayed porous

195

microstructure. To further characterize the porous structures of CSGs, nitrogen

196

adsorption-desorption measurement was carried out. Figure 2a shows a typical

197

hysteresis loop within a relative pressure P/P0 ranging from 0.4 to 1, suggesting the

198

characteristic mesoporous structure.32 The BET surface area of CSGs was determined

199

to be 77 m2/g and the pore size ranged from 3 to 113 nm with the peak value of 23 nm

200

(Figure 2b).

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The structural stability and mechanic strength of the hydrogel microspheres are

202

important for their practical applications. The swelling behaviors of microspheres

203

before and after genipin cross-linking in acidic environment (Figure S6a) indicated

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that the swelling degree of CSG was much lower than that of CS. Nowadays, there is

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no standard test method for the measurement of the mechanical properties of the

206

hydrogel microspheres, thus the compression tests of the chitosan hydrogels

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constructed under the same condition of the preparation of the hydrogel microspheres

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except for the absence of water/oil emulsion were performed to compare their

209

mechanical strength. The typical compressive stress-strain curves (Figure S6b) 10


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indicated that the compressive fracture stress of the chitosan hydrogels from alkaline

211

system (CS-Gel) (4.0 MPa) and genipin cross-linked CS-Gel (CSG-Gel) (3.4 MPa)

212

were much higher than that obtained by the conventional acidic method (ACS-Gel)

213

(0.3 MPa). The compression fracture stress of the chitosan hydrogels was over 10

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times that of the hydrogels prepared by the traditional acid method. It was not hard to

215

imagine that the mechanical strength of the chitosan hydrogel microspheres prepared

216

from alkaline solution system could be significantly improved. With genipin

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crosslinking, CSG-Gel exhibited a higher static compressive modulus judging from

218

the slope of the stress-strain curve linear region. Therefore, the porous and robust

219

CSGs can be used as biosorbents.

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Adsorption, desorption and reusability of CSGs. Herein, heparin was selected as

221

a model sulfated polysaccharide to evaluate the adsorption behaviors of CSGs. Figure

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3a shows the effects of initial pH (2~13) on the adsorption of heparin onto CSGs at

223

the same initial concentration (0.5 mg/mL). Obviously, the adsorption efficiency of

224

heparin remained high (＞90%) at pH ≤7, then dropped rapidly with increasing pH,

225

and finally decreased to almost nil at pH＞10. It is easily understood that the

226

decreased protonation of amino groups on CSGs with increasing pH lead to negative

227

charges of CSGs, confirmed by the zeta potentials of CSGs (ζCSGs) as a function of

228

initial pH in Figure 3b. The ζCSGs decreased as the pH value increased, and the

229

isoelectric point was found to be 7.4. Thus, it could be concluded that high adsorption

230

occurred at a low initial pH, as a result of the enhanced electrostatic interactions 11



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between more protonated −NH2 groups on CSGs and negatively charged heparin

232

molecules.

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Figure 3c shows the adsorption isotherms of CSGs and commercially available Q

234

Sepharose Fast Flow (QFF, a strong anion exchanger chemically attached quaternary

235

ammonium ligands：-O-CH2CHOHCH2OCH2CHOHCH2N+(CH3)3) in 50 mM acetic

236

acid/sodium acetate buffer at pH 4.6 (AC buffer). It was noted that CSGs displayed a

237

remarkably higher adsorption capacity than QFF. To quantify the maximum

238

theoretical sorption capacity of adsorbents for heparin, data points were fitted with

239

three commonly used adsorption models of Langmuir (Eq.(4)), Freundlich (Eq.(5))

240

and Langmuir-Freundlich (Eq.(6))33-34 isotherms as described in the following

241

equations: qe=

qm KL ce (4) 1 + KL ce

qe= KF ce1/n' (5)

qm (KLF ce)n qe = (6) 1 + (KLF ce)n 242

where qm (mg/g) is theoretical maximum adsorption capacity; KL (L/mg), KF

243

(mg/g(L/mg)1/n) and KLF (L/mg) are equilibrium constants of Langmuir, Freundlich

244

and Langmuir-Freundlich, respectively. 1/n' is the Freundlich sorption intensity

245

constant which is usually between 0.1 and 1 pointing out favorable sorption; n is the

246

index of heterogeneity which varies from 0 and 1 (when n =1 the model reduces to the

247

Langmuir isotherm). The estimated model parameters along with correlation

248

coefficients (R2) are summarized in Table S1. Clearly, the Langmuir-Freundlich 12


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249

isotherm which combined both Langmuir and Freundlich features accurately

250

described the equilibrium behavior (RLF2 ＞0.99) over the concentration ranges

251

studied, and was thus chosen to fit the data in Figure 3c. It can be derived from the

252

modeling that the maximum adsorption capacity at monolayer coverage of CSGs was

253

75.7 mg/g, significantly higher than that of QFF (22.8 mg/g). Coinciding with the

254

fitting result from the original Langmuir isotherm, the value of n from QFF was 1,

255

indicating that QFF well followed the Langmuir equation which was based on the fact

256

that the adsorption process occurred in a monolayer that covered the surface of the

257

material. However, the n of CSGs was 0.3, suggesting that the adsorption of heparin

258

onto CSGs was controlled by multiple processes and multilayers of heparin possibly

259

formed around CSGs.35 This was in line with the observation that the adsorption

260

amounts for CSGs was significantly higher than QFF, indicating that CSGs showed

261

highly efficient adsorption for anion molecules such as sulfated polysaccharides.

262

Figure 3d shows the effects of the contact time on adsorption of heparin by CSGs.

263

The heparin adsorption reached equilibrium within 6 h, which was much faster than

264

that of 30 h from modified Sepabeads for fucoidan adsorption.36 The adsorption

265

kinetics of heparin was firstly investigated with the help of linear forms of

266

pseudo-first-order (Eq.(7)) and pseudo-second-order (Eq.(8)) models, namely

267

reaction-based models:37 1 K1 1 1 = + qt qe t qe t 1 1 = t + qt qe K2 qe2

(7) (8) 13



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268

where K1 (h-1) and K2 (g/(mg h)) are the rate constants of pseudo-first-order and

269

pseudo-second-order adsorption, and qt (mg/g) represents the amount of adsorption at

270

various time t. The corresponding kinetic parameters from both models are listed in

271

Table S2. It is important to note that R22 was higher than 0.999 with linear plots of t/qt

272

versus t (the inset in Figure 3d), and qe,cal (31.3 mg/g) value was much closer to the

273

experimental value qe,exp (29.9 mg/g) (Table S2), suggesting that the typical

274

pseudo-second-order kinetic model appeared to be feasible to describe the static

275

adsorption process. Therefore, it can be assumed that the chemisorption was the rate

276

limiting step of CSGs.38 However, the above equations could not give definite

277

mechanism. Consequently, Webber’s intraparticle diffusion model (Eq.(9)), a kind of

278

diffusion-based model, was then introduced to further understand the mechanism of

279

adsorption: qt = ki t 1/2 +C (9)

280

where ki (mg/(g h1/2)) is the intraparticle diffusion rate constant, and C (mg/g) is a

281

constant related to the thickness of the boundary layer. The plot of qt versus t1/2 is

282

shown in Figure S7. Clearly, the heparin adsorption process of CSGs was essentially

283

divided into three steps including film-diffusion (step1), intraparticle diffusion (step 2)

284

and equilibrium (step 3). As illustrated in Figure S7, the slope of step 1 was larger

285

than step 2, indicating that the intraparticle diffusion stage was a gradual process. In

286

addition, the linear plot of the second portion allows to obtain the values of ki and C

287

which are shown in Table S2. The smaller is the value of C, the greater the 14


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288

contribution of intraparticle diffusion is.39 With the C of 17.7 mg/g, the linear plot of

289

step 2 did not pass through the origin, suggesting that intraparticle diffusion was not

290

the rate-limiting step, and that external mass transfer of heparin molecules onto CSGs

291

described by film diffusion was remarkable in the adsorption process.39-40

292

Owing to the dominant electrostatic interaction between the heparin and CSGs

293

surface, the pH and ionic strength would play a key role in the desorption process.

294

Thus, two kinds of different potential stripping eluents, namely 4 M NaCl solution at

295

pH 4.6 and alkaline aqueous solution at pH 13, were subjected to investigate the

296

elution of heparin from CSGs. The pH values of solutions were adjusted with 0.1 M

297

NaOH or 0.1 M HCl aqueous solution. Desorption kinetics of heparin from CSGs is

298

presented in Figure 3e. The rapid desorption of heparin stripping from CSGs occurred

299

and reached equilibrium within 30 min both for NaCl and alkaline eluents. Moreover,

300

the quantitative desorption efficiencies from NaCl and alkaline eluents were 98.9%

301

and 88.3%, respectively, suggesting that electrostatic shielding effect from NaCl was

302

superior to deprotonation effect from alkaline solution to strip heparin from CSGs.

303

The reusability was checked by following adsorption-desorption process for five

304

cycles and the adsorption efficiency in each cycle was analyzed as shown in Figure 3f.

305

Clearly, after five cycles, the adsorption efficiency remained constant above 75%,

306

indicating that CSGs can be subjected to sustainable utilization. According to the

307

above results, CSGs adsorbents exhibited good stability and recycling.

308

Selective adsorption of CSGs. In practical purification of polysaccharides, 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

309

proteins are one of the undesirable impurities. Particularly, the strong electrostatic

310

interaction between anionic polysaccharides and proteins and their competitive

311

adsorption onto adsorbents lead to the hardship of deproteinization. As shown above,

312

CSGs possessed an excellent adsorption capacity for heparin with pH-responsive

313

behavior due to the change of surface charge of CSGs at different pH values.

314

Coincidentally, the surface charges carried on amphoteric proteins were also

315

pH-dependent. As shown in Figure 3b, the zeta potentials (ζ) of CSGs was positive at

316

low pH due to protonation of –NH2 (region 1 and 2), but became negative at higher

317

pH, showing isoelectric point at pH 7.4. While for BSA, ζBSA became negative for pH

318

＞4.9, showing about −7 mV of ζBSA in region 2. In comparison, several sulfated

319

polysaccharides such as heparin, fucoidan and carrageenan were negatively charged

320

over the whole investigated pH ranges and saturated at roughly −25 mV with high

321

charge density. In region 1 with pH lower than 4.9, BSA exhibits higher positive

322

charges, and the electrostatic attraction between BSA and sulfated polysaccharides

323

with negative charges was strong enough to form precipitate.41 Therefore, selective

324

adsorption of sulfated polysaccharides and BSA onto CSGs could only be discussed in

325

region 2 of pH 4.9-7.4, as a result of their significantly different surface charge

326

density. Figure 4a shows the competitive adsorption of heparin and BSA onto the

327

surface of CSGs around this region. It was noted that the adsorption efficiency of BSA

328

suddenly decreased when the pH value was higher 6.3, and dipped to only 12% for pH

329

7.4, at which the adsorption of heparin still retained ∼90%. When pH exceeded 7.4, 16


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330

the adsorption of heparin began to decrease. It has been reported that, at pH 6.5~7.0,

331

the intrinsic binding constants of BSA to heparin markedly decrease with an increase

332

of pH due to the increasing negative charge of BSA and the resultant increasing

333

repulsion between heparin and BSA.42 Therefore, it is not hard to understand that, at

334

pH 6.3~7.4, the adsorption of BSA sharply decreased but without change of heparin,

335

indicating the increasing electrostatic repulsion between heparin and BSA, along with

336

strong electrostatic interaction between heparin and CSGs. Thus, the separation of

337

heparin in the presence of BSA could be readily achieved by controlling the acidity of

338

the sample solution at pH 7.4. In principle, salt concentration is of prime importance

339

for influencing interactions of polyanion-polycation pair.19 The effect of ionic strength

340

on the adsorption of heparin and BSA onto CSGs was investigated by addition of

341

certain amount of NaCl at pH 4.6 (Figure 4b). Obviously, with an increase of the

342

concentration of NaCl within the range of up to 2 M, the adsorption of BSA was

343

significantly decreased from 66.4% to 15%. However, for heparin, the adsorption was

344

hardly changed until the NaCl concentration exceeded 2 M, showing good selectivity

345

for heparin adsorption against BSA in the range of 0.5-2 M. Accordingly, based on the

346

strong electrostatic interaction between CSGs and sulfated polysaccharides with high

347

charge density, it was able to control the solution pH (4.6-7.4) containing salt (0.5 M

348

NaCl) to achieve selective adsorption for sulfated polysaccharides against proteins.

349

It had been reported that functionalized chitosan in a more rigid matrix of silica gel

350

shows more efficient chromatographic separation procedures.43 As mentioned above, 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

351

CSG-Gel displayed higher compressive stress and modulus than ACS-Gel prepared by

352

traditional method. Therefore, column behavior of CSGs was investigated to further

353

examine the selectivity and understand the separation mechanism. Figure 5 shows the

354

chromatogram patterns of the adsorption and desorption for BSA, heparin,

355

heparin-BSA mixture and FN1-BSA mixture with AC buffers containing 1, 2, and 4 M

356

NaCl as the eluents. Firstly, breakthrough of BSA was initiated by elution with the

357

loading buffer, namely AC buffer (pH 4.6) containing 0.5 M NaCl. Clearly, there was

358

few BSA retained on the column after elution by the loading buffer (Figure 5a),

359

whereas BSA failed to be thoroughly washed from the column in the absence of NaCl

360

in AC buffer (Figure S8). These results clearly suggested that electrostatics screening

361

effect of 0.5 M NaCl resisted the electrostatic interactions between BSA and the

362

surface of CSGs, preventing the BSA from adsorption onto CSGs. By contrast,

363

heparin was not recovered after elution by the loading buffer until NaCl concentration

364

arrived at 4 M (Figure 5b), indicating that heparin was far more strongly retained on

365

CSGs owing to its higher negative charge density. Next, 3 mL of an aqueous 1:1

366

heparin-BSA mixture was loaded to the column, and stripping was then initiated by

367

stepwise elution with AC buffers containing 1, 2 and 4 M NaCl. Indeed, BSA could

368

not be adsorbed onto the column in the loading buffer, and heparin was stripped by 4

369

M NaCl (Figure 5c), confirming the selectivity. Similar observations were also found

370

for FN1-BSA mixtures (Figure 5d), demonstrating that CSGs-packed column was

371

able to effectively separate sulfated polysaccharides from BSA dissolved in AC buffer 18


Page 18 of 33

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372

containing 0.5 M NaCl. Consequently, CSGs were able to selectively separate

373

sulfated polysaccharides as adsorbents.

374

Purification and fractionation of fucoidan by CSGs. On the basis of the

375

conditions for selective separation of heparin and fucoidan, the CSGs were applied to

376

purify the crude fucoidan (FN0) in large-scale using the batch adsorption method. The

377

typical purification process using CSGs is shown in Figure 6. 30 g FN0 was first

378

dissolved in 3 L of AC buffer containing 0.5 M NaCl (Figure 6d), and then was

379

stirring with CSGs for 6 h to facilitate equilibrium adsorption (Figure 6e). The CSGs

380

carrying the trapped fucoidan were separated from the buffer with a filter to remove

381

the undesired protein, and then dipped into AC buffer containing 4 M NaCl (Figure 6f)

382

to strip fucoidan. After 30 min, the collected eluents (Figure 6g) were dialyzed and

383

lyophilized to get the purified fucoidan, coded as FN-CS with yield of 44.6%. To

384

compare the performance of CSGs, the present procedure was performed in parallel

385

with the commercially QFF and the resulting fucoidan was named as FN-QS.

386

Moreover, FN1 obtained from the Sevag method was also used for comparison (see

387

Materials section). The protein contents, deproteinization ratios and decoloration

388

ratios of FN-CS, FN-QS and FN1 are shown in Figure 7a. Clearly, FN-CS exhibited

389

the lowest protein content (1.2%), showing a favorable deproteinization ratio of 83%.

390

As shown in Figure 7b, FN-CS solution was colorless with decolorization ratio of

391

94%, which was comparable to that of FN1 (91%) decolored by H2O2 and far higher

392

than FN-QS (87%) by QFF. All these results demonstrated the high performance and 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

393

time saving of CSGs to remove proteins and pigments from the sulfated

394

polysaccharide just by one step.

395

Additionally, fractionation of fucoidan could be simply achieved by stepwise

396

adsorption following above steps. In brief, as the purification progress went to stepⅡ

397

(Figure 6e-f), the CSGs capturing fucoidan were stepwise eluted with 1.5 and 2.5

398

NaCl in AC buffer, respectively. After dialysis, the eluent from 2.5 NaCl (denoted as

399

CF2.5) was again adsorbed onto CSGs and stripped successively with 0.5, 1 and 2 M

400

Na2SO4, respectively, to obtain the CF2.5-0.5, CF 2.5-1 and CF2.5-2 fractions

401

according to the eluting order. The three fractions were presented colorless. The

402

contents of protein, uronic acid, ester sulphate and neutral sugars composition of

403

CF2.5 and its fractions were analyzed, and the results are summarized in Table 1.

404

Similar to the FN1 fractions from chromatographic method (SF0.5, SF1 and SF2),

405

protein and uronic acids contents of fractions from CSGs decreased distinctly with the

406

progress of fractionation, and the ester sulphate and Fuc contents (Fucose, the major

407

neutral sugar) increased. It is worth noting that the protein content of CF2.5-2 was just

408

0.3% with deproteinization ratio of 96%, indicating the powerful deproteinization of

409

CSGs by stepwise desorption. The apparent hydrodynamic radius distribution f (Rh) of

410

CF2.5 and its fractions in dilute solutions (≈0.4 mg/mL) at 25℃ and scattering angle

411

θ of 30° is shown in Figure 8a. The peak values reflect the most probable

412

hydrodynamic radius (Rh*) of fractions, which decreased in the order of CF2.5-0.5 to

413

2.5-2, suggesting that the hydrodynamic size of fractions decreased with the 20


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414

enhancement of ionic strength in the eluent. The trend was consistent with FN1

415

fractions from chromatographic separation based on DEAE Sepharose CL-6B (Figure

416

8b). The Rh value was determined according to the Stokes-Einstein relation from DLS,

417

and Mw was calculated from a Zimm plot.7 The experimental results are summarized

418

in Table 1. Clearly, the values of Mw and Rh for the CF2.5 fractions also decreased

419

with fractionation, further demonstrating that CF2.5 was successfully separated into

420

three fractions. The Mw and Rh values from FN1 and its fractions were much lower

421

than those of CF2.5 and its fractions. This could be explained that the strong

422

degradation had occurred by using H2O2 to decolor or the classic Sevag method to

423

deproteinize.44-45 Consequently, CSGs can be used for fractionation along with

424

efficient deproteinization and decoloration of the crude sulfated polysaccharide

425

without the risk of degradation in large-scale. Taken together, an easy scale-up

426

puriﬁcation and fractionation strategy based on CSGs absorbents in this work was

427

simple, green and highly efficient, greatly shortening the purification time.

428 429

Conclusion

430

New hydrogel microspheres cross-linked with genipin (CSGs) were successfully

431

prepared from chitosan in the 8 wt% LiOH/ 7 wt% KOH/ 8 wt% urea aqueous

432

solution at low temperature. This was a simple and "green" pathway for the

433

preparation of robust chitosan microspheres adsorbents. The mechanical strength of

434

the chitosan hydrogels fabricated from the alkaline solution system and cross-linked 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

435

with genipin was over 10 times that of the hydrogels prepared by the traditional acid

436

method. In contrast to the widely used QFF, CSGs exhibited mesoporous structure

437

and higher adsorption capacity for heparin. The adsorption equilibrium could be well

438

described by Langmuir-Freundlich isotherm, confirming the heterogeneity of CSGs

439

surface. The adsorption kinetics data were well fitted by pseudo-second-order model,

440

revealing that chemisorption was the determining step during adsorption process in

441

which the intraparticle diffusion was not the rate-limiting step. The reusability of

442

CSGs could be easily realized by 4 M NaCl solution which played a role in the

443

electrostatic shielding effect. In the purification of the crude fucoidan, CSGs

444

equilibrated at pH 4.6 containing 0.5 M NaCl exhibited high clearance of proteins and

445

pigments, which was superior to the conventional Sevag method and H2O2 bleaching.

446

In addition, fractionation by CSGs adsorbents was comparable to chromatographic

447

separation. Taken together, this work established an alternative one-step method with

448

high performance to purify the sulfated polysaccharides by using CSGs adsorbents,

449

which would has great potential in large-scale separation engineering applications.

450 451

Acknowledgements

452

This work was supported by the Major Program of National Natural Science

453

Foundation of China (21334005), Major International Joint Research Project

454

(21620102004), the National Natural Science Foundation of China (21574102,

455

21274114 and 20874079), Special National Key Research and Development Program 22


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456

of China (2016YFD0400202), and New Century Excellent Talents Program of

457

Education Ministry (NCET-13-0442).

458 459

Supporting Information

460

Characterizations of FTIR, swelling ratio and the compression tests; elution profile of

461

fucoidan on conventional column; chemical structure of genipin and heparin;

462

morphology and structure of QFF; preparation process of CSGs; FTIR, swelling ratio

463

and the compression tests results; intra-particle diffusion plot; adsorption and

464

desorption processes for BSA; Langmuir, Freundlich and Langmuir-Freundlich

465

isotherm parameters; and kinetic parameters.

466 467

References

468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486

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extraction of gastrodin from the aqueous extract of Gastrodia elata. J. Chromatogr. A 2014, 1342, 1-7. (38) Ho, Y. Review of second-order models for adsorption systems. J. Hazard. Mater. 2006, 136 (3), 681-689. (39) Xiong, L.; Yang, Y.; Mai, J.; Sun, W.; Zhang, C.; Wei, D.; Chen, Q.; Ni, J. Adsorption behavior of methylene blue onto titanate nanotubes. Chem. Eng. J. 2010, 156 (2), 313-320. (40) Al-Jubouri, S. M.; Holmes, S. M. Hierarchically porous zeolite X composites for manganese ion-exchange and solidification: Equilibrium isotherms, kinetic and thermodynamic studies. Chem. Eng. J. 2017, 308, 476-491. (41) Fang, Y.; Li, L.; Inoue, C.; Lundin, L.; Appelqvist, I. Associative and segregative phase separations of gelatin/κ-carrageenan aqueous mixtures. Langmuir 2006, 22 (23), 9532-9537. (42) Hattori, T.; Kimura, K.; Seyrek, E.; Dubin, P. L. Binding of bovine serum albumin to heparin determined by turbidimetric titration and frontal analysis continuous capillary electrophoresis. Anal. Biochem. 2001, 295 (2), 158-67. (43) Roosen, J.; Binnemans, K. Adsorption and chromatographic separation of rare earths with EDTA- and DTPA-functionalized chitosan biopolymers. J. Mater. Chem. A 2014, 2 (5), 1530-1540. (44) Hou, Y.; Wang, J.; Jin, W.; Zhang, H.; Zhang, Q. Degradation of Laminaria japonica fucoidan by hydrogen peroxide and antioxidant activities of the degradation products of different molecular weights. Carbohydr. Polym. 2012, 87 (1), 153-159. (45) Hsu, W.; Hsu, T.; Lin, F.; Cheng, Y.; Yang, J. P. Separation, purification, and α-glucosidase inhibition of polysaccharides from Coriolus versicolor LH1 mycelia. Carbohydr. Polym. 2013, 92 (1), 297-306.

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597

Graphic for manuscript

598 599

Figure 1. SEM images (a) and the size distribution (b) of CSGs. SEM images of the

600

representative surface (c, d) and cross-section (e) of CSGs.

601 602

Figure 2. Nitrogen adsorption-desorption isotherms (a) and the pore size distribution

603

of CSGs (b).

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

604 605

Figure 3. Influence of pH on the adsorption of heparin by CSGs (a). Zeta potentials of

606

CSGs, BSA (5 mg/mL) and sulfated polysaccharides (5 mg/mL) as a function of pH

607

(b). Equilibrium adsorption isotherms of heparin with CSGs and QFF at initial pH 4.6,

608

both of which were fitted with the Langmuir-Freundlich model (c). Kinetic of heparin

609

(initial concentration of 1 mg/mL) adsorption to CSGs and its pseudo-second-order

610

kinetics (inset) at initial pH 4.6 (d). Kinetics of desorption of heparin from CSGs in 28


Page 28 of 33

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611

aqueous 4 M NaCl solution at pH 4.6 and alkaline aqueous solution adjusted at pH 13,

612

respectively

613

adsorption/desorption cycles (f).

(e).

The

adsorption

efficiency

for

CSGs

in

consecutive

614 615

Figure 4. The pH dependent adsorption behaviors of heparin and BSA onto the

616

surface of CSGs (a). The dependence of heparin and BSA adsorption on the variation

617

of NaCl concentrations within a range of 0-4 M at pH 4.6 (b).

618 619

Figure 5. The chromatogram patterns of the adsorption and desorption for BSA (a) , 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

620

heparin (b), heparin-BSA mixture (c) and fucoidan-BSA mixture (d) on the surface of

621

the CSGs eluted stepwise with AC buffer containing 1, 2 and 4 M NaCl. The loading

622

buffer was AC buffer containing 0.5 M NaCl.

623 624

Figure 6. The typically schematic process for purification of crude fucoidan by using

625

CSGs as adsorbents. Pictures of crude fucoidan (a), CSGs carrying the trapped

626

fucoidan in AC buffer containing 0.5 M NaCl (b), the purified fucoidan (c) coupled

627

with the schematic illustration of the purification process (d-g). 30


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629 630

Figure 7. Deproteinization and decolorization ratios together with protein contents of

631

FN-CS (based on CSGs), FN-QS (based on commercially QFF) and FN1 (from Sevag

632

method) (a). Pictures of FN-QS, FN1 and FN-CS aqueous solutions at the

633

concentration of 40 mg/mL (b).

634 635

Figure 8. Rh distribution of fucoidan fractions from CSGs (a) and DEAE Sepharose

636

CL-6B (b) in 0.15 M NaNO3 at 25 ℃ (scattering angle θ of 30°).

637 638 639 31



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Table 1. Contents of protein, uronic acids, ester sulphate and neutral sugars

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composition together with Mw and Rh in fucoidan fractions purified by CSGs

643

adsorbents, compared with the conventional chemical method followed by

644

chromatographic separation. Samples

645

Mw×10-4

Rh

Gal

(g/mol)

(nm)

3.9

16.8

161.6 a

146

17.4

10.3

10.7

181.5 a

165

61.1

10.8

3.0

20.4

123.2 a

153

1.7

82.2

5.1

0.6

10.3

61.2 a

120

27.6

-

-

-

-

-

91.1 b

130

30.0

16.7

7.0

32.8

39.8

12.5

7.9

43.4 b

111

0.9

13.2

21.2

6.7

49.3

21.5

4.8

17.6

16.3 b

79

2.0

7.5

22.5

2.3

67.5

4.0

1.4

24.7

6.7 b

71

Protein

Uronic

Ester

Neutral sugars (mol%)

(%)

acids (%)

sulphate (%)

Rha

Fuc

Man

Glc

CF2.5

0.6

11.9

29.2

4.8

62.8

11.7

CF2.5-0.5

1.6

16.1

19.9

10.2

51.5

CF2.5-1

1.0

14.8

21.9

4.7

CF2.5-2

0.3

10.9

23.3

FN1

1.8

15.0

SF0.5

2.7

SF1

SF2

a: the dn/dc was 0.122 mL/g; b: the dn/dc was 0.137 mL/g.

646 647

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For Table of Contents Use Only

649 650

Highly Efficient One-step Purification of Sulfate Polysaccharides via Chitosan

651

Microspheres Adsorbents

652

Xueqin Wei, Jiangjiang Duan, Xiaojuan Xu*, Lina Zhang*

653

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072,

654

China

655

656 657 658 659

Synopsis: Chitosan microspheres were constructed as highly efficient adsorbents for purification of sulfated polysaccharides by one step with easy scale-up and recycling.

660

33


Highly Efficient One-Step Purification of Sulfated Polysaccharides via

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