Highly Efficient One-Step Purification of Sulfated Polysaccharides via

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Research Article pubs.acs.org/journal/ascecg

Highly Efficient One-Step Purification of Sulfated Polysaccharides via Chitosan Microspheres Adsorbents Xueqin Wei, Jiangjiang Duan, Xiaojuan Xu,* and Lina Zhang* College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China

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S Supporting Information *

ABSTRACT: Sulfated polysaccharides from marine biomass exhibit various biological activities, however, traditional purification approaches are time and money consuming, particularly involving the usage of toxic chemical reagents. Herein, novel chitosan microspheres cross-linked by genipin (CSGs) were prepared from chitosan solution in an alkali/urea aqueous system as multifunctional adsorbents for deproteinization and decoloration by one step. The compression fracture stress of chitosan hydrogels fabricated from the alkaline solution system was over 10 times that of the hydrogels prepared by a traditional acid method, suggesting a significantly improved mechanical strength. The robust CSGs with mean diameter of 100 μm exhibited mesoporous structure. The adsorption/desorption performance of CSGs for sulfated polysaccharides was investigated by using heparin as a model, showing significantly high adsorption capacity, over 3-fold of that from commercial Q Sepharose Fast Flow. Moreover, CSGs selectively adsorbed heparin in the presence of BSA at pH 4.6 by adding 0.5 M NaCl to screen electrostatics between CSGs and proteins. Furthermore, CSGs adsorbents were practically applied in purification and fractionation of crude fucoidan by stepwise elution with higher salt concentrations, showing a maximum deproteinization ratio of 96% and decolorization ratio of 94%. This work provided a simple, green, and highly efficient approach for purification and fractionation of sulfated polysaccharides. KEYWORDS: Chitosan, Scale-up purification, Fractionation of fucoidan, Green approach, Multifunctional adsorbent

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INTRODUCTION With nearly three years expedition, the Tara Oceans consortium, a team of multinational scientists, has found many unidentified microorganisms’ genes in the world oceans, suggesting the mysteries of marine biomass, and more efforts should be focused on them.1 Nowadays, it has been recognized that natural sulfated polysaccharides, such as fucoidan from brown algae, are one of the most promising glycan-based drugs candidates,2 which reportedly display a wide range of biological activities, such as antitumor, antiviral, anticoagulant, and immunomodulatory effects.3,4 However, they are still far from clinical applications due to the ambiguous structure− activity relationships resulting from impurities, unknown fine structures, and so forth.5 In our laboratory, we have accumulated experiences on the purification, chain conformation, and structure−bioactivity relationship of fungus6,7 and yeast polysaccharides.8,9 The issues of purification of polysaccharides should be first resolved. However, routine purification protocols including the Sevag method with a mixture of 1-butanol/chloroform to remove proteins and 30% H2O2 to decolor10,11 are not only toxic but also may cause degradation and structure alteration, thereby influencing bioactivities.12 Although chromatography packed with ion-exchange or gel-filtration materials have been widely used, the time and money consumption coupled with low capacity are not ignorable.13 Therefore, seeking an efficient, green, and cost-efficient © 2017 American Chemical Society

purification method by using novel adsorbents is extremely urgent. Adsorbents for selective adsorption of target biomolecules are sustaining innovations.14,15 In our laboratory, novel absorbents based on chitin for removal of metal ions, dyes, and oil have been reported.16−18 For negatively charged sulfated polysaccharides, polycation would be a good candidate for their adsorption.19 Chitosan, with a cationic nature, is produced from the deacetylation of chitin, consisting of β-(1−4)-linked D-glucosamine and N-acetyl-D-glucosamine. Owing to its abundant hydroxyl and amino groups, chitosan has been proved as an eco-friendly adsorbent material for environmental and biomedical applications.20,21 However, it is an undeniable fact that chitosan materials prepared from a traditional acid system suffer from poor mechanical properties.22,23 In our recent findings, high strength chitosan hydrogels with a nanofibrous structure obtained from chitosan solution in an alkali/urea aqueous system at low temperature via physical cross-linking display an unexpectedly high compression fracture stress about 100 times as much as that prepared from traditional acidic solvent.24 Therefore, we believe that chitosan fabricated in an alkali system should be an ideal material for Received: December 7, 2016 Revised: February 3, 2017 Published: February 14, 2017 3195

DOI: 10.1021/acssuschemeng.6b02975 ACS Sustainable Chem. Eng. 2017, 5, 3195−3203

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(FE-SEM, Zeiss, SIGMA). Surface area and pore size distribution were evaluated using nitrogen adsorption (Micromeritics, AsAp2020, U.S.A.). The specific surface area was calculated from the nitrogen adsorption isotherm using the Branauer−Emmett−Teller (BET) equation and the pore size distribution using the Barrett−Joyner− Halenda (BJH) model. The zeta potentials of CSGs were measured by a Zetasizer NanoZS (ZEN 3600, Malvern, U.K.) following the method described by Yan et al.25 Adsorption Performance of CSGs. Herein, heparin, the complex mixture of highly sulfated glycosaminoglycan,26 was used as a model of sulfated polysaccharide to investigate the adsorption characteristics of CSGs. Generally, 0.66 g of drained CSGs were mixed with 20 mL heparin solution. The mixed solutions were then kept shaking at ambient temperature for 12 h. The amount of heparin in the supernatant was measured by acid oxidation with 90% H2SO4 containing 25 mM borax27 and then monitored by a UV−vis recording spectrophotometer (UV-61, Meipuda, Shanghai, China) at 298 nm. For the batch adsorption experiment, the amount of heparin adsorbed onto CSGs (qe, mg/g) at equilibrium was calculated by the following formula:

selective adsorption of sulfated polysaccharides through electrostatic interactions between protonated −NH2 groups on chitosan and negatively charged sulfate groups from sulfated polysaccharides. In the present work, novel chitosan microspheres adsorbents were prepared from a chitosan solution in a LiOH/KOH/urea aqueous system at low temperature using W/O emulsion and then cross-linked by genipin to improve stability. The adsorption equilibrium and kinetics of heparin by chitosan microspheres (CSGs) were first tested. The selectivity of CSGs was studied and discussed with the separation of heparin from a model heparin−BSA mixture. Finally, the application of CSGs in purification of crude fucoidan was evaluated and compared to that of a chemical and chromatographic method. This work would provide a simple, green, and highly efficient method to separate and purify sulfated polysaccharides in large scale.

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EXPERIMENTAL SECTION

Materials. Commercial grade chitosan (degree of deacetylation = 89%) was purchased from Ruji Biotechnology Co., Ltd. (Shanghai, China) and used without further purification. Crude commercial fucoidan (coded as FN0) from Laminaria japonica was purchased from Rizhao Jiejing Ocean Biotechnology Development Co., Ltd. To improve the purity, FN0 was purified by using the classical methods as follows. First, 20 g of FN0 powder was subjected to the Sevag method to remove proteins and treated with 30% H2O2 to eliminate pigments followed by exhaustively dialyzing, concentrating, and finally lyophilizing to gain the purified fucoidan named as FN1. Next, 4.4 g of FN1, dissolved in 250 mL of ultrapure water, was subjected to fractionation by anion-exchange chromatography on the DEAE Sepharose CL-6B column (φ5 cm × 30 cm) and stepwise elution with 0.5, 1, and 2 M NaCl to obtain three fractions coded as SF0.5, SF1, and SF2, respectively (Figure S1). Genipin was attained from Linchuan Zhixin Biotechonlogy Co., Ltd. (Jiangxi, China). Heparin (sodium salt with activity 160 U/mg, Mw = 6000−20000) was bought from Toyobo (Japan). The chemical structures of genipin and heparin are shown in Figure S2. Q Sepharose Fast Flow, with a mean diameter of 90 μm, BET surface area of 195 m2/g, and mesoporous structure (Figure S3), was bought from Pharmacia (Sweden). Other chemical reagents from commercial sources were of analytical grade and used without further purification. Preparation of Chitosan Microspheres. To prepare the chitosan solutions, chitosan powder was first dispersed into the alkaline aqueous solvent of LiOH/KOH/urea/H2O (8:7:8:77 by weight) and frozen at −35 °C for 6 h. Then, the frozen solid was thoroughly thawed by stirring vigorously at ambient temperature. After freezingthawing twice, 4 mL of epichlorohydrin as a cross-linker was added into 200 g of the chitosan solution at −20 °C with stirring for 2 h. The obtained solution was centrifuged at 7000 rpm for 10 min at 5 °C to remove air bubbles, and the transparent chitosan solution with the concentration of 4 wt % was thus obtained, labeled as TCS solution. Subsequently, a well-mixed suspension containing 450 mL isooctane and 4.5 mL Span 85 was dispersed in a three-necked round-bottomed flask at 0 °C and stirred for 10 min. Then, 150 mL of the TCS solution was rapidly dropped into the suspension which was kept stirring for 30 min in a water bath at 0 °C to achieve stable water/oil emulsion droplets. After removing the water bath, the suspension was continuously stirred for 4 h at room temperature. Afterward, the suspension was promptly poured into a solution of ethanol/water (3:2 by volume) and stirred for 1 h to obtain chitosan microspheres coded as CSs. The microspheres were then washed repeatedly with ethanol and deionized water to remove all residual reagent. For further cross-linking CSs, 100 mL of the drained microspheres were introduced into 40 mL of 5 mg/mL genipin solution, which was stirred at 40 °C for 3 h until microspheres started to turn dark blue. The final chitosan microspheres were cleaned with distilled water and coded as CSGs. Characterization of CSGs. The structure and morphology of CSGs were characterized by field-emission scanning electron microscopy

qe =

Vi(c0 − ce) mad

(1)

where c0 and ce are the initial and equilibrium concentrations of heparin in the solution, respectively (mg/L). Also, Vi is the volume of the heparin solution (L), and mad is the mass of adsorbent (g). For the adsorption kinetics experiment, CSGs were mixed with 1 mg/mL of aqueous heparin and then shaken at ambient temperature. At desired time intervals, the remaining amount of heparin in the aqueous solution was then determined. Separation by Column Packed with CSGs. Using the slurry packing method, CSGs suspended in the loading buffer were packed in a glass column (φ10 mm × 600 mm) to form a 15 cm bed, which was eluted with the loading buffer at a flow rate of 1 mL/min for 24 h at room temperature for equilibrium and stability. The sample solution was added onto the top of the column, followed by an additional 150 mL of loading buffer. Stripping of the sample was next initiated by stepwise elution with buffers containing 1, 2, and 4 M NaCl, respectively. Fractions of 3 mL were collected and monitored by UV−vis spectrophotometer at 280 nm for BSA and 298 nm (acid oxidation with 90% H2SO4) for polysaccharides. Characterization of Sulfated Polysaccharide. To characterize the weight-average molecular weight (Mw) and hydrodynamic radius (Rh) of sulfated polysaccharides in 0.15 M NaNO3 aqueous solution, static and dynamic light scattering (SLS and DLS) measurements were carried out on a light scattering spectrometer (ALV/CGS-3, ALV GmbH, Germany).28 The specific refractive index increments (dn/dc) were determined with a differential refractometer (Optilab T-rEX, Wyatt Technology Co.) after the sulfated polysaccharides were dialyzed against the solvent for 72 h. Protein content was quantified using a bicinchoninic acid (BCA) assay with bovine serum albumin (BSA) as the standard.29 The deproteinization ratio was calculated by the following equation:

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

(2)

where m0 and me are the contents of proteins before and after deproteinization, respectively. The decoloration ratio was determined by the following equation:

Decoloration ratio (%) = (A 0 − Ae)/A 0 × 100

(3)

where A0 and Ae are the absorbance of the polysaccharides dissolved in 27% acetone at 340 nm determined by UV−vis spectrophotometer before and after decoloration, respectively. Ester sulfate content was measured after acid hydrolysis (1 M HCl at 100 °C for 6 h,) using the turbidimetric method.30 The total uronic acid content was colorimetrically determined using the m-hydroxydiphenyl assay31 with glucuronic acid as the standard. Neutral sugars composition analysis of the final samples was performed by gas chromatography (6890N, Agilent, U.S.A.).8 3196

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RESULTS AND DISCUSSION Morphology, Structure, and Properties of CSGs. The chitosan microspheres (CSs) prepared from the chitosan solution were further cross-linked by genipin (CSGs), resulting in the color change of microspheres from light yellow to dark blue (Figure S4). The cross-linking of CSGs was confirmed by FTIR (Figure S5). As shown in Figure 1a, CSGs exhibited a

(Figure S6b) indicated that the compressive fracture stress of the chitosan hydrogels from the alkaline system (CS-Gel) (4.0 MPa) and genipin cross-linked CS-Gel (CSG-Gel) (3.4 MPa) were much higher than that obtained by the conventional acidic method (ACS-Gel) (0.3 MPa). The compression fracture stress of the chitosan hydrogels was over 10 times that of the hydrogels prepared by the traditional acid method. It was not hard to imagine that the mechanical strength of the chitosan hydrogel microspheres prepared from an alkaline solution system could be significantly improved. With genipin cross-linking, CSG-Gel exhibited a higher static compressive modulus judging from the slope of the stress−strain curve linear region. Therefore, the porous and robust CSGs can be used as biosorbents. Adsorption, Desorption, and Reusability of CSGs. Herein, heparin was selected as a model sulfated polysaccharide to evaluate the adsorption behaviors of CSGs. Figure 3a shows the effects of initial pH (2−13) on the adsorption of heparin onto CSGs at the same initial concentration (0.5 mg/mL). Obviously, the adsorption efficiency of heparin remained high (>90%) at pH ≤ 7, then dropped rapidly with increasing pH, and finally decreased to almost nil at pH > 10. It is easily understood that the decreased protonation of amino groups on CSGs with increasing pH leaded to negative charges of CSGs, confirmed by the zeta potentials of CSGs (ζCSGs) as a function of initial pH in Figure 3b. The ζCSGs decreased as the pH value increased, and the isoelectric point was found to be 7.4. Thus, it could be concluded that high adsorption occurred at a low initial pH, as a result of the enhanced electrostatic interactions between more protonated −NH2 groups on CSGs and negatively charged heparin molecules. Figure 3c shows the adsorption isotherms of CSGs and commercially available Q Sepharose Fast Flow (QFF, a strong anion exchanger chemically attached quaternary ammonium ligands: −O−CH2CHOHCH2OCH2CHOHCH2N+(CH3)3) in 50 mM acetic acid/sodium acetate buffer at pH 4.6 (AC buffer). It was noted that CSGs displayed a remarkably higher adsorption capacity than QFF. To quantify the maximum theoretical sorption capacity of adsorbents for heparin, data points were fitted with three commonly used adsorption models of Langmuir (eq 4), Freundlich (eq 5), and Langmuir−Freundlich (eq 6)33,34 isotherms as described in the following equations:

Figure 1. SEM images (a) and size distribution (b) of CSGs. SEM image of the representative surface (c, d) and cross-section (e) of CSGs.

spherical shape with a mean diameter of 100 μm, and both the surface and cross-section (Figure 1c−e) of the CSGs displayed porous microstructures. To further characterize the porous structures of CSGs, a nitrogen adsorption−desorption measurement was carried out. Figure 2a shows a typical hysteresis loop within a relative pressure P/P0 ranging from 0.4 to 1, suggesting the characteristic mesoporous structure.32 The BET surface area of CSGs was determined to be 77 m2/g, and the pore size ranged from 3 to 113 nm with the peak value of 23 nm (Figure 2b). The structural stability and mechanic strength of the hydrogel microspheres are important for their practical applications. The swelling behaviors of microspheres before and after genipin cross-linking in an acidic environment (Figure S6a) indicated that the swelling degree of CSGs was much lower than that of CSs. Nowadays, there is no standard test method for the measurement of the mechanical properties of the hydrogel microspheres, thus the compression tests of the chitosan hydrogels constructed under the same condition of the preparation of the hydrogel microspheres except for the absence of water/oil emulsion were performed to compare their mechanical strength. The typical compressive stress−strain curves

qe =

qmKLce 1 + KL ce

qe = KFce1/ n ′

(4)

(5)

Figure 2. Nitrogen adsorption−desorption isotherms (a) and pore size distribution of CSGs (b). 3197

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Figure 3. Influence of pH on the adsorption of heparin by CSGs (a). Zeta potentials of CSGs, BSA (5 mg/mL), and sulfated polysaccharides (5 mg/mL) as a function of pH (b). Equilibrium adsorption isotherms of heparin with CSGs and QFF at initial pH 4.6, both of which were fitted with the Langmuir−Freundlich model (c). Kinetic of heparin (initial concentration of 1 mg/mL) adsorption to CSGs and its pseudo-second-order kinetics (inset) at initial pH 4.6 (d). Kinetics of desorption of heparin from CSGs in aqueous 4 M NaCl solution at pH 4.6 and alkaline aqueous solution adjusted at pH 13. (e). Adsorption efficiency for CSGs in consecutive adsorption/desorption cycles (f).

qe =

qm(KLFce)n n

1 + (KLFce)

controlled by multiple processes, and multilayers of heparin possibly formed around CSGs.35 This was in line with the observation that the adsorption amounts for CSGs were significantly higher than QFF, indicating that CSGs showed highly efficient adsorption for anion molecules such as sulfated polysaccharides. Figure 3d shows the effect of the contact time on adsorption of heparin by CSGs. The heparin adsorption reached equilibrium within 6 h, which was much faster than that of 30 h from modified Sepabeads for fucoidan adsorption.36 The adsorption kinetics of heparin was first investigated with the help of linear forms of pseudo-first-order (eq 7) and pseudosecond-order (eq 8) models, namely, reaction-based models:37

(6)

where qm (mg/g) is theoretical maximum adsorption capacity; KL (L/mg), KF (mg/g(L/mg)1/n), and KLF (L/mg) are equilibrium constants of Langmuir, Freundlich, and Langmuir− Freundlich, respectively. Here, 1/n′ is the Freundlich sorption intensity constant which is usually between 0.1 and 1 pointing out favorable sorption; n is the index of heterogeneity which varies from 0 and 1 (when n = 1 the model reduces to the Langmuir isotherm). The estimated model parameters along with correlation coefficients (R2) are summarized in Table S1. Clearly, the Langmuir−Freundlich isotherm which combined both Langmuir and Freundlich features accurately described the equilibrium behavior (RLF2 > 0.99) over the concentration ranges studied and was thus chosen to fit the data in Figure 3c. It can be derived from the modeling that the maximum adsorption capacity at monolayer coverage of CSGs was 75.7 mg/g, significantly higher than that of QFF (22.8 mg/g). Coinciding with the fitting result from the original Langmuir isotherm, the value of n from QFF was 1, indicating that QFF well followed the Langmuir equation which was based on the fact that the adsorption process occurred in a monolayer that covered the surface of the material. However, the n of CSGs was 0.3, suggesting that the adsorption of heparin onto CSGs was

⎛ K ⎞⎛ 1 ⎞ 1 1 = ⎜⎜ 1 ⎟⎟⎜ ⎟ + ⎝ ⎠ qt qe ⎝ qe ⎠ t

(7)

⎛1⎞ t 1 = ⎜⎜ ⎟⎟t + qt K 2qe 2 ⎝ qe ⎠

(8)

−1

where K1 (h ) and K2 (g/(mg h)) are the rate constants of pseudo-first-order and pseudo-second-order adsorption, respectively, and qt (mg/g) represents the amount of adsorption at various time t. The corresponding kinetic parameters from both 3198

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Figure 4. pH-dependent adsorption behaviors of heparin and BSA onto the surface of CSGs (a). The dependence of heparin and BSA adsorption on the variation of NaCl concentrations within a range of 0−4 M at pH 4.6 (b).

models are listed in Table S2. It is important to note that R22 was higher than 0.999 with linear plots of t/qt versus t (inset in Figure 3d), and the qe,cal (31.3 mg/g) value was much closer to the experimental value qe,exp (29.9 mg/g) (Table S2), suggesting that the typical pseudo-second-order kinetic model appeared to be feasible to describe the static adsorption process. Therefore, it can be assumed that the chemisorption was the rate-limiting step of CSGs.38 However, the above equations could not give a definite mechanism. Consequently, Webber’s intraparticle diffusion model (eq 9), a kind of diffusion-based model, was then introduced to further understand the mechanism of adsorption:

qt = k it 1/2 + C

efficiency in each cycle was analyzed as shown in Figure 3f. Clearly, after five cycles, the adsorption efficiency remained constant above 75%, indicating that CSGs can be subjected to sustainable utilization. According to the above results, CSGs adsorbents exhibited good stability and recycling. Selective Adsorption of CSGs. In practical purification of polysaccharides, proteins are one of the undesirable impurities. Particularly, the strong electrostatic interaction between anionic polysaccharides and proteins and their competitive adsorption onto adsorbents lead to the hardship of deproteinization. As shown above, CSGs possessed an excellent adsorption capacity for heparin with pH-responsive behavior due to the change in surface charge of CSGs at different pH values. Coincidentally, the surface charges carried on amphoteric proteins were also pH-dependent. As shown in Figure 3b, the zeta potentials (ζ) of CSGs was positive at low pH due to protonation of −NH2 (regions 1 and 2) but became negative at higher pH, showing an isoelectric point at pH 7.4. While for BSA, ζBSA became negative for pH > 4.9, showing about −7 mV of ζBSA in region 2. In comparison, several sulfated polysaccharides such as heparin, fucoidan, and carrageenan were negatively charged over the whole investigated pH ranges and saturated at roughly −25 mV with high charge density. In region 1 with pH lower than 4.9, BSA exhibited higher positive charges, and the electrostatic attraction between BSA and sulfated polysaccharides with negative charges was strong enough to form precipitate.41 Therefore, selective adsorption of sulfated polysaccharides and BSA onto CSGs could only be discussed in region 2 of pH 4.9−7.4, as a result of their significantly different surface charge density. Figure 4a shows the competitive adsorption of heparin and BSA onto the surface of CSGs around this region. It was noted that the adsorption efficiency of BSA suddenly decreased when the pH value was higher 6.3 and dipped to only 12% for pH 7.4, at which the adsorption of heparin still retained ∼90%. When pH exceeded 7.4, the adsorption of heparin began to decrease. It has been reported that at pH 6.5−7.0 the intrinsic binding constants of BSA to heparin markedly decrease with an increase in pH due to the increasing negative charge of BSA and the resultant increasing repulsion between heparin and BSA.42 Therefore, it is not hard to understand that at pH 6.3−7.4 the adsorption of BSA sharply decreased but without change in heparin, indicating the increasing electrostatic repulsion between heparin and BSA, along with a strong electrostatic interaction between heparin and CSGs. Thus, the separation of heparin in the presence of BSA could be readily achieved by controlling the acidity of the sample solution at pH 7.4. In principle, salt concentration is of prime importance for influencing interactions of polyanion−polycation pairs.19 The effect of ionic

(9)

where ki (mg/(g h1/2)) is the intraparticle diffusion rate constant, and C (mg/g) is a constant related to the thickness of the boundary layer. The plot of qt versus t1/2 is shown in Figure S7. Clearly, the heparin adsorption process of CSGs was essentially divided into three steps including film diffusion (step1), intraparticle diffusion (step 2), and equilibrium (step 3). As illustrated in Figure S7, the slope of step 1 was larger than step 2, indicating that the intraparticle diffusion stage was a gradual process. In addition, the linear plot of the second portion allowed us to obtain the values of ki and C which are shown in Table S2. The smaller is the value of C, the greater the contribution of intraparticle diffusion is.39 With a C of 17.7 mg/g, the linear plot of step 2 did not pass through the origin, suggesting that intraparticle diffusion was not the rate-limiting step and that external mass transfer of heparin molecules onto CSGs described by film diffusion was remarkable in the adsorption process.39,40 Owing to the dominant electrostatic interaction between the heparin and CSGs surface, the pH and ionic strength would play a key role in the desorption process. Thus, two kinds of different potential stripping eluents, namely, 4 M NaCl solution at pH 4.6 and alkaline aqueous solution at pH 13, were subjected to investigate the elution of heparin from CSGs. The pH values of solutions were adjusted with 0.1 M NaOH or 0.1 M HCl aqueous solution. Desorption kinetics of heparin from CSGs is presented in Figure 3e. The rapid desorption of heparin stripping from CSGs occurred and reached equilibrium within 30 min for both NaCl and alkaline eluents. Moreover, the quantitative desorption efficiencies from NaCl and alkaline eluents were 98.9% and 88.3%, respectively, suggesting that the electrostatic shielding effect from NaCl was superior to the deprotonation effect from the alkaline solution to strip heparin from CSGs. The reusability was checked by following the adsorption−desorption process for five cycles, and the adsorption 3199

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Figure 5. Chromatogram patterns of the adsorption and desorption for BSA (a), heparin (b), heparin−BSA mixture (c), and fucoidan−BSA mixture (d) on the surface of the CSGs eluted stepwise with AC buffer containing 1, 2, and 4 M NaCl. The loading buffer was an AC buffer containing 0.5 M NaCl.

NaCl. Indeed, BSA could not be adsorbed onto the column in the loading buffer, and heparin was stripped by 4 M NaCl (Figure 5c), confirming the selectivity. Similar observations were also found for FN1−BSA mixtures (Figure 5d), demonstrating that the CSGs-packed column was able to effectively separate sulfated polysaccharides from BSA dissolved in AC buffer containing 0.5 M NaCl. Consequently, CSGs were able to selectively separate sulfated polysaccharides as adsorbents. Purification and Fractionation of Fucoidan by CSGs. On the basis of the conditions for selective separation of heparin and fucoidan, the CSGs were applied to purify the crude fucoidan (FN0) in large scale using the batch adsorption method. The typical purification process using CSGs is shown in Figure 6. Thirty grams of FN0 was first dissolved in 3 L of AC buffer containing 0.5 M NaCl (Figure 6d) and then stirred with CSGs for 6 h to facilitate equilibrium adsorption (Figure 6e). The CSGs carrying the trapped fucoidan were separated from the buffer with a filter to remove the undesired protein and then dipped into AC buffer containing 4 M NaCl (Figure 6f) to strip fucoidan. After 30 min, the collected eluents (Figure 6g) were dialyzed and lyophilized to get the purified fucoidan and coded as FN-CS with a yield of 44.6%. To compare the performance of CSGs, the present procedure was performed in parallel with the commercial QFF, and the resulting fucoidan was named FN-QS. Moreover, FN1 obtained from the Sevag method was also used for comparison (see Materials section). The protein contents, deproteinization ratios, and decoloration ratios of FN-CS, FN-QS, and FN1 are shown in Figure 7a. Clearly, FN-CS exhibited the lowest protein content (1.2%), showing a favorable deproteinization ratio of 83%. As shown in Figure 7b, the FN-CS solution was colorless with a decolorization ratio of 94%, which was comparable to that of FN1 (91%) decolored by H2O2 and far higher than FN-QS (87%) by QFF. All these results demonstrated the high performance and time savings of CSGs to remove proteins and pigments from the sulfated polysaccharide just by one step. Additionally, fractionation of fucoidan could be simply achieved by stepwise adsorption following the above steps. In brief, as

strength on the adsorption of heparin and BSA onto CSGs was investigated by the addition of certain amounts of NaCl at pH 4.6 (Figure 4b). Obviously, with an increase in the concentration of NaCl within the range of up to 2 M, the adsorption of BSA was significantly decreased from 66.4% to 15%. However, for heparin, the adsorption was hardly changed until the NaCl concentration exceeded 2 M, showing good selectivity for heparin adsorption against BSA in the range of 0.5−2 M. Accordingly, based on the strong electrostatic interaction between CSGs and sulfated polysaccharides with high charge density, it was able to control the solution pH (4.6−7.4) containing salt (0.5 M NaCl) to achieve selective adsorption for sulfated polysaccharides against proteins. It had been reported that functionalized chitosan in a more rigid matrix of silica gel shows more efficient chromatographic separation procedures.43 As mentioned above, CSG-Gel displayed higher compressive stress and modulus than ACSGel prepared by the traditional method. Therefore, column behavior of CSGs was investigated to further examine the selectivity and understand the separation mechanism. Figure 5 shows the chromatogram patterns of the adsorption and desorption for BSA, heparin, heparin−BSA mixture, and FN1− BSA mixture with AC buffers containing 1, 2, and 4 M NaCl as the eluents. First, breakthrough of BSA was initiated by elution with the loading buffer, namely, AC buffer (pH 4.6) containing 0.5 M NaCl. Clearly, there were few BSA retained on the column after elution by the loading buffer (Figure 5a), whereas BSA failed to be thoroughly washed from the column in the absence of NaCl in the AC buffer (Figure S8). These results clearly suggested that the electrostatics screening effect of 0.5 M NaCl resisted the electrostatic interactions between BSA and the surface of CSGs, preventing BSA from adsorption onto CSGs. By contrast, heparin was not recovered after elution by the loading buffer until NaCl concentration arrived at 4 M (Figure 5b), indicating that heparin was far more strongly retained on CSGs owing to its higher negative charge density. Next, 3 mL of an aqueous 1:1 heparin−BSA mixture was loaded onto the column, and stripping was then initiated by stepwise elution with AC buffers containing 1, 2, and 4 M 3200

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to obtain the CF2.5-0.5, CF 2.5-1, and CF2.5-2 fractions according to the eluting order. The three fractions were presented as colorless. The contents of the protein, uronic acid, ester sulfate, and neutral sugars composition of CF2.5 and its fractions were analyzed, and the results are summarized in Table 1. Similar to the FN1 fractions from chromatographic method (SF0.5, SF1, and SF2), the protein and uronic acids contents of fractions from CSGs decreased distinctly with the progress of fractionation, and the ester sulfate and Fuc contents (Fucose, the major neutral sugar) increased. It is worth noting that the protein content of CF2.5-2 was just 0.3% with a deproteinization ratio of 96%, indicating the powerful deproteinization of CSGs by stepwise desorption. The apparent hydrodynamic radius distribution f (Rh) of CF2.5 and its fractions in dilute solutions (≈0.4 mg/mL) at 25 °C and scattering angle θ of 30° is shown in Figure 8a. The peak values reflect the

Figure 6. Typical schematic process for purification of crude fucoidan by using CSGs as adsorbents. Pictures of crude fucoidan (a), CSGs carrying the trapped fucoidan in AC buffer containing 0.5 M NaCl (b), purified fucoidan (c), and coupled with the schematic illustration of the purification process (d−g). Figure 8. Rh distribution of fucoidan fractions from CSGs (a) and DEAE Sepharose CL-6B (b) in 0.15 M NaNO3 at 25 °C (scattering angle θ of 30°).

most probable hydrodynamic radius (Rh*) of fractions, which decreased in the order of CF2.5-0.5 to 2.5-2, suggesting that the hydrodynamic size of the fractions decreased with the enhancement of ionic strength in the eluent. The trend was consistent with FN1 fractions from chromatographic separation based on DEAE Sepharose CL-6B (Figure 8b). The Rh value was determined according to the Stokes−Einstein relation from DLS, and Mw was calculated from a Zimm plot.7 The experimental results are summarized in Table 1. Clearly, the values of Mw and Rh for the CF2.5 fractions also decreased with fractionation, further demonstrating that CF2.5 was successfully separated into three fractions. The Mw and Rh values from FN1 and its fractions were much lower than those of CF2.5 and its fractions. This could be explained as that the strong degradation had occurred by using H2O2 to decolor or the classic Sevag

Figure 7. Deproteinization and decolorization ratios together with protein contents of FN-CS (based on CSGs), FN-QS (based on commercially QFF), and FN1 (from Sevag method) (a). Pictures of FN-QS, FN1, and FN-CS aqueous solutions at a concentration of 40 mg/mL (b).

the purification progress went to step II (Figure 6e,f), the CSGs capturing fucoidan were stepwise eluted with 1.5 and 2.5 NaCl in AC buffer, respectively. After dialysis, the eluent from 2.5 NaCl (denoted as CF2.5) was again adsorbed onto CSGs and stripped successively with 0.5, 1, and 2 M Na2SO4, respectively,

Table 1. Contents of Protein, Uronic Acids, Ester Sulfate, and Neutral Sugars Composition Together with Mw and Rh in Fucoidan Fractions Purified by CSGs Adsorbents, Compared with the Conventional Chemical Method Followed by Chromatographic Separation neutral sugars (mol %)

a

sample

protein (%)

uronic acid (%)

ester sulfate (%)

Rha

Fuc

Man

Glc

Gal

Mw × 10−4 (g/mol)

Rh (nm)

CF2.5 CF2.5-0.5 CF2.5-1 CF2.5-2 FN1 SF0.5 SF1 SF2

0.6 1.6 1.0 0.3 1.8 2.7 0.9 2.0

11.9 16.1 14.8 10.9 15.0 30.0 13.2 7.5

29.2 19.9 21.9 23.3 27.6 16.7 21.2 22.5

4.8 10.2 4.7 1.7 − 7.0 6.7 2.3

62.8 51.5 61.1 82.2 − 32.8 49.3 67.5

11.7 17.4 10.8 5.1 − 39.8 21.5 4.0

3.9 10.3 3.0 0.6 − 12.5 4.8 1.4

16.8 10.7 20.4 10.3 − 7.9 17.6 24.7

161.6a 181.5a 123.2a 61.2a 91.1b 43.4b 16.3b 6.7b

146 165 153 120 130 111 79 71

The dn/dc was 0.122 mL/g. bThe dn/dc was 0.137 mL/g. 3201

DOI: 10.1021/acssuschemeng.6b02975 ACS Sustainable Chem. Eng. 2017, 5, 3195−3203

Research Article

ACS Sustainable Chemistry & Engineering method to deproteinize.44,45 Consequently, CSGs can be used for fractionation along with efficient deproteinization and decoloration of the crude sulfated polysaccharide without the risk of degradation in large scale. Taken together, an easy scaleup purification and fractionation strategy based on CSGs absorbents in this work was simple, green, and highly efficient, greatly shortening the purification time.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Major Program of National Natural Science Foundation of China (21334005), Major International Joint Research Project (21620102004), National Natural Science Foundation of China (21574102, 21274114 and 20874079), Special National Key Research and Development Program of China (2016YFD0400202), and New Century Excellent Talents Program of Education Ministry (NCET-13-0442).

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CONCLUSION New hydrogel microspheres cross-linked with genipin (CSGs) were successfully prepared from chitosan in the 8 wt % LiOH/7 wt % KOH/8 wt % urea aqueous solution at low temperature. This was a simple and “green” pathway for the preparation of robust chitosan microspheres adsorbents. The mechanical strength of the chitosan hydrogels fabricated from the alkaline solution system and cross-linked with genipin was over 10 times that of the hydrogels prepared by the traditional acid method. In contrast to the widely used QFF, CSGs exhibited mesoporous structure and higher adsorption capacity for heparin. The adsorption equilibrium could be well described by the Langmuir−Freundlich isotherm, confirming the heterogeneity of CSGs surface. The adsorption kinetics data were well fitted by the pseudo-second-order model, revealing that chemisorption was the determining step during adsorption process in which the intraparticle diffusion was not the rate-limiting step. The reusability of CSGs could be easily realized by a 4 M NaCl solution which played a role in the electrostatic shielding effect. In the purification of the crude fucoidan, CSGs equilibrated at pH 4.6 containing 0.5 M NaCl exhibited high clearance of proteins and pigments, which was superior to the conventional Sevag method and H2O2 bleaching. In addition, fractionation by CSGs adsorbents was comparable to chromatographic separation. Taken together, this work established an alternative one-step method with high performance to purify the sulfated polysaccharides by using CSGs adsorbents, which would have great potential in large-scale separation engineering applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b02975. Characterizations of FTIR, swelling ratio, and the compression tests; elution profile of fucoidan on conventional column; chemical structure of genipin and heparin; morphology and structure of QFF; preparation process of CSGs; FTIR, swelling ratio, and the compression tests results; intraparticle diffusion plot; adsorption and desorption processes for BSA; Langmuir, Freundlich, and Langmuir−Freundlich isotherm parameters; and kinetic parameters. (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-27-87219274. Fax: +86-27-68762005. E-mail: xuxj@ whu.edu.cn (X. Xu). *Tel.: +86-27-87219274. Fax: +86-27-68762005. E-mail: [email protected] (L. Zhang). ORCID

Lina Zhang: 0000-0003-3890-8690 3202

DOI: 10.1021/acssuschemeng.6b02975 ACS Sustainable Chem. Eng. 2017, 5, 3195−3203

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