Colloids and Surfaces B: Biointerfaces 115 (2014) 340–348

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Polypropylene non-woven meshes with conformal glycosylated layer for lectin affinity adsorption: The effect of side chain length Xiang-Yu Ye, Xiao-Jun Huang, Zhi-Kang Xu ∗ MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China

a r t i c l e

i n f o

Article history: Received 13 September 2013 Received in revised form 6 December 2013 Accepted 10 December 2013 Available online 21 December 2013 Keywords: Polypropylene non-woven mesh Conformal grafting Surface glycosylation Affinity membrane Lectin adsorption

a b s t r a c t The unique characteristics of polypropylene non-woven meshes (PPNWMs), like random network of overlapped fibers, multiple connected pores and overall high porosity, make them high potentials for use as separation or adsorption media. Meanwhile, carbohydrates can specifically recognize certain lectin through multivalent interactions. Therefore glycosylated PPNWMs, combing the merits of both, can be regarded as superior affinity membranes for lectin adsorption and purification. Here, we describe a versatile strategy for the glycosylation of PPNWMs. Two hydrophilic polymers with different side chain length, poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(oligo(ethylene glycol) methacrylate) (POEGMA), were first conformally tethered on the polypropylene fiber surface by a modified plasma pretreatment and benzophenone (BP) entrapment UV irradiation process. Then glucose ligands were bound through the reaction between the hydroxyl group and acetyl glucose. Chemical changes of the PPNWMs surface were monitored by FT-IR/ATR. SEM pictures show that conformal glucose ligands can be achieved through the modified process. After deprotection, the glycosylated PPNWMs became superhydrophilic and had high specific recognition capability toward Concanavalin A (Con A). Static Con A adsorption experiments were further performed and the results indicate that fast adsorption kinetics and high binding capacity can be accomplished at the same time. We also found that increasing the side chain length of polymer brushes had positive effect on protein binding capacity due to improved chain mobility. Model studies suggest a multilayer adsorption behavior of Con A. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Polypropylene non-woven meshes (PPNWMs) are one kind of the most popular membrane materials due to their excellent mechanical properties, high thermal and chemical stabilities, and comparatively low-cost. In addition, the random network of overlapped fibers offers many unique characteristics, including relatively large specific surface area, engineered interconnected pores and overall high porosity. Computational fluid dynamics (CFD) simulation results reveal that such disordered fibrous structure endows non-woven meshes with high permeability, low pressure drop and reduced mass transfer/diffusion resistance [1]. Therefore, PPNWMs exhibit promising prospects for comprehensive applications in air/liquid filtration or adsorption media, biomedical textile and protective cloth [2–6].

∗ Corresponding author. Tel.: +86 571 8795 2605; fax: +86 571 8795 1773. E-mail address: [email protected] (Z.-K. Xu). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.025

Among these promising applications of PPNWMs, membrane chromatography for protein bioseparation has captured growing attention in recent years [3,7–10]. Industrial protein purification process is conventionally carried out using packed bed or column chromatography, which has several major limitations such as slow intraparticle diffusion, large pressure drop, high cost and hence low protein binding capacity/productivity and difficulty in scale-up. The unique structural characteristics of PPNWMs make them the most promising alternative materials to replace traditional packedbed resins [11,12]. For example, PPNWMs grafted with conformal anion exchange ligands and hydrophilic spacers were prepared through a UV pretreatment-UV grafting process, and it was found that such activated meshes had high bovine serum albumin binding capacity and permeability coefficient [9]. Even when stacking 150 layers in a column, the pressure drop was still acceptable and it helped to create sharp chromatographic elution profiles [13]. It was also reported that the presence of a molecular spacer between the ligand and the membrane matrix could be beneficial for protein adsorption [14]. Further attempts to increase the binding capacity led to the creation of PPNWMs supported/macroporous gel based 3D binding domain composite membrane [10]. However,

X.-Y. Ye et al. / Colloids and Surfaces B: Biointerfaces 115 (2014) 340–348

the separation mechanism was mainly based on non-specific interactions like electrostatic attraction/repulsion, hydrophobic interaction and Vander Waals forces. It is a logical expectation that the introduction of affinity ligands with specific interaction with target proteins can result in high separation efficiency and purity. Carbohydrates are widely regarded as energy sources and building elements. With the development of glycobiology, their structurally and functionally diverse roles are gradually discovered [15]. Carbohydrate–protein recognition mediates various biological processes and is the first step in cell–cell interactions via multivalent interactions, or glycocluster effect [16]. Lectin, as a kind of carbohydrate-binding proteins or glycoproteins, can specifically recognize certain carbohydrate moieties [17]. Meanwhile, carbohydrates are highly hydrophilic molecules that non-specific protein adsorptions can be greatly reduced or eliminated. Therefore, glycosylated PPNWMs are reasonably expected as superior affinity membranes for lectin adsorption and purification. In the literature, a few studies have reported the fabrication of glycosylated poly(ethylene terephthalate) fibers [18–20]. However, it is rarely addressed on the bioseparation performance of glycosylated non-woven meshes. In this work, our goal is to develop a facile method for the construction of glycosylated PPNWMs and further evaluate their application prospects as affinity membranes for lectin purification. First, a modified plasma pretreatment and benzophenone entrapment UV irradiation process was employed to graft conformal and uniform poly(oligo(ethylene glycol) methacrylate) and poly(2hydroxyethyl methacrylate) (POEGMA and PHEMA) brushes on the fiber surfaces of PPNWMs. Then surface glycosylation was accomplished by using different acetyl glycosyl donors in the presence of boron trifluoride diethyl etherate and subsequent deprotection. The surface wettability, qualitative fluorescein-labeled lectin and quantitative static Con A adsorption were characterized. Furthermore, the effect of side chain length on adsorption capacity was also evaluated.

2. Experimental 2.1. Materials Commercial PPNWMs (Jiangyin Golden Phoenix Special Textile Co., Ltd, China) used in this work were produced with a melt-blown process. The fiber diameter was in the range of 5–10 ␮m. The density and porosity were about 35 g/m2 and 80%, respectively. The samples were cut into rotundity with a diameter of 2.5 cm (S = 4.91 cm2 ), washed with acetone and dried in a vacuum oven at 40 ◦ C. 2-Hydroxyethyl methacrylate (HEMA, 97%) and oligo(ethylene glycol) methacrylate (OEGMA, Mn = 360, 99%) were supplied by Sigma–Aldrich and passed through neutral Al2 O3 flash column chromatography to remove inhibitors. Benzophenone (BP) was recrystallized from cold ethanol. Boron trifluoride diethyl etherate (BF3 ·Et2 O) was purified by vacuum distillation. Dichloromethane was distilled from phosphorus pentoxide immediately before use. Trichloroacetonitrile, 2,3,4,6-tetraacetyl-␤-d-glucose and ␤-d-glucose pentaacetate were bought from Beijing Chemsynlab Pharmaceutical Science & Technology Co. Ltd. and used as received. Concanavalin A (Con A), fluorescein-labeled Con A (FL-Con A) and peanut agglutinin (FL-PNA) (Vector, USA) were purchased and used directly. All the other chemicals like sodium methoxide, heptane, petroleum ether (60–70 ◦ C), potassium carbonate, and 2-[4-(2-hydroxyethyl)1-piperazinyl]ethanesulfonic acid (HEPES) were of analytical grade and used without further purification. Water used in all experiments was deionized and ultrafiltrated to 18 M using an ELGA LabWater system (ELGA Classic UF, France).

341

2.2. Graft polymerization of OEGMA and HEMA onto PPNWMs The experimental procedure is schematically illustrated in Fig. 1. Dielectric barrier discharge plasma was used to pretreat the nascent PPNWM at atmospheric pressure by a plasma apparatus (Nanjing Suman Electronics Co., Ltd., China). Two quartz glass plates with a gap of 2 mm served as the dielectric layer and argon (99%)/air (1%) was used as the discharge gas. Firstly, the sample was irradiated at 35 V and 10 kHz for a given time and exposed in the air for 10 min. After that, the pretreated PPNWMs were immersed in 20 mM BP heptane solution for 45 min to immobilize the photoinitiator in the surface layer of the polypropylene fiber and then dried in the air. Thereafter, the PPNWMs were presoaked with acetone and immediately dipped into OEGMA or HEMA solutions in petri dishes and fixed between two sheets of filter paper. Finally, UV grafting polymerization on the surfaces of PPNWMs was carried out under a homemade high pressure mercury lamp (232–400 nm, intensity 3 mW/cm2 ) for a predetermined time. The grafted PPNWMs were washed thoroughly with ethanol overnight to remove unreacted monomer and homopolymer, and then dried in a vacuum oven at 40 ◦ C. They were weighed with an analytical balance to a precision of 0.01 mg (XP105DR, Mettler Toledo, Switzerland). The grafting density (GD, ␮g/cm2 ) was calculated by the following equation: GD =

W1 − W0 S

(1)

where W0 and W1 are the mass of the nascent and the POEGMA/PHEMA grafted PPNWMs, respectively. S represents the surface area of each sample. 2.3. Coupling of glucose ligands to hydroxyl groups of the POEGMA/PHEMA grafted PPNWMs Surface glycosylation reaction was conducted as reported in our previous work [21]. Three pieces of POEGMA/PHEMA grafted PPNWMs were fully immersed in 6 mL freshly dried dichloromethane solution containing 20 equiv glucose pentaacetate and 100 equiv BF3 ·Et2 O. The reaction was sealed in a Schlenk tube and carried out at 0 ◦ C for 2 h followed by 20 h at room temperature. After that, the samples were washed extensively with ethanol and dried in a vacuum oven at 40 ◦ C. Glycosyl trichloroacetimidate exhibiting excellent glycosyl donor properties was also tested, and the general synthesis procedure was as follows [22]. 2,3,4,6-Tetraacetyl-␤-d-glucose (2 g) was dissolved in freshly dried dichloromethane (20 mL), and treated with trichloroacetonitrile (4 mL) and finely powdered K2 CO3 (2 g). The solution was stirred at room temperature and monitored by thin layer chromatography (TLC) till the complete consumption of the raw material. The reaction mixture was filtered and concentrated. Crude product was further purified by column chromatography [ethy1 acetate–petroleum ether (1:2, v/v)] to afford 2,3,4,6-tetraacetyl-␣-d-glucopyranosy trichloroacetimidate as a yellow syrup in yield of 80.3% ([␣]20 D = +92.5 (CHCl3 ), 1 H NMR (500 MHz, CDCl3 ) ı (ppm):8.69 (s, 1H, C(NH)CCl3 ), 6.56 (d, 1H, J = 3.6 Hz, H-1), 5.56 (t, 1H, H-3), 5.18(t, 1H, H-4), 5.13 (dd, 1H, H2), 4.25–4.12 (m, 3H, H-5, H-6), 2.07(s, 3H, oAc), 2.04 (s, 3H, oAc), 2.03(s, 3H, oAc), 2.01(s, 3H, oAc)). The glycosylation procedure was the same as described before. The binding density (BD, ␮g/cm2 ) of glucose ligands and the reaction ratio (R, %) of hydroxyl groups were calculated according to the following equations: BD = R=

W2 − W1 S

Mn (W2 − W1 ) × 100% 330(W1 − W0 )

(2)

(3)

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Fig. 1. Schematic representation for the glycosylation of PPNWMs and the lectin affinity adsorption process.

where W1 and W2 are the mass of POEGMA/PHEMA grafted and corresponding acetyl glucose modified PPNWMs, respectively. Mn represents the molecular weight of OEGMA (360) or HEMA (130). The acetyl groups were deprotected by dipping the samples in 50 mg/mL sodium methoxide/methanol solution for 90 min at room temperature and washed with de-ionized water several times. The as-prepared glycosylated PPNWMs were assigned as PPNWMPOEGMA-Glu and PPNWM-PHEMA-Glu, respectively. Each of the grafting and glycosylation data reported was an average of at least three parallel experiments. 2.4. Characterization FT-IR/ATR spectra were carried out on Nicolet Nexus 6700 equipped with an ATR accessory (ZnSe crystal, 45◦ ). Thirty two scans were taken for each spectrum at a normal resolution of 4 cm−1 . Surface morphology of PPNWMs was observed using a field-emission scanning electron microscope (FESEM, Hitachi S4800, Japan) after samples were sputtering coated with gold. Water contact angle (WCA) was measured to characterize the surface hydrophilicity of PPNWMs on a CTS-200 contact angle system (Mighty Technology Pvt. Ltd., China) at room temperature by sessile drop method. Briefly, a water drop (2 ␮L) was carefully dropped onto the sample surface with a microsyringe. Then images of the water droplet were recorded and contact angles were calculated with system software in equal time intervals.

(10 mM, pH 7.5, containing 0.1 mM Ca2+ , 0.15 M Na+ , 0.01 mM Mn2+ (not for PNA)). Subsequently, they were immersed in 200 ␮L of FL-Con A and FL-PNA HEPES buffer solutions with a concentration of 50 ␮g/mL for 2 h at 25 ◦ C. The samples were then rinsed with fresh HEPES buffer solution 6 times by gentle shaking, each time using 200 ␮L solution for 10 min. After being dried under vacuum at room temperature, fluorescent images of the sample surfaces were captured by fluorescence microscopy (Nikon ECLIPES Ti-U, Japan). Blue light was adopted to excite the fluorophore FITC and exposure time was 100 ms. Bradford method was further used to quantitatively determine the lectin binding capacity and adsorption kinetics of nascent PPNWMs, PPNWM-POEGMA-Glu and PPNWM-PHEMA-Glu using Con A as the protein standard, on a UV spectrophotometer (UV-2450, Shimadzu, Japan). The amount of adsorbed Con A was estimated from the differences of Con A concentration before and after incubation at 25 ◦ C. Prior to adsorption experiments, the samples were soaked in ethanol for 120 min and washed completely by HEPES buffer solution just as before. The dynamic Con A adsorption process was measured by immersing a piece of the glycosylated PPNWMs in 15 mL Con A solution with a concentration 0.1 mg/mL, and then 0.3 mL solution was taken out every 10 min until the concentration reached a constant value. The Con A adsorption isotherms were determined by incubating the glycosylated PPNWMs in 3 mL Con A solutions with different concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL) and the adsorption time was set for 3 h to ensure equilibrium adsorption.

2.5. Static protein adsorption

3. Results and discussion

The bound glucose amount on the PPNWM surface was fixed at 0.3 mg for comparison in the following experiments. Firstly, fluorescein-labeled lectin adsorption assays were performed to qualitatively evaluate the specific recognition capability of the glycosylated PPNWMs. Samples with diameter of 3 mm were wetted by ethanol for 30 min and replaced by HEPES buffer solution

3.1. Conformal grafting of POEGMA and PHEMA by the plasma-pretreatment and BP-entrapment UV irradiation process As shown in Fig. 1, the glycosylation procedure of PPNWMs includes two steps: the surface grafting of functional polymer chains, and the reaction between the functional groups and

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Plasma Pretratment Time (s)

600

0 10 20 40 60

2

GD ( g/cm )

500 400 300 200 100

(a) 0 5

10

15

20

25

UV Time (min) 1200 Plasma 0 s Plasma 40 s

1000 800 2

GD ( g/cm )

saccharide derivatives. Advantages of this strategy are complex synthesis and purification of vinyl sugar monomers can be avoided and the glycosylation reaction does not affect the bulk properties even at high glycosylation density [23,24]. Here, we chose POEGMA and PHEMA as functional polymer chains because they are widely regarded as hydrophilic polymers that can greatly resist non-specific protein adsorption, which is beneficial to affinity membrane application [25]. Furthermore, the hydroxyl group ( OH) is readily coupled with different acetylated saccharides in the presence of BF3 ·Et2 O and one can compare the effect of side chain length ( CH2 CH2 O ) on protein binding capacity. Surface UV grafting has been commonly employed due to fast reaction rate, simple equipment and versatile for various vinyl monomers [26]. BP is the most widely used photoinitiator for the surface modification of PPNWMs, and different initiating types have been reported including soaking, adsorption, entrapment, and sequential living grafting [27]. BP entrapping method is very simple, based on pre-swelling and subsequent exchanging, the photoinitiators were tightly immobilized in the surface layer of the polypropylene fibers [28]. This process has distinct advantages including the more control of grafted layer structure leading to an improved membrane adsorber performance and less homopolymerization reactions in solution caused by dissolved BP. However, one important issue has to take into consideration is the spatial grafting uniformity of the polymer chains, because UV light decays continuously as passing through the non-woven meshes [9,29]. What’s more, polypropylene fibers of commercial PPNWMs are intact, non-porous and contain small amount of inhibitors or antioxidants. Therefore, a modified UV process is necessary for the uniform surface modification of PPNWMs and BP distribution plays a key role here. A plasma pretreatment was introduced and its effect on UV grafting of OEGMA was carefully studied (Fig. 2). It can be seen that all the grafting kinetic curves have a ∼5 min polymerization induction period which is ascribed to the residual oxygen in the aqueous solutions. After this period, the grafting density increases rapidly with increasing UV irradiation time and then levels off due to the steric inhibition from nearby grafted chains. It is interesting to see that the plasma pretreatment has a significant impact on the UV grafting process, and even when the pretreatment time is as short as 10 s the grafting density increases dramatically. 40 s is the optimum pretreatment time, however, further increasing time to 60 s shows adverse effect as long plasma exposure time would result in decomposition (etching effect) and cross-linking reaction on the polypropylene fiber surface. The plasma pretreatment also increases the grafting density at the same monomer concentration and UV irradiation time. The increased UV grafting density by the introduction of plasma pretreatment may be mainly attributed to three aspects. First, plasma pretreatment can introduce polar groups onto the fiber surface and usually results in increased surface waviness or roughness [30], even sometimes obvious wrinkled or grooved structure appears (Fig. 3b). Both of them tend to promote the adsorption and entrapment of BP to the fiber surface. Second, plasma pretreatment will generate peroxide/hydroperoxide groups on the fiber surface and thus free radicals can be produced under UV irradiation [31–33]. We found that a very small amount of OEGMA could be grafted on PPNWMs even without BP-entrapment step. Third, the plasma pretreatment greatly increases the hydrophilicity of PPNWMs and facilitates the diffusion of hydrophilic monomer from solution to the fiber surface. Furthermore, plasma pretreatment improves the conformity of grafted POEGMA on the fiber surface (Fig. 3c and d). PPNWMs without pretreatment have many ribbon-like fibers while the plasma pretreated one preserve the nascent cylindrical shape after grafting. The improvement in conformity is ascribed to the enhanced BP and monomer distribution

343

600 400 200

(b) 0

4

6

8

10

12

14

16

18

20

22

OEGMA Concentration (% w/v) Fig. 2. Effect of (a) plasma pretreatment time and UV irradiation time (OEGMA concentration was fixed at 15% (w/v)) and (b) OEGMA concentration (UV irradiation time was fixed at 18 min) on the grafting density.

uniformity [9], or else autoacceleration effect and chain transfer reaction would even cause the formation of POEGMA gel [34]. Under the optimum plasma pretreatment time 40 s, the effects of UV irradiation time and monomer concentration on the grafting behavior of HEMA were further conducted and similar results were also found (Fig. S1 in Supplementary material). In short, by using a modified plasma-pretreatment and BP-entrapment UV irradiation process, we achieved conformal grafting of POEGMA and PHEMA on the fiber surfaces of PPNWMs with high grafting density. 3.2. Surface glycosylation and structure characterization The glycosylated PPNWMs were prepared through chemical reaction between the grafted chains and glucose pentaacetate under BF3 ·Et2 O catalysis followed by a deacetylation process. The influences of grafting density of POEGMA or PHEMA on the binding density of glucose ligands and the reaction ratio of hydroxyl groups with excess glucose pentaacetate (20 equiv) and BF3 ·Et2 O (100 equiv) were studied. Typical results are shown in Fig. 4. It can be seen that the binding density increases remarkably with increasing grafting density and ∼140 ␮g/cm2 of acetyl glucose moiety can be reached. The reaction ratios of hydroxyl groups lie in the range of 10–30% and the case of POEGMA only has slightly higher values than that of PHEMA. The glycosylation reaction here is heterogeneous and the steric hindrance effect is predominant. Therefore, it

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Fig. 3. SEM pictures of the pristine and modified PPNWMs: (a) nascent; (b) plasma pretreatment; (c) POEGMA grafted with plasma pretreatment and (d) POEGMA grafted without plasma pretreatment. The insets show the surface morphology of a single polypropylene fiber.

declines at first for the reaction ratio of hydroxyl groups. With further increasement of the grafting density, the reaction ratio slowly increases due to a significant increase in the concentration of reaction substrate and catalyst. 2,3,4,6-Tetraacetyl-␣-d-glucopyranosy trichloroacetimidate was also synthesized and used, as glycosyl trichloroacetimidates are very powerful glycosyl donors under acidic conditions. It was reported that a high degree of lactose conjugation onto the hydroxyl-terminated PEO star and dendritic polymers could be achieved by trichloroacetimidate glycosidation methodology, using BF3 ·Et2 O in homogeneous solution reaction [35]. However, we found that the conjugation ratio of hydroxyl groups by using glycosyl trichloroacetimidate was approximate to glucose pentaacetate (data not shown). This result is mainly because both of them experience a SN 2 type reaction mechanism and form oxocarbenium ion intermediates [36], and thus the reaction ratio of hydroxyl groups is limited by the steric hindrance effect. Finally, the deprotection of acetyl groups of saccharide residues was accomplished to yield glycosylated PPNWMs [21]. FT-IR/ATR spectroscopy was used to monitor the chemical changes of PPNWMs surface (Fig. 5). Compared with the nascent PPNWMs, POEGMA and PHEMA grafted ones exhibit additional intense adsorption peak at 1725 cm−1 , which is ascribed to the C O stretching vibration aroused by the major characteristic carbonyl groups. It should be noted that POEGMA grafted PPNWM has higher peak intensity of C O C (1165 cm−1 ) than PHEMA grafted one as it contains more CH2 CH2 O chain segments. When the acetyl glucose moieties were bound, the O H stretching vibration (3100–3500 cm−1 ) decreases yet not vanishes, demonstrating the existence of partial unreacted hydroxyl groups. Furthermore, peak at 1750 cm−1 can be observed, associated with the acetyl protected glucose groups. After deprotection, the band at 1750 cm−1 disappears in accordance with the mass reduction caused by complete removal of the acetyl groups, and the band ranging from

3100–3500 cm−1 becomes much more intense, which is ascribed to the exposed free hydroxyl groups on the glucose ligands. The surface morphology of the glycosylated PPNWMs was observed by FESEM (Fig. S2 in Supplementary material). Very conformal glucose ligands can be attained on both POEGMA and PHEMA grafted PPNWM surfaces. Besides, the glycosylated PPNWMs can well preserve the nascent cylindrical fiber shape and the pore structure with high glucose binding density, which are important physical factors for protein affinity adsorption. Surface wettability was also evaluated by WCA experiments, and it is found that glycosylation can endow PPNMWs with superhydrophilicity. (Fig. S3 in Supplementary material). 3.3. Lectin affinity adsorption by the glycosylated PPNWMs FL-Con A and FL-PNA were used to qualitatively investigate the specific recognition capability of the glycosylated PPNWMs. The bound glucose amount was fixed at 0.3 mg (ensure glycocluster effect) for comparing the influences of the side chain length. Con A shows specificity for mannose, glucose and acetyl glucosamine with 3-,4-,6-hydroxyl groups while PNA binds preferentially to galactose and acetyl galactosamine [37]. As shown in Fig. 6, slight green fluorescence was detected on the nascent PPNWMs incubating in FL-Con A and FL-PNA solutions, which results from non-specific protein adsorption on the hydrophobic surfaces. In contrast, no fluorescence was observable on the glycosylated PPNWMs surface after exposure to FL-PNA solution while notably green fluorescence appeared after exposure to FL-Con A solution. It indicates that the glycosylated PPNWMs can bind a large amount of Con A and resist non-specific PNA adsorption at the same time. These glycosylated PPNWMs with high specific recognition capability can be used as affinity membranes for Con A purification, and the quantitative Con A binding capacity was

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160

40

(A)

140 2

120 30

100 80 60

Ratio (%)

Glucose BD ( g/cm )

345

20

d

40

c

(a)

20 100

200

300

400

500

600

b a

10 700

3500

3000

2500

2

POEGMA GD ( g/cm )

2000

1500

1000

-1

Wavenumber (cm ) 30

180

2

25

140

(B)

120

20

100 15

80 60

Ratio (%)

Glucose BD ( g/cm )

160

10

40

(b) 20

100

200

300

400

d c b a

5

2

3500

3000

2500

2000

1500

1000

-1

Wavenumber (cm )

PHEMA GD ( g/cm ) Fig. 4. Effect of the GD of (a) POEGMA and (b) PHEMA on the binding density of glucose ligands and reaction ratio of hydroxyl groups. Glucose pentaacetate was 20 equiv and BF3 • Et2 O was 100 equiv relative to the monomer unit grafted on the PPNWMs.

further determined by Bradford method. It should be mentioned that both POEGMA and PHEMA grafted PPNWMs greatly resisted non-specific protein adsorption (Fig. S4 in Supplementary material) and no protein adsorption could be detected using Bradford method. The adsorption kinetics of Con A on the nascent PPNWMs, PPNWM-POEGMA-Glu and PPNWM-PHEMA-Glu were first studied (Fig. 7a). It can be clearly seen that Con A had fast adsorption rate on the studied PPNWMs and the amount of adsorbed Con A tended to reach an equilibrium value after 60 min. The nascent PPNWMs showed about 10 ␮g/cm2 Con A binding capacity due to the non-specific hydrophobic interaction, while the binding capacity of PPNWM-POEGMA-Glu and PPNWM-PHEMA-Glu was around 60 ␮g/cm2 and 40 ␮g/cm2 , respectively. The introduction of glucose ligands greatly increased the Con A binding capacity and the increased side chain length had a positive effect on the Con A adsorption. Mammen and Stenzel suggested it was important to match the distance between the saccharide ligand and the recognition sites of lectin for good binding configuration [38,39]. It is evident here that POEGMA has a flexible (CH2 CH2 O)6 spacer increasing the chain mobility and then enhancing the lectin binding capacity. Compared with our previous glycosylated microporous polypropylene membrane [40], we found that glycosylated PPNWMs had faster adsorption kinetics (60 min vs. 5 h) and higher protein binding capacity (0.3 mg glucose/0.3 mg Con A vs. 0.64 mg

Fig. 5. FT-IR/ATR spectra of (A) POEGMA and (B) PHEMA tethered PPNWMs: (a) nascent; (b) OH grafted; (c) acetyl glucose grafted; (d) glycosylated. The insets show the spectra in the range of 1650–1850 cm−1 .

glucose/0.12 mg Con A) under identical conditions, which can be mainly attributed to the unique inter-connected pore structure that significantly reduces the mass transport and diffusional resistance. The static adsorption isotherm was carried out in different Con A concentrations and the adsorption time was fixed at 3 h to reach equilibrium adsorption. As shown in Fig. 7b, the amount of Con A adsorbed on the glycosylated PPNWMs increased sharply at the very beginning and then leveled off approaching saturation values. The Con A binding capacity decreased in the following sequence: PPNWM-POEGMA-Glu > PPNWM-PHEMA-Glu > the nascent PPNWM, which further demonstrates the roles of glucose ligands and flexible CH2 CH2 O spacer. Langmuir (monolayer) and Freundlich (multilayer) adsorption models were both used to quantitatively analyze the adsorption isotherms, detailed instructions and calculated data are listed in Table 1. We found that both of the two models verified that PPNWM-POEGMA-Glu had the highest adsorption capacity, and the Freundlich model fitted the adsorption isotherm in a better way compared with Langmuir model. This result suggests a multilayer adsorption behavior of Con A on the glycosylated PPNWMs. It can be further theoretically evaluated by concerning the 3D size of Con A (molecular weight 104 000 Da, 6.7 nm × 11.3 nm × 12.2 nm [41]), the total surface area of polypropylene fibers and adsorbed amount of Con A in the sample. In our case, the theoretical saturation capacity is in the range of 0.13–0.23 ␮g/cm2 when Con A is in monolayer adsorption using geometric consideration. For glycosylated

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Fig. 6. Fluorescent pictures of the PPNWMs surfaces after incubating with (a–c) FL-Con A and (a’–c’) FL-PNA: (a, a’) nascent; (b, b’) PPNWM-POEGMA-Glu; (c, c’) PPNWMPHEMA-Glu. The bound glucose amount was fixed at 0.3 mg for comparison. The scale bar is 50 ␮m, 400× magnification.

PPNWMs, the theoretical Con A binding capacity on single fiber can be calculated by the following equation:

q=

W4 D 4W0

(4)

where q is the Con A binding capacity ␮g/cm2 , W0 and W4 are the mass of the nascent PPNWMs (16 mg) and adsorbed Con A, respectively.  is the density of polypropylene (0.91 g/cm3 ) and D is the average diameter of polypropylene fibers (8 ␮m). Calculated results show that there are approximately at least 15 layers of Con A molecules stacked around each fiber. The multilayer adsorption

Table 1 Adsorption behaviors of Con A on the studied PPNWMs. Sample

Nascent PPNWMs PPNWM-PHEMA-Glu PPNWM-POEGMA-Glu

Langmuir adsorptiona

Freundlich adsorptionb

Ka (× 105 )

Qe (␮g/cm2 )

R2

Log Kf

n

R2

3.77 3.25 5.03

57.24 158.98 188.32

0.975 0.903 0.905

1.21 1.63 1.81

0.49 0.49 0.43

0.956 0.969 0.958

a Data were calculated by equation [C]/Q = [C]/Qe + 1/Qe × 1/Ka , where Q is the measured amounts of adsorbed Con A, Qe is the theoretical equilibrium amounts of adsorbed Con A, [C] is the equilibrium concentration of Con A in solution, and Ka is the value of adsorption equilibrium constant. b Data were calculated by equation log Q = log Kf + n log[C], where Kf and n are the Freundlich characteristic constants indicating adsorption capacity and adsorption intensity, respectively.

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and Zhejiang Provincial Innovative Research Team (Grant no. 2009R50004).

70 60

PPNWM-POEGMA-Glu

Appendix A. Supplementary data

2

Q ( g/cm )

50

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.12.025.

40 PPNWM-PHEMA-Glu 30 20

References

10

Nascent

(a)

0 0

20

40

60

80

100

120

Time (min) 160 140

PPNWM-POEGMA-Glu

120

2

Q ( g/cm )

100 PPNWM-PHEMA-Glu

80 60 40 20

Nascent

0 -20

347

(b) 0.0

0.2

0.4

0.6

0.8

1.0

Equilibrium protein concenration (mg/mL) Fig. 7. (a) Adsorption kinetics of Con A (concentration 0.1 mg/mL) and (b) Con A adsorption isotherms (adsorption time 3 h) on PPNWMs at 25 ◦ C. The bound glucose amount was fixed at 0.3 mg for comparison.

behavior of Con A is reasonable, because Con A is a glycoprotein with intermolecular interactions and can diffuse into the inner layer of the flexible polymer brushes [14,42]. Furthermore, such affinity membranes can be easily regenerated by 1 M HAc [42]. 4. Conclusions In summary, glycosylated PPNWMs were successfully prepared by a modified UV grafting and chemical reaction process. The rapid plasma pretreatment and BP entrapment UV irradiation method resulted in conformal and uniform POEGMA/PHEMA brushes on the polypropylene fiber surface with high grafting density. The conjugation ratio between the hydroxyl groups and acetyl glucose ligands was limited by the steric hindrance effect under BF3 ·Et2 O catalysis. After deprotection, the as-prepared glycosylated PPNWMs had superhydrophilicity and high specific recognition capability toward Con A. The introduction of flexible CH2 CH2 O spacer further increased the protein binding capacity due to improved chain mobility. Con A had fast adsorption rate, multilayer adsorption behavior and high binding capacity on the glycosylated PPNWMs, which make them promising affinity membranes for the separation and purification of lectins. Acknowledgments The authors are grateful to the financial support from the National Natural Science Foundation of China (Grant no. 50933006)

[1] S. Jaganathana, H.V. Tafreshib, B. Pourdeyhimi, A realistic approach for modeling permeability of fibrous media: 3-D imaging coupled with CFD simulation, Chem. Eng. Sci. 63 (2008) 244–252. [2] R.S. Barhate, S. Ramakrishna, Nanofibrous filtering media: filtration problems and solutions from tiny materials, J. Membr. Sci. 296 (2007) 1–8. [3] V. Orr, L.Y. Zhong, M.M. Young, C.P. Chou, Recent advances in bioprocessing application of membrane chromatography, Biotechnol. Adv. 31 (2013) 450–465. [4] A. Prudente, C.L.Z. Riccetto, M.M.S.G. Simoes, B.M. Pires, M.G. Oliveira, Impregnation of implantable polypropylene mesh with S-nitrosoglutathione-loaded poly(vinyl alcohol), Colloid. Surf. B: Biointerfaces 108 (2013) 178–184. [5] S. Lee, S.K. Obendorf, Developing protective textile materials as barriers to liquid penetration using melt-electrospinning, J. Appl. Polym. Sci. 102 (2006) 3430–3437. [6] R. Uppal, G. Bhat, C. Eash, K. Akato, Meltblown nanofiber media for enhanced quality factor, Fibers Polym. 14 (2013) 660–668. [7] E. Klein, Affinity membranes: a 10-year review, J. Membr. Sci. 179 (2000) 1–27. [8] H.Y. Liu, Y. Zheng, P.V. Gurgel, R.G. Carbonell, Affinity membrane development from PBT nonwoven by photo-induced graft polymerization, hydrophilization and ligand attachment, J. Membr. Sci. 428 (2013) 562–575. [9] Y. Zheng, H.Y. Liu, P.V. Gurgel, R.G. Carbonell, Polypropylene nonwoven fabrics with conformal grafting of poly(glycidyl methacrylate) for bioseparations, J. Membr. Sci. 364 (2010) 362–371. [10] D.M. Kanani, E. Komkova, T. Wong, A. Mika, R.F. Childs, R. Ghosh, Separation of human plasma proteins HSA and HIgG using high-capacity macroporous gel-filled membranes, Biochem. Eng. J. 35 (2007) 295–300. [11] Z. Ma, S. Ramakrishna, Electrospun regenerated cellulose nanofiber affinity membrane functionalized with protein A/G for IgG purification, J. Membr. Sci. 319 (2008) 23–28. [12] L.J. Zhu, L.P. Zhu, Z. Yi, J.H. Jiang, B.K. Zhu, Y.Y. Xu, Hemocompatible and antibacterial porous membranes with heparinized copper hydroxide nanofibers as separation layer, Colloids Surf. B: Biointerfaces 110 (2013) 36–44. [13] T.J. Menkhaus, H. Varadaraju, L.F. Zhang, S. Schneiderman, S. Bjustrom, L. Liu, H. Fong, Electrospun nanofiber membranes surface functionalized with 3dimensional nanolayers as an innovative adsorption medium with ultra-high capacity and throughput, Chem. Commun. 46 (2010) 3720–3722. [14] W. Guo, E. Ruckenstein, A new matrix for membrane affinity chromatography and its application to the purification of concanavalin A, J. Membr. Sci. 182 (2001) 227–234. [15] T.W. Rademacher, R.B. Parekh, R.A. Dwek, Glycobiology, Annu. Rev. Biochem. 57 (1988) 785–838. [16] J.J. Lundquist, E.J. Toone, The cluster glycoside effect, Chem. Rev. 102 (2002) 555–578. [17] H. Lis, N. Sharon, Lectins: carbohydrate-specific proteins that mediate cellular recognition, Chem. Rev. 98 (1998) 637–674. [18] L. Bech, B. Lepoittevin, A.E. Achhab, E. Lepleux, L.T. Gay, C.B. Laporte, P. Roger, Double plasma treatment-induced graft polymerization of carbohydrated monomers on poly(ethylene terephthalate) fibers, Langmuir 23 (2007) 10348–10352. [19] L. Renaudie, C.L. Narvor, E. Lepleux, P. Roger, Functionalization of poly(ethylene terephthalate) fibers by photografting of a carbohydrate derivatized with a phenyl azide group, Biomacromolecules 8 (2007) 679–685. [20] L. Bech, T. Meylheuc, B. Lepoittevin, P. Roger, Chemical surface modification of poly(ethylene terephthalate) fibers by aminolysis and grafting of carbohydrates, J. Appl. Polym. Sci. 45 (2007) 2172–2183. [21] M.X. Hu, Z.K. Xu, Carbohydrate decoration of microporous polypropylene membranes for lectin affinity adsorption: comparison of mono- and disaccharides, Colloids Surf. B: Biointerfaces 85 (2011) 19–25. [22] R.R. Schmidt, J. Michel, Facile synthesis of ␣-and ␤-O-glycosyl imidates: preparation of glycosides and disaccharides, Angew. Chem. Int. Ed. Engl. 19 (1980) 731–732. [23] Z.W. Dai, L.S. Wan, Z.K. Xu, Surface glycosylation of polymeric membranes, Sci. China. Ser. B: Chem. 51 (2008) 901–910. [24] A.F. Che, X.J. Huang, Z.K. Xu, Polyacrylonitrile-based nanofibrous membrane with glycosylated surface for lectin affinity adsorption, J. Membr. Sci. 366 (2011) 272–277. [25] T.B. Stachowiak, F. Svec, J.M.J. Frechet, Patternable protein resistant surfaces for multifunctional microfluidic devices via surface hydrophilization of porous polymer monoliths using photografting, Chem. Mater. 18 (2006) 5950–5957. [26] J.P. Deng, L.F. Wang, L.Y. Liu, W.T. Yang, Developments and new applications of UV-induced surface graft polymerizations, Prog. Polym. Sci. 34 (2009) 156–193.

348

X.-Y. Ye et al. / Colloids and Surfaces B: Biointerfaces 115 (2014) 340–348

[27] D.M. He, H. Susanto, M. Ulbricht, Photo-irradiation for preparation, modification and stimulation of polymeric membranes, Prog. Polym. Sci. 34 (2009) 62–98. [28] M. Ulbricht, H. Yang, Porous polypropylene membranes with different carboxyl polymer brush layers for reversible protein binding via surface-initiated graft copolymerization, Chem. Mater. 17 (2005) 2622–2631. [29] Y. Zheng, P.V. Gurgel, R.G. Carbonell, Effects of UV exposure and initiator concentration on the spatial variation of poly(glycidyl methacrylate) grafts on nonwoven fabrics, Ind. Eng. Chem. Res. 50 (2011) 6115–6123. [30] Y.J. Hwang, M.G. Mccord, J.S. An, B.C. Kang, S.W. Park, Effects of helium atmospheric pressure plasma treatment on low-stress mechanical properties of polypropylene nonwoven fabrics, Text. Res. J. 75 (2005) 771–778. [31] C. Wang, Y. Fan, M.X. Hu, W. Xu, J. Wu, P.F. Ren, Z.K. Xu, Glycosylation of the polypropylene membrane surface via thiol–yne click chemistry for lectin adsorption, Colloids Surf. B: Biointerfaces 110 (2013) 105–112. [32] S.F. Luan, J. Zhao, H.W. Yang, H.C. Shi, J. Jin, X.M. Li, J.C. Liu, J.W. Wang, J.H. Yin, P. Stagnaro, Surface modification of poly(styrene-b-(ethylene-co-butylene)-bstyrene) elastomer via UV-induced graft polymerization of N-vinyl pyrrolidone, Colloids Surf. B: Biointerfaces 93 (2012) 127–134. [33] C. Zhang, J. Jin, J. Zhao, W. Jiang, J.H. Yin, Functionalized polypropylene nonwoven fabric membrane with bovine serum albumin and its hemocompatibility enhancement, Colloids Surf. B: Biointerfaces 102 (2013) 45–52. [34] Y. Chang, C.Y. Ko, Y.J. Shih, D. Quemener, A. Deratani, T.C. Wei, D.M. Wang, J.Y. Lai, Surface grafting control of PEGylated poly(vinylidene fluoride) antifouling

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

membrane via surface-initiated radical graft copolymerization, J. Membr. Sci. 345 (2009) 160–169. S.M. Rele, W.X. Cui, L.C. Wang, S.J. Hou, G.B. Zarae, D. Tatton, Y. Gnanou, J.D. Esko, E.L. Chaikof, Dendrimer-like PEO glycopolymers exhibit anti-inflammatory properties, J. Am. Chem. Soc. 127 (2005) 10132–10133. R.R. Schmidt, X.M. Zhu, Glycosyl trichloroacetimidates, in: R.B. Fraser, K. Tatsuta, J. Thiem (Eds.), Glycoscience, Springer-Verlag, Berlin, Heidelberg, Germany, 2008, pp. 452–517. I.J. Goldstein, C.E. Hollerman, E.E. Smith, Protein-carbohydrate interaction 2. Inhibition studies on interaction of concanavalin a with polysaccharides, Biochemistry 4 (1965) 876–883. M. Mammen, S.K. Choi, G.M. Whitesides, Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors, Angew. Chem. Int. Ed. 37 (1998) 2755–2794. J. Kumar, L. McDowall, G.J. Chen, M.H. Stenzel, Synthesis of thermo-responsive glycopolymers via copper catalysed azide–alkyne ‘click’ chemistry for inhibition of ricin: the effect of spacer between polymer backbone and galactose, Polym. Chem. 2 (2011) 1879–1886. C. Wang, P.F. Ren, X.J. Huang, J. Wu, Z.K. Xu, Surface glycosylation of polymer membrane by thiol-yne click chemistry for affinity adsorption of lectin, Chem. Commun. 47 (2011) 3930–3932. J. Bouckaert, F. Poortmans, L. Wyns, R. Loris, Sequential structural changes upon zinc and calcium binding to metal-free concanavalin A, J. Biol. Chem. 271 (1996) 16144–16150. M.X. Hu, L.S. Wan, Z.K. Xu, Multilayer adsorption of lectins on glycosylated microporous polypropylene membranes, J. Membr. Sci. 335 (2009) 111–117.