One-Pot Synthesis of Bimodal Gigaporous Polystyrene Microspheres

May 10, 2018 - provided by Institute of Process Engineering, Chinese Academy of. Sciences ..... will promise the specific surface area (separation cap...
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Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

One-Pot Synthesis of Bimodal Gigaporous Polystyrene Microspheres with Hydrophilic Surfaces Jian-Bo Qu,* Yuan Liu, Jun-Yi Liu, Guan-Sheng Huan, Sheng-Nan Wei, Shi-Hai Li, and Jian-Guo Liu* State Key Laboratory of Heavy Oil Processing, Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266580, P. R. China S Supporting Information *

ABSTRACT: Hydrophilic gigaporous polystyrene (HGPS) microspheres with bimodal pore distributions were first synthesized in one step, using amphiphilic diblock glycopolymers (ADG) and toluene as macroporogen and mesoporogen, respectively. The effects of different HLB values, molecular weights, and concentrations of ADG as well as the addition of mesoporogen on the morphology of microspheres were investigated in detail. The HGPS microspheres were characterized to possess two type of pores, i.e., gigapores (300−7000 nm) and mesopores (4−50 nm). As a consequence, the column packed with HGPS microspheres presented low backpressure, good permeability, and high column efficiency, indicating the existence of flowthrough pores in the particle. Compared to native polystyrene microspheres, the nonspecific adsorption of proteins on HGPS microspheres was greatly reduced by 93.2%. The HGPS microspheres can be easily derivatized by classical methods since their surface was hydroxyl-rich glycopolymers. All the results indicate that the HGPS microspheres have great potentials in high-speed protein chromatography, cell culture, and enzyme immobilization.

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INTRODUCTION Porous polymer microspheres have been used extensively in many biotechnology fields such as cell culture and tissue engineering,1−4 cell and enzyme immobilization,5,6 protein chromatography separation,7−9 and drug-controlled release.10 Because of diffusion constraints and/or mass transfer resistance of cell/biomacromolecules, particles with pore sizes larger than 200 nm, usually called gigaporous microspheres,11 have great advantages in these fields. So far, a number of gigaporous particles have been made from natural materials (e.g., agarose, cellulous, and chitosan)12−14 and synthetic polymers (e.g., polystyrene (PS), poly(glycidyl methacrylate) (PGMA), and poly(ethylene glycol dimethacrylate) (PEGDMA)). 15−17 Among them, gigaporous polystyrene microspheres are of increasing interest owing to their excellent mechanical properties and good chemical stability over a wide pH range,18,19 especially as a chromatographic matrix for proteins and antibodies. There are a few methods for fabricating gigaporous PS microspheres, one which is the nanoparticle coagulation method. POROS PS microspheres prepared by this method © XXXX American Chemical Society

have two sets of poresthrough pores (600−800 nm) and diffusive pores (80−150 nm)and give faster protein separation.9,15 However, possibly because of the complicated preparation method of POROS particles there has been little progress with these particles. The high internal phase emulsion (HIPE) method is very popular for the preparation of gigaporous polymeric matrices, and the most widely studied monomer is styrene.20−22 By dispersing HIPE further in the aqueous phase, Li et al. developed polyHIPE microspheres.23,24 Zhou et al. reported a novel and easy methodthe surfactant reverse micelle swelling methodto prepare gigaporous PS microspheres with pore diameters of about 300−500 nm.16,25 Although all these gigaporous PS microspheres mentioned are very promising in biotechnology field, the highly hydrophobic property of PS is a fatal flaw when it comes to biocompatibility.26,27 To avoid this drawback, chemical/ physical surface modification with hydrophilic material is an Received: March 22, 2018 Revised: May 10, 2018

A

DOI: 10.1021/acs.macromol.8b00611 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Scheme 1. Preparation Mechanism of HGPS Microspheres

indispensible process.28,29 However, this approach cannot ensure that the inner pore surface of particles is effectively functionalized, and in most cases it is complex and laborious. Here we describe a bottom-up approach based on a modified surfactant reverse micelle swelling method to prepare HGPS microspheres with bimodal pore distributions (Scheme 1). The macroporgen ADG with a controlled hydrophilic−lipophilic balance (HLB) was first synthesized via atom transfer radical polymerization (ATRP). Taking ADG as an oil phase surfactant, gigaporous microspheres with PS skeleton and glycopolymer surface can therefore be prepared in a one-step process. The formation mechanism of gigapores in HGPS microspheres is similar to that of gigaporous PS microspheres prepared by the surfactant reverse micelle swelling method.16,25 The amphiphilic ADG in the oil phase first forms reverse micelles; then the reverse micelles expand and aggregate to form continuous water channels after absorbing water from the continuous water phase. After further polymerization macrophase separation occurs between water channel−monomer and toluene−monomer in the oil droplets, producing gigapores and mesopores in the particles, respectively. The hydrophobic block of ADG penetrates into the monomer phase and then integrates with the underlying polymer matrix during polymerization, and the hydrophilic block of ADG provides a glycopolymer surface for the underlying PS skeleton during this process. The HGPS microspheres as prepared were characterized by Fourier transform infrared (FTIR) spectra, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), mercury porosimetry measurements (MPM), flow hydrodynamics, column efficiency, and bovine serum albumin (BSA) adsorption experiments to evaluate their potentials as supports in high-speed protein chromatography.

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Synthesis of ADG via the ATRP Method. The ADG with various HLB and molecular weight were synthesized by a three-step reaction. Briefly, sugar-based monomer 3-O-methacryloyldiacetone−D-glucose (MDAGlu) was first synthesized according to a slight modification of the method proposed by Ohno et al.;30 then polystyrene-bpoly(MDAGlu) (PS-b-PMDAGlu) diblock copolymers were prepared by the ATRP method. Finally, ADG was obtained by removing the protective groups of glucose moiety.31 Further preparation details can be found in the Supporting Information. Molecular Weight and HLB Measurements. The monomer conversion and the theoretical molecular weight (Mn) of the copolymers were determined by 1H NMR (Bruker, 400 MHz) with CDCl3 or DMSO-d6 as the solvent. The molecular weight distributions (Mw/Mn) were measured with a gel permeation chromatography (GPC) system (Viscotek, TDA 302, USA) equipped with TSK gel columns GMHHR-L and GMHHR-N. THF was used as the mobile phase, the flow rate was 1.0 mL/min, and temperature was maintained at 30 °C. The HLB values of ADG were evaluated by the Griffin formula. After removal of the isopropylidene group on the glucose moiety, the glucose moiety and CO in PMDAGlu unit can be defined as a hydrophilic block. So the HLB values of ADG can be calculated from formula 1.32 HLB = 20 ×

M n,PMDAGlu × M n,ADG

207 328

(1)

where Mn,PMDAGlu is the molecular weight of PMDAGlu, 207 is the molar mass of glucose moiety and CO, 328 is the molar mass of MDAGlu, and Mn,ADG is the molecular weight of ADG. HGPS Microspheres Fabrication. The HGPS microspheres were prepared by the surfactant reverse micelles swelling method, as shown in Scheme 1. Typically, 0.4 g of ADG and 0.4 g of toluene were added to a mixture of monomers (3 g of St and 1 g of DVB) containing 0.16 g of benzoyl peroxide (BPO) and mixed thoroughly. The monomer phase was then transferred into a continuous aqueous phase (100 mL) containing 2 wt % poly(vinyl alcohol) (PVA), 0.01% HQ, and 0.02% Na2SO4 in a three-neck glass flask equipped with an anchor-type agitator, a condenser, and a nitrogen inlet nozzle. The two-phase system was stirred at 400 rpm to prepare the emulsion. The emulsion was bubbled with nitrogen for 30 min and incubated for another 30 min; then the temperature was elevated to 75 °C for 24 h of polymerization. The HGPS microspheres were washed by hot water and ethanol three times and dried in a vacuum at room temperature. Composition and Structure Analysis. The surface chemical compositions of HGPS microspheres were characterized by X-ray photoelectron spectroscopy (XPS) (VG Scientific ESCALab220i-XL) and Fourier transform infrared spectroscopy (FTIR) (Nicolet6700, USA). Scanning electron microscopy (SEM) (S-4800, Hitachi, Japan) was used to observe the surface morphology of HGPS microspheres. Samples were placed on a metal stub with double-sided conductive adhesive tape and were coated with a thin gold film under reduced pressure below 5 Pa with an ion-sputter coater (Hitachi E-1010, Japan). The diameter and porosity of HGPS microspheres were measured by a BT-9300S laser particle size analyzer (Bettersize,

EXPERIMENTAL SECTION

Materials. The monomers styrene (St, 99%) and divinylbenzene (DVB, 80%) were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). They were washed with an aqueous solution of sodium hydroxide (5 wt %) to remove the inhibitor and distilled under a vacuum prior to use. Ethyl 2-bromoisobutylate (2-EiBBr, 98%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99%), methacrylic anhydride (94%) and diacetone−D-glucose (98%) were ordered from Alfa Aesar (Heysham, UK). Blue Dextran 2000 and bovine serum albumin (BSA) were ordered from Amresco (USA). Hydroquinone (HQ, 99%), benzoyl peroxide (BPO, 99%), and formic acid (88%) were ordered from Sinopharm Chemical Reagent Co. Ltd. (China). Native gigaporous PS (IPE GPS) microspheres were kindly provided by Institute of Process Engineering, Chinese Academy of Sciences (average pore diameter 280 nm, average particle size 55 μm, specific surface area 22.69 m2/g). Other reagents were all of analytical grade from local sources. B

DOI: 10.1021/acs.macromol.8b00611 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules China) and an AutoPore IV 9500 mercury porosimetry (Micromeritics, USA), respectively. The specific surface area of HGPS microspheres was measured by BET nitrogen adsorption measurements using a Micrometrics (ASAP 2020, USA) apparatus. Samples were degassed under vacuum at 70 °C for 8 h before analysis. Permeability and Mechanical Stability of HGPS Microspheres. To test the permeability and mechanical stability of HGPS microspheres, the particles were first sieved by using sieves with nominal aperture of 48 and 150 μm and then were packed into a stainless steel column (100 × 4.6 mm i.d.) by the slurry packing method using a Waters 1525 HPLC system equipped with a 2489 UV/ vis detector (Binary HPLC pump, USA). The column compressibility and permeability were tested by the relationship between flow velocity and column backpressure at 25 °C. The bed void fraction (εb) was obtained by using Blue Dextran 2000 as a tracer, which reflected the void volume of the column. Blue Dextran 2000 was detected at 260 nm. The flow rate was 1.0 mL/min, the concentration of tracer solution was 3 mg/mL, and the injection amount was 50 μL. For a packed column with a length of L, K can also be described by the Darcy’s law in a laminar flow region.33

K=

μuL ΔP

Table 1. ADG with Various Molecular Weight and HLB Valuesa run no.

Mn,PMDAGlub

Mn,ADGb

HLBc

Mw/Mn

1 2 3 4 5 6 7 8 9 10 11

1050 2100 1700 2100 1700 2300 2100 2500 2500 3100 5300

4500 7400 5500 6400 5000 6500 5700 5900 5200 9000 16000

2.9 3.6 4.0 4.1 4.3 4.5 4.6 5.2 6.1 4.3 4.2

1.27 1.19 1.26 1.15 1.27 1.23 1.25 1.26 1.28 1.21 1.25

The monomer concentration for the first block and the second block is 1 and 2 M, respectively. bCalculated from the conversion of monomer, which is determined by 1H NMR. cCalculated from formula 1. a

(2)

where u is the superficial velocity (cm/s), μ the viscosity of the mobile phase (Pa·s), ΔP the column pressure drop (Pa), and L the length of column (cm). Column Efficiency. The column efficiency was evaluated in terms of HETP (the height equivalent to a theoretical plate) on an Ä KTA purifier 100 system (GE Healthcare) under a nonretained condition.7 The mobile phase was 20 mmol/L phosphate buffer (pH 7.0), and BSA was used as a probe protein. After equilibrating the column with 10 CVs of mobile phase, 2 mg/mL BSA (200 μL) was injected, and the chromatogram was recorded at 280 nm. The dead volume of the system was measured by injecting 200 μL of 20% acetone solution via the injection loop. Surface Hydrophilicity of HGPS Microspheres. Hydrophilicity of microspheres was defined as the ratio of the water content of the wet microspheres relative to the original weight. The surface hydrophilicity of HGPS microspheres was also assessed by protein adsorption using BSA as model protein.34 Detailed experimental processes are described in our previous works.28,35

Keeping the amount of ADG with similar molecular weight to 10% w/w of the monomers (St and DVB), the effect of HLB values of ADG on HGPS morphologies was investigated in detail. As shown in Figure 1, the formation of gigapores is closely related to HLB values of ADG (2.9−6.1). When the HLB value of ADG is lower than 3.6, there is nearly no pores on the particle surface. With the increase of HLB values of ADG, both the pore sizes and pore densities of HGPS microspheres increase gradually owing to the increased adsorbing water capacity of the reverse micelles in the oil phase. However, when the HLB value of ADG is further increased (larger than 5.2), excess amount of water adsorbed in the oil droplet will cause serious macrophase separation during polymerization, resulting in the broken particles. (Figure 1f). The HLB value of ADG in the range of 3.6−5.2 is appropriate for the formation of gigapores on the particles. Neither the smaller nor larger HLB value can the gigapores obtain. Effect of ADG Molecular Weight on the Morphology of HGPS Microspheres. Figure 2 shows SEM images of HGPS microspheres prepared with ADG having similar HLB values and different molecular weights. In this case the adding amount of ADG was kept constant. It can be seen that the molecular weights of ADG have a significant impact on the structure of microspheres. The pore sizes of HGPS microspheres increase significantly with the molecular weight of ADG. On the contrary, the pore densities of HGPS microspheres decrease gradually. Considering high molecular weight of ADG means low molar amount when keeping the mass amount of ADG at the same level; this result is reasonable. When the molecular weight of ADG is 5000, the distribution of gigapores in the particles is homogeneous and the diameter of gigapores is around 1−2 μm; when the molecular weight of ADG is 16 000, the distribution of gigapores is heterogeneous and the diameter of some gigapores is over 10 μm. Gigapores have great advantages in reducing the mass transfer resistance of biomacromolecules in the separation process. However, too large pore diameter of microspheres will not improve the mass transfer rate of biomacromolecules anymore. Also, the specific surface area and mechanical strength of particles will decrease significantly. Moreover, the solubility of larger molecular weight of ADG in the oil phase was particularly poor; some ADG failed to form an effective

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RESULTS AND DISCUSSION Characterization of ADG. Generally, surfactants with HLB values between 3 and 8 are suitable for preparation of W/O emulsions.36 Lots of similar molecular weight ADG with HLB values in this range as well as some ADG with the similar HLB value but different molecular weights had been synthesized via the ATRP method to testify their effects on the morphology of HGPS microspheres. Table 1 presents some ADG with various molecular weights and HLB values. The HLB values of ADG prepared can also be determined by the emulsifiability method besides using formula 1, and the results indicate that the two methods have good consistency (Figure S5 and Table S1). It can be seen that all the molecular weight distributions (Mw/ Mn) of ADG are lower than 1.3, which will be advantageous to form homogeneous pore structure of the particles. As shown in Figure S6, GPC traces of PMDAGlu, PS-b-PMDAGlu, and ADG present clean peak shifts, indicating that the polymerization was well controlled. Effect of ADG HLB Value on the Morphology of HGPS Microspheres. Although the addition of macromolecular surfactant ADG was favorable for the formation of gigapores on the microspheres, the gigapore size of microspheres was mainly relied on the adsorbing water capacity and stability of reverse micelles in the oil phase (Scheme 1). The HLB value of the surfactant plays an important role during this process. C

DOI: 10.1021/acs.macromol.8b00611 Macromolecules XXXX, XXX, XXX−XXX

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Figure 1. . Effect of ADG HLB value on the morphology of HGPS microspheres. ADG amount, 10% w/w of the monomers (St and DVB); toluene amount, 10% w/w of the monomers; the HLB values of ADG used in (a)−(f) are 2.9, 3.6, 4.1, 4.5, 5.2, and 6.1, respectively (Table 1).

Figure 2. Effect of ADG molecular weight on the morphology of HGPS microspheres. ADG amount, 10% w/w of the monomers (St and DVB); toluene amount, 10% w/w of the monomers; the molecular weights of ADG used in (a)−(d) are 5000, 6500, 9000, and 16 000, respectively (Table 1).

reverse micelles structure, resulting in the heterogenerous distribution of gigapores on HGPS microspheres. Effect of ADG Amount on the Morphology of HGPS Microspheres. Keeping the molecular weight and HLB value of ADG constant, the effect of ADG (Mn = 5500, HLB = 4.0) amount on the morphology of HGPS microspheres was also

studied. It could be seen from Figure 3 that the pore sizes on microspheres become apparently larger in pace with the addition of ADG dosage. The porosities of microspheres determined by mercury porosimetry in Figures 3a, 3b, and 3c are also increased with the addition of ADG, i.e., 50.9%, 54.2%, and 57.4%, respectively. When the amount of ADG is 10%, the D

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Figure 3. Effect of ADG amount on the morphology of HGPS microspheres (run 3 ADG in Table 1). Toluene amount, 10% w/w of the monomers; the ADG amounts added in (a)−(d) are 10, 20, 30, and 40 wt %, respectively.

Figure 4. Effect of toluene amount on the morphology of HGPS microspheres (run 7 ADG in Table 1). ADG amount, 10% w/w of the monomers (St and DVB); the toluene amounts in (a)−(d) are 10, 30, 50, and 100% w/w of the monomers, respectively.

size of gigapores in microspheres is around 1−2 μm. When it is increased to 30%, the gigapore size increases to around 5−7 μm. In addition, some cracks appear clearly on the particles, indicating the mechanical strength of HGPS microspheres began to decrease. Similar with HGPS microspheres prepared with ADG having HLB value of 6.1, the microspheres were broken during polymerization when the amount of ADG further increased to 40%. Both resulted from the excess water content adsorbed by ADG in the oil droplet. Moreover, excess ADG addition not only makes it difficult to dissolve in the oil phase but also increases the viscosity of the oil phase, which will increase the dispersion resistance of the oil phase in the aqueous phase. Correspondingly, the uniformity of microspheres becomes unsatisfactory. Effect of Toluene Amount on the Morphology of HGPS Microspheres. An ideal bioseparation medium should

have a certain amount of mesopores besides gigapores, which will promise the specific surface area (separation capacity) of the medium. Toluene is a common and useful mesoporogen in the preparation of polymer materials, especially for styrene and DVB system. Figure 4 shows the magnified surface morphology of HGPS microspheres with different toluene amounts in the oil phase. Compared to the rough surface of IPE GPS microspheres (Figure S7), HGPS microsphere’s surface is apparently smooth and hydrogel-like, ascribing to the hydrophilic glycopolymers on ADG chains. Because the hydrophilic block of ADG tends to spread on the outer surface of the microspheres during polymerization, the increase of toluene amount will also increase the degree of macrophase separation. As a result, the surface roughness of microspheres gradually increased with the toluene amount. It can also be seen from Figure 4 that the amount and size of mesopores increased with E

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Figure 5. Pore size distribution curve (a) and BET isotherm (b) of HGPS microspheres prepared with run 7 ADG. ADG amount, 10% w/w of the monomers (St and DVB); toluene amount, 30% w/w of the monomers.

the amount of toluene. Accordingly, the specific surface areas of HGPS microspheres in Figure 4a−d were 70.12, 76.97, 86.67, and 106.74 m2/g, respectively. However, excessive toluene in the oil phase will lead to the heterogeneous surface structure and decrease the chromatographic efficiency and mechanical strength of the particles. Pore Size and Structure Analysis. Figure 5a shows a typical pore diameter distribution of HGPS microspheres determined by mercury porosimetry. The microspheres prepared with both ADG and toluene as the porogens exhibit porosity of 52.3% and a clearly bimodal pore size distribution. The gigapores are in the range of approximately 0.3−7 μm with a distinct maximum around 2 μm; the mesopores are in the range of 4−50 nm with the greatest incremental pore volume occurring at a pore diameter of near 15 nm. Nitrogen adsorption/desorption isotherms of HGPS microspheres are shown in Figure 5b. It is clear that the isotherms exhibit typical reversible type IV adsorption isotherms with H1 type hysteresis loop closed at relative pressure around 0.5, indicating the presence of well-ordered and connected mesopores on HGPS microspheres.37 As described in Scheme 1, the formation of gigapores and mesopores can be attributed to the contribution of ADG and toluene in the oil phase, respectively. The former can effectively reduce stagnant mobile phase mass transfer resistance during separation of biomacromolecules,7,15 and the latter might contribute significantly to the specific surface area. Both are equal important to the successful application of HGPS microspheres in high-speed protein chromatography. Surface Chemical Composition Verification of HGPS Microspheres. XPS is an efficient method to characterize the surface chemical composition of materials. Figure 6 shows the XPS C1s and O1s spectra of HGPS microspheres prepared with run 4 ADG. There are three peaks at 284.6, 286.0, and 288.8 eV in the C1s spectrum of microspheres (Figure 6a), representing C−C/C−H, C−O−C/C−OH, and O−CO bonds, respectively. The appearance of C−O−C/C−OH and O−CO bonds on the surface of HGPS microspheres can be attributed to ether bonds/hydroxyls and ester bonds in the hydrophilic block of ADG. The O1s spectrum of HGPS microspheres presents two peaks at 532.9 eV (C−O−C/C−OH) and 531.7 eV (O−CO), confirming the existence of ADG on HGPS microspheres surface again. In addition, the peak area ratios of peak 3 (288.8 eV)/peak 2 (286.0 eV) in the C1s spectrum and peak 2 (531.7 eV)/peak 1 (532.9 eV) in the O1s spectrum are 0.149 and 0.167, respectively (see Table 2). They are close to

Figure 6. C1s (a) and O1s (b) XPS spectra of HGPS microspheres prepared with run 4 ADG. ADG amount, 10% w/w of the monomers (St and DVB); toluene amount, 30% w/w of the monomers.

the ratio of C−O−C/C−OH and O−CO on the glycopolymer (1:6). This further proved that ADG had been integrated into HGPS microspheres skeleton during polymerization. The FT-IR spectrum of HGPS microspheres also confirmed the existence of ADG on the particles (Figure S8). Column Permeability and Column Efficiency. Column permeability and column efficiency are important factors for chromatographic packing materials. Figure 7a shows good linear relationships between column backpressure and flow velocity up to 6140 cm/h for HGPS columns, and the backpressure of HGPS columns under 6140 cm/h is only 1.21 MPa, indicating the good mechanical strength of the particles. The bed permeability (K) calculated from eq 2 for HGPS column is 4.07 × 10−10 m2. As a chromatographic matrix, higher permeability means better mass transfer rate. The HETP of BSA (nonretained) in HGPS column is presented in Figure 7b as a functional of mobile phase velocity. As shown in Figure 7b, HETP exhibits a typical curve defined by the Van Deemter equation when the flow velocity is lower than 360 cm/h, suggesting longitudinal diffusion occupies the dominant effect in this region. When the mobile phase flow velocity is higher than 360 cm/h, the HETP of HGPS column only increases slightly with flow velocity up to 5000 cm/h, which is similar to that of POROS supports. It is proposed that there was intraparticle convective flow at high mobile phase velocity due to the presence of flow-through pores in HGPS microspheres.38 Although the larger particle diameter (89 μm, see Figure S9) of HGPS microspheres was contributed to the low backpressure and high bed permeability of HGPS column to some F

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Macromolecules Table 2. Peak Area Ratio of C1s and O1s for HGPS Microspheres Prepared with Run 4 ADG sample

peak 1

peak 2

peak 3

peak 2/peak 1

peak 3/peak 2

C1s O1s

C−C, C−H C−O−C, C−OH

C−O−C, C−OH O−CO

O−CO

0.463 0.167

0.149

Figure 7. Column backpressure (a) and column efficiency (b) versus flow velocity at 25 °C. Column, 100 mm × 4.6 mm i.d.; mobile phase, 20% ethanol solution (a) and 20 mM phosphate buffer, pH 7.0 (b). 2 mg/mL BSA as a probe protein of HETP. HGPS microspheres were prepared with run 4 ADG. ADG amount, 10% w/w of the monomers (St and DVB); toluene amount, 30% w/w of the monomers.

Figure 8. Hydrophilicity (a) and BSA adsorption isotherms (b) of HGPS microspheres prepared with different amounts of run 3 ADG. Blank refers to IPE GPS microspheres; protein adsorption experiment was conducted in 0.05 M phosphate buffer (pH 7.4, 25 °C).

extent, the presence of flow-through pores in the particles also played a significant role.39 According to the theoretical model reported by Afeyan et al.,38 the existence of flow-through pores in HGPS microspheres was also estimated, and the average linear velocity of mobile phase through a 2.5 μm flow-through pore in a 89 μm HGPS microsphere was around 5.1% of the average linear velocity through the column bed (see details in the Supporting Information). An expected convective flow of mobile phase in the through pores will significantly reduce the flow resistance when purifying biomacromolecules, especially in high-speed operation. Surface Hydrophilicity of HGPS Microspheres. Glycopolymer, the hydrophilic block of ADG, will automatically spread on the water channel surface of reverse micelles during polymerization, resulting in the prepared HGPS microspheres having glycopolymer surface and PS skeleton. Figure 8a shows the hydrophilicity of HGPS microspheres prepared with different amounts of run 3 ADG. Compared to IPE GPS microspheres, the hydrophilicity of HGPS microspheres was greatly improved. It can be seen that the H of HGPS microspheres prepared using 30 wt % ADG is 4 times over that of IPE GPS microspheres. As also expected, the hydrophilicity

of HGPS microspheres increased gradually with the adding amount of ADG. The H of HGPS microspheres prepared using 30 wt % ADG is only slightly higher than that of particles prepared using 20 wt % ADG, indicating the hydrophobic PS skeleton of HGPS microspheres prepared in this case was shielded sufficiently by glycopolymers. Hydrophobic interaction between styrene based microspheres and proteins dominates the adsorption behavior of proteins.34 BSA adsorption isotherms of HGPS and IPE GPS microspheres are shown in Figure 8b. Compared to IPE GPS microspheres (89.55 mg/g dry microspheres), the adsorbed amount of BSA on HGPS microspheres (30 wt % ADG) at the plateau (6.09 mg/g dry microspheres) is reduced by 93.6%, suggesting the hydrophobic PS skeleton was well shielded by glycopolymers. In accordance with the H of HGPS microspheres, the adsorbed amount of BSA on HGPS microspheres (20 wt % ADG) (6.82 mg/g dry microspheres) is nearly the same as that on HGPS microspheres (30 wt % ADG). Compared to IPE GPS microspheres with agarose physical coating (1.18 mg/g dry microspheres), the adsorbed amount of BSA on HGPS microspheres is slightly higher.28 The possible G

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Province (No. ZR2017MB019), and the fund of State Key Laboratory of Heavy Oil Processing (No. SLKZZ-2017010).

reason lies in the formation of hydrogen bonding between BSA and glycopolymer (ester bonds) on ADG. Stability of ADG inside HGPS Microspheres. As a chromatographic matrix, the stability of ADG inside HGPS microspheres is of great importance to ensure the surface hydrophilicity of particles. To test the stability of ADG integrated into HGPS microspheres skeleton, the particles (50 mg) were washed with 5 mL of 2% sodium dodecyl sulfate solution (SDS), 1.0 M NaOH, and 1.0 M HCl at 25 °C for 24 h. The suspension was filtered through a sintered glass funnel. The leaked ADG or glucose produced by hydrolysis of ester bond in the filtrate was determined according to the anthrone method by UV spectroscopy at 625 nm.40 The results suggest there was no leakage of ADG or hydrolysis of ester bond on ADG during these processes, indicating the stability of ADG inside HGPS microspheres is satisfactory (the limit of detection is 15.7 ng/L).

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CONCLUSIONS Novel HGPS microspheres with hydrophilic surface and bimodal pore distributions have been first synthesized in one step, which saves the complex and laborious postfunctionalization processes in the practical application. It was found the appropriate HLB, molecular weight, and concentration of ADG were crucial factors for fabricating HGPS microspheres with homogeneous gigapores. The addition of toluene can produce mesopores and promise the microspheres with satisfied specific surface area. Nonspecific adsorption of protein on HGPS microspheres can be neglected owing to the presence of glycopolymer surface. The HGPS microspheres packed into a steel column exhibit low backpressure, high bed permeability, and high column efficiency, which are evidence for the presence of flow-through pores in particles. Besides high-speed protein chromatography, it can be predicted that the HGPS microspheres should be promising in many other biotechnology fields such as cell culture, enzyme immobilization, etc.

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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/acs.macromol.8b00611. NMR spectra; SEM images; particle size analysis; HLB calibration curve; GPC traces; FTIR spectrum; and synthesis scheme of diblock glycopolymer (PDF)

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REFERENCES

(1) Gümüsḑ erelioglu, M.; Ç akmak, S.; Timuçin, H. O.; Ç akmak, A. S. Thermosensitive PHEMA Microcarriers: ATRP Synthesis, Characterization, and Usabilities in Cell Cultures. J. Biomater. Sci., Polym. Ed. 2013, 24 (18), 2110−2125. (2) Nienow, A. W.; Rafiq, Q. A.; Coopman, K.; Hewitt, C. J. A Potentially Scalable Method for the Harvesting of hMSCs from Microcarriers. Biochem. Eng. J. 2014, 85, 79−88. (3) Naidoo, K.; Rolfes, H.; Easton, K.; Moolman, S.; Chetty, A.; Richter, W.; Nilen, R. An Emulsion Preparation for Novel MicroPorous Polymeric Hemi-Shells. Mater. Lett. 2008, 62 (2), 252−254. (4) Sun, L.; Xiong, Z.; Zhou, W.; Liu, R.; Yan, X.; Li, J.; An, W.; Yuan, G.; Ma, G.; Su, Z. Novel Konjac Glucomannan Microcarriers for Anchorage-Dependent Animal Cell Culture. Biochem. Eng. J. 2015, 96, 46−54. (5) Huang, J.; Jin, N.; Katsuda, T.; Fukuda, H.; Yamaji, H. Immobilization of Escherichia Coli Cells Using PolyethyleneimineCoated Porous Support Particles for L-Aspartic Acid Production. Biochem. Eng. J. 2009, 46 (1), 65−68. (6) Li, Y.; Gao, F.; Wei, W.; Qu, J. B.; Ma, G. H.; Zhou, W. Q. Pore Size of Macroporous Polystyrene Microspheres Affects Lipase Immobilization. J. Mol. Catal. B: Enzym. 2010, 66 (1−2), 182−189. (7) Qu, J. B.; Wan, X. Z.; Zhai, Y. Q.; Zhou, W. Q.; Su, Z. G.; Ma, G. H. A Novel Stationary Phase Derivatized from Hydrophilic Gigaporous Polystyrene-Based Microspheres for High-Speed Protein Chromatography. J. Chromatogr. A 2009, 1216 (37), 6511−6516. (8) Jungbauer, A. Chromatographic Media for Bioseparation. J. Chromatogr. A 2005, 1065 (1), 3−12. (9) Whitney, D.; McCoy, M.; Gordon, N.; Afeyan, N. Characterization of Large-Pore Polymeric Supports for Use in Perfusion Biochromatography. J. Chromatogr. A 1998, 807 (2), 165−184. (10) Moglia, R.; Whitely, M.; Brooks, M.; Robinson, J.; Pishko, M.; Cosgriff-Hernandez, E. Solvent-Free Fabrication of polyHIPE Microspheres for Controlled Release of Growth Factors. Macromol. Rapid Commun. 2014, 35 (14), 1301−1305. (11) Chen, S.; Gao, F.; Wang, Q.; Su, Z.; Ma, G. Double EmulsionTemplated Microspheres with Flow-through Pores at Micrometer Scale. Colloid Polym. Sci. 2013, 291 (1), 117−126. (12) Wang, D. M.; Hao, G.; Shi, Q. H.; Sun, Y. Fabrication and Characterization of Superporous Cellulose Bead for High-Speed Protein Chromatography. J. Chromatogr. A 2007, 1146 (1), 32−40. (13) Gustavsson, P. E.; Axelsson, A.; Larsson, P. O. Superporous Agarose Beads as a Hydrophobic Interaction Chromatography Support. J. Chromatogr. A 1999, 830 (2), 275−284. (14) Chavda, H.; Patel, C.; Karen, H. Preparation and Characterization of Chitosan-Based Superporous Hydrogel Composite. J. Young Pharm. 2009, 1 (3), 199−204. (15) McCoy, M.; Kalghatgi, K.; Regnier, F. E.; Afeyan, N. Perfusion Chromatographycharacterization of Column Packings for Chromatography of Proteins. J. Chromatogr. A 1996, 743 (1), 221−229. (16) Zhou, W. Q.; Gu, T. Y.; Su, Z. G.; Ma, G. H. Synthesis of Macroporous Poly(glycidyl Methacrylate) Microspheres by Surfactant Reverse Micelles Swelling Method. Eur. Polym. J. 2007, 43 (10), 4493−4502. (17) Gong, X.; Wen, W.; Sheng, P. Microfluidic Fabrication of Porous Polymer Microspheres: Dual Reactions in Single Droplets. Langmuir 2009, 25 (12), 7072−7077. (18) Huang, X.; Zhang, S.; Schultz, G. A.; Henion, J. SurfaceAlkylated Polystyrene Monolithic Columns for Peptide Analysis in Capillary Liquid Chromatography-Electrospray Ionization Mass Spectrometry. Anal. Chem. 2002, 74, 2336−2344. (19) Lee, D. P. Reversed-Phase HPLC from pH 1 to 13. J. Chromatogr. Sci. 1982, 20, 203−208. (20) Viswanathan, P.; Johnson, D. W.; Hurley, C.; Cameron, N. R.; Battaglia, G. 3D Surface Functionalization of Emulsion-Templated Polymeric Foams. Macromolecules 2014, 47 (20), 7091−7098.

AUTHOR INFORMATION

Corresponding Authors

*(J.-B.Q.) Fax +86 532-86981133, e-mail [email protected]. *(J.-G.L.) Fax +86 532-86981133, e-mail [email protected]. cn. ORCID

Jian-Bo Qu: 0000-0002-9538-1701 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the financial support of National Natural Science Foundation of China (Nos. 21776310, 21176257, and 21473256), the Natural Science Foundation of Shandong H

DOI: 10.1021/acs.macromol.8b00611 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules (21) Li, X.; Zhu, C.; Wei, Y.; Lu, Z. Fabrication of Macroporous Foam and Microspheres of Polystyrene by Pickering Emulsion Polymerization. Colloid Polym. Sci. 2014, 292 (1), 115−122. (22) Mercier, A.; Deleuze, H.; Mondain-Monval, O. Preparation and Functionalization of (Vinyl)polystyrene polyHIPE. Short Routes to Binding Functional Groups through a Dimethylene Spacer. React. Funct. Polym. 2000, 46, 67−79. (23) Li, N.-H.; Benson, J. R.; Kitagawa, N. Polymeric Microbeads and Methods of Preparation. US Patent 5 653 922, 1996. (24) Li, N.-H.; Benson, J. R.; Kitagawa, N. Polymeric Microbeads. US Patent 5 863 957, 1999. (25) Zhou, W. Q.; Gu, T. Y.; Su, Z. G.; Ma, G. H. Synthesis of Macroporous Poly(styrene-Divinyl Benzene) Microspheres by Surfactant Reverse Micelles Swelling Method. Polymer 2007, 48, 1981−1988. (26) Barbetta, A.; Dentini, M.; Zannoni, E. M.; De Stefano, M. E. Tailoring the Porosity and Morphology of Gelatin-Methacrylate polyHIPE Scaffolds for Tissue Engineering Applications. Langmuir 2005, 21 (26), 12333−12341. (27) Li, Y.; Lee, M. L. Biocompatible Polymeric Monoliths for Protein and Peptide Separations. J. Sep. Sci. 2009, 32 (20), 3369− 3378. (28) Qu, J. B.; Zhou, W. Q.; Wei, W.; Su, Z. G.; Ma, G. H. An Effective Way to Hydrophilize Gigaporous Polystyrene Microspheres as Rapid Chromatographic Separation Media for Proteins. Langmuir 2008, 24 (23), 13646−13652. (29) Qu, J. B.; Huan, G. S.; Chen, Y. L.; Zhou, W. Q.; Liu, J. G.; Huang, F. Coating Gigaporous Polystyrene Microspheres with CrossLinked Poly(vinyl Alcohol) Hydrogel as a Rapid Protein Chromatography Matrix. ACS Appl. Mater. Interfaces 2014, 6 (15), 12752−12760. (30) Ohno, K.; Tsujii, Y.; Fukuda, T. Synthesis of a Well-Defined Glycopolymer by Atom Transfer Radical Polymerization. J. Polym. Sci., Part A: Polym. Chem. 1998, 36, 2473−2481. (31) Meng, J.-Q.; Du, F.-S.; Liu, Y.-S.; Li, Z.-C. Atom Transfer Radical Polymerization of 6-O-Methacryloyl-1,2;3,4-Di-O-Isopropylidene-D-Galactopyranose in Solution. J. Polym. Sci., Part A: Polym. Chem. 2005, 43 (4), 752−762. (32) Griffin, W. C. Calculation of HLB Values of Non-Ionic Surfactants. J. Soc. Cosmet. Chem. 1954, 5 (4), 249−256. (33) Chenou, C.; Loureiro, J. M.; Rodrigues, A. E. Mass Transfer Mechanisms in Hyper D Media for Chromatographic Protein Separation. Biochem. Eng. J. 1998, 1, 11−23. (34) Yoon, J. Y.; Kim, J. H.; Kim, W. S. The Relationship of Interaction Forces in the Protein Adsorption onto Polymeric Microspheres. Colloids Surf., A 1999, 153 (1−3), 413−419. (35) Qu, J.; Shao, H.; Li, J.; Jing, G.; Liu, J.; Zhou, W. Reactive & Functional Polymers Hydrophilization of Gigaporous Polystyrene Microspheres with Saccharide as High-Speed Protein Chromatography Base Support. React. Funct. Polym. 2012, 72 (9), 606−612. (36) Utama, R. H.; Stenzel, M. H.; Zetterlund, P. B. Inverse Miniemulsion Periphery RAFT Polymerization: A Convenient Route to Hollow Polymeric Nanoparticles with an Aqueous Core. Macromolecules 2013, 46 (6), 2118−2127. (37) Sing, K. S. W. Reporting Physisorption Data for Gas/solid Systems with Special Reference to the Determination of Surface Area and Porosity. Pure Appl. Chem. 1985, 57 (4), 603−619. (38) Afeyan, N. B.; Yang, Y. B.; Regnier, F. E.; Mazsaroff, I.; Varady, L.; Fulton, S. P. Flow-through Particles for the High-Performance Liquid Chromatographic Separation of Biomolecules: Perfusion Chromatography. J. Chromatogr. 1990, 519, 1−29. (39) Sun, G.; Shi, Q.; Sun, Y. Novel Biporous Polymeric Stationary Phase for High-Speed Protein Chromatography. J. Chromatogr. A 2004, 1061, 159−165. (40) Scott, T. A.; Melvin, E. H. Determination of Dextran with Anthrone. Anal. Chem. 1953, 25 (11), 1656−1661.

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DOI: 10.1021/acs.macromol.8b00611 Macromolecules XXXX, XXX, XXX−XXX