Temperature and pH Dual-Responsive Core-Brush Nanocomposite

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Temperature and pH Dual-Responsive Core-Brush Nanocomposite for Enrichment of Glycoproteins Lingdong Jiang,† Maria E. Messing,‡ and Lei Ye*,† †

Division of Pure and Applied Biochemistry, Department of Chemistry, Lund University, Box 124, 221 00 Lund, Sweden Division of Solid State Physics and NanoLund, Department of Physics, Lund University, Box 118, 221 00 Lund, Sweden

‡

S Supporting Information *

ABSTRACT: In this report, we present a novel modular approach to the immobilization of a high density of boronic acid ligands on thermoresponsive block copolymer brushes for effective enrichment of glycoproteins via their synergistic multiple covalent binding with the immobilized boronic acids. Specifically, a two-step, consecutive surface-initiated atom transfer radical polymerization (SI-ATRP) was employed to graft a flexible block copolymer brush, pNIPAm-bpGMA, from an initiator-functionalized nanosilica surface, followed by postpolymerization modification of the pGMA moiety with sodium azide. Subsequently, an alkyne-tagged boronic acid (PCAPBA) was conjugated to the polymer brush via a Cu(I)catalyzed azide−alkyne cycloaddition (CuAAC) click reaction, leading to a silica-supported polymeric hybrid material, Si@ pNIPAm-b-pBA, with a potent glycol binding affinity. The obtained core-brush nanocomposite was systematically characterized with regard to particle size, morphology, organic content, brush density, and number of immobilized boronic acids. We also studied the characteristics of glycoprotein binding of the nanocomposite under different conditions. The nanocomposite showed high binding capacities for ovalbumin (OVA) (98.0 mg g−1) and horseradish peroxidase (HRP) (26.8 mg g−1) in a basic buffer (pH 9.0) at 20 °C. More importantly, by adjusting the pH and temperature, the binding capacities of the nanocomposite can be tuned, which is meaningful for the separation of biological molecules. In general, the synthetic approach developed for the fabrication of block copolymer brushes in the nanocomposite opened new opportunities for the design of more functional hybrid materials that will be useful in bioseparation and biomedical applications. KEYWORDS: atom transfer radical polymerization, boronic acid, block copolymer brush, glycoprotein, bioseparation, nanocomposite

1. INTRODUCTION Protein glycosylation plays key roles in numerous biological events, including molecular recognition, signal transduction, and immune response. 1 Besides, altered and aberrant glycosylated proteins have long been considered closely associated with the occurrence of various types of cancer.2 Large-scale systematic profiling of these glycoproteins not only facilitates the discovery of new diagnostic biomarkers but also provides important information for therapeutic treatment.3 However, the inherent low-abundance of glycoproteins along with large amount of interfering substances in biological samples4 causes considerable difficulty in glycoprotein analysis. Therefore, the selective enrichment of target proteins is a critical step for the analysis of glycoproteins. Hydrazide chemistry5−7 is a traditional method for the selective enrichment of glycoproteins; however, the reaction step is timeconsuming and may lead to possible side reactions. Lectins8 and antibodies9 are two important molecular tools for selective enrichment of glycoprotein. Unfortunately, these biomolecules are associated with obvious disadvantages, including high cost and poor stability, which limit their widespread applications. © 2017 American Chemical Society

In recent years, boronate affinity materials, including macroporous monoliths,10 molecularly imprinted polymers,11−13 and magnetic materials14−16 have aroused enormous interest for the enrichment of glycoproteins because of their unique affinity for biomolecules containing cis-diol moieties. Boronate affinity materials can capture cis-diol-containing biomolecules in mildly basic aqueous media. More importantly, the target biomolecules can be released in acidic media resulting from the dissociation of the boronate ester bond. All these properties have been main driving forces for the rapid development of boronate affinity materials in separation science. However, as glycoprotein adsorbents, boronic acids have one apparent drawback that is due to the relatively weak boronate ester bond: it is a challenging task for single boronic acid to capture biomacromolecules like glycoproteins efficiently. Much effort has been devoted to overcome this disadvantage, and among the efforts the use of synergistic, Received: November 30, 2016 Accepted: February 27, 2017 Published: February 27, 2017 8985

DOI: 10.1021/acsami.6b15326 ACS Appl. Mater. Interfaces 2017, 9, 8985−8995

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ACS Applied Materials & Interfaces

Scheme 1. Preparation of Si@pNIPAm-b-pBA Core-Brush Nanocomposite via the Combination of SI-ATRP with CuAAC Click Reaction, and the Schematic of Glycoprotein Binding to the Boronic Acid-Functionalized Polymer Brushes

multiple covalent bonds has proven an effective strategy.17 Liu and co-workers reported a dendrimeric boronic acid-functionalized magnetic nanoparticle, which exhibited significantly enhanced binding strength to glycoproteins.18 In this case, highly branched dendrimers were employed to provide a large number of boronate groups. However, the dendrimers involved are associated with apparent disadvantages, such as high cost and high rigidity. Zhang and co-workers combined distillation− precipitation polymerization and click reaction to prepare a core−shell hybrid composite with a high density of boronic acid ligands on the particle surfaces.19 The click reaction used is characterized by many remarkable merits such as high specificity, nearly quantitative yields, moderate reaction conditions, and limited side reactions.20 Deng and co-workers prepared boronic acid functionalized Fe3O4@C@Au nanoparticles via a layer-by-layer self-assembly method for the selective enrichment of glycoproteins and glycopeptides.21 However, both the core−shell and the multilayer structures have severe steric hindrance owing to the short spacers between the solid surface and the boronic acid ligands.22 Therefore, to realize more efficient separation of glycoproteins, it is necessary to explore soft boronate affinity materials that have a high density of boronic acids with a low steric hindrance. In the past decades, the development of controlled radical polymerization (CRP)23,24 has rendered surface-initiated atom transfer radical polymerization (SI-ATRP)25,26 as an indispensable approach to the controlled synthesis of polymer brushes from various types of functionalized substrates, including silica,27,28 iron oxide,29 gold,30,31 quantum dots,32 as well as polymer microspheres.33 SI-ATRP provides access to previously inaccessible hybrid systems and polymeric materials with tailored micro/nano-structures, complex molecular architectures, tunable sequences, and a wide variety of functionalities, which are important characters for a variety of biological and biomedical applications. Furthermore, as the chain ends of the grafted polymer brushes via SI-ATRP are terminated with an “active” or “living” halogen atom, the

initially grown polymer can be used as a macroinitiator for further chain extension, resulting in a block copolymer brush. 34,35 Using SI-ATRP, Schö n herr and co-workers synthesized well-defined block copolymer brushes of poly(acrylamide), poly(oligo(ethylene glycol)methyl ether methacrylate), and poly(acrylic acid) on initiator-functionalized gold surfaces.30 “Smart” or “intelligent” nanostructured materials can respond to external stimuli such as pH,36,37 temperature,38,39 light,40 and magnetic field.41 These materials are finding extensive applications in areas spanning bioseparations,42 biosensors,42 and controlled gene/drug delivery.31,43 Boronic acid belongs to one of the most popular pH-responsive ligands that can recognize cis-diol-containing molecules. Poly(N-isopropylacrylamide) (pNIPAm) is a widely studied thermoresponsive polymer,44 exhibiting a lower critical solution temperature (LCST) around 32 °C in aqueous solution. It adopts a random coil structure below the LCST but forms a more collapsed globular structure above the LCST. Liu and co-workers synthesized pNIPAm-grafted silica nanoparticles via SI-ATRP and investigated the thermal behavior of the composite particles.45,46 Nagase and co-workers prepared poly(3-acrylamidopropyl trimethylammonium chloride)-b-polyNIPAm modified silica beads via SI-ATRP and applied the Si-supported polymer brushes as chromatographic matrices for protein separations.47 Recently, we reported a nanohybrid material by grafting random copolymer brushes on silica nanoparticles for glycoprotein separation.48 The long and flexible polymer brushes were found to be beneficial for glycoprotein binding due to the reduced steric hindrance.22 These results motivated us to further explore the feasibility of preparing block copolymer brushes with different monomer sequences and to investigate the impact of polymer structure on its performance of glycoprotein binding. Herein, we report a synthetic protocol, as graphically depicted in Scheme 1, that utilizes a two-step SI-ATRP combined with click chemistry for the synthesis of a new 8986


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2.2. Preparation of Silica Nanoparticles. Silica nanoparticles were prepared using a one-step Stöber procedure.50 Briefly, to a flask equipped with a magnetic stirrer, H2O (33 mL), methanol (100 mL), and ammonia (25%, 22.4 mL) were charged, followed by the addition of 13.8 mL TEOS dissolved in 130 mL methanol. After 8 h of stirring at ambient temperature, the silica nanoparticles formed were isolated by centrifugation at 12000 rpm, washed with water and methanol thoroughly, and dried in a vacuum desiccator. 2.3. Preparation of Amino-Functionalized Silica Nanoparticles (Si@NH2). Silica nanoparticles (3.0 g) were immersed into 1% APTES solution (1 mL APTES in 99 mL anhydrous toluene) and stirred for 24 h at reflux temperature. After the reaction, the particles were isolated, thoroughly washed with acetone and methanol and dried in a vacuum desiccator. The obtained particle is denoted as Si@NH2. 2.4. Preparation of Initiator-Functionalized Silica Nanoparticles (Si@initiator). Si@NH2 (0.5 g), triethylamine (0.61 mL, 4.35 mmol), and THF (12 mL) were added into a flask. The mixture was stirred and cooled in an ice−water bath. To the suspension, BIBB (0.62 mL, 5.0 mmol) was added slowly. The reaction mixture was warmed to room temperature and stirred overnight. The particles were isolated and purified following the procedures used for the synthesis of the silica nanoparticles. The obtained particle is denoted as Si@ initiator. 2.5. Preparation of pNIPAm Brushes Grafted on Silica Nanoparticles (Si@pNIPAm). Si@initiator (150 mg), NIPAm (510 mg, 4.5 mmol), CuBr (7.2 mg, 0.05 mmol), CuBr2 (1.1 mg, 0.005 mmol), and 2-propanol (3 mL) were added into a 25 mL flask. The mixture was deoxygenated by bubbling with nitrogen gas for 15 min. Me6TREN (11.5 mg, 0.05 mmol) was then added to form the CuBr/ CuBr 2/Me 6TREN ATRP catalyst. The reaction mixture was deoxygenated by bubbling with nitrogen gas for another 15 min and was sealed, and then magnetically stirred at room temperature for 12 h. After the reaction, the mixture was exposed to air to terminate the polymerization. The composite particles were isolated, washed with 5% EDTA solution (5 wt %), water, and methanol, and vacuum-dried. The obtained particle is denoted as Si@pNIPAm. To determine molecular weight, pNIPAm brushes were cleaved from the silica core by etching the composite particles with an aqueous solution of hydrofluoric acid (HF, 5%). The polymer brushes were collected by precipitation in hexane and were analyzed using 1H NMR and GPC measurements (Caution: HF is extremely corrosive, and all operation should be conducted under suitable protection). 2.6. Preparation of Block Copolymer Brushes Grafted on Silica Nanoparticles (Si@pNIPAm-b-pGMA). Si@pNIPAm (100 mg), GMA (640 mg, 4.5 mmol), CuBr (7.2 mg, 0.05 mmol), CuBr2 (1.1 mg, 0.005 mmol), and DMF (3 mL) were added into a 25 mL flask. The reaction mixture was deoxygenated by bubbling with nitrogen gas for 15 min. After addition of PMDETA (8.7 mg, 0.05 mmol) to form the CuBr/CuBr2/PMDETA ATRP catalyst, the mixture was bubbled with nitrogen gas for another 15 min, sealed and magnetically stirred at 90 °C for 24 h. The composite particles formed were isolated and purified following the same procedures used for the synthesis of the Si@pNIPAm particles. The obtained particle is denoted as Si@pNIPAm-b-pGMA. 2.7. Preparation of Silica-Supported Copolymer Brushes Containing Pendant Azide Groups (Si@pNIPAm-b-pN3). Si@ pNIPAm-b-pGMA (100 mg), sodium azide (52 mg, 0.80 mmol), ammonium chloride (43 mg, 0.80 mmol), and DMF (5 mL) were added into a glass vial and sealed. After sonication for 5 min, the glass vial was rotated slowly in a hybridization oven for 24 h at 60 °C. After the reaction, the particles were isolated and washed with water and methanol and vacuum-dried. The product is denoted as Si@pNIPAmb-pN3. 2.8. Preparation of Silica-Supported Copolymer Brushes Containing Pendant Boronic Acids (Si@pNIPAm-b-pBA). Si@ pNIPAm-b-pN3 (80 mg) and PCAPBA (54 mg, 0.23 mmol) were dispersed in a solution of methanol-H2O (v/v, 1/1, 6 mL). The mixture was bubbled with nitrogen gas for 15 min, followed by addition of CuSO4 solution (20 μL, 100 mM) and sodium ascorbate

nanocomposite material for glycoprotein separation. Starting from the initiator-functionalized silica nanoparticles, the consecutive grafting of NIPAm and glycidyl methacrylate (GMA) monomers resulted in nanosilica coated with pNIPAmb-pGMA brushes. Taking advantage of the SI-ATRP, different types of block copolymer brushes with various properties can be prepared by selecting different monomers and adjusting the reaction conditions (e.g., to control the length of each polymer blocks). In this work, we have optimized the length of each polymer block by varying the monomer concentration in order to retain the thermoresponsive property of pNIPAm while keeping a sufficiently high density of epoxide groups from pGMA. The epoxide groups in the pGMA block can serve as an intermediate platform for the synthesis of new affinity adsorbents through further modification, for example by conjugation with boronic acids, aptamers, and antibodies. To the best of our knowledge, this is the first time that block copolymer brush, pNIPAm-b-pGMA, is synthesized via a twostep, consecutive SI-ATRP. After postfunctionalization of the pGMA block with sodium azide, we successfully introduced a high density of alkyne-tagged boronic acid to the polymer brushes via Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC) click reaction and obtained a new boronate affinity nanocomposite, Si@pNIPAm-b-pBA. The nanocomposites were characterized to determine their particle sizes, compositions, density of polymer brushes and affinity ligands, and their thermal responses using dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), elemental analysis, thermogravimetric analysis (TGA), 1 H NMR, and gel permission chromatography (GPC). The core-brush nanocomposites were then applied to enrich glycoproteins [ovalbumin (OVA) and horseradish peroxidase (HRP)] under different pH and temperature conditions. The flexible polymer brushes contain a high density of boronic acid ligands, which significantly improved the binding strength toward the target glycoproteins via simultaneous multivalent interactions. In addition, the optimal composite material was employed successfully to enrich glycoproteins in a real complex sample (egg white). The synthetic approach and the composite material developed in this work are envisaged to have a great potential for the development of more efficient adsorbents for biological samples.

2. EXPERIMENTAL SECTION 2.1. Materials. Tetraethylorthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), triethylamine (TEA), 2-bromoisobutyryl bromide (BIBB), N-isopropylacrylamide (NIPAm), glycidyl methacrylate (GMA), tris(2-dimethylaminoethyl)amine (Me6TREN), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA), CuBr, CuBr2, ethylenediaminetetraacetic acid (EDTA), Alizarin Red S (ARS), CuSO4, sodium ascorbate, sodium azide, bovine serum albumin (BSA), ovalbumin (OVA), horseradish peroxidase (HRP), sodium dodecyl sulfate (SDS), methanol, ammonia, toluene, acetone, tetrahydrofuran (THF), isopropanol, and N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich or Fisher and were used as received unless otherwise noted. 2,2′-Azinobis[3-ethylbenzthiazoline-6-sulfonic acid] diamonium salt (1-Step ABTS) was from Thermo Scientific. Polyacrylamide gels (4−20%, Mini-protean) used in SDS− PAGE were purchased from Bio-Rad. CuBr was stirred overnight in acetic acid, centrifuged, washed with water and methanol, and vacuumdried before use. 3-(Prop-2-ynyloxycarbonylamino)phenylboronic acid (PCAPBA) was synthesized according to our previously published procedure.49 8987


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ACS Applied Materials & Interfaces solution (200 μL, 100 mM). After another 15 min of nitrogen bubbling, the reaction mixture was sealed and rotated slowly in a hybridization oven for 24 h at room temperature. The particles were isolated and purified following the procedures as described above. The obtained product is denoted as Si@pNIPAm-b-pBA. 2.9. Protein Binding with Si@pNIPAm-b-pBA Particles. Protein binding at 20 °C: Si@PNIPAm-b-pBA particles (2 mg) were added to 1 mL of phosphate-buffered saline (PBS) (20 mM phosphate, pH 9.0 or pH 7.4, containing 0.5 M NaCl) and sonicated for 5 min, followed by addition of different concentrations of protein solution (0.4−2.0 mg mL−1 for BSA and OVA, 40−240 μg mL−1 for HRP) (1 mL, in PBS). The mixture was slowly rotated in a hybridization oven at 20 °C for 4 h. The particles were removed by centrifugation, and the protein concentration in the supernatant was determined by measuring the UV−vis absorption at 280 nm for OVA and BSA. To quantify the concentration of HRP, ABTS, a watersoluble ELISA substrate, was used to measure the enzyme activity of HRP. Briefly, diluted HRP solution (0.5∼2.5 μg mL−1, 100 μL) were mixed with ABTS solution (200 μL) and shaken for 5 min before a stop solution (1% SDS, 400 μL) was added to quench the enzymatic reaction. After 1 h, the UV−vis absorption at 650 nm was measured. On the basis of the concentration difference between the initial protein solution and the supernatant solution, the amount of protein bound to the composite particles was calculated. The experimental data are presented as the amount of bound proteins per unit mass (g) of the Si@pNIPAm-b-pBA particles. The bound protein (Q) is calculated using the equation:

Q=

instrument (Thermo Scientific). The morphologies of particles were investigated with a scanning electron microscope (SEM) (Hitachi SU8010) and a transmission electron microscope (TEM) equipped with a field-emission gun (JEOL, model 3000F). The hydrodynamic diameters of the nanoparticles were measured by dynamic light scattering using a temperature-controlled particle size analyzer (Zetasizer Nano ZS, Malvern Instruments, U.K.). Thermal gravimetric analysis (TGA) was carried out in synthetic air. The samples were heated at a rate of 10 °C/min. Elemental analysis (C, H, N, Br, and B) was performed by Mikroanalytisches Laboratorium Kolbe (Germany). UV−Vis absorption spectra were recorded with a Cary 60 UV/vis spectrophotometer (Agilent Technologies). Fluorescence emission was measured using a QuantaMaster C-60/2000 spectrofluorometer (Photon Technology International, Lawrenceville, NJ). 1H NMR spectrum was measured on a Bruker DR X400 spectrometer at 400.13 MHz using CDCl3 (δ = 7.26 ppm) as a solvent. Gel permeation chromatography (GPC) was performed on a Viscothek GPCmax instrument equipped with a PFG column (300 × 8 mm, 5 μm particle size) and a refractive index detector from PSS (Mainz, Germany). DMF containing 5 mM LiBr was used as the mobile phase at a flow rate of 0.75 mL min−1 at 60 °C. Molecular weight was calculated from a calibration curve obtained from polystyrene standards.

3. RESULTS AND DISCUSSION The synthetic procedure used to prepare the boronate affinity nanocomposite, Si@pNIPAm-b-pBA, is shown in Scheme 1. Silica nanoparticles, Si@NH2 and Si@initiator particles, were prepared according to our previous work with minor modification.48 Briefly, bare silica nanoparticles were prepared via a one-step Stöber process. The modification of silica with APTES afforded Si@NH2 nanoparticles, which were then subjected to an acylation reaction with BIBB, leading to Si@ initiator particles. Successful immobilization of the aminopropyl groups and the ATRP initiator on the silica nanoparticles was verified by elemental analysis and TGA. The block copolymer brushes were grafted sequentially on the surface of Si@initiator using a two-step SI-ATRP method. Regarding the sequence of pNIPAm and pGMA, we started from synthesizing both pNIPAm-b-pGMA and pGMA-bpNIPAm brushes. When the first polymer block was synthesized on Si@initiator particles using SI-ATRP, both pNIPAm and pGMA could be grafted on the particles to give Si@pNIPAm and Si@pGMA, respectively. When it came to the second step SI-ATRP, it turned out that pGMA could be successfully grafted from the Si@pNIPAm particles; however, it was difficult to graft pNIPAm from the Si@pGMA particles. As shown in Figure S1, the IR signals corresponding to the amide I and amide II structures did not increase obviously even when the Si@pGMA particles were reacted with a high concentration of NIPAm for a prolonged reaction time. The low efficiency of grafting pNIPAm block from Si@pGMA can be explained by the fact that substituted acrylamides such as NIPAm are intrinsically more difficult to polymerize by ATRP compared to other kinds of monomers such as (meth)acrylates, as has been reported in the literature.51,52 After the first step of grafting pGMA brushes on the surface of Si@initiator, the density of “living” halogen atoms may have decreased, which also made it more difficult to grow pNIPAm brushes from Si@pGMA. Therefore, we chose to focus on Si@pNIPAm-b-pGMA particles in our remaining investigation. As block length plays an important role on the properties of a block copolymer, effective control of each block length is required to achieve an optimal function of a block copolymer and to gain a better understanding of structure−property relationship of block copolymer brushes. In this work, we optimized the length of

(C0 − Ct )V 3 10 m

where C0 (mg mL−1) is the initial concentration of protein, Ct (mg mL−1) is the equilibrium concentration of protein, V (mL) is the volume of the protein solution, and m (mg) is the mass of the particles. Protein binding at 40 °C: two incubation conditions were employed to test the effect of temperature on protein binding. Under condition 1, 1 mL of protein sample (2 mg mL−1 for OVA and BSA, 240 μg mL−1 for HRP, pH 9.0) and 1 mL of Si@pNIPAm-b-pBA particle solution (2 mg mL−1, pH 9.0) were incubated separately at 40 °C in a hybridization oven for 30 min. After that, the protein solution and the particle solution were mixed and allowed to rotate at 40 °C for 4 h. The amount of bound protein was calculated as described above. Under condition 2, a solution of HRP protein (240 μg mL−1, 1 mL, pH 9.0 or pH 7.4) and a solution of Si@pNIPAm-b-pBA particles (2 mg mL−1, 1 mL, pH 9.0 or pH 7.4) were mixed and allowed to rotate at 20 °C for 4 h. After the sample was centrifuged, 10 μL of supernatant was collected. The particles were redispersed in the same tube and allowed to rotate for another 4 h at 40 °C. After this step, the sample was centrifuged to allow the supernatant to be collected. After the collected supernatant cooled down to room temperature, 10 μL of the supernatant was collected. HRP concentration in the two collected supernatants was quantified using the same enzyme assay as described above. From the results of the enzyme assay, the protein binding to the composite particles at the different temperatures was calculated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS− PAGE) analysis was employed to evaluate the efficiency of glycoprotein separation. Briefly, Si@pNIPAm-b-pBA solution (6 mg mL−1, 1 mL; 20 mM PBS, pH 9.0) were added into BSA solution (1 mg mL−1, 1 mL; 20 mM PBS, pH 9.0), OVA solution (1 mg mL−1, 1 mL; PBS, 20 mM, pH 9.0), and 100-fold diluted egg white (1 mL; 20 mM PBS, pH 9.0 or pH 7.4), respectively. The mixtures were incubated at room temperature for 4 h and then centrifuged. The particles were washed with the same PBS before the bound proteins were eluted in sodium acetate buffer (HAc-NaAc, 20 mM, pH 4.0, 0.2 mL). The eluted proteins were lyophilized and dissolved in PBS (20 mM, pH 9.0, 0.1 mL), and were analyzed by SDS−PAGE. 2.10. Characterization. Attenuated total reflection (ATR) infrared spectra were recorded in the range of 4000−525 cm−1 with a resolution of 4 cm−1 and 16 scans using a Nicolet iS5 FT-IR 8988


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Figure 1. Hydrodynamic particle size of Si@pNIPAm (a) and Si@pNIPAm-b-pGMA (b) measured in deionized water.

the pNIPAm block by varying the monomer concentration to achieve the desired thermal sensitivity. After grafting the second block, the obtained Si@pNIPAm-b-pGMA particles were subjected to a ring-opening reaction with sodium azide to introduce multiple azide groups in the outer segment. After the introduction of alkyne-tagged boronic acid (PCAPBA) via click reaction, we obtained the target composite particles that contain a temperature-responsive segment extended to a boronic acid-rich block, Si@pNIPAm-b-pBA. 3.1. Sensitivity of Grafted Polymer Brushes to Temperature. Hydrodynamic diameters and size distributions of the different particles were monitored by DLS. As shown in Figure S2, at 20 °C, the mean hydrodynamic diameters of silica core, Si@NH2, and Si@initiator particles were 225, 227, and 242 nm, respectively. It is known that pNIPAm undergoes a rapid chain dehydration and aggregation as its environmental temperature increases above its LCST (ca. 32 °C). The aggregated pNIPAm has more hydrophobic character than its extended form.53,54 This reversible property endows pNIPAmgrafted particle system the ability to tune its architecture and chemistry through appropriate adjustment of its environmental temperature. In our work, the length of the pNIPAm brush was optimized based on the thermal sensitivity of the Si@pNIPAm particles. As shown in Figure S3, when the amount of NIPAm monomer added in the SI-ATRP reaction mixture was 1.5 mmol, the difference in the hydrodynamic diameter of the Si@ pNIPAm particle between 20 and 40 °C was around 22 nm, which was considered not large enough. Therefore, the NIPAm monomer was increased to 4.5 mmol in order to grow longer pNIPAm brushes. Figure 1a shows that after increasing the NIPAm concentration, the hydrodynamic diameter of the obtained Si@pNIPAm reached 370 nm at 20 °C. Upon heating to 40 °C, its hydrodynamic diameter decreased to 287 nm. The new Si@pNIPAm obtained therefore displayed an obvious response to temperature variation, which was used to initiate the second step of the SI-ATRP reaction. As shown in Figure 1b, the hydrodynamic diameters of Si@pNIPAm-b-pGMA particles at 20 and 40 °C were found to be 627 and 535 nm, respectively, suggesting that the block copolymer brushes also have a satisfactory thermoresponsiveness. Along with the stepwise modification of the particles, the size distribution of the particles also increased slightly. 3.2. Characterization of Particles using SEM and TEM. The morphologies and sizes of Si@initiator, Si@pNIPAm, and Si@pNIPAm-b-pGMA particles were characterized by SEM and TEM. As seen from Figure 2 (panels a and b), the Si@ initiator nanoparticles were spherical and had similar diameters

Figure 2. SEM images of (a) Si@initiator, (c) Si@pNIPAm, and (e) Si@pNIPAm-b-pGMA. TEM images of (b) Si@initiator, (d) Si@ pNIPAm, and (f) Si@pNIPAm-b-pGMA.

(∼200 nm). After grafting pNIPAm brushes from the Si@ initiator surface, the Si@pNIPAm particles exhibited a core− shell structure where the silica core and the surrounding polymer shell (∼5 nm) could be distinguished in the TEM image (Figure 2, panel d). After further grafting the pGMA block, the thickness of the polymer shell in the obtained Si@ pNIPAm-b-pGMA particles increased to ∼30 nm (Figure 2, panels e and f). The diameters of the nanocomposites observed by SEM and TEM were smaller than that measured by DLS, due to that the polymer brushes collapsed when the composite particles were dehydrated during the electron microscopy analyses. 3.3. Characterization of Particles by Compositional Analyses. The chemical composition of the composite particles was analyzed by FT-IR (Figure 3). The most distinct absorption peak at 1062 cm−1 is ascribed to the asymmetric vibration of Si−O−Si (Figure 3, curve a). After the acylation reaction with BIBB, two weak amide signals corresponding to 8989


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ACS Applied Materials & Interfaces Table 1. Elemental Analysis Results entry

sample name

%C

%H

%N

% Br

%B

1 2 3 4 5 6

Si@NH2 Si@initiator Si@pNIPAm Si@pNIPAm-b-pGMA Si@pNIPAm-b-pN3 Si@pNIPAm-b-pBA

1.32 2.05 9.31 34.42 32.24 34.83

1.99 2.03 3.07 5.56 5.54 5.31

0.28 0.14 1.62 0.97 3.55 3.74

− 0.8 0.35 − − −

− − − − − 0.29

particles were consumed to immobilize the ATRP initiator. After the first step SI-ATRP, the content of C, H, and N increased significantly (entry 3), which resulted from the successful grafting of the pNIPAm brushes. Compared with the Si@pNIPAm particles, the content of C and H in Si@pNIPAmb-pGMA particles further increased and the content of N decreased (entry 4). After the introduction of azide groups into the polymer brushes, the content of N increased significantly (entry 5). On the basis of the content of boron (entry 6), the density of boronic acid ligands in the Si@pNIPAm-b-pBA particles was estimated to be 0.27 mmol g−1. The organic content in the different particles (Si, Si@NH2, Si@initiator, Si@pNIPAm, Si@pNIPAm-b-pGMA, and Si@ pNIPAm-b-pBA) was further investigated by TGA analysis. In Figure 4, the weight loss below 250 °C can be attributed to the

Figure 3. FT-IR spectra of (a) Si@NH2, (b) Si@initiator, (c) Si@ pNIPAm, (d) Si@pNIPAm-b-pGMA, (e) Si@pNIPAm-b-pN3, and (f) Si@pNIPAm-b-pBA.

amide I (1634 cm−1, CO stretching) and amide II (1534 cm−1, N−H stretching) appeared (Figure 3, curve b). After grafting the pNIPAm brushes, the intensity of the two amide signals (at 1646 and 1541 cm−1) increased significantly (Figure 3, curve c). For the Si@pNIPAm-b-pGMA particles, the IR bands at 1722, 1388, and 1453 cm−1 are associated with the stretching vibration of the ester carbonyl groups, the antisymmetric stretching of the carbonyl groups, and the methylene scissoring, respectively (Figure 3, curve d). These new bands indicated the successful grafting of the pGMA block. The success of sequential grafting of block copolymer brushes confirmed the living nature of the polymer chains synthesized in the first step ATRP reaction. After reacting the Si@pNIPAmb-pGMA particles with NaN3, a new IR band at 2103 cm−1 was observed from the Si@pNIPAm-b-pN3 particles (Figure 3, curve e). This IR signal is characteristic for the pendant azide groups, which were derived from the epoxide groups after reaction with sodium azide. The introduction of alkyne-tagged boronic acid via click reaction led to almost complete disappearance of the azide band (Figure 3, curve f), which confirmed the nearly quantitative conversion enabled by the high efficiency click reaction. ARS is a generally used diol-containing dye which displays dramatic change in color and fluorescence intensity after forming esters with boronic acids.55 Therefore, ARS was employed as a fluorescent reporter to detect the boronic acid ligands in the Si@pNIPAm-b-pBA particles. As shown in Figure S4, when Si@pNIPAm-b-pBA particles were mixed with ARS, the resulting complex displayed an orange color. The ARStreated particles also emitted a strong fluorescence at 600 nm (Ex: 469 nm), as shown in Figure S5. In comparison, when the silica nanoparticles and Si@pNIPAm-b-pN3 particles were treated with ARS, none of the particles exhibited color change or fluorescence emission. All these results confirmed the successful synthesis of the target nanocomposite, Si@pNIPAmb-pBA, and the expected responsiveness of the nanocomposite toward cis-diol molecules. The element contents of the different composite particles were determined by elemental analysis as listed in Table 1. On the basis of the nitrogen content in particle Si@NH2 (entry 1) and the bromine content in particle Si@initiator (entry 2), the density of the aminopropyl group and the initiator on the silica particles were calculated to be 0.207 and 0.105 mmol g−1, respectively. About 51% of the amino groups on the silica

Figure 4. TGA analysis of the (a) silica core, (b) Si@NH2, (c) Si@ initiator, (d) Si@pNIPAm, (e) Si@pNIPAm-b-pGMA, and (f) Si@ pNIPAm-b-pBA particles.

evaporation of residual organic solvent and water. TGA reveals ∼1.6 wt % difference in weight retentions at 750 °C between bare silica and Si@NH2 particles (curves a and b), which is attributed to the modification with APTES. If we regard the conversion of the polycondensation reaction between the silica core and APTES to be 100% and no ethoxyl group is left, the density of aminopropyl group on silica would be ∼0.310 mmol g−1. On the basis of the result obtained from the elemental analysis, the efficiency of the polycondensation reaction can be estimated to be 67%. The difference in weight loss between Si@NH2 and Si@initiator particles (curves b and c) is ∼1.4 wt %, which is attributed to the immobilized initiator molecules. If the mass retention of Si@initiator at 750 °C is used as a reference, the amount of initiator immobilized on silica is ∼0.106 mmol g−1. This value is in agreement with the result obtained from the elemental analysis (0.105 mmol g−1). 8990


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Figure 5. (a) Protein binding isotherm measured at pH 9.0 for OVA (▲) and BSA (●). (b) Protein binding isotherm measured at pH 9.0 for HRP (■). The concentration of the Si@pNIPAm-b-pBA composite particles was 1 mg mL−1.

NIPAm. As the isolated pNIPAm-b-pGMA has very low solubility in the common solvents used for 1H NMR and GPC analysis, we were not able to measure its molecular weight directly. However, based on the molar ratio between pNIPAm and pGMA (1/5) obtained from the TGA analysis, the average molecular weight of the pGMA block can be estimated to be approximately 71600 g mol−1. 3.5. Protein Binding with Si@pNIPAm-b-pBA Particles. The affinity of boronate materials toward glycoproteins is based on the esterification reaction between boronic acid and cis-diols of glycans in an alkaline solution. To determine the selectivity and efficiency of the prepared Si@pNIPAm-b-pBA particles for protein separation, two glycoproteins OVA, HRP, and a nonglycoprotein BSA were employed as models. The model proteins dissolved in phosphate-buffered saline (PBS, 0.02 M; pH 9.0, containing 0.5 M NaCl) were incubated with the composite materials. NaCl was added to minimize nonspecific adsorptions caused by electrostatic interactions between the proteins and the negatively charged boronic acid groups. As can be seen from the equilibrium binding isotherms (Figure 5, panels a and b), the binding capacities of the composite material Si@pNIPAm-b-pBA toward the two glycoproteins are 98.0 and 26.8 mg g−1 for OVA and HRP, respectively. These values are much higher than the capacities achieved in our previous work.48 By contrast, the binding of the nonglycoprotein (BSA) was much less (6.7 mg g−1). The high adsorption capacity and selectivity are mainly attributed to the multiple site interaction of boronic acid ligands immobilized on the long and flexible polymer brushes, which substantially reduced steric hindrance and increased the possibility of forming covalent bonds between boronic acids and the target glycoproteins. Moreover, due to the presence of the amide structure in the boronic acid molecule, PCAPBA is more hydrophilic and easier to combine with glycoproteins compared to the commonly used vinylphenylboronic acid (VPBA). As an example, Zhang and co-workers prepared poly(VPBA) chain grafted polymer beads that had a binding capacity of 31.4 mg g−1 for OVA.56 Furthermore, compared with previously reported bulk boronate affinity matrices such as macroporous cryogel57 (with a binding capacity below 25 mg g−1 for OVA) and surface imprinted polymer nanospheres58 (with a binding capacity below 10 mg g−1 for both OVA and HRP), the new core-brush structured composite with multiple boronic acid ligands in the polymer brushers displayed significantly higher capacity of glycoprotein binding.

For Si@pNIPAm, Si@pNIPAm-b-pGMA, and Si@pNIPAmb-pBA particles, a large weight loss occurred when the temperature increased from 250 to 750 °C (curves d, e, and f). This large weight loss is due to the decomposition of the organic polymer on the surface of these core-brush composite particles. On the basis of the mass retention of the TGA curve (curves d and e), the amount of the pNIPAm brushes and pGMA brushes grafted on silica are calculated to be ∼0.196 and ∼1.3 g g−1 silica, respectively. Considering that the molecular weight of GMA is 1.25 times of that of NIPAm, we can calculate that the molar ratio of NIPAm to GMA in the block copolymer brushes is around 1:5. The relatively long pGMA block allows for the immobilization of abundant boronic acid ligands, which is beneficial for achieving a high binding capacity for glycoproteins. On the basis of the mass retention of the Si@ pNIPAm-b-pBA particles (29.6%, curve f) and the density of boronic acid (0.27 mmol g−1) measured by elemental analysis, we can estimate that the density of boronic acid ligand immobilized on the composite particles to be ∼0.91 mmol g−1 silica, which is 2 times higher than that achieved in our previous work.48 3.4. Characterization of Molecular Weight and Brush Density. To determine the molecular weight of the grafted polymer brushes, the composite particles Si@pNIPAm and Si@ pNIPAm-b-pGMA were treated with HF to etch the silica core. The polymer brushes were collected by dialysis and freezedried. The molecular weight of pNIPAm brushes was measured using both 1H NMR and GPC analysis. When the purified pNIPAm was analyzed by 1H NMR in CDCl3, the proton signal from the isopropyl group at 4.03 ppm was compared with the signal located at 3.3 ppm due to the terminal C−CH2−N group. The comparison led to an estimated molecular weight of 10800 g mol−1. The GPC analysis of the purified pNIPAm revealed Mn = 11800 g mol−1 and Mw/Mn = 1.30 (Figure S6). The nearly symmetric GPC trace and the relatively narrow dispersity suggest that the surface-initiated ATRP has been conducted in a controlled manner. As the mass of the pNIPAm grafted on silica core was ∼0.196 g g−1 silica, the density of the pNIPAm brushes can be calculated to be ∼0.017 mmol g−1 silica, which corresponds to ∼0.7 chains per nm2 on silica surface, if the density of the silica nanoparticles (200 nm in diameter) is assumed to be identical to that of bulk silica (2.07 g cm−3). Considering that the density of ATRP initiator immobilized on silica was ∼0.106 mmol g−1, about 16% of the immobilized initiator actually initiated the polymerization of 8991


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Figure 6. (a) Protein binding isotherm measured at pH 7.4 for OVA (▲) and BSA (●). (b) Protein binding isotherm measured at pH 7.4 for HRP (■). The concentration of Si@pNIPAm-b-pBA particles was 1 mg mL−1.

At high pH (pH 9.0), boronic acid exists in the form of a tetragonal anion that can react with cis-diols to form cyclic esters easily. However, as reported before,59 an alkaline pH condition to operate affinity separation can increase the risk of degradation of labile biomolecules. A neutral or physiological binding pH would be more favorable for biological samples. In previous works, sulfonyl-substituted phenylboronic acid,60 Wulff-type boronic acid,61 improved Wulff-type boronic acid,62 and azide-tagged boronic acid48,63,64 have been reported to be able to capture cis-diol-containing biomolecules under neutral conditions. In this work, the protein binding offered by the Si@ pNIPAm-b-pBA particle toward the glycoproteins and the nonglycoprotein at physiological condition (pH 7.4) was also investigated. As shown in Figure 6, at pH 7.4, the binding of glycoprotein to Si@pNIPAm-b-pBA was lower than that measured at pH 9.0. However, the core−shell particles were still able to bind OVA and HRP with a capacity of 54.2 and 17.6 mg g−1, respectively, which are significantly higher than the binding of the nonglycoprotein BSA. This result may be explained by that due to the triazole nitrogen located in the vicinity of the phenylboronic acid, the pKa of the boronic acid was decreased. The triazole group can help keep the B atom in a sp3 configuration even under neutral pH conditions.63−65 For these reasons, the new composite particle is suitable for separation of glycoproteins from biological samples under physiological pH condition. 3.6. Modulation of Protein Binding by Temperature. Temperature responsive materials offer a possibility to modulate their structures and properties via temperature control. As discussed earlier, the particle size of the nanocomposites changed when the temperature of the media was altered. On the basis of this response, we further investigated the temperature effect on protein binding to the Si@pNIPAmb-pBA particles. Two incubation conditions were investigated to study the effect of increasing temperature. In the first binding experiment, the particle solution and protein solution were heated to 40 °C separately before they were mixed. As shown in Figure 7, compared to glycoprotein binding measured at 20 °C, at 40 °C, the binding of OVA and HRP to Si@pNIPAm-b-pBA decreased to 55.8 and 13.4 mg g−1, respectively, indicating that the increase in temperature indeed reduced the glycoprotein binding. As discussed previously, the nanocomposite prepared in this work had a relatively high grafting density (0.7 chains per nm2), which was achieved by the “grafting from” technique (SI-ATRP). Dense polymer chains tended to stretch away from

Figure 7. Effect of temperature on protein binding to Si@pNIPAm-bpBA particles at pH 9.0. The concentration of OVA, BSA, and HRP are 1 mg mL−1, 1 mg mL−1, and 120 μg mL−1, respectively.

the solid surface to form “brushes” instead of “mushroom” structures.24,25 Vancso and co-workers probed the thermal collapse of grafted pNIPAm chains with different grafting densities (0.69, 0.27, and 0.03 chains per nm2) by quantitative in situ ellipsometry measurements.66 Their results showed that high density chains exhibited a sharp phase transition different from low density chains. Notably, pNIPAm of high grafting density could undergo a phase transition. On the basis of these results, the temperature effect on glycoprotein binding of the nanocomposite can be easily understood from the aspect of structure change of the block copolymer brushes. Above the LCST of pNIPAm, the pNIPAm block became dehydrated and collapsed, making the appended boronic acids to locate closer to the silica core and thus become less accessible to the glycoproteins. In contrast to the binding of the two glycoproteins, the binding of the nonglycoprotein BSA did not show a difference when the temperature was increased from 20 to 40 °C. The temperature effect on glycoprotein binding can be evaluated in another way (incubation condition 2). The glycoprotein binding was first established at 20 °C for 4 h, followed by increasing the incubation temperature to 40 °C. After each binding step reached equilibrium, the concentration of the free protein was determined. As shown in Figure 8, at pH 8992


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Figure 9. SDS−PAGE of protein fractions after treatment with Si@ pNIPAm-b-pBA particles in pH 9.0. Lane M, protein marker; lane 1, BSA before adsorption; lane 2, remaining BSA after adsorption; lane 3, BSA eluted from the particles; lane 4, OVA before adsorption; lane 5, remaining OVA after adsorption; lane 6, OVA eluted from the particles; lane 7, 100-fold diluted egg white; lane 8, remaining 100-fold diluted egg white after adsorption; lane 9, eluted 100-fold diluted egg white.

Figure 8. Effect of temperature on HRP binding to Si@pNIPAm-bpBA particles at pH 9.0 and pH 7.4. The concentration of HRP was 120 μg mL−1.

9.0, when the temperature was increased from 20 to 40 °C, the bound HRP remained to be captured by the boronic acids appended in the polymer brushes. At pH 7.4, when the incubation temperature was increased from 20 to 40 °C, the bound HRP decreased from 17.6 to 11.4 mg g−1, suggesting that ∼35% of the bound HRP was released. This result can be explained as follows: at pH 9.0, boronic acid exists in the form of a tetragonal anion, making the covalent bonds formed between the boronic acids and HRP strong enough to retain the bound glycoprotein even when the inner pNIPAm block collapsed. However, at pH 7.4, the boronic acids tend to adopt a trigonal configuration leading to a weaker boronic acidglycoprotein complex, which could be more easily disrupted by the chain collapsing when the temperature was raised to above the LCST. 3.7. Separation of Glycoproteins from Complex Samples. In order to further demonstrate the applicability of our new boronate affinity material for purification of complex samples, a diluted egg white sample was applied to test the efficiency of glycoprotein separation. Pure OVA and nonglycoprotein BSA were used as control. The egg white was 100fold diluted first with PBS (pH 9.0 or pH 7.4). The different protein samples were first incubated with the Si@pNIPAm-bpBA particles. After equilibrium binding, the particles were collected and washed with the binding buffer before an acidic buffer was added to release the bound proteins. SDS−PAGE was used to analyze all the protein fractions. Figure 9 shows the efficiency of the nanocomposite for protein separation at pH 9.0. The strong band in lane 1 corresponded to BSA (65 kDa) before being treated with the particles. The band hardly changed after the treatment with the composite particles (lane 2), and no BSA band was observed in the eluate (lane 3). This result demonstrates that the composite material did not have affinity toward BSA. In contrast, after being processed by the composite particles, the OVA band (46 kDa) faded (lane 5) compared to the original OVA solution (lane 4), and a clear OVA band emerged in the lane of the eluted fraction (lane 6), indicating that the composite material did bind OVA in the basic buffer, and the bound glycoprotein could be released by eluting with the acidic buffer. When the diluted egg white was treated with the same affinity particles, similar glycoprotein binding results were

observed. It is known that ovalbumin comprises 60−65% of the total protein in egg white.67 In the diluted egg white, three abundant protein bands located at 76.7, 46, and 14.4 kDa were observed (lane 7). These bands represented the glycoproteins ovotransferrin (OVT), OVA, and the nonglycoprotein lysozyme (Lyz), respectively. It was obvious that the bands of the glycoproteins OVT and OVA faded after they were treated by Si@pNIPAm-b-pBA particles, while the band corresponding to Lyz (14.4 kDa) remained almost unchanged (lane 8). After the adsorbed proteins were eluted, the bands of the adsorbed glycoproteins appeared in lane 9. Moreover, the glycoproteins in diluted egg white could also be separated using the nanocomposite at neutral pH (Figure S7, lane 3). These results confirm that the composite material can selectively capture and separate glycoproteins directly from complex samples.

4. CONCLUSIONS In summary, a novel dually stimuli-responsive nanostructured core-brush composite, Si@pNIPAm-b-pBA, has been synthesized for bioseparation via the combination of SI-ATRP and click chemistry. The two-step consecutive SI-ATRP provides a facile approach to the fabrication of block copolymer brushes with well-controlled chain architectures. The click reaction was proved to be an efficient coupling method to obtain a high degree of boronic acid ligands immobilized on flexible polymer brushes, which endowed the new nanocomposite excellent affinity toward glycoproteins via synergistic multiple binding with the boronic acids. Most importantly, the nanocomposite can retain high binding capacities in physiological pH conditions and the pNIPAm segment in the block copolymer brushes can serve as an intelligent “spring” to tune the structure of the polymer brushes, thus regulating the binding capacity to glycoproteins. We envision that the synthetic method developed in this work will be of generic use in fabricating multifunctional polymer brush-based materials, including antibody-based affinity materials, immobilized metal-ion affinity chromatography (IMAC) materials, and responsive carriers for controlled gene/drug delivery. 8993


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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/acsami.6b15326. Additional FT-IR, SEM, DLS, fluorescence, GPC, and SDS−PAGE results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 00 46 46 2229560. ORCID

Lei Ye: 0000-0002-3646-4072 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Swedish Research Council FORMAS (Grant 212-2013-1350) and the Swedish Foundation for Strategic Research (Grant RBP14-0055). L.D.J. thanks the China Scholarship Council (CSC) for a Ph.D. fellowship.

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REFERENCES

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Temperature and pH Dual-Responsive Core-Brush Nanocomposite

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