Phase Behavior of Poly (sulfobetaine methacrylate)-Grafted Silica

ARTICLE pubs.acs.org/Langmuir

Phase Behavior of Poly(sulfobetaine methacrylate)-Grafted Silica Nanoparticles and Their Stability in Protein Solutions Zhixin Dong, Jun Mao, Muquan Yang, Dapeng Wang, Shuqin Bo, and Xiangling Ji* State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Changchun 130022, People’s Republic of China

bS Supporting Information ABSTRACT: Biocompatible and zwitterionic poly(sulfobetaine methacrylate) (PSBMA) was grafted onto the surface of initiator-modified silica nanoparticles via surface-initiated atom transfer radical polymerization. The resultant samples were characterized via nuclear magnetic resonance, Fourier transform infrared spectroscopy, transmission electron microscopy, and thermogravimetric analysis. Their molecular weights and molecular weight distributions were determined via gel permeation chromatography after the removal of silica by etching. Moreover, the phase behavior of these polyzwitterionic-grafted silica nanoparticles in aqueous solutions and stability in protein/PBS solutions were systematically investigated. Dynamic light scattering and UV
visible spectroscopy results indicate that the silica-g-PSBMA nanoparticles exhibit an upper critical solution temperature (UCST) in aqueous solutions, which can be controlled by varying the PSBMA molecular weight, ionic strength, silica-g-PSBMA nanoparticle concentration, and solvent polarity. The UCSTs shift toward high temperatures with increasing PSBMA molecular weight and silica-g-PSBMA nanoparticle concentration. However, increasing the ionic strength and solvent polarity leads to a lowering of the UCSTs. The silica-g-PSBMA nanoparticles are stable for at least 72 h in both negative and positive protein/PBS solutions at 37 °C. The current study is crucial for the translation of polyzwitterionic solution behavior to surfaces to exploit their diverse properties in the development of new, smart, and responsive coatings.

’ INTRODUCTION Recently, synthetic polymers containing zwitterionic structures, such as phosphobetaine, carboxybetaine, and sulfobetaine, have attracted considerable attention as biomedical materials because of their excellent hemocompatibility and biocompatibility. The chemical modification of solid substrates with zwitterionic functionalities has become an interesting subject both in research and industry. Examples are the surface-immobilized polymers bearing zwitterionic side groups that prove beneficial in the creation of ultralow fouling surfaces,1
5 in the design of ideal lubricant surfaces,6 in the construction of effective blood-compatible interfaces,7
9 in providing control over electroosmotic flow in microfluidics,10 and as the stationary phase in chromatography.11,12 Self-assembled monolayers containing zwitterionic terminal groups are also used in the fabrication of efficient protein-resistant substrates.13
15 The extremely high dipole moment of zwitterionic groups in polymeric structures endows a range of unique properties because of the strong inter- and intramolecular dipolar interactions that give rise to the selfassociation of the polymer chains.16 Poly(sulfobetaine methacrylate) (PSBMA), which has a methacrylate main chain and a pendant group consisting of an analogue of the taurine betaine [CH2CH2N+(CH3)2CH2CH2CH2SO3
], has been an extensively studied zwitterionic polymer because of its easy synthesis.3,7,8,15,17
20 Previous studies have shown that surfaces modified with PSBMA are ideal for the synthesis of effective and stable superlow fouling materials when the surface density and chain length of the zwitterionic groups are controlled.3,4,18,19,21 r 2011 American Chemical Society

Despite many detailed studies on the properties of surfaces modified with PSBMA, the behavior of the dense layers of these polymers on surfaces is still not well understood. Thus, Huck et al.22 studied the phase transition mechanism of PSBMA brushes grafted from gold substrates and found that the bulk or solution behavior of sulfobetaines has a counterpart on the surface. The self-associated regime develops hydrophobic surface characteristics after passing through the hydrophilic, nonassociated regime, and the ionically cross-linked, superdense conformation is driven by an avalanchetype self-association process. In addition, the self-associated state can be reversed by increasing the temperature. They further investigated the controllable synthesis of PSBMA brushes from the initiator-modified gold substrates and the properties of these brushes as a function of the grafting density and thickness.23 They found that low-molecular weight, dilute brushes are always hydrophilic. Higher-molecular weight, dilute (and/or polydisperse) brushes are also hydrophilic, until the chains stretch away from the surface and acquire a sufficient molecular weight, sufficient density, and monodispersion to form interchain associations. A more recent study by Polzer24 et al. reported that the zwitterionic PSBMA shell on the cross-linked poly(styrene) (PS) surface exhibited a reversible swelling upon heating which was enhanced upon the salt addition. Further efforts have also been taken to Received: September 30, 2011 Revised: November 6, 2011 Published: November 07, 2011 15282

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Langmuir propose a model for the PSBMA brush by a combination of dynamic light scattering (DLS), transmission electron microscopy (TEM), and cryoTEM measurements. In the model, the shell is mostly collapsed near the core surface whereas only a small portion of the shell is in a dilute swollen state. However, the study on PSBMA coatings onto oxide nanoparticles and on the behavior of the grafted polymer brushes on the “hard” curved surface in solutions is less reported. Understanding the phase behavior of PSBMA-grafted nanoparticles in the solution is important, and the results from such studies would enable the rational design of zwitterionic polymer-grafted nanoparticles for biomedical applications. In the current study, a series of silica-g-PSBMA nanoparticles with different molecular weights and similar grafting densities was designed and prepared. The study aims to (1) demonstrate the phase behavior of silica-g-PSBMA nanoparticles, (2) illustrate the effect of the PSBMA molecular weight, ionic strength, silica-g-PSBMA nanoparticle concentration, and solvent polarity on the phase transition temperatures (upper critical solution temperatures, UCSTs), (3) propose a possible schematic illustration for the phase behavior, and (4) investigate the stability of these polyzwitterionic-grafted silica nanoparticles in protein/ PBS solutions.

’ EXPERIMENTAL SECTION Materials. [2-(Methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)ammonium hydroxide (sulfobetaine methacrylate, SBMA, 97%, Aldrich, USA), tetraethoxysilane (TEOS, 98%, Alfa, USA), 3-aminopropyltrimethoxysilane (APTMS, 97%, Aldrich, USA), 2-bromoisobutyl bromide (BIBB, 98%, Aldrich, USA), 2,20 -bipyridine (bpy, 99%, Aldrich, USA), and copper(II) bromide (CuBr2, 99%, Shanghai Chemical Reagent Co., China) were used without further purification. Copper(I) bromide (CuBr, 98%, Shanghai Chemical Reagent Co., China) was treated with glacial acetic acid, washed with ethanol, and then dried in a vacuum oven. Toluene and triethylamine (Et3N) were refluxed with sodium under nitrogen and distilled prior to use. Initiator-functionalized silica nanoparticles were synthesized according to literature procedures25 (Supporting Information). Bovine serum albumin (BSA, fraction V) was purchased from J&K Scientific Ltd. (China). Lysozyme was obtained from Aladdin Chemistry Co. Ltd. (China). All other solvents and chemicals were used as received. For dialysis, regenerated cellulose membranes (MWCO 3400) were used. Phosphate-buffered saline (PBS; 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4) was used at a 0.15 M concentration. Deionized water was purified using a Millipore water purification system with a minimum resistivity of 18.2 MΩ 3 cm. Surface-Initiated Atom Transfer Radical Polymerization (SI-ATRP) of SBMA from Initiator-Functionalized Silica Nanoparticles. A typical synthesis of silica-g-PSBMA nanoparticles

is described as follows: initiator-functionalized silica nanoparticles (50 mg, 0.011 mmol of the initiating sites), CuBr2 (1.2 mg, 0.0055 mmol), bpy (18.9 mg, 0.121 mmol), SBMA (1.2 g, 4.4 mmol), NaCl (0.26 g, 4.4 mmol), and methanol/water (v/v = 4/1, 2.44 mL) were added to a polymerization tube equipped with a magnetic stirring bar. The initiator nanoparticles were dispersed after ultrasonication for 5 min. The mixture was deoxygenated via two freeze
pump
thaw cycles, backfilled with argon. CuBr (7.9 mg, 0.055 mmol) was introduced into the tube under an argon flow. Finally, the reaction mixture was degassed with two additional freeze
pump
thaw cycles, and the tube was immersed into an oil bath at 26 °C for polymerization. After 12 h reaction, the tube was removed from the bath and the reaction mixture was dialyzed against distilled water for 72 h. The product was collected via freeze
drying.

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Cleavage of the PSBMA Chains from the Silica Nanoparticles. The silica-g-PSBMA nanoparticles (60 mg) and an aqueous

hydrofluoric acid (HF) solution (1.0 mL, ∼10 wt %) were added in a polyethylene (PE) tube equipped with a magnetic stirring bar. The mixture was stirred overnight at room temperature and dried in vacuum. The aqueous HF solution (0.5 mL, ∼10 wt %,) was added again. After stirring for 2 h, the mixture was dried in vacuum. This cycle was repeated three times. The mixture was then neutralized by adding an aqueous Na2CO3 solution (1 mL, 5 wt %). Finally, the solution was subjected to dialysis against distilled water for 72 h to remove impurities. The recovered PSBMA was harvested via freeze
drying prior to gel permeation chromatography (GPC).

Characterization of the Silica-g-PSBMA Nanoparticles. Nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker-600 MHz NMR instrument using D2O as the solvent and tetramethylsilane as the internal reference standard. Fourier transform infrared (FTIR) spectra were recorded on a Bruker VECTOR-22 IR spectrometer. The samples were pressed into KBr pellets. The spectra were collected at 64 scans with a spectral resolution of 4 cm
1. Thermogravimetric analysis (TGA) was performed from room temperature to 800 °C at a heating rate of 10 °C/min under a nitrogen atmosphere using a Perkin-Elmer Diamond TG/DTA instrument. TEM was conducted using a JEOL JEM-1011 electron microscope at an acceleration voltage of 100 kV. The samples were mixed with CsI solution to increase the contrast of the shell. Therefore, the silicag-PSBMA nanoparticle dispersion was diluted with CsI solution to reach a solid content of 0.4 wt % of silica-g-PSBMA nanoparticles and the desired concentration of CsI at 0.1 M. Then, the sample was deposited a drop of the foregoing suspension onto a copper grid with a carbon film and dried prior to visualization. The molecular weights and molecular weight distributions of the PSBMA chains were determined using GPC equipped with a Waters 515 pump, Waters 717 plus autosampler, Shodex OHpak SB-804 HQ (300 mm  8.0 mm, 10 μm), and Waters 2414 differential refractive index detector at 35 °C, using monodispersed poly(ethylene oxide) as the calibration standard. An aqueous NH4NO3 (1.0 M) solution was used as the eluent, at a flow rate of 1.0 mL/min.

Determination of the Silica-g-PSBMA Nanoparticle UCSTs. The phase transition temperatures of the silica-g-PSBMA nanoparticles in aqueous solutions at various PSBMA molecular weights, ionic strengths, silica-g-PSBMA nanoparticle concentrations, and solvent polarities were determined using a Malvern Zetasizer Nano ZS90 DLS instrument with a He
Ne laser (633 nm) and 90° collecting optics. The grafted zwitterionic polymer nanoparticles with a given molecular weight were dissolved in an aqueous solution at a concentration of 0.2 g/L at 70 °C. The temperature first rapidly decreased from 70 to 2 °C and then gradually increased from 2 to 58 °C at a rate of 1 °C/min, followed by a decrease from 58 to 2 at 1 °C/min. The UCST in a solution is defined as the temperature at which the maximum slope of the hydrodynamic radius (Rh) versus the temperature curve. The phase transition temperatures were also determined by measuring the absorbance of the silica-g-PSBMA nanoparticle solutions at 550 nm using a UV
visible spectrometer (Shimadzu UV
2401PC) equipped with a temperature controller (Shimadzu S-1700) at a heating rate of 0.5 °C/min. Zeta-Potential Measurements. The zeta-potential measurements were performed with a Malvern Zetasizer Nano ZS90 equipped with a 4 mW He
Ne Laser (633 nm) using folded capillary cells with gold electrodes. One can determine the value of the zeta-potential via the Henry equation, which is UE ¼

2εζf ðkaÞ 3η

where ζ is the zeta-potential, UE is electrophoretic mobility, ε is dielectric constant, and η is viscosity. And f(ka) is the Henry’s function, 15283

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Langmuir which is generally used as approximations either 1.5 or 1.0. Herein the determination of zeta-potential is made in aqueous solutions, so in this case f(ka) is 1.5. The Smoluchowski model is used to calculate ζ from UE.

Stability of the Silica-g-PSBMA Nanoparticles in Protein Solutions. The determination of the particle stability in a protein solution was conducted using the Malvern Zetasizer Nano ZS90 DLS instrument. The experiments were performed by resuspending the modified nanoparticles in a protein/PBS solution (pH 7.4). The concentrations of the modified nanoparticles and proteins were fixed at 0.2 and 10 g/L, respectively. The Rh change of the nanoparticles during the incubation at 37 °C was detected via DLS.

Scheme 1. Synthesis of Silica-g-PSBMA Nanoparticles via SI-ATRP

Figure 1. FTIR spectra of (a) silica, (b) silica-Br, and (c) silica-g-PSBMA nanoparticles.

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’ RESULTS AND DISCUSSION SI-ATRP of SBMA from the Initiator-Functionalized Silica Nanoparticles. The ATRP of SBMA on the surface of the

initiator-functionalized silica nanoparticles was conducted in methanol/water, using silica-Br as the initiator and CuBr/CuBr2/ bpy as the catalyst (Scheme 1). Synthesis of Initiator-Functionalized Silica Nanoparticles. Initiator-functionalized silica nanoparticles were synthesized according to previously reported procedures,25 which are summarized as follows: bare silica nanoparticles were prepared via the St€ober process and then modified with APTMS to afford aminofunctionalized silica nanoparticles. Initiator-functionalized silica nanoparticles were prepared via the amidation of the aminofunctionalized silica nanoparticles with BIBB. The as-prepared nanoparticles were characterized via FTIR analysis. The presence of amide moieties was confirmed by the presence of the amide band (1540 cm
1, N
H stretching) (Figure 1b), as compared to that of bare silica nanoparticles (Figure 1a). The average diameter of the initiator-functionalized silica nanoparticles was determined as approximately 50 nm via TEM (Figure 2b). TGA reveals an approximately 2.9 wt % difference between the weight retentions of the amino- and 2-bromoisobutyrate-functionalized silica nanoparticles at 800 °C (Figure 3b and c). If the mass retention of the amino-functionalized silica nanoparticles at 800 °C is taken as a reference and the density of the silica nanoparticles is assumed identical to that of bulk silica (2.07 g/cm3), the grafting density of the ATRP initiators on the surface of the silica nanoparticles can be estimated as ∼4.54 initiator/nm2. Synthesis of PSBMA-Grafted Silica Nanoparticles. PSBMA chains grafted on silica nanoparticles were prepared via SI-ATRP of SBMA from the initiator-functionalized silica nanoparticles in a mixture of methanol and water. CuBr2 (10 mol % relative to CuBr) was added to ensure an efficient exchange between the dormant and active species to control the polymerization. In addition, NaCl was deliberately added to the catalytic ingredients to control the polymerization rate, as suggested in literature.7,26,27 From Table 1 and Figure 4, the molecular weight of PSBMA, which was cleaved from the silica nanoparticles, decreases with the increase in NaCl concentration. The molecular weight of PSBMA decreases from 13.1  104 to 5.53  104 g/mol when the NaCl concentration is increased from 0 to 1.8 M, which corresponds to samples 5 and 1, respectively. The addition of some alkaline metal salts reportedly increase the initial polymerization rate of SBMA in aqueous solutions, and the polymerization is hastened when the salt concentration is increased.28 The acceleration of the initial polymerization can consume monomers in a shorter time, resulting in a

Figure 2. TEM images of (a) silica, (b) silica-Br, and (c) silica-g-PSBMA nanoparticles. Scale bar: 200 nm. 15284

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rapid increase in the viscosity, the induction of more chain transfer reactions, and a decrease in their molecular weights.28 Furthermore, when the volume ratio of methanol/water decreases from 4 to 1 (corresponding to samples 1 and 4), the molecular weight of PSBMA increases from 5.53  104 to 9.76  104 g/mol, and the polydispersity index (PDI) increases from 1.46 to 2.33, respectively. Baker et al.29 reported that water can accelerate the polymerization of 2-hydroxyethyl methacrylate on gold substrates via ATRP. Polymerization from the surface rapidly occurred at room temperature in aqueous media, resulting in the formation of 700 nm thick polymer films in just 12 h. However, control experiments using a neat monomer and catalyst (no water) yielded films with thicknesses of only 6 nm.29 Hence, the decrease in the methanol/water ratio in the current experiment leads to an increase in the polymer molecular weight at the same polymerization time, and the poor control of polymerization results in the higher PDI of the grafted PSBMA. The FTIR spectrum of the silica-g-PSBMA nanoparticles (Figure 1c) shows the appearance of the characteristic absorption peaks associated with PSBMA, namely, the peaks at 1730 cm
1 (the carbonyl group) and 1033 cm
1 (the sulfonate group), indicating the successful grafting of the PSBMA chains on the surface of the silica nanoparticles via SI-ATRP. The 1H NMR spectrum of the silica-g-PSBMA nanoparticles is shown in Figure 5. 1 H NMR (600 MHz, D2O), δ (ppm) = 1.97 (CH2C(CH3)COO), 2.26 (CH2CH2CH2SO3
), 2.96 (CH2CH2SO3
), 3.21 (CH2N+ (CH3)2CH2), 3.57
3.60 (CH2CH2N+(CH3)2), 3.79 (CH2N+ (CH3)2CH2CH2O), 4.48 (CH2CH2OCO). In addition, the 1H NMR spectrum of the SBMA monomer is shown in Figure S1 in the Supporting Information. According to the characteristic peaks of PSBMA segments and the disappearance of vinyl double bonds at δ of 5.7 and 6.1 ppm, we can confirm the successful polymerization of the SBMA monomer on the silica nanoparticle surfaces.

The morphology of the silica-g-PSBMA nanoparticles was observed using TEM and the contrast of the zwitterionic PSBMA shell was increased by adding CsI to the suspension. As shown in Figure 2c, the dark silica cores in the structure of the silicag-PSBMA nanoparticles are surrounded by a light gray polymer shell. The average size of the core nanoparticles is about 50 nm in diameter. Measurements of the shell thickness in the dried state according to Figure 2c reveal the value of about 28.6 nm, and the overall radius of the silica-g-PSBMA nanoparticles is about 53.6 nm. According to the following DLS measurements, the Rh of the dispersed silica-g-PSBMA nanoparticles in water at 54 °C is 61.2 nm, which is larger than 53.6 nm in dried state since PSBMA chains are extended in water. The grafting density (σ) of the tethered polymer chains, calculated using TGA data, is an important parameter that determines the property of the polymer-grafted surface. As shown in Figure 3, the weight retention of the silica-g-PSBMA nanoparticles at 800 °C is approximately 15.4%. Using the weight

Figure 4. GPC curves, samples with different polymerization conditions in Table 1 and PSBMA chains were cleaved from silica nanoparticles via etching with HF.

Figure 3. TGA curves of (a) silica, (b) silica-NH2, (c) silica-Br, and (d) silica-g-PSBMA nanoparticles. TGA was performed under nitrogen atmosphere at a heating rate of 10 °C/min.

Figure 5. 1H NMR spectrum of the silica-g-PSBMA nanoparticles.

Table 1. Experimental Conditions and Results of Silica-g-PSBMA Nanoparticles Prepared via SI-ATRP at 26 °Ca no.

solvent (MeOH/water)

[SBMA] (M)

[NaCl] (M)

reaction time (h)

Mw (104 g/mol)b

PDI (Mw/Mn)b

σ (chains/nm2)c

Rg/Dd,e

1 2

4 1

1.8 1.8

1.8 1.8

12 4

5.53 6.74

1.46 2.34

0.38 0.40

6.3 12.8

3

1

1.8

1.8

8

7.75

2.25

0.35

10.9

4

1

1.8

1.8

12

9.76

2.33

0.34

11.8

5

4

1.8

0

12

1.90

0.32

11.2

13.1

a

The ratio of initiator/CuBr/CuBr2/bpy was 1:5:0.5:10. b Apparent values determined by conventional GPC with 1.0 M NH4NO3 aqueous solution as the eluent and PEO standards as calibration. PSBMA chains were cleaved from silica nanoparticles via etching with HF. c Grafting density determined by TGA. d Rg: radius of gyration estimated by static light scattering (SLS). e Distance between neighboring grafted chains D = σ
1/2. 15285

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Figure 6. Phase transition of silica-g-PSBMA nanoparticles in aqueous solutions during (a) heating process and (b) one heating-and-cooling cycle; Rh distributions of silica-g-PSBMA nanoparticles in aqueous solutions during (c) heating and (d) cooling process.

retention of the silica-Br nanoparticles at 800 °C (82.1 wt %) as a reference, the PSBMA weight content relative to that of the silica cores was calculated as ∼86.7%. The number average molecular weight (Mn) of the grafted PSBMA chain was determined as 37 900 using GPC analysis; the grafting density of the PSBMA chain on the surface of a silica nanoparticle is estimated as ∼0.38 chain/nm2; and the number of grafted PSBMA chains on the surface of one silica nanoparticle is approximately 2983. Brooks et al.30 reported that the grafted polymer layers are in the brush regime and are highly strained when the Rg/D value is greater than 2 (Rg/D > 2; D is the average distance between the grafted chains and Rg is the radius of gyration of the polymer in solution). In the current system, the Rg/D values for the synthesized polymer-grafted nanoparticles are given in Table 1, where D is denoted as σ
1/2 and Rg is measured using static light scattering. The Rg/D values of all the samples are much higher than 2, indicating that the grafting density of PSBMA on the surface of the silica nanoparticles synthesized via SI-ATRP is in the brush regime. Phase Behavior of PSBMA Grafted on Silica Nanoparticles. Figure 6a shows a typical phase transition (UCST) of silicag-PSBMA nanoparticles during the heating process measured using the UV
visible spectrometer and DLS. The transmittance and Rh of the silica-g-PSBMA nanoparticles in the aqueous solution vary with temperature. At temperatures below 26 °C, the silica-g-PSBMA nanoparticles exist in an aggregated state, with a transmittance of 24%. A phase transition occurs in the 26
54 °C temperature range, and the transmittance of the silicag-PSBMA solution increases with increasing temperature. The transmittance of the silica-g-PSBMA nanoparticle solution reaches 100% when the temperatures are above 54 °C, indicating a good dispersion of the nanoparticles in the solution without polymer chain associations from the different silica cores. Meanwhile, the Rh of the silica-g-PSBMA nanoparticles changes with temperature. Below 26 °C, the Rh is approximately 134.3 nm. The Rh then gradually decreases when the temperature is increased

from 26 to 54 °C. Finally, above 54 °C, the Rh remains stable at approximately 61.2 nm. Figure 6b shows the change in the Rh of the silica-g-PSBMA nanoparticles during the cooling process. The Rh curve during cooling is below that during the heating process, and the Rh during cooling is smaller than that during heating almost in the entire temperature range of 2
58 °C. Comparison between the initial Rh below 26 °C during heating and that after a heating-and-cooling cycle reveals an Rh of 134.3 and 88.6 nm, respectively, indicating that the nanoparticle size cannot return to its initial state as the environment cools down. However, the phase transition temperature during heating is 41 °C, which is the same as that during cooling. Figure 6c and d shows the Rh distributions of silica-g-PSBMA nanoparticles during the heating and cooling process. The Rh distributions are 0.182 at 15 °C and 0.133 at 55 °C during the heating process. When the temperature cools down, the Rh distributions are 0.221 at 15 °C and 0.185 at 55 °C. The above results imply a narrow size distribution for nanoparticles during the heating and cooling process. And the size distributions in the aggregated state are broader than those in the individual dispersed state. PSBMA, like other zwitterionic polymers, generally exhibits an UCST in an aqueous solution.31 Polzer et al.24 found an increase in shell thickness of PSBMA brush upon heating, and the results for the cooling and reheating show a good reproducibility. According to the model proposed by Polzer et al., the shell of the PSBMA brush undergoes a phase separation into a condensed phase near the core surface and a dilute swollen layer of the shell which extends far into the solution. When upon heating or cooling, the shell changes due to the UCST behavior of the PSBMA chains. Obviously, the different phenomenon is observed in the current study. Very strong electrostatic interactions exist in the intrachain association of zwitterionic groups on the same chains and in the interchain association between neighboring polymer chains in the silica-g-PSBMA nanoparticle system. Figure 6 shows the changes in the transmittance and Rh of the silica-g-PSBMA nanoparticles with the temperature. First, at 15286

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Figure 7. Simple model for the phase transition of the silica-g-PSBMA nanoparticles in aqueous solution during one heating-and-cooling cycle.

temperatures below 26 °C, the strong intra- and interchain electrostatic attraction by ion pairings between the ammonium cation and the sulfo-anion of the zwitterionic sulfobetaine groups induces the aggregation of the silica-g-PSBMA nanoparticles, and then results in the formation of a translucent physical gel. Second, when the temperature is increased from 26 to 54 °C, the obtained thermal energy gradually weakens the mutual electrostatic attraction, which leads to a phase transition followed the increase in the transmittance and the decrease in Rh. In the third stage (above 54 °C), the electrostatic interactions of polymer chains from the different silica cores are fully destroyed and disappear. Finally, the silica-g-PSBMA nanoparticles show an excellent dispersed state, with a transmittance up to 100%; these nanoparticles are also stable, with an Rh of 61.2 nm. During the cooling process (58
2 °C), the same three Rh stages are found and the UCST is detected at 41 °C, which is the same as that during the heating process. The difference between the heating and cooling processes is that the Rh cannot return to the initial state after cooling; this phenomenon may be due to the rearrangement of the silica-g-PSBMA nanoparticles. The PSBMA chains are densely tethered to the curved silica surface and cannot easily aggregate and disaggregate like free chains. The PSBMA chains from the same and different silica cores gradually entangle and again interpenetrate when the environmental temperature cools down. These chains cannot keep the same number of aggregates compared to the initial state under heating. Hence, during cooling, they cannot return to the same initial Rh as that during heating. Figure 7 shows a simple model for the phase transition of the silica-g-PSBMA nanoparticles in an aqueous solution during one heating-and-cooling cycle. Effect of the PSBMA Molecular Weight, Ionic Strength, Silica-g-PSBMA Nanoparticle Concentration, and Solvent Polarity on the UCSTs of PSBMA Brushes in Solutions. Samples 1, 2, 3, 4, and 5 have similar grafting densities, σ (0.36 ( 0.04 chain/nm2), and the changes in their phase transition behavior with the molecular weight, ionic strength, concentration, and solvent polarity in aqueous solutions were investigated. As shown in Figure 8a, the zwitterionic PSBMA brushes in the aqueous solution almost exhibit a linear increase in UCSTs, from 20 to 52 °C, which correspond to the molecular weights of the grafted PSBMA (from 5.53  104 to 13.1  104 g/mol, respectively). The Rh of zwitterionic polymer brushes generally increases with increasing molecular weight and contributes to the formation of

more intra- and interchain electrostatic attractions (i.e., more ionic pairings of opposite charges between zwitterionic groups). Therefore, the phase transition of high-molecular weight PSBMA brushes in aqueous solutions requires a high temperature, that is, sufficient thermal energy to break the electrostatic bonding between ionic pairs and to produce individual polymer-grafted nanoparticles dispersed in the aqueous solution. The chain expansion of a zwitterionic polymer in an aqueous solution occurs upon the addition of an electrolyte, exhibiting the so-called antipolyelectrolyte effect.32,33 This behavior is typical of most zwitterionic polymers composed of sulfobetaines with three methylene units between the cationic and anionic groups.17,34,35 In Polzer’s study,24 the extension of the PSBMA shell on the PS surface upon the addition of salt have been investigated and the shell showed a reversible swelling upon heating both in water and salt solutions. Nevertheless, in our system, the effect of the salt concentration on the UCST values of silica-g-PSBMA nanoparticles is discussed. The ionic strength of the aqueous solution was adjusted by dissolving the electrolyte NaCl in deionized water at concentrations ranging from 0 to 0.03 M. The UCST dependence of sample 4 on the NaCl concentration is shown in Figure 8b. The UCST of the silica-g-PSBMA nanoparticles exhibits a rapid decrease from 41 to 28 °C when the NaCl concentration increases from 0 to 0.005 M, followed by a gradual decrease to 22 °C when the NaCl concentration is further increased to 0.03 M. The silica-g-PSBMA nanoparticles definitely exhibit an unusual antipolyelectrolyte effect in the presence of salt ions, which notably increases with the ionic strength of the aqueous solution. The UCST dependence of PSBMA brushes on NaCl concentration is attributed to the screening of the net attractive electrostatic interactions by Na+ and Cl
ions between the zwitterionic polymer chains, resulting in a polymer chain expansion and a lower thermal energy requirement. Thus, the UCST of the silica-g-PSBMA nanoparticles decreases from 41 to 22 °C when the NaCl concentration is increased from 0 to 0.03 M. The dependence of UCSTs on the concentration of silica-gPSBMA nanoparticles in aqueous solutions was further investigated. In Figure 8c, the UCST for sample 1 rapidly increases from 20 to 37 °C when the concentration is increased from 0.2 to 2 g/L and then exhibits a gradual increase from 40 to 45 °C when the concentration is increased from 3 to 6 g/L. The distance between the silica-g-PSBMA nanoparticles is normally reduced as the 15287

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Figure 8. Effect of (a) the PSBMA molecular weight, (b) ionic strength, (c) silica-g-PSBMA nanoparticle concentration, and (d) methanol content on the UCST of the silica-g-PSBMA nanoparticle solutions.

concentration increases, causing the generation of more intermolecular electrostatic attractions of ionic pairings between zwitterionic groups. Therefore, at high nanoparticle concentration in water, a high thermal energy is needed to destroy the electrostatic interaction between interchain ionic pairs and to form a well-dispersed nanoparticle solution. As reported in literature, solvent polarity also affects the UCST values of polymers. In the current study, the mixtures of methanol and water serve as solvents with variable polarities. Figure 8d shows a nearly linear relationship between the UCST and the methanol content. The UCST of sample 1 increases from 20 to 48 °C when the solvent polarity is decreased; this increase corresponds to an increase in the methanol content, from 0% to 30%. Generally, as the solvent polarity is reduced, the surrounding dielectric property of the solution medium decreases, resulting in increased intra- and intermolecular electrostatic interactions of opposite charges between zwitterionic groups. Thus, Figure 8d shows that in an environment with low polarity, a high temperature is required to provide more thermal energy to disrupt the intra- and intermolecular electrostatic interactions of PSBMA brush associations. The UCSTs of silica-g-PSBMA nanoparticles under different conditions are also listed in the table of Supporting Information (Table S1). Furthermore, in order to describe the net charge of the silicag-nanoparticles, zeta-potential measurements have been conducted at 25 °C. As shown in Figure 9, the silica-g-PSBMA nanoparticles (sample 4) have negative zeta-potential of
7.43 mV at salt-free state. With the NaCl concentration increasing from 0.001 to 0.01 M, the zeta-potential exhibits a slight increase from
7.49 to
4.88 mV. Then the zeta-potential decreases from
5.7 to
6.45 mV when the NaCl concentration is increased from 0.05 to 0.15 M. Overall, there is slight fluctuation in the zeta-potential. The results are different from those of Polzer et al.24 They found a

Figure 9. Zeta-potential of silica-g-PSBMA nanoparticles in solution at different NaCl concentrations.

negative zeta-potential (
35 mV) of PSBMA brush particles at the lowest KCl concentration, which increases obviously toward zero with increasing salt content. Theoretically, the zeta-potential of silica-g-PSBMA nanoparticles should be zero due to the zwitterionic nature of PSBMA. Polzer24 et al. reported that in their system, the negative charge is due to the incorporation of potassium peroxodisulfate and anionic surfactant residues during the synthesis of the core particles. In present study, the preparation of silica-g-PSBMA nanoparticles is also conducted in neutral medium. Based on 1H NMR results, there is no hydrolysis of PSBMA chains. Thus, the negative zeta-potential of silica-g-PSBMA nanoparticles is due to the excess negative charge of silica nanoparticle surface. On the basis of the study of Mary et al.,35 Na+ interacts more strongly with
SO3
than that of Cl
with
(CH3)2N+
. Therefore, the PSBMA chains complex preferentially with Na+ and the Na+ positive charges compensate for the net anionic charge of silica nanoparticle surface, which leads to a slight increase of the zeta-potential. 15288

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Figure 10. Rh change of silica-g-PSBMA nanoparticles in (a) PBS, (b) BSA/PBS, and (c) lysozyme/PBS solution at 37 °C.

Figure 11. Rh and the corresponding distribution of silica-g-PSBMA nanoparticles in PBS (a
c), in BSA/PBS (d
f), and in lysozyme/PBS solution (g
i).

Stability in a Protein Solution. DLS was used to monitor the changes in the size of the silica-g-PSBMA nanoparticles during their incubation in protein solutions to evaluate their stability.36,37 BSA and lysozyme, which represent the negatively and positively charged proteins at neutral pH, respectively, were used for the protein binding tests. Sample 1 has no an UCST, but samples 4, and 5 exhibit UCST at 15 and 33 °C in PBS solutions (Table S2, Supporting Information), respectively. The stability of the silica-g-PSBMA

nanoparticles in a protein/PBS solution was tested at 37 °C. Figure 10a shows the Rh change of the PSBMA-grafted silica nanoparticles in the PBS solution under 72 h. The average Rh values of the three samples are 68.8, 76.3, and 116.4 nm, respectively, and the corresponding Rh distributions are 0.095, 0.093, and 0.055 (Figure 11a
c). According to Figure 10b, the average Rh values of the three samples in the BSA/PBS solution during the 72 h incubation period are 67.6, 75.8, and 117.0 nm, respectively, 15289

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Langmuir and the corresponding Rh distributions are 0.083, 0.076, and 0.062 (Figure 11d
f). The size change of the silica-g-PSBMA nanoparticles during incubation in a lysozyme/PBS solution is shown in Figure 10c. The average Rh values of the three samples after 72 h of incubation are 68.0, 73.3, and 114.9 nm, and the Rh distributions are 0.002, 0.094, and 0.018, respectively (Figure 11g
i). Thus, no obvious size and size distributions changes were observed before and after incubation. Moreover, three silica-g-PSBMA nanoparticles show prominent stabilities during the 72 h incubation in both BSA/PBS and lysozyme/PBS solutions (Figure 10b and c). The additional information is provided by zeta-potential measurements. As seen in Figure S2 in the Supporting Information, the zetapotentials of samples 1, 4, and 5 in BSA/PBS and lysozyme/PBS solution exhibit no apparent changes at 37 °C, which confirm the good stability of three silica-g-PSBMA nanoparticles in protein/PBS solutions. The excellent stability of the samples in both negative and positive protein solutions indicates that silica-g-PSBMA nanoparticles can effectively resist nonspecific protein binding. This stability may facilitate their future application.

’ CONCLUSIONS A series of silica-g-PSBMA nanoparticles was successfully synthesized via SI-ATRP in methanol/water using silica-Br as the initiator and CuBr/CuBr2/bpy as the catalytic system. The phase behavior of the prepared polyzwitterionic brushes in aqueous media was determined to illustrate the interplay between the intra- and interchain associations, which strongly depend on the PSBMA molecular weight, ionic strength in solution, hybrid nanoparticle concentration, and solvent polarity. Increasing the PSBMA molecular weight and silica-g-PSBMA nanoparticle concentration enhances the mutual intra- and interchain associations of the sulfobetaine groups in aqueous solutions, resulting in an increase in their UCSTs. When the ionic strength of the solution and the solvent polarity are increased, the UCSTs of the silica-g-PSBMA nanoparticles shift to a lower temperature because of the screening of the net attractive electrostatic interactions and the increase in the dielectric property of the solution medium. Furthermore, silica-g-PSBMA nanoparticles with 5.53  104, 9.76  104, and 13.1  104 g/mol molecular weights are stable at 37 °C for at least 72 h in both negative and positive protein/PBS solutions. Therefore, the tunable phase behavior and stability in protein solutions, combined with the reported biocompatibility and hemocompatibility of polyzwitterionic polymers, make these polysulfobetaine-grafted silica nanoparticles to be potential candidates for new types of adaptive surfaces, and apply in diagnostic biosensors, controlled release, smart throughput membranes, implanted devices, and other biotechnologies. ’ ASSOCIATED CONTENT

bS

Supporting Information. Procedure for the synthesis of initiator-functionalized silica nanoparticles, UCSTs of silicag-PSBMA nanoparticles under different conditions, UCSTs of sample 1, 4, and 5 in PBS solutions, 1H NMR spectrum of the SBMA monomer, and the zeta-potential of the sample 1, 4, and 5 in BSA/PBS and lysozyme/PBS solution at 37 °C. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Telephone: +86 431 85262876. Fax: +86 431 85685653. E-mail: [email protected].

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