Dual Stimuli-Responsive Phenylboronic Acid-Containing Framboidal

Jun 19, 2015 - Phenylboronic acid-containing nanomaterials have found applications in various fields including biomedical engineering due to their uni...
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Dual Stimuli-Responsive Phenylboronic Acid-Containing Framboidal Nanoparticles by One-Step Aqueous Dispersion Polymerization Urara Hasegawa,*,†,‡ Tomoki Nishida,∥ and André J. van der Vlies‡,§ †

Frontier Research Base for Young Researchers, Graduate School of Engineering, ‡Department of Applied Chemistry, Graduate School of Engineering, and §Frontier Research Center, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan ∥ Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, 7-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan S Supporting Information *

ABSTRACT: Phenylboronic acid-containing nanomaterials have found applications in various fields including biomedical engineering due to their unique stimuli-responsive characteristics. Contrary to the many reports on spherical nanoparticles, we are interested in nanostructures with different morphology which could potentially exhibit additional morphology-related effects. Here, phenylboronic acid-containing nanoparticles (PBANPs) in the size range of 80−250 nm in diameter were synthesized via aqueous dispersion polymerization of N-acryloyl-3-aminophenylboronic acid (PBAAM) using methoxy poly(ethylene glycol) acrylamide (PEGAM) as a polymerizable dispersant and N,N′-methylenebis(acrylamide) (MBAM) as a cross-linker. Microscopic analysis revealed that PBANPs were clusters, composed of smaller primary nanoparticles of ∼20 nm in diameter, possessing a framboidal morphology. The size of the PBANPs was significantly affected by the concentrations of PBAAM and PEGAM. Furthermore, PBANPs showed reversible swelling behavior in response to the changes in pH and fructose concentration. PBANPs could be used for fructose detection by the PBA-Alizarin Red S displacement assay. The unique framboidal morphology together with the characteristic properties of phenylboronic acid groups may be useful in biosensing applications.

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INTRODUCTION Multifunctional polymeric nanoarchitectures have attracted growing attention as important platforms for a wide variety of applications.1 Considerable efforts have been made to engineer polymeric nanomaterials to undergo dynamic changes in their structure and physicochemical properties in response to external triggers such as pH, temperature, light, and magnetic field.2 In addition to the chemical nature of materials, their morphology is also a crucial factor to realize desired functionalities.3 For this reason, polymeric nanostructures with various morphologies such as spheres, vesicles, rods, and other structures have been applied to drug delivery systems, sensing techniques, and electrophotonic engineering.4 Phenylboronic acids (PBAs) are a unique class of functional building blocks used to design stimuli-responsive materials.5 PBAs are Lewis acids having pKa values in the range of 6−10 depending on substituents on the phenyl ring. In aqueous media, PBAs exist in a pH-dependent equilibrium between a neutral trigonal form and an anionic tetrahedral boronate form so that materials having PBA moieties show pH-dependent hydration. In addition, PBAs are able to form reversible covalent complexes with 1,2- or 1,3-diol-containing compounds, such as saccharides, by forming cyclic boronate esters.6 Because of these unique characteristics, PBA-containing materials undergo dynamic structural changes in response to changes in pH and saccharide concentrations.7 Furthermore, © XXXX American Chemical Society

PBAs have been extensively used as chemical building blocks in organic synthesis such as Suzuki−Miyaura cross-coupling reaction for C−C bond formation.8 This versatile reactivity allows PBAs to be attractive functional groups for modification of nanomaterials. Polymeric nanomaterials have been prepared by various methods including self-assembly of block copolymers,9 emulsion/dispersion polymerization,10 and electron beam lithography.11 Among them, aqueous dispersion polymerization is an attractive and promising technique that enables to prepare nanostructures with diverse functionality and morphology in a single batch process.10b In this method, monomers, which are miscible with water but the corresponding polymers are aqueous insoluble, are polymerized in the presence of dispersants such as surfactants, amphiphilic polymers, and polymers with a polymerizable end group, also called macromonomers, to provide steric stabilization. Recent reports show that by careful choice of monomers, polymeric nanostructures with different morphologies such as spheres, vesicles, rods, and other anisotropic structures can be formed in situ by the aqueous dispersion polymerization technique, either Received: March 18, 2015 Revised: June 8, 2015

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Scheme 1. Synthesis of Phenylboronic Acid-Containing Nanoparticles (PBANPs) via Aqueous Dispersion Polymerization

with 10 μL of 2 mM ARS in phosphate buffer (pH 8.0), and 180 μL of 0, 11.7, and 58.9 mM D-fructose in phosphate buffer (pH 8.0) was added. After incubation at RT for 1.5 h, the fluorescence intensity was measured using a Tecan infinite M200 well plate reader (λex 485 nm, λem 616 nm). Synthesis of PBA-Containing Spherical Nanoparticles (SNPs). N-Isopropylacrylamide (61.4 mg), MBAM (2.7 mg), and sodium dodecyl sulfate (SDS, 2.9 mg) were dissolved in 9.6 mL of Milli-Q water. This solution was mixed with 0.2 mL of methanol containing PBAAM (26.7 mg) and filtered through a 0.2 μm syringe filter. The solution was heated at 70 °C and degassed with N2 for 1 h. APS (6.8 mg) in 0.2 mL of Milli-Q water was added to the solution under stirring. After 6 h, the reaction mixture was dialyzed against deionized water (MWCO 2 kDa). Detection of D-Fructose with PBANPs and SNPs. First, 20 μL of PBANPs or SNPs (2.1 mM of PBA moieties) and 190 μL of ARS (0.10 mM) were mixed in 0.1 M phosphate buffer (pH 8.0) and kept at RT for 30 min. To the solution was added 20 μL of D-fructose solution (final concentration: 0−25 mM), and fluorescence intensity was measured using a Tecan infinite M200 well plate reader (λex 485 nm, λem 616 nm).

by free radical polymerization or in combination with living radical polymerization techniques.12 Here, we prepared phenylboronic acid-containing nanoparticles (PBANPs) with framboidal morphology using the aqueous dispersion polymerization technique. The effect of monomer, dispersant, and cross-linker concentrations on the size and morphology of PBANPs was investigated. Furthermore, the swelling behavior of PBANPs in response to the changes in pH and saccharide concentration was studied.

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EXPERIMENTAL SECTION

Synthesis of Phenylboronic Acid-Containing Nanoparticles (PBANPs) via Aqueous Dispersion Polymerization. A typical procedure is as follows: N-acryloyl-3-aminophenylboronic acid (PBAAM, 9.5 mg, 50 μmol), methoxypoly(ethylene glycol) acrylamide (PEGAM, 72.4 mg, 12.5 μmol), and N,N′-methylenebis(acrylamide) (MBAM, 0.77 mg, 5 μmol) were dissolved in 20 mL of 0.1 M phosphate buffer (pH 7.0) and placed in a Schlenk tube. The solution was degassed under nitrogen bubbling for 30 min followed by three vacuum−nitrogen purge cycles and incubated at 70 °C for 30 min. A solution of ammonium persulfate (APS, 1.14 mg, 5 μmol) in deionized water was added under nitrogen flow followed by three vacuum− nitrogen purge cycles. The reaction mixture was stirred at 70 °C for 24 h before being exposed to air and cooled down to RT. The solution was dialyzed against deionized water (MWCO 2000 Da), lyophilized, and stored at RT. Yield: 80.6 mg (98%). To follow the reaction, the polymerization was stopped at different time points by dipping in liquid nitrogen and exposing to air, and the crude reaction mixture was analyzed by HPLC. Quantification of Phenylboronic Acid Moieties within PBANPs Using Alizarin Red S (ARS). First, 10 μL of 1.66 mg/mL PBANP in distilled water was mixed with 10 μL of 1.11 mM ARS in distilled water and 180 μL of phosphate buffer (pH 8.0) and incubated at RT for 1.5 h. The fluorescence intensity of the ARZ−PBA complex was measured using a Tecan infinite M200 well plate reader (λex 485 nm, λem 616 nm). (3-Propionamidophenyl)boronic acid was dissolved in distilled water and used to make a standard curve. Swelling Behavior of PBANPs in Response to pH Change. PBANPs were dissolved in acetate buffer (pH 5.0) and incubated at RT for 3 h. This solution was measured with DLS. To exchange buffer, the PBANP solution was transferred to an Amicon filter (MWCO 3 kDa) and centrifuged at 9600 rpm for 30 min. To the concentrated solution was added 1 mL of carbonate buffer (pH 10.0) and centrifuged at 9600 rpm for 30 min. The concentrated solution was again added 1 mL of carbonate buffer and centrifuged at 9600 rpm for 30 min. After the addition of 1 mL of carbonate buffer, the solution was kept at RT for 3 h and measured by DLS. Thereafter, the buffer was exchanged back to acetate buffer (pH 5.0) using an Amicon filter as mentioned above, and the sample was measured by DLS. This process was repeated three times. Swelling Behavior of PBANPs upon the Addition of DGlucose and D-Fructose. PBANPs (0.5 mg/mL) were dissolved in phosphate buffer (pH 8.0) containing 0, 10, and 50 mM of D-glucose or D-fructose and incubated at RT for 1 day. The hydrodynamic diameter of PBANPs was determined by DLS. Detection of the Binding of D-Fructose to PBANPs Using ARS. First, 20 μL of 4.4 mg/mL PBANP in distilled water was mixed

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RESULTS AND DISCUSSION Synthesis of Phenylboronic Acid-Containing Nanoparticles. N-Acryloyl-3-aminophenylboronic acid (PBAAM), a water-soluble monomer which becomes immiscible with water after polymerization, was polymerized via aqueous dispersion polymerization in the presence of the polymerizable dispersant, methoxypoly(ethylene glycol) acrylamide (PEGAM), and the cross-linker, N,N′-methylenebis(acrylamide) (MBAM), as shown in Scheme 1. Dispersion copolymerization of PBAAM (5.0 mM), PEGAM (1.25 mM), and MBAM (0.5 mM) was carried out in phosphate buffer (pH 7.0) at 70 °C using ammonium persulfate (APS) as a radical initiator. After 24 h, the resulting solution was dialyzed against water. No precipitation was observed during polymerization and dialysis. Dynamic light scattering (DLS) revealed the formation of nanoparticles with average diameter of 151 nm. The polydispersity index (PDI = μ2/Γ2, μ2 = the second cumulant of the decay function, Γ = the average decay rate) was 0.08, indicating a narrow size distribution. Interestingly, the resulting nanoparticles were clusters composed of smaller primary nanoparticles (∼20 nm in diameter) as shown by atomic force microscopy (AFM) and field emission-scanning electron microscopy (FE-SEM) (Figure 1a,b). This framboidal morphology was also confirmed by transmission electron microscopy (TEM) after negatively staining of the particles with 2 wt % sodium phosphotungstate aqueous solution (Figure 1c). Furthermore, the size of these nanoparticles does not change after sonication for 20 min (Figure S1), indicating that the framoidal structure was covalently stabilized by the MBAM cross-linker. To confirm the incorporation of PBAAM, the number of the PBA moieties within PBANPs was quantified using Alizarin B

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Effect of the Reaction Mixture Composition on the Size and Morphology of PBANPs. To study the effect of the concentration of PBAAM on particle size and morphology, we first prepared PBANPs at different PBAAM concentrations while fixing the molar ratio of PBAAM:PEGAM:MBAM at 10:2.5:1. The diameter of PBANPs becomes larger at higher PBAAM concentrations as determined by DLS (Figure 3a). Up

Figure 1. Morphology of PBANPs observed by (a) AFM, (b) FESEM, and (c) TEM. Concentrations of PBAAM, PEGAM, and MBAM for PBANP synthesis were 5, 1.25, and 0.5 mM, respectively. (a, b) PBANPs were adsorbed onto a fresh mica surface and air-dried. Scale bars: 200 nm. (c) PBANPs were negatively stained with 2 wt % sodium phosphotungstate solution. Scale bars: 200 nm (left); 80 nm (right).

Red S (ARS). ARS is an anthraquinone derivative that becomes strongly fluorescent when complexed with boronic acids.6 We used (3-propionamidophenyl)boronic acid as a model compound to quantify the concentration of PBA moieties. As shown in Figure S2, PBANPs contained 605 μmol/g of PBA moieties, which is in good agreement with the feed ratio of PBAAM (604 μmol/g). This data showed that PBAAM was incorporated quantitatively within the particles. To explore the kinetics of particle formation, we followed particle growth as a function of time by DLS (Figure 2a). The

Figure 3. Effect of the PBAAM concentration on size and morphology of PBANPs. The molar ratio of PBAAM:PEGAM:MBAM was fixed at 10:2.5:1. (a) Hydrodynamic diameter of PBANPs as measured by DLS. The mean diameter (open circle) and PDI (closed circle) were calculated by the cumulant method. (b−d) AFM images of PBANPs prepared at different PBAAM concentrations. (b) 2.5, (c) 10, and (d) 20 mM. Scale bars: 400 nm.

to 10 mM, the PDI values were below 0.1, indicating monodisperse size distributions, while the PDI value slightly increased to 0.14 for particles prepared at 20 mM of PBAAM. Furthermore, below 10 mM, PBANPs possessed framboidal spherical structures according to the AFM, FE-SEM, and TEM images (Figure 3b,c and Figures S3a,b and S4a,b). On the other hand, branched worm-like structures were observed for PBANPs prepared at 20 mM of PBAAM showing that heterogeneous aggregation of primary particles occurred under this condition (Figure 3d and Figure S3c). We next investigated the effect of the molar ratio of PBAAM, PEGAM, and MBAM in the reaction mixture on particle size and morphology. First, PBANPs were prepared at different PEGAM concentrations keeping the PBAAM and MBAM concentrations constant (5.0 and 0.5 mM, respectively). As shown in Figure 4a, the PEGAM concentration significantly affected particle size. By decreasing the concentration from 1.9 to 1.0 mM, the diameter of PBANPs increased from 80 to 255 nm. AFM and TEM showed that these PBANPs also possessed framboidal structures (Figure 4c,d and Figure S4c). When the PEGAM concentration was further decreased to 0.675 mM, we observed the formation of a precipitate. These data clearly show that the size of PBANPs can be controlled by the concentration of PEGAM. The effect of the concentration of MBAM in the reaction mixture was also studied by fixing the PBAAM and PEGAM concentrations at 5.0 and 1.25 mM, respectively. Contrary to

Figure 2. Formation of PBANPs as a function of time. (a) Change in diameter as measured by DLS. The mean diameter (open circle) and PDI (closed circle) were calculated by the cumulant method. (b) Conversion of PBAAM (circle), PEGAM (triangle), and MBAM (square) as determined by HPLC.

particle size rapidly increased to 125 nm within 10 min followed by a gradual increase up to 60 min. The conversion of PBAAM, PEGAM, and MBAM was also determined by HPLC as a function of time. As shown in Figure 2b, 51% of PBAAM was polymerized within 10 min, while the conversions of PEGAM and MBAAM were 11 and 24%, respectively. This result revealed that initially PBAAM-rich particles were formed and that thereafter PEGAM and MBAM were incorporated into the particles to provide steric and structural stabilization. It should be noted that full conversion was confirmed after 60 min for PBAAM, PEGAM, and MBAM. C

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Figure 5. Swelling behavior of PBANPs in response to changes in pH and saccharide concentrations. (a) Scheme showing the pH-dependent boronate formation and complexation with diols. Ktet > Ktrig. (b) Change in diameter of PBANPs as a function of pH. (c) Reversible swelling behavior upon pH change between pH 5 and pH 10 as measured by DLS. The mean diameter (open circle) and PDI (closed circle) were calculated by the cumulant method. (d) Diameter change of PBANPs upon the addition of D-glucose (triangle) and D-fructose (circle) as measured by DLS.

Figure 4. Effect of the PEGAM and MBAM concentrations on size and morphology of PBANPs. Concentration of PBAAM was fixed at 5.0 mM. (a, b) Hydrodynamic diameter of PBANPs prepared at different concentrations of (a) PEGAM (MBAM: 0.5 mM) and (b) MBAM (PEGAM: 1.25 mM) as measured by DLS. The mean diameter (open circle) and PDI (closed circle) were calculated by the cumulant method. (c, d) AFM images of PBANPs prepared at different PEGAM concentrations. (c) 1.0 and (d) 1.9 mM. Scale bars: 400 nm.

to the boronate form, which makes the polymer more hydrophilic and water-soluble. Because of this pH-dependent ionization, PBA-containing polymers behave as pH-responsive polymers that reversibly change their water miscibility upon pH change. Another interesting characteristic is their interaction with diol-containing compounds. PBAs form boronate esters with 1,2- or 1,3-diol groups via reversible covalent bond formation.14 This reaction is favored when PBAs are in the tetrahedral boronate form while the complexes between PBAs in a trigonal form and diol groups are generally hydrolytically unstable. Given these interesting ionization and diol complexation equilibria, PBA-containing polymeric materials have been used as artificial lectins, pH- and diol-responsive drug carriers, and biosensors.5d,15 With these characteristics in mind, we first investigated the swelling behavior of PBANPs at different pH. In this experiment, we used PBANPs prepared at 5.0, 1.25, and 0.5 mM of PBAAM, PEGAM, and MBAM, respectively. The diameter of PBANPs at different pH in the range of 5−10 was measured by DLS. As shown in Figure 5b, PBANPs increased their size with the increase of pH. Since the pKa value of polymerized PBAAM is reported to be 8.5,16 the amount of PBA moieties in a boronate form was estimated to be 82% at pH 10 while only 3% of PBA moieties are in the boronate form at pH 5. Thus, the increased ionization of PBA moieties at higher pH may result in significant swelling of PBANPs. It should be noted that the PDI value was below 0.1 at all pH tested, showing that the PBANPs were monodisperse at each pH. This implies that the increase in particle size at higher pH was not due to the aggregation of PBANPs. Furthermore, we also confirmed the reversibility of this pH-dependent swelling. PBANPs were first dissolved in acetate buffer at pH 5, and the pH was increased to 10 by exchanging acetate buffer with carbonate buffer. After that the pH was adjusted back again to 5. This cycle was continued three times, and the diameter was measured. Figure 5c clearly shows that the pH-dependent

PEGAM, the MBAM concentration did not show obvious effects on the particle size (Figure 4b). Although the mechanism for the formation of the framboidal morphology is not clearly understood at this moment, one possible explanation is a steric effect of the PEG dispersant on the surface of the nuclei formed in the initial stage of dispersion polymerization. Generally, dispersion polymerization proceeds in the following steps.13 In the initial stage of polymerization, the monomers are polymerized to form oligomer radicals which precipitate from the continuous phase to form unstable nuclei when reaching a critical length. These nuclei coalesce to form larger particles which eventually are stabilized by dispersants. At the same time, the monomers are incorporated and polymerized within these particles, which results in further growth of the particles.13c In this study, we used PEGAM as the dispersant, which can be copolymerized with PBAAM to cover the surface of nuclei. Since it is likely that the nuclei formed at the beginning of the polymerization contained PEG to some extent, the hydrated PEG brush may prevent complete fusion of these nuclei, resulting in the unique observed framboidal morphology. This explanation is supported by the observed effect of the PEG dispersant on particle size as shown in Figure 4a. In this model, an increase of the dispersant concentration will lead to a higher PEG density on the nuclei, which in turn will further slow down the coalescence of these nuclei, resulting in smaller particles, as observed in Figure 4a. Swelling Behavior of PBANPs in Response to Changes in pH and Saccharide Concentration. Polymeric materials containing PBA moieties are known to exhibit unique stimuliresponsive characteristics.5c,6 As shown in Figure 5a, in aqueous media, PBA moieties exist in both neutral/hydrophobic trigonal and anionic/hydrophilic tetrahedral (boronate) forms.14 At low pH, PBAs are mainly in trigonal form so that the PBAcontaining polymers, in general, become immiscible in water. However, with the increase of pH, PBA moieties are converted D

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Figure 6. Detection of D-fructose with PBANPs and SNPs. (a) Fluorescence intensity of the PBA−ARS complex at different Dfructose concentrations. PBANPs (circle) or SNPs (triangle) were reacted with ARS in 0.1 M phosphate buffer (pH 8.0) to form the PBA−ARS complex and mixed with D-fructose solutions. After 2 h, the fluorescence intensity was measured (λex = 485 nm, λem = 616 nm). (b) Ratio of decrease in fluorescence intensity, (Io − Ix)/Io, as a function of D-fructose concentration. Io: fluorescence intensity of the PBA−ARS complex. Ix: fluorescence intensity of PBA−ARS complex in the presence of x mM D-fructose.

SNPs. In the case of PBANPs, we could detect D-fructose as a low as 1.5 mM while in the case of the SNPs the detection limit was 12.5 mM (Figure 6b). Therefore, the framboidal nanoparticles would have potential for highly sensitive detection of diol-containing compounds in biosensing applications. In this study, we found that simple aqueous dispersion polymerization of a PBA-containing monomer in the presence of a polymerizable PEG dispersant resulted in unique framboidal nanostructures. Polymeric framboidal particles have attracted attention in many research area including biomimetic science,18 surface chemistry,19 and photonic science.20 So far, several methods have been developed to engineer such polymeric structures. In most cases, small nanoparticles were first prepared and assembled into colloidal clusters by evaporation-driven self-assembly in emulsion droplets,21 adsorption onto larger particles,19,22 or cross-linking under diluted conditions.23 On the other hand, there are few reports on the methods to prepare framboidal nanostructures directly from monomer solutions without using preformed nanoparticles. Kaneko et al. reported formation of framboidal particles by dispersion terpolymerization of styrene, acrylonitrile, and PEG monomethacrylate in ethanol−water solution. The resulting particles possessed a solvent-immiscible styrene core and an acrylonitrile/PEG corona having several projections.18 Recently, Chambon et al. showed that the combination of aqueous dispersion polymerization, reversible addition− fragmentation chain transfer (RAFT) polymerization, and seeded emulsion polymerization resulted in in situ formation of framboidal morphologies. In this approach, water-immiscible monomers were polymerized in the presence of diblock copolymer vesicles prepared via RAFT aqueous dispersion polymerization, which leads to morphological changes from vesicular to framboidal structures by microphase separation.24 In these dispersion polymerization methods, the reactions can be carried out in one pot without need for special instruments or techniques so that preparation of the particles is much easier compared to the other methods. However, since the formation of characteristic morphology relies on microphase separation of two or more monomers as well as a polymeric dispersant during polymerization, incorporation of desired functional moieties may be difficult due to the limited choice of monomers and dispersants. In this aspect, our method may offer advantages in flexible design of framboidal nanostructures with various functionalities. Apart from the characteristic responsiveness to pH and diol-containg compounds, PBA moieties are known to exhibit versatile reactivity and can be easily converted to other functional groups.25 Therefore, PBANPs would be a promising platform which allows further modification with desired functional groups. The modification of PBANPs by Suzuki−Miyaura cross-coupling reaction is currently under investigation.

about 5 times higher fluorescence intensity in the absence of Dfructose compared to SNPs, meaning that the PBA moieties were more accessible to ARS in the case of PBANPs compared to SNPs. We believe that this is due to the higher surface areato-volume ratio of the framboidal particles, which increases the amount of PBA moieties exposed to the aqueous media, resulting in the formation of higher amount of PBA−ARS complex. Importantly, the framboidal PBANPs significantly increased the sensitivity of fructose detection compared to the

CONCLUSIONS PBA-containing framboidal nanoparticles were synthesized via one-step aqueous dispersion polymerization of PBAAM in the presence of a polymerizable PEGAM dispersant and a MBAM cross-linker. The diameter of PBANPs could be controlled by varying the concentrations of PBAAM and PEGAM in the reaction mixture. Furthermore, PBANPs showed significant swelling at basic pH and upon the addition of D-fructose due to the hydration of polymer chains within PBANPs. PBANPs

swelling/shrinking was fully reversible and that PBANPs aggregation did not occur after three cycles according to the PDI values. The swelling behavior in the presence of diol-containing compounds, D-glucose and D-fructose, was also studied as shown in Figure 5d. The addition of D-fructose increased particle size in a concentration-dependent manner. On the other hand, the particle size did not change upon the addition of D-glucose up to 50 mM. This result correlates with the difference in affinity of both saccharides with PBA. It has been reported that the association constants for boronate ester formation with PBA are 310 M−1 for D-fructose and 7.2 M−1 for 6,17 D-glucose at pH 8.0. Thus, D-fructose has a much higher affinity to PBA than D-glucose. Therefore, more D-fructose binding may result in hydration of polymer chains within PBANPs, thereby causing higher swelling of PBANPs. The interaction between D-fructose and PBA was further confirmed by the decrease in fluorescence of the PBA−ARS complex upon the addition of D-fructose as reported (Figure S5).6 To further explore the potential in biosensing applications, PBANPs were used to detect D-fructose by the PBA−Alizarin Red S (ARS) displacement assay.6 In this assay, the fluorescent PBA−ARS complex is first prepared and used as a fluorescent reporter which becomes nonfluorescent upon the addition of cis-diol-containing compounds such as D-fructose. In order to evaluate the effects of the unique framboidal morphology, we also prepared spherical PBA-containing nanoparticles (SNPs) by precipitation copolymerization of N-isopropylacrylamide and PBAAM in the presence of sodium dodecyl sulfate (SDS).26 The diameter of the SNPs was 147 nm (PDI: 0.02) according to DLS, which is comparable in size with PBANPs of 151 nm (PDI: 0.08). As shown in Figure 6a, PBANPs showed

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(9) Blanazs, A.; Armes, S. P.; Ryan, A. J. Macromol. Rapid Commun. 2009, 30 (4−5), 267−277. (10) (a) van der Vlies, A. J.; O’Neil, C. P.; Hasegawa, U.; Hammond, N.; Hubbell, J. A. Bioconjugate Chem. 2010, 21 (4), 653−662. (b) Warren, N. J.; Armes, S. P. J. Am. Chem. Soc. 2014, 136 (29), 10174−10185. (11) Juhasz, R.; Elfström, N.; Linnros, J. Nano Lett. 2005, 5 (2), 275−280. (12) (a) Sugihara, S.; Blanazs, A.; Armes, S. P.; Ryan, A. J.; Lewis, A. L. J. Am. Chem. Soc. 2011, 133 (39), 15707−13. (b) Jia, Z.; Bobrin, V. A.; Truong, N. P.; Gillard, M.; Monteiro, M. J. J. Am. Chem. Soc. 2014, 136 (16), 5824−5827. (c) Delaittre, G.; Save, M.; Charleux, B. Macromol. Rapid Commun. 2007, 28 (15), 1528−1533. (d) Ali, A. M. I.; Pareek, P.; Sewell, L.; Schmid, A.; Fujii, S.; Armes, S. P.; Shirley, I. M. Soft Matter 2007, 3 (8), 1003−1013. (13) (a) Yıldız, U.; Hazer, B. Angew. Makromol. Chem. 1999, 265 (1), 16−19. (b) Kawaguchi, S.; Ito, K. Dispersion Polymerization. In Polymer Particles; Okubo, M., Ed.; Springer: Berlin, 2005; Vol. 175, pp 299−328. (c) Cao, K.; Li, B.-F.; Huang, Y.; Li, B.-G.; Pan, Z.-R. Macromol. Symp. 2000, 150 (1), 187−194. (14) Yan, J.; Springsteen, G.; Deeter, S.; Wang, B. Tetrahedron 2004, 60 (49), 11205−11209. (15) Ancla, C.; Lapeyre, V.; Gosse, I.; Catargi, B.; Ravaine, V. Langmuir 2011, 27 (20), 12693−12701. (16) Alexeev, V. L.; Sharma, A. C.; Goponenko, A. V.; Das, S.; Lednev, I. K.; Wilcox, C. S.; Finegold, D. N.; Asher, S. A. Anal. Chem. 2003, 75 (10), 2316−2323. (17) Cannizzo, C.; Amigoni-Gerbier, S.; Larpent, C. Polymer 2005, 46 (4), 1269−1276. (18) Kaneko, T.; Hamada, K.; Chen, M. Q.; Akashi, M. Macromolecules 2004, 37 (2), 501−506. (19) Telford, A. M.; Hawkett, B. S.; Such, C.; Neto, C. Chem. Mater. 2013, 25 (17), 3472−3479. (20) Moon, J. H.; Yi, G. R.; Yang, S. M.; Pine, D. J.; Park, S. B. Adv. Mater. 2004, 16 (7), 605−609. (21) Cho, Y.-S.; Kim, S.-H.; Yi, G.-R.; Yang, S.-M. Colloids Surf., A 2009, 345 (1−3), 237−245. (22) Mammen, L.; Deng, X.; Untch, M.; Vijayshankar, D.; Papadopoulos, P.; Berger, R.; Riccardi, E.; Leroy, F.; Vollmer, D. Langmuir 2012, 28 (42), 15005−15014. (23) Hasegawa, U.; Sawada, S.-i.; Shimizu, T.; Kishida, T.; Otsuji, E.; Mazda, O.; Akiyoshi, K. J. Controlled Release 2009, 140 (3), 312−317. (24) Chambon, P.; Blanazs, A.; Battaglia, G.; Armes, S. P. Macromolecules 2012, 45 (12), 5081−5090. (25) Hall, D. G. In Boronic Acids; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, 2011; pp 1−133. (26) Lapeyre, V.; Gosse, I.; Chevreux, S.; Ravaine, V. Biomacromolecules 2006, 7, 3356−3363.

would be an attractive multifunctional platform for various applications such as drug delivery and biosensing.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details, size of PBANPs after sonication, Alizarin Red S (ARS) complexation assay, FE-SEM and TEM images, binding of PBA to fructose. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b00574.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel +81-66879-7365; Fax +81-6-6879-7367 (U.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Kazunari Akiyoshi (Kyoto University, Japan) for his help with TEM measurements and Prof. Hiroshi Uyama (Osaka University, Japan) for his help with DLS, AFM, and HPLC measurements. We also thank Prof. Françoise M. Winnik (University of Montreal, Canada) for her advice for nanoparticle characterization. This work was partially supported by Grant-in-Aid for Young Scientists (B), No. 25750175, from the Japan Society for the Promotion of Science (JSPS), Japan, Research Grant from the Ogasawara Foundation for the Promotion of Science & Engineering, Japan, and “Nanotechnology Platform” (project No. 12024046) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

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ABBREVIATIONS PBA, phenylboronic acid; PBANP, phenylboronic acidcontaining nanoparticle; PEGAM, methoxypoly(ethylene glycol) acrylamide; MBAM, N,N′-methylenebis(acrylamide); APS, ammonium persulfate.

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REFERENCES

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DOI: 10.1021/acs.macromol.5b00574 Macromolecules XXXX, XXX, XXX−XXX