Thermoresponsive Nanospheres with a Regulated Diameter and Well

Dec 2, 2013 - Takuya Matsuyama , Ayaka Kimura , Taka-Aki Asoh , Takuma Suzuki , Akihiko Kikuchi. Colloids and Surfaces B: Biointerfaces 2014 123, 75-8...
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Thermoresponsive Nanospheres with a Regulated Diameter and Well-Defined Corona Layer Takuya Matsuyama, Hironori Shiga, Taka-Aki Asoh, and Akihiko Kikuchi* Department of Materials Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, Japan S Supporting Information *

ABSTRACT: In the present work, we prepared core−coronatype nanospheres bearing a thermoresponsive polymer with a controlled chain length on their surface. The corona layers were composed of poly(N-isopropylacrylamide) (PNIPAAm) chains (Mn = 3000−18 000) with a narrow polydispersity index prepared by atom-transfer radical polymerization (ATRP). Nanospheres were prepared by dispersion copolymerization of styrene with the PNIPAAm macromonomer in a polar solvent. The obtained nanospheres were monodisperse in diameter. The diameter of the nanospheres was regulated either by the number or chain length of the PNIPAAm macromonomers. In fact, the nanosphere diameter was regulated from ca. 100 to 1000 nm. When two types of PNIPAAm macromonomers are used, the obtained nanospheres have two different kinds of PNIPAAm on their surface. The surface of the nanospheres was observed to be thermoresponsive nanosphere in 0, 50, 100 mmol L−1 NaCl aqueous solution. The nanosphere diameter and the surface-grafted polymer were concurrently adjusted for use in biomedical applications.

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INTRODUCTION Polymeric nanospheres with an extremely large surface area are useful in technological and biomedical fields. Nanospheres can be defined as colloidal systems with a diameter smaller than 1000 nm.1 Many kinds of nanospheres have been synthesized and utilized for technological and biological applications. Nanospheres have been used for bioimaging,2 diagnosis,3,4 and drug-delivery systems (DDS).5 Various types of polymeric nanoparticles have been designed, such as spheres,6 tubes,7 micelles,5 gels,8 polymersomes,9 and hybrid particles.10 Among them, many studies have been conducted on microspheres and nanospheres.3,11 For example, polymer was grafted onto a nanosphere surface to make functional nanospheres.3 However, latex nanospheres have some disadvantages; in particular, the nonspecific adsorption of proteins or other analyte molecules on their surface is a serious problems. Polymer grafting has been utilized to inhibit such nonspecific adsorption and to improve the biocompatibility. Controlled size and shape are key factors in nanoparticles used as biomaterials. In particular, the selection of nanosphere diameter and surface function is important for applications in diagnosis and DDS because a macrophage is known to recognize and phagocytose nanospheres of certain diameters.12,13 In other cases, to obtain an effective enhanced permeation and retention (EPR) effect, nanospheres of less than 200 nm in diameter are crucial. The polystyrene sphere size can be controlled by the polymerization conditions.14 However, the diameters of gold nanoparticles or silica nanoparticles are well-controlled, though it is necessary to © 2013 American Chemical Society

modify these surfaces to avoid the irreversible adsorption of biomolecules and nanoparticle aggregation.15−17 However, both the diameter and surface design of the spheres are difficult to control at the same time. Most importantly, nanosphere characterization is determined by many factors, not only the core but also the polymer grafted to the nanosphere surface. Many kinds of polymers, such as N-alkyl acrylamide-type polymer18,19 and ethylene glycol-based polymer,20 have thermoresponsive properties. Poly(N-isopropylacrylamide) (PNIPAAm) is well known as a thermoresponsive polymer exhibiting a lower critical solution temperature (LCST) at 32 °C.19 PNIPAAm is soluble in water below its LCST, but above this critical temperature, the chain undergoes a coil-to-globule transition and becomes insoluble. PNIPAAm is widely used in the biomedical and biochemical fields. The conjugation of PNIPAAm to an antibody was investigated for diagnosis and affinity separation.21 The grafting of PNIPAAm polymer brushes onto solid surfaces has also been used in a range of applications, such as liquid chromatography,22 cell culture,23 permeation-controlled filters,24 and protein adsorption.25 Our research group studied the thermoresponsive chromatography system, which was affected by the separation of biomolecule compounds with modified polymer chain lengths.26 Moreover, the designed PNIPAAm chain was controlled via interaction Received: September 6, 2013 Revised: October 22, 2013 Published: December 2, 2013 15770

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volume was 20 μL. 1H NMR spectra were recorded in either CDCl3 or DMSO-d6 using a JNM-ECP500 (500 MHz, Jeol, Tokyo, Japan). The composition ratio of St and PNIPAAm was measured by 1H NMR. The diameter and morphology of the nanospheres were observed by scanning electron microscopy (SEM, JSM-6060, Jeol, Tokyo, Japan) at an operating voltage of 15 kV. The samples were dried onto brass stubs and sputter-coated with a thin layer of platinum prior to examination to prevent sample charging. The diameter of the nanospheres was measured by dynamic light scattering (DLS, ELS-Z 1000S, Otsuka Electronics Co., Ltd., Osaka, Japan). The diameter of the nanospheres was observed in aqueous media below and above the LCST. The thermoresponsive behavior of nanospheres was observed by microscopy (Bio-Zero8100, Keyence, Osaka, Japan). Synthesis of Tris(2-(N,N-dimethylamino)ethyl)amine (Me6TREN). An ATRP ligand, tris(2-(N,N-dimethylamino)ethyl)amine (Me6TREN), was synthesized from tris(2-aminoethyl)amine (TREN), according to a previous report.35 Briefly, to a mixture of formaldehyde (71 mL) and formic acid (85 mL) an aqueous TREN solution (25 g of TREN, 15 mL of water) was added dropwise at 0 °C. The solution was then stirred at reflux (95 °C) overnight. After cooling to 25 °C, the reactant solution was evaporated to remove residual formaldehyde and formic acid. Aqueous sodium hydroxide (10 M, 100 mL) was added to the residual solution, followed by extraction with methylene chloride (600 mL) at least three times. The organic phase was collected and dried over anhydrous magnesium sulfate overnight. The magnesium sulfate was removed by filtration, followed by evaporation of the remaining filtrate to obtain the crude product as a yellow-brownish oil. Me6TREN was then obtained by distillation at 0.67 kPa and 110 °C (yield ca. 70%). Synthesis of PNIPAAm by ATRP. The general procedure employed for the preparation of PNIPAAm was as follows. For example, in case of polymerization of ca. Mn 10000 of PNIPAAm, a mixture containing NIPAAm (10.18 g, 90 mmol), Me6TREN (241 μL, 0.9 mmol), and DMF (30 mL) was deoxygenated by bubbling with nitrogen gas for at least 30 min. CuCl (89 mg, 0.9 mmol) was introduced under a flow of N2 gas to protect CuCl from oxidation. The reaction mixture was stirred for approximately 10 min to allow the formation of a CuCl/Me6TREN complex. N-(Chloromethyl)phthalimide (CMP) (176 mg, 0.09 mmol) was then added to initiate the polymerization. The reaction was carried out at 25 °C and allowed to stir under a nitrogen atmosphere for a predetermined amount of time. The polymerization reaction was stopped by opening the flask and exposing the catalyst to air. The solution was concentrated by evaporation, and the reactant was poured into a large amount of diethyl ether to precipitate the polymer. The polymer was further purified by repeated precipitation from THF solution into diethyl ether twice. The sediments were collected and redissolved in THF and passed through a neutral alumina column using THF as the eluent to remove the copper catalyst. Again, the polymer was precipitated into diethyl ether. After the solvent was decanted, the polymer was dried under vacuum overnight. The molecular weight and polydispersity were determined by GPC. The number-average molecular weight of PNIPAAm was controlled from Mn = 3000 to 18 000 by changing the monomer/initiator ratio in the ATRP. The polymerization conversion was measured directly with 1H NMR spectroscopy in CDCl3 by comparing the peak areas of the phthalimide group signal at 7.8 ppm (four protons) with the signal of the polymer methyne proton on the NIPAAm side chain at 4.0 ppm (one proton) after correcting for the contribution due to the monomer. Synthesis of the PNIPAAm Macromonomer. The phthalimide end group of PNIPAAm was transformed into a primary amino group by hydrazine decomposition. PNIPAAm with a phthalimide end group (3 g, 0.3 mmol) and hydrazine monohydrate (1.65 mL, 30 mmol) were dissolved in ethanol (30 mL), and the mixture was stirred at room temperature under nitrogen for 18 h. The mixture was dialyzed in regenerated cellulose tubing (MWCO 1000) for 3 days with the water being changed every 12 h. The aqueous solution in the dialysis tube was collected and then freeze dried. The PNIPAAm macromonomer was prepared by reacting acryloyl chloride with the amino end groups. Briefly, 2 g of PNIPAAm with a primary amino end group

with various biomolecules in aqueous media as a function of its temperature dependence. Therefore, several reports exist on the fabrication of thermoresponsive composite materials for DDS.27,28 In addition, grafted polymer chain lengths in the solid state are very important to the biomedical and biochemical fields. The surface properties of nanospheres can be altered by grafting PNIPAAm. Therefore, the characteristics of the nanospheres, such as colloidal dispersity and the prevention of biomolecular adsorption on their surfaces, were temperaturedependent. In general, both the diameter and surface design of the spheres were difficult to control at the same time. The nanosphere surface thus requires a functional polymer with a controlled chain length. Although thermoresponsive nanospheres prepared by the self-assembly of graft copolymers have been reported, the diameters of nanospheres over wide ranges were not regulated.38 However, functional polymeric nanospheres have been prepared during polymerization using a hydrophilic macromonomer surrounding the nanosphere surface.29,30 Furthermore, this preparation method for functional nanospheres is easier and is a versatile technique for nanosphere surface design. Recently, several research groups reported on grafted polymers with controlled chain lengths on nanosphere surfaces, such as mixed polymer brushs16,17,31 and thermoresponsive32 and pH-responsive polymers.33 In particular, grafted high-density polymer brushes were utilized for biomedical and biochemical applications.4 However, controlling the nanosphere diameter concurrently with its surface properties has not been reported in the literature. A concurrently regulated diameter and polymer chain length on the surface would result in the successful preparation of nanospheres with the desired functions. In the present study, we focused on the synthesis of nanospheres and the functionalization of their surface with controlled chain lengths of PNIPAAm by combining ATRP and the macromonomer method. Therefore, the nanospheres were synthesized by the macromonomer method in which the self-assembly of the nanospheres occurred during polymerization in a selective solvent.34 Here, we regulated the size of the polymeric nanospheres with controlled chain lengths of PNIPAAm. In fact, the nanosphere diameter was regulated from ∼100 to 1000 nm by the PNIPAAm macromonomer chain length and/or amount. We also investigated the aggregation of the nanospheres and its dependence on temperature. The prepared nanospheres could be utilized for diagnostic tests.

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EXPERIMENTAL SECTION

Materials. N,N-Dimethylformamide (DMF), tetrahydrofuran (THF), ethanol, hydrazine monohydrate, triethylamine (TEA), CuCl, N-isopropylacrylamide (NIPAAm), acryloyl chloride, styrene (St), and 2,2′-azobis(isobutyronitrile) (AIBN) were all purchased from Wako Pure Chemical Industries (Osaka, Japan). N-(Chloromethyl)phthalimide (CMP) was purchased from Aldrich (St. Louis, MO, USA). NMR solvents D2O, CDCl3, and DMSO-d6 were purchased from Acros Organics (NJ, USA). Polymer Characterization. The molecular weight and polydispersity of the various PNIPAAm macromonomers were determined by gel permeation chromatography (GPC) [Tosoh column TSK-gel G3000HHR+ TSKgel G4000HHR at 45 °C using DMF containing 10 mM LiCl as an eluent, a Jasco system that consisted of a PU-2080Plus intelligent HPLC pump, a UV-2075Plus intelligent UV/vis detector, an RI-2031Plus intelligent RI detector, and a CO-2060Plus column oven (Jasco, Tokyo, Japan)]. PEG was used as a molecular weight standard. A sample polymeric solution was 1 mg/mL, and the injection 15771

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was dissolved in THF, and 36 mol equiv of triethylamine with respect to the primary amino end group was added. The solution was cooled to 0 °C, followed by the dropwise addition of 30 mol equiv of acryloyl chloride relative to the primary amino end group of PNIPAAm. The solution was stirred for 14 h at room temperature. After the reaction, the solution was dialyzed against distilled water. Finally, the PNIPAAm macromonomer was obtained by freeze drying. Transformation of the terminal group of PNIPAAm at each step was confirmed by 1H NMR spectroscopy in DMSO-d6. Synthesis of the Monodisperse Core−Corona Nanospheres. PNIPAAm macromonomer (0.02 mmol) as an equivalent vinyl group and styrene (2.0 mmol) were added to a flask, together with 1 mol % AIBN relative to the styrene monomer as the initiator and 2.0 mL of ethanol/water (7/3 v/v). The solution was degassed by freeze−thaw cycles on a vacuum pump and then placed in a shaking water bath at 60 °C, and radical polymerization proceeded for 14 h. The obtained nanospheres were refined by three centrifugation/redispersion cycles in methanol and dialyzed in pure water using regenerated cellulose tubing (MWCO 50000) to remove unreacted monomer and macromonomer. The resulting thermoresponsive core−corona-type nanospheres were analyzed by 1H NMR, SEM, and dynamic light scattering (DLS) in water. Nanosphere diameter was measured from SEM images. The averaged diameter and size distribution of nanospheres were calculated from SEM images (n = 30). Error bars show the size distribution of nanosphere. We also investigated the aggregation of thermoresponsive nanospheres in aqueous media by the effect of salt concentration. We observed the temperature dependence of the aggregation of nanospheres by microscopy.

When the molar ratio of monomer to initiator, [M]/[I], was changed from 25 to 200, the molecular weight of PNIPAAm increased with a constant polymerization time of 4 h. Thus, PNIPAAm with twice the molecular weight was prepared by halving the [M]/[I] ratio. In fact, the number-average molecular weight of PNIPAAm was controlled from 3000 to 18 000 by controlling the [M]/[I] ratio. The PNIPAAm terminal group was then transformed. The terminal phthalimide group of PNIPAAm was analyzed by 1H NMR measurements. Figure 1a shows that the peak of the phthalimide group appeared at 7.8 ppm. The phthalimide group of PNIPAAm was transformed into a primary amino group through the Gabriel reaction (Scheme 1). After hydrazine decomposition, the peak of the phthalimide group disappeared from the 1H NMR spectrum (Figure 1b). This disappearance confirmed the successful synthesis of the terminal primary amino group. Finally, the PNIPAAm macromonomer was prepared by reacting acryloyl chloride with the amino end group. After the reaction, new peaks appeared at 5.5−6.2 ppm in the 1H NMR spectrum, which was assigned to the vinyl group (Figure 1c). Thus, the conversion of the phthalimide group to a vinyl group was calculated to be about 80% by 1H NMR spectroscopy. We obtained the PNIPAAm macromonomer with controlled chain lengths from 3000 to 18 000. Preparation and Characterization of Thermoresponsive Core−Corona-Type Nanospheres. Previously, thermoresponsive nanospheres were prepared as functional particles.28,32,37 Other research groups reported that the nanosphere diameter is influenced by the initiator concentration, polymerization temperature, macromonomer and St concentrations, and solvent composition.34 We attempted to regulate the nanosphere diameter by controlling the PNIPAAm macromonomer amount and the chain length. Monodisperse nanospheres consisting of a polystyrene (PSt) core and PNIPAAm branches on their surface were prepared by free radical copolymerization of the PNIPAAm macromonomer and St at 60 °C in ethanol/water in the presence of AIBN (Scheme 2). The copolymerization proceeded heterogeneously in ethanol/water to form spheres. The nanospheres were formed by the self-assembly of the amphiphilic polymer structure, owing to the difference in affinity for the solvent between their hydrophilic and hydrophobic moieties. The PSt core diameter and the graft density of PNIPAAm on the PSt sphere surfaces were controlled by varying the reaction parameters. The nanospheres were composed of PSt and PNIPAAm. The chemical structure of the nanospheres was measured by 1H NMR spectroscopy in CDCl3. The integration of the aromatic proton signal was compared to the other signals to calculate the copolymer composition (Figure S1). The composition of the nanospheres was also calculated using the 1H NMR spectrum. In addition, the nanospheres surface was analyzed in D2O by 1 H NMR spectroscopy. PNIPAAm was detected by the methyne proton at 4.0 ppm and the methyl proton at 1.3 ppm at 25 °C. This result suggested that PNIPAAm was present on the surface of the nanospheres (Figure S2). Thermoresponsive core−corona-type nanospheres were synthesized with PNIPAAm of a controlled chain length. First, the core−corona nanospheres were prepared with an St/ PNIPAAm molar feed ratio of 100:1. The molecular weight of the PNIPAAm macromonomer used ranged from Mn = 3300 to 11 700. In all cases, spherical and monodisperse nanospheres were observed by SEM, as shown in Figure 2. The diameter of

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RESULTS AND DISCUSSION Preparation and Characterization of the PNIPAAm Macromonomer. The synthesis route for the PNIPAAm macromonomer is outlined in Scheme 1. PNIPAAm was Scheme 1. Synthesis of the PNIPAAm Macromonomer Used in This Work

synthesized by ATRP of NIPAAm at 25 °C using CMP as the initiator and CuCl/Me6TREN as the catalyst system. The synthesized PNIPAAm has one phthalimide group as the initiator residue. PNIPAAm with the phthalimide terminus was analyzed by 1H NMR. This substituent was a very convenient 1 H NMR label for the determination of the number-average molecular weight. The monomer conversions were monitored by 1H NMR based on the integrated areas of the resonance peaks at 4.0 ppm (1H, characteristic of an NIPAAm monomer) with respect to the phthalimide resonance peak at 7.8 ppm (4H). The number-average molecular weight and PDI were obtained by GPC analysis, and these values are summarized in Table 1. The molecular weight of PNIPAAm increased with increasing reaction time, and the polydispersity of the PNIPAAm chains remained below 1.15. After a short induction period of approximately 30 min, the polymerization proceeded rapidly, with around 65% conversion being attained in 4 h. 15772

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Table 1. Syntheses of PNIPAAm Using a Phthalimide Derivative as an ATRP Initiatora sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

[M]0/[1]0 25:1 25:1 25:1 50:1 50:1 50:1 50:1 50:1 50:1 50:1 100:1 100:1 100:1 100:1 100:1 100:1 100:1

time (h) 2 4 6 0.5 1 2 3 4 5 6 0.5 1 2 3 4 5 6

conv. (%)

Mn (Th.)b

c

c

c

c

c

c

25.9 44.1 53.5 68.9 74.0 72.4 75.2 23.1 34.6 54.8 63.6 65.4 58.8 73.7

1500 2500 3000 3900 4200 4100 4200 2600 3900 6200 7200 7400 6600 8300

Mn (1H NMR)

Mn (GPC)

PDI

yield (%)

2800 3100 3200 2800 3800 4300 5400 6400 5700 6200 4200 5700 9000 10 000 11 000

3900 4500 5000 3800 5500 6500 8400 9600 9100 9600 6500 9100 14 600 16 400 17 000 16 000 17 100

1.14 1.13 1.13 1.17 1.16 1.11 1.11 1.11 1.13 1.12 1.40 1.20 1.10 1.09 1.09 1.10 1.09

10.8 19.2 40.0 0.46 26.7 28.3 46.0 55.6 50.7 51.2 6.94 18.0 27.6 45.3 46.0 37.7 51.0

c

11 300

Reaction temp = 25 °C, [ligand]/[catalyst]/[initiator] = 1:1:1 mol/mol/mol, [NIPAAm] = 3 mol/L, DMF = 90 mL. bMn (Th.)= [NIPAAm]/ [initiator]0 × Conv. × 113 (NIPAAm MW). cNot determined. a

Figure 1. 1H NMR spectra of PNIPAAm in DMSO-d6 with different end groups: (a) phthalimide, (b) amine, and (c) vinyl.

Scheme 2. Synthesis of the Thermoresponsive Core− Corona-Type Nanospheres by Dispersion Polymerization Figure 2. SEM images of nanospheres prepared by dispersion polymerization with different PNIPAAm macromonomers: (a) Mn = 11 700, (b) Mn = 7600, (c) Mn = 5900, and (d) Mn = 3300.

shown in Figure 3. As the PNIPAAm macromonomer feed ratio increased, the diameter of the nanospheres decreased. As

the nanospheres was 240 nm when the PNIPAAm macromonomer with an Mn of 11 700 was used (Figure 2a). The nanospheres prepared using PNIPAAm macromonomer with a molecular weight ranging from 3300 to 11 700 showed average diameters ranging from 800−250 nm, increasing as the molecular weight of the PNIPAAm macromonomer decreased. We then investigated the effect of the monomer feed composition on the nanosphere diameter. The data are

Figure 3. Effect of the PNIPAAm macromonomer chain length and/or amount on the nanosphere size. 15773

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summarized in Table 2, as the PNIPAAm macromonomer feed amount increased, the PNIPAAm content in the copolymer Table 2. Summary of the Prepared Nanosphere Composition

a

St/PNIPAAm (in feed) (molar ratio)

PNIPAAm (Mn)

St/ PNIPAAma

macromonomer content (wt %)

100:0.5 100:1 100:2 100:0.5 100:1 100:2 100:0.5 100:1 100:2

3300 3300 3300 6000 6000 6000 9800 9800 9800

1453:1 1326:1 906:1 2722:1 2011:1 907:1 3386:1 1650:1 600:1

0.28 2.34 3.30 3.38 2.08 5.98 2.71 5.40 13.57

Figure 4. Effect of two different chain lengths of PNIPAAm macromonomer on the nanosphere size. The total PNIPAAm macromonomer contents is 1 mol% relative to the styrene monomer.

As determined by 1H NMR.

prepared, but these surfaces have different types of coronas. Furthermore, the longer-chained PNIPAAm resulted in more stably dispersed nanospheres in water. Both the PNIPAAm amount and chain length affected the size of the nanospheres (Figure 5). The average chain length of PNIPAAm was

also increased. The diameter of the nanospheres decreased from 380 nm for St/PNIPAAm = 100:0.5 to 150 nm for St/ PNIPAAm = 100:2 when using the PNIPAAm macromonomer with a molecular weight of 9800. A similar trend was observed regardless of the PNIPAAm chain length, though with a decreasing PNIPAAm chain length the obtained nanosphere diameter became larger. When the amount of macromonomer exceeded 2 mol %, no observable change in the diameter occurred in all cases. The PNIPAAm macromonomer acts as a dispersant during the polymerization of styrene. Thus, the diameter of the nanospheres was determined by the amount of PNIPAAm macromonomer and/or the PNIPAAm macromonomer chain length. Recently, Armes et al. reported the preparation of PNIPAAm macromonomer via RAFT polymerization.32 In their systems, however, with an increasing macromonomer degree of polymerization, the obtained nanosphere diameters increased. Conversely, the nanosphere diameter decreased with increasing macromonomer chain length. A previous report by Akashi et al. suggested that the molecular weight and amount of PEG were the same but that the size of their obtained nanosphere was different depending on the solvent mixing ratio.30 In addition, the diameter of the nanospheres was dependent on the solvent polarity and/or the chemical structure of macromonomer. However, when looking at the surface functionalization of nanospheres, several research groups attempted to graft two kinds of polymers with different chain lengths and/or different polymer systems by utilizing the “grafting from” and/or “grafting to” method.17,31 Therefore, to regulate the size of the nanospheres, we used PNIPAAm macromonomers with different chain lengths and/or a different feed ratio of PNIPAAm. The occupied area per chain on the nanospheres would increase with increasing polymer chain length but decreasing polymer density. Thus, two kinds of PNIPAAm with different chain lengths were grafted onto the nanosphere surfaces. Figure 4 indicates that the nanosphere diameter was controlled by two kinds of PNIPAAm feed ratios. In this experiment, PNIPAAm macromonomers with Mn = 3300 and 9800 were used. The diameter of the nanospheres decreased with an increasing feed amount of the longer-chained PNIPAAm. Such a diameter change, as indicated in Figure 4, suggested that the PNIPAAm macromonomers with different chain lengths were simultaneously introduced on the nanosphere surfaces. Therefore, the nanosphere size was regulated by the PNIPAAm amount as well as the chain length. By utilizing this method, nanospheres with the same diameter were

Figure 5. Effect of the PNIPAAm macromonomer chain length on the size of the nanospheres. The feed ratio is St/PNIPAAm 100:1 (molar ratio). The error bar shows the particle size distribution.

calculated from the number-average molecular weight and the feed mixture ratio. For example, when Mn = 3300 and 9800 were used with a 0.5:0.5 feed ratio, the number-average molecular weight was calculated to be Mn = 6500. Therefore, the nanosphere diameter was regulated by using the average chain length from two different sizes of PNIPAAm macromonomer. In addition, the longer PNIPAAm macromonomer (Mn = 10 000) is a more effective dispersant. Therefore, using long-chained PNIPAAm resulted in nanospheres with a smaller diameter. Moreover, we considered that the occupied area per PNIPAAm chain was dependent on the chain length. Thus, when PNIPAAm with different chain lengths was grafted onto the nanosphere surface, the surfaces have high-density polymer brushes, which can prevent nonspecific protein adsorption.16 As a result of using two kinds of PNIPAAm, a high-density corona was prepared on the nanosphere surfaces with a regulated nanosphere size. Thermoresponsive Behavior. The thermoresponsive nanospheres were analyzed by dynamic light scattering (DLS) at different temperatures. As shown in Figure 6, the diameter of the nanosphere decreased above the PNIPAAm LCST (Figure 6). The nanosphere diameter was determined from SEM images and DLS. As shown in Figure 6, the average nanosphere diameters were 280 ± 80 nm at 25 °C and 230 ± 60 nm at 40 15774

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Figure 6. Size distribution and change in the nanosphere size at 20 and 40 °C.

°C from DLS in a dilute suspension. In addition, the average nanosphere diameter calculated from SEM images (Figure 2a) was 240 ± 40 nm. The average diameter was almost the same from SEM (solid state) and DLS (dehydration state) of a dilute water suspension. Because the PSt core did not show apparent changes in diameter in this temperature region because PSt has a Tg of around 80 °C, this diameter change arose from the random coil−globule conformational change of the PNIPAAm corona on the nanosphere surface. The theoretical value of the PNIPAAm chain length was calculated from the C−C bond distance. For example, a PNIPAAm macromonomer with a degree of polymerization of 103 is approximately 25 nm in its extended form. From the DLS data, the change in the nanosphere diameter was equivalent to the theoretical value of the diameter, as shown in Figure 6. Generally, in the case of a random copolymer of NIPAAm with a hydrophobic comonomer, the LCST shifts to a lower temperature with increasing composition of the hydrophobic comonomer because incorporating the hydrophobic comonomer facilitates chain dehydration and aggregation, resulting in a decrease in the LCST; this opposite tendency was also observed by the incorporation of a hydrophilic comonomer.40 However, in our system the LCST did not change for prepared nanospheres to that of sole PNIPAAm because PNIPAAm grafts and the PSt domain were phase-separated from each other so that the nanosphere surfaces were occupied only by PNIPAAm chains. Thus, the nanosphere would have an LCST at around 32 °C.22,38 We also investigated the aggregation of the nanospheres in an aqueous droplet with respect to the effect of salt concentration and temperature. Aggregation of the nanospheres was observed by optical microscopy. The thermoresponsive behavior in an aqueous dispersion of the obtained nanospheres (1 wt %) was investigated over the temperature range from 20 to 40 °C, and the data are shown in Figure 7. We observed the edge of an aqueous droplet that put a drop of 10 μL on the slide glass at warmed predefined temperature. Nanosphere aggregation was obvious above the LCST, aggregating at 35 °C, where the grafted PNIPAAm became dehydrated. Armes et al. reported that prepared nanospheres were colloidally stable in aqueous dispersions at 20 °C, with macroscopic aggregation being observed upon heating to above 35 °C.32 Thus, prepared thermoresponsive nanospheres started to aggregate at around 32 °C as a result of grafted PNIPAAm on their surface. Also, in the case of 50 mmol L−1 NaCl, nanospheres were slightly aggregated at 30 °C by the effect of salt, leading to a decrease in the PNIPAAm LCST below 32 °C. Similarly, in the case of 100 mmol L−1, aggregation of the nanospheres was apparent at 25

Figure 7. Microscopy images of the thermoresponsive behavior of a nanosphere suspension at 0, 50, and 100 mmol L−1 NaCl concentrations. The nanosphere concentration is 1 wt % in an aqueous droplet, and the exposure time is 1000 s−1. All scale bars are 200 μm. Preparation conditions of nanospheres: St/PNIPAm = 100:1 (molar ratio) and PNIPAAm Mn = 9800.

°C. These results suggested that the aggregation of nanospheres occurred by a salting-out effect and decreasing LCST. The PNIPAAm LCST decreased the LCST by the added amount of salt.39 Moreover, the aggregation of the nanosphere suspension was increased with increasing temperature. Therefore, in the case of a sodium chloride suspension, nanospheres were obviously aggregated at 30 °C. With decreasing temperature, these aggregates disappeared and a homogeneous suspension was observed. These results thus suggested that the thermoresponsive nanospheres changed not only their diameter in aqueous media but also their dispersion stability as a result of grafted PNIPAAm on the nanosphere surfaces as a result of their temperature response. Therefore, the dispersion and aggregation of thermoresponsive nanosphere are reversible. Thus, the prepared thermoresponsive nanospheres are colloidally stable below the PNIPAAm LCST in aqueous media. Moreover, the dispersion stability of nanospheres could be controlled under physiological conditions. We are trying to control protein adsorption using thermoresponsive nanospheres with controlled chain lengths; this research is now in progress.

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CONCLUSIONS We successfully prepared well-defined core−corona-type thermoresponsive nanospheres with a regulated diameter. The prepared thermoresponsive polymeric nanospheres consisted of hydrophobic PSt cores and hydrophilic PNIPAAm corona layers on their surfaces. The nanosphere size was regulated by controlling the PNIPAAm macromonomer chain length and/or amount. In fact, the diameter of the nanosphere was controlled from 200 to 800 nm by the macromonomer chain length. Furthermore, we regulated the nanosphere diameter range from 150 to 900 nm by the PNIPAAm macromonomer chain length and/or amount. We prepared nanospheres with the same core size but different coronas. In addition, two different chain lengths of the PNIPAAm 15775

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(9) Anraku, Y.; Kishimura, A.; Oba, M.; Yamasaki, Y.; Kataoka, K. Spontaneous formation of nanosized unilamellar polyion complex vesicles with tunable size and properties. J. Am. Chem. Soc. 2010, 132, 1631−1636. (10) Li, D.; Sheng, X.; Zhao, B. Environmentally responsive “hairy” nanoparticles: mixed homopolymer brushes on silica nanoparticles synthesized by living radical polymerization techniques. J. Am. Chem. Soc. 2005, 127, 6248−6256. (11) Kawaguchi, H. Functional polymer microspheres. Prog. Polym. Sci. 2000, 25, 1171−1210. (12) Walkey, C. D.; Olsen, J. B.; Guo, H.; Emili, A.; Chan, W. C. W. Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake. J. Am. Chem. Soc. 2012, 134, 2139−2147. (13) Champion, J. A.; Walker, A.; Mitragotri, S. Role of particle size in phagocytosis of polymeric microspheres. Pharm. Res. 2008, 25, 1815−1821. (14) Lok, K. P.; Ober, C. K. Particle size control in dispersion polymerization of polystyrene. Can. J. Chem. 1985, 63, 209−216. (15) Werne, T. V.; Patten, T. E. Preparation of structurally welldefined polymer-nanoparticle hybrids with controlled/living radical polymerizations. J. Am. Chem. Soc. 1999, 121, 7409−7410. (16) Yuan, X.; Yoshitomi, K.; Nagasaki, Y. High-performance immunolatex possessing a mixed-PEG/antibody coimmobilized surface: highly sensitive ferritin immunodiagnostics. Anal. Chem. 2009, 81, 1549−1556. (17) Rungta, A.; Natarajan, B.; Neely, T.; Dukes, D.; Schadler, L. S.; Benicewicz, B. C. Grafting bimodal polymer brushes on nanoparticles using controlled radical polymerization. Macromolecules 2012, 45, 9303−9311. (18) Idziak, I.; Avoce, D.; Lessard, D.; Gravel, D.; Zhu, X. X. Thermosensitivity of aqueous solutions of poly(N,N-diethylacrylamide). Macromolecules 1999, 32, 1260−1263. (19) Heskins, M.; Guillet, J. E. Solution propaties of poly(Nisopropylacrylamide). J. Macromol. Sci., Part A 1968, 2, 1441−1455. (20) Kessel, S.; Schmidt, S.; Muller, R.; Wischerhoff, E.; Laschewsky, A.; Lutz, J.-F.; Uhlig, K.; Lankenau, A.; Duschl, C.; Fery, A. Thermoresponsive PEG-based polymer layers: surface characterization with AFM force measurements. Langmuir 2010, 26, 3462−3467. (21) Hoffman, A. S. Bioconjugates of intelligent polymers and recognition proteins for use in diagnostics and affinity separations. Clin. Chem. 2000, 46, 1478−1486. (22) Kikuchi, A.; Okano, T. Intelligent thermoresponsive polymeric stationary phases for aqueous chromatography of biological compounds. Prog. Polym. Sci. 2002, 27, 1165−1193. (23) Kikuchi, A.; Okuhara, M.; Karikusa, F.; Sakurai, Y.; Okano, T. Two-dimensional manipulation of confluently cultured vascular endothelial cells using temperature-responsive poly(N-isopropylacrylamide)-grafted surfaces. J. Biomater. Sci. Polym. Ed. 1998, 9, 1331− 1348. (24) Park, Y. S.; Ito, Y. Y.; Imanishi, Y. Permeation control through porous membranes immobilized with thermosensitive polymer. Langmuir 1998, 14, 910−914. (25) Silva, C. S. O.; Baptista, R. P.; Santos, A. M.; Martinho, J. M. G.; Cabral, J. M. S.; Taipa, M. A. Adsorption of human IgG on to poly(Nisopropylacrylamide)-based polymer particles. Biotechnol. Lett. 2006, 28, 2019−2025. (26) K. Nagase, K.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Kanazawa, H.; Okano, T. Interfacial property modulation of thermoresponsive polymer brush surfaces and their interaction with biomolecules. Langmuir 2007, 23, 9409−9415. (27) Kawano, T.; Niidome, Y.; Mori, T.; Katayama, Y.; Niidome, T. PNIPAM gel-coated gold nanorods for targeted delivery responding to a near-infrared laser. Bioconjugate Chem. 2009, 20, 209−212. (28) Nakayama, M.; Okano, T.; Miyazaki, T.; Kohori, F.; Sakai, K.; Yokoyama, M. Molecular design of biodegradable polymeric micelles for temperature-responsive drug release. J. Controlled Release 2006, 115, 46−56.

macromonomer were grafted onto the surface. By controlling the chain length, the nanosphere size and surface were controlled simultaneously. These well-defined nanospheres will prevent protein adsorption on nanosphere surface, which is attractive for diagnosis and/or DDS application.

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ASSOCIATED CONTENT

S Supporting Information *

Characterization of prepared core−corona-type nanospheres and their surfaces as analyzed by 1H NMR spectroscopy in CDCl3 and D2O. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-3-5876-1415. Fax: +81-3-5876-1415. E-mail: [email protected]. Author Contributions

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for a grant-in-aid for scientific research on innovative area “Molecular Soft Interface Science” (20106004) from the ministry of education, culture, sports, science and technology (MEXT), Japan. T.M. also acknowledges a grant for graduate research from Tokyo University of Science, 2012.

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REFERENCES

(1) Akagi, T.; Baba, M.; Akashi, M. Preparation of nanoparticles by the self-organization of polymers consisting of hydrophobic and hydrophilic segments: potential applications. Polymer 2007, 48, 6729− 6747. (2) Kamimura, M.; Kanayama, N.; Tokuzen, K.; Soga, K.; Nagasaki, Y. Near-infrared (1550 nm) in vivo bioimaging based on rare-earth doped ceramic nanophosphors modified with PEG-b-poly(4-vinylbenzylphosphonate). Nanoscale 2011, 3, 3705−3713. (3) Slomkowski, S.; Basinska, T. Polymer nano- and microparticle based systems for medical diagnostics. Macromol. Symp. 2010, 295, 13−22. (4) Sakuma, S.; Kataoka, M.; Higashino, H.; Yano, T.; Masaoka, Y.; Yamashita, S.; Hiwatari, K.; Tachikawa, H.; Kimura, R.; Nakamura, K.; Kumagai, H.; Gore, J.; Pham, W. A potential of peanut agglutininimmobilized fluorescent nanospheres as a safe candidate of diagnostic drugs for colonoscopy. Eur. J. Pharm. Sci. 2011, 42, 340−347. (5) Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada, Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.; Nishiyama, N.; Kataoka, K. Accumulation of sub-100 nm polymeric micelles in poorly permeable tumours depends on size. Nat. Nanotechnol. 2011, 6, 815−823. (6) Fujibayashi, T.; Okubo, M. Preparation and thermodynamic stability of micron-sized, monodisperse composite polymer particles of disc-like shapes by seeded dispersion polymerization. Langmuir 2007, 23, 7958−7962. (7) Kondo, K.; Kida, T.; Ogawa, Y.; Arikawa, Y.; Akashi, M. Nanotube formation through the continuous one-dimensional fusion of hollow nanocapsules composed of layer-by-layer poly(lactic acid) stereocomplex films. J. Am. Chem. Soc. 2010, 9, 8236−8237. (8) Ryu, J. H.; Chacko, R. T.; Jiwpanich, S.; Bickerton, S.; Babu, R. P.; Thayumanavan, S. Surface-functionalizable polymer nanogels with facile hydrophobic guest encapsulation capabilities. J. Am. Chem. Soc. 2010, 132, 17227−17235. 15776

dx.doi.org/10.1021/la4034468 | Langmuir 2013, 29, 15770−15777

Langmuir

Article

(29) Akashi, M.; Kirikihira, I.; Miyauchi, N. Synthesis and polymerization of a styryl terminated oligovinylpyrrolidone macromonomer. Angew. Makromol. Chem. 1985, 132, 81−89. (30) Chen, M. Q.; Serizawa, T.; Kishida, A.; Akashi, M. Graft copolymers having hydrophobic backbone and hydrophilic branches. XXIII. Particle size control of poly(ethylene glycol)-coated polystyrene nanoparticles prepared by macromonomer method. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 2155−2166. (31) Uchida, K.; Otsuka, H.; Kaneko, M.; Kataoka, K. A reactive poly(ethylene glycol) layer to achieve specific surface plasmon resonance sensing with a high S/N ratio: the substantial role of a short underbrushed PEG layer in minimizing nonspecific adsorption. Anal. Chem. 2005, 77, 1075−1080. (32) McKee, J. R.; Ladmiral, V.; Niskanen, J.; Tenhu, H.; Armes, S. P. Synthesis of sterically-stabilized polystyrene latexes using well-defined thermoresponsive poly(N-isopropylacrylamide) macromonomers. Macromolecules 2011, 44, 7692−7703. (33) Fujii, S.; Aono, K.; Suzaki, M.; Hamasaki, S.; Yusa, S.; Nakamura, Y. pH-responsive hairy particles synthesized by dispersion polymerization with a macroinitiator as an inistab and their use as a gas-sensitive liquid marble stabilizer. Macromolecules 2012, 45, 2863− 2873. (34) Serizawa, T.; Chen, M. Q.; Akashi, M. Unusual size formation of polymeric nanospheres synthesized by free radical polymerization in ethanol-water mixed solvents. Langmuir 1998, 14, 1278−1280. (35) Idota, N.; Kikuchi, A.; Kobayashi, J.; Akiyama, Y.; Sakai, K.; Okano, T. Thermal modulated interaction of aqueous steroids using polymer-grafted capillaries. Langmuir 2006, 22, 425−430. (36) Xu, J.; Ye, J.; Liu, S. Synthesis of well-defined cyclic poly(Nisopropylacrylamide) via click chemistry and its unique thermal phase transition behavior. Macromolecules 2007, 40, 9103−9110. (37) Santis, S. D.; Ladogana, R. D.; Diociaiuti, M.; Masci, G. Pegylated and thermosensitive polyion complex micelles by selfassembly of two oppositely and permanently charged diblock copolymers. Macromolecules 2010, 43, 1992−2001. (38) Wang, S.; Cheng, Z.; Zhu, J.; Zhang, Z.; Zhu, X. Synthesis of amphiphilic and thermosensitive graft copolymers with fluorescence P(St-co-(p-CMS))-g-PNIPAAM by combination of NMP and RAFT methods. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 5318−5328. (39) Kanazawa, H.; Yamamoto, K.; Matsushima, Y.; Takai, N.; Kikuchi, A.; Sakurai, Y.; Okano, T. Temperature-responsive chromatography using poly(N-isopropylacrylamide)-modified silica. Anal. Chem. 1996, 68, 100−105. (40) Feil, H.; Han, Y.; Bae Feijen, J.; Kim, S. W. Effect of comonomer hydrophilicity and ionization on the lower critical solution temperature of N-isopropylacrylamide copolymers. Macromolecules 1993, 26, 2496−2500.

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