Fabrication of Thermoresponsive Nanoactinia Tentacles by a Single

Oct 6, 2015 - Center for Collaborative Research, Anan National College of Technology, 265 Aoki Minobayashi, Anan, Tokushima 774-0017, Japan ... We pre...
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Fabrication of Thermoresponsive Nanoactinia Tentacles by a Single Particle Nanofabrication Technique Masaaki Omichi,*,†,‡,# Hiromi Marui,† Vikas S. Padalkar,§ Akifumi Horio,†,§ Satoshi Tsukuda,∥ Masaki Sugimoto,⊥ and Shu Seki*,†,§ †

Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita 565-0871, Japan Center for Collaborative Research, Anan National College of Technology, 265 Aoki Minobayashi, Anan, Tokushima 774-0017, Japan § Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto, 615-8510, Japan ∥ Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan ⊥ Japan Atomic Energy Agency, Takasaki Advanced Radiation Research Institute, 1233 Watanuki-machi Takasaki, Gunma 370-1292, Japan ‡

S Supporting Information *

ABSTRACT: Nanowires that are retractable by external stimulus are the key to fabrication of nanomachines that mimick actinia tentacles in nature. A single particle nanofabrication technique (SPNT) was applied over a large area to the fabrication of retractable nanowires (nanoactinia tentacles) composed of poly(N-isopropylacrylamide) (PNIPAM) and poly(vinylpyrrolidone) (PVP), which are thermoresponsive and hydrophilic polymers. The nanowires were transformed with increasing temperature from rod-like- to globule-forms with gyration radii of ∼1.5 and ∼0.7 μm, respectively. The transformation of the nanowires was reversible and reproducible under repeated cycles of heating and cooling. The reversible transformation was driven by hydration and dehydration of PNIPAM, the thermoresponsive segments, resulting in coil-to-globule transformation of the segments. The nanoactinia tentacle systems trapped the nanoparticles as a model of living cells under thermal stimulation, and the trapping was controlled by temperature. We present herein a unique nanomachine system which can be applicable to nanoparticle filtering/ sensing systems and expandable to large-area functionalization and demonstrate polymer-based nanoactuators via scaling of molecular level coil-to-globule transformation into micron-sizes.

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efficiency are still shortcomings of polymer-based actuators.25 The capability of polymers to show reversible/reproducible change in their volume at the nanoscale level after heating/ cooling has attracted tremendous interest from a product engineering point of view.19,20 This reversible transformation was driven by hydration and dehydration of the used polymer. The actinia, which are captivating motifs of nanomachines in the living world, have a large number of retractable tentacles that catch prey effectively and protect the organism from exposure to enemies. For biomimicking of actinia, their retractable tentacles were downsized from millimeter to nanometer with thermoresponsive polymeric nanowires, which recognize and trap bacteria and specific cells, such as immune system cells, on the functional surfaces (Scheme 1). The fabrication of size-controlled nanowires by MeV-order high-energy charged particles is an efficient technique for

INTRODUCTION Nano- and microactuators are the important components of nanomachines, such as the working assembly of nanomotors, and have been extensively studied in recent years.1−5 Their high-tech applications have also been proposed and demonstrated across a broad range of fields such as medical devices,6 microrobotics,7,8 tissue and cell engineering,9,10 artificial muscle,11 molecular switches,12−14 environmental sensing,2 etc. These nano- and microactuators have been designed based on a variety of organic, inorganic, polymeric, and biomolecular materials.15−18 Polymer-based actuators are more advantageous and attractive over the others because of the extremely wide tunability of stimuli-sensitive properties, which reflects their dynamic backbone configurational changes associated with rapid side-chain responses depending on the functional groups.18 There are plenty of excellent nano- or microscale polymeric actuators based on changes in different properties such as swelling, melting, anisotropic expansion, and surface forces that have been reported to date.18−24 However, slow response, short life cycle, and low energy conversion © 2015 American Chemical Society

Received: August 11, 2015 Revised: October 3, 2015 Published: October 6, 2015 11692

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such as poly(N-isopropylacrylamide) (PNIPAM), for stimuliresponsive nanowires.39,40 PNIPAM is a thermoresponsive polymer with a lower critical solution temperature (LCST) of approximately 32 °C in water.41,42 Statistical analysis of backbone configuration of PNIPAM has been well analyzed in terms of coil-to-globule transition with substantial changes in the gyration radius of a single chain in aqueous solution via intramolecular dehydration upon heating, giving the LCST phase diagram.43,44 The PNIPAM-based nanowires get transformed from the nonaggregated form to the aggregated form with increasing temperature.40 However, the transformation is irreversible, due to the strong interaction between the PNIPAM nanowires and the silicon (Si) substrate. Reduction of the interaction is expected to make nanowires retractable like actinia tentacles. Poly(vinylpyrrolidone) (PVP) is a biocompatible hydrophilic polymer that has been widely used in biomedical field.45 The fabrication of retractable nanowires controlled by thermal stimulation, by inserting PVP nanowires as a spacer between the PNIPAM nanowires and the Si substrate, is reported in this research article.

Scheme 1. Schematic Image of Concept under Study

nanowire-fabrication (single particle nanofabrication technique: SPNT). We have developed a method for the fabrication of homogeneous nanowires over a large area using a SPNT.26,27 High-energy charged particles induce nonhomogeneous condensation reactions in nanometer-scale cylindrical areas of the polymers and organic molecules in their condensed phases along their trajectories,28−30 leading to one-dimensional (1D) linear nanostructures with a high aspect ratio via crosslinking31−36 and/or polymerization reactions.37,38 Recently, the SPNT has been extended to stimuli-responsive polymers,

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RESULTS AND DISCUSSION SPNT is an easy method of providing homogeneous nanowires composed of two different polymers which are difficult to fabricate.32,35 The PNIPAM and PVP bilayer film was prepared

Figure 1. (a) Schematic representation of the formation of PNIPAM−PVP connected nanowires by SPNT from a PNIPAM layer (PNIPAM/ MBAAM = 100/20) (upper) and a PVP layer (PNIPAM/MBAAM = 100/20) (lower) bilayer. (b, c) AFM topological image of PNIPAM−PVP connected nanowires with 0.1 and 0.5 μm thicknesses of PVP layer treated in water at 25 °C (left) and then 50 °C (right). PNIPAM−PVP connected nanowires were formed by irradiation of the bilayer films (b: PNIPAM layer/PVP layer = 0.5 μm/0.1 μm, c: PNIPAM layer/PVP layer = 0.5 μm/0.5 μm) with a 490 MeV 192Os30+ ion beam at a fluence of 5.0 × 107 ions cm−2. 11693

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Figure 2. (a) AFM topological image of the PNIPAM−PVP connected nanowires with the short PVP portion repeatedly treated in water at 25 °C and 50 °C. (b) Length of PVP (blue) and PNIPAM (red) segments in the nanowires as a function of the cycle of temperature change (n = 40). (c) Schematic image showing the reversible transformation of the PNIPAM−PVP connected nanowires by thermal stimulation.

nanowire cross section was r = 13.5 ± 2.0 nm for the thick portion and r = 5.4 ± 1.5 nm for the thin portion (averaged over n = 50). In the 0.5 μm thickness of the PVP layer, the radii were r = 11.3 ± 1.9 nm and r = 4.6 ± 0.8 nm, respectively (n = 50). Each average radius of the thick and thin portion was about the same size, regardless of the thickness of PVP layer. The r values have been reported to obey the following theoretical equation with a parameter reflecting average energy deposition by an incident high energy charged particle as linear energy transfer (LET, eV nm−1):26,27,46

by spin-coating a PNIPAM layer from its tetrahydrofuran solution onto a spin-cast PVP layer without significant intermixing. The bilayer film was exposed to 490 MeV 192 Os30+ ions and developed using H2O (Figure 1a). Addition of the N,N′-methylene bis-acrylamide (MBAAM) cross-linker to PNIPAM and PVP increased the cross-linking efficiency of these polymer systems under irradiation.40 Figure 1b,c shows the atomic force microscopy (AFM) topology image of the PNIPAM−PVP connected nanowires formed by ion beam irradiation of the bilayer films with 0.1 and 0.5 μm thickness of PVP layer (PNIPAM layer: 0.5 μm). The nanowire consists of two components: a thick portion and a thin portion. The length of the thick portion increased in proportion to the thickness of PVP layer, indicating that the thick portion is composed of PVP and the thin portion is composed of PNIPAM. In the 0.1 μm thickness of the PVP layer, the average radius (r) of the

⎡ ⎛ 1/2 ⎞⎤−1 LET × G(x)mN ⎢ ⎜ e rp ⎟⎥ ln r = ⎢ ⎜⎝ rc ⎟⎠⎥ 400πρA ⎣ ⎦ 2

(1)

where A is Avogadro’s number, e is Napier’s constant, m is the mass of a monomer unit, ρ (g nm−3) is the density of the 11694

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Figure 3. (a) AFM topological image of PNIPAM−PVP connected nanowires with the long PVP portion repeatedly treated in water at 25 °C and 50 °C.

polymer, N is the degree of polymerization, G(x) (100 eV)−1 is the number of cross-links induced by a radiation-deposited energy of 100 eV, and rc and rp are the radii of the core and penumbra areas, defined from the equipartition theorem of collisions of charged particles with matter.47 The estimate of r derived from eq 1 depends on the density and molecular weights of PNIPAM and PVP, respectively, in the present case. The value of LET is calculated as 12 000 and 13 000 eV nm−1 for 490 MeV 192Os30+ in PNIPAM (ρ = 1.1 g cm−3) and PVP (ρ = 1.2 g cm−3) films, respectively, and the value is almost constant (less than 1% decrease) over the trajectories of incident particles.48 This gives homogeneous double adjoined 1D nanowires. The cross-linking efficiency of G(x) is also derived from eq 1 from observed values of r, estimated as G(x) = 1.2 and 3.9 (100 eV)−1 for PNIPAM and PVP, suggesting that effective cross-linking reactions in the PVP layer is providing a stable base of nanostructures to immobilize the connected segments of PNIPAM. The G(x) of PNIPAM is a bit lower than the reported value (∼2.5).49 Radiolytic yield (G) of radical species in condensed media has been reported to be decreased by the self-quenching effect via enhanced secondorder reactions in a high density of reactive sites.28,29,50−52 This gives the drop of G(x) in the present radiation induced by cross-linking reactions in an ion track. The PNIPAM portions were folded and transformed to globular shape in both cases by changing temperature from 25 °C to 50 °C (Figure 1b and 1c, right). To examine whether transformation of the PNIPAM−PVP connected nanowires with the short PVP portion is reversible,

changing temperatures between 25 °C and 50 °C were repeated and the lengths of the nanowires at each steps were estimated from AFM observation. No matter how many times the temperature of the water changed, the PNIPAM portion was repeatedly unfolded and folded by thermal stimulation (Figure 2a). The average lengths of these nanowires at 25 °C were 2.4 μm (Initial), 2.6 μm (first cycle), 2.4 μm (second cycle), 2.6 μm (third cycle), and 2.3 μm (fourth cycle), and the lengths at 50 °C were 0.5 μm (initial), 0.7 μm (first cycle), 0.9 μm (second cycle), 0.5 μm (third cycle), and 0.8 μm (fourth cycle) (Figure S1, Supporting Information). Despite almost equal solubility of parameters of PVP (δ = 11.4)53 and PNIPAM (δ = 11.2)54 at room temperature, where the hydrophilic nature of PNIPAM is preserved by hydrogen bonding via amide side chains, the polymers are immiscible with each other in the whole range of composition (Figure S2, Supporting Information)55 because of weak interchain interactions due to spatial hindrance and preferential intrachain hydrogen bonding in PNIPAM, rather than ineffective interchain ones due to the proton-accepting nature of PVP as a Lewis base. This allows a cross-sectional trace of each segregated PNIPAM or PVP segment in a nanowire on the substrate, showing a clear contrast in the thickness (Figure S3, Supporting Information). Considerable mismatches of the solubility parameters of both polymers above the LCST limit of PNIPAM at 32 °C will lead to stabilization of segregated structures of the segments. Taking particular note of PNIPAM segments, the average lengths of PNIPAM segments at 25 °C were 1.9 μm (Initial), 2.1 μm (first cycle), 2.0 μm (second cycle), 2.1 μm (third cycle), and 2.0 μm 11695

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Figure 4. Statistical analysis of in-plane gyration radii of nanowires observed in each step of heating and cooling procedures.

(fourth cycle), and the lengths at 50 °C were 0.3 μm (initial), 0.4 μm (first cycle), 0.3 μm (second cycle), 0.4 μm (third cycle), and 0.3 μm (fourth cycle) (Figure 2b red, n = 40). These results clearly indicate that the transformation of the nanowires, especially PNIPAM segments, was reversible and reproducible under repeated cycles of heating and cooling (Figure 2c), which could be attributed to hydration and dehydration of the PNIPAM portions under and over the LCST of 32 °C, respectively. The PNIPAM portion of the PNIPAM−PVP connected nanowire with a long PVP portion (PVP layer: 0.5 μm) was also reversibly unfolded and folded by thermal stimulation in a manner similar to that with a short PVP portion (Figure 3). It is likely that the inserted PVP portion of the nanowires works as a spacer to block contact of the PNIPAM portion with the Si substrate, leading to reversible transformation by thermal stimulation. Diblock copolymer systems of PNIPAM-b-PVP, a single chain analogue of the present PNIPAM−PVP connected nanowires, have been of interest as a motif for thermoresponsive polymer self−assembly systems giving a variety of aggregated structures.41 The aggregate has been developed as a drug delivery system,56 catalyst carrier,57 etc., and the spherical shape of the aggregates (micelles) has been well controlled by the design of multiblock copolymer segments.58,59 The key to morphological control is the change in the hydrodynamic/ gyration radii of thermoresponsive segments; however, it has been complicated to access the temperature-induced changes in single chain levels quantitatively, spinning off the effects of aggregation with rapid dehydration dynamics. As seen in Figure 2, the PNIPAM nanowires exhibit long enough relaxation time

of transformation via dehydration because of the concerted structural changes of PNIPAM chains incorporated into a nanowire and strong interaction between nanowire and the substrate surface,33 allowing us to assess the scaled coil-toglobule transition of PNIPAM segments in the present system. The transformation was statistically analyzed by the in-plane gyration radius of the nanowires with short reference PVP segments. More intuitive change is evident from the statistics of gyration radii of the nanowires as shown in Figure 4. The average radii of the initial nanowires was 1.51 μm with small standard deviation (σ) of 0.28 μm, while that of annealed nanowires under water at 50 °C was decreased to 0.69 μm (σ = 0.11 μm) as a result of the dehydrated shape of the nanowires. Concomitantly, the distribution of the gyration radii was widened again to σ = 0.22 μm for the next step of cooling, and the shrunk-stretched cycles were reproduced perfectly under temperature control up to the repeated cycles. These characterizations strongly support our claim on temperatureswitchable mechanical control of the present nanowire systems. To the best of our knowledge, this is the first report of retractable nanowires showing reversible and uniform transformation by external stimulus over a large area. The factor of Rg(25 °C)/Rg(50 °C) is estimated as ∼1.9, which is far smaller than the hydrodynamic radius change of diblock copolymer systems via self-aggregation in solutions60−62 but almost identical to the change of Rg for isolated PNIPAM chains in water,63−65 suggesting the present nanowire transformation via the scaled coil-to-globule transition of PNIPAM molecules. Nano/microfibers are widely used as adsorbent modules for separating nano- and microparticles owing to their large surface areas with applications including leukocyte removal filter.66−69 11696

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Figure 5. (a) AFM topological image of the high aspect PNIPAM−PVP connected nanowires treated in water at 30 °C (upper) and 40 °C (lower). (b) AFM topological (left) and phase (right) images of the high aspect PNIPAM−PVP connected nanowires after the adsorption of PS nanoparticles for 30 min at 30 °C (upper) and 40 °C (lower). (c) Schematic image showing the adsorption of the PS nanoparticles on PNIPAM− PVP connected nanowires at 30 °C and 40 °C.

connected nanowires. The PS nanoparticles were adsorbed on the PNIPAM portion of the PNIPAM−PVP connected nanowires due to hydrophobic interaction. At 30 °C, the PNIPAM portion covers the whole surface. On the other hand, at 40 °C, the PNIPAM portion was folded and surface coverage of the PNIPAM portion became smaller compared to that at 30 °C. The PS nanoparticles were adsorbed on the PNIPAM portion of the PNIPAM−PVP connected nanowires due to hydrophobic interactions. While the interactions between the hydrophobic PS nanoparticles and the hydrophilic PVP portion and Si substrate are considered to be weak, actually the PS nanoparticles were hardly adsorbed on the PVP portion and Si substrate. Therefore, the amount of the adsorbed PS nanoparticles depended largely on temperature. Trapping of the PS nanoparticles by the PNIPAM−PVP connected nanowires can be controlled by changing the temperature. The nanowires seem like retractable actinia tentacles. Actinia spreads its tentacles to catch prey and retracts them to protect the organism from exposure to an enemy. The “nanoactinia tentacles” extend to catch the PS nanoparticles and retract to avoid contact with them as needed. The PNIPAM−PVP connected nanowires are thus considered to be a kind of nanomachine. Previous studies on various functional nanowires, such as fluorescent nanowires and protein nanowires with enzymatic activity, have been reported by us. 34,35 A combination of stimulus-responsive nanowires and the abovediscussed nanowires will lead to fabrication of complex and multifunctional nanomachines.

The surface areas of the nanoactinia vary greatly depending on the transformation of PNIPAM-based segments in the connected nanowires. Therefore, the nanoactinia have been applied to control trapping of the nanoparticles as a model of cells. Increase in the length of the PNIPAM portion should lead to a large change in surface areas by the transformation. It is difficult to increase the thickness of the PNIPAM layer, because the bilayer has been fabricated by spin-casting to avoid dissolving the PVP layer. To fabricate the higher aspect PNIPAM−PVP connected nanowires without increasing the thickness of the PNIPAM−MBAAM layer, the bilayer was irradiated by ion beam at an irradiation angle of 30°. The length of the nanowires formed was twice that of normal ion beam irradiation. To reduce the influence of temperature to a considerable extent, the adsorption of polystyrene (PS) nanoparticles onto the surface of the PNIPAM−PVP connected nanowires was evaluated at 30 °C and 40 °C instead of 25 °C and 50 °C. At 30 °C and 40 °C, the PNIPAM portion was unfolded and folded in a similar manner to that at 25 °C and 50 °C (Figure 5a). Figure 5b shows AFM topology and phase image of the high aspect PNIPAM−PVP connected nanowires after the adsorption of PS nanoparticles. In the AFM topology image, the PS nanoparticles and the globular shape of the PNIPAM were only observed because the diameter of the PS nanoparticles (82 nm) is considerably larger than the diameter of the nanowires. At 30 °C, the PS nanoparticles were adsorbed on the larger surface, while their adsorption was suppressed at 40 °C (Figure 5b, left). The amount of the adsorbed PS nanoparticles was estimated to be 11.5 particles μm−2 and 2.9 particles μm−2 at 30 °C and 40 °C, respectively. In the AFM phase image, the PS nanoparticles had an overlap with the PNIPAM portion in both cases, suggesting that the PS nanoparticles were adsorbed on the PNIPAM portion (Figure 5b, right). Figure 5c is the illustration showing that the PS nanoparticles were adsorbed on PNIPAM−PVP

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CONCLUSION Retractable nanowires connecting PNIPAM and PVP segments, namely, nanoactinia tentacles, were successfully fabricated by application of SPNT to a PNIPAM and PVP bilayer film. The length of the PVP portion increased in proportion to the thickness of the PVP layer. PNIPAM−PVP connected nanowires showed reversible transformation by thermal 11697

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respective temperature (30 °C or 40 °C) for 1 h prior to AFM observation.

stimulation, regardless of the thicknesses of the PVP layer. The inserted PVP portion worked as a spacer to block the contact of the PNIPAM portion with the Si substrate, resulting in reversible transformation of the nanowires. Furthermore, the nanoactinia tentacles could control trapping of the PS nanoparticles by thermal stimulation. Application of SPNT to multilayer films, one of which involves stimulus−responsive polymers, is very effective for the development of polymer nanoactuators.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02962. Experimental details and Figures S1 (changes in total length of PNIPAM−PVP nanowires), S2 (AFM images of PNIPAM/PVP binary mixture), and S3 (AFM crosssectional traces of nanowire segments) (PDF)

EXPERIMENTAL SECTION

Materials. Poly(N-isopropylacrylamide) (Mw: 20 000−25 000 g mol−1) and N,N′-methylene bis-acrylamide were purchased from Sigma-Aldrich Corporation (USA). Poly(vinylpyrrolidone) (K30) (Mw: 40 000 g mol−1) was purchased from Tokyo Chemical Industry Co., Inc. (Japan). Polybeads polystyrene 0.10 μm microspheres (diameter of 82 ± 6 nm) were purchased from Polysciences Inc. (USA). All other reagents were purchased from Wako Pure Chemical Industries, Ltd. (Japan). Preparation of PNIPAM−PVP Connected Nanowires. PVP− MBAAM blend films on a Si substrate (1.5 × 1.5 cm2) were prepared by spin-casting with PVP and MBAAM (PVP/MBAAM = 100/20) in methanol. The thickness of the films was adjusted to 0.1 and 0.5 μm and confirmed by a Dektak 150 Stylus Surface Profiler (ULVAC, Inc., Japan). PNIPAM−MBAAM blend films were deposited upon the PVP−MBAAM blend films by spin-casting with PNIPAM and MBAAM (PNIPAM/MBAAM = 100/20) in tetrahydrofuran. The thickness of the bilayer films was adjusted to 0.6 and 1.0 μm and confirmed by a Dektak 150 Stylus Surface Profiler. The bilayer films on Si substrate were irradiated in a vacuum chamber (