Naokazu Idota

Shape-Memory Surface with Dynamically Tunable Nano-Geometry Activated by Body Heat

Shape-memory surfaces with on-demand, tunable nanopatterns are developed to observe time dependent changes in cell alignment using temperature-responsive poly(ϵ-caprolactone) (PCL) films. Temporary grooved nanopatterns are easily programmed on the films and triggered to transition quickly to permanent surface patterns by the application of body heat. A time-dependent cytoskeleton remodeling is also observed under biologically relevant conditions.

Photo-switchable control of pH-responsive actuators via pH jump reaction

We propose a new approach to fabricate reversible self-bending actuators utilizing a photo-triggered pH jump reaction. A photo-initiated proton-releasing agent of o-nitrobenzaldehyde (NBA) was successfully integrated into bilayer hydrogels composed of a polyacid layer, poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) (P(NIPAAm-co-CIPAAm)) and a polybase layer, poly(N-isopropylacrylamide-co-N,N′-dimethylaminopropylacylamide) (P(NIPAAm-co-DMAPAAm)), where the adhesion of both layers was achieved via electrophoresis of semi-interpenetrating polyelectrolyte chains. The NBA-integrated bilayer gels demonstrated quick proton release upon UV irradiation, allowing the pH within the gel to decrease below the volume phase transition pH in 30 seconds. By controlling the NBA concentration and the gel thickness, the degrees and the kinetics of bending were easily controlled. Reversible bending was also studied with respect to the NBA concentration in response to ‘on–off’ UV irradiation. Additionally, self-bending of the non-UV irradiated region of the gel was also achieved because the generated protons gradually diffused toward the non-irradiated region. The proposed system can be potentially applied in the fields of mechanical actuators, controlled encapsulation and drug release, robotics and microfluidic technologies because control over autonomous motion by both physical and chemical signals is essential as a programmable system for real biomedical and nano-technological applications.

The use of electron beam lithographic graft-polymerization on thermoresponsive polymers for regulating the directionality of cell attachment and detachment.

A simple process for nano-patterned cell culture substrates by direct graft-polymerization has been developed using an electron beam (EB) lithography system requiring no photo-masks or EB-sensitive resists. The compound N-isopropylacrylamide (IPAAm) was locally polymerized and grafted directly by EB lithographic exposure onto hydrophilic polyacrylamide (PAAm)-grafted glass surfaces. The size of the surface grafted polymers was controlled by varying the area of EB dose, and a minimal stripe pattern with a 200 nm line-width could be fabricated onto the surface. On the stripe-patterned surfaces, above the lower critical solution temperature (LCST), the cells initially adhered and spread with an orientation along the pattern direction. The magnitude of the spreading angle and elongation of adhered cells depended on the pattern intervals of the grafted PIPAAm. When culture temperature was lower than the LCST, cultured cells detached from the surfaces with strong shrinkage along the pattern direction, and sometimes folded and became parallel with the stripe pattern. This patterned cell recovery technique may be useful for the construction of muscle cell sheets with efficient shrinkage/relaxation in a specific direction and spheroidal 3D cell structures, with application to tissue engineering and microfluidic cellular devices.

Novel temperature-responsive polymer brushes with carbohydrate residues facilitate selective adhesion and collection of hepatocytes

Abstract Temperature-responsive glycopolymer brushes were designed to investigate the effects of grafting architectures of the copolymers on the selective adhesion and collection of hypatocytes. Homo, random and block sequences of N-isopropylacrylamide and 2-lactobionamidoethyl methacrylate were grafted on glass substrates via surface-initiated atom transfer radical polymerization. The galactose/lactose-specific lectin RCA120 and HepG2 cells were used to test for specific recognition of the polymer brushes containing galactose residues over the lower critical solution temperatures (LCSTs). RCA120 showed a specific binding to the brush surfaces at 37 °C. These brush surfaces also facilitated the adhesion of HepG2 cells at 37 °C under nonserum conditions, whereas no adhesion was observed for NIH-3T3 fibroblasts. When the temperature was decreased to 25 °C, almost all the HepG2 cells detached from the block copolymer brush, whereas the random copolymer brush did not release the cells. The difference in releasing kinetics of cells from the surfaces with different grafting architectures can be explained by the correlated effects of significant changes in LCST, mobility, hydrophilicity and mechanical properties of the grafted polymer chains. These findings are important for designing ‘on–off’ cell capture/release substrates for various biomedical applications such as selective cell separation.

Rewritable and shape-memory soft matter with dynamically tunable microchannel geometry in a biological temperature range

Here we present on-demand switchable microchip materials that display potent rewritable and shape-memory properties which are shown to contribute to fluidic control as pumps and valves. Semi-crystalline poly(e-caprolactone) (PCL) was chemically crosslinked to show shape-memory effects over its melting temperature (Tm) because the crosslinking points set the permanent shape and the crystalline domains serve as thermally reversible mobile phase. The Tm was adjusted to nearly biologically relevant temperatures by crosslinking two and four branched PCL macromonomers with different ratios. The Tm decreased proportionally with increasing four branched PCL content because an increase in crosslinking density imposes restrictions on chain mobility and reduces the crystallization. The sample with 50/50 wt% mixing ratio of two- and four-PCL had a Tm around 33 °C. Permanent surface patterns were first generated by crosslinking the macromonomers in a mold, and temporary surface patterns were then embossed onto the permanent patterns. From the cross-sectional profiles, almost 100% recovery of the permanent pattern was successfully achieved after shape-memory transition. The effects of dynamic geometric changes of the shape-memory channels on the microfluidic flow were also investigated and shape-memory channel closing was achieved by the application of heat. The proposed system can be potentially applied as a new class of microfluidic control techniques, which enables portable microfluidic based diagnostic tools for biomedical applications and environmental monitoring allowing on-site analysis.

Cultivation and recovery of vascular endothelial cells in microchannels of a separable micro-chemical chip.

Various micro cell culture systems have recently been developed. However, it is extremely difficult to recover cultured cells from a microchannel because the upper and lower substrates of a microchip are permanently combined. Therefore, we developed a cell culture and recovery system that uses a separable microchip with reversible combining that allows separation between closed and open channels. To realize this system, two problems related to microfluidic control-prevention of leakage and non-invasive recovery of cultured cells from the substrate-must be overcome. In the present study, we used surface chemistry modification to solve both problems. First, octadecyltrimethoxysilane (ODTMS) was utilized to control the Laplace pressure at the liquid/vapor phase interface, such that it was directed toward the microchannels, which suppressed leakage from the slight gap between two substrates. Second, a thermoresponsive polymer poly(N-isopropyl acrylamide) (PNIPAAm) was used to coat the surface of the ODTMS-modified microchannel by UV-mediated photopolymerization. PNIPAAm substrates are well known for controlled cell adhesion/detachment by alteration of temperature. Finally, the ODTMS- and PNIPAAm-modified separable microchips were subjected to patterning, and human arterial endothelial cells (HAECs) were cultured in the resulting microchannels with no leakage. After 96 h of the culture, the HAECs were detached from the microchips by decreasing the temperature and were then recovered from the microchannels. This study is the first to demonstrate the recovery of living cells cultured in a microchannel, and may be useful as a fundamental technique for vascular tissue engineering.

A ‘smart’ approach towards the formation of multifunctional nano-assemblies by simple mixing of block copolymers having a common temperature sensitive segment

A smart approach has been developed to form nanoassemblies with tunable shells functions by simply mixing three different block copolymers with a common temperature-responsive segment at 25 °C. The formation of nanoassemblies and their thermodynamic stability are driven by the hydrophobic interaction of the common poly(N-isopropylacrylamide-co-N-(isobutoxymethyl)acrylamide) (P(NIPAAm-co-BMAAm)) segment which has a lower critical solution temperature (LCST) below 25 °C. Three series of copolymers with different polymer structures, acrylamide-type P(NIPAAm-co-N-(hydroxymethyl)acrylamide (HMAAm))-b-P(NIPAAm-co-BMAAm), poly(ethylene oxide) (PEO)-b-P(NIPAAm-co-BMAAm), and methacrylate-type poly(2-lactobionamidoethyl methacrylate) (PLAMA)-b-P(NIPAAm-co-HMAAm)-b-P(NIPAAm-co-BMAAm), were successfully polymerized by reversible addition–fragmentation chain transfer (RAFT) polymerization. Regardless of the block copolymer types the selected block copolymers successfully formed a stable core–shell assembly with the collapsed common segments forming the inner core by simple mixing in aqueous solutions. The size distributions were monodisperse and relatively narrow when all or two of these three block copolymers were mixed, while addition of free-copolymers without the common segment did not affect the assembly formation. The ratio of functional segments into shell could be easily tuned by changing the mixing ratio of three block copolymers. This system is highly expected to find use as smart nano-carriers for encapsulation, targeting, and triggered release of drug under control through a combination of temperature-responsive chains, accessible functionality, and choice of sugar moiety.

The taming of the cell: shape-memory nanopatterns direct cell orientation.

We report here that the direction of aligned cells on nanopatterns can be tuned to a perpendicular direction without use of any biochemical reagents. This was enabled by shape-memory activation of nanopatterns that transition from a memorized temporal pattern to the original permanent pattern by heating. The thermally induced shape-memory nanopatterns were prepared by chemically crosslinking semi-crystalline poly(ε-caprolactone) (PCL) in a mold to show shape-memory effects over its melting temperature (Tm = 33°C). Permanent surface patterns were first generated by crosslinking the PCL macromonomers in a mold, and temporary surface patterns were then embossed onto the permanent patterns. The temporary surface patterns could be easily triggered to transition quickly to the permanent surface patterns by a 37°C heat treatment, while surface wettability was independent of temperature. To investigate the role of dynamic and reversible surface nanopatterns on cell alignment on the PCL films before and after a topographic transition, NIH 3T3 fibroblasts were seeded on fibronectin-coated PCL films with a temporary grooved topography (grooves with a height of 300 nm and width of 2 μm were spaced 9 μm apart). Interestingly, cells did not change their direction immediately after the surface transition. However, cell alignment was gradually lost with time, and finally cells realigned parallel to the permanent grooves that emerged. The addition of a cytoskeletal inhibitor prevented realignment. These results clearly indicate that cells can sense dynamic changes in the surrounding environments and spontaneously adapt to a new environment by remodeling their cytoskeleton. These findings will serve as the basis for new development of spatiotemporal tunable materials to direct cell fate.

Light-induced spatial control of pH-jump reaction at smart gel interface

We proposed here a smart control of an interface movement of proton diffusion in temperature- and pH-responsive hydrogels using a light-induced spatial pH-jump reaction. A photoinitiated proton-releasing reaction of o-nitrobenzaldehyde (NBA) was integrated into poly(N-isopropylacrylamide-o-2-carboxyisopropylacrylamide) (P(NIPAAm-co-CIPAAm)) hydrogels. NBA-integrated hydrogels demonstrated quick release of proton upon UV irradiation, allowing the pH inside the gel to decrease below the pK(a) of P(NIPAAm-co-CIPAAm) within a minute. The NBA-integrated gel was shown to shrink rapidly upon UV irradiation without polymer skin layer formation due to a uniform decrease of pH inside the gel. Spatial control of gel shrinking was also created by irradiating UV light to a limited region of the gel through a photomask. The interface of proton diffusion (active interface) gradually moved toward non-illuminated area. The apparent position of active interface, however, did not change remarkably above the LCST, while protons continuously diffused outward direction. This is because the active interface also moved inward direction as gel shrank above the LCST. As a result, slow movement of the apparent interface was observed. The NBA-integrated gel was also successfully employed for the controlled release of an entrapped dextran in a light controlled manner. This system is highly promising as smart platforms for triggered and programmed transportation of drugs.

Modulation of graft architectures for enhancing hydrophobic interaction of biomolecules with thermoresponsive polymer-grafted surfaces

This paper describes the effects of graft architecture of poly(N-isopropylacrylamide) (PIPAAm) brush surfaces on thermoresponsive aqueous wettability changes and the temperature-dependent hydrophobic interaction of steroids in silica capillaries (I.D.: 50 μm). PIPAAm brushes were grafted onto glass substrates by surface-initiated atom transfer radical polymerization (ATRP) that is one of the living radical polymerization techniques. Increases in the graft density and chain length of PIPAAm brushes increased the hydration of polymer brushes, resulting in the increased hydrophilic properties of the surface below the transition temperature of PIPAAm at 32 °C. More hydrophobic surface properties were also observed on surfaces modified with the block copolymers of IPAAm and n-butyl methacrylate (BMA) than that with IPAAm homopolymer-grafted surfaces over the transition temperature. Using PBMA-b-PIPAAm-grafted silica capillaries, the baseline separation of steroids was successfully achieved by only changing temperature. The incorporation of hydrophobic PBMA chains in grafted PIPAAm enhanced the hydrophobic interaction with testosterone above the transition temperature. The surface modification of hydrophobicity-enhanced thermoresponsive polymers is a promising method for the preparation of thermoresponsive biointerfaces that can effectively modulated their biomolecule and cell adsorption with the wide dynamic range of hydrophilic/hydrophobic property change across the transition temperature.