Takahito Kawano

PNIPAM Gel-Coated Gold Nanorods for Targeted Delivery Responding to a Near-Infrared Laser

Gold nanorods can be used as photothermal converters, permitting near-infrared (NIR) light to be transmitted deep into tissues without causing damage. We prepared hybrid nanorods with a core-shell structure, i.e., a single gold nanorod encapsulated in a poly (N-isopropylacrylamide) nanogel. Hybrid nanorods demonstrated remote, reversible, pulsatile phase transition and in vivo action after irradiation using a NIR laser.

Poly(ethylene glycol)-Modified Gold Nanorods as a Photothermal Nanodevice for Hyperthermia

Gold nanorods, which have a strong surface plasmon band at the near-infrared region, absorb light energy which is then converted to heat. Since near-infrared light can penetrate deeply into tissue, gold nanorods are expected to be useful as photosensitizers for photothermal therapy. In this study, the length of the poly(ethylene glycol) (PEG) chain was optimized in order to stabilize the gold nanorods in the blood circulation after intravenous injection. PEG5000- and PEG10000-modified gold nanorods showed higher stability in the blood circulation compared with PEG2000- and PEG20000-modified gold nanorods. As a demonstration of photothermal tissue damage, PEG5000-modified gold nanorods were injected into the muscle in the hind limbs of a mouse, and then irradiated with near-infrared pulsed laser light. Significant tissue damage was observed only in the presence of gold nanorods and laser irradiation. We next injected the gold nanorods directly into subcutaneous tumors in mice, and then irradiated the tumor with near-infrared pulsed laser light. Significant suppression of tumor growth was observed. In the case of the intravenous injection of gold nanorods, the suppression of tumor growth was weaker than for the case of direct injection, indicating that the targeted delivery of gold nanorods to the tumor tissue is an important key to improve the therapeutic effect.

Elasticity boundary conditions required for cell mechanotaxis on microelastically-patterned gels

Directional cell migration induced by a mechanical gradient on a substrate surface toward a harder region, so-called mechanotaxis or durotaxis, has recently drawn attention not only in the field of mechanobiology but also for possible cell manipulation in biomedical engineering. Before we can use mechanotaxis to control cell migration on a biomaterial surface, quantitative design criteria for a microelasticity gradient should be established. To clarify the conditions required to induce mechanotaxis, the effects of a microelasticity boundary on cell culture hydrogels have been systematically assessed with regard to fibroblast migration based on a custom-built reduction projection-type photolithographic microelasticity patterning system with elasticity-tunable photocurable styrenated gelatins, which is a thoroughly-improved system of our previous simple photomasking method [41]. As a result, the conditions required to induce mechanotaxis were found to include a certain threshold jump in elasticity (30-40 kPa) and a sufficiently narrow width of the elasticity boundary (50 μm) comparable to a single cells adhered area, i.e., a sufficiently high gradient strength (30-40 kPa/50 μm in our gelatinous gel system). A significant asymmetric distribution of the number and size of focal adhesions across the elasticity boundary was confirmed to be one of the driving factors of mechanotaxis by indirect immunofluorescence microscopy, and mechanistic considerations in the design criteria are discussed.

In vivo monitoring of intravenously injected gold nanorods using near-infrared light

Gold nanorods showing surface plasmon (SP) bands in the near-IR region are used as bioimaging probes that respond to near-IR light in mice. The SP bands of intravenously injected polyethylene glycol-modified gold nanorods are directly monitored from the mouse abdomen by using a spectrophotometer equipped with an integrating sphere. The absorbance at 900 nm from the gold nanorods immediately increases after injection and reaches a plateau. The injection of phosphatidylcholine-modified gold nanorods also increases the absorbance at 900 nm, but the absorbance decreases single exponentially with a 1.3-min half-life. In vivo spectral changes of gold nanorods depend on the surface characteristics, and can be observed in real time using simple spectroscopic measurements.

Active accumulation of gold nanorods in tumor in response to near-infrared laser irradiation

Gold nanorods, rod-shaped gold nanoparticles, have strong absorbance in the near-infrared region, and the absorbed light energy can be converted to heat, the so-called photothermal effect. The gold nanorods were coated with thermoresponsive polymers, which have different phase transition temperatures that were controlled by adding comonomers, N,N-dimethylacrylamide (DMAA) or acrylamide (AAm) to N-isopropylacrylamide (NIPAM). The phase transition temperatures of poly(NIPAM-DMAA) and poly(NIPAM-AAm)-coated gold nanorods were 38 and 41 °C, respectively, while polyNIPAM-coated gold nanorods showed phase transition at 34 °C. Irradiation of the coated gold nanorods using the near-infrared laser induced a decrease in their sizes due to a phase transition of the polymer layers. Poly(NIPAM-AAm)-coated gold nanorods stably circulated in the blood flow without a phase transition after intravenous injection. Irradiation of near-infrared light at a tumor after the injection resulted in the gold specifically accumulating in the tumor. This novel accumulation technique which combines a thermoresponsive polymer and the photothermal effect of the gold nanorods should be a powerful tool for targeted delivery in response to light irradiation.

Fabrication of mussel-inspired highly adhesive honeycomb films containing catechol groups and their applications for substrate-independent porous templates.

Porous surface patterns are used in a wide variety of practical applications. Honeycomb-patterned porous polymer films are good templates for preparing porous surfaces due to their simple fabrication and the arrangement of pores on the surface. Catechol groups include in adhesive protein of mussels have attracted much attention due to their highly and substrate-independent adhesive properties. In this paper, highly and substrate-independent adhesive honeycomb-patterned porous polymer films are prepared by using amphiphilic copolymer having catechol moieties. Furthermore, porous surface patterns are transferred on various organic or inorganic substrates by wet etching with using adhesive honeycomb films as templates.

Basic fibroblast growth factor-treated adipose tissue-derived mesenchymal stem cell infusion to ameliorate liver cirrhosis via paracrine hepatocyte growth factor

Recent studies show that adipose tissue‐derived mesenchymal stem cells have potential clinical applications. However, the mechanism has not been fully elucidated yet. Here, we investigated the effect of basic fibroblast growth factor‐treated adipose tissue‐derived mesenchymal stem cells infusion on a liver fibrosis rat model and elucidated the underlying mechanism.

Morphology and adhesion strength of myoblast cells on photocurable gelatin under native and non-native micromechanical environments.

We have quantitatively determined how the morphology and adhesion strength of myoblast cells can be regulated by photocurable gelatin gels, whose mechanical properties can be fine-tuned by a factor of 10(3) (0.1 kPa ≤ E ≤ 140 kPa). The use of such gels allows for the investigation of mechanosensing of cells not only near the natural mechanical microenvironments (E ~ 10 kPa) but also far below and beyond of the natural condition. Optical microscopy and statistical image analysis revealed that myoblast cells sensitively adopt their morphology in response to the substrate elasticity at E ~ 1-20 kPa, which can be characterized by the significant changes in the contact area and order parameters of actin cytoskeletons. In contrast, the cells in contact with the gels with lower elastic moduli remained almost round, and the increase in the elasticity beyond E ~ 20 kPa caused no distinct change in morphology. In addition to the morphological analysis, the adhesion strength was quantitatively evaluated by measuring the critical detachment pressure with an aid of intensive pressure waves induced by picosecond laser pulses. This noninvasive technique utilizing extremely short pressure waves (pulse time width ~100 ns) enables one to determine the critical pressure for cell detachment with reliable statistics while minimizing the artifacts arising from the inelastic deformation of cells. The adhesion strength also exhibited a transition from weak adhesion to strong adhesion within the same elasticity range (E ~ 1-20 kPa). A clear correlation between the cell morphology and adhesion strength suggests the coupling of the strain of the substrate and the mechanosensors near focal adhesion sites.

Design and function of engineered protein nanocages as a drug delivery system for targeting pancreatic cancer cells via neuropilin-1

We describe the development of neuropilin 1-binding peptide (iRGD)-nanocages that specifically target human pancreatic cancer cells in which an iRGD is joined to the surface of naturally occurring heat shock protein (HSP) cages. Using a genetic engineering approach, the iRGD domain was joined to the C-terminal region of the HSP cage using flexible linker moieties. The characteristics of the interdomain linkages between the nanocage and the iRGD domain play an important role in the specificity and affinity of the iRGD-nanocages for their target cells. An engineered L30-iRGD-nanocage with 30 amino acid linkers, (GGS)10, showed greater binding affinity for pancreatic cancer cells relative to that of other linkers. Furthermore, a moderately hydrophobic anticancer drug, OSU03012, was successfully incorporated into the L30-iRGD-nanocage by heating the mixture. The OSU03012-loaded L30-iRGD-nanocage induced cell death of pancreatic cancer cells by activating the caspase cascade more effectively than the same concentrations of free OSU03012. The iRGD-nanocages show great potential as a novel nanocarrier for pancreatic cancer-targeted drug delivery.

Rigidity Matching between Cells and the Extracellular Matrix Leads to the Stabilization of Cardiac Conduction

Biomechanical dynamic interactions between cells and the extracellular environment dynamically regulate physiological tissue behavior in living organisms, such as that seen in tissue maintenance and remodeling. In this study, the substrate-induced modulation of synchronized beating in cultured cardiomyocyte tissue was systematically characterized on elasticity-tunable substrates to elucidate the effect of biomechanical coupling. We found that myocardial conduction is significantly promoted when the rigidity of the cell culture environment matches that of the cardiac cells (4 kiloPascals). The stability of spontaneous target wave activity and calcium transient alternans in high frequency-paced tissue were both enhanced when the cell substrate and cell tissue showed the same rigidity. By adapting a simple theoretical model, we reproduced the experimental trend on the rigidity matching for the synchronized excitation. We conclude that rigidity matching in cell-to-substrate interactions critically improves cardiomyocyte-tissue synchronization, suggesting that mechanical coupling plays an essential role in the dynamic activity of the beating heart.