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Controlled release of oxygen from myoglobin was achieved by modulating autoxidation of oxymyoglobin using ascorbic acid as a reductant by temperature variation. Long-term storage, prompt release and re-storage of oxygen were also available with this system. Furthermore, 20 nm thick nanofilms composed of oxymyoglobin and type I collagen containing ascorbic acid could successfully show autoxidation of oxymyoglobin in response to environmental temperature. The ultrathin nanofilms will be useful as oxygen-controlled releasable scaffolds for tissue engineering application.

Liquid-ordered (Lo)-phase domains, a cholesterol-rich area on lipid bilayers, have attracted significant attention recently because of their relevance to lipid rafts, the formation/collapse of which is associated with various kinds of information exchange through the plasma membrane. Here, we demonstrate that the formation/collapse of Lo-phase domains in cell-sized liposomes, that is, giant unilamellar vesicles (GUVs), can be controlled with bioactive plasmonic nanoparticles and light. The nanoparticles were prepared by surface modification of gold nanorods (AuNRs) using a cationized mutant of high-density lipoprotein (HDL), which is a natural cholesterol transporter. Upon the addition of surface-engineered AuNRs to GUVs with the mixed domains of Lo and liquid-disorder (Ld) phases, the Lo domains collapsed and solid-ordered (So)-phase domains were formed. The reverse phase transition was achieved photothermally, with the AuNRs loaded with cholesterol. During these transitions, the AuNRs appeared to be selectively localized on the less fluidic domain (Lo or So) in the phase-mixed GUVs. These results indicate that the phase transitions occur through the membrane binding of the AuNRs followed by spontaneous/photothermal transfer of cholesterol between the AuNRs and GUVs. Our strategy to develop bioactive AuNRs potentially enables spatiotemporal control of the formation/collapse of lipid rafts in living cells.

Drug delivery systems (DDS) have been studied in an effort to reduce side effects by increasing the accumulation of anticancer drugs in cancer cells. However, the transport efficiency is still low due to the blocking by surrounding stromal tissues and the multiple intracellular drug transportation processes required to get the drug to a target cytosol. Thus, improving the efficiency of cancer therapy is still a major challenge. Here, a drug-free cancer microenvironment-targeting therapy using molecular blocks (MBs) is demonstrated, which is designed for efficient blood circulation and penetration through the stromal tissues as either a single molecule or a few molecules. When the MBs moved to a cancer microenvironment by the enhanced permeability and retention effect, they formed a self-assembled aggregate on the cancer cell surfaces in response to the weak acid (pH ∼ 6.5) condition leading to subsequent cancer cell death by membrane disruption. This strategy avoids multiple intracellular transportation processes and also stimulates cell membrane disruption by self-assembly of the MB VIA hydrophobic interactions. Deoxycholic acid (DCA) was selected as a cancer microenvironment-responsive unit because its pKa = 6.6. The DCA conjugated 4-arm poly(ethylene glycol) (4-MB) showed self-assembly phenomena on cancer cell membranes and subsequently significant cytotoxicity was clearly observed. Moreover, they clearly showed efficient accumulation in the tumor and the effective suppression of tumor growth in in vivo experiments. This MB therapy will be a new strategy for addressing the current issues of DDS.

In vitro construction of highly organized three-dimentional (3D) tissues is still a key challenge for tissue engineering. In this study, we fabricated multilayered tissues composed of extracellular matrix (ECM) layer and cell layer by stacking cell-seeded ECM papers. A paperlike scaffold was prepared by simply casting dispersion of microfibered ECM. We showed the paperlike scaffold is a superior material for constructing 3D tissue because of its high permeability and cell migration ability, and our method can control the thickness and component of ECM in multilayered 3D tissues. It can contribute to construction of normal and disease tissue models.

Construction of organized three-dimensional (3D) tissue with extracellular matrix (ECM) and multiple types of cells is important for tissue engineering to enable tissue function and enhance cellular function. However, the concentration of ECM and the thickness of the 3D tissue have been limited in previous methods due to a lack of permeability to nutrients and oxygen. Besides, it is difficult to use matured natural ECM as a cell scaffold without chemical modification due to its insolubility. In this article, we focus on multi-layered structure, which is commonly found in living tissue such as skin, blood vessels, and other organs. Here, we describe the preparation of a paper-like scaffold (ECM paper) from micro-fibered natural ECM and the construction of 3D multi-layered tissue composed of cell layers and ECM layers by stacking cell-seeded ECM papers. The thickness and components of the ECM layers are easily controllable by changing the composition of the ECM papers, and the fibrous structure of ECM paper shows high permeability and permits cell migration. Additionally, the ECM microfiber, which is physically defiberized from natural ECM, has a high ECM concentration equal to that of living tissue and high stability under physiological conditions. Therefore, this set of protocols enables construction of multi-layered 3D tissue composed of precisely controlled ECM layers and cell layers that may contribute to the assembly of tissue models. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of extracellular matrix paper Basic Protocol 2: Evaluation of cellular function of cells on extracellular matrix paper Basic Protocol 3: Construction of multi-layered 3D tissue.

Success of tumor photothermal immunotherapy requires a system that induces heat stress in cancer cells and enhances strong anti-tumor immune responses. Here, we designed a composite-type immunostimulatory DNA hydrogel consisting of a hexapod-like structured DNA (hexapodna) with CpG sequences and gold nanoparticles. Mixing of the properly designed hexapodna and oligodeoxynucleotide-modified gold nanoparticles resulted in the formation of composite-type gold nanoparticle-DNA hydrogels. Laser irradiation of the hydrogel resulted in the release of hexapodna, which efficiently stimulated immune cells to release proinflammatory cytokines. Then, EG7-OVA tumor-bearing mice received an intratumoral injection of a gold nanoparticle-DNA hydrogel, followed by laser irradiation at 780 nm. This treatment increased the local temperature and the mRNA expression of heat shock protein 70 in the tumor tissue, increased tumor-associated antigen-specific IgG levels in the serum, and induced tumor-associated antigen-specific interferon-γ production from splenocytes. Moreover, the treatment significantly retarded the tumor growth and extended the survival of the tumor-bearing mice.

Two features of meso-Aryl-substituted expanded porphyrins suggest suitability as theranostic agents. They have excellent absorption in near infrared (NIR) region, and they offer the possibility of introduction of multiple fluorine atoms at structurally equivalent positions. Here, hexaphyrin (hexa) was synthesized from 2,6-bis(trifluoromethyl)-4-formyl benzoate and pyrrole and evaluated as a novel expanded porphyrin with the above features. Under NIR illumination hexa showed intense photothermal and weak photodynamic effects, which were most likely due to its low excited states, close to singlet oxygen. The sustained photothermal effect caused ablation of cancer cells more effectively than the photodynamic effect of indocyanine green (a clinical dye). In addition, hexa showed potential for use in the visualization of tumors by 19 F magnetic resonance imaging (MRI), because of the multiple fluorine atoms. Our results strongly support the utility of expanded porphyrins as theranostic agents in both photothermal therapy and 19 F MRI.

Controlling cell functions using external photoresponsive nanomaterials has enormous potential for the development of cell-engineering technologies and intractable disease therapies, but the former currently requires genetic modification of the target cells. We present a method using plasma-membrane-targeted gold nanorods (pm-AuNRs) prepared with a cationic protein/lipid complex to activate a thermosensitive cation channel, TRPV1, in intact neuronal cells. Highly localized photothermal heat generation mediated by the pm-AuNRs induced Ca(2+) influx solely by TRPV1 activation. In contrast, the use of previously reported cationic AuNRs that are coated with a conventional synthetic polymer also led to photoinduced Ca(2+) influx, but this influx resulted from membrane damage. Our method provides an optogenetic platform without the need for prior genetic engineering of the target cells and might be useful for novel TRPV1-targeted phototherapeutic approaches., 光を使って神経細胞の「痛み」感知を制御する手法を開発 -新しい鎮痛療法の可能性-. 京都大学プレスリリース. 2015-08-14.

A hydrophobic gadolinium bis(naphthalocyanine) sandwich complex (GdSand) possessing several absorbances across visible and infrared wavelengths (up to 2500 nm) was solubilized in aqueous solution by uptake into a nascent mutant high-density lipoprotein (HDL) nanocarrier. The HDL nanocarrier was additionally functionalized with a trans-activator of transcription peptide sequence to promote efficient cell penetration of the drug delivery system (cpHDL). The dye-loaded nanocarrier (GdSand@cpHDL) exhibited photothermal heat generation properties upon irradiation with near-infrared (NIR) laser light, with controllable heat generation abilities as a function of the incident laser light power. Comparison of the photothermal behavior of the dyes GdSand and the well-explored molecular photothermal agent indocyanine green (ICG) in the cpHDL nanocarrier (i.e., ICG@cpHDL) revealed two significant advantages of GdSand@cpHDL: (1) the ability to maintain elevated temperatures upon light absorption for extended periods of time, with a reduced degree of self-destruction of the dye, and (2) exclusive photothermal heat generation with no detectable singlet oxygen production leading to improved integrity of the cpHDL nanocarrier after irradiation. Finally, GdSand@cpHDL was successfully subjected to an in vitro study against NCI-H460 human lung cancer cells, demonstrating the proof-of-principle utility of lanthanide sandwich complexes in photothermal therapeutic applications.

Two features of meso-Aryl-substituted expanded porphyrins suggest suitability as theranostic agents. They have excellent absorption in near infrared (NIR) region, and they offer the possibility of introduction of multiple fluorine atoms at structurally equivalent positions. Here, hexaphyrin (hexa) was synthesized from 2,6-bis(trifluoromethyl)-4-formyl benzoate and pyrrole and evaluated as a novel expanded porphyrin with the above features. Under NIR illumination hexa showed intense photothermal and weak photodynamic effects, which were most likely due to its low excited states, close to singlet oxygen. The sustained photothermal effect caused ablation of cancer cells more effectively than the photodynamic effect of indocyanine green (a clinical dye). In addition, hexa showed potential for use in the visualization of tumors by

Semiconducting and metallic single-walled carbon nanotubes (s-SWNTs and m-SWNTs) were enriched by agarose gel chromatography and their photothermal and photodynamic effects were compared in H(2)O. Under near-infrared laser irradiation, s-SWNTs generated reactive oxygen species (ROS) more than m-SWNTs, whereas m-SWNTs produced heat more efficiently than s-SWNTs. More importantly, cancer cell killing by PDE of s-SWNTs has been disclosed for the first time.

Surface engineering of mesoscopic metal nanoparticles to increase biocompatibility and cell interaction is important for improvement of their therapeutic properties. Here, we describe a strategy to stabilize mesoscopic metal nanoparticles and to enhance their cell interaction by stepwise addition of (Z)-9-octadecenoate (oleate) and a cell-penetrating peptide-fused high-density lipoprotein (cpHDL). Oleate replaces a cytotoxic dispersant on the surface of gold nanorods (AuNRs), which enables subsequent cpHDL binding without causing aggregation. Notably, these two lipidic dispersants are probably intercalated on the surface. This procedure was also used to stabilize 20 nm spherical gold nanoparticles and 40 nm aggregates of 10 nm magnetite nanoparticles. cpHDL-bound AuNRs were internalized greater than 80 times more efficiently than poly(ethylene glycol)-conjugated AuNRs and were able to elicit cancer cell photoablation.

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The investigation was carried out in order to elucidate the reason for the reduction in nutritive value of browned protein, by using labeled casein as a model protein. Goat casein preparation in which lysine residues had been labeled with 14C was browned by amino-carbonyl reaction with glucose at 37°C. Browned or non-browned casein was ingested by growing rats by spaced feeding. When the rats ingested the browned casein the experimental group, higher radioactivity was found in TCA-soluble fraction in the small intestine as compared with that in the control group, while radioactivity was scarecely found in feces for 22 hr. Along with absorption delay, considerably high radioactivity was found in urine. The recovery of radioactivity in expired air of rats fed the labeled casein (browned and non-browned) was measured. In the experimental group, expired 14CO2 came out slower than the control group. From these results, it is suggested that the main reason for the reduction in nutritive value by browning reaction may be the formation of a lysine derivative in a protein, which remains in the small intestinal lumen as an absorption-delayed material and is finally excreted in urine.

The investigation was carried out in order to elucidate the reason for the reduction in nutritive value of browned protein, by using labeled casein as a model protein. Goat casein preparation in which lysine residues had been labeled with 14C was browned by amino-carbonyl reaction with glucose at 37°C. Browned or non-browned casein was ingested by growing rats by spaced feeding.When the rats ingested the browned casein the experimental group, higher radioactivity was found in TCA-soluble fraction in the small intestine as compared with that in the control group, while radioactivity was scarecely found in feces for 22 hr. Along with absorption delay, considerably high radioactivity was found in urine. The recovery of radioactivity in expired air of rats fed the labeled casein (browned and non-browned) was measured. In the experimental group, expired 14CO2 came out slower than the control group. From these results, it is suggested that the main reason for the reduction in nutritive value by browning reactio...