Yuki Hiruta

Near IR Emitting Red-Shifting Ratiometric Fluorophores Based on Borondipyrromethene

Two distyryl-BODIPY-based NIR red-shifting ratiometric fluorescent probes are reported: KBHR-1 for pH and KBAHgR-1 for Ag(+) and Hg(2+). KBHR-1 showed a red-shifting ratiometric response to pH in the NIR region. The identical fluorophore core structure applied to KBAHgR-1 with a different recognition moiety resulted in a ratiometric response to Ag(+) and Hg(2+) in the NIR region.

Highly Durable Double Sol–Gel Layer Ratiometric Fluorescent pH Optode Based on the Combination of Two Types of Quantum Dots and Absorbing pH Indicators

Long-term stable ratiometric fluorescent optical pH sensors (optodes) based on double sol-gel silica layers are prepared with the first layer embedding two types of quantum dots (QDs) and the second layer embedding light-absorbing pH indicators. The sensors are fabricated by a simple general sol-gel spin-coating method. The resulting double-layer pH optodes are designed as having long Stokes shift as well as ratiometric fluorescence emission response to pH in aqueous solutions of varying pH values. This optode has high durability against continuous light exposure, even under severe acidic condition (1 M HCl), and the storage stability is over a period of more than 6 months. These results indicate that the double-layer ratiometric fluorescence-based pH optode allows for long-term pH sensing. When two pH indicators of different pK(a) values with an optimized mixing ratio are embedded into the second layer, a double-layer pH optode with reproducible linear response in a wide pH range of over 6 pH units (from pH 4 to 10) can be designed and fabricated.

Design of Environmentally Responsive Fluorescent Polymer Probes for Cellular Imaging

We report the development of environmentally responsive fluorescent polymers. The reversible temperature-induced phase transition of copolymers composed of N-isopropylacrylamide and a fluorescent monomer based on the fluorescein (FL), coumarin (CO), rhodamine (RH), or dansyl (DA) skeleton was used as a molecular switch to control the fluorescence intensity. The poly(N-isopropylacrylamide) (PNIPAAm) chain showed an expanded coil conformation below the lower critical solution temperature (LCST) due to hydration, but it changed to a globular form above the LCST due to dehydration. Through the combination of a polarity-sensitive fluorophore with PNIPAAm, the synthetic fluorescent polymer displayed a response to external temperature, with the fluorescence strength dramatically changing close to the LCST. Additionally, the P(NIPAAm-co-FL) and P(NIPAAm-co-CO) polymers, containing fluorescein and coumarin groups, respectively, exhibited pH responsiveness. The environmental responsiveness of the reported polymers is derived directly from the PNIPAAm and fluorophore structures, thus allowing for the cellular uptake of the fluorescence copolymer by RAW264.7 cells to be temperature-controlled. Cellular uptake was suppressed below the LCST but enhanced above the LCST. Furthermore, the cellular uptake of both P(NIPAAm-co-CO) and P(NIPAAm-co-RH) conjugated with a fusogenic lipid, namely, l-α-phosphatidylethanolamine, dioleoyl (DOPE), was enhanced. Such lipid-conjugated fluorescence probes are expected to be useful as physiological indicators for intracellular imaging.

The effects of anionic electrolytes and human serum albumin on the LCST of poly(N-isopropylacrylamide)-based temperature-responsive copolymers

Poly(N-isopropylacrylamide) (PNIPAm) is one of the most widely studied temperature-responsive polymers among those that have been applied to biomaterials science and technology. Here, we investigated the importance of interactions between PNIPAm-based copolymers and biological factors. The effects of a series of major anionic electrolytes in biological environments and of human serum albumin (HSA) on the lower critical solution temperature (LCST) of homo-PNIPAm and PNIPAm copolymers were studied, using either a hydrophobic monomer or a cationic monomer. We synthesized P(NIPAm-co-BMA3%) with butyl methacrylate (BMA) as a hydrophobic monomer and P(NIPAm-co-DMAPAm2%) with N,N-dimethylaminopropyl acrylamide (DMAPAm) as a cationic monomer. The LCST of PNIPAm and P(NIPAm-co-DMAPAm2%) decreased with increasing salt concentrations, and the effects of anions on each polymer corresponded to the Hofmeister series. The LCST of P(NIPAm-co-DMAPAm2%) was greatly affected by anionic electrolytes compared with those of homo-PNIPAm and P(NIPAm-co-BMA3%). While the LCST of homo-PNIPAm was not affected by HSA, the LCST of P(NIPAm-co-DMAPAm2%) decreased non-linearly with increasing HSA concentrations. These effects were due to the electrostatic interactions between the positively charged polymer chains and the negatively charged HSA, as well as the stabilization of polymer aggregations with HSA. Under physiological buffer conditions, the LCST of P(NIPAm-co-DMAPAm2%) was not significantly affected by the HSA concentration. These results indicated that depending on the types of copolymers used for biological applications, it is necessary to take into account the effect of biological media while designing polymers.

Temperature-responsive molecular recognition chromatography using phenylalanine and tryptophan derived polymer modified silica beads

Temperature-responsive polymers incorporating molecular-recognition sites were developed as stationary phases for high-performance liquid chromatography (HPLC). The grafted stationary phases consisted of functional copolymers composed of N-isopropylacrylamide (NIPAAm) and N-acryloyl aromatic amino acid methyl esters, i.e., phenylalanine and tryptophan methyl esters (Phe-OMe and Trp-OMe). Three novel temperature-responsive polymers, P(NIPAAm-co-Phe-OMe5), P(NIPAAm-co-Phe-OMe10), and P(NIPAAm-co-Trp-OMe5), were synthesized. These copolymers exhibited a reversible hydrophilic/hydrophobic phase transition at their lower critical solution temperatures (LCSTs). The polymers were grafted onto aminopropyl silica using an activated ester-amine coupling method, and were packed into a stainless steel column, which was connected to an HPLC system. Temperature-responsive chromatography was conducted using water as the sole mobile phase. More hydrophobic analytes were retained longer, and the retention times of aromatic steroids and aromatic amino acids were dramatically increased. This indicated that π-π interactions occurred between the phenyl or indole moieties of phenylalanine or tryptophan, respectively, and the aromatic compounds. Furthermore, the retention times of compounds with hydrogen bond acceptors were higher with P(NIPAAm-co-Trp-OMe5), which contained indole as a hydrogen bond donor, than with P(NIPAAm-co-Phe-OMe5). This indicated that hydrogen bonding occurred between the stationary phase and the analytes. These results indicate that hydrophobic, π-π, and hydrogen bonding interactions all affected the separation mode of the temperature-responsive chromatography, and led to selective separation with molecular recognition. Both temperature-response and molecular recognition characteristics are present in the proposed separation system that utilizes a temperature-responsive polymer bearing aromatic amino acid derivatives.

Temperature-responsive Solid-phase Extraction Column for Biological Sample Pretreatment

We have developed a novel solid-phase extraction (SPE) system utilizing a temperature-responsive polymer hydrogel-modified stationary phase. Aminopropyl silica beads (average diameter, 40 - 64 μm) were coated with poly(N-isopropylacrylamide) (PNIPAAm)-based thermo-responsive hydrogels. Butyl methacrylate (BMA) and N,N-dimethylaminopropyl acrylamide (DMAPAAm) were used as the hydrophobic and cationic monomers, respectively, and copolymerized with NIPAAm. To evaluate the use of this SPE cartridge for the analysis of drugs and proteins in biological fluids, we studied the separation of phenytoin and theophylline from human serum albumin (HSA) as a model system. The retention of the analytes in an exclusively aqueous eluent could be modulated by changing the temperature and salt content. These results indicated that this temperature-responsive SPE system can be applied to the pretreatment of biological samples for the measurement of serum drug levels.

A Fluorous Biphasic Solvent Extraction System for Lanthanides with a Fluorophilic β-Diketone Type Extractant

The properties of a fluorous solvent extraction system for trivalent lanthanide metal ions are reported. A fluorinated extractant, 4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluoro-1-(2-thienyl)-1,3-nonanedione, and HFE-7200 (C4F9OC2H5) as the extraction solvent were chosen. With this fluorous extractant/solvent combination, higher extraction ratios and separation factors compared to a conventional organic solvent system (thenoyltrifluoroacetone in CHCl3) were achieved for 5 heavy lanthanide ions (Lu, Yb, Tm, Er and Ho). On the other hand, light lanthanide ions (Nd, Pr, Ce and La) are hardly extracted, therefore enabling the mutual separation of light lanthanides from middle or heavy lanthanide ions.

Effects of terminal group and chain length on temperature-responsive chromatography utilizing poly(N-isopropylacrylamide) synthesized via RAFT polymerization

Poly(N-isopropylacrylamide) with two chain lengths (MW: ca. 5000, and ca. 20000 g mol−1) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, using 2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid as the chain transfer agent. The derivatization of terminal dodecyl groups to maleimide groups was performed through the reduction of terminal trithiocarbonate moieties. These polymers were grafted onto aminopropyl silica using an activated ester–amine coupling method. Five types of polymer modified silica beads were prepared, i.e., long chain length, short chain length, and mixed chain length functionalized with a dodecyl terminal group, and long chain length and short chain length with a maleimide terminal group. The surface properties of the modified polymer silica beads were evaluated by employing them as the stationary phase in HPLC setups and examining the temperature-dependent elution profiles of five steroids. The retention factor of steroids became smaller when the terminal dodecyl group was substituted with a maleimide group. A hydrophobic dodecyl moiety on the outermost surface strongly affected the retention of steroids, and the retention factor of steroids on short chains was larger than that on long chains below the lower critical solution temperature. These results indicate that the chain length and terminal functional group on the outermost surface of the polymer have a critical effect on the characteristics of polymer modified silica bead interfaces.

Dual temperature- and pH-responsive polymeric micelle for selective and efficient two-step doxorubicin delivery

We report a polymeric micelle drug delivery system, which enables selective intracellular uptake with external thermal stimulation, and effective release of a drug at internal acidic endosomal pH. We developed a dual temperature- and pH-responsive polymeric micelle composed of a temperature-responsive corona segment with poly(N-isopropylacrylamide-co-dimethylacrylamide) [P(NIPAAm-co-DMAAm)] and a pH-responsive core segment with poly[2-(diisopropylamino)ethyl methacrylate] (PDPA). A dual temperature- and pH-responsive amphiphilic diblock copolymer was synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization. The dithiobenzoate end-group was removed with radical-induced ester exchange. This copolymer formed nano-sized micelles in aqueous solution, and encapsulated anti-cancer agent, doxorubicin (DOX). The resultant micelles exhibited a temperature-dependent phase transition at a temperature slightly higher than body temperature, and intracellular uptake of encapsulated DOX was accelerated above the phase transition temperature. A transition from neutral to positive charge, leading to micelle disassembly, accelerated the release of Nile red as a model drug. The cytotoxicity of doxorubicin (DOX)-loaded dual temperature- and pH-responsive micelles against human cervical cancer HeLa cells was significantly greater at 42 °C than at 37 °C, while no significant temperature-dependent cytotoxicity was observed with DOX-loaded micelles that were only temperature-responsive. This proof-of-concept synergistic two-step delivery system with enhanced intracellular uptake upon external thermal stimulation and rapid release of DOX at internal acidic endosomal pH was effective against tumor cells in vitro.

Intracellular localization and delivery of plasmid DNA by biodegradable microsphere-mediated femtosecond laser optoporation

Micro-/nanosphere-mediated femtosecond laser cell perforation is one of the high throughput technologies used for macro-molecule-delivery into multiple cells. We have demonstrated the delivery of plasmid-DNA/liposome complexes into cells using biodegradable polymer microspheres and a femtosecond laser and investigated the intracellular localization of the complexes by delivering fluorescence-labeled plasmid-DNA/liposome complexes into cells. The utilization of liposomes increases the number of complexes delivered into the cytoplasm by laser illumination, which contributed to the increased transfection rate. In the experiment involving polystyrene (PS) microspheres of different diameters, the fluorescence of the complexes was detected in the nucleus as well as cytoplasm after laser illumination for PS microspheres of 3.0 μm diameter. The direct delivery of complexes into the nucleus is probably attributed to the enhancement of the nuclear membrane permeability by the enhanced optical field obtained close to the nucleus. These revelations on the intracellular localization of foreign DNA would provide effective laser-based transfection. Picture: Intranuclear delivery of plasmid-DNA/liposome complexes by utilizing dielectric microspheres and a femtosecond laser.