Yuta Nakamura

A protein kinase assay based on FRET between quantum dots and fluorescently-labeled peptides

A novel protein kinase assay was developed, based on FRET between QDs and fluorescently-labeled substrate peptides. The negatively charged QDs recognize the change in net charge of the peptide upon phosphorylation. Despite its simple mechanism, this assay is sensitive and robust enough to be applied to the evaluation of protein kinase inhibitors.

Stabilization of cancer-specific gene carrier via hydrophobic interaction for a clear-cut response to cancer signaling.

Here, we developed a new gene carrier, comprising a linear polyethylenimine (LPEI) grafted with a hydrophobically modified cationic peptide containing a long alkyl chain, for use in cancer-specific gene delivery. The cationic peptide is a substrate of protein kinase Cα (PKCα), which is known to be activated specifically in cancer cells. The hydrophobically modified LPEI-peptide conjugate (LPEI-C10-peptide) could form a polyplex with DNA through electrostatic and hydrophobic interactions between the anionic DNA strands and the cationic peptide substrate. The hydrophobic modification of the peptide did not affect the reactivity of the peptide toward PKCα, while the polyplex showed improved intracellular uptake. Because of the efficient endosomal escape and enhanced stability, the polyplex significantly improved the transgene regulation responding to intracellular PKCα activity.

Histidinylated poly-L-lysine-based vectors for cancer-specific gene expression via enhancing the endosomal escape.

In this work, we synthesized a series of poly-L-lysine (PLL)-based polymers for gene delivery, by modifying the PLL with both cationic peptide and histidine. The peptide moieties serve as cationic centers for polyplex formation, and also as substrates for protein kinase Cα (PKCα), which is specifically activated in many types of cancer cells, to achieve cancer-specific gene expression. The histidine groups serve as buffering moieties to increase the ability of the plasmid DNA (pDNA)-polymer complex (polyplex) to escape the endosome and thus to promote expression of the pDNA in the transfected cells. The facile synthesis of the polymers proceeded by modifying the PLL with side-group-protected peptide and protected histidine, followed by deprotection of the functional groups. The synthesized polymers showed significant buffering capacity over the neutral to acidic pH range and showed less cytotoxicity in vitro compared with histidine-unmodified polymers. The polyplexes successfully showed PKCα-responsive gene expression immediately after their introduction into cancer cells and the gene expression continued for at least 24 h. These PLL-based carriers thus show promise for cancer-targeted gene therapy.

Development of one-sided directional thin planar antenna for 5GHz wireless communication applications

In this paper, the one-sided directional slot dipole antenna with for 5 GHz-band application have been designed and experimentally verified. The antenna size is 22 mm × 29 mm, which is smaller than a patch antenna at the same frequency. The measured radiation patterns are in the good agreement with the simulation which shows the effectiveness of the presented technique to realize on-sided directional antenna. Such antennas are very advantageous than that of a patch antenna for mounting 3-dimensional packages or layer structures in miniaturized RF-front end in thinner substrate.

Design and performance of electrically small planar antennas with matching circuit at 2.4GHz band

In this paper, we designed and tested ESAs with impedance matching circuits having different gain. We succeeded in implementing the circuit which matches the small radiation resistance of ESA to amplifier. Measured results are close to the simulation value. So we proved possibility to design and fabricate ESA with high radiation efficiency.

Modification of ligands for serum albumin on polyethyleneimine to stabilize polyplexes in gene delivery

Abstract In this work, we have developed a new technique to stabilize a ternary complex composed of plasmid DNA, a linear polyethyleneimine (LPEI) and serum albumin. A stearoyl group was conjugated to LPEI as a specific ligand for serum albumin. The resultant ternary complex has excellent stability in physiological saline conditions, maintaining its initial diameter and preventing aggregation of red blood cells. The ternary complex has equivalent transfection ability to and significantly lower cytotoxicity than unmodified LPEI. Therefore, the ternary complex is potentially useful as a new gene carrier, possessing high blood stability.

Cancer-specific gene carriers responding to cancer microenvironment: Acidosis and hyper-activated protein kinases

Protein kinase (PK)-responsive gene carriers modified with polyethylene glycol (PEG) chains using an acid-labile linker were developed. These carriers were obtained by modifying the PEG chains and substrate peptides for the PKs (PKA or PKCα) on the branched polyethyleneimine main chain. Polyplexes formed from these carriers and plasmid DNA (pDNA) were stably dispersed under neutral pH medium. The polyplexes were also taken up by cells on the release of the PEG chains under the slightly acidic extracellular pH associated with cancer cells. The polyplexes taken up by cells resulted in gene expression when the substrate peptides were phosphorylated by the intracellular PKs to release pDNA from the polyplexes. These novel gene carriers are expected to be promising for cancer-specific gene therapy via intravenous administration.

Tumor accumulation of protein kinase-responsive gene carrier/DNA polyplex stabilized by alkanethiol for intravenous injection

We synthesized polymeric gene carriers consisting of poly-L-lysine (PLL) main chain modified both with substrate peptide for protein kinase Cα (PKCα) and alkanethiol (pentadecanethiol). Due to the grafted substrate peptide, the polyplex prepared from these carriers is expected to show gene expression triggered by the phosphorylation of the peptide by intracellular PKCα. The modified alkanethiol on the main chain stabilized the polyplex both via disulfide crosslinking and hydrophobic interaction. The polyplex found to show gene expression in vitro when the alkanethiol content in the main chain was enough low (4-mol%-modification of PLL’s ε-amine group) to minimize cytotoxic effect. Even though the content of alkanethiol is low, the polyplex had significant stability in a model serum solution and showed longer blood circulation in vivo. The polyplex clearly accumulated in tumor after intravenous injection.

Branched polyethylenimine-based PKCα-responsive gene carriers

We examined in vitro performance of the branched polyethylenimine (bPEI)-based gene carriers which respond to cancer-specific activation of protein kinase Cα (PKCα) to express plasmid DNA. The carriers were synthesized straightforward by using amide bond formation between a peptide terminal carboxyl and a primary amine group of bPEI. To examine the effect of the peptide contents in the carrier, we prepared several carriers with various peptide contents. The obtained polymers form polyplexes with tighter condensation of plasmid DNA than our previous gene carriers. After internalization of the polyplexes via endocytosis, the polyplexes effectively escaped from the endosome into cytosol. Then, the polyplexes showed a clear-cut response to PKCα to release plasmid DNA for gene expression. We determined the optimum contents of the peptides in carriers as 5 mol% to achieve the clear-cut response to PKCα.