ATP2B4 is an essential gene for epidermal growth factor-induced macropinocytosis in A431 cells.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.

Involvement of protein disulfide isomerase in subtilase cytotoxin-induced cell death in HeLa cells.

Subtilase cytotoxin (SubAB) is a member of bacterial AB5 toxin produced by certain enterohemorrhagic E. coli strains which cleaves host chaperone BiP in endoplasmic reticulum (ER), leading to ER stress-mediated cytotoxicity. Previous study suggested that protein disulfide isomerase (PDI), an enzyme which catalyzes the formation and breakage of disulfide bonds in proteins, regulates AB5 toxin such as cholera toxin by unfolding of A subunit, leading to its translocation into cytosol to induce disease. Although SubAB targets ER and has similar A subunit to that of other AB5 toxins, it is unclear whether PDI can modulate the SubAB function. Here we determined the role of PDI on SubAB-induced BiP cleavage, ER stress response and cytotoxicity in HeLa cells. We found that PDI knockdown significantly suppressed SubAB-induced BiP cleavage and eIF2α phosphorylation. The accumulation of SubAB in ER was perturbed upon PDI knockdown. Finally, cell viability assay showed that PDI knockdown and PDI inhibitor canceled the SubAB-induced cytotoxicity. Present results suggested that SubAB, after cellular uptake, translocates into ER and interacts with BiP that might be modulated by PDI. Identification of pivotal role of host proteins on bacterial toxin to elicit its pathogenesis is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of toxin-producing bacterial infections.

Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages.

Poly(D,L-lactide-co-glycolic) acid (PLGA) is a synthetic copolymer that has been used to design micro/nanoparticles as a carrier for macromolecules, such as protein and nucleic acids, that can be internalized by the endocytosis pathway. However, it is difficult to control the intracellular delivery to target organelles. Here we report an intracellular delivery system of nanoparticles modified with bacterial cytotoxins to the endoplasmic reticulum (ER) and anti-inflammatory activity of the nanoparticles. Subtilase cytotoxin (SubAB) is a bacterial toxin in certain enterohemorrhagic Escherichia coli (EHEC) strains that cleaves the host ER chaperone BiP and suppresses nuclear factor-kappaB (NF-κB) activation and nitric oxide (NO) generation in macrophages at sub-lethal concentration. PLGA-nanoparticles were modified with oligo histidine-tagged (6 × His-tagged) recombinant SubAB (SubAB-PLGA) through a pH-sensitive linkage, and their translocation to the ER in macrophage cell line J774.1 cells, effects on inducible NO synthase (iNOS), and levels of tumor necrosis factor (TNF)-α cytokine induced by lipopolysaccharide (LPS) were examined. Compared with free SubAB, SubAB-PLGA was significantly effective in BiP cleavage and the induction of the ER stress marker C/EBP homologous protein (CHOP) in J774.1 cells. Furthermore, SubAB-PLGA attenuated LPS-stimulated induction of iNOS and TNF-α. Our findings provide useful information for protein delivery to macrophages and may encourage therapeutic applications of nanoparticles to the treatment of inflammatory diseases.

Spatially-targeted laser fabrication of multi-metal microstructures inside a hydrogel.

The spatially-targeted fabrication of bimetallic microstructures coexisting in the supporting hydrogel is demonstrated by multi-photon photoreduction. Microstructures composed of gold and silver were fabricated along a predefined trajectory by taking advantages of the hydrogel's ionic permeability. Different resonant wavelengths of optical absorption were obtained for gold, silver, and their bimetallic structures. Transmission electron microscopy and energy dispersive X-ray analysis revealed that the optical properties are attributable to the formation of bimetallic structure consisted of core-shell nanoparticles. The fabrication of dissimilar metal structures within hydrogel is a promising technique for optically driven actuators in soft robotics and sensing applications by allowing for site-selective optical properties.

Accumulation of gold nano-rods in the failing heart of transgenic mice with the cardiac-specific expression of TNF-α.

Gold nano-rods, rod-shaped gold nanoparticles, act as contrast agents for in vivo bioimaging, drug delivery vehicles and thermal converters for photothermal therapy. Pro-inflammatory cytokines play critical roles in the development of heart failure. We examined the delivery of GNRs into the failing heart of a transgenic (TG) mouse model of inflammatory cardiomyopathy with the cardiac-specific overexpression of TNF-α. We modified GNRs with polyethylene glycol (PEG) to avoid cytotoxicity and reduce the rapid clearance of nanoparticles from blood. PEG-modified GNRs (4.5 mM as gold atoms, 200 µL) were administered intravenously to TG (n = 7) and wild-type (WT) mice (n = 5). These were killed 24 h later, and the heart, lung, liver, kidney and spleen were excised. A quantitative analysis of gold was performed using inductively coupled plasma mass or optical emission spectrometry. The amount of gold (ng) in the TG heart (3.24 ± 1.56 ng/mg heart weight) was significantly greater than that in the WT heart (1.01 ± 0.19; p < 0.05). No significant differences were observed among the other organs of TG and WT mice. The amount of gold in the TG heart was significantly and positively correlated with the ratio of the ventricular weight to body weight, which is known to be an index of ventricular hypertrophy. In conclusion, PEG-modified GNRs accumulated in the inflammatory TG heart in proportion with the severity of ventricular hypertrophy.

Glycol Chitosan-Docosahexaenoic Acid Liposomes for Drug Delivery: Synergistic Effect of Doxorubicin-Rapamycin in Drug-Resistant Breast Cancer.

Marine ecosystems are the most prevalent ecosystems on the planet, providing a diversity of living organisms and resources. The development of nanotechnology may provide solutions for utilizing these thousands of potential compounds as marine pharmaceuticals. Here, we designed a liposomal glycol chitosan formulation to load both doxorubicin (DOX) and rapamycin (RAPA), and then evaluated its therapeutic potential in a prepared drug-resistant cell model. We explored the stability of the drug delivery system by changing the physiological conditions and characterized its physicochemical properties. The electrostatic complexation between DOX-glycol chitosan and docosahexaenoic acid RAPA-liposomes (GC-DOX/RAPA ω-liposomes) was precisely regulated, resulting in particle size of 131.3 nm and zeta potential of -14.5 mV. The well-characterized structure of GC-DOX/RAPA ω-liposomes led to high loading efficiencies of 4.1% for DOX and 6.2% for RAPA. Also, GC-DOX/RAPA ω-liposomes exhibited high colloidal stability under physiological conditions and synergistic anti-cancer effects on DOX-resistant MDA-MB-231 cells, while showing pH-sensitive drug release behavior. Our results provided a viable example of marine pharmaceuticals with therapeutic potential for treating drug-resistant tumors using an efficient and safe drug delivery system.

Gold Coating of Silver Nanoplates for Enhanced Dispersion Stability and Efficient Antimicrobial Activity against Intracellular Bacteria.

Silver nanoparticles have antibacterial activity. However, the nanoparticles are unstable and easily form aggregates, which decreases their antibacterial activity. To improve the dispersion stability of silver nanoparticles in aqueous media and to increase their effectiveness as antibacterial agents, we coated triangular plate-like silver nanoparticles (silver nanoplates, Ag NPLs) with one or two layers of gold atoms (Ag@Au1L NPLs and Ag@Au2L NPLs, respectively). These gold coatings improved the dispersion stability in aqueous media with high salt concentrations. Ag@Au1L NPLs showed stronger antibacterial activity on pathogenic bacteria than Ag NPLs and Ag@Au2L NPLs. Furthermore, the Ag@Au1L NPLs decreased the number of bacteria in RAW 264.7 cells. The Ag@Au1L NPLs displayed no cytotoxicity towards RAW 264.7 cells and could be used as antibacterial agents for intracellular bacterial infections.

Gold Nanorods as Nanodevices for Bioimaging, Photothermal Therapeutics, and Drug Delivery.

Gold nanorods are promising metals in several biomedical applications such as bioimaging, thermal therapy, and drug delivery. Gold nanorods have strong absorption bands in near-infrared (NIR) light region and show photothermal effects. Since NIR light can penetrate deeply into tissues, their unique optical, chemical, and biological properties have attracted considerable clinical interest. Gold nanorods are expected to act not only as on-demand thermal converters for photothermal therapy but also as mediators of a controlled drug-release system responding to light irradiation. In this review, we discuss current progress using gold nanorods as bioimaging platform, phototherapeutic agents, and drug delivery vehicles.

Fluorescent polyion complex nanoparticle that incorporates an internal standard for quantitative analysis of protein kinase activity.

We demonstrate a polyion complex (PIC) nanoparticle that contains both a responsive fluorophore and an "internal standard" fluorophore for quantitative measurement of protein kinase (PK) activity. The PK-responsive fluorophore becomes more fluorescent with PK-catalyzed phosphorylation of substrate peptides incorporated in the PIC, while fluorescence from the internal standard remains unchanged during phosphorylation. This new concept will be useful for quantitative PK assays and the discovery of PK inhibitors.

Short peptide motifs for long-lasting anchoring to the cell surface.

A rational design strategy has been developed for the construction of stable peptide-based anchors for the efficient modification of cell surfaces. Six types of peptide composed of five residues with divalent hydrophobic groups have been designed using this new strategy. Among them, a peptide with a sequence of NBD-Lys-Lys(X)-Lys-Lys-Lys(X)-NH2 (NBD: fluorophore, Lys(X): N-ε-palmitoyl-l-lysine) was found to show the highest modification efficacy and longevity in culture medium. The good performance of this peptide was attributed to (1) its high aqueous solubility, which allowed it to partition from the medium to the cell surface, and (2) the high binding affinity of the saturated palmitoyl groups to the cell membrane. We found that the distribution of the peptide was affected by recycling endosome, which enabled the representation of the peptide following its endocytotic disappearance from the cell membrane. Biotin was also presented on the cell surface using this peptide-based anchor to examine its recognition by streptavidin. The efficacy of the recognition process increased as the length of the oligoethylene glycol spacer increased, indicating that it was necessary for the biotin tag to move away from the membrane glycoproteins on the cell surface to facilitate its efficient recognition by streptavidin.

Combination of bubble liposomes and high-intensity focused ultrasound (HIFU) enhanced antitumor effect by tumor ablation.

Ultrasound (US) is used in the clinical setting not only for diagnosis but also for therapy. As a therapeutic US technique, high-intensity focused ultrasound (HIFU) can be applied to treat cancer in a clinical setting. Microbubbles increased temperature and improved the low therapeutic efficiency under HIFU; however, microbubbles have room for improvement in size, stability, and targeting ability. To solve these issues, we reported that "Bubble liposomes" (BLs) containing the US imaging gas (perfluoropropane gas) liposomes were suitable for ultrasound imaging and gene delivery. In this study, we examined whether BLs and HIFU could enhance the ablation area of the tumor and the antitumor effect. First, we histologically analyzed the tumor after BLs and HIFU. The ablation area of the treatment of BLs and HIFU was broader than that of HIFU alone. Next, we monitored the temperature of the tumor, and examined the antitumor effect. The temperature increase with BLs and HIFU treatment was faster and higher than that with HIFU alone. Moreover, treatment with BLs and HIFU enhanced the antitumor effect, which was better than with HIFU alone. Thus, the combination of BLs and HIFU could be efficacious for cancer therapy.

Bacterial membrane vesicles combined with nanoparticles for bacterial vaccines and cancer immunotherapy.

Similar to mammalian cells, most bacteria can release nano-sized membrane vesicles (MVs) into the extracellular environment. MVs contain lipids, bioactive proteins, nucleic acids, and metabolites, and play important roles in microbial physiology. MVs have great potential for immunotherapeutic applications, such as bacterial vaccines and cancer immunotherapy. However, because of the diversity in content and heterogeneity in size of MVs, the clinical application of MVs has been limited. Recently, the use of MVs combined with nanoparticles (NPs) has been shown to be effective in improving the homogeneity, stability and function of MVs. In this review, we focus on studies of MVs combined with NPs (MV-NPs) and describe the use of these MV-NPs in biotechnology, especially in bacterial vaccine and cancer immunotherapy.

Fluoride-treated rare earth-free magnesium alloy ZK30: An inert and bioresorbable material for bone fracture treatment devices.

Bone fractures represent a common health problem, particularly in an increasingly aging population. Bioresorbable magnesium (Mg) alloy-based implants offer promising alternatives to traditional metallic implants for the treatment of bone fractures because they eliminate the need for implant removal after healing. The Mg-Y-rare-earth (RE)-Zr alloy WE43, designed for orthopedic implants, has received European Conformity mark approval. However, currently, WE43 is not clinically used in certain countries possibly because of concerns related to RE metals. In this study, we investigated the use of a RE-free alloy, namely, Mg-Zn-Zr alloy (ZK30), as an implant for bone fractures. Hydrofluoric acid (HF) treatment was performed to improve the corrosion resistance of ZK30. HF-treated ZK30 (HF-ZK30) exhibited lower corrosion rate and higher biocompatibility than those of WE43 in in vitro experiments. After implanting a rod of HF-ZK30 into the fractured femoral bones of mice, HF-ZK30 held the bones and healed the fracture without deformation. Treatment results of HF-ZK30 were comparable to those of WE43, indicating the potential of HF-ZK30 as a bioresorbable and safe implant for bone repair.

Modification of Silver Nanoplates with Cell-Binding Subunit of Bacterial Toxin and Their Antimicrobial Activity against Intracellular Bacteria.

Intracellular bacteria are able to survive and grow in host cells and often cause serious infectious diseases. The B subunit of the subtilase cytotoxin (SubB) found in enterohemorrhagic Escherichia coli O113:H21 recognizes sialoglycans on cell surfaces and triggers the uptake of cytotoxin by the cells, meaning that Sub B is a ligand molecule that is expected to be useful for drug delivery into cells. In this study, we conjugated SubB to silver nanoplates (AgNPLs) for use as an antibacterial drug and examined their antimicrobial activity against intracellularly infecting Salmonella typhimurium (S. typhimurium). The modification of AgNPLs with SubB improved their dispersion stability and antibacterial activity against planktonic S. typhimurium. The SubB modification enhanced the cellular uptake of AgNPLs, and intracellularly infecting S. typhimurium were killed at low concentrations of AgNPLs. Interestingly, larger amounts of SubB-modified AgNPLs were taken up by infected cells compared with uninfected cells. These results suggest that the S. typhimurium infection activated the uptake of the nanoparticles into the cells. SubB-modified AgNPLs are expected to be useful bactericidal systems for intracellularly infecting bacteria.

Alkyl gallates inhibit serine O-acetyltransferase in bacteria and enhance susceptibility of drug-resistant Gram-negative bacteria to antibiotics.

A principal concept in developing antibacterial agents with selective toxicity is blocking metabolic pathways that are critical for bacterial growth but that mammalian cells lack. Serine O-acetyltransferase (CysE) is an enzyme in many bacteria that catalyzes the first step in l-cysteine biosynthesis by transferring an acetyl group from acetyl coenzyme A (acetyl-CoA) to l-serine to form O-acetylserine. Because mammalian cells lack this l-cysteine biosynthesis pathway, developing an inhibitor of CysE has been thought to be a way to establish a new class of antibacterial agents. Here, we demonstrated that alkyl gallates such as octyl gallate (OGA) could act as potent CysE inhibitors in vitro and in bacteria. Mass spectrometry analyses indicated that OGA treatment markedly reduced intrabacterial levels of l-cysteine and its metabolites including glutathione and glutathione persulfide in Escherichia coli to a level similar to that found in E. coli lacking the cysE gene. Consistent with the reduction of those antioxidant molecules in bacteria, E. coli became vulnerable to hydrogen peroxide-mediated bacterial killing in the presence of OGA. More important, OGA treatment intensified susceptibilities of metallo-ß-lactamase-expressing Gram-negative bacteria (E. coli and Klebsiella pneumoniae) to carbapenem. Structural analyses showed that alkyl gallate bound to the binding site for acetyl-CoA that limits access of acetyl-CoA to the active site. Our data thus suggest that CysE inhibitors may be used to treat infectious diseases caused by drug-resistant Gram-negative bacteria not only via direct antibacterial activity but also by enhancing therapeutic potentials of existing antibiotics.

Gene carrier showing all-or-none response to cancer cell signaling.

In this work we designed a novel nano carrier, a linear polyethylenimine (LPEI)-peptide conjugate, for cancer-specific expression of transgenes. The conjugate was easily synthesized by using a click chemistry scheme orthogonal to the reactive side groups of the peptide, which is the substrate of protein kinase Cα (PKCα). Polyplexes of the conjugates with plasmid DNA (pDNA) were intact and stably dispersed even in the presence of cell lysate. Despite this stability, the polyplexes readily dissociated upon phosphorylation of the grafted peptides by PKCα. Because of its endosomal escape ability and adequate susceptibility to PKCα, the polyplexes showed an all-or-none type response to PKCα activity in transgene expression in vitro. The polyplexes achieved cancer tissue-specific transgene expression even for a tumor with a relatively low PKCα activity. Thus the LPEI-peptide conjugate has high potential as a nanocarrier for cancer-targeted gene therapy.

Fluorometric detection of protein kinase Cα activity based on phosphorylation-induced dissociation of a polyion complex.

Here, we report the fluorometric detection of protein kinase Cα (PKCα) activity in a cancerous cell lysate using a polyion complex (PIC) composed of a quencher (BHQ3)-modified chondroitin sulfate [CS(X)] and a dendrimer modified with a cationic peptide substrate (FKKQGSFAKKK-NH(2)) and a near infrared (NIR) fluorophore (Cy5.5) (polymer 1). When polymer 1 formed the PIC with CS(X) through electrostatic interactions, the NIR fluorescence was quenched effectively via Förster resonance energy transfer (FRET) between Cy5.5 and BHQ3. However, this quenched fluorescence was recovered when the pendant peptides in polymer 1 were phosphorylated with PKCα due to the dissociation of the PIC. When PKCα was added to the PIC dispersion, a significant increase in fluorescence intensity was observed, whereas its fluorescence increase was inhibited with a PKCα inhibitor in a concentration-dependent manner. Furthermore, our PIC was robust enough to measure PKCα activity in a cancerous cellular lysate without purification. The PKCα-responsive PIC offers a simple, rapid, sensitive, and robust approach to detect PKCα activity in crude cellular lysates that would be suitable for drug screening formats and cancer diagnosis using crude cellular lysates.

Correlation between phosphorylation ratios by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and radioactivities by radioactive assay.

To investigate the correlation between the counts per minute (CPM) by radioactivity assay and the phosphorylation ratio by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, we prepared 136 peptide substrates. The correlation coefficient of phosphorylation ratios to CPM was 0.77 for all samples. However, the more the numbers of positively charged amino acids increased, the more the correlation coefficient increased. Although positively charged amino acids can have an effect on the correlation results, MALDI-TOF MS analysis is a useful means for monitoring phosphorylated peptide and protein kinase activity instead of radioactivity assays.

Folate-PEG-appended dendrimer conjugate with α-cyclodextrin as a novel cancer cell-selective siRNA delivery carrier.

We previously reported that of the various polyamidoamine (PAMAM) STARBURST dendrimer (generation 3, G3) (dendrimer) conjugates with cyclodextrins (CyDs), the dendrimer (G3) conjugate with α-CyD having an average degree of substitution of 2.4 (α-CDE (G3)) has the greatest potential for a novel carrier for siRNA in vitro and in vivo. To improve the siRNA transfer activity and the lack of target specificity of α-CDE (G3), we prepared folate-polyethylene glycol (PEG)-appended α-CDEs (G3) (Fol-PαCs) with various degrees of substitution of folate (DSF) and evaluated their siRNA transfer activity to folate receptor (FR)-overexpressing cancer cells in vitro and in vivo. Of the three Fol-PαCs (G3, DSF 2, 4 and 7), Fol-PαC (G3, DSF 4) had the highest siRNA transfer activity in KB cells (FR-positive). Fol-PαC (G3, DSF 4) was endocytosed into KB cells through FR. No cytotoxicity of the siRNA complex with Fol-PαC (G3, DSF 4) was observed in KB cells (FR-positive) or A549 cells (FR-negative) up to the charge ratio of 100/1 (carrier/siRNA). In addition, the siRNA complex with Fol-PαC (G3, DSF 4) showed neither interferon response nor inflammatory response. Importantly, the siRNA complex with Fol-PαC (G3, DSF 4) tended to show the in vivo RNAi effects after intratumoral injection and intravenous injection in tumor cells-bearing mice. The FITC-labeled siRNA and TRITC-labeled Fol-PαC (G3, DSF 4) were actually accumulated in tumor tissues after intravenous injection in the mice. In conclusion, the present results suggest that Fol-PαC (G3, DSF 4) could potentially be used as a FR-overexpressing cancer cell-selective siRNA delivery carrier in vitro and in vivo.

Creating a unique environment for selecting reactive enzymes with DNA: 'Sticky' binding of oligocation-grafted polymers to DNA.

To provide colloidally stable polyplexes formed between pDNA and cationic polymers, cationic polymers have been modified with hydrophilic polymers to form a hydrophilic shell. Block copolymers of cationic and hydrophilic polymers and cationic polymers grafted with hydrophilic polymers are representative designs of such polymers. Here, we report a new design of cationic polymers and oligocationic peptide-grafted polymers. We synthesized 15 kinds of graft copolymers by varying the number of cationic charges of the peptides and their grafting density. We found that graft copolymers with less cationic peptides and less grafting density formed colloidally stable polyplexes. Interestingly, the less cationic graft copolymers bind to excess amounts of pDNA. We also found that the graft copolymers showed selectivity toward reactive enzymes affording the reaction of pDNA with nucleases, while suppressing both the replication of DNA by DNA polymerase and gene expression. The suppression of the replication and expression is considered to result from the high capacity of the graft copolymers for binding with pDNA. The polynucleotides produced by DNA polymerase or RNA polymerase would be captured by the graft copolymers to impede these enzymatic reactions.