Hypoxia-responsive expression of vascular endothelial growth factor for induction of angiogenesis in artificial three-dimensional tissues.

Constructing three-dimensional (3D) tissues is an important process to improve cellular functions in tissue engineering. When transplanting artificially constructed tissues, a poor vascular network restricts oxygen and nutrient supplies to the tissue cells, which leads to cell death and reduced rates of tissue engraftment. Therefore, it is necessary to develop a system that builds a vascular network within 3D tissues. Here, we developed a hypoxia-responsive gene expression system for production of an angiogenic factor, vascular endothelial growth factor (VEGF), to improve hypoxia and nutrition deficiencies inside artificial 3D tissues. We demonstrated that cells into which the hypoxia-responsive VEGF gene expression system had been introduced autonomously controlled VEGF expression in a hypoxic stress-dependent manner. Next, we confirmed that VEGF expression within a 3D cell sheet was induced in response to a hypoxic environment in vitro. The genetically modified cell sheet was subcutaneously transplanted into mice to evaluate the feasibility of the hypoxia-responsive VEGF gene expression system in vivo. The results suggest that the hypoxia-responsive VEGF gene expression system is promising to prepare artificial 3D tissues in regenerative medicine.

Magnetically triggered transgene expression in mammalian cells by localized cellular heating of magnetic nanoparticles.

To develop a remote control system of transgene expression through localized cellular heating of magnetic nanoparticles, a heat-inducible transgene expression system was introduced into mammalian cells. Cells were labeled with magnetic nanoparticles and exposed to an alternating magnetic field. The magnetically labeled cells expressed the transgene in a monolayer and multilayered cell sheets in which cells were heated around the magnetic nanoparticles without an apparent temperature increase in the culture medium. Magnetic cells were also generated by genetically engineering with a ferritin gene, and transgene expression could be induced by exposure to an alternating magnetic field. This approach may be applicable to the development of novel gene therapies in cell-based medicine.

Correction: Development of Novel Faster-Dissolving Microneedle Patches for Transcutaneous Vaccine Delivery. Pharmaceutics, 2017, 9(3), 27.

The authors wish to make a change to their published paper [1].[...].

Development of novel double-decker microneedle patches for transcutaneous vaccine delivery.

Microneedle (MN) patches have great potential as transcutaneous vaccine delivery devices because MNs can effectively deliver vaccine antigen into the skin through the micropores formed in the stratum corneum by low-invasive and painless skin puncturing. This study aims to develop novel double-decker MN patches which have not only high safety and efficacy but also broad applicability to various vaccine antigens. We developed two types of MN patches (PGA-MN and Nylon-MN) that are made from polyglycolic acid and Nylon-6. In pre-clinical studies, both MN patches could demonstrably deliver antigens into resected human dermal tissue, prolong antigen deposition and increase antigen-specific IgG levels after vaccination compared with conventional injections. We demonstrated both MN patches could be safely applied to human skin because no broken MNs or significant skin irritation were observed after applications in the clinical research. PGA-MN was suggested to be superior to Nylon-MN regarding human skin puncturability based on measurements of transepidermal water loss and needle failure force. A high content of tetravalent influenza hemagglutinin antigens loaded on PGA-MN could stably maintain HA titers at 35°C for 1year. Overall, double-decker MN patches can reliably and safely puncture human skin and are promising as effective transcutaneous vaccine delivery devices.

Development of Novel Faster-Dissolving Microneedle Patches for Transcutaneous Vaccine Delivery.

Microneedle (MN) patches are promising for transcutaneous vaccination because they enable vaccine antigens to physically penetrate the stratum corneum via low-invasive skin puncturing, and to be effectively delivered to antigen-presenting cells in the skin. In second-generation MN patches, the dissolving MNs release the loaded vaccine antigen into the skin. To shorten skin application time for clinical practice, this study aims to develop novel faster-dissolving MNs. We designed two types of MNs made from a single thickening agent, carboxymethylcellulose (CMC) or hyaluronan (HN). Both CMC-MN and HN-MN completely dissolved in rat skin after a 5-min application. In pre-clinical studies, both MNs could demonstrably increase antigen-specific IgG levels after vaccination and prolong antigen deposition compared with conventional injections, and deliver antigens into resected human dermal tissue. In clinical research, we demonstrated that both MNs could reliably and safely puncture human skin without any significant skin irritation from transepidermal water loss measurements and ICDRG (International Contact Dermatitis Research Group) evaluation results.

Hypoxia-responsive transgene expression system using RTP801 promoter and synthetic transactivator fused with oxygen-dependent degradation domain.

Precise control of gene expression using an artificial gene circuit is a major challenge in the application of synthetic biology. Here, we designed a hypoxia-responsive transgene expression system by combining a hypoxia-inducible RTP801 promoter and a tetracycline-responsive transactivator fused with an oxygen-dependent degradation domain (TA-ODD). The reporter gene expression was highly induced by hypoxia when a transactivator-expression plasmid, pRTP801/TA-ODD, harboring a TA-ODD gene driven by the RTP801 promoter, was cotransfected with a reporter plasmid, pTRE/EGFP, harboring an EGFP gene controlled under the transactivator-responsive promoter. A stable cell line into which the expression units RTP801/TA-ODD and TRE/EGFP had been introduced responded to hypoxia and expressed the reporter gene in an oxygen-concentration-dependent manner. Moreover, the cells demonstrated potential as sensors to detect hypoxic conditions in a three-dimensional tissue culture in vitro. These results indicate that the hypoxia-responsive transgene expression system is useful for constructing cell-based hypoxia detection systems.

Electrochemical reduction of CO2 to ethylene glycol on imidazolium ion-terminated self-assembly monolayer-modified Au electrodes in an aqueous solution.

Imidazolium ion-terminated self-assembled monolayer (SAM)-modified electrodes achieve CO2 conversion while suppressing hydrogen evolution. Immobile imidazolium ion on gold (Au) electrodes reduce CO2 at low overpotential. The distance between electrode and imidazolium ion separated by alkane thiol affects CO2 reduction activity. CO2 reduction current depends on the tunnel current rate. Although the product of CO2 reduction at the bare Au electrode is CO, SAM-modified electrodes produce ethylene glycol in aqueous electrolyte solution without CO evolution. The faradaic efficiency reached a maximum of 87%. CO2 reduction at SAM-modified electrodes is unaffected by reduction activity of Au electrode. This phenomenon shows that the reaction field of CO2 reduction is not the electrode surface but the imidazolium ion monolayer.

DNA damage-responsive transgene expression mediated by the p53 promoter with transcriptional amplification.

We constructed a DNA damage-responsive transgene expression system mediated by the p53 promoter. We incorporated a transactivation system to generate transcriptional amplification via a positive feedback loop. Higher levels of DNA damage-responsive transgene expression were observed when transactivation was active.

Heat-inducible gene expression system by applying alternating magnetic field to magnetic nanoparticles.

By combining synthetic biology with nanotechnology, we demonstrate remote controlled gene expression using a magnetic field. Magnetite nanoparticles, which generate heat under an alternating magnetic field, have been developed to label cells. Magnetite nanoparticles and heat-induced therapeutic genes were introduced into tumor xenografts. The magnetically triggered gene expression resulted in tumor growth inhibition. This system shows great potential for controlling target gene expression in a space and time selective manner and may be used for remote control of cell functions via gene expression.