Optogenetic Calcium Ion Influx in Myoblasts and Myotubes by Near-Infrared Light Using Upconversion Nanoparticles.

Bioactuators made of cultured skeletal muscle cells are generally driven by electrical or visible light stimuli. Among these, the technology to control skeletal muscle consisting of myoblasts genetically engineered to express photoreceptor proteins with visible light is very promising, as there is no risk of cell contamination by electrodes, and the skeletal muscle bioactuator can be operated remotely. However, due to the low biopermeability of visible light, it can only be applied to thin skeletal muscle films, making it difficult to realize high-power bioactuators consisting of thick skeletal muscle. To solve this problem, it is desirable to realize thick skeletal muscle bioactuators that can be driven by near-infrared (NIR) light, to which living tissue is highly permeable. In this study, as a promising first step, upconversion nanoparticles (UCNPs) capable of converting NIR light into blue light were bound to C2C12 myoblasts expressing the photoreceptor protein channelrhodopsin-2 (ChR2), and the myoblasts calcium ion (Ca2+) influx was remotely manipulated by NIR light exposure. UCNP-bound myoblasts and UCNP-bound differentiated myotubes were exposed to NIR light, and the intracellular Ca2+ concentrations were measured and compared to myoblasts exposed to blue light. Exposure of the UCNP-bound cells to NIR light was found to be more efficient than exposure to blue light in terms of stimulating Ca2+ influx.

A Robust Nanoparticle-based Magnetic Separation Method for Intact Lysosomes.

Lysosome isolation is a preresiquite for identifying lysosomal protein composition by mass spectroscopic analysis, to reveal lysosome functions, and their involvement in some diseases. Magnetic nanoparticle-based fractionation has received great attention for lysosome isolation, owing to its high efficiency, purity, and preservation of lysosomal structures. Understanding the intracellular trafficking of magnetic probes is the key point of this technique, to determine the appropriate time for magnetic isolation of lysosomes, because this parameter changes depending on different cell lines used. The traditional magnetic probes, such as superparamagnetic iron oxide nanoparticles (SPIONs), require surface modification by fluorescent dyes to enable the investigation of their intracellular trafficking, which has some disadvantages, including the possible alternation of their bio-interaction, and the instability of fluorescence properties in the lysosomal environment. To overcome those limitations, we present a protocol that employs magnetic-plasmonic nanoparticles (MPNPs) to investigate intracellular trafficking using their intrinsic imaging capability, followed by quick lysosome isolation using a magnetic column. This protocol can be easily applied to isolate the intact lysosomes of any adherent cell lines. Graphical abstract.

Quick and Mild Isolation of Intact Lysosomes Using Magnetic-Plasmonic Hybrid Nanoparticles.

Rapid and efficient isolation of intact lysosomes is necessary to study their functions and metabolites by proteomic analysis. We developed a swift and robust nanoparticle-based magnetic separation method in which magnetic-plasmonic hybrid nanoparticles (MPNPs) conjugated with amino dextran (aDxt) were targeted to the lumen of lysosomes via the endocytosis pathway. For well-directed magnetic separation of the lysosomes, it is important to trace the intracellular trafficking of the aDxt-conjugated MPNPs (aDxt-MPNPs) in the endocytosis pathway. Therefore, we analyzed the intracellular transport process of the aDxt-MPNPs by investigating the time-dependent colocalization of plasmonic scattering of aDxt-MPNPs and immunostained marker proteins of organelles using the threshold Manders' colocalization coefficient (Rt). Detailed analysis of time variations of Rt for early and late endosomes and lysosomes allowed us to derive the transport kinetics of aDxt-MPNPs in a cell. After confirming the incubation time required for sufficient accumulation of aDxt-MPNPs in lysosomes, the lysosomes were magnetically isolated as intact as possible. By varying the elapsed time from homogenization to complete isolation of lysosomes (tdelay) and temperature (T), the influences of tdelay and T on the protein composition of the lysosomes were investigated by polyacrylamide gel electrophoresis and amino acid analysis. We found that the intactness of lysosomes could become impaired quite quickly, and to isolate lysosomes as intact as possible with high purity, tdelay = 30 min and T = 4 °C were optimal settings.

Enhancing the Sensitivity of Lateral Flow Immunoassay by Magnetic Enrichment Using Multifunctional Nanocomposite Probes.

For lateral flow immunoassay (LFIA), it is an important challenge to enhance the detection sensitivity to the same level as polymerase chain reaction or enzyme-linked immunosorbent assay to make LFIA pervasive in the field of on-site environmental analysis. We recently demonstrated that the LFIA sensitivity is dramatically enhanced by using Pt-nanoparticle-latex nanocomposite beads (Pt-P2VPs) as probes for the detection of the influenza A (H1N1) antigen compared with using conventional Au colloids as probes. Here, to further enhance the LFIA sensitivity using Pt-P2VPs, superparamagnetic iron oxide nanoparticles (SPIONs) were chemically conjugated to Pt-P2VPs (Pt-P2VP@SPION) to give them magnetic separation capability (enrichment and/or purification). To investigate the effect of magnetic enrichment on the LFIA sensitivity in a sandwich format, the C-reactive protein (CRP) was chosen as a model analyte and anti-CRP antibody (CRPAb)-conjugated Pt-P2VP@SPION (Pt-P2VP@SPION-CRPAb) beads were used as probes. The visual limit of detection (LOD) of LFIA was successfully lowered by increasing the magnetic enrichment factor φ. The minimum LOD under the present experimental conditions was 0.08 ng/mL for φ = 40, which is 26-fold lower than that of the standard Au-nanoparticle-based LFIA. In theory, the LOD can be unlimitedly decreased by just increasing φ. However, the times required for both the antigen-antibody binding reaction and magnetic separation dramatically increase with φ. We also propose solutions to overcome this drawback.

Disinfection of various bacterial pathogens using novel silver nanoparticle-decorated magnetic hybrid colloids.

Silver nanoparticles (AgNPs) have long been considered a powerful disinfectant for controlling pathogenic microorganisms. However, AgNPs might have adverse effects on both human health and our ecosystems due to their potential cytotoxicity and the difficulty in recovering them after their release into the environment. In this study, we characterized the antimicrobial efficacy caused by a novel micrometer-sized magnetic hybrid colloid (MHC) containing 7, 15, or 30nm sized monodispersed AgNPs (AgNP-MHCs), which can be re-collected from the environment using simple procedures, such as a magnet or centrifugation. We evaluated the antibacterial capabilities of AgNP-MHCs against target bacteria (Legionella pneumophila, Bacillus subtilis, Escherichia coli, and Clostridium perfringens) and compared them with the inactivation efficacy of AgNPs ~30nm in diameter (nAg30s). Among the different AgNP-MHCs composites evaluated, Ag30-MHCs had the greatest antibacterial effect. After 1h of exposure, more than a 4-log10 reduction of L. pneumophila and 6-log10 reduction of B. subtilis was achieved by 4.6×109particles/mL of Ag30-MHCs and Ag30-MHC-Ls. In addition, Ag30-MHC-Ls maintained their strong antibacterial capabilities under anaerobic conditions. Our results indicate that AgNP-MHCs can be considered excellent tools for controlling waterborne bacterial pathogens, with a minimal risk of release into the environment.