Chiaki Yoshikawa

Zwitterionic polymer with long-term durability has been widely explored for the construction of bio-inactive surfaces.

Nonspecific protein absorption is an ongoing problem in the development of highly sensitive magnetic particle (MP)-based diagnostic assays, whereby the MP surface undergoes biofouling, significantly reducing the limit of detection and sensitivity of the device. In this study, a bioinert, concentrated polymer brush (CPB) composed of poly[poly(ethylene oxide) methyl ether methacrylate] (PPEGMA) was employed to reduce the absorption of protein onto the MP surfaces; and an anti-human serum albumin antibody (Ab) was then immobilized onto the brush layer by click chemistry to demonstrate its application as an immunoassay platform. The amount of antibody grafted onto the ends of the brush coating was quantified by a BCA assay with a high grafting efficiency (∼0.80 antibody per chain). Furthermore, when using the antibody-conjugated CPB-coated MPs (MP-PPEGMA-Ab) as an immunoassay platform, we were able to determine the capture efficacy of human serum albumin (HSA) in both a buffered solution and diluted human serum by colorimetric analysis, and further confirmation was achieved via liquid chromatography/mass spectrometry, in which our MPs showed a high selectivity toward the targeted analyte. Lastly, we demonstrated that MP-PPEGMA-Ab was capable of detecting human serum albumin at a concentration as low as 6.4 ng mL^-1, 30% more sensitive than the unmodified MP, demonstrating the impact of CPBs on diagnostic assays. Due to their high selectivity and sensitivity, our CPB-based MPs are expected to be applicable for a wide range of immunoassay applications by employing different bioinert polymers and biofunctional groups.

Nestin is a type VI intermediate filament protein and a well-known neural stem cell marker. It is also expressed in high-grade cancer cells, forming copolymerized filaments with vimentin. We previously showed that nestin inhibits the binding of vimentin’s tail domain to actin filaments (AFs) by steric hindrance through its large nestin tail domain (NTD), thereby increasing three-dimensional cytoskeleton network mobility, enhancing cell flexibility, and promoting cancer progression. Further, we found that nestin itself stably binds to AFs via the NTD. We therefore hypothesized that the NTD may form a flexible cytoskeletal structure by extending with weak force. In vitro tensile tests using atomic force microscopy were performed to assess the mechanical properties of NTDs. The C-terminus of the NTD bound AFs by bringing the AFM tip modified with the NTD into contact with the AFs on the substrate. NTDs were elongated to approximately 80% of their maximum length at weak forces < 150 pN. Repeated tensile tests revealed that the NTD refolded quickly and behaved like a soft elastic material. We speculate that nestin stably binds AFs, and the NTD extends with weak force, contracting quickly upon load release. Thereby, nestin would absorb mechanical load and maintain cytoskeletal integrity.

Synthetic cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) are promising candidates for vaccine adjuvants, because they activate immune responses through the Toll-like receptor 9 (TLR9) pathway. However, unmodified CpG ODNs are quickly degraded by serum nucleases, and their negative charge hinders cellular uptake, limiting their clinical application. Our group previously reported that guanine-quadruplex (G4)-forming CpG ODNs exhibit enhanced stability and cellular uptake. G4 structures can form in parallel, anti-parallel, or hybrid topologies, depending on strand orientation, but the effects of these topologies on CpG ODNs have not yet been explored. In this study, we designed three distinct G4 topologies as scaffolds for CpG ODNs. Among the three topology, the parallel G4 CpG ODN demonstrated the highest serum stability and cellular uptake, resulting in the strongest immune response from macrophage cells. Additionally, we investigated the binding affinities of the different G4 topologies to macrophage scavenger receptor-1 and TLR9, both of which are key to immune activation. These findings provide valuable insights into the development of CpG ODN-based vaccine adjuvants.

Manipulating the three-dimensional (3D) structures of cells is important for facilitating to repair or regenerate tissues. A self-assembly system of cells with cellulose nanofibers (CNFs) and concentrated polymer brushes (CPBs) has been developed to fabricate various cell 3D structures. To further generate tissues at an implantable level, it is necessary to carry out a large number of experiments using different cell culture conditions and material properties; however this is practically intractable. To address this issue, we present a graph-neural network-based simulator (GNS) that can be trained by using assembly process images to predict the assembly status of future time steps. A total of 24 (25 steps) time-series images were recorded (four repeats for each of six different conditions), and each image was transformed into a graph by regarding the cells as nodes and the connecting neighboring cells as edges. Using the obtained data, the performances of the GNS were examined under three scenarios (i.e., changing a pair of the training and testing data) to verify the possibility of using the GNS as a predictor for further time steps. It was confirmed that the GNS could reasonably reproduce the assembly process, even under the toughest scenario, in which the experimental conditions differed between the training and testing data. Practically, this means that the GNS trained by the first 24 h images could predict the cell types obtained 3 weeks later. This result could reduce the number of experiments required to find the optimal conditions for generating cells with desired 3D structures. Ultimately, our approach could accelerate progress in regenerative medicine.

Manipulating the three-dimensional (3D) structures of cells is important for facilitating to repair or regenerate tissues. A self-assembly system of cells with cellulose nanofibers (CNFs) and concentrated polymer brushes (CPBs) has been developed to fabricate various cell 3D structures. To further generate tissues at an implantable level, it is necessary to carry out a large number of experiments using different cell culture conditions and material properties; however this is practically intractable. To address this issue, we present a graph neural network-based simulator (GNS) that can be trained by using assembly process images to predict the assembly status of future time steps. A total of 24 (25 steps) time-series images were recorded (four repeats for each of six different conditions), and each image was transformed into a graph by regarding the cells as nodes and the connecting neighboring cells as edges. Using the obtained data, the performances of the GNS were examined under three scenarios (i.e. changing a pair of the training and testing data) to verify the possibility of using the GNS as a predictor for further time steps. It was confirmed that the GNS could reasonably reproduce the assembly process, even under the toughest scenario, in which the experimental conditions differed between the training and testing data. Practically, this means that the GNS trained by the first 24 hour images could predict the cell types obtained three weeks later. This result could reduce the number of experiments required to find the optimal conditions for generating cells with desired 3D structures. Ultimately, our approach could accelerate progress in regenerative medicine.

Cationic liposomes, specifically 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes, serve as successful carriers for guanine-quadruplex (G4) structure-based cytosine-guanine oligodeoxynucleotides (CpG ODNs). The combined benefits of CpG ODNs forming a G4 structure and a non-viral vector carrier endow the ensuing complex with promising adjuvant properties. Although G4-CpG ODN-DOTAP complexes show a higher immunostimulatory effect than naked G4-CpG ODNs, the effects of the complex composition, especially charge ratios, on the production of the pro-inflammatory cytokines interleukin (IL)-6 and interferon (IFN)-α remain unclear. Here, we examined whether charge ratios drive the bifurcation of cytokine inductions in human peripheral blood mononuclear cells. Linear CpG ODN-DOTAP liposome complexes formed micrometer-sized positively charged agglomerates; G4-CpG ODN-DOTAP liposome complexes with low charge ratios (0.5 and 1.5) formed ~250 nm-sized negatively charged complexes. Notably, low-charge-ratio (0.5 and 1.5) complexes induced significantly higher IL-6 and IFN-α levels simultaneously than high-charge-ratio (2 and 2.5) complexes. Moreover, confocal microscopy indicated a positive correlation between the cellular uptake of the complex and amount of cytokine induced. The observed effects of charge ratios on complex size, surface charge, and affinity for factors that modify cellular-uptake, intracellular-activity, and cytokine-production efficiency highlight the importance of a rational complex design for delivering and controlling G4-CpG ODN activity.

Unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) induce inflammatory cytokines and type I interferons (IFNs) to activate the immune system. To apply CpG ODNs as vaccine adjuvants, the cellular uptake and stability of phosphodiester-based, non-modified ODNs require further improvement. Previously developed new CpG ODNs forming guanine-quadruplex (G4) structures showed higher nuclease resistance and cellular uptake than linear CpG ODNs; however, the complex formation of G4-CpG ODNs with antigen proteins is necessary for their application as vaccine adjuvants. In this study, we utilized a cationic polymer, ε-poly-_L-lysine (ε-PLL), as a carrier for G4-CpG ODNs and antigen. The ε-PLL/G4-CpG ODN complex exhibited enhanced stability against nucleases. Cellular uptake of the ε-PLL/G4-CpG ODN complex positively correlated with the N/P ratio. In comparison to naked G4-CpG ODNs, the ε-PLL/G4-CpG ODN complex induced extremely high levels of interleukin (IL)-6, IL-12, and IFN-β. Relative immune cytokine production was successfully tuned by N/P ratio modification. Mice with the ε-PLL/G4-CpG ODN/ovalbumin (OVA) complex showed increased OVA-specific immunoglobulin (Ig)G, IgG1, and IgG2c levels, whereas total IgE levels did not increase and weight gain rates were not affected. Therefore, ε-PLL can serve as a safe and effective phosphodiester-based, non-modified CpG ODN delivery system, and the ε-PLL/G4-CpG ODN/antigen complex is a highly promising candidate for vaccine adjuvants and can be further used in clinical research.