Ji Yu Choi

Cytocompatible Polymer Grafting from Individual Living Cells by Atom-Transfer Radical Polymerization

A cytocompatible method of surface-initiated, activator regenerated by electron transfer, atom transfer radical polymerization (SI-ARGET ATRP) is developed for engineering cell surfaces with synthetic polymers. Dopamine-based ATRP initiators are used for both introducing the ATRP initiator onto chemically complex cell surfaces uniformly (by the material-independent coating property of polydopamine) and protecting the cells from radical attack during polymerization (by the radical-scavenging property of polydopamine). Synthetic polymers are grafted onto the surface of individual yeast cells without significant loss of cell viability, and the uniform and dense grafting is confirmed by various characterization methods including agglutination assay and cell-division studies. This work will provide a strategic approach to the generation of living cell-polymer hybrid structures and open the door to their application in multitude of areas, such as sensor technology, catalysis, theranostics, and cell therapy.

Layer‐by‐Layer‐Based Silica Encapsulation of Individual Yeast with Thickness Control

In the area of cell-surface engineering with nanomaterials, the metabolic and functional activities of the encapsulated cells are manipulated and controlled by various parameters of the artificial shells that encase the cells, such as stiffness and elasticity, thickness, and porosity. The mechanical durability and physicochemical stability of inorganic shells prove superior to layer-by-layer-based organic shells with regard to cytoprotection, but it has been difficult to vary the parameters of inorganic shells including their thickness. In this work, we combine the layer-by-layer technique with a process of bioinspired silicification to control the thickness of the silica shells that encapsulate yeast Saccharomyces cerevisiae cells individually, and investigate the thickness-dependent microbial growth.

Control over Neurite Directionality and Neurite Elongation on Anisotropic Micropillar Arrays

Control over neurite orientation in primary hippocampal neurons is achieved by using interrupted, anisotropic micropillar arrays as a cell culture platform. Both neurite orientation and neurite length are controlled by a function of interpillar distance.

Axon-First Neuritogenesis on Vertical Nanowires

In this work, we report that high-density, vertically grown silicon nanowires (vg-SiNWs) direct a new in vitro developmental pathway of primary hippocampal neurons. Neurons on vg-SiNWs formed a single, extremely elongated major neurite earlier than minor neurites, which led to accelerated polarization. Additionally, the development of lamellipodia, which generally occurs on 2D culture coverslips, was absent on vg-SiNWs. The results indicate that surface topography is an important factor that influences neuronal development and also provide implications for the role of topography in neuronal development in vivo.

Tissue-based metabolic labeling of polysialic acids in living primary hippocampal neurons

Significance The roles of cell-surface glycans remain elusive compared with those of proteins or lipids because of their diverse and dynamic nature. Metabolic incorporation of unnatural monosaccharides in the biochemical synthesis of glycans as a chemical reporter has been a successful method to investigate the functions of cell-surface glycans but has also left an issue of cytotoxicity for certain cells. In this work, we developed a tissue-based strategy for metabolic incorporation of a chemical reporter to primary neurons. We let an unnatural monosaccharide be metabolized by hippocampal tissues before dissociation into individual cells, and thereby, we could eliminate cytotoxicity. We used this method to describe, for the first time to our knowledge, the real-time distribution of polysialic acids on the membranes of neurons. The posttranslational modification of neural cell-adhesion molecule (NCAM) with polysialic acid (PSA) and the spatiotemporal distribution of PSA-NCAM play an important role in the neuronal development. In this work, we developed a tissue-based strategy for metabolically incorporating an unnatural monosaccharide, peracetylated N-azidoacetyl-d-mannosamine, in the sialic acid biochemical pathway to present N-azidoacetyl sialic acid to PSA-NCAM. Although significant neurotoxicity was observed in the conventional metabolic labeling that used the dissociated neuron cells, neurotoxicity disappeared in this modified strategy, allowing for investigation of the temporal and spatial distributions of PSA in the primary hippocampal neurons. PSA-NCAM was synthesized and recycled continuously during neuronal development, and the two-color labeling showed that newly synthesized PSA-NCAMs were transported and inserted mainly to the growing neurites and not significantly to the cell body. This report suggests a reliable and cytocompatible method for in vitro analysis of glycans complementary to the conventional cell-based metabolic labeling for chemical glycobiology.

Structure Modulation of Silica Microspheres in Bio-Inspired Silicification: Effects of TEOS Concentration

Silica nanoand microspheres have applications in various areas, such as in photonic crystals, catalysis, biosensors, bioassay, and drug delivery. Numerous synthetic methods have been developed and been modified to meet the demands of the applications mentioned above. In particular, the Stcber method is considered as a basic platform for the chemical synthesis of silica spheres: it generally uses ammonia as a catalyst and silicon alkoxide as a precursor in the water/ethanol co-solvent system. Although the conventional Stcber method is quite useful for sizeand shape-control of silica structures, the reaction conditions are harsh owing to the use of ammonia (pH>12) and cannot be applied to biological systems, such as living cells. In this respect, biomimetic (or bio-inspired) silicification, inspired by diatom and glass sponges, was suggested as an alternative approach for the mild and biocompatible formation of silica structures, because it proceeds under physiologically mild conditions (i.e., neutral pH, room temperature, and ambient pressure). The bio-inspired silicification has successfully been applied to the coating of individual living cells without deterioration of cell viability by us. We also have recently reported that individually separated silica microspheres were formed under relatively mild conditions in the presence of cetyltrimethylammonium bromide (CTAB) by using cysteamine (HSCH2CH2NH2) as a biomimetic hydrolysis catalyst, inspired by silicatein, a silica-forming protein, found in glass sponge. It is noteworthy that the diameter of the formed silica spheres was on the micrometer-scale, because microspheres had barely been found in both Stcber and modified Stcber methods. Understanding how the bioinspired silicification was affected by reaction parameters in the cysteamine/CTAB system, such as reactant concentrations and solvents, was required for the detailed elucidation of mechanisms and the morphological control of silica. However, there have been few reports on the effects of the concetrations of silica precursors on silica morphogenesis. Herein, we systematically investigated the effects of the concentration of tetraethyl orthosilicate (TEOS) in detail, along with the ratio of water and ethanol. The synthetic procedure was as follows (Figure 1). The final concentrations of cysteamine and CTAB were fixed to be 50 mm and 5 mm, respectively, after optimization for silica formation. We varied the water/ethanol ratio from 0.6:1 to 1:1 (v/v), and the concentration of TEOS from 80 to 140 mm in 20 mm-intervals. Although the observable changes in the reaction could be seen after 45 minutes, the silicification was performed for 3 hours for comparative studies. The resulting silica precipitates were washed with ethanol several times using centrifugation, dispersed in ethanol, and characterized by attenuated total reflectance infrared (ATR-IR) spectroscopy and field-emission scanning electron microscopy (FE-SEM). The IR spectra showed the characteristic peaks at 1049 (Si O Si asymmetric stretching), 955 (Si O stretching), and 784 cm 1 (Si O Si symmetric stretching) after silicification (for the representative IR spectrum, see the Supporting Information, Figure S1). To investigate the effects of TEOS concentration, we first varied only the concentration of TEOS in the 0.65:1 water/ ethanol system, while keeping the other parameters (the concentrations of cysteamine and CTAB) the same. Of interest, the silica morphology was found to be affected greatly by the concentration of TEOS, as shown in the FE-SEM micrographs (Figure 2a). Specifically, interconnected aggre[a] J. H. Park, J. Y. Choi, T. Park, S. H. Yang, S. Kwon, Prof. Dr. H.-S. Lee, Prof. Dr. I. S. Choi Molecular-Level Interface Research Center Department of Chemistry KAIST Daejeon 305-701 (Korea) Fax: (+82)42-350-2810 E-mail : ischoi@kaist.ac.kr hee-seung_lee@kaist.ac.kr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201100265.

Neuro-Compatible Metabolic Glycan Labeling of Primary Hippocampal Neurons in Noncontact, Sandwich-Type Neuron–Astrocyte Coculture

Glycans are intimately involved in several facets of neuronal development and neuropathology. However, the metabolic labeling of surface glycans in primary neurons is a difficult task because of the neurotoxicity of unnatural monosaccharides that are used as a metabolic precursor, hindering the progress of metabolic engineering in neuron-related fields. Therefore, in this paper, we report a neurosupportive, neuron-astrocyte coculture system that neutralizes the neurotoxic effects of unnatural monosaccharides, allowing for the long-term observation and characterization of glycans in primary neurons in vitro. Polysialic acids in neurons are selectively imaged, via the metabolic labeling of sialoglycans with peracetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz), for up to 21 DIV. Two-color labeling shows that neuronal activities, such as neurite outgrowth and recycling of membrane components, are highly dynamic and change over time during development. In addition, the insertion sites of membrane components are suggested to not be random, but be predominantly localized in developing neurites. This work provides a new research platform and also suggests advanced 3D systems for metabolic-labeling studies of glycans in primary neurons.

Nanotopography-Promoted Formation of Axon Collateral Branches of Hippocampal Neurons

Axon collateral branches, as a key structural motif of neurons, allow neurons to integrate information from highly interconnected, divergent networks by establishing terminal boutons. Although physical cues are generally known to have a comprehensive range of effects on neuronal development, their involvement in axonal branching remains elusive. Herein, it is demonstrated that the nanopillar arrays significantly increase the number of axon collateral branches and also promote their growth. Immunostaining and biochemical analyses indicate that the physical interactions between the nanopillars and the neurons give rise to lateral filopodia at the axon shaft via cytoskeletal changes, leading to the formation of axonal branches. This report, demonstrates that nanotopography regulates axonal branching, and provides a guideline for the design of sophisticated neuron-based devices and scaffolds for neuro-engineering.

Accelerated Development of Hippocampal Neurons and Limited Adhesion of Astrocytes on Negatively Charged Surfaces

This work examines the development of primary neurons and astrocytes on thoroughly controlled functional groups. Negatively charged surfaces presenting carboxylate (COO-) or sulfonate (SO3-) groups prove beneficial to neuronal behavior, in spite of their supposed repulsive electrostatic interactions with cellular membranes. The adhesion and survival of primary hippocampal neurons on negatively charged surfaces are comparable to or slightly better than those on positively charged (poly-d-lysine-coated) surfaces, and neuritogenesis and neurite outgrowth are accelerated on COO- and SO3- surfaces. Moreover, such favorable influences of the negatively charged surfaces are only seen in neurons but not for astrocytes. Our results indicate that the in vitro developmental behavior of primary hippocampal neurons is sophisticatedly modulated by angstrom-sized differences in chemical structure or the charge density of the surface. We believe that this work provides new implications for understanding neuron-material interfaces as well as for establishing new ways to fabricate neuro-active surfaces.

Multiplexed Metabolic Labeling of Glycoconjugates in Polarized Primary Cerebral Cortical Neurons

The spatial distribution of cell-surface glycoconjugates in the brain changes continuously, reflecting neurophysiology especially in the developing phase, but their functions and fates mostly remain unexplored. Their spatiotemporal distribution is particularly important in polarized neuronal cells, such as cerebral cortical neurons composed of a soma and neurites. In this work, we dually labeled sialic acid (Sia5Ac) and N-acetylgalactosamine/glucosamine (GalNAc/GlcNAc) by a neurocompatible strategy of metabolic glycan labeling, metabolism-by-tissues (MbT), and obtained the multiplexed information on their spatiotemporal distribution on polarized cortical neurons. The analyses showed the preferentially distinct distribution of each saccharide set at the late developmental stage after randomized, heterogeneous distribution at the early stage, suggesting that Sia5Ac and GalNAc/GlcNAc are translocated anisotropically during neuronal development.