Eun Hyea Ko

Bioinspired Functionalization of Silica-Encapsulated Yeast Cells†

Cell-surface modification is usually achieved by sophisticated but complicated methods, such as the introduction of nonbiogenic functional groups by metabolic or genetic engineering. Although such methods have evolved into biocompatible and bioorthogonal strategies, the possibility that the direct insertion of functional moieties causes significant perturbations to cell membranes still remains. For a decade, encapsulation methods have been developed as an alternative, indirect approach to cell-surface modifications, as it is thought that the cell integrity would not be perturbed by the encapsulation methods where functional moieties are introduced onto the cell surface without any direct contact with cell membranes. For example, the noncovalent adsorption of macromolecules, mostly by layer-by-layer (LbL) processes, has been utilized to introduce various functionalities, including fluorescent and magnetic properties, catalytic moieties, and supporting templates, to the living cells. On the other hand, recently reported artificial shells, which robustly encapsulate individual living cells, have attracted a great deal of attention as a new approach to cell-surface modifications and formation of artificial spores, because the artificial shells were reported to enhance cell viability and also to control cell division; these factors would be beneficial in the development of biosensor circuits, lab-ona-chip systems, and bioreactors, as well as for fundamental studies in cell biology. It is therefore anticipated that the synergistic combination of the protective encapsulation and the cell-surface functionalization would make a significant step towards the aforementioned applications. Despite the advantages of physically protective shells, the utilization of the artificial shells for practical applications still remains a challenge. The mechanical robustness and chemical inertness of the artificial shells prove beneficial for protecting living cells, but, contradictorily, these properties limit chemical functionalizations of the shells in terms of reactivity. For example, calcium carbonate or calcium phosphate shells lack chemical reactivity. Although the chemistry of silicon is well established, the functionalization of silica shells requires harsh conditions, such as high pH values and harmful solvents. Therefore, it is a prerequisite for any application that the functionalizabilty of the artificial shells is ensured along with the mechanical robustness of the protective shells. Herein we report a bioinspired method for the encapsulation of individual living yeast cells with functionalizable silica shells. Specifically, we used biomimetic silicification, which was inspired by the biosilicification of diatoms. Biomimetic silicification is achieved by specific interactions between silicic acid derivatives and cationic polyamines, such as natural and synthetic peptides, and synthetic polymers: the self-assembled structure of polyamines is thought to act as a catalytic template for the in vivo polycondensation of silicic acid derivatives. We reasoned that chemical functional groups would be introduced directly to the biomimetically formed silica by adding silanol derivatives that contain functional groups in the course of biomimetic polycondensation of silicic acid derivatives. (3-Mercaptopropyl)trimethoxysilane (MPTMS) was selected as a model additive because it was reported to be polycondensed simultaneously with silicic acid under physiologically mild conditions. 12] The functionalizable silica shells formed in this work would expand the utility of artificial shells, because the thiol group in the silica shell can be used for introducing various functions through specific reactions of the thiol moiety with maleimide derivatives under biocompatible conditions (aqueous solution, pH 7.4; Figure 1). The polyelectrolyte multilayer of poly(ethyleneimine) (PEI, Mw: 750 000) and poly(sodium 4-styrenesulfonate) (PSS, Mw: 70000) was used as a catalytic template for biomimetic silicification because previous studies indicated that PEI was biocompatible and acts as a catalyst for biomimetic silica formation. PEI and PSS were alternately deposited onto the surface of Saccharomyces cerevisiae (S. cerevisiae ; baker s yeast). The layer-by-layer processes were initiated with PEI so that electrostatic interactions occur with the negatively charged cell surfaces, and terminated with PEI so that catalytic interactions occur with silicic acid derivatives at the outer interface. For the individual encapsulation of yeast cells with thiol-functionalized silica (SiO2 ; i.e., formation of yeast@SiO2 ), the PEI/PSS multilayercoated cells were placed for 30 min in a silicic acid derivative solution (100 mm), which had been prepared by adding [*] Dr. S. H. Yang, E. H. Ko, 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 Homepage: http://cisgroup.kaist.ac.kr

Nanocoating of single cells: from maintenance of cell viability to manipulation of cellular activities.

The chronological progresses in single-cell nanocoating are described. The historical developments in the field are divided into biotemplating, cytocompatible nanocoating, and cells in nano-nutshells, depending on the main research focuses. Each subfield is discussed in conjunction with the others, regarding how and why to manipulate living cells by nanocoating at the single-cell level.

Interfacing Living Yeast Cells with Graphene Oxide Nanosheaths

The first example of the encapsulation of living yeast cells with multilayers of GO nanosheets via LbL self-assembly is reported. The GO nanosheets with opposite charges are alternatively coated onto the individual yeast cells while preserving the viability of the yeast cells, thus affording a means of interfacing graphene with living yeast cells. This approach is expanded by integrating other organic polymers or inorganic nanoparticles to the cells by hybridizing the entries with GO nanosheets through LbL self-assembly. It is demonstrated that incorporated iron oxide nanoparticles can deliver magnetic properties to the biological systems, allowing the integration of new physical and chemical functions for living cells with a combination of GO nanosheets.

Cytocompatible Encapsulation of Individual Chlorella Cells within Titanium Dioxide Shells by a Designed Catalytic Peptide

The individual encapsulation of living cells has a great impact on the areas of single cell-based sensors and devices as well as fundamental studies in single cell-based biology. In this work, living Chlorella cells were encapsulated individually with abiological, functionalizable TiO(2), by a designed catalytic peptide that was inspired by biosilicification of diatoms in nature. The bioinspired cytocompatible reaction conditions allowed the encapsulated Chlorella cells to maintain their viability and original shapes. After formation of the TiO(2) shells, the shells were postfunctionalized by using catechol chemistry. Our work suggests a bioinspired approach to the interfacing of individual living cells with abiological materials in a controlled manner.

Bioinspired, Cytocompatible Mineralization of Silica–Titania Composites: Thermoprotective Nanoshell Formation for Individual Chlorella Cells†

Hard-shell case: Using a (RKK)4 D8 peptide allows mineralization to occur under cytocompatible conditions. Thus individual Chlorella cells could be encapsulated within a SiO2 -TiO2 nanoshell with high cell viability (87 %). The encapsulated Chlorella showed an almost threefold increase in their thermo-tolerance after 2 h at 45 °C.

Cytoprotective Alginate/Polydopamine Core/Shell Microcapsules in Microbial Encapsulation

Chemical encapsulation of microbes in threedimensional polymeric microcapsules promises various applications, such as cell therapy and biosensors, and provides a basic platform for studying microbial communications. However, the cytoprotection of microbes in the microcapsules against external aggressors has been a major challenge in the field of microbial microencapsulation, because ionotropic hydrogels widely used for microencapsulation swell uncontrollably, and are physicochemically labile. Herein, we developed a simple polydopamine coating for obtaining cytoprotective capability of the alginate capsule that encapsulated Saccharomyces cerevisiae. The resulting alginate/ polydopamine core/shell capsule was mechanically tough, prevented gel swelling and cell leakage, and increased resistance against enzymatic attack and UV-C irradiation. We believe that this multifunctional core/shell structure will provide a practical tool for manipulating microorganisms inside the microcapsules.

Cytoprotective Encapsulation of Individual Jurkat T Cells within Durable TiO2 Shells for T‐Cell Therapy

Lymphocytes, such as T cells and natural killer (NK) cells, have therapeutic promise in adoptive cell transfer (ACT) therapy, where the cells are activated and expanded in vitro and then infused into a patient. However, the in vitro preservation of labile lymphocytes during transfer, manipulation, and storage has been one of the bottlenecks in the development and commercialization of therapeutic lymphocytes. Herein, we suggest a cell-in-shell (or artificial spore) strategy to enhance the cell viability in the practical settings, while maintaining biological activities for therapeutic efficacy. A durable titanium oxide (TiO2 ) shell is formed on individual Jurkat T cells, and the CD3 and other antigens on cell surfaces remain accessible to the antibodies. Interleukin-2 (IL-2) secretion is also not hampered by the shell formation. This work suggests a chemical toolbox for effectively preserving lymphocytes in vitro and developing the lymphocyte-based cancer immunotherapy.

Modulation of Heterotypic and Homotypic Cell-Cell Interactions via Zwitterionic Lipid Masks

Since the pioneering work by Whitesides, innumerable platforms that aim to spatio-selectively seed cells and control the degree of cell-cell interactions in vitro have been developed. These methods, however, have generally been technically and methodologically complex, or demanded stringent materials and conditions. In this work, we introduce zwitterionic lipids as patternable, cell-repellant masks for selectively seeding cells. The lipid masks are easily removed with a routine washing step under physiological conditions (37 °C, pH 7.4), and are used to create patterned cocultures, as well as to conduct cell migration studies. We demonstrate, via patterned cocultures of NIH 3T3 fibroblasts and HeLa cells, that HeLa cells proliferate far more aggressively than NIH 3T3 cells, regardless of initial population sizes. We also show that fibronectin-coated substrates induce cell movement akin to collective migration in NIH 3T3 fibroblasts, while the cells cultured on unmodified substrates migrate independently. Our lipid mask platform offers a rapid and highly biocompatible means of selectively seeding cells, and acts as a versatile tool for the study of cell-cell interactions.

CHAPTER 8:Artificial Spores

Recent progresses on cytocompatible encapsulation of living cells have witnessed their new role for constructing artificial spores. Their cell-in-shell structure emulated essential features of natural endospores that are able to survive under harsh environmental conditions, such as malnutrition, osmotic pressure, lytic enzyme, heat, and UV radiation, as well as the control over cell division. The field of artificial spores is not limited to the mimicry behavior of natural endospores, but also includes shell functionalization for developing cell-based sensor, and provides a basic platform for studying single-cell biology.