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