Chemical sporulation and germination of mammalian cells

Abstract

Nature finds ways of protecting cellular components and preserving genetic information against external stresses such as nutrient deprivation, desiccation, high temperatures, radiation, and caustic chemicals. For example, bacteria and fungi are covered with a cell wall or robust membrane composed of polysaccharides or peptidoglycans. Certain unicellular organisms, such as diatoms, foraminifera, coccoliths, and rhabdoliths, are encased within exoskeletal shells made of silica or calcium carbonate. However, most mammalian cells do not have a robust membrane or exoskeleton. Instead, they are enclosed in a lipid bilayer, which is fluidic and vulnerable to changes in its environments. Because the surfaces of mammalian cells are not protected or reinforced by a tough coat, it is more difficult to treat mammalian cells in vitro than microbial cells. In this thesis, we report a cytoprotective degradable nanocoat for mammalian cells. Three types of mammalian cells (HeLa cells, NIH 3T3 fibroblasts, and Jurkat cells) were individually coated within metal polyphenol and silica shells, inspired by biomimetic silicification. To maintain the viability of the mammalian cells, we performed the whole processes under strictly physiological culture conditions, and carefully selected nontoxic materials, established with toxicity tests. Bioinspired silicification has attracted a great deal of attention as a cytocompatible method for encapsulating proteins and living cells, because it does not require harsh conditions that harm proteins or cells. Although bioinspired silicification has successfully been used to encapsulate individual microbial cells, which have durable cell walls, the conditions required to coat mammalian cells with silica are yet to be optimized because mammalian cells are inherently fragile. We systematically varied the silicification conditions and optimized them to coat HeLa cells, the model cells, with silica. We found that the use of Dulbecco’s modified Eagle’s medium (DMEM) greatly increased cell viability during silicification, and also optimized the reagent concentrations and reaction time for coating individual HeLa cells with cytocompatible silica. The HeLa cells, in suspension, were then individually coated with silica, in a cytocompatible way with bioinspired silicification. The silica coating greatly enhanced the resistance of the HeLa cells to enzymatic attack by trypsin and the toxic compound poly-(allylamine hydrochloride), and suppressed cell division in a controlled fashion. This bioinspired cytocompatible strategy for coating single cells was also applied to NIH 3T3 fibroblasts and Jurkat cells. Individual HeLa cells were also coated with titania $(TiO_2)$ using a specifically designed peptide that was both cytocompatible and catalytic for the formation of $TiO_2$. After the cytocompatibility of the catalytic templates was established, HeLa cells were encapsulated by immersing them alternately in solutions of $(RKK)_4D_8$ and titanium bis(ammonium lactato)dihydroxide. The viability of the coated HeLa cells was maintained and the cells showed greatly enhanced resistance to ultraviolet C (UV-C) radiation. Tannic acid (TA), a type of polyphenol containing 1,2,3-trihydroxybenzoic acid (gallic acid), can chelate $Fe^{III}$ in seconds (~10 s) under biocompatible conditions (aqueous solution, neutral pH), generating a high-ly stable metal organic nanofilm. The nanofilm formed of TA and $Fe^{III}$ (TA- $Fe^{III}$) is structurally rigid and degradable by external stimuli under mild conditions, and can therefore be used to cytocompatibly nanocoat mammalian cells. Taking advantage of the process that forms the TA-$Fe^{III}$ complex, three types of mammalian cells (HeLa, NIH 3T3, and Jurkat T cells) were coated with TA-$Fe^{III}$. Briefly, a suspension of cells was prepared in serum-free DMEM (pH 7.4) as the reaction medium. Solutions of $FeCl_3$ (0.1 mg/mL^{-1})$ and TA $(0.4 mg/mL^{-1})$ were added sequentially to the cells and incubated for 10 s. The TA-$Fe^{III}$ nanocoat effectively protected the coated mammalian cells against UV-C radiation and a toxic compound. More importantly, cell proliferation was controlled by the programmed formation and degradation of the TA-$Fe^{III}$ nanocoat, mimicking the sporulation and germination processes found in nature. The chemical control of cell division has attracted much attention in the fields of single-cell-based biology, high-throughput screening platforms, and clinical applications. For example, the loss of growth control is a characteristic of many disease states, including cancer. A mussel-inspired cytocompatible encapsulation method to control cell division with cross-linked layer-by-layer (LbL) shells is developed. Catechol-grafted polyethyleneimine and hyaluronic acid were chosen as the polyelectrolytes for the LbL process, and were cross-linked at pH 8.5. Cell division was controlled by the number of LbL nanolayers and the cross-linking reaction. Cells replicate and segregate their genetic material in specific phases of the cell cycle. Because the growth of the coated cells was retarded, we investigated the changes in the cell cycles of the TA-$Fe^{III}$ -coated HeLa cells. Before the HeLa cells were coated with TA-$Fe^{III}$, we synchronized the cell cycle of the cells to M/G1 or G1//S using the double thymidine block method, and the state of the cell cycle was confirmed with Fucci cell-cycle staining and propidium iodide.