A bilayered nanoshell for durable protection of single yeast cells against multiple, simultaneous hostile stimuli

Abstract

Single cell surface engineering provides the most efficient, non-genetic strategy to enhance cell stability. However, it remains a huge challenge to improve cell stability in complex artificial environments. Here, a soft biohybrid interfacial layer is fabricated on individual living-cell surfaces by their exposure to a suspension of gold nanoparticles and l-cysteine to form a protecting functional layer to which porous silica layers were bound yielding pores with a diameter of 3.9 nm. The living cells within the bilayered nanoshells maintained high viability (96 ± 2%) as demonstrated by agar plating, even after five cycles of simultaneous exposure to high temperature (40 °C), lyticase and UV light. Moreover, yeast cells encapsulated in bilayered nanoshells were more recyclable than native cells due to nutrient storage in the shell.

Fig. 1. Bilayered nanoshell formation on a single yeast cell. (A) Exposure of a cell (i) to a solution containing l-cysteine-coated gold nanoparticles, (ii) yielding a cell with a biohybrid layer, followed by self-assembly of silica, (iii) yielding a bilayered nanoshell. Artificial colors were used in the SEM micrographs (the scale bar equals 1 μm). Authentic micrographs can be found in Fig. S1. (B) Sequential steps in the formation of a bilayered nanoshell: (i) a yeast cell surface possessing abundant hydroxyl groups and sparsely distributed amino groups and carboxyl groups, (ii) a biohybrid layer composed of amino-covered gold nanoparticle groups and carboxyl groups (yellow dots and blue layers represent gold nanoparticles and l-cysteine molecules, respectively), and (iii) the silica outer surface, exposing hydroxyl groups. Red dashed lines represent hydrogen bonding between functional groups on the cell surface with the biohybrid layer and functional groups of the biohybrid layer with the amorphous silica layer, as indicated in the lower part.

Fig. 2. Characterization of bilayered nanoshells, encapsulating a single S. cerevisiae cell. (A) TEM micrograph of individual gold nanoparticles (denoted as GNP). (B) Nanopores in the biohybrid layer. (C) High-magnification SEM micrograph of a cross-section of the bilayer around a yeast cell. (D) ¹³C solid-state NMR spectra of different components making up the bilayered nanoshell. (E) TEM micrographs of an encapsulated yeast cell with a bilayered nanoshell (indicated by the black square), together with a higher magnification image of the biohybrid layer (top left inset; the scale bar equals 200 nm) and the boundary of the inner and outer layers indicated (top right inset; the scale bar equals 200 nm). The inner and outer layers are indicated by arrows while their boundary is indicated for clarity by the red dashed line. (F) N₂ adsorption/desorption isotherm and the corresponding pore-size distribution (inset) of silica layers. Measurements were taken at standard temperature and pressure (STP) of 1 atmosphere and 0 °C.

Fig. 3. Viability of S. cerevisiae after multiple cycles of simultaneous or single hostile stimuli, assessed using agar plating. (A) Yeast cell viability after multiple cycles of simultaneous hostile stimuli with lyticase, high temperature (40 °C) and UV light. (B) Under similar conditions, but with lyticase exposure alone. (C) Under similar conditions, but with high temperature exposure alone. (D) Under similar conditions, but with UV light exposure alone. Data indicate the percentage of colony forming units on agar plates, with error bars indicating standard deviations over five separate experiments with different yeast cultures. 100% viability represents pre-stimulus CFU levels. (E) Proposed mechanisms of protection by bilayered nanoshells against the different single stimuli applied: (i) lyticase, (ii) thermal stress, and (iii) UV radiation. Arrows indicate the penetration of stimuli through the outer silica layer and inner biohybrid layer.

Fig. 4. Protection of S. cerevisiae cells encapsulated in bilayered nanoshells against lyticase, high temperature and UV light exposure. (A) SEM images of (i) native cells, (ii) cells encapsulated in a biohybrid layer alone (the scale bar equals 1 μm) and (iii) cells encapsulated in a bilayered nanoshell (the scale bar equals 5 μm) after exposure to a temperature of 40 °C for 12 h. (B) Simulated (i) attenuation of thermal stress and (ii) temperature distribution through the silica (grey shaded) and biohybrid layer (blue shaded) to the cell (green shaded). The scale bar equals 200 nm. (C) Surface temperature of differently encapsulated yeast cells exposed to a range of different surrounding temperatures (36–54 °C). Data represent averages with standard deviations over 3 separate yeast cultures. (D) UV absorption spectra of (i) biohybrids and (ii) silica. The inset shows the percentage reflection of light through the amorphous silica layer. (E) Simulated (i) attenuation of UV light and (ii) intensity distribution through the outer silica layer (grey shaded) and inner biohybrid layer (blue shaded). The scale bar equals 200 nm. The arrow indicates the direction of light propagation.

Fig. 5. Post-functionalization of S. cerevisiae cells encapsulated in bilayered nanoshells. (A) Schematic of post-functionalized, bilayered nanoshell encapsulated cells with graphene and magnetic nanoparticles. (B) SEM image of a yeast cell with a graphene-based bilayered nanoshell (the scale bar equals 5 μm). (C) Electrical conductivity of differently encapsulated yeast cells and yeast cells with a graphene-based bilayered nanoshell. (D) SEM image of Fe₃O₄-based bilayered nanoshell encapsulated cells and EDX line scan for elemental Fe (inset). The scale bar equals 5 μm. (E) Magnetic separation of yeast cells encapsulated with magnetic iron oxide nanoparticles. The scale bar equals 0.5 cm. (F) Viability of yeast cells encapsulated in bilayered nanoshells with post-functionalities after multiple cycles of simultaneous hostile stimuli with lyticase, high temperature (40 °C) and UV light. Note that cells in bilayered nanoshells with and without graphene or Fe₃O₄ demonstrate comparable viability after recycling (see also Fig. 3A).