Construction of silica-enhanced S-layer protein cages

The work presented here shows for the first time that it is possible to silicify S-layer coated liposomes and to obtain stable functionalized hollow nano-containers. For this purpose, the S-layer protein of Geobacillus stearothermophilus PV72/p2 was recombinantly expressed and used for coating positively charged liposomes composed of dipalmitoylphosphatidylcholine, cholesterol and hexadecylamine in a molar ratio of 10:5:4. Subsequently, plain (uncoated) liposomes and S-layer coated liposomes were silicified. Determination of the charge of the constructs during silicification allowed the deposition process to be followed. After the particles had been silicified, lipids were dissolved by treatment with Triton X-100 with the release of previously entrapped fluorescent dyes being determined by fluorimetry. Both, ζ-potential and release experiments showed differences between silicified plain liposomes and silicified S-layer coated liposomes. The results of the individual preparation steps were examined by embedding the respective assemblies in resin, ultrathin sectioning and inspection by bright-field transmission electron microscopy (TEM). Energy filtered TEM confirmed the successful construction of S-layer based silica cages. It is anticipated that this approach will provide a key to enabling technology for the fabrication of nanoporous protein cages for applications ranging from nano medicine to materials science.