A multi-functional polyhydroxybutyrate nanoparticle for theranostic applications.

Biopolymer-based multi-functional nanoparticles have been developed through a one-step enzymatic polymerization reaction using engineered polyhydroxyalkanoate (PHA) synthase fused with green fluorescent protein (GFP) and a single chain variable fragment antibody (A33scFv) specific to colon cancer. PHA synthase possesses unique catalytic characteristics, namely covalent catalysis, by which the synthesized polyhydroxybutyrate chain remains covalently attached to the enzyme. The amphiphilic nature of the resulting protein-polymer hybrid gives rise to spontaneous self-assembly into a micellar structure with GFP and A33scFv displayed on the surface (AGPHB nanoparticle). A model compound, Nile red, was loaded into the hydrophobic core of the AGPHB nanoparticle during the polymerization and self-assembly process. The specificity of the fluorescent multi-functional AGPHB nanoparticle towards the colon cancer cell lines SW1222 (A33+) and HT29 (A33-) was confirmed and analysed quantitatively in vitro. This new biological approach provides a simple means of producing nanocarriers with a range of surface functionality and the sizes desired for imaging and targeted drug delivery.

Removal of pathogenic factors from 2,3-butanediol-producing Klebsiella species by inactivating virulence-related wabG gene.

Klebsiella species are the most extensively studied among a number of 2,3-butanediol (2,3-BDO)-producing microorganisms. The ability to metabolize a wide variety of substrates together with the ease of cultivation made this microorganisms particularly promising for the application in industrial-scale production of 2,3-BDO. However, the pathogenic characteristics of encapsulated Klebsiella species are considered to be an obstacle hindering their industrial applications. Here, we removed the virulence factors from three 2,3-BDO-producing strains, Klebsiella pneumoniae KCTC 2242, Klebsiella oxytoca KCTC1686, and K. oxytoca ATCC 43863 through site-specific recombination technique. We generated deletion mutation in wabG gene encoding glucosyltransferase which plays a key role in the synthesis of outer core lipopolysaccharides (LPS) by attaching the first outer core residue D-GalAp to the O-3 position of the L,D-HeppII residue. The morphologies and adhesion properties against epithelial cells were investigated, and the results indicated that the wabG mutant strains were devoid of the outer core LPS and lost the ability to retain capsular structure. The time profile of growth and 2,3-BDO production from K. pneumoniae KCTC 2242 and K. pneumoniae KCTC 2242 ΔwabG were analyzed in batch culture with initial glucose concentration of 70 g/l. The growth was not affected by disrupting wabG gene, but the production of 2,3-BDO decreased from 31.27 to 22.44 g/l in mutant compared with that of parental strain. However, the productions of acetoin and lactate from wabG mutant strain were negligible, whereas that from parental strain reached to ~5 g/l.

Tumor-specific hybrid polyhydroxybutyrate nanoparticle: surface modification of nanoparticle by enzymatically synthesized functional block copolymer.

A new approach to functionalize the surface of hydrophobic nanocarrier through enzymatic polymerization was demonstrated. The effective coupling between the hydrophobic surface of PHB nanoparticle and PHB chain grown from the enzyme fused with a specific ligand provided a simple way of functionalizing nanoparticles with active protein layers in aqueous environment. PHB nanoparticles loaded with model drug molecule, Nile red, were prepared through oil-in-water emulsion solvent evaporation method and the surface of nanoparticles were functionalized with tumor-specific ligand, RGD4C, fused with PHA synthase that drove the coupling reaction. The functionalized PHB nanoparticles showed a specific affinity to MDA-MB 231 breast cancer cells indicating that the tumor-specific ligand, RGD4C, was effectively displayed on the surface of PHB nanoparticles through enzymatic modification and confers targeting capability on the drug carrier.