Highly secretory expression of recombinant cowpea chlorotic mottle virus capsid proteins in Pichia pastoris and in-vitro encapsulation of ruthenium nanoparticles for catalysis.

The applications of viral protein cages have expanded rapidly into the fields of bionanotechnology and materials science. However, the low-cost production of viral capsid proteins (CPs) on a large scale is always a challenge. Herein, we develop a highly efficient expression system by constructing recombinant Pichia pastoris cells as a "factory" for the secretion of soluble cowpea chlorotic mottle virus (CCMV) CPs. Under optimal induction conditions (0.9 mg/mL of methanol concentration at 30 °C for 96 h), a high yield of approximately 95 mg/L of CCMV CPs was harvested from the fermentation supernatant with CPs purity >90%, which has significantly simplified the rest of the purification process. The resultant CPs are employed to encapsulate Ruthenium (Ru) nanoparticles (NPs) via in-vitro self-assembly to prepare hybrid nanocatalyst, i.e. Ru@virus-like particles (VLPs). The catalytic activity over Ru@VLPs was evaluated by reducing 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). The results indicate that, with the protection of protein cages, Ru NPs were highly stabilized during the catalytic reaction. This results in enhanced catalytic activity (reaction rate constant k = 0.14 min-1) in comparison with unsupported citrate-stabilized Ru NPs (Ru-CA) (k = 0.08 min-1). Additionally, comparatively lower activation energy over Ru@VLPs (approximately 32 kJ/mol) than that over Ru-CA (approximately 39 kJ/mol) could be attributed to the synergistic effect between Ru NPs and some functional groups such as amino groups (-NH2) on CPs that weakened the activation barrier of 4-NP reduction. Therefore, enhanced activity and decreased activation energy over Ru@VLPs demonstrated the superiority of Ru@VLPs to unsupported Ru-CA.

PEGylated reduced graphene oxide as a superior ssRNA delivery system.

Single stranded ribonucleic acid (ssRNA) acts as a probe, antisense (AS), miRNA analog and inhibitor, and is promising for gene therapy and molecular diagnosis. However, free ssRNA exhibits poor cellular uptake due to its negative charges, and enzyme instability, which have largely limited the practical applications of ssRNA in biomedicine. To address these issues, we have developed a PEGylated reduced graphene oxide (PEG-RGO) nanovector for efficient delivery of ssRNA. We have demonstrated that PEG-RGO exhibits superior ssRNA loading and delivery capability, compared to the widely studied PEGylated graphene oxide (PEG-GO). Computational simulation further suggested that PEG-RGO binds ssRNA much stronger than PEG-GO, consistent with the experimental results. These results will have implications in designing RGO-based biocompatible and efficient ssRNA delivery systems.

Rotavirus capsid surface protein VP4-coated Fe(3)O(4) nanoparticles as a theranostic platform for cellular imaging and drug delivery.

The development of a theranostic nanoplatform based on rotavirus structural protein VP4-coated Fe(3)O(4) nanoparticles (NPs) for dual modality magnetic resonance/fluorescence cellular imaging and drug delivery is reported. VP4 protein was obtained from Escherichia coli approach, and then chemically conjugated to Fe(3)O(4) NPs premodified with meso-2,3-dimercaptosuccinnic acid (DMSA) in the presence of 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC). Next, the VP4-coated Fe(3)O(4) NPs were loaded with doxorubicin (DOX), a typical anticancer drug, via formation of amide bond through the EDC approach. Prussian blue staining analysis reveals that the VP4-coated Fe(3)O(4) NPs can be internalized efficiently by MA104 and HepG2 cells, thereby significantly improving cellular MRI sensitivity, compared with dextran- and BSA-coated Fe(3)O(4) NPs. In addition, DOX loaded on the VP4-coated Fe(3)O(4) NPs exhibits significant cytotoxicity to the cancer cells (HepG2). The current work provides a general approach toward the rational design and synthesis of a versatile theranostic nanoplatform based on functional protein-coated magnetic NPs with good biocompatibility, biodegradability, and capability of simultaneously performing multimodality imaging and therapy for optimal clinical outcomes.

Self-assembled virus-like particles from rotavirus structural protein VP6 for targeted drug delivery.

Proteins of viral capsid may self-assemble into virus-like particles (VLPs) that can find many biomedical applications such as platform for drug delivery. In this paper, we describe preparation of VLPs by self-assembly of VP6, a rotavirus capsid protein that was chemically conjugated with doxorubicin (DOX), an anticancer drug. VP6 was first highly expressed in E. Coli, followed by purification and renaturation. DOX was then covalently attached to VP6 to form DOX-VP6 (DVP6) conjugates, which were subsequently self-assembled into VLPs under appropriate condition. Next, lactobionic acid (LA) was chemically linked to the surface of the VLPs. We demonstrated that the aforementioned nanosystem shows specific targeting to hepatoma cell line HepG2. The chemically functionalized VLPs, a kind of biological nanoparticles with excellent biocompatibility and biodegradability, can be prepared in large scale from E. Coli through our method, which may find practical applications in biomedicine.

Functional graphene oxide as a nanocarrier for controlled loading and targeted delivery of mixed anticancer drugs.

A simple synthetic route for the preparation of functional nanoscale graphene oxide (NGO), a novel nanocarrier for the loading and targeted delivery of anticancer drugs, is reported. The NGO is functionalized with sulfonic acid groups, which render it stable in physiological solution, followed by covalent binding of folic acid (FA) molecules to the NGO, thus allowing it to specifically target MCF-7 cells, human breast cancer cells with FA receptors. Furthermore, controlled loading of two anticancer drugs, doxorubicin (DOX) and camptothecin (CPT), onto the FA-conjugated NGO (FA-NGO) via pi-pi stacking and hydrophobic interactions is investigated. It is demonstrated that FA-NGO loaded with the two anticancer drugs shows specific targeting to MCF-7 cells, and remarkably high cytotoxicity compared to NGO loaded with either DOX or CPT only. Considering that the combined use of two or more drugs, a widely adopted clinical practice, often displays much better therapeutic efficacy than that of a single drug, the controlled loading and targeted delivery of mixed anticancer drugs using these graphene-based nanocarriers may find widespread application in biomedicine.

Full genomic analysis of human rotavirus strain TB-Chen isolated in China.

A G2P[4]/NSP4[A] rotavirus strain TB-Chen was isolated from a 2-year-old patient hospitalized with acute gastroenteritis in Kunming, China. The strain TB-Chen was demonstrated having group A-specific antigenicity, a "short" (subgroup II) electropherotype. To investigate its overall genomic relatedness and to determine which group it belonged, the complete genome of strain TB-Chen was determined. Genomic comparison based on amino acid sequence identity and phylogenetic analysis revealed that all 11 gene segments of strain TB-Chen were highly identical (>91.80%) with the representative G2P[4]/NSP4[A] human strains DS-1, S2, NR1 and IS2, suggesting that this rotavirus strain was derived from human host. Besides, almost all the available representative rotavirus gene segments among group A were analyzed and identified within 15 G-types, 28 P-types, and 6 NSP4 genotypes. This is the first report of group A rotavirus genomic analyses in China and the findings have important implications for rotavirus vaccine development.