Takuya Shishido

Specific Protein Delivery to Target Cells by Antibody-displaying Bionanocapsules

Bionanocapsules (BNCs) are nanoparticles with a high biocompatibility composed of the L protein of the hepatitis B virus surface antigen. BNC can deliver bioactive molecules to hepatocytes efficiently and specifically. However, delivery is limited to hepatocytes and incorporation of proteins into BNC is quite troublesome. Here, in order to alter the specificity of BNC and to achieve efficient protein delivery, we developed engineered BNC displaying the ZZ domain of protein A and incorporating enhanced green fluorescent protein (EGFP) inside the particles using an insect cell expression system. The ZZ domain displayed on the surface of BNC binds to anti-epidermal growth factor receptor (EGFR) antibodies, allowing specific delivery of EGFP to HeLa cells. The engineered BNCs are a promising and powerful tool for efficient and cell-specific protein delivery.

Affibody-displaying bionanocapsules for specific drug delivery to HER2-expressing cancer cells

A novel HER2-targeted carrier was developed using bionanocapsules (BNCs). Bionanocapsules (BNCs) are 100-nm hollow nanoparticles composed of the L-protein of hepatitis B virus surface antigen. An affibody of HER2 was genetically displayed on the BNC surface (Z(HER2)-BNC). For the investigation of binding affinity, Z(HER2)-BNC was incubated with the cancer cell lines SK-BR-3 (HER2 positive), and MDA-MB-231 (HER2 negative). For analysis of HER2 targeting specificity, Z(HER2)-BNC or Z(WT)-BNC (without affibody) was incubated with both SK-BR-3 and MDA-MB-231 cells by time lapse and concentration. For the delivery of encapsulated molecules (calcein), fluorescence of Z(HER2)-BNC mixed with liposomes was also compared with that of Z(WT)-BNC and nude liposomes by incubation with SK-BR-3 cells. As a result, Z(HER2)-BNC-liposome complex demonstrated the delivery to HER2-expressing cells (SK-BR-3) with a high degree of specificity. This indicates that genetically engineered BNCs are promising carrier for cancer treatment.

Biotinylated Bionanocapsules for Displaying Diverse Ligands Toward Cell-specific Delivery

Bionanocapsule (BNC) is hollow nanoparticle composed of the l-protein of the hepatitis B virus surface antigen. BNC allows targeted delivery of either genes or drugs only to hepatocytes, but not to other cell types. In this study, we attempted to alter the specificity of BNC by insertion of biotin-acceptor peptide (BAP), which is efficiently biotinylated using biotin ligase BirA from Escherichia coli. Using streptavidin as a linker, biotinylated BNC could be display various biotinylated ligands that are otherwise difficult to fuse with BNC, such as antibodies, synthetic peptides and functional molecules. BAP-fused BNC was efficiently biotinylated and effectively displayed streptavidin. Furthermore, we demonstrated that biotinylated BNC was internalized into targeted cells via biotinylated Nanobody displayed on the BNC surface. Biotinylated BNC permit display of diverse ligands, and thus have potential as a versatile carrier for drug delivery to a variety of target cells.

Construction of arginine-rich peptide displaying bionanocapsules.

Bionanocapsule (BNC) is a hollow nanoparticle composed of L-protein of the hepatitis B virus surface antigen. BNC can deliver genes or drugs into specific human hepatocytes, but delivery is limited to hepatocytes. In this study, we attempted to alter the specificity of BNCs by genetically introducing cell-penetrating peptides (CPPs), such as arginine-rich peptides, into BNCs. The CPP-fused BNC was efficiently internalized into various cell lines in a short period without significant cytotoxicity. These results show that CPP-BNC could be applied as an efficient carrier for gene and drug delivery.

Protein-encapsulated bio-nanocapsules production with ER membrane localization sequences

Bio-nanocapsules (BNCs) are hollow nanoparticles composed of the L protein of hepatitis B virus (HBV) surface antigen (HBsAg), which can specifically introduce genes and drugs into various kinds of target cells. Although the classic electroporation method has typically been used to introduce highly charged molecules such as DNA, it is rarely adopted for proteins due to its very low efficiency. In this study, a novel approach to the preparation of BNC was established whereby a target protein was pre-encapsulated during the course of nanoparticle formation. Briefly, because of the process of BNC formation in a budding manner on the endoplasmic reticulum (ER) membrane, the association of target proteins to the ER membrane with lipidation sequences (ER membrane localization sequences) could directly generate protein-encapsulating BNC in collaboration with co-expression of the L proteins. Since the membrane-localized proteins are automatically enveloped into BNCs during the budding event, this method can be protect the proteins and BNCs from damage caused by electroporation and obviate the need for laborious consideration to study the optimal conditions for protein encapsulation. This approach would be a useful method for encapsulating therapeutic candidate proteins into BNCs.