Masayasu Mie

Growth Factor Tethering to Protein Nanoparticles via Coiled-Coil Formation for Targeted Drug Delivery

Protein-based nanoparticles are attractive carriers for drug delivery because they are biodegradable and can be genetically designed. Moreover, modification of protein-based nanoparticles with cell-specific ligands allows for active targeting abilities. Previously, we developed protein nanoparticles comprising genetically engineered elastin-like polypeptides (ELPs) with fused polyaspartic acid tails (ELP-D). Epidermal growth factor (EGF) was displayed on the surface of the ELP-D nanoparticles via genetic design to allow for active cell-targeting abilities. Herein, we focused on the coiled-coil structural motif as a means for noncovalent tethering of growth factor to ELP-D. Specifically, two peptides known to form a heterodimer via a coiled-coil structural motif were fused to ELP-D and single-chain vascular endothelial growth factor (scVEGF121), to facilitate noncovalent tethering upon formation of the heterodimer coiled-coil structure. Drug-loaded growth factor-tethered ELP-Ds were found to be effective against cancer cells by provoking cell apoptosis. These results demonstrate that tethering growth factor to protein nanoparticles through coiled-coil formation yields a promising biomaterial candidate for targeted drug delivery.

Targeting of EGF-displayed protein nanoparticles with anticancer drugs.

The development of protein-based carriers for drug delivery has been well studied. We previously constructed a protein-based nanoparticle consisting of genetically engineered elastin-like polypeptides (ELPs) with a fused poly-aspartic acid tail (ELPD ). The size of the self-assembled ELPD nanoparticles was regulated by charged repulsion of the poly-aspartic acid chains. In the present study, epidermal growth factor (EGF) was genetically fused to the C-terminus of ELPD to impart an active targeting ability to the ELPD nanoparticles. We examined the nanoparticle formation with EGF as well as its targeting ability. ELPD with fused EGF was found to form nanoparticles that displayed multivalent EGFs on their surface. EGF-displayed nanoparticles loaded with the anti-cancer drug paclitaxel were internalized into cells overexpressing the EGF receptor, and induced cell death.

Construction of nanoscale protein particle using temperature-sensitive elastin-like peptide and polyaspartic acid chain.

Temperature-responsive monodisperse spheres are useful for various in vivo and in vitro applications. Size, response temperature and biocompatibility are particularly important consideration with in vivo applications. In this work, we constructed fusion proteins of low antigenic elastin-like peptide (ELP) and a polyaspartic acid chain, and studied the particles that had a favorable size and temperature of formation of particle. From DLS analysis, we confirmed that some of them formed particles with less than 100nm in diameter around 37 degrees C, while the diameter of ELPs alone is larger than 1microm in diameter. The (PGVGV)(160)D(22), which is composed of a short aspartic acid chain and a long ELP region, had a tendency to form large particles. The temperature of formation and collapse of the protein particle were dependent on the length of the ELP and the polyaspartic acid chain, and the concentration of proteins. The direct observation with TEM indicated that the morphologies of the particles were spherical except when (PGVGV)(160)D(22) was used. The intensities of the environment-sensitive hydrophobic fluorescence increased at 37 degrees C more than 1.5 times as much as at 25 degrees C both in free form and modified at the ELP region. These results indicated that the polarity of the environment surround the fluorescence decreased or the movement of fluorescence was limited, and thus, implied that the ELP formed a more hydrophobic or rigid region and could hold hydrophobic drugs. These results suggest that a temperature-responsive protein particle with favorable size and temperature of formation can be constructed that is suitable for any in vitro or in vivo application.

Construction of multi-functional extracellular matrix proteins that promote tube formation of endothelial cells

We developed artificial extracellular matrix proteins designed to have collagen-binding activity and active functional units that promote network formation of vascular endothelial cells. We engineered a laminin-derived IKVAV sequence, which stimulates capillary network formation of vascular endothelial cells, to incorporate into an elastin-derived structural unit. The designed fusion protein also had a cell-adhesive RGD sequence and a collagen-binding domain derived from fibronectin. The resultant fusion protein could bind to collagen type I and promote angiogenic activity of collagen gel. The collagen-binding domain also had slight angiogenic activity; however, the designed fusion protein also enhanced cellular migration activity. The engineering strategy of designing multi-functional ECM proteins has a possibility for supporting current tissue engineering techniques.

Ribosome display for selection of active dihydrofolate reductase mutants using immobilized methotrexate on agarose beads

Ribosome display was applied to the selection of an enzyme. As a model, we selected and amplified the dihydrofolate reductase (DHFR) gene by ribosome display utilizing a wheat germ cell‐free protein synthesis system based on binding affinity to its substrate analog, methotrexate, immobilized on agarose beads. After three rounds of selection, the DHFR gene could be effectively selected and preferentially amplified from a small proportion in a mixture also containing competitive genes. Active enzymes were expressed and amplified and by sequence analysis, four mutants of DHFR were identified. These mutants showed as much activity as the wild‐type enzyme.

The promotion of angiogenesis by growth factors integrated with ECM proteins through coiled-coil structures

An appropriate method to bind extracellular matrix (ECM) proteins and growth factors using advanced protein engineering techniques has the potential to enhance cell proliferation and differentiation for tissue regeneration and repair. In this study we developed a method to co-immobilize non-covalently an ECM protein to three different types of growth factors: basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and single-chain vascular endothelial growth factor (scVEGF121) through a coiled-coil structure formed by helixA/helixB in order to promote angiogenesis. The designed ECM was established by fusing two repeats of elastin-derived unit (APGVGV)(12), cell-adhesive sequence (RGD), laminin-derived IKVAV sequence and collagen-binding domain (CBD) to obtain CBDEREI2. HelixA was fused to each growth factor and helixB to the engineered ECM. Human umbilical vein endothelial cells (HUVECs) were cultured on engineered ECM and growth factors connected through the coiled-coil formation between helixA and helixB. Cell proliferation and capillary tube-like formation were monitored. Moreover, the differentiated cells with high expression of Ang-2 suggested the ECM remodeling. Our approach of non-covalent coupling method should provide a protein-release control system as a new contribution in biomaterial for tissue engineering field.

Construction of multifunctional proteins for tissue engineering: Epidermal growth factor with collagen binding and cell adhesive activities

The development of different techniques based on natural and polymeric scaffolds are useful for the design of different biomimetic materials. These approaches, however, require supplementary steps for the chemical or physical modification of the biomaterial. To avoid such steps, in the present study, we constructed a new multifunctional protein that can be easily immobilized onto hydrophobic surfaces, and at the same time helps enhance specific cell adhesion and proliferation onto collagen substrates. A collagen binding domain was fused to a previously constructed protein, which had an epidermal growth factor fused to a hydrophobic peptide that allows for cell adhesion. The new fusion protein, designated fnCBD-ERE-EGF is produced in Escherichia coli, and its abilities to bind to collagen and promote cell proliferation were investigated. fnCBD-ERE-EGF was shown to keep both collagen binding and cell growth-promoting activities comparable to those of the corresponding unfused proteins. The results obtained in this study also suggest the use of a fnCBD-ERE-EGF as an alternative for the design of multifunctional ECM-bound growth factor based materials.

Osteogenesis Coordinated in C3H10T1/2 Cells by Adipogenesis-Dependent BMP-2 Expression System

A novel tissue engineering for bone formation has been proposed, to make osteoblast differentiation balanced by transfecting the mesenchymal stem cells with a gene encoding human bone morphogenetic protein-2 (hBMP-2) under the control of adipocyte specific lipoprotein lipase (LPL) promoter. Due to the promoter specificity, the initiation of BMP transcription is dependent on adipogenesis. For 14-day culture in the presence of ascorbic acid (asc) and beta-glycerophosphate (gly), nontransfected mouse embryonic fibroblast C3H10T1/2 (10T1/2) cells showed extensive accumulation of lipid droplets and adipocyte specific enzyme glycerol-3-phosphate dehydrogenase (G3PDH) mRNA expression, but exhibited neither BMP-2 expression, high alkaline phosphatase (ALP) activity which reflects osteoblast phenotype. On the other hand, transfected 10T1/2 cells showed hBMP-2 expression, high ALP activity and low level of G3PDH. mRNA expression accompanied with minimal lipid droplets. These results indicate that 10T1/2 cells are proved to be differentiated with maintaining coordinated balance of adipogenesis and osteogenesis, when they are transfected by the gene encoding hBMP-2 under the control of LPL promoter.

Non-destructive monitoring of rpoS promoter activity as stress marker for evaluating cellular physiological status

To monitor the extent of cellular physiological stress, the activity of the rpoS promoter was evaluated as a marker of the stress pathway. A reporter plasmid was constructed by inserting the GFPuv gene under the rpoS promoter and used to transform Escherichia coli cells. The fluorescence of the GFPuv protein was measured in intact cells in a non-destructive manner. The physiological status of the cells could be conveniently monitored using the rpoS-GFPuv reporter gene with respect to the cellular growth phase and to elevated ethanol and NaCl concentrations as two examples of environmental stress factors. Comparison of the response of different E. coli strains demonstrated an essential role of the relA gene in the induction of the rpoS-GFPuv reporter gene.

Induction of neural differentiation by electrically stimulated gene expression of NeuroD2

Regulation of cell differentiation is an important assignment for cellular engineering. One of the techniques for regulation is gene transfection into undifferentiated cells. Transient expression of NeuroD2, one of neural bHLH transcription factors, converted mouse N1E-115 neuroblastoma cells into differentiated neurons. The regulation of neural bHLH expression should be a novel strategy for cell differentiation. In this study, we tried to regulate neural differentiation by NeuroD2 gene inserted under the control of heat shock protein-70 (HSP) promoter, which can be activated by electrical stimulation. Mouse neuroblastoma cell line, N1E-115, was stably transfected with expression vector containing mouse NeuroD2 cDNA under HSP promoter. Transfected cells were cultured on the electrode surface and applied electrical stimulation. After stimulation, NeuroD2 expression was induced, and transfected cells adopt a neuronal morphology at 3 days after stimulation. These results suggest that neural differentiation can be induced by electrically stimulated gene expression of NeuroD2.