Use of biosilica to improve loading and delivery of bone morphogenic protein 2.

The clinical utility of bone morphogenetic protein 2 (BMP2) is limited because of the poor attraction between BMP2 and carriers, resulting in low loading efficiency and initial burst release. Here, the high binding affinity of BMP2 to the biosilica surface was utilized to overcome this limitation. Atomic force microscopy revealed that BMP2 bound nearly 8- and 2-fold more strongly to biosilica-coated hydroxyapatite than to uncoated and plain silica-coated hydroxyapatite, respectively. To achieve controlled release, collagen was introduced between the silica layers on hydroxyapatite, which was optimized by adjusting the collagen concentration and number of layers. The optimal biosilica/collagen formulation induced sustained BMP2 release without compromising loading efficiency. BMP2 combined with the mentioned formulation led to an increase in osteogenesis, as compared to the combination of BMP2 with either biosilica-coated or non-coated hydroxyapatite in vitro. In rat calvarial defect models, the biosilica/collagen-coated hydroxyapatite with 1 µg BMP2 showed 26 % more bone regeneration than the same dose of BMP2-loaded hydroxyapatite and 10.6 % more than hydroxyapatite with 2.5-fold dose of BMP2. Using BMP2 affinity carriers coated with biosilica/collagen allows for more efficacious in situ loading and delivery of BMP2, making them suitable for the clinical application of growth factors through a soaking method.

Chimeric protein-mediated dual mineral formation on biopolymer: Non-segregated and well-distributed deposition of CaCO3 and silica particles.

Incorporation of biogenic or biocompatible synthetic polymers with inorganic mineral components have been suggested for the preparation of more bioactive materials. However, when two different inorganic minerals such as Ca- and Si-based minerals are introduced onto organic polymers, each mineral is deposited in a segregated form. Here, we presented a biomolecule-mediated preparation method for dual mineral-deposited polymer, in which two inorganic minerals were well-deposited on organic polymer with the aid of biological molecules. A chimeric bio-macromolecules, a fusion protein (CA-SFP) of carbonic anhydrase (CA) and silica-forming peptide (SFP), was designed and used. Surface-immobilized CA-SFP enabled the deposition of CaCO3 and silica nanoparticles on biopolymer without any segregated aggregation. SEM, EDS, FTIR, and swelling ratio analysis indicated that in the developed dual mineral-deposited polymer, each mineral was well-distributed across the polymer surfaces. Investigation by MTS assays, fluorescent imaging, and RT-qPCR revealed that the dual mineral-deposited polymer, when used as bone scaffolds, led to better cell proliferation and differentiation without any significant cytotoxicity compared to the counterparts. These results show that our mineral-deposition method mediated by biomolecules not only overcomes mineral-segregation involving multi-mineral formations, but also facilitates the preparation of highly-bioactive composite materials.

Bone Graft Biomineral Complex Coderived from Marine Biocalcification and Biosilicification.

Bone graft materials have been mainly developed based on inorganic materials, including calcium phosphate. However, these graft materials usually act as osteoconductive rather than osteoinductive scaffolds. To improve bone reconstruction, a combination of several materials has been proposed. However, there are still no alternatives that can completely replace the existing animal-derived bone graft materials. In this work, a marine-inspired biomineral complex was suggested as a potential bone graft material. The proposed biosilicified coccolithophore-derived coccoliths using bioengineered mussel adhesive proteins show osteopromotive ability through the synergistic effects of osteoconductivity from calcium carbonate and osteoinductivity from silica. Its possibility of use as a bone substitute was determined by evaluating the in vitro osteogenic behaviors of multipotent mesenchymal stem cells and in vivo bone regeneration in a rat calvarial defect model. Therefore, the marine-inspired biomineral complex developed in this study could be successfully used for bone tissue engineering.

Silaffin-3-derived pentalysine cluster as a new fusion tag for one-step immobilization and purification of recombinant Bacillus subtilis catalase on bare silica particles.

Bio-catalysis by enzymes on solid surfaces has been implemented in several practical applications. However, the current methods for efficient enzyme immobilization with retained activity need further development. Herein, a simple, rapid, and economical, bio-affinity-based approach was developed for the direct immobilization with high activity recovery of the Bacillus subtilis catalase (CAT), recombinantly expressed in Escherichia coli. Silaffin-3-derived pentalysine cluster (Sil3K) from Thalassiosira pseudonana and its mutant variant (penta-arginine peptide; Sil3R) were used for the first time in the non-covalent immobilization of the recombinant enzyme on silica particles. The fusion proteins CAT-Sil3K and CAT-Sil3R were selectively loaded from the cell lysates onto the silica surface. Unexpectedly, the Lys-based tag (Sil3K) was the superior to Arg-based tag (Sil3R) or tag-less system for the high recovery of CAT activity upon immobilization; an 8.4-fold and 1.5-fold increase in the catalytic activity was observed for CAT-Sil3K compared with the tag-less CAT and CAT-Sil3R, respectively. Furthermore, the CAT-Sil3K immobilized on silica particles exhibited improved thermal, pH and storage stabilities, and retained 72% of the initial activity after five reaction cycles. Moreover, CAT-Sil3K was released with approximately 85% recovery and 91% purity, in a biologically active form when free lysine solution was used as the eluent. Our data proved that Sil3K-tag, 12-mer peptide, can be a highly promising silica-affinity tag for effective enzyme immobilization with preserved activity. Additionally, the novel findings obtained here may open a new route not only for cost-effective enzyme immobilization approaches but also for high recovery of enzyme activity.

Self-encapsulation and controlled release of recombinant proteins using novel silica-forming peptides as fusion linkers.

Recently, the potential use of biomimetic silica as smart matrices for the auto-encapsulation and controlled release of functional proteins has gained increased interest because of the mild synthesis conditions. Inspired by biological silicification, in this study, we studied novel silica-forming peptides (SFPs), Volp1 and Salp1, to mediate the generation of silica hybrids in vitro. The fusion of SFPs to model fluorescent proteins directed their auto-encapsulation in wet sol-gel silica materials. Furthermore, the SFPs served as affinity linkers for the immobilization of recombinant proteins in silica. Interestingly, the SFP fusion proteins modulated silicic acid polycondensation and allowed for the self-immobilization of SFP fusion proteins in two distinct silica formulations depending on the ionic strength-precipitated silica particles or wet silica gel. The controlled release of Salp1/Volp1 fusion proteins from silica matrices was significantly greater than that of the silaffin R5 fusion proteins. Subsequently, we showed that multiple SFP-tagged proteins homogenously entrapped within a silica matrix could be separately released following pre-incubation with different concentrations of l-arginine solution. These new findings provide a simple and reproducible route for silica hybrid formation for in situ stable auto-encapsulation and the sustained release of recombinant proteins with potential applications in biotechnology.

Highly effective detection of inflamed cells using a modified bradykinin ligand labeled with FITC fluorescence.

Detection of inflammation in live cells is important because long-lasting inflammation is considered to be a primary cause of several diseases. However, few reports have been published on imaging analysis of inflammation in live cells. In this study, we developed an effective imaging system for detection of inflamed cells using a bradykinin ligand (BK) or a modified BK (mBK), which has specific affinity with the cellular B1R receptor. Synthetic BK or mBK labeled with FITC at the N-terminus was employed for discriminating between inflamed and normal cells; this method was found to be effective for detection of inflammation in live cells. In addition, using the mBK-based cell imaging system, we successfully performed flow-based analysis of live cell inflammation on a micro-chip channel, composed of a Starna flow cell and PDMS (Polydimethylsiloxane) walls. The BK-based cell imaging methods designed here would be a useful platform for development of a high-throughput live cell analysis system for investigating the factors underlying inflammation or for screening of anti-inflammation candidate drugs.

Specific detection of inflamed cells using TLR1 antibody and its secondary antibody-conjugated nano-beads.

Chronic inflammation can lead to several diseases, thus analysis of the inflammation state of live cells may have important clinical applications. However, there is not currently well-established method for specific detection of inflamed cells via live cell imaging. In this study, we developed an effective antibody (Ab)-based cell imaging method for the detection of inflamed cells using Ab-conjugated nano-beads. Several receptors were tested as potential biomarkers for cell inflammation, and corresponding fluorescence-labeled Abs and/or Ab-conjugated nano-beads were used to detect inflamed cells via fluorescence imaging. Interestingly, when we employed sequential use of TLR1 primary Ab and size-optimized nano-beads conjugated with secondary Ab, we were able to clearly discriminate inflamed cells from normal ones. The Ab-based cell-imaging method described herein provides an important basis for the development of high-throughput analysis of cell inflammation, potentially leading to the identification of factors involved in inflammation and anti-inflammatory drug candidates.

Surface immobilization of protein via biosilification catalyzed by silicatein fused to glutathione S-transferase (GST).

Silicatein from Suberites domuncula was known to catalyze silica deposition in vitro under near neutral pH and ambient temperature conditions. In this study, we employed GST-glutathione (GSH) interaction system to increase the production of silicatein and develop an efficient protein immobilization method. Recombinant silicatein fused with GST (GST-SIL) was produced in E. coli and the GST-SIL protein was employed on GSH-coated glass plate. GST-SIL bound surface or matrix can catalyze the formation of silica layer in the presence of tetraethyl orthosilicate as a substrate at an ambient temperature and neutral pH. During silicatein-mediated silicification, green fluorescent protein (GFP) or horseradish peroxidase (HRP) can be efficiently immobilized on the silica surface. Immobilized GFP or HRP retained their activity and were released gradually. This biocompatible silica coating technique can be employed to prepare biomolecule-immobilized surfaces or matrixes, which are useful for the development of biocatalytic, diagnostic and biosensing system, or tissue culture scaffolds.