Hogyun Cheong

Mussel‐Inspired Protein Nanoparticles Containing Iron(III)–DOPA Complexes for pH‐Responsive Drug Delivery

A novel bioinspired strategy for protein nanoparticle (NP) synthesis to achieve pH-responsive drug release exploits the pH-dependent changes in the coordination stoichiometry of iron(III)-3,4-dihydroxyphenylalanine (DOPA) complexes, which play a major cross-linking role in mussel byssal threads. Doxorubicin-loaded polymeric NPs that are based on Fe(III)-DOPA complexation were thus synthesized with a DOPA-modified recombinant mussel adhesive protein through a co-electrospraying process. The release of doxorubicin was found to be predominantly governed by a change in the structure of the Fe(III)-DOPA complexes induced by an acidic pH value. It was also demonstrated that the fabricated NPs exhibited effective cytotoxicity towards cancer cells through efficient cellular uptake and cytosolic release. Therefore, it is anticipated that Fe(III)-DOPA complexation can be successfully utilized as a new design principle for pH-responsive NPs for diverse controlled drug-delivery applications.

Highly purified mussel adhesive protein to secure biosafety for in vivo applications

BackgroundUnique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult.ResultsIn the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151.ConclusionsThrough this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications.

Accelerated skin wound healing using electrospun nanofibrous mats blended with mussel adhesive protein and polycaprolactone

Nanofibrous scaffolds have been assessed as one of many promising tissue engineering scaffolds to be utilized for wound-healing applications. Previously, we reported multi-functionalized electrospun nanofibrous scaffolds blended with mussel adhesive protein (MAP) and polycaprolactone (PCL), which provide durable mechanical strength, cell-friendly environments, and a substantial ability to capture diverse bioactive molecules without any surface modifications. In the present work, we applied the blended nanofibrous mats of MAP and PCL for in vivo skin wound healing. The nanofibrous mats showed accelerated regeneration in a rat skin wound-healing model, which might be attributed to a highly compatible environment for keratinocyte cell growth, an ability to capture inherent growth factors, and an efficient exudate absorption capacity. Thus, this work would suggest that adhesive property of scaffold could be a factor of successful application for wound healing. The MAP-blended nanofibers could also be potentially exploited for diverse tissue regeneration applications.

Recent developments and applications of bioinspired silicification

Bioinspired synthesis of silica has attracted attention from a wide range of researchers as novel route for fabrication of various nanomaterials. Proteins including silaffins and silicateins as well as polyamines from marine diatoms and sponges are key biomolecules in these biomimetic silicification processes. These methods allow silica mineralization from various silica precursors under mild, biologically compatible conditions in an unprecedentedly fast and facile manner. Notably, the silica polycondensation entails the concomitant encapsulation of other molecules in the reaction solutions. Due to the efficient encapsulation and synergetic effects brought by the encapsulated molecules and the characteristics of biomimetic silica synthesis as well as the mechanical and chemical properties of silica itself, the silica- biomolecule nanocomposites have broad applications in biocatalysis, biosensor, and biomedical areas. Introduction and combination of novel template, precursors, inorganics, or enzymes with the previously used strategies will allow construction of more efficient, purpose-optimized silica nanomaterials with controlled size, composition, and morphology.

Engineered mussel bioglue as a functional osteoinductive binder for grafting of bone substitute particles to accelerate in vivo bone regeneration

Xenograft bone substitutes, such as deproteinized bovine bone mineral (DBBM), have been widely employed as osteoconductive structural materials for bone tissue engineering. However, the loss of xenograft bone substitute particles in defects has been a major limitation, along with a lack of osteoinductive function. Mussel adhesive protein (MAP), a remarkable and powerful adhesive biomaterial in nature, can attach to various substrates, even in wet environments. Its adhesive and water-resistant abilities are considered to be mainly derived from the reduced catechol form, 3,4-dihydroxyphenylalanine (DOPA), of its tyrosine residues. Here, we evaluated the use of DOPA-containing MAP as a functional binder biomaterial to effectively retain DBBM particles at the defect site during in vivo bone regeneration. We observed that DOPA-containing MAP was able to bind DBBM particles easily to make an aggregate, and grafted DBBM particles were not lost in a defect in the rat calvaria during the healing period. Importantly, grafting of a DOPA-containing MAP-bound DBBM aggregate resulted in remarkably accelerated in vivo bone regeneration and even bone remodeling. Interestingly, we found that the DOPA residues in the modified MAP had an osteoinductive ability based on clear observation of the in vivo maturation of new bones with a similar bone density to the normal bone and of the in vitro osteogenic differentiation of osteoblast cells. Collectively, DOPA-containing MAP is a promising functional binder biomaterial for xenograft bone substitute-assisted bone regeneration with enhanced osteoconductivity and acquired osteoinductivity. This mussel glue could also be successfully utilized as a potential biomaterial for general bone tissue engineering.

Expression and N-glycan analysis of human 90K glycoprotein in Drosophila S2 cells

Human 90K (h90K; Mac-2-binding protein) glycoprotein is a potential pharmaceutical due to its inhibitory activity against cancer metastasis and expansion. Here, h90K glycoprotein was produced in insect Drosophila S2 cell system, and its N-glycan pattern was analyzed. A plasmid encoding h90K gene, fused with a hexahistidine tag under the control of Drosophila metallotionein promoter, was stably transfected into S2 cells. After copper sulfate induction, transfected S2 cells secreted recombinant h90K at a good expression level of 28mg/L in a 150-mL spinner flask culture. The purified recombinant h90K showed an apparent molecular weight of ∼78kDa which was much smaller than that (∼97kDa) of the natural h90K. Because de-N-glycosylated h90K appeared at ∼60kDa protein band, it was suggested that the recombinant h90K from S2 cells has small N-glycans with about half the molecular weight (∼18kDa) of N-glycans of the natural h90K. Through detail analyses using high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the S2-derived recombinant h90K was confirmed that it has simple paucimannosidic structures containing two or three mannose residues with core fucose as the major (∼79%) N-glycans.

Mussel-Derived Bioadhesives

Marine mussels use MAPs (MAPs) for their adhesion. MAPs have fascinating properties, including strong adhesion to various material substrates, water displacement, biocompatibility, and controlled biodegradability. In this work, among six types of MAPs, including fp-1–fp-6, biosynthetic constructs of MAPs are considered; hybrid type recombinant MAPs are designed to improve productivity and purification. Hybrid recombinant fp-151, which comprises six decapeptide repeats of fp-1 at both N and C-termini of fp-5, was successfully overexpressed in a bacterial system, showing approximately ≈ 1 g / L Open image in new window production yield in a pilot scale fed-batch bioreactor culture. For industrial applications, it was attempted to use MAPs in tissue engineering fields as coating extracellular matrix (ECM ) through surface modification and constructing nanofibrous scaffolds. As a result, the MAP-based coating strategy could be generally applied for facile and efficient surface modification of negatively charged bioactive molecules for tissue engineering. The use of MAP-based nanofibers could provide bioactive peptides efficiently onto the scaffold surface, enhancing the cell attachment and proliferation on the nanofibers fabricated using RGD peptide-conjugated MAPs compared with bare polycaprolactone (PCL ) polymer nanofibers as well having a four times higher mechanical strength. Also, easy fabrication through blending with diverse types of synthetic polymers and significant bone regeneration was observed. In addition, there was a trial for utilization of MAPs in pharmaceutics, cosmetics, and food industries with encapsulating active molecules such as chemical drugs, proteins, cells, and flavor ingredients through a complex coacervation technique based on MAPs.