Novel fabrication of fluorescent silk utilized in biotechnological and medical applications

doi:10.1016/j.biomaterials.2015.08.025

Biomaterials

Volume 70, November 2015, Pages 48-56

https://doi.org/10.1016/j.biomaterials.2015.08.025 Get rights and content

Abstract

Silk fibroin (SF) is a natural polymer widely used and studied for diverse applications in the biomedical field. Recently, genetically modified silks, particularly fluorescent SF fibers, were reported to have been produced from transgenic silkworms. However, they are currently limited to textile manufacturing. To expand the use of transgenic silkworms for biomedical applications, a solution form of fluorescent SF needed to be developed. Here, we describe a novel method of preparing a fluorescent SF solution and demonstrate long-term fluorescent function up to one year after subcutaneous insertion. We also show that fluorescent SF labeled p53 antibodies clearly identify HeLa cells, indicating the applicability of fluorescent SF to cancer detection and bio-imaging. Furthermore, we demonstrate the intraoperative use of fluorescent SF in an animal model to detect a small esophageal perforation (0.5 mm). This study suggests how fluorescent SF biomaterials can be applied in biotechnology and clinical medicine.

Introduction

Silk fibroin (SF), a natural fibrous protein produced by Bombyx mori, has been used for biomedical and biotechnological applications [1]. For example, applications of silk in tissue engineering, wound dressing [2], enzyme immobilization matrices [3], vascular prostheses and structural implants [4], [5] have been reported. Depending on its application, SF can be processed into different forms, including film, gel, membrane, powder and porous sponge. However, processing SF into these various forms relies on preparing a solution form of SF as a precursor. To suit a wide range of applications, SF has been integrated with various materials or chemically modified [6]. For example, coupling reactions, amino acid modifications and grafting reactions were used for the chemical modification of silk fibroin. Genetically modified silks produced from transgenic silkworms have recently been reported [7]. Transgenic silkworms can easily be proliferated and retained once the silkworm strain is established and recently, fluorescent transgenic silkworms developed using various transformation vectors [7], [8], [9]. Moreover, the transgene inserted into the silkworm genome permits the acquisition of specific desirable characteristics by modifying the silk protein [10], [11].

As is commonly known, green fluorescent protein (GFP), first identified in the aquatic jellyfish Aequorea victoria, has been the subject of continued interest since it was cloned in 1992 [12]. Over the decades, fluorescent proteins have become a favorable biotechnological tool that scientists use to investigate the function of genes of interest by directly visualizing, monitoring and quantifying protein expression in living cells. However, there have not been any reports on the biomedical and biotechnological applications using fluorescent silk fibroin. Here, we developed the first method of preparing fluorescent silk fibroin solution in order to produce various fluorescent SF materials.

Section snippets

Materials and methods

Silkworm strains. The B. mori bivoltine strain, Kumokjam (Jam140 × Jam125), was obtained from the National Academy of Agricultural Science (Suwon, Korea). The silkworms were grown at 25 °C and fed with mulberry leaves and an artificial diet. DNA-injected eggs were maintained at 25 °C in moist Petri dishes. The hatched larvae were fed on an artificial diet and reared in groups under standard conditions.

Plasmid DNA construction. The transition vector pBac-3xP3-DsRed2-FibH was constructed as

Preparation and characterization of fluorescent SF solution

The structure of the vector for producing fluorescent SF is shown in Supplementary Fig. 1a. The fluorescent color protein, fused with the N-terminal and C-terminal domains of the silk fibroin H chain, is expressed in the silkworm. For the production of EGFP, mKate2 and EYFP fluorescent silk fibroin, transgenic silkworm strains were generated by injecting the vector DNA shown in Supplementary Fig. 1a with a helper plasmid into pre-blastoderm embryos. A prospective structure of the fluorescent

Conclusion

The first fluorescent SF solution produced by our lab was only the foundation for producing several forms of fluorescent SF. We also can produce fluorescent SF solution as film, gel, membrane, powder and porous sponge forms. In addition, the fluorescent silk fibroin that results from the transgenic cocoons can be modified by conjugating it to other proteins such as antibodies, enzymes, or tumor markers. Therefore, fluorescent SF can be applied as a bioimaging tool or a controlled release drug

Acknowledgments

This work was supported by the Hallym University Research Fund, Cooperative Research Program for Agriculture Science & Technology Development (Project No.PJ011214022015), Rural Development Administration, and Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1D1A3A01020100), Republic of Korea.

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Fluorescent silk-based materials have been applied in biomedical engineering, optics, and photonics [97,98]. In general, fluorescent silk fibers can be produced by the genetic modification of silkworms or via dying treatment [99,100]. However, transferring the transgene to the next generation remains a problem, while dying often results in unstable fluorescence.
Many natural fibers are lightweight and display remarkable strength and toughness. These properties originate from the fibers’ hierarchical structures, assembled from the molecular to macroscopic scale. The natural spinning systems that produce such fibers are highly energy efficient, inspiring researchers to mimic these processes to realize robust artificial spinning. Significant developments have been achieved in recent years toward the preparation of high-performance bio-based fibers. Beyond excellent mechanical properties, bio-based fibers can be functionalized with a series of new features, thus expanding their sophisticated applications in smart textiles, electronic sensors, and biomedical engineering. Here, recent progress in the construction of bio-based fibers is outlined. Various bioinspired spinning methods, strengthening strategies for mechanically strong fibers, and the diverse applications of these fibers are discussed. Moreover, challenges in reproducing the mechanical performance of natural systems and understanding their dynamic spinning process are presented. Finally, a perspective on the development of biological fibers is given.
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¹: These authors contributed equally to this work.

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Novel fabrication of fluorescent silk utilized in biotechnological and medical applications

Abstract

Introduction

Section snippets

Materials and methods

Preparation and characterization of fluorescent SF solution

Conclusion

Acknowledgments

Biomaterials

Biomaterials

Bone

Ann. Thorac. Surg.

In vitro evaluation of the inflammatory potential of the silk fibroin

J. Biomed. Mater Res.

Performance evaluation of a silk protein-based matrix for the enzymatic conversion of tyrosine to L-DOPA

Biotechnol. J.

Biomedical applications of chemically-modified silk fibroin

J. Mater. Chem.

Gene targeting in the silkworm by use of a baculovirus

Genes Dev.

High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system

Mol. Biol. Rep.

Production of high quality silks having different fluorescent colors using transgenic silkworms

J. Aff. Res.

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector

Nat. Biotechnol.