Novel fabrication of fluorescent silk utilized in ...

Biomaterials 70 (2015) 48e56

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Novel fabrication of fluorescent silk utilized in biotechnological and medical applications Dong Wook Kim a, 1, Ok Joo Lee a, 1, Seong-Wan Kim b, Chang Seok Ki c, Janet Ren Chao d, Hyojong Yoo e, Sung-il Yoon f, Jeong Eun Lee a, Ye Ri Park a, HaeYong Kweon b, Kwang Gill Lee b, David L. Kaplan g, Chan Hum Park a, h, * a

Nano-Bio Regenerative Medical Institute, Hallym University, 1, Hallymdaehak-gil, Chuncheon, Gangwon-do, 200-702, Republic of Korea Department of Agricultural Biology, National Academy of Agricultural Science, Rural Development Administration, 166, Nongsaengmyeong-ro, Iseo-myeon, Wanju-gun, Jeollabuk-do 565-851, Republic of Korea c Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-921, Republic of Korea d School of Medicine, George Washington University, Washington, D.C., 20037, USA e Department of Chemistry, Hallym University, 1, Hallymdaehak-gil, Chuncheon, Gangwon-do, 200-702, Republic of Korea f Department of Systems Immunology, School of Biomedical Sciences, Kangwon National University, 1, Kangwondaehak-gil, Chuncheon, Gangwon-do, 200-701, Republic of Korea g Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA h Department of Otorhinolaryngology-Head and Neck Surgery, Chuncheon Sacred Heart Hospital, School of Medicine, Hallym University, 77, Sakju-ro, Chuncheon, Gangwon-do, 200-704, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 August 2015 Received in revised form 10 August 2015 Accepted 14 August 2015 Available online 17 August 2015

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. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Fluorescent Silk fibroin Biomaterials Medical application

1. 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

* Corresponding author. Department of Otorhinolaryngology-Head and Neck Surgery, Chuncheon Sacred Heart Hospital, School of Medicine, Hallym University, 77, Sakju-ro, Chuncheon, Gangwon-do, 200-704, Republic of Korea. E-mail address: [email protected] (C.H. Park). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2015.08.025 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

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 [7e9]. Moreover, the transgene inserted into the silkworm genome permits the acquisition of specific desirable characteristics by modifying the silk protein [10,11].

D.W. Kim et al. / Biomaterials 70 (2015) 48e56

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. 2. 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. DNAinjected 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-3xP3DsRed2-FibH was constructed as follows. The DsRed2 cDNA was used as a marker and amplified by PCR using specific primers with NheI/AflII sites from pDsRed2-C1 (NheI-DsRed2-F: 50 -GCTAGCATGGCCTCCTCCGAGAAC-30 and DsRed2-AflII-R: 50 -CTTAAGCTACAGGAACAGGTGGTGGCG-3'; Clontech, Mountain View, CA, USA). The PCR product was cloned into the pGEM-T-easy vector (Promega, Fitchburg, WI, USA) and named pGEM-DsRed2. The DsRed2 gene was excised from pGEM-DsRed2 with NheI/AflII and replaced with an EGFP gene from pBac-3 P3-EGFP to generate pBac-3xP3DsRed2. From the genomic DNA of B. mori, the DNA fragment (GenBank Accession No. AF226688, nt. 61,312e63,870), including the promoter domain (1124 bp), N-terminal region 1 (NTR-1, 142 bp), first intron (871 bp), and N-terminal region 2 (417 bp, NTR2), was amplified by PCR using specific primers with the AscI/NotI sites (pFibHN-F: 50 -AGGCGCGCCGTGCGTGATCAGGAAAAAT-30 and pFibHN-R: 50 -GCGGCCGCTGCACCGACTGCAGCACTAGTGCTGAA-30 ). The resultant DNA fragment was cloned into the pGEM-T Easy Vector System (Promega, Fitchburg, WI, USA) and named pGEMTpFibH-NTR. The DNA fragment (GenBank Accession No. AF226688, nt. 79,021e79,500) including the C-terminal region (179 bp, CTR) and the poly(A) signal region (301 bp) of the H-chain was amplified by PCR using specific primers with the SalI/SbfI/FseI sites (pFibHC-F: 50 -CCTGCAGGAAGTCGAC AGCGTCAGTTACGGAGCTGGCAGGGGA-30 and pFibHC-R: 50 -GGCCGGCCTATAGTATTCTTAGTTGAGAAGGCATA-30 ). The resultant DNA fragment was cloned into the pGEM-T Easy Vector System (Promega, Fitchburg, WI, USA) and named pGEMT-CTR. The pFibH-NTR fragment was excised from pGEMT-pFibH-NTR with ApaI/SalI and subcloned into a pBluescriptII SK() vector (Stratagene, La Jolla, CA, USA) that had been digested with ApaI/SalI to generate pFibH-NTR-null. Next, a CTR fragment was excised from pGEMT-CTR with SalI/SacI and subcloned into a pFibH-NTR-null that had been digested with SalI/ SacI to generate pFibHNC-null. Fluorescent genes (EGFP, mKate2, EYFP) were synthesized and purchased from the BIONEER corporation (Korea). The N- and C-terminals had the NotI and SbfI restriction sites, respectively. The fluorescent genes were digested with NotI/SbfI and subcloned into a pFibHNC-null that had been digested with NotI/SbfI to generate pFibHNC-EGFP, pFibHNCmKate2, and pFibHNC-EYFP. These vectors were then digested with AscI/FseI and subcloned into separate pBac-3xP3-DsRed2. The resulting vectors pBac-3xP3-DsRed2-FibH-EGFP, pBac-3xP3DsRed2-FibH-mKate2, and pBac-3xP3-DsRed2-pFibH-EYFP were generated. pBac-3 P3-EGFP25 and the helper vector pHA3PIG7

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were provided by Dr. M. Jindra (Academy of Sciences of the Czech Republic, Prague, Czech Republic). Each vector was purified with an EndoFree Plasmid Maxi Kit (QIAGEN Gmbh, Hilden, Germany) and used to generate transgenic silkworms. Transgenesis and screening of silkworms. For egg preparation, male and female moths were allowed to mate for at least 4 h at 25 C. The mating moths were stored overnight at 4 C. The female moths were placed on a plastic sheet and left in dark boxes for 1 h. Laid eggs were immersed in HCl (specific gravity 1.0955, 25 C) for 30 min at 25 C, rinsed with distilled water, and dried. The transition vectors (pBac-3xP3-DsRed2-pFibH-EGFP, pBac-3xP3-DsRed2pFibH-mKate2, and pBac-3xP3-DsRed2-pFibH-EYFP) and the helper vector pHA3PIG were dissolved in 5 mM KCl and 0.5 mM phosphate buffer (pH 7.0) at a concentration of 0.2 mg/mL and mixed at a ratio of 1:1. Approximately 5e10 nL of this mixture were injected using an IM300 microinjector (Narishige Scientific Instrument Lab., Tokyo, Japan) into pre-blastoderm embryos at 2e8 h after oviposition. Injected embryos were allowed to develop at 25 C in moist chambers. G1 embryos and larvae were screened under a fluorescence stereomicroscope equipped with a red filter (Leica, Wetzlar, Germany). Preparation of fluorescent silk fibroin solution. The fluorescent cocoons were heated overnight at 60 C in an aqueous solution of 3% NaHCO3 with alcalase (1.5 ml/L) and then washed with distilled water several times to remove the glue-like sericin proteins. Subsequently, the extracted silk was dissolved in a 9.5 M LiBr solution with 1 mM DTT at 40 C. Then, this solution was filtered through a miracloth (Calbiochem, San Diego, CA, USA) and dialyzed with distilled water for 2 days to remove the salt. The final concentration of the aqueous silk fibroin solution was 4 wt. %. The SF solutions were stored at 4 C before use to avoid premature precipitation or freeze-dried to obtain regenerated silk fibroin. Fluorescence studies. The fluorescence spectrum of each cocoon and solution was captured using the LS-55 (Perkin Elmer, Santa Clara, CA, USA) fluorescence spectrophotometer. The fluorescence was recorded at excitation wavelengths of 488 nme588 nm and emission wavelengths of 507 nme633 nm. Preparation of silk fibroin materials. To obtain SF membranes, the SF solution, prepared as described above, was cast on a polystyrene plate at 0.4 ml/cm, followed by drying overnight. SF sponges were obtained from SF solution in a mold, frozen at 80 C overnight, and freeze-dried for 2 days to completely remove the solvent. Cell study. The fibroblast cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 mg/ml penicillin and 100 mg/ml streptomycin. The membranes were sterilized by soaking the samples in 70% ethanol for 30 min. The wells were seeded at a density of 10,000 cells/well onto fluorescent silk fibroin membranes. The media was changed two times per week. To examine the cell attachment on the SF membrane, cells (10,000 cells/well) were allowed to grow for 24 h. The SF membranes were fixed in 4% glutaraldehyde in 0.1 M phosphate buffer for 30 min, washed repeatedly with PBS buffer, and stained with DAPI (Vector Lab., Burlingame, CA, USA) and rhodamine phalloidin (Invitrogen Co., San Diego, CA, USA). The membranes were analyzed under a fluorescence microscope (Eclipse 80i, Nikon Co., Japan). Animals. 8-week- old male nude mice (BALB/c), 8-week- old male mice (ICR) and 8-week- old male rats (SD) were purchased from Dooyeol Biotech (Seoul, Korea) and the animal center of Hallym University in Korea, respectively. This study was approved by the Institutional Review Board of Hallym University, Chuncheon, Korea. All animals were observed in separate cages with free access to food and water. All surgical procedures were performed under general anesthesia using intraperitoneal xylazine (5e10 mg/kg) and intramuscular ketamine (40e80 mg/kg).

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Whole-body imaging. Fluorescent SF sponges were 3 mm thick and cut into 8 mm diameter disks using a tissue punch. For the in vivo observation of fluorescence, they were implanted into dorsal regions of nude mice and fluorescence images were captured using the Luminescence and Fluorescence Animal Imaging System (IVIS 200, Perkin Elmer, Santa Clara, CA, USA). Histological examination. The mice were sacrificed 2 weeks after implantation. SF sponges were dissected from the mice, including the surrounding soft tissue. For histological evaluation, the harvested tissues were frozen and stored at 80 C. Each specimen was embedded with Optimum Cutting Temperature (OCT) compound (Tissue Tek, Elkhart, IN, USA) and sectioned into 10-mm-thick slides. Then, the sections were stained with DAPI (Vector Lab., Burlingame, CA, USA) and rhodamine phalloidin (Invitrogen Co., San Diego, CA, USA) and analyzed under a fluorescence microscope (Eclipse 80i, Nikon Co., Japan). In vitro imaging of EGFP fluorescent SF-p53 labeling. The fluorescent SF solution was mixed NHS-PEG4-Biotin solution (EZ-Link NHS-PEG4-Biotinylation Kit, Thermo Fischer Scientific, Waltham, MA, USA) and incubated for 4 h on ice. The solution was passed through a column to prepare a biotinylated-fluorescent SF. Then p53 antibody (Abcore Inc, Ramona, CA, USA) was mixed with the biotinylated-fluorescent SF and the mixture was incubated for 2 h resulting in fluorescent SF-labeled p53 antibody. Fluorescentlabeled p53 antibodies treated in HeLa cells for 24 h and analyzed under a fluorescence microscope (Eclipse 80i, Nikon Co., Japan). Observation of EGFP fluorescent SF particle in rat gastric mucous membrane. The fluorescent SF particle (1 g) was mixed with water

and fed to a rat. After 24 h, the gastric mucous membrane was harvested. We used SF particle from B. mori as control. For histological evaluation, the harvested tissues were frozen and stored at 80 C. Each specimen was embedded with Optimum Cutting Temperature (OCT) compound (Tissue Tek, Elkhart, IN, USA) and sectioned into 10-mm-thick slides. Then, the sections were stained with DAPI (Vector Lab., Burlingame, CA, USA) and rhodamine phalloidin (Invitrogen Co., San Diego, CA, USA) and analyzed under a fluorescence microscope (Eclipse 80i, Nikon Co., Japan). Intraoperative detection of esophageal fistula by EGFP fluorescent SF solution. 1 ml of the EGFP fluorescent SF solution was slowly injected into the esophagus after making of small fistula (0.5 mm) at upper esophagus in rats. The leakage of fluorescent SF solution was monitored using a fluorescence stereomicroscopy (SMZ25, Nikon Co., Japan). All images were captured per 0.2 s. 3. Results and discussion 3.1. 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 silk fibroin is

Fig. 1. Multicolored fluorescent cocoons and silk fibroin fibers. The fluorescent cocoons produced by transgenic silkworms had various fluorescent proteins including EGFP, mKate2 and EYFP. Cocoons were exposed with a blue LED, and their pictures were taken through a night filter (Embi Tec, CA, USA). The SF fibers were observed under confocal microscopy.


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Fig. 2. Schematic of fluorescent SF extraction procedure. Fluorescent silk was degummed with 0.5% (w/w) Na2CO3 with 1 mM DTT solution at 45 C for overnight and then washed with distilled water. Degummed fluorescent SF was dissolved in 9.5 M LiBr for 4 h at 45 C and dialyzed to remove salts in a cellulose tube against distilled water for 2 days at room temperature.

Fig. 3. Fluorescence excitation and emission spectra of cocoon and SF solution. (a, b) cocoon, (c, d) silk fibroin solution. The fluorescence studies were conducted at excitation wavelengths of 488 nm (EGFP), 514 nm (EYFP) and 588 nm (mKate2); maximum emission wavelengths were observed at 507 nm (EGFP), 527 nm (EYFP) and 633 nm (mKate2). Similar excitation and emission behaviors were observed for the cocoons and solution.

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shown in Supplementary Fig. 1b. SF is typically purified from sericins by boiling silk cocoons in an alkaline solution. This degumming method makes it is difficult to acquire an SF solution with fluorescent properties because fluorescence stability is not maintained at high temperatures or high pH values (greater than 9). Therefore, an alternate method for the preparation of an SF solution with fluorescent colors is necessary. Prior to preparing our solution form of fluorescent SF, we produced fluorescent silk fibers from transgenic silkworms with DsRed2 fluorescent protein, enhanced green fluorescent protein (EGFP), monomeric far-red fluorescent protein (mKate2), and enhanced yellow fluorescent protein (EYFP) (Fig. 1) [13,14]. We used fluorescent cocoons from transgenic silk worms and prepared them with alternate degumming and dissolving methods for the production of silk materials that can maintain fluorescent colors. The conventional degumming method includes processing by drying and cooking the cocoons near 100 C which denatures the fluorescent protein and causes it to lose fluorescence. Alternatively, we used a degumming method with alcalase enzyme treatment. To prepare fluorescent silk fibroins, cocoons were treated with 0.5% (w/w) Na2CO3 and alcalase solution at 45 C overnight. After the degumming step, we observed that silk fibers did not lose fluorescent colors (in the web version) as shown in Fig. 1. The SF solution was then fabricated from fluorescent silk fibers. Because the fluorescence protein undergoes denaturation in the solvent, a reductase, such as dithiothreitol (DTT), is required for the renaturation of the protein structure [15]. DTT is frequently used to reduce the disulfide bonds of proteins and peptides. It prevents intramolecular and intermolecular disulfide bonds from forming

between cysteine residues of proteins. As shown in Fig. 2, SF fibers were dissolved in a 9.5 M LiBr solution with 1 mM DTT at 45 C. After incubating the silk fiber in a solution and dialyzing, we obtained an SF solution with green, red and yellow fluorescent colors. We analyzed fluorescent spectra of the cocoons and the SF solution (Fig. 3). Although fluorescent SF fibers have been made previously, this is the first time a fluorescent SF has been successfully produced in solution form. Upon confirming the fluorescent capability of our new SF solution, we fabricated different forms of materials, such as sponges, membranes and electrospun mats. Using three different fluorescent-colored SF membranes, cell growth and adhesion was observed (Fig. 4). Cell viabilities of the fluorescent SF membranes were evaluated using CCK assays at days 1, 2, and 3 (Supplementary Fig. 2). Compared with the wild type SF membrane, the optical density (OD) values were similarly increased in all SF membranes, suggesting that these membranes were suitable for cell proliferation. Fluorescent electrospun fibers were successfully generated from all fluorescent SF solutions (Supplementary Fig. 3). 3.2. Biocompatibility of fluorescent SF materials To evaluate the biocompatibility and fluorescent duration of SF materials, fluorescent SF sponges were subcutaneously inserted in rats. Fig. 5 shows images at 7 days after implantation. All fluorescence colors were detected at the sites where the sponges were located. The intensity of the mKate2 fluorescent SF sponge was higher than those of the EGFP and EYFP fluorescent SF sponges. The EYFP fluorescent SF sponge had the lowest measured intensity. One year after implantation, all SF sponges retained fluorescence

Fig. 4. Confocal laser micrographs of NIH3T3 fibroblast cells cultured on fluorescent SF membranes. Images of cells cultured on (b, f, j, n) EGFP, (c, g, k, o) mKate2 and (d, h, l, p) YFP fluorescent membranes. Cells were stained with (eeh) DAPI and (iel) rhodamine phalloidin. (a, e, i, m) B. mori represents a control with no fluorescent. Cell cultures were observed under confocal laser microscopy Scale bars: 20 mm.


confirming the long-term biocompatibility of our fluorescent SF sponge. Due to the biocompatibility and a controlled degradation rate of fluorescent SF through its crystallinity, silk materials are suitable for use as controlled release drug delivery systems (DDS). As a result of adding long-term fluorescent capability to silk protein, we can expand the use of fluorescent SF to targeted therapy drugs. Having a fluorescent DDS enables us the advantage of being able to visually track the movement and degradation of the DDS. 3.3. Tumor visualization with fluorescent SF Silk fibroin is easily modified due to chemically reactive basic amino groups such as lysine, arginine, and histidine [16]. Therefore, fluorescent SF solution can be conjugated to other proteins modifying the surface properties of the material. Here, fluorescent SF solution was successfully conjugated to p53 antibody. Fluorescent-labeled p53 antibodies were used to detect p53 expression in HeLa cells derived from cervical cancer cells in vitro (Fig. 6). We did not observe any non-specific binding of fluorescent SF to the HeLa cells, confirming the specificity of our fluorescenteSFep53-antibody. Previous studies have reported the

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possibility that p53 protein accumulation is a very early event in human carcinogenesis, particularly in esophageal squamous cell carcinoma. Although additional work is needed to further prove the role of p53 as a biomarker in early esophageal tumors, these studies suggest that our fluorescenteSFep53-antibody could be used to detect p53 overexpression in cancer cells and therefore to detect and remove the primitive esophageal cancer lesions in their early stages. It was also recently reported that folate receptor-alpha (FR-a) targeted fluorescent agent was used to image ovarian cancer cells [17]. This study demonstrates the advantages and utility of intraoperative fluorescence imaging to detect cancer cells in humans. Our study suggests that fluorescenteSFep53-antibody could be used to surgically visualize the cancerous esophageal tumor margins as well as small metastatic nodules, allowing surgeons to selectively remove the malignant cells while saving the undeterred tissue. Radical esophagostomy is often used to ensure that all the damaged tissue is removed when margins cannot be visualized. In the process, such destructive dissections remove and can damage normal tissue. Therefore, patient outcomes could be significantly improved by increasing the capability to detect and visualize tumor margins and metastatic nodules through fluorescent SF labeled biomarkers.

Fig. 5. aed) Whole-body imaging of fluorescent SF sponges in dorsal region of nude mouse at 7 days. Fluorescent SF sponges were implanted in dorsal region of nude mouse. The color bar indicates relative signal intensity. All SF sponges retained their fluorescent expressions. (a) reflectance, (b) EGFP, (c) mKate2, (d) EYFP. eej) Histological analysis of fluorescent SF porous sponges. Analysis was performed after 2 weeks and one year in vivo. Cross-sections of fluorescent SF porous sponges were stained with DAPI and rhodamine phalloidin. All SF porous sponges still had fluorescent colors. Confocal laser micrographs of (eeg) 2 weeks, (hej) one year. Scale bars: 20 mm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

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Fig. 6. Detection of p53 localization using EGFP fluorescent SF labeled p53 antibody in HeLa cell. (aed) Control, (eeh) EGFP fluorescent SF solution, (iel) EGFP fluorescent SF labeled p53. (b, f, j) Fluorescent-labeled p53 antibodies were analyzed under fluorescence microscopy. HeLa cells were stained with (a, e, i) DAPI and (c, g, k) Rhodamine Phalloidin and imaged under fluorescence microscopy, (d, h, l) DAPI, EGFP, and Rhodamine Phalloidin images were merged.

Although we demonstrated and utilized conjugation of our fluorescent SF with the p53 antibody in this paper, fluorescent SF can just as easily be conjugated to many other antibodies or proteins. As more tumor biomarkers are identified in future research studies, this fluorescent SF approach can be applied to detect these biomarkers as well and utilized in intraoperative tumor visualization. We suggest further clinical studies utilizing fluorescent SF as a tumor marker and imaging tool to aid surgeons in delineating the extent of tumor spread. 3.4. In vivo fluorescent SF image Fluorescent SF materials can also be applied in clinical medicine. In this paper, we show that fluorescent SF biomaterials are acidresistant and therefore applicable to detecting lesions in the stomach. The fluorescent SF powder was mixed with water and fed to a mouse. After 24 h, gastric mucosa histology of the mouse

revealed fluorescence particles throughout the stomach's epithelial surface, revealing the acid-resistant property of the fluorescent SF solution. A control powder of SF particles was also fed to the mouse which no fluorescent characteristic (Fig. 7). Furthermore, we apply a fluorescent SF solution to detect a small esophageal perforation. Esophageal perforation is a potentially fatal medical complication that remains difficult to diagnose and manage due to its variability in presentation. Despite surgical advances and improved medical technology, mortality rates approached 20% in 2004 [18]. Small fistulas of the gastrointestinal (GI) tract can be difficult or impossible to detect by direct visual observation. The use of intraesophageal dye as a method to intraoperatively detect esophageal perforation has been studied [19]. We suggest the use of fluorescent SF solution as an alternative method of detecting esophageal perforation. A small esophageal perforation (0.5 mm) in a rat model was clearly observed using EGFP fluorescent SF solution leakage (Fig. 8). EGFP fluorescent SF can improve surgical inspection and

Fig. 7. Absorption image of EGFP fluorescent SF particle in the gastric mucous membrane of a rat. Images of gastric mucosa membrane of a rat fed with SF particles and fluorescent SF particles. The left panel shows control powder of only SF particles. The right panel shows the fluorescent SF particles. Images were observed under fluorescence microscopy. The electrospun mats were analyzed at lex/lem ¼ 488/507 for EGFP. Scale bars: 100 mm.


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Fig. 8. Intraoperative application image of EGFP fluorescent SF solution at rat model with esophageal fistula. (a) Esophageal fistula (0.5 mm) image with white light reflectance, (beg) EGFP fluorescent captured images per 0.2 s. EGFP fluorescent was detected at esophageal fistula according to time passed, (h) Image of leaked EGFP fluorescent SF from fistula with white light reflectance image.

offers superior accuracy and sensitivity compared to visual inspection. By the addition of the fluorescent gene into silk cocoons using transformation vectors, a considerable amount of biocompatible SF solution was produced from transgenic silkworms. Using this solution, fluorescent silk materials can be manufactured in various forms. Although other studies have conjugated materials such as silicon to fluorescent peptides, the advantage of using a transgenic silk is that the genetic modification of the silk cocoons allows the cocoons to be harvested with an inherent fluorescent property [20]. As a result, there is no need to go through a conjugation step to add a fluorescent peptide. Furthermore, the biocompatible nature of silk itself makes fluorescent silk an attractive biomaterial for biotechnological and clinical applications.

4. 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 targeting system in clinical medicine. There are numerous potential clinical applications for fluorescent SF. We demonstrate the visual inspection of esophageal

perforation using EGFP fluorescent SF solution to image the surgical field intraoperatively in an animal model. Using this as a prototype, fluorescent SF solution can shift the paradigm of surgical inspection by enabling localization of a variety GI fistula lesions. In addition, peptide-conjugated fluorescent silicon nanoparticles have been studied to simultaneous track and destroy cancer cells [20]. Through simple surface modifications, fluorescent SF could improve surgical inspection, tumor visualization and treatment by conjugating it to antibodies and tumor markers. Previous studies have reported intraoperative use of fluorescence imaging to detect ovarian cancer [17], meningeal sarcomas [21], and metastasis to lymph nodes [22]. In future animal studies, we will examine the potential of fluorescent SF as a cancer detection and treatment modality. 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 (NRF2015R1D1A3A01020100), Republic of Korea. Appendix A. Supplementary data Supplementary data related to this article can be found at http://

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