Silk-sericin degummed wastewater solution-derived and nitrogen enriched porous carbon nanosheets for robust biological imaging of stem cells

https://doi.org/10.1016/j.ijbiomac.2017.10.093Get rights and content

Highlights

  • Highly fluorescent sericin based carbon nanoparticles was prepared from industrial wastewater containing silk-sericin as a precursor for carbon particles, and utilized as bio-imaging application for oral fat stem cells.

  • The obtained hydrothermal carbons exhibited strong fluorescence properties due to the presence of strong polar groups, such as carboxyl, amino and amide groups in the surface.

  • The obtained SCNs demonstrated low toxicity and their suitability for bio-imaging applications was demonstrated to the oral fat stem cells.

  • Overall, sericin degumming wastewater from the silk textile industry can be utilized for the production of SCNs for stem cells bio-imaging applications.

Abstract

Appreciated raw materials like silk-sericin can be recovered from silk-textile industrial waste for the production of novel functional nanomaterials. In this study, highly fluorescent sericin based carbon nanosheets (SCN) were produced from industrial wastewater containing silk-sericin as a precursor, and was applied as bio-imaging application for oral fat stem cells. A simple one-pot, hydrothermal carbonization method was used to produce SCN at a 180 °C. The obtained hydrothermal carbons exhibited strong fluorescence properties due to the presence of strong polar groups, such as carboxyl, amino and amide groups in the surface. Heteroatom functionalization of the SCN leads to the property of fluorescence due to enriched nitrogen and was confirmed by X-ray photoelectron and Fourier transform infrared spectroscopy. The plate-like morphology of SCN about 35 nm in size was evaluated by transmission electron microscopy. The carbon 13 nuclear magnetic resonance results revealed that nano-sized fluorescent SCN formed during carbonization and functionalization occurred through dehydration of the sericin protein. Moreover, the prepared SCNs demonstrated low toxicity and their suitability for bio-imaging applications was demonstrated to the oral fat stem cells. Overall, sericin degumming wastewater from the silk textile industry can be utilized for the production of SCNs for stem cells bio-imaging applications.

Introduction

Stem cell research has been receiving extensive attention in the fields of medicine and dentistry and has offered a wide range of treatments for many hitherto incurable diseases [1], [2]. Of particular interest are the adipose stem cells (ASCs), which are multipotent cells found in any type of white adipose tissue within the whole body, including buccal fat pad, subcutaneous and omental fat etc [3]. The multipotent nature of ASCs gives them great potential for regenerative medical applications, and the development of new cures for various human diseases as well as dental treatments [4]. Especially, Oral fat stem cells derived from buccal fat pad show excellent osteogenesis and are expected to be an important candidate for bone regeneration in dentistry [5]. Recently, the development of methods for the monitoring of the efficiency and assessment of the safety of stem cell grafts has become an important feature of stem cell-based research [6]. For this purpose, live cell imaging is an efficient method well-suited to dynamically tracking the location of the grafted stem cells [7]. A new class of optical nanomaterials, such as fluorescent organic dyes and semiconductor quantum dots, which can be used for live imaging, are being developed, because the low photobleaching thresholds and toxic nature of the materials used in the past has limited the observation times [7], [8]. Indeed, the preparation of long-lasting imaging reagents with high sensitivity is important in-vivo and in vitro cell imaging and tracking methods. Recently, it has been shown that fluorescent carbonaceous nanomaterials (FCN) are superior in terms of the stability of their photoluminescence (PL), exceptional biocompatibility and low cytotoxicity when compared to luminescent dye molecules, luminescent proteins and semiconductor QDs [9].

Over the last few decades, there has been a trend to find ways to prepare fluorescent carbon nanomaterials from the biomass resources produced by industrial activity because of the exhaustion of resources in the natural world and to counteract the generation of pollution and ecological troubles [10]. Moreover, such biomass resources are inexpensive, abundant and nontoxic offering a versatile approach for the production of multifunctionalized nanoparticles. Nowadays, hydrothermal carbonization technique is feasible method to prepared multifunctional carbon based nanoparticles from biomass with controlled sized and chemical properties in water medium, thus consider as environmental friendly and may not require toxic reagents such as a strong acid, solvent and organic reagent [11]. Hence, the exploration of cheap and sustainable sources of raw material must be considered for hydrothermal carbonization process to obtain fluorescent carbon materials in bulk level production. Thus, bio-waste and bio-renewable materials are believed to be an efficient substitute feedstock for production.

The silk cocoon is a typical biomass material produced by the Bombyx mori silkworm, which is composed of fibrous proteins (fibroin) and gum protein (sericin). The sericin covers the fibroin proteins cementing them together [12]. In silk based textile industrial activities, the separation of the sericin from the fibroin fibers demands the usage of large quantities of water, generating a large volume of wastewater, thus having an adverse impact on the environment. In particular, wastewater is released during the degumming process for the removal of the external sericin coating prior to the dyeing process [13]. Sericin, also called silk gum, is a globular protein, represented as a tube coating the outside of the silk fibroin, with a broad molecular weight distribution between 10,000 and 300,000 Da. During the degumming process about 25–27% of the original weight of the sericin is discarded in the wastewater [14]. Nevertheless, sericin has unique functionality with a wide range of molecular weights, a special molecular structure and exceptional absorption properties that make it suitable for a variety of biological activities including antioxidation, tyrosinase activity inhibition and anticancer treatment. Silk sericin has thus found versatile uses in the fields of bio-medical materials, drug delivery, cosmetics, and textile production [15].

However, sericin from silk degumming wastewater is an underutilized man-made renewable resource that is often simply discarded, leading to problems such as the accumulation of geologically different bio-waste residues in the soil. Despite the large number of possible uses, efforts have thus far have failed to meet the challenge of effectively utilizing sericin waste water as a renewable resource [16]. The remarkable biocompatibility of sericin gives it the potential to be converted into low-cost substrates for bio-imaging probes in stem cell research such as for monitoring oral fat stem cells. Thus, this study seeks to develop ways to improve the recovery and utilization of a valuable raw material like sericin from industrial wastewater for the production of novel functional nanocomposite materials. The main purpose is to utilize industrial wastewater from the silk degumming process for the development of environmentally friendly low-load systems. Hence, we demonstrate an eco-friendly, low-cost and simple method for the fabrication of highly fluorescent sericin based carbonaceous nanoparticles (SCN) for bio-imaging probes for stem cells such as oral fat stem cells. This method is a facile one-step hydrothermal carbonization process carried out at low temperature (180 °C). Silk sericin is comprised of water-soluble proteins with strong polar groups, such as carboxyl, amino and hydroxyl groups, acting as side chains, which can easily enhance the fluorescent properties. Nano-sized fluorescent carbon nanoparticles are formed upon carbonization and functionalization occurs through dehydration of the sericin protein. We obtained sericin based carbonaceous nanoparticles with strong fluorescence and a carbon framework with unique properties, which are advantageous to use in cellular imaging, especially in stem cell imaging. The obtained results demonstrate that the SCNs could be simply internalized into the oral fat stem cells without affecting the characteristics of the stem cells, which can facilitate SCN usage for stem cell imaging and tissue regeneration. This could be the first attempt to produce fluorescent carbon nanomaterials from the sericin obtained from silk-industrial degummed wastewater to be used for the labeling of stem cells.

Section snippets

Materials and methods

The Bombyx mori silk cocoon degumming based wastewater was obtained from the silk textile industry in Taiwan (Danee Silk International Company). All the reagents were of analytical grade and were used as received without further purification. Deionized water was used throughout the experiments.

Sericin isolation

The following process prepared the silk sericin-containing wastewater solution, first the Bombyx mori cocoons were boiled for 40 min in an aqueous solution of 3 wt% citric acid, and then washed carefully

Results and discussion

Native silk sericin is enriched by a combination of various proteins which contain nitrogen source of amino acids including serine (Ser), tyrosine (Tyr), glycine (Gly), aspartic acid (Asp) etc [15]. Hence, it presents a reasonable source of inexpensive biomass, readily available from silk textile degumming wastewater. The silk fabric processing wastewater is enriched by a variety of unique amino groups which can serve as heteroatoms allowing the synthesis of biomass derived nanosized carbon

Conclusion

In summary, a method for the effective utilization of silk degumming wastewater has been demonstrated. We explore the promotion of an environmentally friendly low-load system capable of producing novel functional nanomaterials. Sericin based nano-sized carbon particles were prepared by hydrothermal carbonization at 180 °C in an aqueous medium. The demonstrated method for the preparation of functional carbon nanoparticles is a facile, eco-friendly method with potential for effective waste

Conflict of interest

The authors declare no competing financial interests.

Acknowledgement

The authors would like to thank Dr. Juin-Hong Cheng of National Defense Medical Center (Taiwan) for his assistance with the discussion on the imaging of stem cells.

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