In this project, we focus on developing a method to produce synthetic spider silk thin films. Using these films we optimized mechanical properties, lowered cost, and improved the environmental impact using different processing methods. Applications for spider silk films are broad, ranging from physical protection to biocompatible materials. This project was designed to improve mechanical properties and production methods of films made from synthetic forms of MaSp1 and MaSp2 from the dragline silk of Nephila clavipes. We have increased the mechanical stress (200 MPa) to more than 4 times that of similar products with elongations as high as 35%. The films have also been analyzed using NMR, XRD, and AFM or SEM showing that the secondary structure in as-poured films is mainly alpha-helical and after processing this structure turns to an aligned betasheet formation similar to that in spider silk fibers.

A needlestick injury is defined by the Center for Disease Control (CDC) as a percutaneous injury due to accidental handling of a sharp. The CDC estimates that approximately 400,000 needlestick incidences occur each year in United States healthcare facilities, and reports from other developed countries, such as the United Kingdom and Spain, share similar frequencies. Further, the World Health Organization (WHO) estimates two million international healthcare workers are exposed annually to infectious disease as a consequence of a needlestick event, resulting in 37.6% and 39% of hepatitis B and hepatitis C cases, respectively. In the United States, federal and state legislation have greatly reduced incidence rates since the late 1980s, providing education, better protocols and effective post-exposure management. Additionally, the introduction of national surveillance databases led to stronger epidemiological support for the causation of needlestick injury and consequently, a stronger national awareness. In an effort to better protect healthcare workers, corporations such as DuPont and BD have further reduced needlestick incidences in the United States by designing products ranging from safety-engineered syringes to adhesive strips surrounded in strong synthetic materials such as Kevlar® and Lycra®. These devices are instrumental in minimizing the needlestick problem in both the clinic and in the operating room. As part of the current United States legislation, healthcare organizations are mandated to implement and utilize these safety-engineered syringes and needles. Despite the rise in protective equipment, national database surveillance and federal/state legislature, the incidence rate remains high as hundreds of thousands of injuries persist each year. We sought to find other solutions for better protecting healthcare workers through the implementation of golden orb weaver spider silk in personal protective equipment. This silk, gathered from the Nephila clavipes, is one of the strongest and toughest biomaterials in known existence. Its characteristically high energy absorption makes it an ideal material for reinforcing gloves and other protective equipment for healthcare workers. We believe that products made from this silk would serve as strong barriers against needlestick injury and bloodborne pathogen exposure. We are in the process of designing and fabricating such a glove and completed preliminary strength testing to ensure the superiority of our material. Tensile testing conducted at Tufts' Department of Biomedical Engineering suggests that our silk possesses the same mechanical profile as N. clavipes silk found in published literature. We plan on utilizing Fourier-transform infrared (DSC-FTIR) microspectroscopy to study the protein structure and possibly conducting enzyme degradation assays to assess the property changes under unique conditions. This information combined with our patented extraction and reinforcing methodology will provide the groundwork for partnering with industry leaders to make this product a reality and help eliminate the incidence of needlestick injury.

Spider silks are biological materials that have inspired the humankind since its beginning. From raising the interest of ancient philosophers to the practical outcomes in the societies, spider silks have always been part of our culture and, thus, of our scientific development. They are protein-based materials with exceptional mechanical and biological properties that from liquid solutions passes to the solid fibres once extruded from the body of the spiders. Spider silks have deeply been investigated in these decades for their possible outcomes in biomedical technology as a supporting material for drugs delivery or tissues regeneration. Furthermore, spiders build webs with the support of different types of silks to create mechanically efficient structures, which are currently under investigation as models for metamaterials and fabrics with superior mechanical properties. This diversity in materials and structures makes spider silks scientific outcomes potentially infinite. In this work, we present some of the outputs of these three years of PhD. We explored the properties of the native material across different aspects (different species and glands) and trying to find possible derived applications (tissue engineering). Then we explored the mechanical behaviour of the natural structures (such as orb webs or attachment discs) coupled with their biological functions. In order to develop to an industrial level this material, we tried to understand and improve the physical properties of artificial spider silk, which helps also in understanding the ones of the native materials.

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No / X-Radiography is widely used in the investigation of works of art and other culturally significant artefacts to reveal and record details of their construction, modification and state of preservation. Radiography is considered to be a non-destructive technique but its increasing use in the study of historic textiles has prompted the testing of this assumption as X-rays and other forms of electromagnetic radiation, such as light and micro-waves, cause changes in materials which may be detrimental to their physical stability. An experiment was undertaken to test the safety ofradiography for the imaging of silk fabrics as these are particularly susceptible to photodegradation. The results from a series of radiographic exposures of modern and historic fabrics show that excessive exposure to low energy X-rays produced no detectable changes in their mechanical integrity. This indicates that the customary levels of radiographic exposure used in imaging will not be detrimental to textiles.

Cell-surface engineering has been attracting increased interest in the field of biotechnology, tissue engineering, cell therapy, or biosensors/bioelectronics. Thin nanocoatings or sometimes referred as nanoshells allow for modifying and controlling variety of cell properties, specifically retardation of cell division or growth, masking immunological properties, providing chemical and mechanical resistance to external stressors, and ability to further functionalize shells in order to guide cells attachment, their proliferation and function in artificial environment. Bottom-up approach, utilizing layer-by-layer (LbL) assembly of wide variety of different components (synthetic and natural polyelectrolytes, nanoparticles, and other nano-structures) has been introduced and elaborated to modify cell surfaces. Despite successful examples of the LbL-based cell encapsulation with polyelectrolytes, cytotoxicity of their polycation components possesses severe limitations for this approach. Additionally, by constructing rigid non-permeable shells can suppress the essential properties of cells. In this view, the goal of this research is to explore the formation of cyto-compatible ultrathin coatings from synthetic and natural polymers through utilization of non-cationic counterparts, with possibility to actively control cell division, provide protection from external environment, and temper shell properties in order to elicit or change specific cell response.

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Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In this study we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus , whose DNA coding sequences have not been reported. Based upon solubility differences in 8 M guanidine hydrochloride, it is demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining that the egg case silk is relatively complex at the molecular level, containing a large number of proteins with differing molecular weights. Protein components of egg case silk with a size about 100 kDa were obtained by a solubilization time course study, which indicates these proteins are likely to be embedded in the silk filament. Peptides in these 100 kDa proteins were released by tryptic in-gel and in-solution digestion. The peptides were sequenced using a MALDI tandem TOF mass spectrometer. Some of the de novo sequences were confirmed using a linear ion trap mass spectrometer equipped with a nanospray ion source. Combining the peptide sequences obtained, reverse genetics was employed to trace silk genes encoding proteins containing these de novo peptides. Three silk protein coding sequences were successfully discovered, which encode silk proteins named 3B, T1 and ECSP-1, respectively. 3B and T1 show the standard fibroin protein pattern. Amino acid repeat patterns were observed in these two silk clones. But the amino acid compositions of 3B and T1 show differences with the total amino acid composition of egg case silk, and also, the peptide sequences cannot be found in the primary amino acid sequences of 3B and T1. ECSP-1 protein represents one of the egg case silk proteins with a size of about 100 kDa. A number of peptide sequences obtained by mass spectrometric de novo sequencing were successfully located in ECSP-1's primary amino acid sequence. Sequence analysis demonstrates ECSP-1 represents a new class of silk proteins, with fibroin-like properties. The expression pattern of ecsp-1 is largely restricted to the tubuliform gland inside of the L. hesperus spider, with lower levels detected in the major and minor ampullate glands, which also confirms the identity of ECSP-1. It is also demonstrated that ECSP-1 assembles into higher aggregate structures through the formation of disulfide bonds. Peptide sequences from silk proteins from the Tarantula spider Grammostola rosea were also obtained. These sequences will be beneficial in obtaining genes encoding the silk from this spider species.