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ELECTROSPUN SILKWORM SILK FIBROIN - INDOCYANINE GREEN BIOCOMPOSITE FIBERS: FABRICATION, CHARACTERIZATION AND APPLICATION TOWARDS HEMORRHAGE CONTROL
Title
ELECTROSPUN SILKWORM SILK FIBROIN - INDOCYANINE GREEN BIOCOMPOSITE FIBERS: FABRICATION, CHARACTERIZATION AND APPLICATION TOWARDS HEMORRHAGE CONTROL
Creator
Siddiqua, Ayesha
Date
2022
Description
Silk fibroin (SF), a structural protein found in the Bombyx mori cocoons has gained attention in several biomedical applications as tissue...
Show moreSilk fibroin (SF), a structural protein found in the Bombyx mori cocoons has gained attention in several biomedical applications as tissue engineering scaffolds and wound dressings owing to its properties such as biocompatibility, water vapor permeability and biodegradability. Indocyanine Green (ICG) is an FDA approved tricarbocyanine dye used in medical diagnostics due to its unique photothermal and fluorescent properties. Electrospinning is a highly efficient, easy, and inexpensive technique used to generate nanometer to micrometer thick fibers. In this study, SF and ICG were co-spun to generate flexible microfibers with high surface area to volume ratios. Pure silk, SF-ICG (0.1%) and SF-ICG (0.4%) were chosen for the purpose of this study. Since, as-spun fibers are unstable in aqueous solutions, post treatment methods were explored to enhance the durability of the fibers and to minimize ICG leaching. It was found that ethanol vapor treatment (EVT) not only induced β-sheet formation in SF but also improved the SF-ICG interaction thereby reducing ICG leaching from the composite fibers. Ethanol vapor treated SF-ICG fibers showed less ICG leaching than liquid ethanol treated (LET) SF-ICG fibers indicating the efficacy of the EVT. The increase in SF solution viscosity with ICG concentration suggested a strong silk-ICG interaction which was further confirmed by DSC. The 1h water uptake and the three-day mass loss experiments indicated that the fibers are stable and highly absorbent material. Heat evolution was evaluated by measuring the temperature change in water of a fixed volume after irradiation with a 500 mW, 808 nm diode laser. The heat evolved by the flat fiber scaffolds was higher than the 3D fiber balls, indicating improved light penetration in the former. Pure silk produced negligible heat and it was used as a control. With 14.9 W/cm2 irradiation, the post-treated SF-ICG (0.4%) 3D fibrous ball of 2-3 mg dry weight, solidified a drop of bovine blood in 40 s. In contrast, a single layer fiber matrix required 3 min. to achieve the same clotting effect. Fibers folded into flat scaffolds were able to solidify a blood drop in 25 s. Pure silk fibers in all the cases showed negligible change after irradiation. The results suggest that a larger contact area of fibers is desirable for faster blood clotting, and EVT prompted better ICG retention in SF fibers. Based on the above results, SF-ICG (0.4%) fibers were utilized in a device developed to mimic blood flowing at a rate of 0.5 mL/h through a damaged blood vessel. It was found that irradiation of SF-ICG locally placed at the “damage” region effectively stopped “bleeding” whereas irradiated pure silk was unable to control the blood flow, which demonstrated the success of our SF-ICG fibers towards hemorrhage control.
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Stimulation-Responsive Materials for the Treatment of Disordered Tissues
Title
Stimulation-Responsive Materials for the Treatment of Disordered Tissues
Creator
Clutter II, Elwin Dean
Date
2022
Description
Microenvironments offer physical and chemical cues to cells that affect their behavior. These cues can be bioactive chemicals such as drugs...
Show moreMicroenvironments offer physical and chemical cues to cells that affect their behavior. These cues can be bioactive chemicals such as drugs and cytokines, matrix cues like stiffness and composition, and electrical signals as seen in membrane potentials and epithelial wound healing. Controlling these cues can drive cell behavior toward apoptosis, proliferation, or changes in protein expression. In this thesis research, three components were used to model changes in cell behavior: the near infrared dye indocyanine green (ICG), silk fibroin (SF) from Bombyx mori silkworm cocoons, and carbon nanotubes (CNT). The mechanism of ICG photobleaching was studied for the efficacy to kill neuroblastoma cancer cells. ICG was twice as effective at killing neuroblastoma than fibroblast cells. Albumin stabilized monomeric ICG to enhance photobleaching by improving light interaction, and photodegradation of ICG into α,β-unsaturated aldehydes led to significant reduction of proliferation in neuroblastoma cells by targeting cell signaling components such as protein transcription factors. SF-ICG composite materials were developed into films and electrospun fibers. These composite materials were examined as light-activated wound coverings to control bleeding in hemorrhage, using ICG’s photothermal effect. ICG added an absorbance peak to SF at 805 nm. Irradiation decreased this peak, produced a new absorbance peak at 352 nm, and an increased fluorescence peak around 490 nm, showing photochemical changes that may be useful in sensor design. Increased heat production from irradiation of SF-ICG occurred in the films with 0.2% w/w ICG in SF during 1 min irradiation, whereas SF-ICG fibers required improvement of processing by ethanol vapor treatment (EVT) to reduce loss of ICG during preparation. EVT improved ICG retention in SF fibers during sterilization with 70% ethanol solution thus improving heat generation in the SF-ICG fibers. Heat evolved from SF-ICG 0.2% film solidified bovine blood within 42 s, with visible changes after the first 6 s. SF electrospun fibers were investigated to optimize silk preparation to reduce diameters and increase alignment to mimic local native cell environments with and without CNT to add conductivity for enhancement of electrical stimulation. CNT reduced SF fiber diameters below 1% and effected alignment differently as the concentration increased. Longer degumming times decreased SF fiber diameters and alignment of the same concentration. The optimized conditions were 1 h degumming time with 1.5 ml/h flow rate at a concentration of 100 mg/ml spinning solution with 0.25% CNT to form fibers with 1.36 (± 0.09) µm diameter and 0.31 (± 0.01) a.u. alignment. Additionally, SF fibers were used as a long-term cell growth scaffold to compare with decellularized native tissue. Decellularized tissue decreased fibroblast mRNA expression of collagen type 1, lysyl oxidase-like 1 and matrix metalloproteinase 9, while all other genes expression was the same as cells on plastic. The SF fiber scaffold reduced fibroblast expression of collagen type 3 compared to growth on plastic, and both collagen types 1 and 3 increased over growth time on SF. Immunofluorescence staining showed both collagens newly deposited on the SF scaffold and improved over time. In conclusion, proteins stabilized ICG monomers to improve light interaction, efficacy of photobleaching, and heat evolution to induce apoptosis in neuroblastoma and agglutination of blood in SF composites. Irradiation of ICG enhanced changes in optical properties and may have sensor applications. The decrease of genes may be due to each scaffold having lower stiffness compared to plastic. ICG and SF are both materials with applications as biological scaffolds in pelvic organ prolapse and treatment of neuroblastoma and hemorrhage.
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