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3-D Silk Fibroin Porous Particles Created by the Ouzo Effect for Biomedical Applications UNLV Theses, Dissertations, Professional Papers, and Capstones 5-1-2020 3-D Silk Fibroin Porous Particles Created by the Ouzo Effect for Biomedical Applications Ashley Nicole Lamb Follow this and additional works at: https://digitalscholarship.unlv.edu/thesesdissertations Part of the Biomechanical Engineering Commons, Biomedical Commons, and the Biomedical Devices and Instrumentation Commons Repository Citation Lamb, Ashley Nicole, "3-D Silk Fibroin Porous Particles Created by the Ouzo Effect for Biomedical Applications" (2020). UNLV Theses, Dissertations, Professional Papers, and Capstones. 3914. http://dx.doi.org/10.34917/19412108 This Thesis is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in UNLV Theses, Dissertations, Professional Papers, and Capstones by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected]. 3-D SILK FIBROIN POROUS PARTICLES CREATED BY THE OUZO EFFECT FOR BIOMEDICAL APPLICATIONS By Ashley Lamb Bachelor of Science in Engineering – Mechanical Engineering University of Nevada, Las Vegas 2018 A thesis submitted in partial fulfillment of the requirements for the Master of Science – Biomedical Engineering Department of Mechanical Engineering Howard R. Hughes College of Engineering The Graduate College University of Nevada, Las Vegas May 2020 Thesis Approval The Graduate College The University of Nevada, Las Vegas April 10, 2020 This thesis prepared by Ashley Lamb entitled 3-D Silk Fibroin Porous Particles Created by the Ouzo Effect for Biomedical Applications is approved in partial fulfillment of the requirements for the degree of Master of Science – Biomedical Engineering Department of Mechanical Engineering Hui Zhao, Ph.D. Kathryn Hausbeck Korgan, Ph.D. Examination Committee Chair Graduate College Dean Jeremy Cho, Ph.D. Examination Committee Member Kwang Kim, Ph.D. Examination Committee Member Shengjie Zhai, Ph.D. Examination Committee Member Hui Zhang, Ph.D. Graduate College Faculty Representative ii Abstract Due to its high biocompatibility and biodegradability, silk fibroin – produced from Bombyx mori (B. mori) cocoons – has been at the forefront of research for many biomedical application formats: hydrogels, films, microspheres, and porous sponges/scaffolding, to name a few. For drug delivery, in particular, porous particles are desirable for their large surface area, uniform and tunable pore structure, and high porosity. This thesis focuses on the fabrication of porous particles from silk fibroin by the very interesting Ouzo effect. The Ouzo effect, so named because of the Greek beverage ouzo, describes the phenomenon of an ethanol + anethole oil solution turning milky-white in color once water is added in due to the spontaneous nucleation of oil droplets. Using the Ouzo effect to fabricate porous particles solves the numerous issues of typical colloidal droplet formation by not requiring energy nor a surfactant, which is cost effective and environmentally friendly; the Ouzo effect also tackles the so-called “coffee ring effect” of previous particle fabrication, in which a solution’s suspension medium travels to the edge of a droplet and leaves a residual ring. An Ouzo droplet is able to self-lubricate at the droplet’s edge and form an oil ring that forces the suspension medium to form a 3-D particle with tunable pore shape. By using the Ouzo effect to fabricate these particles from silk fibroin, the result is consistent macro-porous (pore diameter being greater than 50 nm) structures with relative 2-D porosity values greater than 70%. These features make the particles ideal for drug loading and delivery. iii Table of Contents Abstract ............................................................................................................................ iii Table of Contents ............................................................................................................ iv List of Tables .................................................................................................................... vi List of Figures ................................................................................................................. vii Chapter 1: Introduction.................................................................................................... 1 1.1 Fibroin Protein Structure ........................................................................................... 1 1.2 Mechanical Properties .............................................................................................. 2 1.3 Silk Materials for Drug Delivery ................................................................................ 3 Chapter 2: Silk Fibroin Preparation ............................................................................... 5 2.1 Degumming Procedure............................................................................................. 6 2.1.1 Extraction ........................................................................................................... 7 2.1.2 Dissolution in Lithium Bromide .......................................................................... 8 2.2 Condensing Procedure ............................................................................................. 9 2.3 Dissolution in Formic Acid ........................................................................................ 9 Chapter 3: Porous Particles as a Vehicle for Drug Delivery ..................................... 11 3.1 Previous Work on Creating General Porous Particles .......................................... 12 3.2 Previous Work on Creating Porous Structures from Silk Fibroin .......................... 13 Chapter 4: Fabricating 3-D Porous Particles from Silk Fibroin ................................ 16 4.1 Ouzo Effect Introduction ......................................................................................... 16 4.2 Ouzo Effect Working Principle ............................................................................... 16 4.3 Fabricating 3-D Particles via the Ouzo Effect ........................................................ 18 4.4 Silk Fibroin Porous Particles .................................................................................. 19 iv 4.4.1 Experimental Procedure .................................................................................. 19 4.4.2 Results and Discussion ................................................................................... 23 4.4.3 Future Work: Drug Loading and Delivery ........................................................ 35 Chapter 5: Conclusion ................................................................................................... 38 Appendix.......................................................................................................................... 40 Copyright Permission .................................................................................................... 40 References ...................................................................................................................... 43 Curriculum Vitae ............................................................................................................. 49 v List of Tables Table 1: Mechanical properties of B. mori silk from literature [17] – [19]. ...................... 3 Table 2: Unique combination and desirable properties of silk for sustained drug delivery. Reprinted with copyright permission from [20]. ................................................ 4 Table 3: Classification of porous particles based on pore diameter. ........................... 11 Table 4: Composition of Ouzo + SF solution with 9:1 ethanol to formic acid at differing oil percentages, ranging from 6 to 10%. ........................................................................ 22 Table 5: Average 2-D relative porosities of particles containing 7, 8, and 9 percent oil concentration, respectively; calculated by ImageJ. ....................................................... 34 Table 6: Concentration versus absorbance values for Amoxicillin measured from UV- Vis spectrophotometer at 272 nm, with corresponding correlation coefficient. ............ 36 Table 7: Concentration versus absorbance values for Metronidazole measured from UV-Vis spectrophotometer at 340 nm, with corresponding correlation coefficient. ...... 37 vi List of Figures Figure 1: Representation of the repetitive sequence in B. mori silk fibroin (GenBank AF226688). ....................................................................................................................... 2 Figure 2: Full degumming process of silk fibroin ending in an aqueous silk solution ready to use for experiments............................................................................................ 5 Figure 3: Freshly prepared formic acid (aq) silk solution (left) and solution after 1 month has elapsed (right). The visible darkening tint means the solution can be unstable. ... 10 Figure 4: Process of creating porous particles using porogens and
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