3-D Silk Fibroin Porous Particles Created by the Ouzo Effect for Biomedical Applications

UNLV Theses, Dissertations, Professional Papers, and Capstones

5-1-2020

Ashley Nicole Lamb

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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

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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

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

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.

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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

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

References ...... 43

Curriculum Vitae ...... 49

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

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 templating agents

Reprinted from Ref. [26]...... 12

Figure 5: Process of self-forming microspheres. Reprinted from Ref. [26]...... 13

Figure 6: SEM images of porous scaffolds (left) and microspheres (right). Reprinted with copyright permission from [20]...... 15

Figure 7: Generic phase diagram showing where the Ouzo region lies whilst depicting ethanol as the solvent and oil as the solute. Reprinted from Ref. [37]...... 17

Figure 8: Effects of non-diluted SF in Ouzo solution immediately after mixing, which lead to condensing of the fibroin...... 21

Figure 9: Ouzo + SF solutions with varying oil concentrations, ranging from 6 to 10%, immediately after vortex mixing and 15 minutes later...... 22

Figure 10: Drying patterns of 5 different Ouzo + SF solutions. The 9% oil content droplet exhibited the most compact and dense drying pattern...... 24

Figure 11: Compound microscope images of 9% oil concentration droplet illustrating the uniform oil ring at different positions of the droplet...... 25

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Figure 12: Particle with 7% oil content under SEM; full particle view (top) and zoomed in view (bottom)...... 26

Figure 13: Histogram of pore size for 8% oil content from Figure 14 (bottom)...... 27

Figure 14: Particle with 8% oil content under SEM; full particle view (top) and zoomed in view (bottom); bottom image used for pore size distribution analysis...... 28

Figure 15: Histogram of pore size for 9% oil content from Figure 16 (bottom)...... 29

Figure 16: Particle with 9% oil content under SEM; full particle view (top) and zoomed in view (bottom); bottom image used for pore size distribution analysis...... 30

Figure 17: Particle with 9% oil content under SEM; underside view (top) of the particle and cross-sectional view (bottom)...... 31

Figure 18: Behavior of 9% oil concentration droplet being formed by the Ouzo effect under compound microscopy. Top image shows particle surrounded by oil; bottom image shows fully dried particle...... 33

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Chapter 1: Introduction

Bombyx mori (B. mori) silk has been at the forefront of recent research as a biomaterial for many different applications in the engineering, biochemistry, pharmaceutical, cosmetic, and textile industries due to its many desirable properties; though, it’s been utilized as a suture material for centuries. B. mori silk has been touted for its high biocompatibility and biodegradability, which make it an intriguing material to study for in vivo applications, as well as its ability to be easily chemically modified [1] –

[5]. For medical applications, proper extraction and preparation of the silk fibroin must be executed. This is because sericin, the protein that coats the fibroin, has been shown to cause an immune response [6]. In vitro studies done on rats show that silk fibroin causes an inflammatory response comparable to collagen and tissue culture plastic controls. While in vivo studies show that silk fibroin causes a lower inflammatory response than collagen and poly (L-lactic acid) [7]. For mechanical property comparisons, silk fibroin has an ultimate tensile strength of 740 MPa, while collagen ranges from 0.9-7.4 MPa and poly (L-lactic acid) from 28-50 MPa [6].

1.1 Fibroin Protein Structure

The silk fibroin component of B. mori silk is impressive from a biochemistry standpoint. The fibroin is composed of a light chain (~26 kDa) and a heavy chain (~390 kDa) which are linked by a disulfide bond [8]. The heavy chain is comprised of two large hydrophobic blocks and two large hydrophilic blocks at its N and C termini [9, 10]. The primary structure of silk fibroin is dominated by repetitive (GAGAGS)n sequences

(Figure 1), which allow for tight packing of stacked sheets of the hydrogen-bonded

antiparallel chains that forms the -sheet secondary structure [8, 9]. Stacking of these sheets occurs when the methyl and hydrogen groups of opposing sheets interact; strong hydrogen bonds and van der Waals forces create a structure that is thermodynamically stable [8, 11].

Figure 1: Representation of the repetitive sequence in B. mori silk fibroin (GenBank AF226688).

B. mori fibroin is the most -sheet rich structure when compared to other silkworm varieties [12, 13], which is due to its abundance (~53%) of (GAGAGS)n repetitive sequences. This repetitive structure makes up the bulk of crystalline/semi-crystalline regions in the silk fibroin [14].

1.2 Mechanical Properties

The predominance of -sheet structures in B. mori attributes to its high strength in comparison to other silks. As previously mentioned, B. mori has an ultimate tensile strength of 740 MPa, but its breaking strain is lower than other silks [15]. Studies also show that -sheet content is directly proportional to the Young’s Modulus of the silks;

implying that B. mori silk has a higher Young’s Modulus than other silks, as well.

Indeed, with B. mori silk having approximately a 50% -sheet content, it’s Young

Modulus was experimentally found to be 12 GPa [15]. Table 1 summarizes other mechanical properties of B. mori found in literature.

Table 1: Mechanical properties of B. mori silk from literature [16] – [18]. Tensile Strength Young’s Modulus Toughness Breaking Strain (%) (MPa) (GPa) (MJ/m3) 690  20 38.5  6.4 9.6  0.6 187  33 460  80 24.1  7.7 7.2  1.7 81  34

1.3 Silk Materials for Drug Delivery

There has been an increased interest in using silk-based biomaterials for drug delivery due to its unique properties, mentioned previously. Currently, synthetic polymers, such as polylactide-co-glycolide acid (PGLA), comprise the majority of sustained drug-delivery formulations [19]. However, while being deemed safe by the

FDA, the inherent properties and processing requirements of synthetic polymers hinder its use in certain applications [19]. Table 2 summarizes the unique and desirable properties that silk-based materials possess that make it a promising and better alternative over synthetic polymers for sustained drug delivery.

Table 2: Unique combination and desirable properties of silk for sustained drug delivery. Reprinted with copyright permission from [19]. Structure Predominantly hydrophobic, block copolymeric and modifiable sequence Self-assembly into -sheet rich supramolecular structures Strong intra-/intermolecular physical interactions Stimuli-responsive crystal polymorphism High and tunable molecular weight Processing Aqueous-based ambient purification and processing capabilities Versatile material forms Suitability with common sterilization techniques Physicochemical Controllable network density, hydration resistance and properties swelling Controllable surface charge through sequence modifications High thermal stability Robust mechanical properties Tunable aqueous solubility Biological properties Low inflammatory/cytotoxic/immunogenic potential Enzymatic, surface mediated biodegradation Slow, controllable biodegradation rates Non-toxic, neutral biodegradation products (amino acids and peptides) Pharmacological Tunable release rates via diffusion- and biodegradation- properties controlled release Encapsulation of poorly soluble drugs Drug stabilization

Pritchard et al. has studied, at length, the various formats that B. mori silk could be used for drug delivery: microspheres, hydrogels, silk-coated solid reservoirs, nanofilms/layer-by-layer coatings, bulk-loaded monolithic films, and 3-D scaffolds/sponges [20]. This thesis explores the fabrication of silk fibroin porous particles through an interesting method, the Ouzo effect.

Chapter 2: Silk Fibroin Preparation

The raw fiber from Bombyx mori cocoon is comprised of two main components: sericin (~20-30%) and silk fibroin (~70-80%) with trace amounts of carbohydrates and waxes [21]. Sericin - which is a glue-like protein - coats the double-stranded fiber called silk fibroin [22]. Of the B. mori cocoon, only the silk fibroin is used in biomaterial applications because sericin can initiate inflammatory responses [6]. To remove residual sericin, the cocoon undergoes a degumming process, which is summarized in Figure 2.

Degummed silk fibroin is resistant to being solubilized [23]. The next sections will cover the procedure of degumming the fiber and how to prepare the silk fibroin to be used in experiments mentioned in Chapter 4.

Figure 2: Full degumming process of silk fibroin ending in an aqueous silk solution ready to use for experiments.

2.1 Degumming Procedure

The process of degumming the silk fiber was adopted from Rockwood et al. with some modifications [24]. The modified degumming procedure used in this thesis spans two days – cutting out dialysis and centrifugation in favor of a different method since a formic acid solution is utilized instead of a water solution – and each step is critical to successfully remove the sericin. The following equipment list is required for degumming:

• Titanium scissors

• Vinyl gloves

• Large stir bar

• Spatula

• 1 L and 2 L glass beakers

• Petri dish

• Glass stirrer

• Timer

• Hot plate

• Conductivity meter

• Small and large weigh boats

• Scale with precision to 1 decimal place and 4 decimal places

• 50 mL graduated cylinder

• Water bath

• Drainer

2.1.1 Extraction

The first day of the procedure is dedicated to extracting the fibroin from B. mori cocoons. Rockwood et al.’s procedure calls for measuring out 5 grams for cocoon, but the modified procedure used for the purposes of this thesis halves the value to 2.5 grams. The lowered amount also allows for an easier degumming process.

First, a 2 L glass beaker should be filled with 1 L of ultrapure water and put on a hot plate to boil; covering the beaker with aluminum foil and adding in a large stir bar will speed up the process. Then, measure out 2.5 g of silkworm cocoons in a large weigh boat on the 1 decimal precision scale. Note: the initial weighing process can allow about an error of about +0.2 grams due to debris on the cocoons; this extra mass will be lost either in the cutting process or during boiling. Next cut up the silkworm cocoons with titanium scissors into small pieces – generally each cocoon should be cut into four equal size pieces. Re-weigh the cocoon pieces to make sure that mass was not lost in the cutting process; final mass before boiling should be 2.5 g.

Measure out 2.12 grams of sodium carbonate (Na2CO3) in a small weigh boat on the scale with 4 decimal place precision. The weight of 2.12 grams for Na2CO3 is decided upon the need for its molarity to be 0.02. The Na2CO3 will then be added to the water on the hot plate. When the water is boiling and the Na2CO3 is dissolved, add in the cocoon pieces. Cook the cocoon pieces for exactly 30 minutes (using a timer) by stirring with a glass stir rod; stirring promotes better dispersion for the fibroin to be effectively degummed.

After 30 minutes, remove the silk fibroin and begin rinsing it with ultrapure water.

Rinsing should be repeated until all the Na2CO3 are removed from the silk fibroin. To

make sure that all residual Na2CO3 is removed from the fibroin, use the conductivity meter to compare the readings of sole ultrapure water versus the silk fibroin in ultrapure water; once the silk fibroin in ultrapure water is the same or very close to the reading of sole ultrapure water effective rinsing has been completed. Squeeze out any excess water from the silk fibroin and gently pull apart the fibroin to promote even drying. Place the silk fibroin in a clean petri dish and into the fume hood to dry overnight. For assurance that the fibroin was extracted properly, the resulting mass should be about

30% less than the starting mass of 2.5 grams.

2.1.2 Dissolution in Lithium Bromide

The second day of procedure involves dissolving the silk fibroin in lithium bromide (LiBr). The molarity required of the LiBr is 9.3. For effective dissolution, a ratio

1 part silk to 4 parts LiBr is required. The mass (in grams) needed of LiBr is calculated in equation (2.1), where X is the volume of water needed in mL (can be any amount, generally the procedure in this thesis called for 12 mL).

푔 푚표푙 1 퐿 (86.85 ) (9.3 ) ( ) (푋) = ___푔 표푓 퐿푖퐵푟 (2.1) 푚표푙 퐿 1000 푚퐿

Then pack the silk fibroin in a 50 mL conical tube and add in the LiBr; LiBr must be added to the fibroin and not the other way around. Then put the tube in a water bath set to 60C for 4 hours. Completely dissolved silk fibroin will appear to have a pinkish hue and be transparent while having a viscous consistency.

2.2 Condensing Procedure

Silk fibroin needs to be condensed in order to create the solution used in the experiments in Chapter 4. Condensing immediately follows dissolution from 2.1.2.

The first step in condensing is to measure ~500 mL of ethanol in a 1 L glass beaker. Then slowly pour the dissolved silk fibroin solution into the ethanol solution. To reclaim the condensed silk fibroin, stir the mixture while pouring and the silk fibroin will spin around the glass rod stirrer. Once all the dissolved silk fibroin has been condensed, rinse the silk fibroin in ultrapure water and make sure all residual LiBr is removed in a similar manner as described in 2.1.1. Squeeze out the water from the silk fibroin and pull apart the fibroin into very small pieces. The smaller the pieces are, the easier it will be to dissolve the silk fibroin in formic acid discussed in the next section. Place the pieces in a clean petri dish, place in a dryer hood, and let dry overnight.

2.3 Dissolution in Formic Acid

Formic acid is one of the only acids to dissolve silk fibroin while remaining a solution [25]. Silk fibroin in high concentrations will not dissolve in formic acid so for ease - generally in a 1 mL centrifuge tube - 10 w/v% (or ~0.100 g) of the small silk fibroin pieces from 2.2 was measured using the 4-decimal place precision scale. Hand mixing of the silk fibroin and formic acid solution is done by dispersing the silk fibroin pieces in the centrifuge tube with a small spatula. For an extra measure, the centrifuge tube was quickly vortexed. Finally, the centrifuge tube is to be placed on a rocker over night for complete dissolution.

While formic acid – and other acetic acids – is one of the few to dissolve silk fibroin, it has its own drawbacks. Namely, the solution is not stable for more than a month, which is visibly noted by the solution turning a dark golden or brown hue (Figure

3). The instability can impact its biological, chemical, and mechanical properties. It is advised to not use the silk fibroin and formic acid solution once it turns the golden/brown hue.

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.

Chapter 3: Porous Particles as a Vehicle for Drug Delivery

Emerging in the twentieth century as a novel method, porous particles were developed to improve drug delivery, amongst other uses in the pharmaceutical discipline [26]. Porous particles provide a large surface area while being classified as low-density solids [27]. The classification of porous particles is broken down in Table 3.

Porous particles have large surface area, uniform and tunable pore structure, and high porosity in contrast to common particles [28]. Porous particles are able to achieve increased adsorbability and compactibility which are paramount for effective drug loading [26].

Table 3: Classification of porous particles based on pore diameter [29].

Macroporous particles are paramount in the process of adsorption rather than transport, of which microporous and mesoporous particles are generally used [30].

3.1 Previous Work on Creating General Porous Particles

Zhou et al summarized a variety of ways to design porous particles. They discuss at length the mechanism of each method.

A common method of preparing porous particles is injecting and removing porogens or templating agents into particles in a two-step process. Though, the use of porogens or templating agents can be a tedious process. Both processes can be seen in Figure 4. In the porogen route, a droplet with core material undergoes thermal treatment and then the porogen is removed by volatilization or producing gas. In the templating agent route, a droplet with core material undergoes water evaporation and the agent is then removed with ethanol washing [26].

Figure 4: Process of creating porous particles using porogens and templating agents Reprinted from Ref. [26].

A second method to create porous particles removes the tediousness of porogens or templating agents. Wei et al. novelized the process – illustrated in Figure 5

- that can self-form porous microspheres. The pores are caused by (1) hydrophilic polyethylene glycol segments absorbing water molecules followed by a water evaporation process and (2) local explosion of microspheres due to fast evaporation of dichloromethane [31].

Figure 5: Process of self-forming microspheres. Reprinted from Ref. [26].

A third method involves using electrochemical dissolution of silicon to create porous particles by synthesis. This method seems the most complex of the three since it requires monitoring current or voltage to control porosity of the particle [32]. There are other ways to create porous particles, such as spray freeze drying or vacuum freeze drying; furthermore, each technique has its own advantages and disadvantages [33].

3.2 Previous Work on Creating Porous Structures from Silk Fibroin

Not much work has been done on creating porous particles with silk fibroin, but it has been at the center of porous scaffold research. However, Sun et al. synthesized silk

fibroin particles in use of pH-responsive drug delivery. Their procedure prepares the particles by the salting out method, which is a method of using a porogen that was discussed in 3.1. Per their results, the majority of the pores had a diameter around 500 nanometers [34].

Pritchard et al. discussed multiple ways to use silk fibroin in drug delivery but the most noteworthy are porous three-dimensional sponges and scaffolds, as well as microspheres. Figure 6 shows the differing morphology between three-dimensional sponges and microspheres. The three-dimensional sponges can be generated by salt leaching, gas foaming, or freeze-drying [35, 36]. These methods can be tedious, expensive, or impractical depending on the resources needed. However, while sponges and scaffolds have been mostly used for tissue engineering they show great promise in drug delivery as well. Pore size and morphology were dependent on which method was used to create the sponges and scaffolds; this is also true for the duration of drug release [20]. In Figure 6, the three-dimensional sponge is large in overall size and its pores vary greatly in their diameter size. Pritchard et al. also discuss the use of silk fibroin microspheres in drug delivery. However, they do not touch on any porous properties of the microspheres; they just mention the total diameter size of the microsphere which can range from 300 nanometers to 440 micrometers depending on the method used to create the microspheres.

Figure 6: SEM images of porous scaffolds (left) and microspheres (right). Reprinted with copyright permission from [20].

Chapter 4: Fabricating 3-D Porous Particles from Silk Fibroin

4.1 Ouzo Effect Introduction

The “ouzo effect” is a special kind of emulsification process that does not require energy nor a surfactant [37]. “Ouzo effect” gets its name from the Grecian drink, ouzo, which is a mixture of anise (anethole) oil and alcohol that then gets diluted with water to create a solution that’s milky-white in color. While the term “ouzo effect” is used throughout this thesis, this phenomenon goes by other names [38] such as

“spontaneous emulsification” or “nanoprecipitation.”

Emulsifying a solution is desirable in cases – for example, drug delivery – where different immiscible liquids need to form a stable dispersion into each other. The stability of the solution allows for a way to carry compounds through an aqueous fluid [37].

Using an emulsion to fabricate porous particles by means of the ouzo effect is under great consideration for biomedical applications because it is cost-effective and environmentally friendly.

4.2 Ouzo Effect Working Principle

An ouzo solution gets its milky-white color due to the instantaneous formation of micrometer size oil droplets – due to the oil becoming supersaturated thus scattering visible light – when the water gets added to the oil + ethanol mixture. These droplets become visible via Ostwald Ripening, in which they grow in size from nano- to micro- to macro-droplets during a short period of time. The spontaneous formation of the droplets does not rely on pH or stirring interference. Anethole oil is soluble in ethanol but

insoluble in water, in turn, ethanol is soluble in water; thus, explaining why oil droplets form only when water is added to the solution. The ouzo solution defies classical thermodynamic equilibrium since the solution creates an emulsion rather than a multi- phase solution. The ouzo region on a ternary phase diagram – Figure 7 – differs from the binodal and spinodal lines, which further shows that an ouzo solution does not follow the classical theory. The binodal line corresponds to the miscibility limit regarding composition of the solution while the spinodal line corresponds to the limit of thermodynamic stability [37].

Figure 7: Generic phase diagram showing where the Ouzo region lies whilst depicting ethanol as the solvent and oil as the solute. Reprinted from Ref. [37].

4.3 Fabricating 3-D Particles via the Ouzo Effect

Tan et al. has done extensive work on the Ouzo effect and specifically for using the Ouzo effect to create so called “Ouzo drops” – droplets that have a lifetime dictated by the Ouzo effect [39, 40]. While the Ouzo effect is still vaguely understood, Tan et al. reveal the physics that govern the life phases of an Ouzo drop. While the ethanol evaporates in the droplet, the oil nucleates at the rim of the droplet where there is a large surface tension. This process drives fast Marangoni flow within the drop and convection flows inward – which is in contrast to a pure liquid droplet where the flow goes outward. Furthermore, the nucleated oil becomes supersaturated where then the oil can coalesce and form the oil ring around the droplet [39]. In their initial work, Tan et al. researched Ouzo droplets without a suspension medium. They later researched the

Ouzo effect to create porous supraparticles with nanoparticles as the suspension medium.

Tan et al. attribute the fabrication of their porous supraparticles to the notion of self-lubrication in the Ouzo drop. An issue with typical colloidal droplets is that they suffer from a pinned contact line at the rim – where the colloidal particles are carried to the edge of the droplet as opposed to being densely packed. Prior solutions to the pinned contact line – colloquially known as the coffee ring effect – was to use surfactants or high cost surfaces, which is not ideal. By using their previous work, Tan et al. proposed using the Ouzo effect to form porous particles that solve the issue of the pinned contact line of colloidal droplets. The oil ring that forms in this phenomenon lubricates the nanoparticles so that they don’t travel towards the edge of the droplet, but rather towards the center as the oil ring’s diameter shrinks to form the supraparticle.

This process is considered self-lubrication since there are no outside forces or mechanisms that cause this to happen. They tout this process as being reliable and repeatable, while also being tunable, to mass produce porous supraparticles for various applications. They were also able to fabricate supraparticles that had high porosities ranging from 77 to 92%; with increasing porosity being attributed to increased oil to nanoparticle ratios [40].

4.4 Silk Fibroin Porous Particles

The following subchapters go over the main objective of this thesis, including experimental procedures, results and discussions, and future work.

4.4.1 Experimental Procedure

The method of fabricating silk fibroin porous particles is achieved by a quaternary liquid Ouzo solution comprised of ultrapure water, ethanol, trans-anethole oil, and formic acid with a suspension medium of dissolved silk fibroin. The addition of formic acid – and thus creating a quaternary liquid as opposed to a ternary liquid in previous works – is paramount in introducing silk fibroin to the solution as formic acid is one of the only acids to be able to completely dissolve silk fibroin; it also has a similar volatility as ethanol so that it will evaporate at a similar rate and not impede the Ouzo effect. Since ethanol and formic acid play a similar role in the evaporation process, it was imperative to find a critical ratio that would optimize the amount of ethanol (without condensing the silk fibroin, which was discussed in 2.2) in the Ouzo solution while incorporating formic acid which carries silk fibroin. The following ratios of ethanol to formic acid (without silk fibroin) were analyzed and were chosen for their ease to manipulate the volumes of the

liquid: 1 to 9; 2 to 8; 3 to 7; 4 to 6; 5 to 5; 6 to 4; 7 to 3; 8 to 2; 9 to 1. These ratios were tested using the volume for titration number 7 outlined in [39, Tab. S1]; the titration’s volume was chosen for its ease to manipulate the ethanol counterpart (1 mL) for analyzing the effect of the aforementioned ratios and because it exhibited the Ouzo effect according to Tan et al. After analyzing each ratio’s effect under a compound microscope, it was noted that ratios where ethanol’s component was equal to or lower than formic acid did not exhibit the Ouzo effect as outlined by Tan et al. It was ultimately determined that an ethanol to formic acid ratio of 9:1 is used for the experimental procedure since it best kept the identity of the behavior of the Ouzo effect to create the particles. For the experimental procedure, each Ouzo solution with silk fibroin made had a standard volume of 1 mL, with a constant ultrapure water volume of 35% for consistency. The formic acid and silk fibroin solution (abbreviated as SF from here on) was diluted 4 times to lower the chances of the silk fibroin condensing with the higher amount of ethanol, since a non-diluted SF solution condensed immediately when added into the Ouzo solution (Figure 8).

Figure 8: Effects of non-diluted SF in Ouzo solution immediately after mixing, which lead to condensing of the fibroin.

Multiple batches of Ouzo + SF solution were made with differing oil volumes to analyze the effect of oil concentration in creating the silk fibroin particles. Table 4 summarizes the composition of these solutions, while Figure 9 shows what the compositions look like. Figure 9 illustrates that there is a limit to how much oil can be in an Ouzo solution.

Too much oil (i.e. 10% concentration) may cause phase separation in the centrifuge tube. While it is easy enough to vortex the solution again to get an emulsion, it implies high oil concentration leads to low stability and increased agitation of the solution can cause premature condensing of the silk fibroin.

Table 4: Composition of Ouzo + SF solution with 9:1 ethanol to formic acid at differing oil percentages, ranging from 6 to 10%.

Figure 9: Ouzo + SF solutions with varying oil concentrations, ranging from 6 to 10%, immediately after vortex mixing and 15 minutes later.

The Ouzo + SF solutions were fabricated, first, by mixing the trans-anethole oil into ethanol so that it would dissolve; then adding in the ultrapure water - which turns the solution milky-white per the Ouzo effect – and vortexing the ternary liquid for 15 minutes; finally, the SF was added in and the quaternary liquid was vortexed for less than 1 minute to prevent fibroin condensing.

In order to fabricate the silk fibroin porous particles, a hydrophobic surface was created; polydimethylsiloxane (PDMS) was chosen as it's a well-known hydrophobic type silicone that is transparent and non-toxic. The PDMS substrate was poured onto glass microscope slides to hold up its structural integrity as a base for the Ouzo + SF droplets to dry on. The majority of the results was based on 1 L droplets manually placed on the PDMS surface by a micropipette. Larger volume droplets could potentially incur gravitational effects, thus affecting the self-lubrication process of fabricating the particles. Self-lubrication (i.e. the Ouzo effect) was observed under a compound microscope.

4.4.2 Results and Discussion

Fully dried particles (from droplets of 1 L in volume) made of the solution compositions from Table 4 are shown in Figure 10. The drying patterns for 6% and 7% oil content were not uniform and exhibited cracking; this was consistent throughout multiple trials.

Figure 10: Drying patterns of 5 different Ouzo + SF solutions. The 9% oil content droplet exhibited the most compact and dense drying pattern.

The non-uniformity of the drying patterns of the lower oil percentage solutions could be attributed to the lower oil content itself; there was not enough oil in the solution to form a uniform oil ring around the droplet. Conversely, the solution with a 10% oil content was prone to layer separation in the centrifuge tube, which alludes to too much oil concentration. The notion of a uniform oil ring around the droplet is illustrated in Figure

11. The oil ring affects the shape of the resulting particle, thus it’s imperative that it appears around the whole particle – which wasn’t consistently occurring for lower oil concentrations. It is also important to note that the droplet placed on the PDMS surface is already an emulsion, i.e. an Ouzo solution, right at the onset; the Ouzo Effect is being enhanced while on the surface.

Figure 11: Compound microscope images of 9% oil concentration droplet illustrating the uniform oil ring at different positions of the droplet.

Particles of 7%, 8%, and 9% were analyzed under scanning electron microscopy

(SEM). Figure 12 depicts a full and zoomed in view of the particle with 7% oil concentration. It is visibly less porous than what will be shown in later figures.

Figure 12: Particle with 7% oil content under SEM; full particle view (top) and zoomed in view (bottom).

The particle created with 7% oil content further confirms the Ouzo effect’s tunable nature; therefore, different oil concentrations yield different results. While lower porosity is not necessarily a bad quality, the application for the particle must be taken into consideration.

The particle created with 8% oil content (Figure 14) is considerably more porous than that of the 7% oil concentration particle – visibly so. Though, its relative 2-D porosity is still less than that of the particle created by 9% oil content. The pore size was analyzed by ImageJ using the binary segmentation technique; the data was then generated into a histogram using MATLAB. Figure 13 shows a histogram of the various pore sizes from Figure 14 (bottom). The average pore size from this data set is 4.257.

The data shows the two highest frequencies in for the two smallest pore size bins. The frequency trend starts off decreasing, but increases for the sixth pore size bin where the frequency trend decreases sharply thereafter.

Figure 13: Histogram of pore size for 8% oil content from Figure 14 (bottom).

Figure 14: Particle with 8% oil content under SEM; full particle view (top) and zoomed in view (bottom); bottom image used for pore size distribution analysis.

Finally, a particle with 9% oil content is shown in Figure 16. This particle exhibits the highest relative 2-D porosity of the 3 droplets analyzed under SEM. The pore size

was analyzed by ImageJ using the binary segmentation technique; the data was then generated into a histogram using MATLAB. Figure 15 shows a histogram of the various pore sizes from Figure 16 (bottom). The average pore size from this data set is 4.951.

The data shows the highest frequency for the smallest pore size bin. The frequency decreases as pore size increases.

Figure 15: Histogram of pore size for 9% oil content from Figure 16 (bottom).

Figure 16: Particle with 9% oil content under SEM; full particle view (top) and zoomed in view (bottom); bottom image used for pore size distribution analysis.

Figure 17 illustrates how the pores of the particle are not just surface level but are interspersed throughout the particle – adding to its true porosity. The top image shows a view of the underside of the particle – the surface that comes into contact with the

PDMS surface first; whereas the bottom image shows a cross-sectional view of the particle.

Figure 17: Particle with 9% oil content under SEM; underside view (top) of the particle and cross-sectional view (bottom).

Figure 18 further illustrates the Ouzo effect under compound microscopy for the 9% oil concentration particle. The top image shows the silk fibroin surrounded by oil which agrees with results found in [40, Fig. 2]. The bottom image shows the fully dried particle, which becomes more densely packed as the oil ring further shrinks.

Figure 18: Behavior of 9% oil concentration droplet being formed by the Ouzo effect under compound microscopy. Top image shows particle surrounded by oil; bottom image shows fully dried particle.

Porosity is a key parameter to measure for porous particles, which can determine if the particle meets the needs of a specific application. Future work with these particles

entails drug loading and delivery, so superior porosity is desired. Table 5 outlines the relative 2-D average porosities of the three types of particles discussed earlier. These porosities are considered relative and two-dimensional because only a region of interest on the surface of the particles are analyzed; the porosity of the measurement doesn’t take into its three-dimensional properties throughout the particle. The porosity measurements are meant to compare the particles with each other and not meant as the true porosity of the particle. ImageJ was used to calculate the porosity and the procedure was done three times – and then averaged – to ensure consistency. The

SEM images used in the procedure are from Figures 12, 13, and 14, respectively.

Table 5: Average 2-D relative porosities of particles containing 7, 8, and 9 percent oil concentration, respectively; calculated by ImageJ.

The low relative porosity for the particle with 7% is expected since it is visibly less porous than the other two particles. Indeed, this ratio is higher for the particle with 9% oil concentration (with silk fibroin as the suspension medium) than it is for the particle with 8% oil concentration. This result is to be expected as a higher oil content proportionally influences the porosity of the particle.

4.4.3 Future Work: Drug Loading and Delivery

The process to generate numerous silk fibroin particles is simple and timely; many particles can be stored and are easily removed/washed from the PDMS substrate without ruining their structure. To move forward with the generated particles, it is imperative to remove the oil residue from the particle or else the residue could impact adsorption; this can be done by washing the particles with ethanol, methanol, or isopropyl alcohol. The particles fabricated for this thesis were done so with drug loading and delivery as the next step in research. The antibiotics, Amoxicillin and

Metronidazole, were proposed as starting points for drug loading and delivery research.

Preliminary work was done with these antibiotics to linearly fit concentration versus absorbance to create an equation that could be used to determine unknown concentrations in future work. This procedure was done by using a UV-Vis spectrophotometer to determine absorbance of the drug at varying, known concentrations. Tables 8 and 9 show the concentrations with their corresponding absorbance values (calculated by the UV-Vis) for Amoxicillin and Metronidazole, respectively. The values were measured at the antibiotics’ maximum absorbance wavelength, which is 272 nm for Amoxicillin (found by trial and error) and 340 nm for

Metronidazole [41].

Table 6: Concentration versus absorbance values for Amoxicillin measured from UV- Vis spectrophotometer at 272 nm, with corresponding correlation coefficient.

With a correlation coefficient so close to 1, meaning the data points have high linearity, a linear fit equation can be made. Equation (4.1) illustrates the linear relationship between concentration and absorbance for Amoxicillin, which can be used to determine unknown concentrations during the loading and delivery processes.

푦 = 0.0033358푥 + 0.00257 (4.1)

The correlation coefficient and equation were determined by the software, Origin.

Table 7: Concentration versus absorbance values for Metronidazole measured from UV-Vis spectrophotometer at 340 nm, with corresponding correlation coefficient.

Likewise, since the correlation coefficient is so close to 1, there is confidence in

Equation (4.2) to depict the linear relationship for the data points and using the equation to determine unknown concentrations.

푦 = 0.035334푥 + 0.02558 (4.2)

These results segue all the work done on the silk fibroin porous particles to be able to research their ability to load and deliver Amoxicillin and Metronidazole.

Chapter 5: Conclusion

While silk fibroin used for drug delivery can be fabricated in many formats, the methods to do so are often time consuming and expensive. Fabricating porous particles using the Ouzo effect not only is cost-effective and requires minimal procedural time, it is a reliable method that can produce a multitude of particles in a short time frame. The particles can be stored for long periods of time as well. When comparing the particles generated from 7%, 8%, and 9% oil content, their relative, 2-D porosities increase as oil content increases. Since the particles generated in this thesis have a pore size in microns, they can be classified as macroporous. Macroporous particles are ideal for absorbance of solutions, adding to the effectiveness for drug loading and delivery.

Porous particles have large surface area, uniform and tunable pore structure, and high porosity, which makes them an intriguing prospect for silk-based drug delivery research.

With the advent of simpler and more cost-effective methods to create porous particles – and, in particular, from silk fibroin – more research should be on the horizon.

Due to its high molecular weight of around 400 kDA (or 400,000 g/mol), adjustments to the ouzo + SF solution needed to be made. This is because the silk fibroin has a molecular weight 5,000 times larger than that of the nanoparticles used in the study from Tan et al. Thus, there had to be a significant increase in oil content for the Ouzo effect to work. Despite early troubles in finding a proper oil concentration for the ouzo + SF solution, the principles of the Ouzo effect indeed worked to create porous particles out of silk fibroin. The biggest worry was having the ethanol component condense the silk fibroin prematurely, but multiple trials were done to optimize ethanol

and fibroin concentration to avoid this. In doing so, the ouzo + SF solutions have been stable for months without fibroin condensing.

The next steps for this work are to use the fabricated particles for drug loading and delivery for the antibiotics, Amoxicillin and Metronidazole. Preliminary work has been done to create linear fit equations that will help identify unknown concentrations in the loading and delivery processes. The UV-Vis spectrophotometer is a tool that will aid in this work by measuring absorbance of solutions.

Appendix

The following is the copyright permission agreement to reprint Table 2 and Figure

6 in this thesis. Figures 4, 5, and 7 fall under a Creative Commons Attribution-

NonCommerical 3.0 Unported Licence and do not need copyright permission as long as proper and correct acknowledgement is given with the reproduced material and is not used for commercial purposes.

ELSEVIER LICENSE TERMS AND CONDITIONS Mar 29, 2020

This Agreement between Ashley Lamb ("You") and Elsevier ("Elsevier") consists of your license details and the terms and conditions provided by Elsevier and Copyright Clearance Center. License Number 4798111019514 License date Mar 29, 2020 Licensed Content Elsevier Publisher Licensed Content Journal of Controlled Release Publication Licensed Content Title Silk-based biomaterials for sustained drug delivery Licensed Content Author Tuna Yucel,Michael L. Lovett,David L. Kaplan Licensed Content Date Sep 28, 2014 Licensed Content Volume 190 Licensed Content Issue n/a Licensed Content Pages 17 Start Page 381 End Page 397 Type of Use reuse in a thesis/dissertation Portion figures/tables/illustrations Number of 2 figures/tables/illustrations Format electronic Are you the author of this No Elsevier article? Will you be translating? No Title Student Institution name University of Nevada Las Vegas Expected presentation date Apr 2020 Portions Table 1 and Figure 3 Ashley Lamb 2058 Poetry Ave Requestor Location

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United States Attn: Ashley Lamb Publisher Tax ID 98-0397604 Total 0.00 USD

References

[1] A. Murphy and D. Kaplan, "Biomedical applications of chemically-modified silk

fibroin.," J Mat Chem, vol. 19, pp. 6443-6450, 2009.

[2] A. Murphy, P. John and D. Kaplan, "Modification of silk fibroin using diazonium

coupling chemistry and effects on hMSC proliferation and differentiation.,"

Biomaterials, vol. 29, pp. 2829-2838, 2008.

[3] E. Wenk, A. Murphy, D. Kaplan, L. Meinel, H. Merkle and e. al, "The use of

sulfonated silk fibroin derivatives to control binding, delivery and potency of FGF-2

in tissue regeneration.," Biomaterials, vol. 31, pp. 1403-1413, 2010.

[4] C. Vepari, D. Matheson, L. Drummy, R. Naik and D. Kaplan, "Surface modification

of silk fibroin poly(ethylene glycol) or antiadhesion and antithrombotic

applications," J Biomed Mat Res A, vol. 93A, pp. 595-606, 2010.

[5] S. Sofia, M. McCarthy, G. Gronowicz and D. Kaplan, "Functionalized silk-based

biomaterials for bone formation," J Biomed Mat Res, vol. 54, pp. 139-148, 2001.

[6] G. Altman, F. Diaz, C. Jakuba, T. Calabro, R. Horan and e. al, "Silk-based

biomaterials," Biomaterials, vol. 24, pp. 401-416, 2003.

[7] L. Meinel, S. Hofmann, V. Karageorgiou, C. Kirker-Head, J. McCool and e. al, "The

inflammatory responses to silk films in vitro and in vivo," Biomaterials, vol. 26, pp.

147-155, 2005.

[8] C. Vepari and D. Kaplan, "Silk as a biomaterial," Prog Poly Sci, vol. 32, pp. 991-

1007, 2007.

[9] A. Matsumoto, J. Chen, A. Collette and e. al, "Mechanisms of silk fibroin sol-gel

transitions," J Phys Chem B, vol. 110, pp. 21630-21638, 2006.

[10] U. -J. Kim, J. Park, C. Li and e. al, "Structure and properties of silk hydrogels,"

Biomacromolecules, vol. 5, pp. 786-792, 2004.

[11] D. Kaplan, C. Mello, S. Arcidiacono and e. al, Silk, Boston: Birkhäuser, 1997.

[12] T. Lefev're, M. Rousseau and M. Pe'zolet, "Protein Secondary Structure and

Orientation in Silk as Revealed by Raman Spectromicro-scopy," vol. 92, no. 8, pp.

2885-2895, 2007.

[13] F. Pacquet-Mercier, T. Lefev’re, M. Auger, M. Pe’zolet, "Evidence by Infrared

Spectroscopy of the Presence of Two Types of B-Sheets in Major Ampullate

Spider Silk and Silkworm Silk," Soft Matter, vol. 9, no. 1, pp. 208-215, 2013.

[14] T. Asakura, M. Okonogi, Y. Nakazawa and K. Yamauchi, "Structural Analysis of

Alanine Tripeptide with Antiparallel and Parallel B-Sheet Structures in Relation to

the Analysis of Mixed B-Sheet Structures in Samia Cynthia Ricini Silk Protein Fiber

Using Solid-State NMR Spectroscopy," J. Am. Chem. Soc, vol. 128, no. 18, pp.

6321-6238, 2006.

[15] C. Guo, J. Zhang, J. Jordan, X. Wang, R. Henning and J. Yarger, "Structural

Comparison of Various Silkworm Silks: An Insight into the Structure-Property

Relationship," Biomacromolecules, vol. 19, no. 3, pp. 906-917, 2018.

[16] G. Fang, S. Sapru, S. Behera, J. Yao, Z. Shao, S. Kundu and X. Chen,

"Exploration of the Tight Structural-Mechanical Relationship in Mulberry and Non-

Mulberry Silkworm Silks," J. Mater. Chem., vol. 4, pp. 4337-4347, 2016.

[17] B. Swanson, T. Blackledge, B. J. and C. Hayashi, "Variation in the Materials

Properties of Spider Dragline Silk Across Species," Appl. Phys. A: Mater. Sci.

Process, vol. 82, no. 2, pp. 213-218, 2006.

[18] C. Fu, D. Porter, X. Chen, F. Vollrath and Z. Shao, "Understanding the Mechanical

Properties of of Antheraea Pernyi Silk - From Primary Structure to Condensed

Structure of the Protein," Adv. Funct. Mater, vol. 21, no. 4, pp. 729-737, 2011.

[19] T. Yucel, M. Lovett and D. Kaplan, "Silk-based biomaterials for sustained drug

delivery," Journal of Controlled Release, vol. 190, pp. 381-397, 2014.

[20] E. Pritchard and D. Kaplan, "Silk fibroin biomaterials for controlled release drug

delivery," Expert Opinion on Drug Delivery, vol. 8, no. 6, pp. 797-811, 2011.

[21] J. Matthews, The textile fibres: their physical, microscopal and chemical properties,

New York: J. Wiley & Sons, 1913, pp. 63-96.

[22] L. Wray, X. Hu, J. Gallego, I. Georgakoudi, F. Omenetto, D. Schmidt and D.

Kaplan, "Effect of processing on silk-based biomaterials: Reproducibility and

biocompatibility," J Biomed Mater Res Part B, vol. 99, no. 1, pp. 89-101, 2011.

[23] H. Yamada, H. Nakao, Y. Takasu and K. Tsubouchi, "Preparation of undegraded

native molecular fibroin solution from silkworm cocoons.," Mater Sci Eng, C, vol.

14, pp. 41-46, 2001.

[24] D. Rockwood, R. Preda, T. Yucel, X. Wang, M. Lovett and D. Kaplan, "Materials

fabrication from Bombyx mori silk fibroin," Nature Protocols, vol. 6, pp. 1612-1631,

2011.

[25] H. Kweon, H. Ha, I. Um and Y. Park, "Physical properties of silk fibroin/chitosan

blend films," Journal of Applied Polymer Science, vol. 80, no. 7, pp. 928-934,

2001.

[26] M. Zhou, L. Shen, X. Lin, Y. Hong and Y. Feng, "Design and pharmaceutical

applications of porous particles," RSC Advances, vol. 7, no. 63, pp. 39490-39501,

2017.

[27] S. Sharma, P. Sher, S. Badve and A. Pawar, "Adsorption of meloxicam on porous

calcium silicante: characterization and tablet formulation," AAPS PharmSciTech,

vol. 6, no. 4, pp. E618-625, 2005.

[28] P. Sher, G. Ingavle, S. Ponrathnam and A. Pawar, "Low density porous carrier

drug adsorption and release study by response surface methodology using

different solvents," Int J Pharm, vol. 331, no. 1, pp. 72-83, 2007.

[29] X. Du and J. He, "Fine-tuning of silica nanosphere structure by simple regulation of

the volume ratio of cosolvents," Langmuir, vol. 26, no. 12, pp. 10057-10062, 2010.

[30] G. Ahuja and K. Pathak, "Porous Carriers for Controlled/Modulated Drug Delivery,"

Indian J Pharm Sci, vol. 71, no. 6, pp. 599-607, 2009.

[31] Y. Wei, Y. Wang, H. Zhang, W. Zhou and G. Ma, "A novel strategy for the

preparation of porous microspheres and its application in peptide drug loading,"

Journal of Colloid and Interface Science, vol. 478, pp. 46-53, 2016.

[32] J. Salonen, A. Kaukonen, J. Hirvonen and V. Lehto, "Mesoporous silicon in drug

delivery applications," J Pharm Sci, vol. 97, no. 2, pp. 632-653, 2008.

[33] W. Yin and M. Yates, "Effect of interfacial free energy on the formation of polymer

microcapsules by emulsification/freeze-drying," Langmuir, vol. 24, no. 3, p. 2008,

2008.

[34] N. Sun, R. Lei, J. Xu, S. Kundu, Y. Cai, J. Yao and Q. Ni, "Fabricated porous silk

fibroin particles for pH-responsive drug delivery and targeting of tumor cells,"

Journal of Materials Science, vol. 54, pp. 3319-3330, 2019.

[35] R. Nazarov, H. Jin and D. Kaplan, "Porous 3-D scaffolds from regenerated silk

fibroin," Biomacromolecules, vol. 5, pp. 718-726, 2004.

[36] J. Hardy, L. Romer and T. Scheibel, "Polymeric materials based on silk proteins,"

Polymer, vol. 49, pp. 4309-4327, 2008.

[37] R. Botet, "The "ouzo effect", recent developments and application to therapeutic

drug carrying," in J. Phys.: Conf. Ser., Aichi, Japan, 2011.

[38] F. Ganachaud and J. Katz, "Nanoparticles and Nanocapsules Created Using the

Ouzo Effect: Spontaneous Emulsification as an Alternative to Ultrasonic and High-

Shear Devices," ChemPhysChem, vol. 6, no. 2, pp. 209-216, 2005.

[39] H. Tan, C. Diddens, P. Lv, J. Kuerten, X. Zhang and D. Lohse, "Evaporation-

triggered microdroplet nucleation and the four life phases of an evaporating Ouzo

drop," PNAS, vol. 113, no. 31, pp. 8642-8647, 2016.

[40] H. Tan, S. Wooh, H. Butt and e. al, "Porous supraparticle assembly through self-

lubricating evaporating colloidal ouzo drops," Nat Commun, vol. 10, no. 2019, p.

478, 2019.

[41] S. Naveed and F. Qamar, "Simple UV Spectrophotometric Assay of

Metronidazole," Open Access Library Journal, vol. 1, no. 6, pp. 1-4, 2014.

Curriculum Vitae

Ashley Lamb

Graduate Assistant [email protected]

Summary

In my Graduate Program Leader role, I honed my abilities in data analysis and project support to provide support to the College of Engineering Tutoring Center and the Academic Success Tutoring Program as a whole. As a graduate student pursuing a Master's degree in Biomedical Engineering, I honed my abilities in engineering analysis and laboratory operation, by combining my Bachelor’s

Degree in Mechanical Engineering along with my minor in Biological Sciences. My collaboration, people-centric nature, and eagerness to get the job done have afforded me excellent leadership skills. As an extroverted and personable communicator with a proven track record in team supervision, my focus on building strong professional relationships has been a valuable asset throughout my career

– both academic and professional.

Qualifications

• Engineer in Training (EIT) – 0T8241

• Minors in mathematics and biological sciences

• Working knowledge of CAD: Solidworks

• Experience with ANSYS software to solve heat transfer problems

• Operation of biology and chemistry laboratory equipment

Experience

August 2019 – Present: UNLV Graduate Assistant

• Managed and analyzed data to develop structured benchmarks to meet

student’s needs and lead to increased involvement by 12% compared to

previous semesters

• Developed, lead, and facilitated presentations, workshops, and curriculum for

student staff to increase productivity and camaraderie

• Conducted one-on-one meetings with student staff for advising and giving

performance evaluations

• Functioned as liaison and developed relationships with other campus

departments to promote and improve the program

• Project lead on campus-wide initiatives for program exposure and growth

September 2016 – August 2019: UNLV ASC Math Tutor

• Assisted students with identifying and closing gaps in course content knowledge

• Worked with students individually and/or groups of up to 10 students daily

• Used proper questioning and communications skills to determine student’s

knowledge base

• Developed intercultural communications skills by working with diverse

population

• Worked with faculty and staff to better serve the student population

Education

• University of Nevada, Las Vegas, Master of Science – Biomedical Engineering,

May 2020

• University of Nevada, Las Vegas, Bachelor of Science in Engineering –

Mechanical Engineering, May 2018

References

Dr. Hui Zhao (Technical Advisor)

Associate Professor

Mechanical Engineering Department

University of Nevada, Las Vegas

Office: SEB 2174

Phone: 702-895-1463

Email: [email protected]

Michael J. Ramirez (Supervisor)

Senior Coordinator of Supplemental Instruction and Tutoring

Academic Success Center

University of Nevada, Las Vegas

Phone: 702-774-4625

Email: [email protected]