Capillary Force Lithography (CFL) is one of the novel techniques that established recently for fabricating biomimetic substratum of tissue engineering. This approach of making nano-scale scaffold allows us to fabricate complex design of polymer substrates at low-cost. One practical challenge to further application of CFL to biomedical science is biocompatibility of the materials used in CFL. Most of the materials used for CFL are only biocompatible in vitro, but not in vivo. 2-hydroxyethyl methacrylate (HEMA) is introduced as a good alternative biomaterial with exceptional biocompatibility. We recently succeeded in fabricating nano-structured pHEMA via CFL. We confirmed the structure of pHEMA nano-substrate with SEM imaging and AFM measurement. We hypothesized that biocompatibility of HEMA will enhance the cell contact with the nano-substrate, which will let cells penetrate through the grooves. Cells penetrating through the nano-grooves can benefit from increased surface area for cells to contact with polymer substrate, and allow cells to efficiently interact with glycan present on the surface to achieve more biomimetic environment.

Currently, various methods of creating engineered heart muscle cell, or cardiomyocyte, patches are being researched. These cardiomyocytes patches have shown therapeutic potential for implantation over injured or killed heart tissue. Current research often focuses on deriving these cardiomyocytes from stem cells, but these stem cell-derived cardiomyocytes are often not as mature as adult cardiomyocytes. Growing these cells on patterned as opposed to flat surfaces, or substrates, also affects the maturity of the cells. This project is the development of automatic video analysis software for cardiomyocytes. This provides a method of automatically quantifying contraction strength and orientation of cardiomyocytes, therefore providing an indicator of the maturity of the analyzed cells. Furthermore, the analysis program can simultaneously measure voltage-sensitive dyes in order to provide information about calcium levels as electrical signals called action potentials travel throughout the cells. This computational analysis will be performed upon human embryonic stem cell (hESC) derived cardiomyocyte tissue, as well as primary adult cardiomyocyte tissue isolated from rat hearts, grown on both flat substrate as well as nanopatterned substrate. Primary cardiomyocyte tissue is anticipated to show greater contractility, higher levels of alignment, and faster action potential propagation than the hESC-derived cardiomyocyte tissue due to its greater maturity. Furthermore, cardiomyocytes grown onto nanopatterned substrate are expected to show greater contractility, alignment, and action potential propagation than those cells plated onto flat substrates. The program in development can be used to automatically create a quantitative measurement of cardiomyocyte maturity levels across a wide array of various experiments. Being able to use the same computational analysis on different experiments both allows for easy comparison of results as well as substantiating the resultant data. Ultimately, this project provides simple automatic quantification of certain key properties of cardiomyocytes based upon videos of their contractions.

Duchenne muscular dystrophy (DMD), an incurable disease that causes severe morbidity and mortality, affects one in every 3600 male children in the United States. Stem cell therapy and cell replacement therapy have been utilized as possible solutions to the disease; however, there has only been limited success due to two major deficiencies: the lack of blood vessels in the regenerating tissues resulting in oxygen deficiency, and the lack of proper structural cues resulting in the disorganization and un-differentiation of the stem cells. Previous research has demonstrated the angiogenic and myogenic potentials of sphingosine-1-phosphate (S1P). Moreover, muscle tissues in human body have highly organized nanoscaled structures that guide the organization and differentiation of muscle cells. We designed nanopatterned muscle patches conjugated with S1P, and proposed if the patches would allow better cell survival, retention, and long-term functionality. We developed biodegradable S1P-conjugated nanopatterned muscle patches with capillary force lithography (CFL), chemical conjugation, and culturing of undifferentiated muscle cells in vitro. The patches’ biochemical, structural, and functional properties were characterized. We also evaluated the force generation, the long-term potential of regeneration, and the synchronization of the engrafted tissue of the patches within the limb muscle of mice. We anticipated proper vascularization of the patches and that the implanted cells would develop into differentiated, mature, and functional skeletal muscle cells due to the bio-mimicking mechanical cues provided by the nanopattterns. This project aimed to develop fully functional muscle cell patches which could be engrafted into patients with DMD and thereby treating the disease. In addition, the experiment would also improve our understanding in the response of muscle cells to topographical cues.

 Affecting nearly 1% of the human population, von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder worldwide.  VWD is caused by impaired platelet adhesion due to low plasma von Willebrand Factor (VWF) concentration, or dysfunction.  The largest plasma glycoprotein, VWF's function is to mediate attachment of platelets to the exposed collagen on blood vessel walls at sites of injury.  This interaction is sensitive to changes in flow forces or shear stress.  To facilitate the study of VWF, the Thomas Lab is developing a single-molecule force measurement platform called magnetic tweezers; a tool capable of performing multiple single-bond force measurements in parallel.  To use magnetic tweezers, VWF protein domains of interest are adsorbed to a glass surface on one end, and to a magnetic bead on the other.  Electromagnets are then placed above the slide, applying an upward force to the VWF domains.  The design and optimization of the VWF domain tethers is the focus of this research project.  At present, a tethering scheme is being tested that utilizes the strength and longevity of the biotin-streptavidin non-covalent bond and the fimH lectin domain to mannose interaction.  An alternative tethering scheme employing histidine tags instead of fimH is also being explored.  While both types of tethers have been expressed successfully, tested in the magnetic tweezers, and shown to bind beads to the surface under force, recent data has shown that non-specific adhesion between protein-functionalized magnetic beads and the coverslip is an issue.  Using immunoassays such as ELISA and the Western Blot, tether designs will be optimized using magnetic tweezers.  With the capability to more accurately mimic physiologic conditions than immunoassays alone, coupled with the intrinsic ability to multiplex, the quantity and quality of data that could be acquired is impressive.

We have developed nanoparticle-based combination antivirals (ARVs) as an effective strategy for oral or topical HIV prevention. Currently, most candidate-microbicides consist mostly of gels, which have demonstrated poor efficacy in clinical trials. Studies have supported the successful impact of highly active antiretroviral therapy (HAART) as a standard of care for HIV/AIDS affected individuals, which has motivated the investigation of the topical delivery of combination antiretroviral drugs. The proposed work has the potential to address the delivery challenges brought upon by the diverse classes of ARV drugs. The primary appeal for this approach lies in the combination delivery of drugs with different properties and mechanisms of actions to minimize chances of resistance and improve efficacy. Here we show that individual ARV drugs can be encapsulated into poly (lactic-co-glycolic acid) PLGA nanoparticles with sizes ranging between 250-300 nm, zeta potentials of about ≥-20 mV, and high drug loading and encapsulation efficiency, as measured by dynamic light scattering (DLS) and high-performance liquid chromatography (HPLC), respectively. Furthermore, we found that these ARV drug-encapsulated nanoparticles demonstrated potent anti-HIV activity against HIV-1 BaL infection of TZM-bl cells. The effect of combination delivery of these ARV drug-encapsulated nanoparticles was evaluated to identify synergistic drug combinations and favorable drug-drug interactions. Our results demonstrate the potential for this drug delivery system as an effective strategy for HIV prevention. Successful outcomes of this endeavor could have profound implications on microbicide research and a potentially viable female-controlled prevention method for HIV.

It has been hypothesized that effective treatment of neurodegenerative diseases, such as Huntington’s Disease and Alzheimer’s, may be possible through the manipulation of neural stem cells to divide and replenish afflicted neuronal cell populations. A crucial component of this treatment would be the creation of a vehicle for the efficient delivery of stimulating genetic material into target cells. Prospective virus-based vectors accomplish this by making use of a recombinant virus and its natural ability for gene transfer. These viral vectors, while inherently effective, have significant safety issues with the possibility of eliciting harmful immune responses. Peptide-based copolymers are a promising non-viral alternative for delivery systems. While safer, these synthetic vectors have thus far proven less efficient. One problem is lack of consistent cell membrane penetration, preventing the therapeutics from reaching areas within the cell where they would come into effect. Our laboratory has recently incorporating the membrane-lytic peptide melittin (a molecule derived from bee venom) into our base DNA-binding peptide-based polymers. The melittin-functionalized materials were shown to increase gene delivery efficiency by at least 10-fold compared to control materials both in vitro and by injection into mouse brain. This current study seeks to develop a multifunctional delivery system by incorporating the neuronal-targeting peptide “Tet1,” for specialized treatment of neurodegenerative diseases. Previous work showed that conjugation of Tet1 to control polymers results in increased transfer specificity to neuronal cells in vivo. Potentially, Tet1 and melittin can synergize to further improve gene delivery to the central nervous system.

The control of infectious diseases in low-resource settings continues to be impacted by the limited availability of low-cost, easy to use diagnostics. However, even in developing nations, smart phones are common and can be used as “instruments” to control diagnostic devices. My senior capstone project aims to use a smart phone to input a sound wave into a microfluidic card and measure a sound wave coming out of the card to determine whether an analyte—the chemical that indicates whether a person is infected—is present in the sample. This is done by creating a network of fluid channels that can be modeled as an electrical circuit containing the fluidic equivalent of resistors, inductors, and capacitors (RLC circuit). When the sound wave is input to the system, it causes the fluid to oscillate and has a resonant frequency that is dependent on the characteristics of the system (the resistance, inductance, and capacitance). The goal is to cause a change in the fluid resistance, fluid inductance, or fluid capacitance through a chemical reaction (e.g. the creation of a bubble would change the capacitance or a change in viscosity would change the resistance). This change in a characteristic shifts the resonant frequency of the fluid circuit, allowing the presence of the analyte to be recognized. This project seeks to integrate a means of detection (like a microphone) into the diagnostic card to measure the analyte-dependent resonant frequency. This card would then be used as the platform for a measurement of blood clotting. Completion of this project would take the system one step closer to bringing simple and effective diagnostic platforms to developing nations.

Artemisinin (ART) is a sesquiterpene lactone (three rings chemical structure), derived from the plant Artemisia annua. Several studies have shown significantly higher cytotoxicity to cancer cells compared to normal cells. However, the mechanism of this is still in debate. The majority of researchers claim that ART reacts with intracellular iron in cancer cells to generate free radicals (species that contain unpaired valence electrons) which are responsible for the cytotoxicity. A recent finding showing ART inhibits cancer cell growth through inhibiting a protein has raised questions about the free radical mechanism for ART to inhibit growth of cancers. We hypothesize that the endoperoxide bridge (two oxygen atoms joined by single bond in a ring structure) in artemisinin forms free radicals by reacting with intracellular iron in cancer cells. To confirm our hypothesis, Molt-4 human leukemia and MDA-MB-231 human breast cancer cell lines will be treated with Deferoxamine (DX) along with Artemisinin. DX is an iron chelating agent, a chemical that could eliminate the iron in cancer cell. Our research shows that the cytotoxicity of ART is DX concentration dependent, lower concentrations (10 µM to 30 µM) reduce the cytotoxicity while higher concentrations (50 µM to 100 µM) increase it. Thus, our results show that lowering iron (to certain levels) by DX inhibits the cytotoxicity of ART. Furthermore, we have also performed experiments about the effect of phenyl-alpha-tert-butyl nitrone, PBN, an antioxidant, on ART anticancer mechanism (data not published). It is our understanding that antioxidant could inhibit the ART anticancer effects. After comparison, we found that the inhibitory effect of DX on ART cytotoxicity is the same as that of PBN. In conclusion, our preliminary results show that iron is necessary for the cytotoxicity of ART and it is free radical mediated.

Bone tissue healing is problematic for more than a fifth of fractures, bone defects, massive bone loss, and osteoporosis and requires regeneration or implantation of bone. The introduction of a therapeutic bone strategy in the body elicits the foreign body immune response which is characterized by the inflammatory cell recruitment and subsequent encapsulation of the foreign material by fibrotic tissue. The formation of the fibrotic tissue is generated by a process in which macrophages adhere to the foreign material and fuse to generate multi-nucleated cells called foreign body giant cells (FBGCs). FBGCs effectively separate the foreign material from surrounding tissue resulting in sustained inflammation and reduced efficiency. This response, known specifically as the chronic inflammatory response, represents a very challenging complication for biomaterial fabrication in tissue engineering applications. Very recently, preliminary evidence has indicated that an optimal pore size exists in porous scaffolds that upon implantation result in the regeneration of a variety of tissues. Simultaneously incident findings in vivo have indicated that differentiated macrophage may have greater plasticity than previously accredited, and may transdifferentiate into various cell types. These advances may aid clinical treatments for bone repair by facilitating bone growth and avoiding stem-cell therapy controversies. The aim of the project was to design a porous scaffold to promote efficient bone tissue regeneration by enabling macrophage transdifferentiation. The proposed biomaterial synthesizes new information on optimal scaffold pore size and macrophage plasticity in conjunction with a well-studied osteoinductive cue, to both control the human body immune response and facilitate bone growth. The approach thus far has entailed an in vitro analysis of macrophage morphology and structural fabrication of the biomaterial.

Medical diagnostic device development has been, in recent history, focused largely on paper-based diagnostics. Such devices offer incredibly low per patient cost with the downside of poor sensitivity and minimal ability to multiplex, or detect many different compounds simultaneously. New technologies which leverage exceedingly efficient silicon manufacturing have the potential to approach the cost of paper diagnostics with increased sensitivity and extensive multiplexing. This project aims to develop one such technology, silicon photonics. Using existing manufacturing technologies, silicon photonic sensing measures analyte mass accumulation through dielectric shift by taking advantage of the evanescent field of electromagnetic radiation passed through a silicon wafer. Aided by unique binding chemistries, silicon photonic devices are capable of highly specific analyte detection. The current stage in this project is focused on developing tools, the most notable being a rapid prototyping setup for testing chip design. The setup automates the interaction of a variety of necessary hardware components including fluidic pumps, movable stages, a microscope, and an infrared laser. By using such a rapid prototyping setup, it is hoped that a chip design that is extremely condusive to medical diagnostics will be developed and brought to cost effective levels.

A major obstacle in the efficient delivery of genes using non-viral vectors is sequestration and subsequent degradation of the vector in the endosomal/lysosomal trafficking pathway. While incorporation of membrane-lytic peptides into gene delivery vectors has been shown to increase transgene expression by disrupting the endosomal vesicles, non-specific membrane-disruption caused by these modified vectors leads to significantly increased toxicity. One approach to mitigating this increased toxicity is to mask the membrane-lytic peptides inside a pH-responsive micellar core that destabilizes during vesicle acidification in the endosomal/lysosomal pathway, revealing the membrane-lytic segments only when internalized. Incorporating pH-responsive imidazole elements in the hydrophobic micelle core which become charged under acidifying conditions can disrupt the micellar structure by electrostatic repulsion, leading to display of the active peptide, membrane lysis, and increased DNA delivery to the cytoplasm. After synthesis of the pH-responsive motif a panel of representative DNA delivery polymers will be evaluated for pH-responsiveness, transfection efficiency, and cytotoxicity.

One of the major intracellular barriers to non-viral gene delivery is degradation through the endosomal/lysosomal pathway. To overcome this barrier, pH-sensitive polymers that swell in acidic pH and ultimately disrupt the endosome can be designed to escape these compartments. Previously, the Pun lab developed HPMA-oligolysine copolymers that have transfection efficiencies approaching that of polyethylenimine (PEI), the gold standard for transfection. In order to increase the copolymers’ transfection efficiency, oligohistidines were incorporated into the copolymer in both statistical and block architectures to impart pH-sensitivity. Transfection studies with these two copolymers showed that statistical incorporation of oligohistidine increased transfection efficiency, while block incorporation did not increase transfection efficiency. To determine if structure-function differences affected cellular uptake and endosomal buffering, we performed uptake inhibitor and endocytic buffering inhibitor studies. Through these studies, we found that both copolymer geometries preferred non-clathrin coated caveolae uptake pathways, and that they differed in endosomal buffering capabilities. The statistical copolymer showed increased endosomal buffering capacity, which was consistent with the overall higher transfection efficiency of the statistical copolymers over the block copolymers. From these two studies, we are better able to understand the relationship between structure and function in gene delivery using polymers.

Cardiovascular disease is the leading cause of death worldwide, and current treatments for heart failure are limited to slowing the disease progression or transplanting a donor heart. One potential approach to restore heart function is to transplant human embryonic stem cell-derived cardiomyocytes (hESC-CMs). An independent and similarly promising approach is to overexpress the enzyme ribonucleotide reductase (RR), the enzyme responsible for the production of the small molecule deoxy-ATP, which has been previously shown to improve contractility and diffuse between cardiomyocytes via gap junctions. We hypothesize that by overexpressing RR in hESC-CMs and then transplanting the latter into infarcted hearts, we will improve global cardiac function by increasing dATP content throughout the myocardium. To begin testing this hypothesis, I will test the efficacy of an RR adenovirus in hESC-CMs in vitro using quantitative PCR and immunostaining, and I will then correlate the subsequent improvement in fractional shortening following in vivo transplantation to the amount of successfully engrafted cells. To date, I have successfully developed an immunofluorescence staining protocol for RR. I am currently working on designing an immunohistochemistry protocol to identify graft tissue and stain for RR and GFP and localize RR in relation to the infarct. After confirming the protocol, I plan to apply it to the fixed rat heart slices to assist in the proof of concept of the transplantation of hESC-CMs overexpressing RR as a novel therapeutic method.

Active drug targeting involves a specific “lock and key” binding interaction, which enables drugs to be delivered exclusively to specific sites in the body thereby reducing the toxic side effects and improving the efficacy of the drug. However, the field of active-targeting lacks an efficient drug carrier. We are proposing to use a RAFT-based glycopolymer as a novel drug carrier. It is composed of three different types of monomers: 1) pyridal-disulfide methacrylate (PDSMA) for drug conjugation to the polymer through a disulfide bond, 2) hydroxyl propyl methacrylate (HPMA) to make the polymer biocompatible and soluble in the body, and 3) carbohydrate monomers that enable active-targeting through the specific carbohydrate-receptor interactions on outer cell membranes. Then the three monomers are polymerized together by reversible addition-fragmentation chain transfer (RAFT) polymerization. The resulting copolymers were characterized by nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The polymer size is around 8.5 kDa with low polydisperisty (<1.1). The polymer composition is 5% PDSMA, 75% HPMA, and 20% carbohydrate. The bioactivity of these polymers has been tested by surface plasmon resonance (SPR). In order to test the binding specificity of the mannose copolymer to ConA, I tested two different copolymers that contained different carbohydrate monomers. One polymer contains mannose, which specifically interacts with ConA. On the other hand, galactose, a stereoisomer of mannose, does not bind to ConA as expected. These results are promising because the mannose copolymer demonstrates good bioactivity, even though it contains a lower composition of mannose. These RAFT-based glycopolymers provide insight into the design of future active-targeting drug delivery systems.

Silicon microring resonator biosensors utilize nanoscale light confinement to detect biomolecules in a real-time and label-free manner with high sensitivity. Detecting the presence of antibodies that bind human tissue and blood antigens is crucial in transplant and transfusion medicine, as mismatched products can impact the clinical outcome of the recipient. The focus of the current research is to improve data analysis methods to perform serologic analysis of human plasma using a silicon photonic chip. Microring resonator chips were prepared by physisorption of streptavidin followed by functionalization with biotin-conjugated histo-blood group antigens. Testing was performed using undiluted human plasma to detect histo-blood group antibodies on the functionalized chip surface. A secondary enhancement step using an anti-immunoglobulin antibody enabled the quantification of the surface bound antibodies. Data analysis was performed using on-chip positive and negative controls, and kinetic analysis was accomplished using association models. The quantitative determination of antibody binding profiles with a model antibody system is discussed to show how these devices can ascertain kinetic behavior. Positive determination of plasma antibody content is shown, proving these devices can be used in this application at current clinical standards. These devices are suited for clinical translation and with proper quantitative models and testing they could surpass current clinical methods.

Stem cell based therapeutics is a promising venture to regenerate a damaged heart after a myocardial infarction and to prevent the progression to heart failure. However, we currently do not have a clear understanding of the maturation processes of cardiomyocytes, and this has been one of the main obstacles limiting progress in the field. Here, we develop a device that simulates the mechanical conditions that human cardiomyocytes experience during their growth phase. The aim of the device is to apply a constant mechanical stress on cardiac tissue to test the effects of creep on cardiomyocyte size and alignment. Cardiac tissue used in this study is obtained by mixing a collagen solution with cardiomyocytes derived from the directed differentiation of human embryonic stem cells and pipetted into PDMS molds held in 6-well plates (6wp). Testing of initial designs incorporating a moving magnet below the 6wp coupled with a metallic base within the plate have resulted in too much friction, leading to uneven stretching of the cardiac tissue and the breakage of all constructs tested within 10 days. In response, a revised design is utilized, where a direct screw-driven platform suspended on top of the 6wp controls the mechanical stress applied to the cardiac tissue. This device effectively stretches the constructs to 110% of their initial length over a period of 14 days, without the breakage of any of the four constructs tested. Here we show that mechanical creep over an extended period of time affects cardiac morphology, where increased cardiomyocyte size and a greater degree of cell alignment within the cardiac tissue are observed with stretch versus non-stretched controls. This project provides insight on the mechanical cues involved in the maturation of cardiomyocytes, with the device offering a platform for in vitro modeling of human cardiac tissue.

Hydrocephalus is a pathology characterized by the accumulation of excess cerebrospinal fluid (CSF) within the brain, which can lead to dangerously high intracranial pressure (ICP) and brain tissue damage. Surgically implanting a cerebral shunt, which keeps ICP under control by mechanically draining the excess CSF, is the most common method of treating hydrocephalus, but these shunts are plagued with problems. Roughly half of all shunts fail within the first two years of implantation, with 98% failing within ten years. Through a collaboration with pediatric neurosurgeon Dr. Sam Browd of Seattle Children's Hospital, Dr. Barry Lutz’s laboratory is developing the “smart shunt” – an electromechanical shunt which uses electronic feedback control of ICP and a simpler valve design to regulate CSF drainage. The goal of my research is to design a testing platform for the smart shunt, which will be used to simulate CSF and ICP dynamics caused by the respiratory and cardiac cycles, changes in patient posture, and other physiological phenomena that are typical of the daily lives of patients. Since the smart shunt must function on battery power for several years, it needs to be tested with a physiologically accurate simulation system in order to produce an optimal balance between ICP control and energy efficiency. To maximize simplicity and flexibility, the system will be computer controlled and operated through virtual representations of ICP and brain compliance modeled from patient data. The physical portion of the simulation system consists of regulators which stabilize pressure within fluidic reservoirs representing the brain and abdomen, speakers which produce physiological pressure oscillations within these reservoirs, and a pump which circulates mock CSF through the system. Apart from testing shunt performance, the ultimate objective of the testing system is enabling physicians to optimize the smart shunt to suit the unique brain physiology of each patient.

Millions of people in developing countries die from infectious diseases(e.g. malaria and tuberculosis), yet many of these deaths could be prevented if tools for accurate diagnosis were available. The current method for diagnosis of infectious diseases in low-resource settings is the lateral flow test (LFT), which has the appropriate usability (i.e. equipment-free, rapid, and easy to use), but lacks the required sensitivity to have clinical utility for some disease targets. Thus there is a need for diagnostic tools with improved clinical sensitivity and with the appropriate characteristics for use in low-resource settings. I have designed a novel two-dimensional paper network (2DPN), which enables more sophisticated chemical processes for improved sensitivity and usability in low-resource settings. The novel device utilizes commercial enhancement solutions for signal amplification via a metal catalytic reaction to increase sensitivity. The set of reagents and reagent volumes have been optimized for sensitivity and incorporated into a 2DPN assay device, where the programmed delivery of reagents is achieved through the design of the paper geometry and a fluidic on-switch. 

The field of tissue engineering is becoming increasingly aware of the role that the extracellular matrix (ECM) plays in driving cellular structure and function, especially in highly anisotropic skeletal muscle. Skeletal muscle is a cell dense tissue composed of bundles of aligned myotubes responsible for lateral contraction. Replication of this organization is critical for engineering physiologically-relevant tissue constructs. The fibrous nature of the ECM has led us to the use of bio-inspired topographical line patterns on the nanoscale. We employ UV-assisted capillary force lithography to generate polyurethane-based substrates which stimulate cell elongation and alignment. By incorporating glycidyl methacrylate into our substrates, we present a novel method for engrafting a brush polymer coating of the temperature responsive polymer p(N-isopropylacrylamide) (NIPAAm) onto nano-topographically defined substrates. Seeded with C2C12 mouse skeletal myoblasts, these substrates allow for the harvest of intact cell sheets without the use of enzymes. Using a gelatin coated plunger, these cell sheets are stacked to form tri-layer thick tissue constructs. This study explores the effects of a 3D microenvironment on mature myotube formation and the ability of our NIPAAm-based system to control myotube dimension in 3D by altering nanotopography dimension as well as layer number and the timing of differentiation, contributing to the understanding of the 3D microenvironment in regulating the skeletal muscle cell niche. In addition to paving the way for clinically-significant cell-based therapies, this work may prove vital to the development of advanced in vitro drug screening platforms for patients suffering from muscular dystrophy.

Polymers represent a versatile class of chemical compounds that have a wide range of impact in the field of medicine as implantable devices, tissue engineering scaffolds, and drug delivery vehicles. Synthetic polymers such as polyurethane acrylate (PUA) have been used to provide mechanistic cues in the form of altered nanotopography to help guide migration and differentiation in stem cells. To promote cell-biomaterial interactions to elicit desired cellular responses, these surfaces still must be modified. Chemical crosslinking reagents have been applied to engineer surfaces to enhance biological performance; however, they have limitations due to non-ideal reaction environments as well as their low coupling efficiencies with bioactive molecules and their potential converse effects under physiological conditions. Therefore, controlling the surface properties remains a challenge to induce enhanced interaction at the biomaterial interface. Material binding peptides were shown to possess specificity and high affinity to several inorganic surfaces such as gold or silica while offering easy conjugation with biomolecules. This peptide-based surface functionalization, if applied to polymers, can provide a more efficient and robust method of providing biological cues to synthetic polymers. Herein, we demonstrate a simple single-step incubation process that promotes noncovalent binding of bifunctional peptides on polymer surfaces. One end of the peptide possesses high binding affinity for the polymer substrate while the other end contains a bio-functional motif such as the RGD domain, which allows integrin-mediated cell adhesion. When combined with the topographical cues of an anisotropically nanopatterned polymer substrate, a flexible platform can be established to pursue many different research thrusts, such as enhanced stem cell differentiation. Specifically, we plan to use the established platform to uncover the underlying mechanisms of adhesion, morphology, and differentiation in C2C12 mouse myoblasts.

One of the most universal problems faced by persons with transtibial amputations is volume fluctuation of their residual limb. Since the shape and size of a prosthetic socket stays the same, fluctuations in limb volume affect the fit of a socket. For example, if a prosthesis is too tight; pressure spots, skin irritation, and muscle atrophy can occur. Conversely, a loose prosthesis creates gait instability and compromises the comfort of a patient’s prosthetic. Prosthetic socks of different materials and thicknesses are worn to compensate for the changes in volume and manage the discomfort created by daily and monthly volume changes. Properly fitting a residual limb to a socket is further confounded since the industry standard measurement of sock thickness is Ply, a non-linear unit of measurement. Our research examines how absolute thickness and material properties of prosthetic socks change with regular use. We measured the thickness and compression response of new out-of-box socks at three distinct points while in a donned condition. Measurement locations corresponded to area traditionally related to high interface stresses. Thickness and compression response were measured by a custom instrument. Participants walked for two hours while wearing a new sock, after which thickness was measured again in all three locations. Afterwards, volunteers took the socks home, along with a activity monitor, for regular use and returned every two days for one week, and then weekly for a month to re-assess sock thickness and response. The compression response after two hours of walking was used to validate cyclic testing conducting on an Instron material testing machine. Thickness response over long periods of normal use was used to characterize sock fatigue. We hope our study will help practitioners create more accurate volume management recommendations for their patients with a new understanding of the sock stiffness and durability.

 A significant challenge in intravaginal vaccination against mucosal pathogens concerns the immune privileged environment in the genital tract, which prevents migration of immune cells in the absence of inflammation. Dendritic cells (DC), the major antigen-presenting cell to T cells in lymph nodes, play a particularly important role in generating an effective immune response to vaccination. Our goal is to explore a new intravaginal vaccination strategy utilizing chemokines - short biological peptides that activate cells - to recruit DCs. We intravaginally administered CpG, MIP-3 Alpha and Beta-Defensin 2 to female C57BI/6J mice (Jackson Laboratories). Mice were euthanized after 24 hours and cells were isolated from both the spleen and reproductive tract. DCs were quantified using fluorescent activated cell sorting (FACS). We have demonstrated that DCs are actively recruited to vaginal mucosal tissues following intravaginal administration of the chemotactic peptides MIP-3 Alpha and Beta-Defensin 2. Future work will involve time course studies of DC recruitment and the delivery of nanoparticle encapsulated chemokines.

There are many challenges to creating high performance diagnostic tests for use in low resource settings. One approach is to use a paper network format. These paper networks operate by capillary flow and do not require dedicated instrumentation for transporting fluids in them. However, in order to carry out more advanced, multi-step, processes in these networks there is a need for well-characterized paper fluidics tools to precisely control fluid transport within the networks. To address this need, we have designed and characterized a cellulose valving mechanism to produce precise fluid delays, over a wide range of delay times, in both a bench-top system and a disposable card format with less than 10% variability in reproducibility. We achieved this by systematically investigating the effects of valve parameters such as valve dimensions and materials, on the delay time and reproducibility. Finally, the cellulose valves are demonstrated in a paper network card that can automatically sequentially deliver a series of fluids.

The pro-apoptotic (cell death inducing) peptide (KLAKLAK)2 (“KLA”) has been recently investigated for tumor killing applications. To increase the efficacy of the peptide, our group has added additional charged amino acids to increase cellular uptake (Gly-Lys-Arg-Lys-(KLAKLAK)2) (“GKRK-KLA”) and synthesized a polymeric construct of this peptide and HPMA (N-(2-hydroxypropyl)methacrylamide) (“p(HPMA-co-GKRK/KLA”). Preliminary studies have shown an increase in cell death per KLA unit in the modified constructs over the unmodified KLA construct. However, the mechanisms of cellular internalization and subcellular distribution of the peptide is unknown. Recently, we showed using fluorescence microscopy that the KLA constructs do not colocalize with mitochondria, the organelle that was believed to be affected. In this work, we applied radiolabeling and organelle separation techniques to investigate the mechanisms behind cellular uptake and subcellular trafficking over time of both the KLA peptide and our modified designs. These insights will allow us to design better materials to increase the efficacy of KLA peptide delivery.

Nicotinamide adenine dinucleotide (NAD) is used in many biological processes such as metabolism and cellular signaling. Recently NAD is found to have an important role in many signaling pathways. As NADs role in cellular pathways is being discovered, it is important to be able to detect and quantify the amount of NAD in vivo and in vitro by nuclear magnetic resonance (NMR) analysis. NAD is found in cells at low concentrations and its peak intensity in a phosphorus spectrum is much smaller than that of other phosphorus containing molecules like ATP. By decoupling and Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) experiments we hope to isolate and increase the signal of the characteristic phosphorus nuclei found in NAD molecules. Isolating and increasing the signal to noise of the NAD peak will prove that we have the right peak and allow us to quantify the amount of NAD in vivo. This will provide information about health risks of a subject.

Characterizing the migratory behavior of tumor cells is essential in understanding cancer progression and the invasive capacity of the cells that could lead to tumor metastasis. Previous research has established a correlation between the signalling protein known as stromal cell-derived factor-1 (CXCL12) and its corresponding CXCR4 receptor, which are expressed by a subset of cancerous glioma stem cells. Glioma cells expressing the CXCR4 receptor will move toward areas of higher CXCL12 concentration. This signalling pathway plays a significant role in glioma development by enhancing cell proliferation and motility. Thus, there is strong therapeutic potential by neutralizing this pathway through the use of CXCR4-inhibiting drugs. Microfluidics offers a suitable platform to optically characterize the migration of glioma stem cells under the influence of these inhibitory drugs while in the presence of controlled CXCL12 gradients. We present a microfluidic device integrated into a porous membrane to create an air-fluid interface that allows for organotypic slice culture and a fluidic chamber below the culture membrane that enables the formation of stable gradients. The integration of a 96-well plate allows for the simple addition of reagents using a standard pipette and circumvents the need for excess tubing and gels necessary for conventional microfluidic gradient assays. This device offers a spatial control of the biochemical gradient in an organotypic slice culture model to study chemotactic migration of glioma cells in a physiological-relevant manner. This model gives insight into the signaling processes related to the invasiveness and proliferation of glioma stem cells, and could potentially lead to new therapeutic strategies that target the CXCL12/CXCR4 pathway.

Skeletal muscle myosin can use a variety of nucleotides to varying effectiveness as substrates for contraction. We previously demonstrated that complete replacement of ATP with 2 deoxy-ATP (dATP) in activation solutions increases contraction of demembranated rabbit skeletal muscle and that enhanced performance results from increased myosin binding and cycling kinetics, especially during sub-maximal calcium activation. Here we report on a transgenic mouse (Tg-RR) that overexpresses the enzyme, ribonucleotide reductase, which converts ADP to dADP, which is rapidly phosphorylated to dATP. This results in a 10-fold increase in dATP content of skeletal muscle, which still constitutes less than 2% of the adenosine triphosphate nucleotide pool. We are examining the contractile and metabolic properties of skeletal muscle in this transgenic model. Preliminary data indicates the phosphocreatine to ATP (PCr:ATP) ratio of Tg-RR mice is significantly elevated relative to wild type mice, suggesting the Tg-RR mice may have an energetic advantage due to increased high energy phosphate reserves. Furthermore, the Tg-RR mice ran for a longer period of time at increased speed and for longer distances in a graded exercise test. Direct in situ stimulation of the gastrocnemius muscle indicates improved resistance to fatigue, consistent with both the exercise and the metabolic (PCr:ATP) findings. Interestingly, our preliminary experiments suggest the Tg-RR mice produce significantly less peak force and show signs of atrophy. Ongoing experiments are examining changes in mitochondrial content and function as well as contractile properties of isolated muscles to determine the mechanisms underlying the increased fatigue resistance of the Tg-RR mice. These results suggest a new direction for developing interventions to improve exercise tolerance in human patients.

The frequency of premature birth has risen to almost one in every eight births. Although most preterm infants survive, there is growing evidence of adverse neurodevelopmental outcomes due to alterations in brain structure and function. However, the relationship between morphometric changes and final neurological outcome is unknown. This project aims to locate specific structural bio-markers of delayed or decreased brain folding that correlate with decreased neurocognitive abilities, by implementing different quantifications of gyrification. Gyrification, or curvature of the brain, has been identified as a promising structural marker for neurodevelopment. Curvature measurements are heavily dependent on size of the surface which drastically increases during this brief developmental time period; therefore our framework includes normalization and ratio based measures for improved consistency. Our image analysis framework proposes a novel atlas-based automatic tissue segmentation which utilizes age-specific tissue probability maps to serve as a source of spatial priors. We propose to adapt an atlas-based segmentation technique from fetal to preterm analysis with the addition of lobe segmentations that increase the precision of segmentation and registration. This will allow for brain surface extraction by tessellation of tissue maps to reconstruct topology correct representations of the inner and outer cortical surfaces, in which our unique vertex-wise gyrification analysis will be mapped onto a population-average surface by volumetric unbiased template-free groupwise registration. Statistical modeling will be performed for each vertex to detect local and regional patterns of folding and asymmetry. This project has the opportunity to directly relate these measures of early brain folding against later measures of developmental outcomes derived from the Bayley's Scale of Infant Development (BSID) with the goal of predicting neurocognitive outcomes. Achieving this goal will allow for the possibility of earlier clinical diagnostics permitting timely neurodevelopmental interventions.

One of the current challenges in developing an effective microbicide is the ability to provide both short-term and long-lasting protection against HIV with a single application. In this project we seek to develop a nanofiber material that will be used to deliver HIV neutralizing antibodies from poly(vinyl) alcohol (PVA) nanofibers of different degree of hydrolysis to be used as a female controlled vaginal microbicide, using bovine serum albumin (BSA) as a model release protein. We hypothesize that the differences in the degree of hydrolysis of PVA will allow for controllable release of the BSA protein, because a higher degree of hydrolysis allows for a more crystalline structure, which may slow release. PVA was chosen for the nanofibers because of its quick-dissolving abilities and biocompatibility. We are using two compositions of PVA, 87-88% and 99+% degree of hydrolysis. We optimized electrospinning parameters to create PVA nanofibers with high mass yield and consistent fiber diameters that will be used to deliver the antibodies. The time to dissolution was measured by visually determining when no distinguishable fiber pieces were present when added to water at 37C. We found the time to dissolution of 87-88% degree of hydrolysis nanofibers to be approximately 15 minutes compared to greater than 3 days for the 99+% degree of hydrolysis. We incorporated 1% and 2% w/v BSA as a model protein in the PVA nanofibers. Using enzyme-linked immunosorbent assay (ELISA) we characterized the protein release of the fibers and compared the relationship between degree of hydrolysis and release patterns of the proteins. We expect that this project will provide a proof of concept for antibody encapsulating nanofibers for vaginal drug delivery.

Drug resistance is a growing problem in the fights against cancer, malaria, and various bacterial and viral pathogens. Recently, strands of Mycobacterium tuberculosis in India, Iran, and Italy have been found to be resistant to nearly all effective drugs, highlighting the need for drug resistance research. The anti-malarial drug artemisinin and its derivatives have been shown to exhibit cytotoxicity and high selectivity in targeting various types of cancer. We selected a strain of MOLT-4 leukemia cells for resistance to the artemisinin derivative dihydroartemisinin (DHA). The project goals were to characterize these cells by analyzing specifics such as growth rate, genetic expression, DNA repair ability, and cell viability across various conditions. Cancer stem cells are known to be involved in drug resistance, thus we hypothesized that the resistant population would contain a higher percentage of stem cells. Techniques employed include the single cell gel electrophoresis assay (Comet Assay), suspension cell culture, and immunoglobulin assays. When applicable, cell samples were counted using a hemocytometer, and statistical tests were conducted using GraphPad Prizm software. We have currently found the resistant cells to exhibit a five times greater LD50 to DHA than the control MOLT-4 cells, a slower growth rate, and the ability to grow in media containing 12.4 μM DHA – an otherwise cytotoxic concentration. Results suggest the mechanisms of resistance include a thicker cellular membrane and improved DNA repair ability, but we have yet to determine whether cytoplasmic iron requital and DHA effusion play a part as well. We expect to find elevated levels of aldehyde dehydrogenase in resistant cells, strongly indicating the presence of stem cells. The information found will be useful for cancer and malaria drug therapy design, and will further the connection between cancer stem cells and drug resistance.

The application of Magnetic Resonance Imaging (MRI) in medicine and research has seen a stable growth in magnetic field strength. It is therefore essential to determine if motion correction algorithms developed for low field MRI show comparable results for higher field MRI, by observing quantifiable parameters like signal and contrast to noise ratios (SNR and CNR). The goal of this research study is to study the variations in signal-to-noise ratio (SNR) in 3 Tesla and 1.5 Tesla fetal brain Magnetic Resonance Images for slice-intersection based motion-correction (SIMC) algorithms. The SIMC approach aligns the individual MRI slices by considering slice-to-slice matching directly and improves the final image alignment accuracy, thereby reducing motion related image artifacts. In order to perform suitable analysis, SNR is first measured in raw MRI data by evaluating the signal levels in transient tissue regions of the brain against background noise and then compared with the SNR for motion-corrected data in 3T and 1.5T fetal brains. Grey and white matter regions from adult brains are used as a reference to determine the transient tissue regions of interest. Since a higher SNR is produced for 3T in comparison to 1.5T fetal brain MRI for raw data, we expect significant increase in the SNR values for motion-corrected 3T data as well. We also expect that the SIMC method significantly improves motion-related image artifacts, while preserving the image quality for high-field data. The results from this study can help establish 3T field strength as one suitable for imaging fetal brains, due to higher signal and higher image contrast. Moreover, it can help ascertain the efficacy of the SIMC method for a range of MRI field strengths.

In order to elicit an effective immunological response against diseases such as HIV, tuberculosis, and malaria, vaccines aimed at combating these diseases need to induce a potent CD8+ cytotoxic T cell response (CTL). CTL activation typically requires antigen delivery to the cytoplasm of antigen presenting cells. Current vaccine delivery vehicles generally lack the ability to elicit such responses, as they cannot efficiently mediate the delivery of antigenic payloads into the cytosol. To address this shortcoming, the Stayton lab has developed a novel micellar, pH-responsive protein-polymer carrier capable of endosomal escape that enables protein delivery to the cytosol. When the carrier is in the low pH environment of the endosomal compartments, the polymer is able to undergo a conformational change that disrupts the endosomal membrane and allows the protein to escape into the cytosol. In order to confirm endosomal escape and cytosolic delivery, this project aims to establish methods to study the intracellular trafficking of the protein-polymer conjugate in a murine dendritic cell line. The project employs two approaches: i) the conjugate’s location in either cytosolic or vesicular compartments is identified via subcellular and differential fractionation, and ii) the conjugate’s location is identified based on compartmental pH by fluorescently labeling the conjugate with pH sensitive and pH in-sensitive dyes, and imaging via live cell fluorescent microscopy. Finally, the project uses the developed methods to characterize the intracellular trafficking of a non-pH responsive and a glycosylated micellar carrier. Results from these intracellular trafficking studies will help identify potential areas for future improvements and optimization of the delivery vehicle.

Recent techniques now allow us to study the developing fetal brain anatomy in 3D from the reconstruction of motion corrected sets of multiple 2D images. The use of iterative, as opposed to interpolation based, reconstruction to form a 3D image from the slice data can provide improved anatomical detail. However, the lack of strategy on deciding the regularization parameter has prevented us from getting the optimal result. Here we implemented the L-curve method, which took into account of the trade-off between the fit and the data error, on choosing the optimal regularization parameter. We reviewed the 3D reconstruction quality from clinically acquired data through the examination of tissue contrast and spatial resolution and compared the results against interpolation methods. In this study, we analyzed a series of T2-weighted MRI scans from the developing fetuses and focused on the main fetal brain sectors –cortical plate (CP), deep gray nuclei (DG), germinal matrix (GMAT), subplate and intermediate zone (SP+IZ), and ventricles (VENT). Through these evaluations, we were able to obtain the optimal iterative reconstructed images that work best with different numbers of image stacks, image resolution, image signal strength as well as presenting clear anatomical details within critical brain regions.

Understanding the complex signaling pathways that lead to tumor formation is essential for developing methods the diagnosis, prognosis and treatment of cancer. As the proteins implicated in these pathways are often overexpressed or constitutively activated, the technology for detecting these proteins is necessary for this area of research. Recently, studies have suggested the importance of subcellular localization of these proteins in diagnosing cancer subtype and survival rate. However, the current analytical methods for detecting specific proteins do not provide enough spatial or quantitative information for single-molecule resolution. Quantum dots are nano-sized fluorescent crystals that can label single molecules and be more accurately quantified than fluorescent dye, which is currently used. Quantum dot labeling technology offers the high spatial and temporal resolution that is needed to study the localization of cancer-relevant proteins. We studied the correlation of the protein Akt, which has previously been suggested to preferentially localize in the nucleus or cytoplasm depending on the breast cancer subtype. We used a combination of quantum dot labeling and cellular compartment staining on a breast cancer cell line, and stimulated with insulin to activate Akt for varying amounts of time. Cells were imaged via fluorescent microscopy and the localization of proteins was conducted through MATLAB. Our results showed increased activation of Akt in the stimulated cells over non-stimulated cells, as measured with our quantum dot probe. We also showed an insulin stimulation-dependent localization of Akt. This project suggests that quantum dots can effectively label Akt in breast cancer cell lines at a high spatial and temporal resolution and offer a new platform for studying the mechanisms of cancer signaling proteins.

Porous membrane-based lateral flow assays have become extremely popular for their inexpensive and user-friendly diagnosis of many conditions. However, commercial lateral flow assays typically only run single-step processes that may lead to inaccurate results for low concentration analytes. Our goal is to create more sophisticated assays that can perform automated multi-step processes to achieve higher sensitivity detection. In order to achieve this goal, porous fluidic valves for the precise regulation of fluid flow in these devices are needed. We have developed a simple shut-off valve that passes a well-defined volume of fluid before permanent shut-off. These dissolvable bridges offer a way to automate the metering of reagent volumes that would otherwise require the user to pipet exact volumes into the device. We have characterized the operation of the valves and demonstrated their tunability using parameters such as geometry and composition of the dissolvable bridge. In addition, we have demonstrated the utility of dissolvable bridges in the important context of automated delivery of multiple volumes from a common source to different locations in an assay for simple device loading and activation. Dissolvable bridges have the potential to help bring advanced testing using porous membrane networks to limited-resource settings.

Heart disease has been the leading cause of death in the United States for nearly 100 years. The term heart disease covers a wide variety of cardiovascular complications but myocardial infarctions (MI) occur most frequently with nearly 1 million Americans projected to have a heart attack every year. The heart undergoes MI when the myocardial cells are starved of oxygen and permanently die. The process results in scarred tissue, which may induce arrhythmias and heart failure. Currently, there is no available therapy to restore the scarred tissue function. Vascularized heart tissue has the potential to replace the infarcted region to help restore normal heart function. There is a need for vasculature that can withstand physiological conditions with a perfusion system. Here, I will generate a collagen-based microvessel network by using sodium alginate as a sacrificial template. Sodium alginate is a hydrogel synthesized from alginic acid (C₆H₈O₆) and calcium chloride (CaCl₂). This gel is unique in the fact that it can be reversibly cross-linked or dissolved by ionic solutions. This property of alginate has proved useful to generate 3D features in collagen-like hydrogels. Sodium alginate constructs of varying sizes of microvasculature will be created from existing molds. These constructs will then be placed in collagen and dissolved by EDTA to generate cylindrical networks. The morphology and long-term stability of the microvessel will be characterized. Afterward, endothelial cells will be seeded into the network to form a 3D microvessel system. In addition to these microvessel systems, a macroscopic vessel will be created with sodium alginate and collagen to serve as the surgical junction tube between this system and in vivo vasculature. The goal of this research is to produce viable 3D microvessel systems in a collagen matrix as infrastructures for building implantable thick cardiac tissues.

This project’s goal is producing microporous scaffold for tissue growth. These scaffolds are made out of crosslinked 2-hydroxyethyl methacrylate (HEMA), a hydrogel. They are formed by polymerizing the HEMA around polymethyl methacrylate (PMMA) beads that are of a constrained and predetermined size. The PMMA is washed away and we’re left with a porous scaffold of precise pore size. These scaffolds are used to encourage tissue growth and reduce inflammation and the foreign body reaction. The purpose of my research is to investigate blood vessel penetration into scaffolds by producing the largest sized scaffolds I can using novel techniques. We are currently limited to tissue scaffolds of a maximum thickness of 1 mm using our current techniques. With a larger thickness, our scaffolds could be used for many more applications that are presently unavailable with a thin scaffold. Our methods are varied and include creating multiple mold designs, polymerizing techniques, and chemical washing techniques. Once I can create these larger scaffolds we will test them in vivo to evaluate the foreign body reaction as well as angiogenesis in the region. There is a large range of possible future applications for these scaffolds. They could be tuned to encourage many different types of tissue growth.

Heart disease is often responsible for morbidity and mortality in the United States. Current treatments aim to slow the progression of the disease by modifying calcium handling in the heart, but do not restore function to healthy levels. Endogenous troponin C (TnC) is the calcium binding subunit of troponin, a complex that regulates contraction in muscle. The amino acid substitution of leucine for glutamine at position 48, L48Q, in cTnC has been shown to increase calcium binding affinity of the troponin complex. We hypothesize that the expression of engineered protein L48Q-cTnC will increase calcium sensitivity of a failing heart and restore function without disrupting calcium handling. The aim of this study is to determine the expression pattern of this engineered protein in vivo in healthy rat hearts after various dosages of adeno-associated viral vectors of serotype 6 (AAV6) containing L48Q-cTnC. In addition, we aim to determine the correlation between vector dosage and changes heart function. We will achieve these goals by administering our AAV6 virus to healthy rats and monitoring whole heart function over the next four weeks. Heart function will be assessed in vivo with non-invasive echocardiography ex vivo on a Neely whole heart apparatus four weeks post-injection. The Neely apparatus allows us to control the environment of the heart while measuring function, which is not possible in vivo. The expression of L48Q-cTnC will be determined by immunohistochemistry and Western blotting. From this data, we will be able to calculate the dosage necessary to achieve maximum expression and increased heart function in our healthy heart model. This initial study it vital to characterizing the expression and efficacy of a potential heart failure therapeutic and will directly impact the design of future studies in disease models.

Because of its unique immune characteristics, the vaginal tract is highly susceptible to attacks from sexually transmitted infections (STIs) and other pathogens. Therefore, there is a need for vaccines that can be delivered directly to the vaginal mucosa and locally protect against STIs. Due to the high vascularization of the vaginal tract, free drug may traffic to other parts of the body before it has the ability to noticeably impact the mucosal immune environment. To address these challenges, we propose utilizing a vaccine encapsulating, lipid nanoparticle that is capable of storing drug, that can be retained in the vaginal tract until it directly interacts with local immune cells capable of mounting a strong local immune response. The Woodrow Lab (at the University of Washington Department of Bioengineering) has been working on developing a protocol to synthesize a stable, biocompatible lipid nanoparticle that can encapsulate a multi-component vaccine. The particles are synthesized by cross-linking together concentric polar-lipid bilayers to form stable nanoparticles. The multi-bilayer cross-linked nature of these nanoparticles increases stability and allows them to be retained within the tissue longer than simple single-bilayer particle systems. Here we show that our particles exhibit better material efficiency and have the potential to encapsulate greater levels of drug than similar lipid particle platforms. Through electron microscopy we have demonstrated that our protocol successfully produces particles with the characteristic uniform multi-bilayer morphology that we expected. We are currently working on optimizing our production process for scale-up production of the particles for larger trials. In future experiments we plan to encapsulate vaccine in the particles and use in vivo mouse models to demonstrate that our particle-vaccine system creates a more potent and longer lasting immune response than free vaccine alone. This method of vaccine delivery may prove effective at preventing several STIs including herpes and HIV.

Glioblastoma Multiforme, the most prevalent form of primary brain tumors, is characterized by its aggressive, invasive nature and short patient lifespan post-diagnosis - approximately 12 months. Through gene analysis of RNA sequencing data, we have found two miRNAs, miR-149 and miR-181, that target gene products whose increased expression has been shown to promote invasion and migration of U-87 human glioblastoma cells. miRNAs are short, non-coding RNAs that bind to messenger RNAs (mRNAs) - the body’s method of transferring genetic information, and target them to be degraded or prevent them from being translated into protein. As the miRNAs and the genes are inversely related, treatment of U-87 cells with the miRNAs would cause a decrease in migration, and blocking the miRNAs would cause an increase in migration. To quantify migration, we have employed a scratch test or wound healing assay, in which glioblastoma cells are grown on a dish and the middle of the plate is scratched, leaving space for the cells to migrate. The cells are treated with the miRNAs, and the percent of the gap filled after 24 hours is measured. We expect changes in miRNAs to affect the amount of migration in the gap compared to control - providing evidence that these miRNAs inhibit migration. miRNAs have been investigated as markers for disease, and the presence or absence of this miRNA in a patient’s glioblastoma may have implications as to the severity of their case. Additionally, characterizing these miRNAs adds to the database of knowledge concerning glioblastoma. The more that is known about this disease, the closer scientists are to finding a practical treatment.

Single-molecule force spectroscopy is a powerful tool for studying mechanical forces on proteins and other biological molecules. Magnetic tweezers (MT), optical tweezers, and atomic force microscopy (AFM) are specific examples of high-resolution tools that allow users to investigate how changes in the biomechanical function of a single protein, for example, can lead to larger effects in the complete organism. We have chosen to develop and implement an MT apparatus due to its unique potential to expand our current capabilities by allowing many single-molecule measurements in parallel. In this system, molecules are tethered to a glass surface and to magnetic beads, and a magnetic field is applied to pull on the beads and thus the molecules. We then used a bead-tracking algorithm to derive quantitative force measurements. We hope that multiplexing will allow for a more efficient study of how mechanical forces regulate molecular structure and function. Ultimately, this knowledge can be applied to both engineering innovative materials and designing therapeutic interventions. For example, we are using our MT to investigate how mutations in a blood clotting protein called von Willebrand factor (VWF) can lead to bleeding disorders. We intend to accomplish this by measuring how VWF’s sensitivity to forces comparable to those in the blood affect its roles in platelet binding. VWF also plays a major role in stroke and myocardial infarction, which we hope to study and quantify using our MT system.