Benjamin Wheeler 1

A Vesicle Based Biosensor for Real Time Identification of Mild Traumatic Brain Injury. Sponsor: Dr. Peter Searson, Materials Science and Engineering

Objective: The overall objective of this project is to design and fabricate a wearable biosensor composed of lipid vesicles suspended in a polymeric biofilm in order to identify subjects at risk for mild traumatic brain injury following head trauma. This sensor will be designed to allow for attachment or insertion into the protective headwear of groups like professional athletes and military personnel. The vesicles will be fabricated in such a way that they will contain both a fluorescent dye and quencher. Under normal conditions, the vesicles will hold both molecules in proximity such that there will be no fluorescence. However, when there is a mechanical disturbance or event that disrupts the vesicles, the two molecules will leak and separate enough to allow for the fluorescence to be observed. Preliminary results measuring vesicle leakage in an electromagnetic shaker indicate the feasibility of this design. Significance: Mild Traumatic Brain injury (mTBI) is defined as a state when there is alteration of minimal duration and severity in the patient’s baseline neurological or mental status after an injury. Annually, there is an estimate of 3.8 million concussions occurring in the United States. Unfortunately, 50% of the injuries go unreported, making it a serious problem for the health care field.1 mTBI is the leading cause of mortality in the active/working population under 35 in the United States,2 and a major source of these injuries is both collegiate and professional sports. For instance, at least 60% of those playing soccer on the collegiate level developed symptoms compatible with a concussion during a season. Additionally, it has been reported that as high as 18% of soldiers returning from Iraq and Afghanistan experience mild traumatic brain injury.3 In both of these sectors, there is particular interest in the development of a functional real-time identification and diagnostic device to aid in the development of functional return-to-action guidelines. The current diagnosis is straightforward through computed topography (CT) and magnetic resonance imaging (MRI); however, these clinical methods are costly, time consuming, and not readily translatable to devices for on-site use.

When there is a significant mechanical impact to the head, there can be differential movement between the brain and the skull. This can lead to deformation of the brain tissue, which is a primary cause of brain damage. Also, deformation can cause structural alterations to neurons and vasculature, generation of oxygen radicals, and/or excessive neural depolarization.4 Specifically, there can be non-specific breakdown of the plasma membrane and the stretching and tearing of nerve fibers and small blood vessels, which can lead to membrane retraction, extrusion of axoplasm, and formation of large reactive swellings.

Studies show that patients who receive diagnosis and information early are less stressed and report fewer symptoms at three months after the injury5. Currently, there are no simple diagnostic tests for mTBI. Analysis of protein biomarkers such as S100B in serum is being explored for diagnostics. However, these assays are not rapid. Therefore, early real-time diagnostic tools and an effective set of “return to play guidelines” as well as other reference materials should be developed for treatment of mTBI. My proposed device will aid medical professionals onsite during the injury to make a rapid evaluation of the situation. Additionally, the use of a vesicle-based device is critical because of the ability to mimic cell membrane disruption, a critical cellular mechanism in mTBI. The time saved and accurate reflection of the physiology can be used to prevent further injury to the head as well as providing further testing and information to the patient to attenuate the negative repercussions of mTBI.

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Figure 1: (A) Shows how the sensor would easily be incorporated into existing helmet design. (B) Shows how the sensor would change fluorescence upon vesicle rupture

  Benjamin Wheeler 2

Originality: Currently in the field the only studies done to characterize the effects of force on the brain at a cellular level have been done in vivo using animal models. These tests usually consist of either fluid perfused through the tissue to create a pressure differential or a weight dropped from a height that impacts the tissue with a predetermined force. Typically in these studies, a pressure of 2 to 5 atmospheres is applied to the tissue to simulate the impact.6 These tests, while critical in understanding the mechanisms of mTBI, cause significant harm to the animals involved and do not readily translate to a testable in vitro model. Compared to the existing systems, the proposed system is unique in its ability to very finely control the magnitude of force applied. Additionally, this design will allow for testing in multiple platforms including various cell culture dishes, microfluidic devices, and cuvettes filled with vesicles in various solutions. This will allow for easy transition to a functional field diagnostic tool. Project Design: The objective of this project is achieved in two aims. The first aim is to characterize the relationship between vesicle leakage and the applied mechanical force, and the second aim is to create a wearable sensor composed of an optimized vesicle-hydrogel matrix, which can be incorporated into protective headwear. Aim 1: The initial aim of the project will be to characterize the relationship between vesicle leakage and the applied mechanical force. The goal of this is to create leakage versus impact calibration curves that will be used in the design of the sensor and for calibration in the field. The vesicles will be made using the standard method of desiccation, re-suspension, and extrusion. In preliminary work, vesicles have been formed from a physiologically relevant composition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), polyethylene glycol (PEG2k), and cholesterol. Encapsulated within these vesicles is a solution of the dye/quencher pair, 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX). This setup is shown in figure 2. To characterize the relationship between vesicle disruption and force, I have obtained an electromagnetic shaker (labworks et-140). The shaker has been assembled with accompanying amplifier, cooling fan, and function generator, as shown in figure 3. In this configuration, the shaker applies a displacement on the order of centimeters with variable amplitude and frequency, and is used to model the impact force. To accompany this setup I have custom designed and fabricated a sample stage to mount the cuvettes holding the vesicle samples to the shaker. This design can be easily modified to allow attachment of a range of sample types, including: cuvettes, 96 well plates, and custom made microfluidic devices. This setup accommodates a high degree of control, and allows an exact application of impact force in agreement with previous studies and data from the literature. I will be working with Dr. Joe Katz in mechanical engineering to relate the output of the electromagnetic shaker to impact force. Aim 2: The second aim of the project is to use the results previously mentioned to design and fabricate a wearable sensor that can be easily incorporated into existing protective headwear. The sensor envisioned

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Figure 2: (A) Mechanical disturbance of the vesicles leads to a change in the fluorescence of the solution. (B) Shows the vesicle in greater detail illustrating how the close proximity of dye and

Figure 3: (A) In preliminary work I have assembled an electromagnetic shaker with a sample stage, coupled to the cooling vacuum and the connection to the amplifier leading out of view. (B) Amplifier, transformer, and function generator shown in functional arrangement. (C) Close up of the custom stage with cuvette holder attached.

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  Benjamin Wheeler 3

is a hydrogel strip composed of vesicles suspended in a polymeric matrix encased in a protective outer layer of flexible plastic to allow for easy interpretation of fluorescence change as well as efficient transduction of force from the impact to the sensor. It makes sense to fabricate this device using vesicles in a gel matrix versus the aqueous suspension used to initially characterize the force -leakage relationship for two reasons. First, and foremost, the gel will ensure even distribution of the vesicles throughout the sensor, which will allow the sensor to be equally sensitive to impact force in any direction. Second, the structural support provided by the gel will increase the longevity and resilience of the sensor. To create the most effective gel for these purposes, gel composition as well as matrix and vesicle density will have to be optimized. As a starting point, a gel composed of 5mg/mL collagen will be tested. This will allow for an ideal balance between porosity and stiffness. To create a matrix that accurately mirrors the mechanical properties of native tissue, the concentration of collagen as well as the introduction of other polymers can be varied. A key aspect of this sensor is the real-time decision making it allows. To do so, it must be easily distinguishable between a positive and negative result. To accommodate this design parameter, the vesicle concentration will also be varied to produce an obvious response when the impact exceeds a predetermined threshold. Beyond the fluorescence protocol previously described, other dyes that have colorimetric function will be investigated to provide the optimal means of producing real-time results. Timetable: Week 1-6: Production of force-leakage calibration curves Week 6 – 10: Optimization of the gel and vesicle composition Week 10-14: Design and fabrication of the actual sensor and quality testing Summary of Expected Results: Preliminary results show the force-leakage relationship can be readily characterized and predicted based upon vesicle composition. As shown in figure 4 below, preliminary results indicate there is at first a linear correlation between duration of force and vesicle leakage. Once the time and steady state responses are characterized, it is expected that the magnitude of the force and vesicle leakage can be characterized in a similar manor. It is expected at first the fluorescence will exhibit a linear correlation with the force applied and eventually reach a plateau, as shown in the time dependent case below at the 100-minute time point. This effect can be visualized in figure 5. Both cuvettes contain 800 µL of 100 nm diameter vesicles composed entirely of POPC. In the picture, the cuvettes are exposed to light at the excitation wavelength of ANTS/DPX. The cuvette on the left contains undisturbed

vesicles, while the cuvette on the right was exposed to mechanical displacement for 100 minutes. The emission of yellow light indicates vesicle leakage, as expected from impact to the vesicles. The combination of these results will allow for easy optimization, design, and fabrication of the sensor.

Figure 5: Demonstration of fluorescence readout of 100 nm POPC vesicles in solution before and after mechanical disturbance Figure 4: Leakage versus time for

POPC vesicles under continuous force

  Benjamin Wheeler 4

Background: I have worked in Dr. Searson’s lab since May 2013, committed to understanding the galvanotaxis of 612 glioblastoma cells under the super vision of PhD candidate Yu-Ja Huang. During my time with the Searson Group I have learned many critical skills through the major aspects of the project such as microfabrication, cell culture, normal light and fluorescence miscroscopy, and various biological assays. This had led to me developing skills in and not limited to the following: photolithography, hydrogel formation, device assembly, media preparation, stem cell culture, confocal microscopy, live and dead cell fixing and staining, real time PCR, and cell tracking. In addition to the hands-on laboratory work, I am also ahead on degree requirements for my junior standing as a BME. I have already completed essential math classes such as Calculus III, Differential Equations, and Probability and Statistics. Additionally, I have modeling experience including MATLAB and completion of the class Models and Simulations along with preliminary course work in Systems Bioengineering. I also have background with biology and bioengineering, as I have completed organic chemistry, molecules and cells, and tissue engineering while currently working on Biomaterials I and Cellular Engineering. Should some parts of this project have complexity beyond the theoretical and practical preparation I have obtained, I will have several experienced researches as mentors. I will be working closely with PhD candidates Chloe Kim and Yu-Ja Huang who combined have over 8 years of research experience. Dr. Peter Searson, the Reynolds Professor of Materials Science and Engineering and the head of the Institute for NanoBioTechnology, will be overseeing the project. I believe the combination of these rich and expansive has given me the ability to successfully complete this project. Presentation and Evaluation: The results of this project will be presented in poster form, including results of experiments carried out and reported weekly to Dr. Searson and Yu-Ja Huang at weekly working group meetings. Project and experimental designs will be created, evaluated, and decided upon at these meetings as well.

  Benjamin Wheeler 5

References:                                                                                                                1 Hodge, Samuel D., Jr. "A Heads Up on Traumatic Brains Injuries in Sports." Journal of Health Care Law & Policy, 1 Mar. 2014. Web. 2 Oct. 2014. 2 Kovesdi, E et al. "  Update on protein biomarkers in traumatic brain injury with emphasis on clinical use in adults 2 Kovesdi, E et al. "  Update on protein biomarkers in traumatic brain injury with emphasis on clinical use in adults and pediatrics." National Center for Biotechnology Information. U.S. National Library of Medicine, Jan. 2010. Web. 1 Oct. 2014. 3  Hoge, Charles W. "Mild Traumatic Brain Injury in U.S. Soldiers Returning from Iraq — NEJM." New England Journal of Medicine. N.p., 31 Jan. 2008. Web. 1 Oct. 2014.  4  Dixon, C. Edward, PhD, Wiliam C. Taft, PhD, and Ronald L. Hayes, PhD. "Mechanisms of Mild Traumatic Brain Injury. : The Journal of Head Trauma Rehabilitation." Mechanisms of Mild Traumatic Brain Injury. : The Journal of Head Trauma Rehabilitation. Journal of Head Trauma Rehabilitation, Sept. 1993. Web. 10 Oct. 2014.  5 Ponsford, J., C. Willmott, A. Rothwell, P. Cameron, A. Kelly, R. Nelms, and C. Curran. "Impact of early intervention on outcome following mild head injury in adults." National Center for Biotechnology Information. U.S. National Library of Medicine, 21 Feb. 0006. Web. 09 Oct. 2014. 6 DeAngelis, M. M. "Traumatic Brain Injury Causes a Decrease in M2 Muscarinic Cholinergic Receptor Binding in the Rat Brain." National Center for Biotechnology Information. U.S. National Library of Medicine, Aug. 1994. Web. 10 Oct. 2014.