Alzheimer's disease (AD) is a progressive, disabling neurodegenerative illness that affects an estimated 5.5 million people in the United States and approximately 10% of the population over the age of 65. Early symptoms of the disease include short-term memory loss, difficulties with language, and mood swings; however, symptoms gradually worsen over time, ultimately leading to dementia and a loss of bodily functions. Traumatic brain injury (TBI) has been identified as a risk factor for the development of AD and AD-related dementias, but little is known about the mechanisms in which TBI contributes to AD. Part of the difficulty in uncovering the link between TBI and AD can be attributed to the significant challenges that exist in the diagnosis of both TBI and AD. A clinical diagnosis of either condition requires the presence of physical or cognitive symptoms. However, symptoms of both TBI and AD mirror those of a number of other neurological conditions. Moreover, by the time cognitive symptoms have emerged, significant irreversible neurological damage has already occurred. As a result, many efforts have been made to identify biomarkers for TBI and AD, as well as to develop diagnostic tools to identify preclinical AD – a stage during which the pathophysiological process of the disease have begun, but symptoms have yet to emerge. While recent advances in neuroimaging (i.e., magnetic resonance imaging, computed tomography, positron emission tomography) have allowed researchers to gain insight into structural and functional changes taking place in the brain, these imaging techniques are expensive and results vary greatly from patient to patient. Biomarkers capable of providing early, molecular insights into TBI and AD would allow patients to begin therapeutic intervention sooner and could potentially improve health outcomes for the more than 360,000 military personnel who have been diagnosed with TBIs since 2000.

In recent years, exosomes (30-150 nm extracellular vesicles) have emerged as a promising biomarker for evaluating neuronal injuries. Exosomes are an interesting target because they are found in abundance in nearly all biological fluids, including blood, urine, saliva, and cerebrospinal fluid (CSF). Accumulating evidence suggests that neuron-derived exosomes may play a crucial role in the pathology of TBI and AD, by helping to promote neuroinflammation and enabling the spread of abnormal, potentially disease-causing, misfolded proteins throughout the brain. Preliminary studies have shown that by analyzing the number of neuron-derived exosomes and their molecular cargo (such as tau, amyloid-beta, and different microRNA levels), information about the severity of TBI sustained can be obtained. In addition, exosome analysis has been used to distinguish early-stage AD patients from healthy controls as well as patients with other neurological diseases. While researchers have made progress in identifying neuron-derived exosomal proteins and RNAs, difficulties surrounding consistent and efficient isolation of exosomes have prevented their widespread use as a biomarker for TBI and AD.

In this US Department of Defense project, we will address this unmet need by developing an acoustofluidic (i.e., the fusion of acoustics and microfluidics) technology for automated, fast, high-yield, high-purity, biocompatible exosome isolation and validating it in analyzing exosomal biomarkers for TBI and AD. Thus far, we have conducted comprehensive proof-of-concept studies that demonstrate the feasibility and utility of the proposed acoustofluidic exosome-separation technology. We will (1) develop all-in-one acoustofluidic prototypes for exosome separation; (2) validate the acoustofluidic exosome separation prototypes by analyzing known and potential TBI/AD biomarkers, including tau, amyloid-beta, and multiple microRNA levels in isolated exosomes from TBI and AD mouse models' blood and CSF; and (3) validate the acoustofluidic exosome separation beta prototypes by analyzing exosomal TBI/AD biomarkers in clinical blood and CSF from TBI and AD patients. The proposed acoustofluidic technology will have the following features: (1) automated exosome isolation, which facilitates simple, convenient operation; (2) fast isolation (<5 min processing time vs. ~8 hrs processing time with benchmark technologies); (3) high exosome yield (>90%) in comparison to benchmark technologies (5%-25%); (4) high exosome purity (>80%) in comparison to benchmark technologies (~33%); (5) less contamination from non-native serum proteins. Not only will our technology allow for lower levels of albumin and immunoglobulin found in isolated exosomes, but it will be the first exosome isolation technique capable of separating exosomes from low-density lipoproteins; and (6) –low cost and point-of-care design. With these features, the proposed acoustofluidic technology has the potential to greatly simplify and revolutionize the diagnosis of TBI and AD. If successful, our acoustofluidic platform could be used to identify TBI patients who are at an increased risk for developing AD and to identify AD patients at early preclinical stages for future disease altering therapies.