(497e) Brain-Derived Extracellular Vesicles: Molecular Probes and Therapeutic Vehicles in the Neonatal Ischemic Brain

Extracellular vesicles (EVs) are unique bio-derived nanoparticles that are produced in every cell type and serve as critical cellular mediators within the body. Surrounded by a lipid bilayer, EVs are capable of carrying a wide repertoire of biomolecules involved in critical physiological pathways. EVs are categorized by their size and pathway of biogenesis: exosomes are small vesicles (30-100nm) that originate from endosomal multivesicular bodies fusing with the plasma membrane, microvesicles (100-1000nm) are shed directly from the plasma membrane, and apoptotic bodies (>1000nm) bleb from the cell during apoptosis. EVs have many strong advantages for the design of therapeutic platforms, including: 1) biocompatibility, 2) small size, 3) low immunogenicity, 4) stability, and 5) inherent targeting capacity.

Though the mechanisms of cell-specific uptake of EVs remain unclear, EVs contain cargo that are horizontally transferred to neighboring cells, acting as an important mode of cellular communication. This unique ability to transfer information between cells in the body elevate EVs as a promising therapeutic platform for areas that have been traditionally difficult to target with other nanomedicine platforms, such as the central nervous system (CNS). While the blood-brain barrier (BBB) serves as a significant obstacle to many nanotherapeutic platforms, EVs have a large advantage in that they are capable of crossing this barrier. Due to the advantages of EVs as native therapeutic vehicles, our group has successfully sequestered brain tissue-derived EVs to study their therapeutic potential in neonatal brain injury. We evaluated the therapeutic potential of EVs using a holistic approach – quantitative data collected from cellular cytotoxicity, gene expression, and bio-barcoding analysis paired with qualitative confocal imaging data tracking changes in microglial morphology and EV fate with quantum dots. Our research goal is to understand the role and potential therapeutic effect of EVs in neonatal Hypoxic-Ischemic Encephalopathy (HIE), which is the leading cause of morbidity and mortality in neonates.

Therapeutic Motivation

HIE is characterized as a reduction of adequate blood flow to the brain that can lead to stroke or permanent brain damage if left untreated. The impact of this brain condition is on a global scale: for every 1000 live births, 1-8 neonates are affected by HIE in high resource countries and this number is as high as 26 neonates in low resource countries. Currently, the standard treatment option for HIE patients is therapeutic hypothermia (TH) by continually cooling newborn infants to 33.5-35°C for 72h. However, TH only offers a 15% absolute reduction in the risk of death and disability and is resource-intensive. Thus, investigating key mediators of HIE response to identify points of therapeutic intervention has significant clinical potential within the neonatal population.

A hallmark of HIE is the immediate and sustained inflammation that occurs as a physiological response to the injured tissue. At this stage, cells are simultaneously releasing pro-inflammatory cytokines that lead to oxidative stress and cell death as well as anti-inflammatory cytokines that help clear debris and rebuild tissue. Though inflammation plays both beneficial and detrimental roles in the injury outcome of HIE, it serves as a vital time period for brain repair and is a major target for the development of therapies. However, a critical obstacle to the development of new therapeutics is a lack of understanding about the EV-mediated responses following HIE. Due to their ability to regulate communication between immune cells, EVs have emerged as important players in injury response, neuronal development, and neuronal proliferation following brain injury in multiple in vitro and in vivo models. Despite recent interest in studying the role of EVs in cerebral ischemia, most studies and all ongoing clinical trials in this field use adult models administered with stem cell culture-derived EVs, which do not recapitulate native EVs found in a 3D tissue environment.

Results & Significance: EV Therapeutic Potential

In contrast to standard in vitro cell culture models, we successfully isolated and characterized endogenous EVs from neonatal postnatal (P)10 rat brain tissue (BEV) using a combination of ultracentrifugation, ultrafiltration, and size exclusion chromatography sequestering techniques. Following successful BEV isolation and characterization, we performed various dose- and time- dependent therapeutic efficacy studies on ex vivo brain slice models of HI. Our work demonstrated that BEVs elicit a significant therapeutic response by decreasing cytotoxicity within oxygen glucose deprivation (OGD) conditioned brain slices. The minimum therapeutic dosage was determined to be 25mg BEVs , and the therapeutic exposure time is 48h across all dosages. The therapeutic application window for treatment with BEVs was determined to be 4-24h post-injury. This result is clinically relevant because it suggests that BEVs remain therapeutically viable even when administered as early as 4 hours and up to 24 hours post-injury onset.

At the minimum therapeutic dosage, BEV treatment increased the gene expression levels of IL-10, a prominent anti-inflammatory cytokine. In particular, IL-10 expression after BEV treatment was comparable to the healthy control, pointing to the role that BEVs play in regulating injury attenuation. Extensive confocal imaging analysis of microglia supplemented our experimental results. Microglia are the resident immune cell of the brain, whose changes in morphology are connected to inflammation, disease onset and progression, and stimuli from the local environment. To supplement experimental results, we performed quantitative computer-aided image analysis on microglia over several key morphologic features on both an individual and population-based level. We observed a considerable morphology shift from an amoeboid, inflammatory phenotype to a restorative, anti-inflammatory phenotype between 24-48h of BEV exposure following OGD injury. We concluded that BEVs significantly attenuate inflammation and cytotoxicity in HIE neonatal models through increases in anti-inflammatory cytokine expression and microglial morphology shifts. From a technology perspective, studying the brain’s response to endogenous EVs following ischemic injury is a first step to understanding how EVs can improve therapeutic payloads for neonatal HIE patients.

Research Innovation: EV Cellular Fate

Following our confirmation of the therapeutic efficacy of BEVs, we next sought to evaluate BEV fate and localization using our ex vivo platform. Understanding the cell-specific uptake and regional distribution of BEVs can inform us of therapeutic targets within the neonatal HIE brain. A two-pronged approach is used to probe our BEVs: 1) tagging BEVs with quantum dots (QD-BEVs) that can be qualitatively tracked using confocal microscopy, and 2) conjugating BEVs with unique oligobarcode sequences (oligo-BEVs) to quantitatively access and compare uptake across different cell types.

Quantum Dot Tagging of BEVs

Though current techniques for labeling EVs exist such as commonly used lipophilic dyes, a considerable disadvantage of these methods is dye aggregation and non-specific labeling that can give false-positive signals. While fluorescent reporter proteins provide signal specificity, this process requires genetic engineering of cells to produce specific proteins, thereby limiting the translational relevancy of this work. Quantum dots (QDs) are excellent candidates for labeling and tracking EVs due to their tunable excitation/emission spectra, photostability, versatile surface chemistry and resistance to photobleaching. Our lab has tagged EVs with QDs through a simple “click chemistry” coupling reaction between 4-formylbenzoate (4FB) and hydrazinoni-cotinate acetone hydrazone (HyNic). We have shown that our QD-EV formulations are effective at tracking BEV localization within microglial BV-2 cells in vitro as well as in ex vivo brain tissue slices. Confocal imaging of QD-BEV conjugates revealed that BEVs preferentially localize to microglia in the corpus collosum and striatal regions of OGD-conditioned ex vivo brain slices. This result supports the role BEVs may play in shifting microglial morphology following BEV treatment.

Oligobarcode Labeling of BEVs

Supplementing qualitative image analysis using QD tagging techniques, we also utilized oligobarcode labeling as a quantitative approach to determining BEV fate. Oligobarcodes are short oligonucleotide strands that can be designed with unique sequences to differentiate between different markers. We conjugated oligobarcodes to BEVs (oligo-BEVs) using a similar click chemistry technique and quantified their localization and internalization within different cell types in the CNS. We used oligobarcodes to determine preferential cell-specific localization of BEVs, giving us insight into the fate of these nanoparticles.

Conclusions

Together, our therapeutic evaluations of BEVs along with our two-pronged labeling approach to determine and quantify BEV fate reflect the important role that BEVs play in attenuating inflammation and cell death in HIE. Future research in BEVs can inform the design and administration of EV-based therapies for improving outcomes for neonatal HIE patients.