Investigating human milk extracellular vesicles as therapeutic agents: a study on their efficacy in in vitro hit models of premature encephalopathy

Abstract

Background. Encephalopathy of prematurity (EoP) pertains to the injury of both white and grey matter in preterm infants. Currently, there are no curative treatments proven for patients. Therefore, novel treatment is needed urgently. Furthermore, human breast milk is recognized already as one of the main contributors to healthy neonatal brain development. One of milk’s poorly understood, regenerative components are milk extracellular vesicles (EVs). EVs are phospholipid bilayer-enclosed nanoparticles that are secreted by all cell types and play a critical role in bodily homeostasis. These vesicles carry a bioactive cargo, including RNAs, lipids, and proteins, with functional proteins on their surface capable of interacting with cell surface receptors of recipient cells. Recent literature indicates that milk EVs might have the potential to influence the phenotype of injured neurons, thereby they may provide neuroprotection against EoP. As of now, the neuroregenerative and neuroprotective effects of milk EVs on neuronal injury have not yet been investigated. Therefore, this study is the first of its kind to explore the protective effects of milk EVs in different in vitro hit models of neuronal injury. Methods. Human breast milk was used to obtain the EVs and EV-depleted controls in this study. Isolation of EVs and EV-depleted controls was performed in a multi-step process involving differential centrifugation, Optiprep density gradient ultracentrifugation and size exclusion chromatography. For the in vitro hit models, SH-SY5Y neuron cell lines were used, and cell viability was measured with an MTT assay. One in vitro hit model explored the pre-treatment and co-treatment effects of milk EVs in an oxidative stress model using H2O2. Additionally, milk EV treatment effect was investigated in an oxygen-glucose deprivation hit model during the reoxygenation phase. Besides, neuron-EV association was assessed using flow cytometry with fluorescently-labeled EVs. Results. Nanoparticle tracking analysis indicated that the concentration range of EVs were measured from 7.33 x 10 to 2.2267 x 109 particles/mL, while the EV-depleted control concentrations were measured within the range of 9.016 x 107 to 3.96 x 108 particles/mL. Pre-treatment of milk EVs cannot increase neuronal cell viability in an oxidative stress model using H2O2. On the other hand, co-treatment of milk EVs indicated increased neuronal cell viability in a dose-response relationship. Milk EV treatment during reoxygenation in the oxygen-glucose deprivation hit model do not prevent neuronal cell death. Flow cytometry using fluorescently-labeled EVs indicated a visible association between SH-SY5Y neurons and EVs after 4h incubation. Conclusion. This pioneering study demonstrates a treatment with milk EV increases neuronal cell survival in a co-treatment in vitro oxidative stress model with H2O2. The treatment effect might be attributed to either EV-SHSY-5Y association or potential scavenging activity of milk EVs for ROS. Future research is needed to understand of milk EVs' mechanisms of action and to investigate their contribution in the protection against neurodevelopmental impairments in preterm infants.