Engineered extracellular vesicles for the delivery of mitochondria and therapeutic proteins to the BBB - The damage borne by brain endothelial cells (BECs) disrupts the structure and function of the blood-brain barrier (BBB) and contributes to poor patient outcomes post-stroke. In this R01 proposal, we will determine whether engineered microvesicles (MVs) can co-deliver innate MV mitochondria and an exogenous 27 kDa heat shock protein (HSP27) to protect the BBB via increasing BEC survival and BEC tight junction integrity in a mouse model of transient cerebral ischemia/reperfusion injury (stroke). This one-two punch strategy using engineered MVs can protect the post ischemic-BBB metabolic function and structural integrity via effects of the innate MV mitochondria and the exogenous HSP27 protein. Mitochondrial ATP regulates actin dynamics and maintains proper organization of the actin cytoskeleton. Further, ATP depletion-induced changes in the actin equilibrium contributes to dysregulation of the tight junctions, eventually leading to BBB disruption and long-term neurological dysfunction. Endothelial but not neuronal expression of HSP27 in a transgenic mouse model suppressed I/R-induced aberrant actin polymerization, stress fiber formation, and junctional protein translocation—that overall ameliorated neurological damage for as long as one month post-stroke. This scientific premise strongly supports the importance of decreasing damage to the BBB to improve post-stroke outcomes. MVs are a subtype of extracellular vesicles that naturally incorporate mitochondria in addition to its constituent lipids, nucleic acids and proteins. HSP27 is a cationic protein and requires a carrier for its delivery across anionic cell membranes. In our feasibility studies, we have detected mitochondria in MVs and engineered HSP27-MVs using a simple formulation process that allows to retain its functional mitochondrial load. To this end, we have demonstrated that (1) the innate mitochondria in naïve MVs can be transferred to recipient primary human BECs and mouse brain cortical and hippocampal slices, (2) MVs but not the smaller exosomes increase mitochondrial function in oxygen-glucose deprived BECs (3) a simple formulation process resulted in >60% loading efficiency of HSP27 into MVs and (4) mice injected with naïve MVs showed nearly a 50% reduction in infarct volume and lower neurological deficit scores compared to vehicle-injected mice after middle cerebral artery occlusion (MCAo). We hypothesize that the delivery of engineered HSP27-MVs, along with the innate MV mitochondria will protect the metabolic function and structural integrity of the BECs lining the BBB—that in combination will ameliorate the BBB disruption-induced changes in stroke. We will test the proposed hypothesis by first determining protective effects of the HSP27-MVs in hypoxic brain slices, OGD BECs and in isolated brain microvessels (Aims 1 and 2), conduct a pharmacokinetics study to determine the optimal MV dose that shows the greatest brain uptake (Aim 2b) and we will determine the therapeutic efficacy and mechanistic effects of HSP27-MVs in aged MCAo mice in Aim 3. Overall, successful completion of these studies will demonstrate if BBB protection via increasing BEC energetics and decreasing BBB permeability improves post-stroke outcomes.
