Homogenized, engineered extracellular vesicles for intracranial targeting

PROJECT SUMMARY/ABSTRACT
The objective of the proposed research is to engineer a targeted biological nanoparticle platform with high
intracranial delivery and glial cell targeting for broad applicability in drug delivery and imaging. A great deal of
work has already been accomplished elucidating the ability of certain extracellular vesicles (EVs) to cross
endothelial barriers, especially the blood-brain barrier (BBB). Other work has established that EVs exhibit
excellent tropism towards particular tissues and cell types. The focus of this proposal is to understand the
mechanisms by which certain EV subpopulations accomplish these feats, and to engineer them into a hybrid
liposome-EV drug delivery platform. Given the plethora of recent research into EV structure and function, it is
well known that they exhibit considerable compositional heterogeneity. But fundamental questions still exist as
to how EV prescribed functions differ across these subpopulations. It is likely that off-target effects and
inefficiencies in capturing native EV functions with engineered mimetics are due to their substantial
heterogeneity. Our first hypothesis is that homogenization of EVs towards a narrow size range with uniform
biomolecular content will result in a more potent and controllable drug delivery platform that maintains native EV
function yet reduces off-target toxicity. Our second hypothesis is that fusion of homogenized EVs and
liposomes with various functions (i.e., efficient BBB permeation through receptor mediated transcytosis) will
deliver an engineered product combining desired functions. We plan on addressing these hypotheses through
rigorous engineering to homogenize EVs (Aim 1) alongside biochemical assays to detangle the mechanisms
important for EV intracranial delivery. We will utilize EVs isolated from gliatropic “experts”, namely a vast library
of glioblastoma (GBM) patient derived primary cell lines, brain-metastasizing breast cancer cells, and other glial
and neuronal cells like astrocytes and neurons. Key molecular players important for intracranial delivery identified
from those studies will feedback into synthesis of engineered EVs (eEVs) via subsequent fusion with carrier EVs
(Aim 2). For the engineered eEV product, we will also incorporate synthetic liposomes decorated with known
ligands to trigger receptor mediated transcytosis through the BBB endothelial layer. To provide the greatest
opportunity to measure efficiency of functional intracranial delivery, we plan to load formulated, labeled, and
homogenized eEVs with a chemotherapeutic payload and determine drug-release profile, biodistribution, and
efficacy in healthy mice with intact BBBs and then an orthotopic GBM model (Aim 3). The proposed work is
important because it seeks to eliminate the highly confounding factor of particle-to-particle variability plaguing
effective application of EVs as potent drug-delivery vehicles. Success in homogenizing eEVs will result in an
increased understanding of their biological function and assist in their application to combat a wide variety of
neurological disorders where current drug delivery approaches are thwarted by low intracranial delivery.

Grant Number: 5R01EB033389-04
NIH Institute/Center: NIH
Principal Investigator: Randy Carney