Abstract
Effective treatments of neurodegenerative diseases require drugs to be actively transported across the blood-brain barrier (BBB). However, nanoparticle drug carriers explored for this purpose show negligible brain uptake, and the lack of basic understanding of nanoparticle-BBB interactions underlies many translational failures. Here, using two-photon microscopy in mice, we characterize the receptor-mediated transcytosis of nanoparticles at all steps of delivery to the brain in vivo. We show that transferrin receptor-targeted liposome nanoparticles are sequestered by the endothelium at capillaries and venules, but not at arterioles. The nanoparticles move unobstructed within endothelium, but transcytosis-mediated brain entry occurs mainly at post-capillary venules, and is negligible in capillaries. The vascular location of nanoparticle brain entry corresponds to the presence of perivascular space, which facilitates nanoparticle movement after transcytosis. Thus, post-capillary venules are the point-of-least resistance at the BBB, and compared to capillaries, provide a more feasible route for nanoparticle drug carriers into the brain.
Introduction
The blood-brain barrier (BBB) is impermeable to most blood-borne substances, protecting the fragile brain environment from potentially harmful insults1. The paracellular entry of molecules from the blood to the brain is barred by junctional complexes between adjoining brain endothelial cells (BEC)2. Diffusion of molecules across BECs is possible but restricted to low-molecular-weight hydrophobic compounds. Most of them, however, including therapeutics, show negligible brain uptake due to rapid outward transport by efflux pumps to the bloodstream3,4. Macromolecules, e.g., proteins, can enter the brain by vesicular transport, i.e., transcytosis, but this route is highly selective and actively suppressed by recently identified homeostatic mechanisms5,6. Consequently, the BBB precludes more than 99% of neuroprotective compounds from reaching the brain, rendering central nervous system (CNS) disorders resistant to most conventional therapies3,7,8.
Drug delivery systems that aim to adapt receptor-mediated transcytosis (RMT) to shuttle therapeutic cargo across the BBB are currently at the forefront of modern therapeutic approaches against brain diseases9,10,11. The flagship ferrying receptor on BECs used for that purpose is the transferrin receptor (TfR)8,12,13. Neuroprotective compounds show enhanced brain delivery when coupled to TfR ligands, e.g., antibody fragments14,15, but require chemical conjugation to the targeting moiety. In comparison, nanoparticle drug carriers are versatile delivery vehicles that can encapsulate large payloads of xenobiotics with a wide range of biophysical characteristics, offering diverse and unique therapeutic opportunities9,10,11. Liposome nanoparticles functionalized with TfR ligands represent a promising drug delivery approach tested currently in preclinical trials in brain cancer, stroke, and Parkinson’s, Alzheimer’s, and Huntington’s disease, but the levels of nanoparticle transport into the brain need to improve to meet dosage requirements and reach clinical significance12,13.
To improve drug delivery, it is crucial to understand how the BBB handles drug nanocarriers in the living brain, but conventional experimental techniques only provide limited mechanistic insights. Whole-brain imaging techniques, such as PET or MRI, are insufficient to resolve the spatio-temporal characteristics of single nanocarrier-BBB interactions in vivo. Consequently, the events between administration and detection of therapeutics in the brain are obscured. Our current knowledge is extrapolated from chemically processed tissue3,13,16, which erases all information on the dynamic processes at the BBB. In addition, the vessel microanatomy differs between arterioles, capillaries, and venules17,18. This principal feature of the brain is overlooked in drug delivery studies, and how it impacts drug delivery is unknown.
Here, to address these issues, we used two-photon in vivo imaging to examine how distinct types of cerebral vessels handle drug nanocarriers in real-time, in the intact brain, in anesthetized and awake mice. We characterized the pharmacokinetics of nanoparticles targeted toward the TfR at the BBB, their intracellular trafficking patterns in vascular BECs, and transcytosis-mediated entry and transit in the brain parenchyma. We report that TfR-targeted nanoparticles bind to BECs at venules and capillaries but not at arterioles. Despite the highest association of nanoparticles to BECs in capillaries, we found that the trafficking to the brain occurs almost exclusively at post-capillary venules, with a negligible contribution of capillaries. The vascular locus of nanoparticle transport was consistent with the presence of perivascular space around post-capillary venules, which facilitates the movement of nanoparticle-sized elements in the CNS. These observations provide insight into therapeutic nanoparticle trafficking and delivery to the brain and challenge the assumed view that capillaries are the hub for effective brain transport of nanoparticle drug carriers. Thus, our findings prompt reconsideration of nanoparticle targeting strategies for improved drug delivery to the brain.
