Drug Delivery through the Blood-Brain Barrier through Use of Vascular Cell Adhesion Molecule 1- Targeted Nanoparticles

The blood brain barrier (BBB) is a semi-permeable membrane separating the brain from the bloodstream, preventing many drugs that treat neurological diseases, such as Alzheimer’s and Parkinson’s, from reaching the brain. Our project aimed to create a novel drug delivery system targeting the brain during neural inflammation. We developed a cationic solid lipid nanoparticle (CSLN) complex composed of cationic nanoparticles, biotin, streptavidin, and anti-vascular cell adhesion molecule-1 (anti- VCAM-1) antibodies. The anti-VCAM-1 antibody is used to target VCAM-1, a cell adhesion protein found on the BBB endothelium. VCAM-1 expression is elevated in the presence of inflammatory molecules, such as tumor necrosis factor-alpha (TNF- α). Through the use of a simple BBB model, results showed that our novel drug delivery system experienced some level of success in targeting the brain inflammation due to increasing TNF-α concentrations. This is promising for drug delivery research and provides support for VCAM-1 targeting using more robust and complex BBB models.

ROLE OF ICAM-1-MEDIATED ENDOCYTOSIS IN ENDOTHELIAL FUNCTION AND IMPLICATIONS FOR CARRIER-ASSISTED DRUG DELIVERY

Intercellular adhesion molecule 1 (ICAM-1) is a transmembrane protein found on the surface of vascular endothelial cells (ECs). Its expression is upregulated at inflammatory sites, allowing for targeted delivery of therapeutics using ICAM-1-binding drug carriers. Engagement of multiple copies of ICAM-1 by these drug carriers induces cell adhesion molecule (CAM)-mediated endocytosis, which results in trafficking of carriers to lysosomes and across ECs. Knowledge about the regulation behind CAM-mediated endocytosis can help improve drug delivery, but questions remain about these regulatory mechanisms. Furthermore, little is known about the natural function of this endocytic pathway. To address these gaps in knowledge, we focused on two natural binding partners of ICAM-1 that potentially elicit CAM-mediated endocytosis: leukocytes (which bind ICAM-1 via β₂ integrins) and fibrin polymers (a main component of blood clots which binds ICAM-1 via the γ3 sequence). First, inspired by properties of these natural binding partners, we varied the size and targeting moiety of model drug carriers to determine how these parameters affect CAM-mediated endocytosis. Increasing ICAM-1-targeted carrier size slowed carrier uptake kinetics, reduced carrier trafficking to lysosomes, and increased carrier transport across ECs. Changing targeting moieties from antibodies to peptides decreased particle binding and uptake, lowered trafficking to lysosomes, and increased transport across ECs. Second, using cell culture models of leukocyte/EC interactions, inhibiting regulatory elements of the CAM-mediated pathway disrupted leukocyte sampling, a process crucial to leukocyte crossing of endothelial layers (transmigration). This inhibition also decreased leukocyte transmigration across ECs, specifically through the transcellular route, which occurs through a single EC without disassembly of cell-cell junctions. Third, fibrin meshes, which mimic blood clot fragments/remnants, bound to ECs at ICAM-1-enriched sites and were internalized by the endothelium. Inhibiting the CAM-mediated pathway disrupted this uptake. Following endocytosis, fibrin meshes trafficked to lysosomes where they were degraded. In mouse models, CAM-mediated endocytosis of fibrin meshes appeared to remove fibrin remnants at the endothelial surface, preventing re-initiation of the coagulation cascade. Overall, these results support a link between CAM-mediated endocytosis and leukocyte transmigration as well as uptake of fibrin materials by ECs. Furthermore, these results will guide the future design of ICAM-1-targeted carrier-assisted therapies.

THE ROLE OF THE MECHANICAL ENVIRONMENT ON CANCER CELL TRANSMIGRATION AND MRNA LOCALIZATION

Most cancer-related deaths are due to metastasis formation, the ability of cancer cells to break away from the primary tumor site, transmigrate through the endothelium, and form secondary tumors in distant areas. Many studies have identified links between the mechanical properties of the cellular microenvironment and the behavior of cancer cells. Cells may experience heterogeneous microenvironments of varying stiffness during tumor progression, transmigration, and invasion into the basement membrane. In addition to mechanical factors, the localization of RNAs to lamellipodial regions has been proposed to play an important part in metastasis. This dissertation provides a quantitative evaluation of the biophysical effects on cancer cell transmigration and RNA localization. In the first part of this dissertation, we sought to compare cancer cell and leukocyte transmigration and investigate the impact of matrix stiffness on transmigration process. We found that cancer cell transmigration includes an additional step, ‘incorporation’, into the endothelial cell (EC) monolayer. During this phase, cancer cells physically displace ECs and spread into the monolayer. Furthermore, the effects of subendothelial matrix stiffness and endothelial activation on cancer cell incorporation are cell-specific, a notable difference from the process by which leukocytes transmigrate. Collectively, our results provide mechanistic insights into tumor cell extravasation and demonstrate that incorporation into the endothelium is one of the earliest steps. In the next part of this work, we investigated how matrix stiffness impacts RNA localization and its relevance to cancer metastasis. In migrating cells, the tumor suppressor protein, adenomatous polyposis coli (APC) targets RNAs to cellular protrusions. We observed that increasing stiffness promotes the peripheral localization of these APC-dependent RNAs and that cellular contractility plays a role in regulating this pathway. We next investigated the mechanism underlying the effect of substrate stiffness and cellular contractility. We found that contractility drives localization of RNAs to protrusions through modulation of detyrosinated microtubules, a network of modified microtubules that associate with, and are required for localization of APC-dependent RNAs. These results raise the possibility that as the matrix environment becomes stiffer during tumor progression, it promotes the localization of RNAs and ultimately induces a metastatic phenotype.