(G-259) Assessing Trans-Endothelial Transport of Nanoparticles for Delivery to Abdominal Aortic Aneurysms

Introduction:
Abdominal Aortic Aneurysms (AAAs) are localized, rupture-prone expansions of the vascular wall in the abdominal aorta. In this disease, structural extracellular matrix (ECM) proteins of the aorta wall, elastic fibers and collagen fibers, that impart elasticity and stiffness respectively, are slowly degraded by overexpressed matrix metalloproteinases (MMPs). Currently, invasive and risky surgery is the only intervention for AAAs and no therapies exist to reverse their pathophysiology. In seeking to deliver new matrix regenerative therapeutics to the AAA wall using polymer nanoparticles (NPs), we aim to determine how NP design (shape, charge, size, etc) impact NP uptake and extravasation/translocation past the endothelial cell (EC) layer from circulation into the medial layer of the AAA wall. We hypothesize that breakdown of tight junctions between cytokine-activated ECs in the AAA segment enhances uptake and translocation of spherical and rod-shaped NPs. The goal of this ongoing study is to elucidate the mechanisms of NP uptake by healthy and diseased ECs to design appropriate active targeting nanotherapeutics for AAAs.
Materials and Methods:
NPs were formulated using a double emulsification method with PLGA and 1% polyvinyl alcohol (PVA) surfactant. An organic solution was created by dissolving 50mg of 0.5-0.7 dL/g Carboxyl Terminated PLGA in 5mL of dichloromethane (DCM) and a 1% w/v PVA surfactant solution was created by dissolving PVA (MW:30- 70K) in water. The organic solution was mixed with 20mL of the surfactant (1% PVA) by sonication and left stirring overnight at 500rpm to evaporate the organic solvent. Nanospheres were collected by ultracentrifugation at 13K RPM. Films were made by mixing the NPs in 10% PVA and leaving the mixture to dry overnight at 60°C in a 100mm diameter petri dish. The resultant films were stretched while placed in a mineral oil bath at 70°C at a rate of 0.2mm/s. Analyses on the NPs were conducted using dynamic light scattering (DLS) with the Zetasizer Nano ZS, scanning electron microscopy (SEM) with the Hitachi 4300, and atomic force microscopy (AFM) with the Bruker/VEECO DI-3000. Targeting studies were conducted using Rat Aortic Endothelial Cells (RAOECs) and verified with immunofluorescence and confocal microscopy.
Results, Conclusions, and Discussions:
Results: Based on DLS, we determined zeta potential of the PLGA NPs to be -30.47±5.3 mV. SEM and AFM were used to verify various length rod-shaped NPs were achieved by the film stretch method. First, we conducted a live/dead assay to verify no cell death occurred in RAOECs with treatment with PLGA NPs. Immunofluorescence imaging indicated that NP uptake by RAOECs were higher in rod-shaped NPs then spherical NPs. Using permeable cell culture inserts, we additionally saw higher rate of translocation by rod-shaped NPs compared to spherical NPs over a confluent EC monolayer. Also, we observed higher uptake and translocation of NPs in cytokine activated ECs compared to healthy controls. Immunofluorescence staining for adhesion receptors Integrin αVβ3 and E-Cadherin show cytokine activation of ECs disrupts cell-matrix and cell-cell junctions to create intracellular gaps for trans-EC transport of NPs, possibly explaining our observed increase in uptake and translocation of NPs in cytokine activated cells.
Conclusions/Discussions: Our results demonstrate the potential of using rod-shaped NPs for uptake by the AAA segment wall. We see rod-shaped NPs can be created based on the degree in which the PVA films are stretched with longer rods being created based on longer stretched films. Our live/dead assay reveals that our PLGA NPs are safe and do not cause cell death. Immunofluorescence staining reveal cytokine activated ECs have loss of adhesion receptors Integrin αVβ3 and E-Cadherin. Using this observation, we showed the disruption to adhesion receptors allow for greater uptake and translocation of NPs. Fluorescence studies show rod-shaped NPs contain high trans-endothelial translocation and little uptake of rod-shaped NPs in cytokine activated ECs compared to normal control cells, arguing higher aspect ratio NPs are better suited to accumulate at the AAA site, potentially from the NP entering via the minor axis. These results show the potential of using shape as a modality for filtering NPs to the AAA wall. These studies are significance to understanding the mechanisms engaged by NPs for AAA wall uptake.
References (Optional):
1) Melrose, J et al. J Vasc Surg 1998, 28 (4), 676-686.
2) Veith, F. J Vasc Surg 2016, 64 (4), 885–890.