Liposomal Nanocarriers Designed for Spatially- Controlled Targeting Under Vascular Sheer Stress
Jonathan S Cudnik, Lauren B Grimsley, Richard K Fisher, III, Raymond A Dieter, III, Joshua D Arnold, Ryan M Buckley, Michael M McNally, Michael B Freeman, Deidra J Mountain, Oscar H Grandas
University of Tennessee Graduate School of Medicine, Knoxville, TN
INTRODUCTION: Vascular interventions inherently disrupt the tunica intima, exposing sub-endothelial matrix proteins, and resulting in dysfunctional vessel remodeling and intimal hyperplasia (IH) development. Spatially-controlled nanoparticles designed to co-localize to exposed matrices could provide a targeted drug delivery system aimed at IH prevention. Here we present our progress in the development of a targeted drug delivery platform based on discovery-driven liposomal nanocarriers designed for preferential collagen IV binding under static and simulated vascular flow conditions. METHODS: Non-targeting PEGylated liposomes (PLP) were formed with bulk DOPC-PEG + 30mol% cholesterol + 0.1mol% Rhodamine-DOPE. Collagen-targeting liposomes (CT-PLP) were formed by reacting DSPE-PEG-DBCO to previously established collagen binding peptides (CBP), via copper-free click chemistry, and inserting 0.5-15mol% CBP-modified lipids to base PLPs at hydration (PreCBP) or via micellar transfer (PostCBP). Liposome binding affinity to collagen IV matrices, dried at 3ug/cm2, was quantified by fluorometry at 0-24hr static incubation. Hemodynamic liposome binding was assayed by fluorescence microscopy after 0-60min simulated physiological flow at ~2 dynes-s/cm2 using a closed parallel-plate flow chamber. RESULTS: CBP-lipid conjugation reaction was confirmed by MALDI-TOF, and standard characterization studies verified PLPs and CT-PLP groups with 0.5-5mol% CBP-modified lipids formed spherical particles at <100nm with a narrow PDI size distribution. Insertion above 5mol% CBP resulted in liposome aggregation and loss of homogeneity (Fig1A). CT-PLPs with 2.5mol% and 5mol% CBP-modified lipids demonstrated a significant increase in collagen binding vs. PLPs and all other stable CT-PLP groups at 2hr static incubation (Fig1B). Using 5mol%, CT-PLPs formed by PreCBP insertion demonstrated a significant increase in collagen binding compared to PostCBP insertion at ≥ 15min static incubation (Fig2A). Under continuous physiological flow, CT-PLPs formed with 5mol% PreCBP insertion demonstrated remarkable binding to collagen matrices at 60min while PLP control binding was negligible (Fig 2B). CONCLUSIONS: CT-PLPs demonstrated an affinity for collagen IV binding in both a static in vitro environment and under simulated hemodynamic flow. Here 5mol% CBP was validated as the modification level for optimal matrix-targeted binding, and PreCBP insertion was validated as the most efficient technique for CT-PLP assembly. CT-PLP nanocarriers established here show promise as the framework for a spatially-controlled drug delivery platform for future application in targeted vascular therapeutics. Ongoing studies aim to optimize CT-PLP binding capacity under elevated sheer stress simulating pathological flow and to demonstrate in situ matrix collagen binding via ex vivo rodent vessel perfusion.
Back to 2018 Abstracts