(316b) In Vivo Flow-Regulated Endothelial Glycocalyx Integrity Leveraged for Targeted Intravenous Nanoparticle Delivery

Statement of Purpose: The cell surface-attached extracellular glycocalyx (GCX) layer, a heterogeneous polysaccharide chain, is a major contributor to endothelial cell (EC) function and EC-dependent vascular health, acting as a first line of defense against vascular diseases including atherosclerosis [1]. It largely consists of cell-linked sialic acid and glycosaminoglycans including heparan sulfate, hyaluronic acid, and chondroitin sulfate, acting as a buffer between endothelium surface and blood-flow derived shear forces. Endothelial surface GCX shedding plays a role in endothelial dysfunction, compromising endothelial barrier function and increasing vascular permeability [2], leading to consequences such as increase in lipid influx. A unique surgical procedure was developed by Nam et al. [3] to perform partial left carotid artery (LCA) ligation in mice. This was characterized as a model of disturbed blood flow with characteristics of low and oscillatory wall shear stress. However, this study did not examine GCX expression after acutely inducing disturbed blood flow. The first part of our study sought to elucidate the impact on GCX integrity post-one week of partial LCA ligation in correlation to endothelial dysfunction. The second part of this study examined vessel wall permeability after GCX shedding by examining resultant macrophage infiltration, related to atherosclerosis onset, as well as nanoparticle infiltration, related to targeted drug delivery for early atherosclerosis intervention.

Methods: For the first study, C57Bl/6-background apolipoprotein-E knockout (ApoE-KO) male mice were subjected to a partial LCA ligation procedure [3] to induce disturbed blood flow patterns in the LCA while the right carotid artery (RCA) of each mouse was not surgically intervened, to provide a control in each mouse. Mice were sacrificed post-one-week ligation surgery, the LCA and RCA were dissected, and the GCX was immunohistochemically stained to determine its percent coverage and thickness. Macrophage infiltration was similarly immunohistochemically detected and quantified. For the second study, C57Bl/6 male mice underwent partial LCA ligation surgery. Ultra-small gold nanospheres (GNS) coated with PEG were fabricated as previously described [4,5]. At day 26 post-ligation, anesthetized mice intravenously received 25 mg of biotin-conjugated PEGylated GNS in 150 µL of sterile phosphate buffer solution (PBS). GNS were then allowed to circulate in the mice for 48 hours. The animals were euthanized, fixed, and dissected after 28 days post-ligation surgery, 2 days after GNS treatment. The carotid arteries were then extracted. Fluorescent staining of biotinylated GNS uptake in vessel wall, and GCX expression on the inner surface of the vessel wall, were performed. All results were expressed as mean ± standard error of mean (SEM). Data sets were compared using Graph Pad Prism software and a two-way ANOVA test to analyze statistical significance between groups with an alpha value of 0.05.

Results: GCX coverage of the endothelium was significantly reduced in the LCAs of mice exposed to disturbed flow by partial LCA ligation, compared to control RCA. No differences were found in GCX coverage of RCAs from all cohorts. Regarding inflammation, no difference in macrophage accumulation in carotid arterial walls was observed when comparing the LCAs and RCAs of mice that did not undergo partial LCA ligation surgery. However, macrophage uptake in vessel walls showed a 14-fold increase in the LCAs exposed to disturbed flow following ligation, when compared to control LCAs, while no such statistical difference was observed between the RCAs of the group. Additionally, GCX degradation in the ligated LCA correlated to approximately 3-fold increase in 10-nm GNS infiltration of the LCA vessel wall in comparison to its control RCA counter-part. This suggests that GCX dysfunction, which coincides with atherosclerosis, can indeed by leveraged and targeted for enhanced nanoparticle-based drug delivery.

Discussion: In summary, it was observed that partial LCA ligation, to acutely disturb blood flow in a mouse vessel, induce blood vessel remodeling, particularly with respect to GCX, and endothelial dysfunction, associated with pro-atherosclerotic recruitment of inflammatory macrophages. Passive targeting of GNS to areas affected by degraded GCX was also observed. The GCX is often neglected even though it acts as a first line of defense against atherosclerosis. Targeting dysfunctional vessels based on GCX shedding offers an innovative approach in early cardiovascular disease therapy and prevention.

Acknowledgements: Funding: National Institutes of Health (KO1 HL125499) and National Science Foundation (DGE-0965843 and CMMI-1846962).

References: [1] Mitra, R., et al., Glycocalyx in Atherosclerosis-Relevant Endothelium Function and as a Therapeutic Target. Curr Atheroscler Rep, 2017. 19(12): p. 63. [2] Mitra, R., et al., The comparative effects of high fat diet or disturbed blood flow on glycocalyx integrity and vascular inflammation. Transl Med Commun, 2018. 3. [3] Nam, D., et al., Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis. Am J Physiol Heart Circ Physiol, 2009. 297(4): p. H1535-43. [4] Cheng, M.J., et al., Endothelial glycocalyx conditions influence nanoparticle uptake for passive targeting. Int J Nanomedicine, 2016. 11: p. 3305-15. [5] Cheng, M.J., et al., Ultrasmall gold nanorods: synthesis and glycocalyx-related permeability in human endothelial cells. Int J Nanomedicine, 2019. 14: p. 319-333.