(I-325) Nanoparticle endocytosis is driven by monocyte phenotype rather than nanoparticle size under high shear flow conditions.

Monocytes are blood-resident innate immune cells important in initiating pro-inflammatory immune responses and migrating to tissue to aid in wound healing. Due to these key features, they are major players in the development and progression of Atherosclerosis (1). Current research has focused on the use of nanoparticles to specifically target the plaques developed in the bloodstream. Monocytes are also highly phagocytic cells making them responsible for a sizable amount of nanoparticle clearance in the blood (2). We are working on coupling the ideas of using nanotherapeutics for atherosclerosis and the nature of monocytes both in nanoparticle interaction and disease pathogenesis. We are also looking to improve in vitro experimental conditions by increasing the number of variables that are physiologically relevant to disease conditions. In this study, we approached these goals by testing the variables of monocyte phenotype, shear stress, and nanoparticle size. ACE2 knockdown THP-1 monocytes were created to mimic one of the dysregulations observed in atherosclerotic patients. These cells and wildtype THP-1s were exposed to two physiological shears (5 dyne/cm² and 40 dyne/cm²) in the presence of various-size polystyrene nanoparticles, and nanoparticle endocytosis, and monocyte activation were measured. This work is the beginning of the development of nanotherapeutics capable of precision dosing.

Wild-type THP-1 (THP-1 wt) cells or THP-1 cells knocked down for ACE2 (THP-1 ACE2^-) were studied under two different shear regimes; 5 dyne/cm² which is relevant to areas of bifurcations in the vessels where atherosclerotic plaques are more likely to develop, and 40 dyne/cm² which is associated with a smaller probability of plaque development. Shear was applied using a double-gap rheometer for twenty minutes. While being sheared cells were also exposed to fluorescent polystyrene nanoparticles of four different sizes (20nm, 100nm, 200nm, and 500nm), and nanoparticle endocytosis and monocyte response was measured. Samples were taken directly after the 20-minute shear was applied (t=0). Nanoparticle endocytosis was evaluated by the number of cells that took up nanoparticles in a sample and the number of particles taken up per cell. To evaluate monocyte response genetic analysis was done using qPCR. qPCR genes were selected by their relevance in the pathogenesis of atherosclerosis in adhesion, migration, and phenotype categories.

At higher shears ACE2- cells had an increased number of cells that took up particles than both the static ACE2- cells, and WT cells at the same shear (Figure 1). This result also appeared to be independent of size as all sizes tested had this finding (Figure 1). Using the mean fluorescence intensity of the cells we determined that at high shear ACE2- and WT cells took up a similar number of particles per cell across all particle sizes (Figure 2 C, G, K). This highlights that while more ACE2- cells took up particles, the cells that did take up particles do not take up significantly more or fewer particles than a WT cell which took up particles. While still not significant lower shear showed more observable differences between the number of particles being taken up, which ACE2- cells showing diminished uptake (Figure 2 A, E, I). When coupled with the genetic analysis ACE2- showed more upregulation in the gene TNF-alpha which is associated with a pro-inflammatory phenotype (Figure 2 B, F, J). Contrastingly at higher shear WT cells saw more pro-inflammatory activation (Figure 2 D, H, L). At higher shear, the finding that more ACE2- cells took up particles than WT may be indicative of a skew to a more homogenous population of phagocytic monocytes in the higher shear regime. This is further supported by the qPCR analysis that saw very little upregulation of pro-inflammatory genes at high shear in ACE2- cells indicating maintenance of the common phagocytic phenotype. Many studies only use wild-type THP-1s to study the effects of nanoparticles on monocyte, but there is information to be gained from understanding how activation and dynamics change in diseased systems. By studying various factors of disease in in vitro models we have highlighted factors that may not have been elucidated in wild-type only studies. This in combination with the large impact that monocytes have on the pathogenesis of atherosclerosis can highlight important factors which can be used to make targeted and precision-dosed therapeutics that can be delivered intravenously into the system.

(1) Mitra, S.; Goyal, T.; Mehta, J. L. Oxidized LDL, LOX-1 and Atherosclerosis. Cardiovasc. Drugs Ther. 2011, 25 (5), 419. https://doi.org/10.1007/s10557-011-6341-5.

(2) Iversen, T.-G.; Skotland, T.; Sandvig, K. Endocytosis and Intracellular Transport of Nanoparticles: Present Knowledge and Need for Future Studies. Nano Today 2011, 6 (2), 176–185. https://doi.org/10.1016/j.nantod.2011.02.003.