(55a) Substrate Stiffness Regulates CD44 Receptor Mediated Endocytosis of Liposomal Nanoparticles

Nanocarrier systems employ both passive and active mechanisms to achieve targeted delivery of therapeutic cargo. While the passive approach has been used extensively to target numerous cancer tumor types via leaky vasculature and subsequent intra-tumor accumulation, the implementation of active targeting has greatly enhanced per-cell selectivity resulting in reduced offsite toxic effects. One of the major hurdles in utilizing a specific cell surface receptor for active targeting and uptake is the intrinsic fact that their expression and functionality are dynamically controlled by both the state of the cell and the surrounding microenvironment. However, to date no studies have examined how physical changes in the tumor microenvironment such as stiffness effects the expression of targeted receptors on the cell surface for active drug delivery. Herein, we investigated how the commonly exploited CD44 receptor changes as a function of stiffness in both normal and breast cancer cells. To do so, we developed a versatile 2D polymer substrate over a physiologically relevant range of stiffness to generate distinct snapshots of breast cancer progression. We applied hyaluronic acid coated lipid nanoparticles (HALNP) as a model nanocarrier to study the influence of cell receptor expression on HALNP uptake in addition to endolysosomal fate. In the breast cancer cells, the uptake of HALNPs was found to increase with increasing substrate stiffness due to corresponding up regulated CD44 protein levels, while both HALNP uptake and CD44 expression was largely unchanged in normal breast cells as a function of substrate stiffness. To directly assess the capacity in which the CD44-HA endocytosis pathway was employed, two inhibitor pre-treatment assays were performed including 1) excess free HA to saturate the available CD44 receptors and 2) clathrin inhibition to quantify non-specific uptake. These assays validated that the primary route of HALNP cellular entry on the stiff substrate occurred via the CD44 receptor, while uptake on the soft substrate was significantly driven by clathrin-dependence. Lastly, we revealed that there is a correlation between substrate stiffness and HALNP intracellular fate due to differing uptake mechanisms. At low substrate stiffness a significant portion of the HALNP population co-localized with lysosomes whereas on the stiff substrate the HALNPs were homogenously distributed in the cellular cytoplasm. We believe this information can be used to create smarter nanocarrier systems that exploit cell surface receptor changes as a function of the local microenvironment to increase drug delivery efficacy in not only cancer, but any disease influenced by aberrant extracellular matrix maintenance during progression.