Intracellular Delivery of Rapamycin From FKBP Elastin-Like Polypeptides Is Consistent With Macropinocytosis

Abstract

Rapamycin (Rapa) is a highly potent drug; however, its clinical potential is limited by poor solubility, bioavailability, and cytotoxicity. To improve Rapa delivery, our team has fused the cognate protein receptor for Rapa, FKBP12, to high molecular weight elastin-like polypeptides (ELPs). One construct, FAF, includes an FKBP domain at each termini of an ELP. In a recent report, FAF/Rapa outperformed a family of related carriers with higher tumor accumulation and efficacy. Despite apparent efficacy, an explanation for how FAF carries Rapa into cells has not been elucidated. This manuscript explores the intracellular fate of FAF in MDA-MB-468, a triple negative (ER-/PR-/HER2-) breast cancer line. Based on a lack of displacement by excess unlabeled FAF, no evidence was found for the involvement of a receptor in cell-surface binding. Cellular association showed no dose-dependent saturation at concentrations up to 100 μM, which is consistent with uptake through fluid phase endocytosis. FAF does colocalize with dextran, a marker of fluid phase endocytosis. Upon internalization, both FAF and dextran target low pH intracellular compartments similarly. Despite likely exposure to lysosomal pH and proteolytic activity, intracellular FAF is eliminated from cells with a relatively long half-life of 17.7 and 19.0 h by confocal microscopy and SDS-PAGE respectively. A split luciferase reporter assay demonstrated that FAF delays the cytosolic access of Rapa in comparison to free drug by 30 min. A specific macropinocytosis inhibitor, amiloride, completely inhibits the cytosolic delivery of Rapa from FAF. Each of these results are consistent with macropinocytosis as the mechanism of cellular uptake necessary for the hand-off of Rapa from FKBP-based drug carriers like FAF to endogenous FKBP12 in the cytosol.

Berunda Polypeptides: Biheaded Rapamycin Carriers for Subcutaneous Treatment of Autoimmune Dry Eye Disease

“…To support this contention, we recently reported that in vitro cellular uptake of FAF-Rapa occurs through macropinocytosis, which differed significantly from simple diffusion of free Rapa. 84 Further studies must now explore if FAF-Rapa exhibits a therapeutic effect at a subtherapeutic dose or frequency of free Rapa. These studies may solve the body weight loss (Table 3) and induction of hyperglycemia (Figure 8) observed during SC FAF-Rapa treatment.…”

“…If true, then it remains plausible that FAF–Rapa will remain associated even while interacting with lymphocytes and normal cells of the LG. To support this contention, we recently reported that in vitro cellular uptake of FAF–Rapa occurs through macropinocytosis, which differed significantly from simple diffusion of free Rapa . Further studies must now explore if FAF–Rapa exhibits a therapeutic effect at a subtherapeutic dose or frequency of free Rapa.…”

Targeted Delivery of a Ligand–Drug Conjugate via Formyl Peptide Receptor 1 through Cholesterol-Dependent Endocytosis

“…In comparison, macromolecule entry into cells is mainly mediated by cell surface receptors through different endocytic mechanisms, including clathrin-mediated endocytosis, clathrin-independent endocytosis (such as caveolae-mediated endocytosis), macropinocytosis, and phagocytosis. − A well-documented example is the transferrin receptor, a dimeric carrier protein for transport of transferrin through clathrin-mediated endocytosis . Another example is the low-density lipoprotein receptor that binds cholesterol apolipoproteins and mediates the transport of LDL into cells through clathrin-dependent endocytosis .…”

Water-Soluble Flexible Organic Frameworks That Include and Deliver Proteins

“…It was found that amiloride inhibited the delivery to the largest extent, by 42% as compared with the nontreated cells, while others did not display apparent inhibition activity, which suggested that the endocytosis of the protein-included FOFs by the cells occurred predominantly via the mechanism of macropinocytosis. 41,42 BSA, GFP, and OFP all bear a large number of hydrophobic side chains, and their isoelectric points are 4.7, 5.8, and 5.8, respectively. Thus, in the weakly alkaline media used for the intracellular…”